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Development and maturation of Philonema species (Nematoda: Philometridae) in salomid hosts with different… Bashirullah, Abul Kashem Mohammed 1966

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THE DEVELOPMENT AND MATURATION OF PHILONEMA SPECIES (NEMATODA: PHILOMETRIDAE) IN SALMONID HOSTS WITH DIFFERENT LIFE HISTORIES by ABUL KASHEM MOHAMMED BASHIRULLAH B. Sc. Dacca U n i v e r s i t y , 1958 M. Sc. Dacca U n i v e r s i t y , 1960 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS OF THE DEGREE OF Doctor of P h i l o s o p h y i n the Department of Zoology We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard The U n i v e r s i t y of B r i t i s h Columbia August, 1966 i n presenting this thesis in pa r t i a l fulfilment of the requirements for an advanced degree at the University of Bri t i s h Columbia, I agree that the Library shall make i t freely available for reference and study,, L further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ZOOLOGY The University of Br i t i s h Columbia Vancouver 8, Canada Date August §1,1966 The University of British Columbia FACULTY OF GRADUATE STUDIES PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY ABUL KASHEM MOHAMMED BASHIRULLAH B.Sc., University of Dacca, 1958 M.Sc., University of Dacca, 1960 MONDAY, AUGUST 29, 1966 AT 3:30 P.M. IN ROOM 3332, BIOLOGY SCIENCES BUILDING External Examiner: Z. Kabata Aberdeen Marine Laboratory Aberdeen, Scotland. Research Supervisor: J. R. Adams of COMMITTEE IN CHARGE Chairman: W. Gage J. R. Adams P. A. Dehnel L. Margolis D. J. Randall W. B. Schofield J. D. McPhail "The Development and Maturation of Philonema Species (Nematoda: Philometridae) in Salmonid Hosts ' with Different Life Histories^." ABSTRACT This study was undertaken to determine the identity of Philonema oncorhynchi from anadromous sockeye and Philonema  agubernaculum from non-anadromous trout, and also to test the hypothesis that the worm in salmon is dependent on hormonal stimulus from the host for synchronization of reproduction. On the basis of differences found in l i f e cycles, cross infection and starch gel electrophoresis, P. oncorhynchi and P. agubernaculum are considered to be different species. The hypothesis was tested experimentally, using salmon pituitary extracts and synthetic s t i l b e s t r o l . Pituitary extracts accelerated the production of larvae in the uterus, of the worm and stilbestrol inhibited the gonadal development of the fish, but had no apparent effect on the worms. The hypothesis was further supported by the results of transplantation of adult but non-larvigerous worms from maturing sockeye into immature trout. Larval development failed to take place in recipient hosts. As -well, larvigerous worms were collected only from sexually mature fish, whether 3, 4 or 5 years old. Rapid development of the worm paralleled.the rapid gonadal.development of.-the fish during the last 6 months of the fish's l i f e . Sexually immature sockeye had only immature worms. GRADUATE STUDIES Field of Study: Zoology Introductory Parasitology Advance Parasitology Experimental Zoology Quantitative Methods of Zoology Ecology Marine Field Course J. R. Adams J. R. Adams Wo S. Hoar J. T. McFadden W. Murdoch P. A. Dehnel Chairman: Professor James R. Adams ABSTRACT This study was undertaken to determine the i d e n t i t y of Philonema oncorhynchi from anadromous sockeye and Philonema  agubernaculum from non-anadromous trout, and also to test the hypothesis that the worm i n salmon i s dependent on hormonal stimulus from the host for synchronization of reproduction. On the basis of differences found i n the l i f e cycles, cross i n f e c t i o n and starch gel electrophoresis, Philonema  oncorhynchi and Philonema agubernaculum are considered to be d i f f e r e n t species. The hypothesis was tested experimentally, using salmon p i t u i t a r y extract and synthetic s t i l b e s t r o l . P i t u i t a r y extract accelerated the production of larvae i n the uterus of the worm and s t i l b e s t r o l i n h i b i t e d the gonadal development of f i s h but had no apparent e f f e c t on the worms. The hypothesis was further supported by the re s u l t s of transplantation of adult but non-larvigerous worms from maturing sockeye into immature trout. Larval development f a i l e d to take place i n the r e c i p i e n t host. As well, the larvigerous worms were c o l l e c t e d only from sexually mature f i s h whether 3, 4 or 5 years old. Rapid development of the worm p a r a l l e l e d the rapid gonadal development of the f i s h during the l a s t 6 months of the f i s h ' s l i f e . Sexually immature sockeye had only immature worms. TABLE O F CONTENTS PAGE GENERAL INTRODUCTION . 1 OUTLINE OF THE PROBLEM 2 SECTION I A comparison of Philonema oncorhynchi i n n a t u r a l l y i n f e c t e d sockeye of C o i t u s Lake and Philonema agubernaculum i n e x p e r i m e n t a l l y i n f e c t e d t r o u t 7 INTRODUCTION . . 7 MATERIALS AND METHODS 7 1. Source of experimental f i s h 7 2. Source of experimental copepods . . . . 9 3. Source of Larvae of Philonema 9 4. I n f e c t i o n and maintenance of Copepods . 10 5. I n f e c t i o n of t r o u t 10 6. F i x a t i o n , S t a i n i n g and Measurements of worms 10 RESULTS . 11 1. D e s c r i p t i o n of t h i r d and f o u r t h stage l a r v a e . . . . . 11 2. Developmental stages of Philonema from sockeye 13 3. Development of P^ agubernaculum i n t r o u t 16 DISCUSSION . . . . . . . . . . . . . . . 18 1. I n f e c t i o n of j u v e n i l e sockeye salmon under n a t u r a l c o n d i t i o n 18 2. Route of i n f e c t i o n of the swimbladder . 21 3. Growth and development of Philonema „ . . 23 4. Escapement of worm from the host . . . . 27 5. V i s c e r a l adhesions 30 TABLE OF CONTENTS (continued) SUMMARY , . . . 31 SECTION II Experimental c r o s s i n f e c t i o n between t r o u t and salmon s t r a i n s . . . . . . . 32 INTRODUCTION . . . . . . . . . . 32 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . 32 1. Source and husbandry of f i s h . . . . . . 32 2. I n f e c t i o n and maintenance of experimental f i s h 33 RESULTS 34 1. I n f e c t i o n of d i f f e r e n t salmonid and gold f i s h with Philonema from sockeye . . . . 34 2. I n f e c t i o n of sockeye, pink, s p r i n g , kokanee and go l d f i s h with t r o u t s t r a i n s 36 DISCUSSION . 33 SECTION I I I Hormonal treatments 47 INTRODUCTION 47 MATERIALS AND METHODS . . . . . . . . . . . . 47 1. P r e p a r a t i o n of salmon p i t u i t a r y e x t r a c t s 43 2. I n j e c t i o n of salmon p i t u i t a r y e x t r a c t s . 48 a) Shuswap Lake f i s h . . . 48 b) Great C e n t r a l Lake f i s h 49 3. I n j e c t i o n of 1 7 - b - e s t r a d i o l 50 4. Implantation of s t i l b e s t r o l p i l l s . . . 50 5. Feeding of s t i l b e s t r o l powder . . . . . 50 RESULTS . . . , 51 1. Shuswap Lake f i s h . . . 51 2. Great C e n t r a l Lake f i s h . „ 51 a) E f f e c t of salmon p i t u i t a r y e x t r a c t s . 51 b) E f f e c t of s t i l b e s t r o l . 53 i v TABLE OF CONTENTS (Continued) DISCUSSION 55 SECTION IV T r a n s p l a n t a t i o n of worms 65 INTRODUCTION 65 MATERIALS AND METHODS 65 1. Worms used f o r t r a n s p l a n t a t i o n . . . . . . . 65 2. R e c i p i e n t hosts 66 3. T r a n s p l a n t a t i o n of a d u l t worms i n t o f i s h 66 4. T r a n s p l a n t a t i o n o f l a r v a l worm i n t o f i s h . 67 5. T r a n s p l a n t a t i o n of a d u l t and l a r v a l worms i n t o frogs . , 67 RESULTS 68 1. T r a n s p l a n t a t i o n of a d u l t P. oncorhynchi from matured sockeye i n t o immature rainbow t r o u t 68 2 ( a ) . T r a n s p l a n t a t i o n of t h i r d stage l a r v a e i n t o the stomachs of s t e e l h e a d , rainbow t r o u t and sockeye salmon 69 2 ( b ) . T r a n s p l a n t a t i o n of t h i r d stage l a r v a e i n t o body c a v i t y of t r o u t 71 3. T r a n s p l a n t a t i o n of f o u r t h stage l a r v a e i n t o the stomach and body c a v i t y of t r o u t 72 4. T r a n s f e r of i n f e c t i o n by p r e d a t i o n . . . 72 5. T r a n s p l a n t a t i o n of a d u l t and f o u r t h stage l a r v a e of P^ _ oncorhynchi i n t o f r o g s . . 73 6. T r a n s p l a n t a t i o n of f o u r t h stage l a r v a e i n t o the abdominal c a v i t y of go l d f i s h . 73 DISCUSSION 74 V TABLE OF CONTENTS (Continued) SECTION V Development of P h i l onema oncorhynchi i n n a t u r a l l y i n f e c t e d sockeye o i R i v e r s I n l e t . 79 INTRODUCTION 79 MATERIALS AND METHODS 80 1. C o l l e c t i o n and measurement of a d u l t Philonema oncorhynchi . . . . 81 RESULTS 82 DISCUSSION 85 GENERAL SUMMARY AND CONCLUSIONS 91 LITERATURE CITED 93 APPENDIX 100 ERRATA p. 4 l a s t l i n e p. 7 Para 5 l i n e 1 p. 13 Para 3 l i n e 10 p. 15 Para 2 l i n e 9 p. 20 Para i l i n e 10 p. 23 Para 3 l i n e 3 p. 27 l i n e 4 p. 31 Para 2 l i n e 6 p. 34 l i n e 3 p. 34 Para 2 l i n e 3 p. 49 l i n e 8 p. 51 l i n e 1 p. 53 Para 3 l i n e 1 p. 57 l a s t l i n e p. 58 Para 2 l i n e 4 p. 75 l i n e 15 p. 77 l i n e 1 p- 78 l a s t para l i n e 1 and 3 I p. 79 Para 3 l i n e 8 p. 84 Para 2 l i n e 6 p. 88 Para 2 l i n e 2 p. 95 Ishida, T and et a l , 1958 va r i a t i o n in for v a r i a t i o n from' inse r t " i n f e c t i o n of trout with, experimentally" a f t e r experimental v'rs'ceral for v i s e r a l i n s e r t " i n diameter" a f t e r 5.5 cm' ins e r t "the l i f e cycle" in place of "that" inse r t "either" a f t e r "due" read Philometra for Philonema read "mature only" for 'is matured' read "uneaten" for 'unfed' inse r t "amount" afte r minimal' inse r t "were" af t e r 'fish' i n s e r t "were" afte r 'fish' read "between 1 and 23 days" for'between 173 days' inse r t "According to Gorshkov" before 'ontogenesis' in s e r t "occurs" aft e r 'this' i n s e r t "Transplanted" before 'fourth stage' and delete "transplanted" from l i n e 16 substitute "tissue" for 'epithelial' delete "had" substitute " i s " for may be (merely)' in s e r t "were calculated also by regression against si z e of the worms" af t e r 'worms' inser t "The reproductive cycle" before 'of oncorhynchi' in s e r t "waters" aft e r 'offshore'' FIGURE LIST OF FIGURES PAGE 1. Development of Philonema oncorhynchi in sockeye salmon of various ages from Cultus Lake 17 2. Development of Philonema agubernaculum in experimen-t a l l y infected trout 19 3. Diagrammatic comparison of the spawning areas and the migrations of trout and salmon with those of P.  agubernaculum and P. oncorhynchi in t h e i r trout and salmon hosts respectively ~ . . 26 4. Development of P. oncorhynchi in experimentally infected trout, IT two years old 35 5. Development of P. oncorhynchi from sockeye salmon in experimentally" infected trout f r y . 37 6. Development of P. oncorhynchi from trout in experimentally infected f r y of sockeye salmon . . 39 7. Hypothetical reproductive i s o l a t i o n of P.  oncorhynchi and P_^  agubernaculum in salmonids (based on 4 year reproductive cycle of sockeye salmon) 41 8. A comparison of starch gel electropherograms of P. agubernaculum from trout and I\_ oncorhynchi From sockeye salmon 46 9. E f f e c t of crude salmon p i t u i t a r y extracts on the production of larvae in P. oncorhynchi 52 10. E f f e c t of synthetic s t i l b e s t r o l on the production of larvae in P_^  oncorhynchi 54 11. E f f e c t of implanted synthetic s t i l b e s t r o l on the progressive growth of testes of sockeye salmon from the Great Central Lake 56 12. Development of P. oncorhynchi in d i f f e r e n t trans-planted salmonids 70 v i FIGURE PAGE 13. R e l a t i o n s h i p between the degree of maturation of sockeye salmon of R i v e r s I n l e t and time of ca t c h . 83 14. R e l a t i o n s h i p between the ovary weight and the p r o d u c t i o n of l a r v a e i n P. oncorhynchi from sockeye salmon of R i v e r s I n l e t '. . '. 7 86 15. R e l a t i o n s h i p between the t e s t i s weight of f i s h and the p r o d u c t i o n of l a r v a e i n P. oncorhynchi from sockeye salmon of R i v e r s I n l e t 87 LIST OF TABLES TABLE PAGE 1. Stages of Philonema oncorhynchi found i n C u l t u s Lake sockeye salmon of v a r i o u s ages 100 2. Measurements of Philonema oncorhynchi of sockeye salmon of d i f f e r e n t ages from C u l t u s Lake . . . 101 3. Development of Philonema agubernaculum i n e x p e r i -m e n t a l l y i n f e c t e d t r o u t 103 4. E f f e c t of 1 7 - b - e s t r a d i o l , salmon p i t u i t a r y e x t r a c t s and s t i l b e s t r o l on Philonema s p e c i e s i n immature 2 year o l d sockeye from Shuswap Lake . 104 5. E f f e c t on p i t u i t a r y e x t r a c t s on female Philonema recovered from the t r e a t e d f i s h . . . ~ ". '. T 105 6. -Effect of salmon p i t u i t a r y on sockeye salmon . . 106 7. E f f e c t of s t i l b e s t r o l on female Philonema r e c o v e r -ed from the implanted f i s h 107 8. E f f e c t of implanted s t i l b e s t r o l on the t e s t e s of "prespawning sockeye from Great C e n t r a l Lake . . 108 9. Recovery of Philonema oncorhynchi from the body c a v i t y of t r a n s p l a n t e d t r o u t . 109 10. Recovery of t r a n s p l a n t e d worms from the salmoids 111 11. Recovery of t r a n s p l a n t e d a d u l t and f o u r t h stage l a r v a e from the body c a v i t y of f r o g s 112 12. Summary of m e r i s t i c measurements of f i s h of d i f f e r e n t age composition of R i v e r s I n l e t , . . 113 13. Summary of m e r i s t i c measurements of Pi" b n c d r h y n c h i o f " d i f f e r e n t age composition of R i v e r s I n l e t . . 114 14. Measurements of Philonema oncorhynchi from R i v e r s I n l e t Sockeye '. '. 115 15. R e s u l t s of r e g r e s s i o n of worms on age of f i s h and time caught 116 ACKNOWLEDGMENT The author i s gr> at e f u l to Professor James R,. Adams for his supervision, for his unbiased criticism,, and for his helpful suggestions in the preparation of t h i s manuscript* Sincere gratitude i s e s p e c i a l l y extended to Dr. Leo Margolis of the Fishe r i e s Research Board of Canada, Nanaimo, from whom the author always received stimulating encouragement, who allowed free access to unpublished data,, and who helped in many more ways than the author can put into words. Many technical d i f f i c u l t i e s during the early phase of this work were overcome and new methods developed through c r i t i c a l discussion with Dr. G. G. Gibson. The author wishes to express his appreciation to Drs. C. C. Lindsay, P. A. Dehnel, I. A. Effo r d , and W. B. Schofield for t h e i r constructive suggestions in the preparation of t h i s t h e s i s . The author wishes to thank Professor W. S. Hoar for providing synthetic s t i l b e s t r o l , Dr. M. Smith for supplying crude salmon p i t u i t a r y extracts, and Dr. H. Tsyuki and Mrs. E. Roberts for assistance with the electrophoresis. Dr. D. J. Randall who was always prepared to supply experimental f i s h and advice, i s acknowledged with much appreciation. The author wishes to thank Mr. Melvin Weisbert for supplying hatchery-reared salmonids. The assistance of Dr. J . T. McFadden in the s t a t i s t i c a l analysis i s acknowledged. Thanks are extended to Dr. M. Shepgard of the Fis h e r i e s Research Board of Canada, Nanaimo, for his interest in Philonema and for suggesting the problem in sockeye salmon of Rivers I n l e t . Mr. T. Bq:lton, Mr. N. Boyce, personnel of the Fish Culture Room, and many others of the B i o l o g i c a l Station provided assistance during the l a t e r phase of t h i s study at Nanaimo. The author acknowledges the contributions of the following organizations who made d i f f e r e n t kinds of f i s h available for th i s study: The B r i t i s h Columbia Fish and Game Branch, International North P a c i f i c Salmon Commission, Department of Fi s h e r i e s , B i o l o g i c a l Station of the Fis h e r i e s Research Board df Canada at Nanaimo, and the B r i t i s h Columbia Research Council. The author extends his sincere appreciation to a l l those who helped him in the f i e l d , and e s p e c i a l l y to Mr. James Adams, Mr. Gordon Halsey, and Mr. B i l l Woodall. Mrs. Z. J u r i c i c took care of experimental f i s h , and her assistance in the laboratory i s acknowledged. Acknowledged with thanks i s the f i n a n c i a l assistance provided by the Canadian Commonwealth Scholarship and Fellows-ship Committee. Thanks are due to a l l those who have given the author help and advice during the course of t h i s study. The author wishes to thank Mrs. R. C o l l i s for her excellent typing of t h i s manuscript. F i n a l l y , the author i s indebted to Professor H. K. Yosufzai for his encouragement. GENERAL INTRODUCTION The l i t e r a t u r e of P a r a s i t o l o g y c o n t a i n s s e v e r a l demonstrations or suggestions that the c o r r e l a t i o n observed between the r e p r o d u c t i o n of a p a r a s i t e and the seasonal r e p r o d u c t i o n of i t s host i s due t o a response of the p a r a s i t e t o the ho s t s ' hormones (Smyth & Mofty, 1964; Barrow, 1962; M i r e t s k i , 1951; R o t h s c h i l d , 1964a). A p a r a s i t i c nematode, Philonema oncorhynchi i s found i n the coelomic c a v i t y of the anadromous sockeye salmon. The p a r a s i t e i n f e c t s the young f i s h d u r i n g t h e i r e a r l y fresh-water l i f e , but develops very s l o w l y . F i n a l maturation of the worm c o i n c i d e s with the maturation of the f i s h h o s t . Spawning salmon c o n t a i n r i p e worms ready t o r e l e a s e t h e i r l a r v a e . Non-anadromous t r o u t , char, and w h i t e f i s h are a l s o p a r a s i t i z e d by a Philonema which may or may not be c o n s p e c i f i c with that i n salmon. T h i s study was undertaken t o e l u c i d a t e the l i f e c y c l e of Philonema with p a r t i c u l a r r e f e r e n c e t o the hypothesis that the worm i n salmon i s dependent on hormonal s t i m u l u s from the host f o r s y n c h r o n i z a t i o n of r e p r o d u c t i o n . O u t l i n e of the Problem Kuitunen-Ekbaum (1933) d e s c r i b e d Philonema oncorhynchi from the abdominal c a v i t y of ad u l t Oncorhynchus nerka from E n g l i s h Bay, B r i t i s h Columbia. Smedley (1933) d e s c r i b e d the same s p e c i e s of worm from the body c a v i t y of sockeye salmon from C u l t u s Lake, B r i t i s h Columbia. Three years l a t e r , Simon and Simon (1936) d e s c r i b e d another s p e c i e s , Philonema agubernaculum from the body c a v i t y and muscles of the abdominal w a l l of Prosopium w i l l i a m s o n i ( G i r a r d ) , S a l v e l i n u s f o n t i n a l i s ( M i t c h e l l ) and Salmo shasta (Jordan) which were c o l l e c t e d i n Wyoming N a t i o n a l F o r e s t . They e r e c t e d t h i s new s p e c i e s mainly on the b a s i s of s m a l l e r s i z e and the r a t i o of a n t e r i o r t o p o s t e r i o r oesophagus. P l a t z e r and Adams ( i n press) worked out the l i f e c y c l e of Philonema oncorhynchi and found Cyclops b i c u s p j d a t u s was an int e r m e d i a t e h o s t . Two molts occur i n the copepod. T h i r d stage l a r v a e are the i n f e c t i v e stage f o r f i s h . Two f u r t h e r molts occur i n the salmon. i Since the t r a n s m i s s i o n of P. oncorhynchi occurs only i n f r e s h water, i t i s obvious that the worms must ad j u s t t o the l o n g r e p r o d u c t i v e c y c l e of the f i s h . Only those worms i which are g r a v i d when the f i s h spawns can perpetuate the s p e c i e s P. agubernaculum on the other hand has been r e p o r t e d from l o c a l i t i e s i n which f i s h h o sts are unable t o make a seaward m i g r a t i o n . L i f e c y c l e must be completed e n t i r e l y i n f r e s h water. A d u l t P^ agubernaculum have been found i n t r o u t l e s s than a year o l d ( T a y l o r , unpublished d a t a ) . 3 P l a t z e r (1964) made a morphometric a n a l y s i s of Philonema taken from salmonoids i n two g e o g r a p h i c a l l y separated areas, C u l t u s and Kootenay Lakes, B r i t i s h Columbia. C u l t u s Lake c o n t a i n e d anadromous f i s h ; Kootenay Lake only l a n d l o c k e d s p e c i e s . A d u l t worms taken from white f i s h , Prosopium  w i l l i a m s o n i , D o l l y Varden, S a l v e l i n u s malma and kamloops t r o u t , Salmo g a i r d n e r i , were g e n e r a l l y s m a l l e r than those from sockeye salmon. Comparisons of t o t a l body l e n g t h , d i s t a n c e t o nerve r i n g , muscular and g l a n d u l a r oesophagus, t a i l and s p i c u l e l e n g t h r e v e a l e d a tremendous v a r i a b i l i t y of those morphometric aspects i n the genus Philonema. P l a t z e r d i d not f i n d any ta x o n o m i c a l l y s i g n i f i c a n t m o rphological d i f f e r e n c e s between specimens of Philonema oncorhynchi Kuitunen-Ekbaum from C u l t u s Lake and the type specimen of Philonema agubernaculum Simon and Simon. He concluded t h a t the two s p e c i e s c o u l d not be separated on morphological grounds, Seven other s p e c i e s of Philonema have been d e s c r i b e d t o date on the b a s i s of t o t a l body l e n g t h and r a t i o of muscular t o g l a n d u l a r oesophagus i n both male and female worms (Richardson 1937; F u j i t a , 1940; Bauer, 1946; Meyer, 1960 and Rumyantsev, 1965). Akhmerov (1955) s t u d i e d Philonema from Oncorhynchus  nerka, Oncorhynchus k e t a , and S a l v e l i n u s leucomaenis and com-pared them with the s p e c i e s t h a t were d e s c r i b e d by F u j i t a , Bauer and Simon and Simon. He s t a t e d t h a t Philonema agubernaculum Simon and Simon, Philonema e l o n g a t a F u j i t a and Coregonema  s i b i r i c a Bauer were synonyms of Philonema oncorhynchi Kuitunen 4 Ekbaum. He d i d not synoriomize F u j i t a ' s other four s p e c i e s , P. tenuicaUda, P. kondai, P. s a l v e l i n i , and P. ochotense s i n c e the d e s c r i p t i o n s were not a v a i l a b l e t o him. Although Akhmerov (1955) d i d not f i n d any morphological d i f f e r e n c e between Philonema oncorhynchi and Coregonema s i b i r i c a , he noted t h a t t h e i r hosts and t h e i r e c ology were d i f f e r e n t . He proposed t h e r e f o r e that the nematode Coregonema s i b i r i c a be c o n s i d e r e d as a subspecies of P. oncorhynchi and named Philonema  oncorhynchi s i b i r i c a . S p a s s k i i e t a l (1958, 1959) and S t r e l k o v and Shulman (1960) accepted Akhmerov's synonyms but a c o n t r a r y o p i n i o n was formed, by Rumyantsev (1965) who reduced the genus Coregonema Bauer, 19^46 i n t o a synonym of the genus Philonema Kuitunen-Ekbaum, 1933, and e s t a b l i s h e d Philonema s i b i r i c a (synonym Coregonema s i b i r i c a ) as an independent s p e c i e s . He s t a t e d t h a t Philonema s i b i r i c a shows some s i m i l a r i t y t o P.  agubernaculum, and c o n s i d e r e d i t p o s s i b l e that they may prove to be the same s p e c i e s (Rumyantsev, 1965). T h i s review of the l i t e r a t u r e i n d i c a t e s both lumping and s p l i t t i n g of the genus Philonema mainly on the grounds of s i z e and d i f f e r e n c e s i n the b i o l o g y of h o s t s . Both i n A s i a and i n North America the Philonema from anadromous salmon appear t o be b i o l o g i c a l l y d i s t i n c t from thostf.< of non-anadromous h o s t s . However, two s p e c i e s can not be separated by morphological c r i t e r i a . Reported d i f f e r e n c e s i n s i z e and p a t t e r n of l i f e c y c l e c o u l d r e s u l t from v a r i a t i o n from the response of a s i n g l e 5 species of Philonema to the environments provided by the ph y s i o l o g i c a l l y d i f f e r e n t hosts. The differences might equally be due to the existence of d i f f e r e n t b i o l o g i c a l species which are genetically d i s t i n c t . The presence of gravid worms only in sexually maturing sockeye salmon (Platzer, 1964), but in trout as young as one year (Taylor, unpublished data), suggest the following hypotheses: i ) That there are two ph y s i o l o g i c a l l y d i f f e r e n t s t r a i n s of Philonema which may deserve the status of di f f e r e n t b i o l o g i c a l species, i i ) that the form in trout (P^ agubernaculum)has a developmental cycle of a year or less in length and i s i n d i f f e r -ent to the hormonal changes in i t s hosts, i i i ) that the form in salmon (P. oncorhynchi) has a long developmental period dependent on hormonal changes in i t s hosts for synchronization of i t s reproduction process to that of the salmon. In support of these hypotheses f i v e l i n e s of invest-igations w i l l be presented. i ) A comparison of the developmental rate and maturation of the two st r a i n s in the i r normal hosts, i i ) the res u l t s of i n f e c t i n g salmon with the trout s t r a i n (Pj_ agubernaculum) and in trout with the salmon s t r a i n (P. oncorhynchi), i i i ) the e f f e c t on the worm in salmon of treatments of the hosts with p i t u i t a r y extracts and gonadal hormones, 6 i v ) the e f f e c t on the worm of t r a n s p l a n t a t i o n from normal a d u l t hosts t o other salmonids and abnormal h o s t s , v) a study of the p a t t e r n of development and maturation o h Philonema i n a n a t u r a l p o p u l a t i o n of salmon comprising f i s h which mature v a r i o u s l y i n t h e i r 3rd, 4th or 5th y e a r s . Each of these i s presented inaseparate s e c t i o n . 7 SECTION I. A comparison of the development of Philonema  oncorhynchi i n n a t u r a l l y i n f e c t e d sockeye of C u l t u s Lake, and of Philonema agubernaculum i n e x p e r i m e n t a l l y i n f e c t e d t r o u t . INTRODUCTION P l a t z e r and Adams ( i n press) d e s c r i b e d the general development of P^ oncorhynchi i n e x p e r i m e n t a l l y i n f e c t e d sockeye salmon. They showed t h a t the t h i r d stage l a r v a e i n h a b i t the t i s s u e of the swimbladder. Fourth stage l a r v a e were found i n the body c a v i t y of n a t u r a l l y i n f e c t e d f i s h a f t e r 23 months. However, the p r e c i s e r a t e of development c o u l d not be s t a t e d because of the in f r e q u e n c y of sampling. The development i n the f i s h i s r e p o r t e d here with g r e a t e r p r e c i s i o n based on sampling of n a t u r a l l y i n f e c t e d sockeye salmon maintained i n s a l t water, some f o r a p e r i o d of over 2 y e a r s . The development of P^ agubernaculum i n t r o u t has never been r e p o r t e d . Meyer (1958, 1960) and V i k (1964) s u c c e s s f u l l y i n f e c t e d Cyclops s p e c i e s with l a r v a e of P.  agubernaculum but were unable t o tr a n s m i t the p a r a s i t e t o young f i s h h o s t . T h i s s e c t i o n a l s o r e p o r t s the experimental i n f e c t e d Cyclops and d e s c r i b e s the development of I\_ agubernaculum 8 over the p e r i o d of 182 days which the f i s h s u r v i v e d . The development of the two forms i n t h e i r normal hosts i s compared. MATERIALS AND METHODS Source of Experimental F i s h a) Downstream migrant sockeye salmon were obtained from C u l t u s Lake i n May, 1965. A l l one year o l d f i s h , exam-in e d at t h i s time, were i n f e c t e d with t h i r d stage l a r v a e of Philonema oncorhynchi. The smolts were maintained i n sea water and examined i n f r e q u e n t l y u n t i l June 1966, t o determine the development of Philonema. b) Three year o l d n a t u r a l l y - i n f e c t e d sockeye from C u l t u s Lake maintained i n s a l t water s i n c e capture as down-stream migrants i n 1963. c) Wild a d u l t p r e c o c i o u s males, and pre-spawning fo u r year o l d male and female- sockeye salmon were captured from Sweltzer Creek at C u l t u s Lake, B r i t i s h Columbia. They p r o v i d e d data on the f i n a l stages of development of P.  oncorhynchi. d) Y e a r l i n g h a t c h e r y - r e a r e d rainbow t r o u t were obtained from the Summerland Hatchery of B. C. F i s h and Game Branch. They were used f o r experimental i n f e c t i o n with P.  agubernaculum. 9 Source of Experimental Copepods Plankton was c o l l e c t e d i n Smith Bay at C u l t u s Lake. Cyclops b i c u s p i d a t u s were i s o l a t e d by methods s i m i l a r t o those of P l a t z e r (1964) and maintained i n a room with a con-o s t a n t temperature of 10 C as stock c u l t u r e f o r i n f e c t i o n . t Source of Larvae of Philonema a) Philonema oncorhynchi: F u l l y g r a v i d females were obtained g e n e r a l l y from female sockeye salmon (Oncorhynchus nerka) from the Sweltzer Creek f i s h t r a p at C u l t u s Lake, B r i t i s h Columbia. Only g r a v i d females c o n t a i n i n g moving f i r s t stage l a r v a e i n the u t e r u s were s e l e c t e d f o r the c o l l e c t i o n of l a r v a e t o i n f e c t copepods. One to f i v e g r a v i d females were t r a n s f e r r e d i n t o a f i n g e r bowl c o n t a i n i n g lake water. Worms bu r s t w i t h i n a minute t o r e l e a s e the l a r v a e . The water con-t a i n i n g l a r v a e and d e b r i s of g r a v i d females were f i l t e r e d through #12 b o l t i n g s i l k . F i r s t stage l a r v a e passed through the b o l t i n g s i l k e a s i l y . The l a r v a e were kept i n a r e f r i g e r a t o r at approximately 8°C before i n f e c t i o n . b) Philonema agubernaculum: i A female rainbow t r o u t , Salmo g a i r d n e r i of t h i r t e e n pounds was c o l l e c t e d from Trout Lake, B r i t i s h Columbia by u s i n g a g i l l net. The f i s h was brought t o the U n i v e r s i t y of B r i t i s h Columbia i n an i c e packed c o o l e r on May 27, 1965. The same procedure was used t o c o l l e c t the f i r s t stage l a r v a e as f o r Philonema oncorhynchi. 10 I n f e c t i o n and Maintenance of Copepods Approximately four hundred copepods were f i l t e r e d out of a stock j a r through a #12 net i n t o each of a s e r i e s of four i n c h f i n g e r bowls. Each d i s h was i n o c u l a t e d with 1600 t o 2000 Philonema l a r v a e and p l a c e d i n the r e f r i g e r a t o r f o r twenty-four or f o r t y - e i g h t hours. The l a t t e r i n t e r v a l was found t o produce 80 t o 100% i n f e c t i o n of copepods with about 2 l a r v a e per copepod. I n f e c t e d copepods were t r a n s f e r r e d t o an 8 in c h s t a c k i n g d i s h and c u l t u r e d at 10°C. Larvae i n copepods reached i n f e c t i v e 3rd stage i n 37 days. I n f e c t i o n of Trout F i f t y copepods were conc e n t r a t e d i n t o a smal l volume of water and q u i c k l y t r a n s f e r r e d t o a #5 g e l a t i n c a p s u l e . The capsule was pushed through a g l a s s tube i n s e r t e d i n t o the stomach of an a n e s t h e t i z e d f i s h . I t took two people t o complete t h i s o p e r a t i o n as the capsule becomes s o f t and wet within a minute. F i s h were tagged and t r a n s f e r r e d i n t o h o l d i n g tanks. A l l the f i s h were f e d standard wet food every a l t e r n a t e day. F i x a t i o n , S t a i n i n g , and Measurements of Worms, L i v e l a r v a e and a d u l t Philonema were f i x e d i n formalin-acetic a c i d and preserved i n e i t h e r FAA or 70% et h a n o l whereas worms from f r o z e n and f o r m a l i n preserved f i s h were t r a n s f e r r e d d i r e c t l y t o 80% e t h a n o l . 11 For the s t a i n i n g and measurement of l a r v a e ^ f o l l o w -i n g m o d i f i c a t i o n s of Goodey's ( F r a n k l i n & Goodey, 1949) and P l a t z e r ' s (1964) techniques were made: 1) A drop or two of 0.0005% co t t o n blue i n l a c t o -phenol was p l a c e d on a c l e a n s l i d e . Larvae were added. 2) G l a s s wool, g l a s s rod or a sm a l l p i e c e of a n a l y t i c a l f i l t e r paper, depending on the t h i c k n e s s of the l a r v a e , were p l a c e d i n the drop as support f o r a c o v e r s l i p . 3) The c o v e r s l i p was a p p l i e d and s e a l e d with gum mastix p a r a f f i n (50 p.c. p a r a f f i n p l u s 50 p.c. gum m a s t i x ) . Larvae were measured under a compound microscope u s i n g an o c u l a r micrometer; somewhat l a r g e r male worms were measured by usingcvscale p r o j e c t o r ; very l a r g e female worms were measured un d e r a b i n o c u l a r microscope. RESULTS D e s c r i p t i o n of T h i r d and Fourth Stage Larvae T h i r d Stage: Ko (1966) d e s c r i b e d t h i r d stage l a r v a e of Philonema  oncorhynchi which were e x p e r i m e n t a l l y r e a r e d i n copepods. These l a r v a e are m o r p h o l o g i c a l l y i d e n t i c a l t o those taken from f i s h e x p e r i m e n t a l l y i n f e c t e d f o r 55 days. T h i r d stage l a r v a e taken from downstream m i g r a t i n g sockeye d i f f e r only s l i g h t l y from those i n copepods. The body i s f i l i f o r m i n shape, t a p e r i n g p o s t e r i o r l y . The mean body l e n g t h i s 1.16 mm,; body width at the oesophageal i n t e s t i n a l j u n c t i o n i s 16.66 /u. The c u t i c l e i s d i l a t e d at 12 the b u c c a l r e g i o n forming two l i p - l i k e s t r u c t u r e s . The t a i l has rounded shoulders near i t s tapered t i p . The nerve r i n g i s 104.3 /u from the a n t e r i o r end. The muscular oesophagus i s 248.9 /u l o n g , s l e n d e r and the g l a n d u l a r oesophagus w e l l developed (480.6 yu l o n g ) , g r a d u a l l y expanding p o s t e r i o r l y . The o e s o p h a g e a l — i n t e s t i n a l v a l v e i s w e l l developed, pro-t r u d i n g i n t o the lumen of the i n t e s t i n e . The g e n i t a l primordium i s o v a l and mostly at the a n t e r i o r p o r t i o n of the i n t e s t i n e . The rectum i s made up of two r e g i o n s : a n t e r i o r r e g i o n with thick w a l l s and narrow lumen and p o s t e r i o r r e g i o n , a f i n e tube connecting to the e x t e r i o r . R e c t a l glands are w e l l developed. The l a t e t h i r d stage p r i o r t o m o l t i n g d i f f e r s from the t h i r d stage i n the f o l l o w i n g ways: 1) Mean body l e n g t h , 3.32 mm.; nerve r i n g , 143.3 from the a n t e r i o r end; muscular oesophagus, 312.7 /u l o n g , s l e n d e r and w e l l separated from the grandular oesophagus, 773.6 /u l o n g . 2) C u t i c l e d i l a t e d at b u c c a l r e g i o n to produce rounded head without l i p s . 3) O e s o p h a g e a l - - i n t e s t i n a l valve becomes more prominent. Fourth Stage: The f o l l o w i n g a d d i t i o n i s made t o the d e s c r i p t i o n of the f o u r t h stage l a r v a e by P l a t z e r and Adams ( i n p r e s s ) : 1) The f o u r t h stage i n the swimbladder averaged 4.1 mm. i n l e n g t h , the nerve r i n g 149 /u from the a n t e r i o r 13 end, muscular oesophagus, 329.8 /u and g l a n d u l a r oesophagus, 881.6 /u l o n g . 2) A n t e r i o r end b l u n t l y rounded. 3) T a i l changed t o p o i n t e d t i p . 4) Sex of the l a r v a e u n d i f f e r e n t i a t e d while i n the t i s s u e of the swimbladder. Those i n the body c a v i t y of l a r g e r s i z e (5.22 - 9.5 mm.) show rudimentary sexual c h a r a c t e r s . Developmental Stages of Philonema from Sockeye In May, 1965, sampling of y e a r l i n g smolts showed only t h i r d stage l a r v a e of Philonema i n the swimbladder. However, two year o l d downstream migrants had t h i r d , l a t e t h i r d , and f o u r t h stage l a r v a e i n the swimbladder and f o u r t h stage l a r v a e and s u b - a d u l t s i n the body c a v i t y (Table I . ) . By August, 1965, a few l a r v a e of Philonema  oncorhynchi showed growth i n the swimbladder of smolts and one f o u r t h stage l a r v a had migrated i n t o the body c a v i t y . One female sub-adult and a f o u r t h stage l a r v a were found i n the p e r i t o n e a l c a v i t y of a f i s h a month l a t e r . F u r t h e r sampling was i m p o s s i b l e u n t i l January, 1966, when the f i s h were twenty months o l d . Some male and female sub-adult P. oncorhynchi were found i n the body c a v i t y and one female worm had grown t o a l e n g t h of 89.9 mm. By A p r i l , 1966, more worms were found i n the body c a v i t y and v i s e r a l adhesions appeared. The m a j o r i t y of the worms present were male.. A l l stages of P. oncorhynchi with the e x c e p t i o n of a d u l t females 14 were found i n f i s h when examined i n June, 1966. More v i s c e r a l adhesion was observed i n these f i s h r e g a r d l e s s of sex. F r e s h l y encased male worms were found when the adhesion was teased a p a r t . During the same p e r i o d , f i s h maintained i n seawater tanks i n the l a b o r a t o r y - grew from an average f o r k l e n g t h of 8.2 cm. t o 20.8 cm. and i n c r e a s e d i n weight from an average 8.1 gm. t o 88.8 gm. Three immature sockeye c o l l e c t e d from C u l t u s Lake as smolts i n 1963 and maintained i n seawater tanks i n the l a b o r a t o r y u n t i l May, 1965 were examined on t h a t date. Fourth stage, sub-adult and a d u l t males of Philonema  oncorhynchi were found i n the body c a v i t y of these f i s h . No t h i r d stage l a r v a e were found i n the swimbladders. V i s e r a l adhesions were present i n the f i s h and dead and encased worms were found i n loose f i b r o u s t i s s u e of the adhered mass. Pre c o c i o u s male sockeye were examined i n October, 1965, f o u r t h stage l a r v a e o c c u r r e d i n the swimbladder; sub-a d u l t and a d u l t ohcorhynchi were present i n the body c a v i t y . An estimated maximum number of 335 f o u r t h stage l a r v a e were found i n the swimbladder of one of these f i s h . A month l a t e r (November, 1965) most of the l a r v a e had migrated from the swimbladder i n t o the body c a v i t y of the f i s h when c o l l e c t e d from the spawning ground. More male than female worms o c c u r r e d i n the body c a v i t y . During the 15 study three g r a v i d female worms were found i n the body c a v i t y . A few sub-adult male worms were c o l l e c t e d from the swimbladder. V i s c e r a l adhesions were present i n 8 of 10 p r e -coc i o u s males. The i n t e n s i t y of adhesion was scored as from absent t o heavy i n October and absent t o l i g h t i n November. The adhesions o c c u r r e d p r i m a r i l y i n the r e g i o n of the p y l o r i c caeca. T h i n s t r a n d s of t i s s u e were found t o bridge the abdominal c a v i t y when the two body w a l l s of f i s h c o n t a i n i n g v i s e r a l adhesion were teased a p a r t . In the f i s h examined i n November, 1965, f r e s h l y e n cysted female worms were found. L i v e females c o u l d be taken out of the incomplete c y s t s . F r e s h c y s t s were f i l l e d with w h i t i s h f l u i d . Thin f i b r o u s l a y e r s of t i s s u e were found i from the end of the p e r i c a r d i u m t o the end of the stomach and t h i s l a y e r of t i s s u e c o u l d be taken out s e p a r a t e l y . Many c y s t s with unrecognizable worms were found attached t o the l i v e r , t o the body w a l l or f r e e i n the body c a v i t y . Cysts measured from 0.5 t o 5.5 cm. Larvae were not g e n e r a l l y found i n the swimbladder, ( a d u l t spawning f i s h ) but one f o u r t h stage l a r v a e was found i n the swimbladder of a 4g male sockeye. . G r a v i d female Philonema oncorhynchi were more common i n female than i n male f i s h . In female f i s h , i n September, 10% of the a d u l t worms harboured were g r a v i d whereas i n November of the same year, 90% of the a d u l t s harboured were g r a v i d . 16 No v i s c e r a l adhesions were observed in spawning female f i s h but very l i g h t adhesions were observed in some spawning male f i s h . Encysted worms occurred i n both_male and female f i s h , but were more common i n the males. Measurements of worms were taken and t h e i r growth i s presented i n Table II and Figure I. Development of Philonema agubernaculum i n trout Thirty-two slow maturing, hatchery-reared rainbow trout, Salmo gairdneri,one year old, were dosed with 50 infected copepods in a ge l a t i n capsule. Three of the f i s h died on the day of infec t i o n , undigested copepods and larvae of Philonema were found i n the stomach of these f i s h but no larvae were found i n the tissue of the swimbladder. Eight other f i s h were found to be uninfected when examined l a t e r i n the experiment. No growth of larvae occurred in the f i r s t 30 days of i n f e c t i o n . The fourth stage larvae were recovered from the body cavi t y of one f i s h on the 58th day a f t e r i n f e c t i o n . After t h i s time, t h i r d stage larvae were found i n the swimbladder of only f i v e f i s h which also contained sub-adult worms i n the body cavity. Sub-adult worms were found in the body cavi t y from the 118th day onward. Mature male and female Philonema agubernaculum were found i n tie coelom from the 151st day but no gravid worms were recovered from the f i s h which survived up to 182 days (Table I I I ) . Loss of f i s h due to a contaminated water supply, terminated the experiment before any worm became gravid. A l l stages of the trout s t r a i n 17 F i g . I Development of Philonema oncorhynchi i n sockeye salmon of v a r i o u s ages from C u l t u s Lake. i LOG LENGTH IN M M . O I I I I O O) o o o o o 01 o o 1 — i — r TT > m o po CO C- c r a-> Pi LAPVAE o JO m E TO IN SWIh _ at J COELO .ADDER 2 O z — i i to IV) 01 IV) CD CO o IV) 18 d e v e l o p i n g i n t r o u t were m o r p h o l o g i c a l l y i d e n t i c a l t o those of sockeye s t r a i n d e v e l o p i n g i n sockeye. The two forms d i f f e r , however i n the developmental r a t e . No v i s c e r a l adhesions were observed i n any e x p e r i -m e n t a l l y i n f e c t e d f i s h . One a d u l t non-gravid female worm was found dead i n the abdominal c a v i t y of a female f i s h on the 160th day of i n f e c t i o n . The growth of Philonema agubernaculum was the same i n both male and female f i s h . The s i z e achieved by the worm over the 182 day d u r a t i o n of the experiment was from 11.8 mm. t o 15.4 mm. f o r males and from 26.mm. t o 70.9 mm. f o r female Philonema agubernaculum ( F i g u r e I I ) . DISCUSSION I n f e c t i o n of j u v e n i l e sockeye salmon under n a t u r a l c o n d i t i o n s P l a t z e r and Adams ( i n press) s u c c e s s f u l l y completed the l i f e c y c l e of Philonema oncorhynchi u s i n g Cyclops  b i c u s p i d a t u s as the i n t e r m e d i a t e host and f o l l o w i n g i t s i n i t i a l development i n the connective t i s s u e of sockeye salmon. In C u l t u s Lake, the sockeye salmon spawn at L i n d e l Beach i n water of one t o s i x metres i n depth from e a r l y November t o mid December ( F o e r s t e r , 1925). The eggs hatched d u r i n g the winter and the f r y are found f r e e swimming i n the lake i n e a r l y May ( F o e r s t e r , 1938). They remain i n the lake f o r e l e v e n and one-half months and o c c a s i o n a l l y two y e a r s . The l a t t e r c o n s t i t u t e s o n l y 26.7% of the t o t a l migrants ( F o e r s t e r 1929). The seaward m i g r a t i o n commences on A p r i l 12 19 F i g . I I Development of Philonema agubernaculum i n e x p e r i m e n t a l l y i n f e c t e d t r o u t . 20 and co n t i n u e s throughout A p r i l and May r e a c h i n g a peak d u r i n g the f i r s t week of May ( F o e r s t e r , 1925)., They feed and grow i n the sea f o r two years r e t u r n i n g t o the lake t o spawn i n the f a l l of t h e i r t h i r d year ( F o e r s t e r , 1938). At the same time, Philonema oncorhynchi i s c a r r i e d by downstream migrants to the sea. They grow very s l o w l y i n the host and mature r a p i d l y d u r i n g the l a s t f o u r t o s i x months of the host's l i f e and reproduce i n f r e s h water when the hosts spawn. There i s a complete p h y s i o l o g i c a l a d a p t a t i o n of Philonema oncorhynchi t o t h a t of i t s anadromous hosts The mean temperature of C u l t u s Lake d u r i n g November and December (average of a l l depths from 0 t o 40 metres) i s 7.7°C. ( R i c k e r , 1937a). Philonema oncorhynchi e x p e r i m e n t a l l y o r e a r e d i n Cyclops b i c u s p i d a t u s at 10 C reached the i n f e c t i v e t h i r d stage by 37 days and s u r v i v e d f o r 85 d a y s , a f t e r which the c u l t u r e of copepods was used up f o r experimental i n f e c -t i o n of f i s h . Ko (p e r s o n a l communication) maintained i n f e c t e d copepods f o r approximately e i g h t months at 4°C. T h i s suggests t h a t i n f e c t e d copepods may l i v e i n c o l d e r l a k e s f o r p e r i o d s of 8 months and d u r i n g t h i s time are capable of t r a n s f e r r i n g i n f e c t i o n of Philonema t o the f i s h . Sockeye salmon spawn i n the o u t l e t of the Babine R i v e r d u r i n g August t o October each year and emerging swimming f r y e n t e r the lake i n the f o l l o w i n g May and June, where they take up a p e l a g i c , z o o p l a n k t o n - e a t i n g h a b i t (Johnson and Groot, 1963). Sockeye f r y of Babine Lake were found i n f e c t e d 21 with t h i r d stage l a r v a e when captured i n J u l y . I n f e c t e d copepods and c l a d o c e r a n s were found i n the stomach contents of these f r y . T h i s means that the f i s h would be exposed t o i n f e c t e d copepods again d u r i n g the winter before t h e i r sea-ward m i g r a t i o n . P l a t z e r (1964) suggested t h a t i n f e c t i o n of sockeye f i n g e r l i n g s takes pla c e d u r i n g the month of January i n C u l t u s Lake. S u r v i v a l of e x p e r i m e n t a l l y i n f e c t e d copepods f o r approximately e i g h t months, and the occurrence of i n f e c t -ed f r y i n Babine Lake i n J u l y suggest that a few i n f e c t e d copepods may s u r v i v e from January t o August t o i n f e c t some of the next age c l a s s of j u v e n i l e sockeye salmon which become free-swimming i n May. T h i s would accountkon the o c c a s i o n a l l a r g e r s i z e d worms found i n one and two year c l a s s e s of down stream migrants i n C u l t u s Lake and Babine Lake. An a l t e r -n a t i v e e x p l a n a t i o n of the two s i z e c l a s s e s of Philonema i s that the f i s h have been i n f e c t e d by both P^ oncorhychi and P. agubernaculum which grow at d i f f e r e n t rates. Route of I n f e c t i o n of the Swimbladder The t h i r d stage l a r v a e of P^ oncorhynchi are most abundant i n the submucosal l a y e r of swimbladder but a l s o occur i n the p a r i e t a l peritoneum and t u n i c a e x t e r n a . Fange (1966) s t a t e s t h a t t u n i c a externa c o n s i s t s of dense con n e c t i v e t i s s u e which i s probably a b a r r i e r a g a i n s t d i f f u s i o n of gases. The sub-mucosa i s a loose j e l l y - l i k e t i s s u e , through which nerves and v e s s e l s pass t o the mucosa. T h i s might be 22 the reason for abundance of larvae i n sub-mucosal layer. Third stage larvae migrate to the sub-mucosal layer of the swimbladder from the stomach within seventeen hours of being eaten by the f i s h i n the infected copepods or in t a c t infected f i s h . Ricker (1937b) stated that f i n g e r -l i n g sockeye salmon take four to eight- hours to digest a small planktonic meal. This shows that larvae may not require too long a time to move to the swimbladder. The larvae probably avoid complicated routes of migration but i t has not been shown experimentally. Kuitunen-Ekbaum (1937) stated that larvae made t h e i r way through the i n t e s t i n a l wall into the abdominal cavity of the f i s h . Platzer (1964) suggested four routes of migration: 1) The larvae may enter the deep v i s c e r a l lymph-a t i c s and proceed to the posterior cardinal veins via the coeliaco-mesenteric and subvertebral lymph trunks. 2) The larvae may enter the hepatic portal system andb»transported to the swimbladder by the coeliaco-mesenteric artery where they could penetrate the vessel walls and enter the tunica adventitia. 3) Active migration through the i n t e s t i n a l wall. 4) A penetration through the wall of the intestine into the coelom and then penetration into the p a r i e t a l peritoneum or tunica adventitia. A further p o s s i b i l i t y i s that the larvae from the stomach migrate into the swimbladder through the pneumatic duct of the f i s h . 23 Larvae are found f r e e i n the pneumatic c a n a l , mucosal and sub-mucosal t i s s u e around the o u t l e t of the canal i n t o the swimbladder of the fish,, t h a t d i e d between 17 and 23 hours a f t e r attempted i n f e c t i o n . No l a r v a e are found i n the p e r i t o n e a l l a y e r or v i s c e r a at that time. Dead l a r v a e , but r a r e l y l i v e ones were found i n the i n t e s t i n a l c o n t e n t s . They were always found trapped i n f e c a l m a t e r i a l s of the i n t e s t i n e and i t i s u n l i k e l y t h a t they c o u l d pene-t r a t e the i n t e s t i n a l w a l l . Udvardy (unpublished) s t a t e d t h a t the stomach of the Salmonidae i s very s t r o n g w a l l e d . P e n e t r a t i o n through t h i s s t r o n g w a l l e d stomach would not be an easy route f o r m i g r a t i o n . In an experiment, l a r v a e were found t o migrate i n t o the swimbladder when the p o s t e r i o r end of the stomach was t i e d t o r e s t r i c t the passage of l a r v a e i n t o the i n t e s t i n e . Physocl- i s t i c g o ld f i s h c o u l d not be i n f e c t e d when l a r v a e were squirted i n t o the stomach or when i n f e c t e d copepods were f e d t o the f i s h . T h i s may be due t o the p h y s i o -l o g i c a l i n c o m p a t a b i l i t y of the host orfbthe c l o s e d pneumatic duct. The open pneumatic duct of salmonids appears t o be the most l i k e l y route of m i g r a t i o n . Growth and Development of Philonema In the present study, l a r v a e of Philonema  oncorhynchi i n the swimbladder began t o grow when the f i s h were 15 months o l d and sub-adult worms appeared i n abundance i n the abdominal c a v i t y by the time f i s h were 20 months o l d . 24 A d u l t male Philonema oncorhynchi appeared i n the body c a v i t y when f i s h were 25 months o l d . A few two-year o l d downstream migrants i n C u l t u s Lake were found t o harbour l a r g e numbers of t h i r d stage l a r v a e i n the swimbladder and g r e a t e r numbers of f o u r t h stage and sub-a d u l t worms i n the coelom than one year o l d migrants. They have been exposed t o the progeny of two generations of Philonema from spawning salmon. Kuitunen-Ekbaum (1937) observed a g r e a t e r percentage of i n f e c t i o n i n two-year o l d smolts than one-year o l d smolts and she a l s o s t a t e d t h a t p a r a s i t e s were not as numerous per host i n the sockeye o c c u r i n g i n f r e s h water as i n the a d u l t sockeye. In 1937 she found two i n f e c t e d f i s h out of 100 one-year o l d smolts examined, and seven of e i g h t two-year o l d s examined i n C u l t u s Lake. Kuitunen-Ekbaum was unaware of the m i c r o s c o p i c t h i r d stage l a r v a e i n the w a l l s of the swimbladder so t h a t a l l of her r e c o r d s r e f e r t o the macroscopic f o u r t h stage or sub-adult worms i n the body c a v i t y . Her r e c o r d s are t h e r e f o r e i n keeping with the f i n d i n g s of t h i s study. No t h i r d stage l a r v a e are found i n swimbladder of pr e c o c i o u s males(29-30 months) and immature three-year o l d sockeye (35 months) from C u l t u s Lake, but by t h i s time they have grown t o the f o u r t h s t a g e . Fourth stage and sub-adult worms appeared i n the body c a v i t y of both f i s h . 1 A d u l t g r a v i d females are found i n the coelom of p r e c o c i o u s male sockeye but not i n immature f i s h of the same age. The o n l y d i f f e r e n c e between these two groups of f i s h i s t h a t p r e c o c i o u s males are young 25 by age but s e x u a l l y matured. Spawning male and female sockeye of C u l t u s Lake which are 40-43 months o l d , g e n e r a l l y harbour only a d u l t worms. Thus i t i s seen t h a t Philonema oncorhynchi i s adapted to the 3-5 year l i f e c y c l e of i t s anadromous sockeye host. On the c o n t r a r y , the l i f e c y c l e of P^ _ agubernaculum i s not synchron-i z e d with i t s non-anadromous t r o u t host (Figure I I I ) . No growth of l a r v a e of P^ agubernaculum occurs d u r i n g the f i r s t 30 days of i n f e c t i o n . T h e r e a f t e r , from the 58th t o the 182nd day of i n f e c t i o n the worms are g e n e r a l l y present as s u b - a d u l t s . However, the worms may o c c a s i o n a l l y occur i n the swimbladder as t h i r d stage l a r v a e . T h i s d i f f e r e n t i a l r a t e of growth of l a r v a e i s not due t o d i f f e r e n c e i n sex of the host and i s t h e r e f o r e a t t r i b u t e d t o some other unknown f a c t o r ( s ) . S i m i l a r o b s e r v a t i o n s are made on the n a t u r a l l y i n f e c t e d a d u l t host of P.  oncorhynchi. P. agubernaculum grows t o the a d u l t stage between 151 and 182 days, and may become g r a v i d i n l e s s than a year as shown by the presence of g r a v i d worms i n n a t u r a l l y i n f e c t e d one-year o l d immature t r o u t from the Lardeau R i v e r of the Kootenay System. The l i f e c y c l e of agubernaculum resembles t h a t of other d r a c u n c u l o i d s i n i t s requirement of l e s s than 1 year f o r completion, but P^ oncorhynchi d i f f e r s from the r e s t i n the requirement of a longer developmental p e r i o d and i n i t s p h y s i o l o g i c a l a d a p t a t i o n f o r the h o s t ' s m a t u r i t y . I Male worms of most d r a c u n c u l o i d s are r a r e when the females are r i p e . In the case of P. oncorhynchi males are found 26 F i g . I l l Diagrammatic comparison of the spawning areas and the m i g r a t i o n s of t r o u t and salmon with those-of Philonema  agubernaculum and Philonema oncorhynchi i n t h e i r t r o u t and salmon hosts r e s p e c t i v e l y . £ spawning area j u v e n i l e m i g r a t i o n i n freshwater \ _ — _ — _ — t o sea D a d u l t spawning m i g r a t i o n ^ f r e e swimming l a r v a e i n f e c t e d copepods i n f e c t e d j u v e n i l e f i s h i n lake L"" t r a n s f e r of i n f e c t i o n SALMO G -SPAWN MORE LIFE HISTORY OF TROUT AIRDNERI .THAN ONCE -LIFE HISTORY OF P.AGUBERNACULUM ONCORHYNC - SPAWN LIFE HISTORY OF SOCKEYE HUS NERKA ONCE-LIFE HISTORY OF P.ONCORHYNCHI TRIBUTARY A • m A • r ffl LAKE • < f \ m -< • — ® < . — ^ t RIVER J • \) m ffl ' ESTUARY j OCEAN 27 commonly with r i p e females i n the body c a v i t y of a d u l t f i s h but they are not as numerous as i n immature f i s h . Male worms prob-a b l y mature much more e a r l i e r than the, female and then d i e o f f . W i e r z b i c k i (1960) has shown t h a t the males of Philonema  sanguinea which d i e a f t e r the f e r t i l i z a t i o n , become encysted and l a t e r are re-absorbed by the organ of the host. T h i s i n t e r -p r e t a t i o n c o u l d probably be a l s o a p p l i e d t o male worms of P.  oncorhynchi. Escapement of Worm from the Host Few suggestions have been made so f a r r e g a r d i n g the escapement of g r a v i d d r a c u n c u l o i d s . N y b e l i n (1931) s t a t e s that the d r a c u n c u l o i d s i n h a b i t i n g the abdominal c a v i t y never leave the l a t t e r , though no exp l a n -a t i o n i s given how the r i p e female p a r a s i t e s reach the e x t e r i o r . Willemoes-Sum (1871) suggests t h a t the r i p e female p a r a s i t e b u r s t w i t h i n the f i s h and the l i b e r a t e d young a c t i v e l y make t h e i r way out. Linstaw (1874) suggested t h a t the r i p e female parasite;; makes i t s way through the i n t e s t i n a l w a l l of the f i s h , thus r e a c h i n g the e x t e r i o r with the f a e c e s . He based h i s statement upon the f a c t t h a t a r i p e female dracuncu,loid was found which had, with i t s e x t e r i o r end, p i e r c e d the i n t e s t i n a l w a l l of the f i s h . The p a r a s i t e was found dead when observed. Meyer (1960) suggested t h a t the g r a v i d Philonema might pass out with the roe d u r i n g spawning. V i k (1964) suggested t h a t the g r a v i d worms l i b e r a t e l a r v a e i n the body c a v i t y which escapes through the m i l t and roes of salmon, as he has found l a r v a e i n the m i l t and roes 28 of s t r i p p e d f i s h . Kuitunen-Ekbaum (1937) suggested that time of maturation of the host c o i n c i d e s with t h a t of the Philonema  oncorhynchi and the p a r a s i t e might reach the e x t e r i o r d u r i n g the spawning a c t i v i t i e s . K e n d a l l (1921) observed t h a t the o v a r i e s of the salmonid f i s h are suspended from a membrane o r i g i n a t i n g i n the d o r s a l peritoneum. T h i s membrance envelopes the e n t i r e s u r f a c e of an immature ovary. As developments proceed, however, the edge of the membrane of the outer s u r f a c e p a r t s from the mem-brane of the inn e r s u r f a c e l e a v i n g a narrow area of the ovary without membranous c o v e r i n g . The s u r f a c e of the ovary, thus uncovered g r a d u a l l y widens and extends forward as the ovary i n c r e a s e s i n s i z e . Udvardy (unpublished) observed i n salmonids t h a t the maturing o v a r i e s arecnot covered m e s i o - v e n t r a l l y and d o r s o - l a t e r a l l y and f u l l y matured o v a r i e s are not covered d o r s a l l y . The author has made s i m i l a r o b s e r v a t i o n s i n matured t r o u t and sockeye salmon. G r a v i d Philonema are observed t o pass out with roe d u r i n g the s t r i p p i n g of r i p e female t r o u t and sockeye. G r a v i d worms are found i n r i p e o v a r i e s and a l s o i n the mesovarium of the o v a r i e s of r i p e sockeye salmon. G r a v i d Philonema are very f r a g i l e and possess no d e n t i c u l a r s t r u c t u r e or any other mech-a n i c a l means t o penetrate the i n t e s t i n a l or body w a l l and t h e r e f o r e the author supports the assumption of Kuitunen-Ekbaum (1937) and Meyer (1960) that the g r a v i d female pass out with roe duringfcspawning a c t . 30 On the other hand there remains another p o s s i b i l i t y . The r i p e i n d i v i d u a l s of Philonema oncorhynchi l y i n g i n the abdominal c a v i t y of the f i s h , may await the decomposition and gradual break up of the body of the f i s h and the l a r v a e become f r e e swimming i n lake when the female b u r s t s i n coelom of dead d i s i n t e g r a t e d host*. T h i s has a l s o been suggested by Kuitunen-Ekbaum (1937). The above assumption c o u l d not be used t o e x p l a i n the means of l i b e r a t i o n of r i p e worms i n s e x u a l l y immature t r o u t or salmon. Philonema agubernaculum i n e x p e r i m e n t a l l y i n f e c t e d immature t r o u t are found to mature i n s i x months. At the same time dead a d u l t worms are a l s o observed i n the body c a v i t y . Dead and encysted Philonema found i n immature sockeye salmon from Shuswap Lake maybe P^ agubernaculum. I t i s assumed from the s t a t e d f a c t s t h a t the l i f e c y c l e of Philonema agubernaculum i s not s u c c e s s f u l i n immature hosts and therby the r e p r o d u c t i v e c y c l e i s a "dead end" i n immature hosts.. V i s c e r a l adhesions V i s c e r a l adhesions are observed i n immature and maturing anadromous sockeye salmon i n f e c t e d with P^ oncorhynchi i n C u l t u s Lake. These occur i n 23 month o l d f i s h , c o r r e s -ponding to the m i g r a t i o n of l a r v a e from the swimbladder i n t o the body c a v i t y . Severe v i s c e r a l adhesions are observed i n f i s h captured i n January and A p r i l , 1965 from the Gulf of Alaska but much l e s s i n August from C o a s t a l f i s h i n g and none i n f i s h caught from the 31 spawning ground. O c c a s i o n a l l y , the a d u l t male and p r e c o c i o u s male sockeye are found t o posses very l i g h t adhesions. The above o b s e r v a t i o n suggest that the wide spread cf. v i s c e r a l adhesions are t r a n s i t o r y . They begin to disappear correspond-i n g t o the gonadal development of the h o s t s . T h i s i s i n f u l l agreement with French (1965) , who noted the. absence of adhesions i n matured sockeye though adhesions are more p r e v e l a n t i n immature Oncorhyrius nerka and Oncorhyrius keta caught i n the B e r i n g Sea and North P a c i f i c Ocean and the m a j o r i t y of the adhered f i s h are males. Summary Philonema oncorhynchi r e q u i r e s 32 t o 35 months t o mature i n i t s anadromous host, whereas Philonema agubernaculum r e q u i r e s 6 t o 12 months i n i t s non-anadromous t r o u t h ost. The developmental stages through which they pass are i d e n t i c a l . The form i n t r o u t can mature i n immature f i s h , but the form i n salmon i s matured i n maturing f i s h . The v a r i a t i o n i n l i f e c y c l e s of the two forms may r e s u l t from p h y s i o l o g i c a l ^ d i f f e r e n t responses of a s i n g l e s p e c i e s i n d i f f e r e n t h o s t s or from the e x i s t e n c e of two b i o l o g i c a l l y d i s t i n c t s p e c i e s . 32 SECTION II Experimental c r o s s i n f e c t i o n between t r o u t and salmon s t r a i n s . INTRODUCTION The longer developmental pattern of Philonema  oncorhynchi i n i t s anadromous host may be e i t h e r because of inherent c h a r a c t e r i s t i c s or due t o a p h y s i o l o g i c a l adjustment Hot' the lo n g l i f e h i s t o r y and anadromous h a b i t s of salmon. T h i s s e r i e s of experiments of c r o s s i n f e c t i o n between Philonema from t r o u t and salmon were c a r r i e d out t o ob t a i n i n f o r m a t i o n r e g a r d i n g the d i f f e r e n c e s i n the l i f e c y c l e s of Philonema from anadromous and non-anadromous hosts. MATERIALS AND METHODS Source and Husbandry of F i s h The f o l l o w i n g d i f f e r e n t s p e c i e s of f i s h were used. a) T r o u t , Salmo g a i r d n e r i i ) Hatchery r e a r e d rainbow t r o u t f r y which were 4 months o l d at the begin n i n g of the experiment, obtained from Abb o t s f o r d Trout Hatchery of the F i s h and Game Branch of B r i t i s h Columbia, Dept. of R e c r e a t i o n and C o n s e r v a t i o n . i i ) Year o l d slow maturing hatchery r e a r e d t r o u t , obtained from the Summerland Hatchery of the above o r g a n i z a t i o n . b) Salmon, Oncorhynchus i ) Sockeye (Oncorhynchus nerka) r e a r e d from eggs c o l l e c t e d from the winter run sockeye from C u l t u s Lake and 33 hatched i n the U n i v e r s i t y of B r i t i s h Columbia Hatchery. The f i s h were s i x and one-half months of age when i n f e c t e d . i i ) S p r i n g (Oncorhynchus tshawytscha) hatched on December 18, 1964 were obtained from the Samish State Hatchery, B u r l i n g t o n , Washington S t a t e , U.S.A. i i i ) Pink (Oncorhynchus gorbuscha) hatched i n the Department of Zoology, U n i v e r s i t y of B r i t i s h Columbia on January 1, 1965 from eggs c o l l e c t e d from Baer R i v e r , t h i r t y m i l e s n o r t h of Campbell R i v e r , Vancouver I s l a n d , B.C. i v ) Chum (Oncorhynchus keta) hatched i n October, 1964 were obtained from the B r i t i s h Columbia Research C o u n c i l . The f i s h were used on January 17, 1965. v) Coho (Oncorhynchus k i s u t c h ) s i x months of age when caught from Cheakamus R i v e r , Squamish, B r i t i s h Columbia i n September, 1964 and u t i l i z e d i n January, 1965. v i ) Kokanee (Oncorhynchus nerka k e n n e r l y i ) , a year o l d , from Oregon F i s h and Game Hatchery, were obtained through the B r i t i s h Columbia Research C o u n c i l . c) Gold F i s h , C a r a s s i u s c a r a s s i u s Gold f i s h were obtained l o c a l l y from a pet shop. I n f e c t i o n and Maintenance of Experimental F i s h F i s h were i n f e c t e d iy, the f o l l o w i n g d i f f e r e n t methods: a) F r y and f i n g e r l i n g f i s h were s t a r v e d f o r two days before t r a n s f e r r i n g them i n t o 8" g l a s s s t o c k i n g d i s h i n three l i t r e s 34 o f d e c h l o r i n a t e d ' w a t e r . T h i r t y i n f e c t e d c o p e p o d s p e r f i s h were p o u r e d i n t o t h e d i s h f o r a l l o w i n g f i s h t o f e e d on c o p e p o d s . The w a t e r was f i l t e r e d t o r e c o v e r u n f e d c o p e p o d s a f t e r t r a n s -f e r r i n g t h e f i s h i n t o h o l d i n g t a n k s . The f i s h were m a r k e d by f i n c l i p p i n g . b) F i n g e r l i n g f i s h were f e d t h i r t y t o f i f t y ( d e p e n d i n g on t h e r a t e o f i n f e c t i o n ) c o p e p o d s p e r f i s h w i t h t h e h e l p o f a p o l y -e t h y l e n e s t o m a c h t u b e i n a m i n i m a l w a t e r . The f i s h were a n a e t h e t i z e d i n MS 222 (1 g . / l i t r e ) b e f o r e c a r r y i n g o u t t h i s o p e r a t i o n . The f i s h were t a g g e d and t r a n s f e r r e d i n t o a h o l d i n g r e c i r c u l a t i n g t a n k . c ) Y e a r l i n g t r o u t were i n f e c t e d by g e l a t i n c a p s u l e a s d e s c r i b e d i n S e c t i o n I . RESULTS I n f e c t i o n o f d i f f e r e n t S a l m o n i d and G o l d F i s h w i t h P h i l o n e m a  f r o m s o c k e y e D e v e l o p m e n t i n n a t u r a l l y i n f e c t e d s a l m o n f r o m C u l t u s L a k e s e r v e d a s t h e c o n t r o l . a) I n f e c t i o n o f R a i n b o w T r o u t , S a l m o g a i r d n e r i F i s h were f o u n d i n f e c t e d w i t h an a v e r a g e o f 9.1 l a r v a e p e r f i s h i n t h e swim b l a d d e r . The f i s h s u r v i v e d f o r f o r t y - s i x d a y s d u r i n g w h i c h no d e v e l o p m e n t o f l a r v a e wase f o u n d . A l l l a r v a e were i n t h e t h i r d s t a g e w i t h a mean l e n g t h o f 0.98 mm. ( F i g u r e I V ) w h i c h WCLCU c o m p a r a b l e t o t h i r d s t a g e l a r v a e i n c o p e p o d s . 35 F i g . IV Development of Philonema oncorhynchi i n e x p e r i m e n t a l l y i n f e c t e d t r o u t of two years o l d . o CP LENGTH IN MM o T en o o a o 0| o o3»<eoa ooo oo #o o o oh 5 a O I L 36 b) I n f e c t i o n of Coho, Oncornyncfaus k i s u t c h S i x t e e n f i n g e r l i n g Coho (Oncorhynchus kisutch)were exposed f o r i n f e c t i o n . A l l the f i s h d i e d a f t e r f i f t y - s i x days of i n f e c t i o n and 81% of the f i s h were found i n f e c t e d with an average of 3.6 l a r v a e per f i s h i n the swimbladder. A l l the l a r v a e were i n the t h i r d stage. c) I n f e c t i o n of Chum (Oncorhynchus keta) The f i s h d i e d e i g h t e e n hours a f t e r i n f e c t i o n and 75% of the f i s h were i n f e c t e d with an average of 3.1 l a r v a e i n the swimbladder. d) I n f e c t i o n of t r o u t f r y Eleven of twelve f r y were found i n f e c t e d with 2.8 l a r v a e per f i s h . A group of fo u r f i s h were a u t o p s i e d - 17, 32, 62, and 84 hours a f t e r p l a c i n g the f i s h i n the d i s h of water with i n f e c t e d copepods. Two, 2, 6, and 11 l a r v a e r e s p e c t i v e l y were recovered from the swimbladder. Both dead and l i v e l a r v a e were found i n the stomach of the f i r s t f i s h . Heavier i n f e c t i o n thus occurs with longer exposure time. No growth of l a r v a e was observed i n 149 days. One f i s h s u r v i v e d 346 days and one f o u r t h stage l a r v a was recovered from i t s swimbladder (Figure V ) . I n f e c t i o n of Sockeye, Pink, S p r i n g , Kokanee and Gold F i s h with  Trout S t r a i n Trout i n f e c t e d with Philonema agubernaculum i n S e c t i o n I (page 16) served as c o n t r o l i n t h i s experiment, a) I n f e c t i o n of Sockeye (Oncorhynchus nerka) f r y 37 F i g . V Development of Philonema oncorhynchi from sockeye salmon i n e x p e r i m e n t a l l y i n f e c t e d t r o u t f r y . o "WW Nl H19N31 38 F i v e of t h i r t y - s e v e n sockeye f r y were i n f e c t e d (13.5%) with Philonema agubernaculum. No growth was observed up t o the 157th day but a sub-adult female of 32.1 mm. l o n g was found i n the body c a v i t y of a sockeye f r y of 8.5 cm. (Figure VI) a f t e r 161 days of i n f e c t i o n . b) I n f e c t i o n of Pink (Oncorhynchus gorbuscha), S p r i n g (0.  tshawytscha) Kokanee (0. nerka k e n n e r l y i ) and Gold F i s h ( C a r a s s i u s c a r a s s i u s ) i ) Two of three pink salmon were i n f e c t e d and t h i r d stage l a r v a e r e covered a f t e r twelve days. i i ) One of three s p r i n g s were i n f e c t e d and t h i r d stage l a r v a e were found i n the swimbladder on the 55th day. i i i ) Three kokanee and g o l d f i s h were found t o be u n i n f e c t e d . DISCUSSION Though a complete s e t of r e c i p r o c a l i n f e c t i o n s was not made due t o u n a v a i l a b i l i t y of m a t e r i a l s at s p e c i f i c times, there i s s u f f i c i e n t data t o show t h a t the d i f f e r e n c e between the t r o u t and salmon ^ s t r a i n s " are in h e r e n t c h a r a c t e r i s t i c s and not the r e s u l t of v a r i a t i o n s due t o h o s t . N e i t h e r P^ oncorhynchi from salmon or P^ agubernaculum from t r o u t i s s p e c i f i c t o i t s t y p i c a l host. The experiments demonstrate t h a t each form i s capable of i n f e c t i n g s e v e r a l salmonids. N e i t h e r s t r a i n was able t o i n f e c t the s i n g l e non-salmonid used i n the experiment. However, the data suggests t h a t P. agubernaculum from t r o u t does not i n f e c t sockeye salmon 39 F i g . VI Development of Philonema agubernaculum from t r o u t i n e x p e r i m e n t a l l y i n f e c t e d f r y of sockeye salmon. > -< CO 81 LENGTH IN MM. cn T CJl 6 T" OJ o <J1 40 as r e a d i l y as other h o s t s . Only 13.5% of 37 sockeye were i n f e c t e d as compared to 65.6% of 32 t r o u t ( I f three f i s h t h a t d i e d w i t h i n 24 hours of i n f e c t i o n are excluded the l a t t e r f i g u r e becomes 74.8%). oncorhynchi from salmon produced the same percentage i n f e c t i o n i n a l l h o s t s , chum salmon 75%, coho 81%, rainbow y e a r l i n g s 82%, rainbow f r y 88%. I t has been shown e x p e r i m e n t a l l y t h a t Philonema  agubernaculum reaches the f o u r t h stage by 58 days and sub-adult and a d u l t stages 118 and 182 days i n immature t r o u t ( S e c t i o n I page 16). The sub-adult stage was reached i n 161 days i n sockeye f r y . C o n v e r s e l y , no growth was observed i n the t h i r d stage l a r v a e of Philonema oncorhynchi i n e i t h e r t r o u t or sockeye d u r i n g the same p e r i o d of time. In n a t u r a l i n f e c t i o n s oncorhynchi r e q u i r e d approximately 240-360 days to produce f o u r t h stage l a r v a e and 360 t o 450 days t o produce su b - a d u l t s ( S e c t i o n I ) . The development of the two s p e c i e s of worms i n t r o u t and salmon i s c o n t r a s t e d i n F i g u r e V I I . The times r e q u i r e d t o molt t o the f o u r t h stage and again t o the sub-adult stage are d i s t i n c t f o r each s p e c i e s and are independant of the host p a r a s i t i z e d . P. agubernaculum grew much f a s t e r than P^ oncorhynchi i n both t r o u t and sockeye salmon. P_._ agubernaculum i n t r o u t had grown t o 3.3 t o 4.1 mm. ( f o u r t h stage) by 58 days. P.  oncorhynchi i n t r o u t d i d not reach the same s i z e (4.1 mm.) and stage u n t i l the 346th day. P^ agubernaculum produced sub - a d u l t s measuring 11.8 t o 15.4 mm. f o r males and 26.8 t o 70.9 mm. f o r females i n 182 days; i n salmon (O. nerka) a sub 41 F i g . VII H y p o t h e t i c a l r e p r o d u c t i v e i s o l a t i o n of Philonema oncorhynchi and Philonema agubernaculum i n salmonid (based on four year r e p r o d u c t i v e c y c l e of sockeye salmon). Sockeye Philonema Philonema  oncorhynchi agubernaculum Trout Philonema Philonema oncorhynchi agubernaculum 3rd stage 4th stage time i n days 30 50 100 3rd stage 10% 4th stage 10% sub a d u l t sub a d u l t g r a v i d ? dead and enc y s t e d 3rd stage 4th stage a d u l t g r a v i d (at l a t e s t ) 150 200 250 300 350 400 450 500 550 600 650 g r a v i d 1100 42 a d u l t female measuring 32.1 mm. was present i n 161 days. The above experimental data explain; s e v e r a l p u z z l -i n g f i e l d o b s e r v a t i o n s . Philonema are g e n e r a l l y found i n non-andromous t r o u t from Trout and Kootenay Lakes, B r i t i s h Columbia which do not have any l i n k with sea. There are a l s o l a n d l o c k e d sockeye, 0^ nerka k e n n e r l y i i n Kootenay Lake but no anadromous sockeye. Only one a d u l t Philonema was found i n one of 200 spawning kokanee examined i n Kootenay Lake. These f a c t s are e a s i l y e x p l a i n e d i f a l l Philonema i n Kootenay Lake are P.  agubernaculum. C u l t u s Lake may be c o n s i d e r e d a type l o c a l i t y f o r Philonema oncorhynchi,. Kuitunen-Ekbaum (1937) , P l a t z e r (1964) , P l a t z e r and Adams ( i n press) and t h i s study have shown t h a t the P. oncorhynchi i s a s p e c i e s w e l l adapted t o the sockeye salmon. Though t r a n s m i t t e d i n f r e s h water, the worms grow and mature s l o w l y and are g r a v i d only when t h e i r hosts are s e x u a l l y mature and r e t u r n t o the lake t o spawn. However, Kuitunen-Ekbaum recorded the presence of a few macroscopic worms i n the body c a v i t y of downstream migrant f i s h approximately one year o l d . P l a t z e r noted sub-adult male worms i n the coelom of 17 month o l d sockeye from C u l t u s Lake. S i m i l a r p r e c o c i o u s Philonema were noted i n t h i s study. T h e i r presence may be e x p l a i n e d by p o s t u l a t i n g two p e r i o d s of i n f e c t i o n ( S e c t i o n I ) , the f i r s t i n May t o J u l y from i n f e c t e d copepods s u r v i v i n g s i n c e the p r e v i o u s salmon spawning. An a l t e r n a t i v e e x p l a n a t i o n i s suggested by the f a c t t h a t C u l t u s Lake c o n t a i n s sma l l p o p u l a t i o n s of rainbow 4 3 t r o u t , Salmo g a i r d n e r i ; D o l l y Varden; char, S a l v e l i n u s malma ; and white f i s h , Prosopium w i l l i a m s o n i • I f these are p a r a s i t i z e d by P^ agubernaculum, the r a p i d l y growing worms i n sockeye salmon may be t h a t s p e c i e s . The Philonema found as l a r v a e i n the swim blad d e r s of migrant sockeye l e a v i n g Shuswap Lake are very probably P.  agubernaculum. Data i n S e c t i o n I I I of t h i s paper show t h a t they develop r a p i d l y i n the f i s h but are destroyed by host r e a c t i o n before r e a c h i n g m a t u r i t y . Shuswap Lake c o n t a i n s s e v e r a l salmonids, as w e l l as white f i s h . One or more of these s p e c i e s c o u l d be r e s p o n s i b l e f o r m a i n t a i n i n g the l i f e c y c l e of the worm. The p o s s i b l e and even probable occurrence of both P.  oncorhynchi and P^ _ agubernaculum i n some l a k e s of B r i t i s h Columbia r i s e s a q u e s t i o n of i n t e r b r e e d i n g of P^ oncorhynchi from anadro-mous sockeye and P. agubernaculum from t r o u t of lake r e s i d e n c e . I t i s presumed t h a t both the j u v e n i l e t r o u t and sockeye may acqu i r e i n f e c t i o n s of P. oncorhynchi and P^ agubernaculum d u r i n g t h e i r f o r a g i n g on p l a n k t o n i c food i n the l a k e . I n t e r -b r e e d i n g may occur which would i n v a l i d a t e the r e c o g n i t i o n of g e n e t i c a l l y d i s t i n c t s p e c i e s . I t i s argued, here, on the b a s i s of a v a i l a b l e i n f o r m a t i o n t h a t i n t e r b r e e d i n g may never occur i n t r o u t or sockeye, even though they c a r r y both s p e c i e s at the same time. F i g u r e VII pr e s e n t s the argument g r a p h i c a l l y . P. agubernaculum i s f a s t maturing whereas P.  oncorhynchi i s very slow. In sockeye salmon when Pj_ aguberna-culum reaches a r e p r o d u c i n g stage, P. oncorhynchi continue t o 44 l i v e i n the swimbladder as t h i r d or f o u r t h stage l a r v a e . A l l P. agubernaculum w i l l d i e and become encysted by the time P.  oncorhynci reaches m a t u r i t y i n matured h o s t s . T h i s i s i n f a c t confirmed by the i n f o r m a t i o n a v a i l a b l e from sockeye of Shuswap Lake ( S e c t i o n I I I ) . For t r o u t , a s i m i l a r argument has been presented on t h e o r e t i c a l grounds (Figure VII) but because the t r o u t e x p e r i -m e n t a l l y i n f e c t e d with Vj_ oncorhynchi d i d not s u r v i v e beyond 346 days the u l t i m a t e f a t e of the worms i s unknown. At 346 days P^ oncorhynchi had reached o n l y the f o u r t h l a r v a l stage. An i n f e c t i o n with P^ agubernaculum superimposed d u r i n g the l a s t 12 months of the 3 year developmental p e r i o d of P.  oncorhynchi, might produce a c o i n c i d e n c e of a d u l t s and c r o s s b r e e d i n g of the two s p e c i e s , but there i s l i t t l e or no p o s s i -b i l i t y of r e a c h i n g a r e p r o d u c i n g stage (page 30). There i s l i t t l e or no p r o b a b i l i t y of i n t e r b r e e d i n g between P^ oncorhynchi and P_^  agubernaculum i n sockeye salmon because of r e p r o d u c t i v e i s o l a t i o n due t o temporal i s o l a t i n g mechanism of these s p e c i e s . I f there i s any occurrence of i n t e r b r e e d i n g between the two forms i n t r o u t , there i s no c o n t r i b u t i o n t o a common gene p o o l , because of an unsuccess-f u l r e p r o d u c t i v e c y c l e i n the non-spawning host. May.ir (1940) d e f i n e d s p e c i e s as " g r o u p s t f a c t u a l l y or p o t e n t i a l l y i n t e r b r e e d i n g n a t u r a l p o p u l a t i o n s which are r e p r o d u c t i v e l y i s o l a t e d from other such groups." and Dobzhansky (1950) d e f i n e d the same as "The l a r g e s t and most i n c l u s i v e . . . 45 r e p r o d u c t i v e community of sexual and c r o s s f e r t i l i z i n g i n d i v i d -u a l s which share i n a common gene p o o l . " In c o n c l u s i o n , the author confirms the v a l i d i t y of Philonema agubernaculum, Simon and Simon, 1936 on the b a s i s of developmental d i f f e r e n c e s from Philonema oncorhynchi, although there are no morphometric c h a r a c t e r i s t i c s by which the two s p e c i e s can be separated. E l e c t r o p h o r e t i c a n a l y s i s of the two s p e c i e s confirms t h e i r d i f f e r e n c e (Figure V I I I ) . 46 F i g . V I I I A comparison of s t a r c h g e l electropherograms of Philonema agubernaculum from t r o u t and Philonema oncorhynchi from sockeye salmon. T = Philonema agubernaculum S = Philonema oncorhynchi 47 SECTION I I I Hormonal Treatments INTRODUCTION I t was demonstrated i n S e c t i o n I t h a t the r e p r o d u c t -i o n of Philonema oncorhynchi c o i n c i d e s with that of i t s anadromous h o s t . The next two s e c t i o n s d e s c r i b e experiments which were undertaken t o t e s t the h y p o t h e s i s t h a t P^ oncorhynchi i s dependent on hormonal s t i m u l a t i o n from the host f o r s y n c h r o n i z a t i o n of r e p r o d u c t i o n . Pre-spawning green sockeye salmon i n f e c t e d with a d u l t but n o n - l a r v i g e r o u s worms were t r e a t e d with s e v e r a l d i f f e r e n t hormones t o see i f any a c c e l -e r a t i o n of development c o u l d be produced. MATERIALS AND METHODS The f o l l o w i n g s t r a i n s of n a t u r a l l y i n f e c t e d sockeye salmon were used as experimental animals: a) Approximately l\ year o l d sockeye salmon were c o l l e c t e d from Shuswap Lake, B r i t i s h Columbia i n May, 1964. At the time of t h e i r capture they were m i g r a t i n g downstream. The s t o c k s were maintained i n sea water at the F i s h e r i e s Research Board f i s h h atchery. b) Pre-spawning sockeye salmon were c o l l e c t e d from Great C e n t r a l Lake, Vancouver I s l a n d , and maintained i n a l a r g e 48 r e c t a n g u l a r tank i n freshwater at the B i o l o g i c a l S t a t i o n of the F i s h e r i e s Research Board, Nanaimo. The f i s h were given a s a l t w a t e r bath three times a week t o prevent f u n g a l i n f e c t i o n . P r e p a r a t i o n of Salmon P i t u i t a r y E x t r a c t s E x t r a c t s from the p i t u i t a r y of Oncorhynchus  tshawytscha were prepared by Mr. P. Schmidt and Dr. M. Smith of the F i s h e r i e s Research Board of Canada, Vancouver, B r i t i s h Columbia, u s i n g p r e v i o u s l y p u b l i s h e d methods (Schmidt, e t a l 1965) . I n j e c t i o n of P i t u i t a r y E x t r a c t a) Shuswap Lake F i s h Twenty f i s h of approximately equal l e n g t h were used i n the experiment. T h i r t y u n t r e a t e d f i s h were r e s e r v e d as a c o n t r o l . Each f i s h r e c e i v e d 0.4 ml. of p i t u i t a r y e x t r a c t . T h i s amount was given i n e i g h t equal i n j e c t i o n s at 0.05 ml. per f i s h a d ministered on every t h i r d day and spread over a p e r i o d of f o u r weeks. P r i o r t o i n j e c t i o n the f i s h were a n a e s t h e t i z e d i n 2 l i t r e s of an aqueous s o l u t i o n of methane s u l p h o n i c a c i d s a l t of m-amino e t h y l benzoate (MS 222, Sandoz) at a concen-t r a t i o n of 0.1 gm. per l i t r e of seawater. Each i n j e c t i o n was made i n t o the d o r s a l muscle (Von T h e r i n g , 1937) with a 0.25 ml. s y r i n g e and a #30 gauge needle. The needle was i n s e r t e d d i a g o n a l l y on e i t h e r s i d e of the d o r s a l f i n . Some 49 time was allowed before the needle was withdrawn from the body. T h i s p r e c a u t i o n ensured t h a t l i t t l e or no hormone was l o s t from the f i s h . D uring and a f t e r an i n j e c t i o n , the muscle of the f i s h was massaged i n an a n t e r i o r d i r e c t i o n t o a s s i s t d i f f u s i o n of the f l u i d . (Burrows et a l , 1952). b) Great C e n t r a l Lake F i s h F i f t e e n f i s h r e c e i v e d hormone i n j e c t i o n s . Fourteen u n t r e a t e d f i s h used as a c o n t r o l . F i s h were a n a e s t h e t i z e d i n 10 l i t r e s of aqueous s o l u t i o n of (MS 222, 0.1 gm. per l i t r e ) . I n j e c t i o n s were made as b e f o r e , and the head was kept submerged i n the a n a e s t h e t i c . T h i s f a c i l i t a t e d a d m i n i s t r a t i o n of the hormone. Each f i s h r e c e i v e d a t o t a l of 4.0 ml. of p i t u i t a r y e x t r a c t over a p e r i o d of two weeks u s i n g two equal doses administered with a l | i n c h #22 hypodermic needle and a 10 ml. SESI p l a s t i c s y r i n g e . The needle was i n s e r t e d about \ i n c h on the s i d e opposite the p o s t e r i o r p o i n t of i n s e r t i o n of the d o r s a l f i n and were d r i v e n a n t e r i o r l y f o r the f u l l \\ inches of the l e n g t h of the needle before i n j e c t i o n was made. As a p r e c a u t i o n a g a i n s t b a c t e r i a l i n f e c t i o n , the hypodermic needle was dipped i n aureomycin powder before each i n j e c t i o n . The same p r e c a u t i o n was taken as i n (a) t o avoid l o s i n g hormone. F i s h were tagged with spear tags and t r a n s f e r r e d t o a h o l d i n g tank along with the c o n t r o l f i s h . 50 I n j e c t i o n of 1 7 - b - E s t r a d i o l Twenty f i s h from Shuswap Lake r e c e i v e d hormone i n j e c t i o n s and 20 u n t r e a t e d f i s h served as c o n t r o l . A suspension of 0.05 mg. of 1 7 - b - e s t r a d i o l per 0.05 ml. of corn o i l suspension was i n j e c t e d i n t r a m u s c u l a r l y , u s i n g a #30 hypodermic needle with a 0.25 ml. s y r i n g e . Each f i s h r e c e i v e d a t o t a l of 0.3 mg. of 1 7 - b - e s t r a d i o l over a 3 week p e r i o d i n s i x equal doses. F u r t h e r i n j e c t i o n s were abandoned because of high m o r t a l i t y d u r i n g a d m i n i s t r a t i o n of the hormone. F i s h were tagged by f i n c l i p p i n g and maintained i n s a l t water. Implantation of S t i l b e s t r o l P i l l s Seventeen f i s h , 4 t o 5 months p r i o r t o spawning, sockeye salmon were obtained from Great C e n t r a l Lake. Seven-teen unteated f i s h used as c o n t r o l . A s m a l l i n c i s i o n of approximately one-half an i n c h was made v e r t i c a l l y on the m i d l i n e about two inches p o s t e r i o r t o the p e c t o r a l f i n s . The f i s h were h e l d v e n t r a l s i d e up i n an aqueous s o l u t i o n of MS 222. Two S t i l b e s t r o l p i l l s (B.D.H.) 25 mg. each were p l a c e d i n the body c a v i t y and pushed away from the i n c i s i o n with the h e l p of a g l a s s r o d . Aureomycin was then spread over the i n c i s i o n as a p r e c a u t i o n a g a i n s t i n f e c t i o n . F i s h were tagged and t r a n s f e r r e d t o h o l d i n g tank. Feeding S t i l b e s t r o l , P o w d e r T h i r t y t a b l e t s of S t i l b e s t r q l (25 mg. each) were mixed i n 1 3/4 pounds of wet food by an e l e c t r i c food g r i n d -i n g machine. F o r t y grams of t h i s food was f e d twice d a i l y 51 t o twenty sockeye salmon over a p e r i o d of 14 days. The f i s h tagged and maintained i n s a l t water f o r 8 weeks. Untreated f i s h of the experiment (3) were used as a c o n t r o l . RESULTS Shuswap Lake F i s h Three groups of s e x u a l l y immature sockeye were t r e a t -ed s e p a r a t e l y with salmon p i t u i t a r y , 1 7 - b - e s t r a d i o l and s y n t h e t i c S t i l b e s t r o l between February 23 and March 19, 1965. No a d u l t worms were found i n any of the experimen-t a l and c o n t r o l groups. Only two sub-adult female Philonema were found i n the experimental f i s h , whereas none were found i n the t h i r t y c o n t r o l f i s h (Table I V . ) . Great C e n t r a l Lake F i s h (Table V. & F i g u r e IX) a) E f f e c t of Crude Salmon P i t u i t a r y E x t r a c t A l l the i n j e c t e d f i s h d i e d between seven and f o r t y -seven days a f t e r treatment. F i f t y a d u l t female Philonema  oncorhynchi were found i n f i f t e e n P i t u i t a r y i n j e c t e d f i s h . S i x t y a d u l t female worms were obtained from the c o n t r o l f i s h . One g r a v i d worm was found i n the c o n t r o l f i s h d u r i n g the tenure of the experiment. F i v e developmental stages c o u l d be r e c o g n i z e d i n the u t e r u s of the female worms: egg, morula, embryo, t a i l l e s s l a r v a e , and t a i l e d l a r v a e ( f r e e swimming). No t a i l e d l a r v a e were found i n the female worms between 7 and 12 days of treatment. Of those f i s h t h a t d i e d between 19 and 47 days 52 F i g . IX E f f e c t of crude salmon p i t u i t a r y extracts on the prod-uction of larva in Philonema oncorhynchi. 0 represents the number of worms. 50|-45f-4 0 3 5 CD 30 0 CCD a m mama o to >-§ 25 20 o LU cam ceo cccco 10 •ceo cr» t I N 0 CONTROL • PITUITARY j i i 1 1 1 NO EGG MOR EMB TAIL TAIL DEVELOPMENTAL STAGES 53 a f t e r treatment, 33 female worms were c o l l e c t e d . E ighteen of these worms had t a i l e d l a r v a e i n t h e i r u t e r i . None of the female worms had eggs i n t h e i r u t e r i a f t e r 12 days of t r e a t -ment (Table V. and F i g u r e IX ) . Jhe, abundance of t a i l e d l a r v a e i n the female worms i s s i g n i f i c a n t at P<^.05 l e v e l i n both combined sexes and female f i s h . The n o n s i g n i f i c a n t r e s u l t i n male f i s h i s probably due t o very low sample number. The changes i n body weight and gonad weight of the t r e a t e d and u n t r e a t e d f i s h were not s i g n i f i c a n t . N u p t i a l c o l o r was observed i n male and female f i s h w i t h i n two weeks a f t e r treatment but no such secondary sexual c h a r a c t e r s were observed i n the u n t r e a t e d f i s h (Table V I . ) . Development of n u p t i a l c o l o r a t i o n was recorded on an a r b i t r a r y b a s i s from + t o +++ ( P i c k f o r d , 1953a). b) E f f e c t of S t i l b e s t r o l A l l treated f i s h d i e d between 173 days. Seventy female worms were found i n 17 of the S t i l b e s t r o l implanted pre-spawning sockeye. No stages beyond the embryo were found i n the u t e r i of the 70 female Philonema oncorhynchi taken from the S t i l b e s -t r o l implanted f i s h . A l s o , no d i f f e r e n c e was found i n the worms from male and female hosts (Table VII.and F i g u r e X ) . One of 60 a d u l t worms recovered from the c o n t r o l f i s h had t a i l e d l a r v a e . There was no d i f f e r e n c e i n body weight or gonad weight between the t r e a t e d and u n t r e a t e d female f i s h . Body 54 F i g . X E f f e c t o f s y n t h e t i c s t i l b e s t r o l on t h e p r o d u c t i o n o f l a r v a e i n P h i l o n e m a o n c o r h y n c h i . 0 r e p r e s e n t s t h e number o f worms. 5 0 r OCD o aoocniD OCD 4 5 4 0 3 5 3 0 a) 25 < 20 UJ 1 ODD I5h COD oocco 0 03) CD 0 CONTROL " • STILBESTROL i i i 1 1 » NO EGG MOR EMB TAIL TAIL DEVELOPMENTAL STAGES 55 weight of implanted male f i s h d i d not change but a notable r e g r e s s i o n of t e s t i s was found i n the implanted male f i s h (Table V I I I . and F i g u r e X I ) . T e s t i s weight and m a t u r i t y index (gonad weight X 100/body weight) of the S t i l b e s t r o l t r e a t e d salmon d i f f e r e d s i g n i f i c a n t l y at P<^.05 l e v e l from the un-t r e a t e d male f i s h . DISCUSSION "In the m a j o r i t y of animals, n e u r o s e c r e t a r y c e l l s form the f i r s t l i n k between the environmental t r i g g e r and the p h y s i o l o g i c a l machinery concerned with r e p r o d u c t i o n " (Scharer and Scharer, 1963). The endocrine r e g u l a t i o n of r e p r o d u c t i o n i n p a r a s i t e s i s p o o r l y known. So f a r , i t has been found t h a t the e f f e c t s of host hormones on p a r a s i t e s (from Protozoan to Arthropod) are of two k i n d s . They i n f l u -ence the number of p a r a s i t e s per host and they c o n t r o l d i r e c t l y or i n d i r e c t l y the growth and r e p r o d u c t i o n of the p a r a s i t e s . S e v e r a l cases of s y n c h r o n i z a t i o n between the l i f e c y c l e of the host and the p a r a s i t e are known and r e p r e s e n t an i n t e r e s t i n g phenomanon. Leucocytozoon simondi, a haemo-s p o r i d i a n p a r a s i t e of ducks, has a l i f e c y c l e that i s s y n c h r o n i z e d with i t s d e f i n i t e and i n t e r m e d i a t e host, S i m u l i a n s p e c i e s (Smyth, 1962). C l e v e l a n d (1957) showed a r e l a t i o n between the r e p r o d u c t i v e c y c l e of the p a r a s i t i c f l a g e l l a t e s and the moulting c y c l e s of t h e i r t e r m i t e h o s t s . Wessenberg (1961) r e s t u d i e d the l i f e c y c l e of O p a l i n a , which l i v e s i n 56 F i g . XI E f f e c t of implanted s y n t h e t i c s t i l b e s t r o l on the p r o g r e s s i v e growth of t e s t e s of sockeye salmon from the Great C e n t r a l Lake. I I I 1 1 1 L. if) O <P O tp O uO |V) O J CVI — — O X3QNI A i i a m v w 57 the rectum of f r o g s and toads. He found t h a t the o p a l i n i d s reproduce a s e x u a l l y by f i s s i o n d u r i n g the summer, autumn and winter and s e x u a l l y d u r i n g the s p r i n g when the hosts move to water t o breed. B i e n i a r z (1950) had suggested t h a t t h i s change was probably induced by the gonadotropic hormones of the host r e l e a s e d by the a n t e r i o r p i t u i t a r y d u r i n g c o p u l a t -i o n . Cehovic (1956) s t a t e d t h a t the c o r r e l a t i o n between host and p a r a s i t e l i f e c y c l e s was r e l a t e d t o seasonal p i t u i t a r y a c t i v i t y , and probably due t o the production of gonadotropin. Mofty and Smyth (1964) showed t h a t the i n d u c t i o n of c y s t formation i n the o p a l i n i d s i s r e l a t e d , not t o the l e v e l of gonadotropic hormones, but to the l e v e l s of gonadal hormones present i n i t s host. Z e l l e r (1876) d e s c r i b e d the l i f e c y c l e of Polystoma  integerrimum, in the u r i n a r y bladder of an European f r o g , Rana temporaria, and he a l s o r e p o r t e d that host and p a r a s i t e develop s i m u l t a n e o u s l y . G a l l i e n (1935) confirmed t h i s obser-v a t i o n . M i r e t s k i (1951) shows t h a t the maturation of Polystoma  integerrimum i s almost dependent on the host^s endocrine c y c l e . Stunkard (1959) t r i e d t o demonstrate the r o l e of p i t u i t a r y i n the maturation of Polystomum s t e l l a i but d i d not suceed because of s c a r c i t y of m a t e r i a l s . Gorshkov (1964) r e p o r t e d t h a t i n nature the f i r s t r e p r o d u c t i o n of Polystomum takes place not at the end of 3rd, but at the end of the 2nd year of i t s l i f e , i . e . one year p r i o r t o the f i r s t spawning of the f r o g . Ontogenesis and annual sexual c y c l e of Polystomum do 58 not depend upon gonadotropins and sexual hormones of the f r o g . Raikova (1959) e x p e r i m e n t a l l y confirmed a c l o s e r e l a t i o n s h i p between the egg of the c o e l e n t e r a t e p a r a s i t e Polypodium hydriforme and sturgeon eggs. The e v e r s i o n of the Polypodium s t o l o n s occurs j u s t before spawning when the female f i s h becomes r i p e . The i n v e r s i o n of the s t o l o n s could be induced by hypophysis i n j e c t i o n s which caused the eggs to pass i n t o t h e i r f i f t h stage of maturation. S z i d a t (1956, 1958) d e s c r i b e d the b i o l o g y of the trematode G e n a r c h e l l a which l i v e s i n the i n t e s t i n e s of c y p r i n i d s t h a t migrate from sea t o f r e s h water. During t h a t time the trematode develops r a p i d l y . He concludes t h i s because of i n c r e a s e d a c t i v i t y and development of the t h y r o i d and hypophysis. R o t h s c h i l d ' s work (1964b, 1965a) on f l e a s of r a b b i t s showed that an e c t o p a r a s i t e may a l s o r e l y on host hormones f o r maturation. She (1965b) has shown t h a t the most e f f e c t i v e hormones t h a t c o n t r o l maturation i n the r a b b i t f l e a s are c o r t i c o s t e r o i d s and e s t r a d i o l . I n j e c t i o n of salmon p i t u i t a r i e s i n t o prespawning sockeye from Great C e n t r a l Lake s t i m u l a t e d development of secondary sexual c h a r a c t e r s . N u p t i a l c o l o r a t i o n was apparent a f t e r ten days and p i t u i t a r y i n j e c t e d f i s h c o u l d r e a d i l y be separated from the r e s t a f t e r 21 days on the b a s i s of n u p t i a l c o l o r . Burrows e t a l (1952) observed spawning c o l o r i n p i t u i t a r y i n j e c t e d a d u l t blueback sockeye, Oncorhynchus nerka 59 after, 12 days of i n j e c t i o n . Palmer e t a l (1954) s t a t e d that i n j e c t i o n of salmon p i t u i t a r y i n t o a d u l t salmon markedly hastened the development of n u p t i a l c o l o r a t i o n and other second-ary sexual c h a r a c t e r i s t i c s and r i p e n e d males as e a r l y as 3 days a f t e r i n j e c t i o n . I n j e c t i o n of salmon p i t u i t a r y has s t i m u l a t e d the development of the l a r v a e of Philonema oncorhynchi. Development was judged on the b a s i s of absence or presence of v i a b l e ' t a i l e d ' l a r v a e i n the u t e r u s of female worms. Development of P. oncorhynchi i n p i t u i t a r y i n j e c t e d f i s h i s very s i g n i f i -cant when compared with worms from u n t r e a t e d c o n t r o l s and from S t i l b e s t r o l t r e a t e d f i s h . L a r v a l development of P^ oncorhynchi may be suppressed by the i m p l a n t a t i o n of S t i l b e s t r o l i n f i s h of both sexes. S t i l -b e s t r o l has a marked e f f e c t on the growth of t e s t e s , but no s i g n i f i c a n t changes i n the o v a r i e s of implanted f i s h . No d i f f -erence was found between body weight of implanted and c o n t r o l f i s h , and m a t u r i t y index of the former d i f f e r s s i g n i f i c a n t l y from the l a t t e r . The r e s u l t s d i s c u s s e d above suggest t h a t the p i t u i t a r y e x t r a c t s t i m u l a t e s the development of Philonema  oncorhynchi d i r e c t l y or i n d i r e c t l y , whereas i t i s suppressed by S t i l b e s t r o l i m p l a n t a t i o n . Experiments u s i n g s e x u a l l y immature sockeye from Shuswap Lake f a i l e d (Table I V ) . T h i s may have r e s u l t e d from l a c k of l i v e a d u l t female worms i n t r e a t e d and u n t r e a t e d f i s h . Sub-adult female worms were found i n the body c a v i t y of approx-i m a t e l y f i f t y percent of the f i s h , upon examination before 60 experiments began. But a l l the worms were found encysted i n both experimental and the c o n t r o l f i s h i n June, 1965. A s i m i l a r s i t u a t i o n was found i n p r e c o c i o u s male a d u l t spawning sockeye i n Shuswap Lake. Only one c y s t was found i n a spawn-i n g f i s h out of 25 examined ( M a r g o l i s , p e r s o n a l communication). The above f a c t s suggest that Philonema i n Shuswap Lake are not Philonema oncorhynchi which i s synchronized with the l i f e c y c l e of the h o s t . Development of P. oncorhynchi i s probably synchronized with seasonal changes i n p h y s i o l o g y and behavior of sockeye. Hoar (1965a) d i s c u s s e d the seasonal changes i n d e t a i l s . Sockeye spawn once d u r i n g t h e i r l i f e c y c l e and t h i s i s g e n e r a l l y on the f o u r t h year but i n some stock i n the f i f t h year i n f r e s h water, and P. oncorhynchi f o l l o w s i t c l o s e l y . There are two molts i n Philonema l a r v a e i n f i s h : the f i r s t occurs d u r i n g the f i r s t autumn i n the sea a f t e r which the l a r v a e move out of the swim-bladder t i s s u e i n t o the p e r i t o n e a l c a v i t y . The t i m i n g of the second molt c o u l d not be determined but i t probably occurs d u r i n g the f i r s t s p r i n g i n the sea or soon a f t e r the f i r s t m o l t i n g p e r i o d , a f t e r which P^ _ oncorhynchi grows r a p i d l y . The p r o d u c t i o n of l a r v a e i n P^ oncorhynchi p a r a l l e l s gonadal growth i n the f i s h . G r a v i d P^ _ oncorhynchi are e m i t t e d along with f i s h eggs i n t o the water, and f i r s t stage l a r v a e then become f r e e -swimming. They are eaten by Cyclops b i c u s p i d a t u s which are a v a i l a b l e i n abundance d u r i n g t h i s p e r i o d . Reproduction of P.  oncorhynchi i s seasonal and i s c o r r e l a t e d with the c y c l e of 61 sockeye salmon. I t i s an in s t a n c e of the general statement made by Hoar (1966), " I f r e p r o d u c t i o n i s seasonal or occurs only once i n the animals l i f e c y c l e , i t must be p r e c i s e l y timed so th a t young appear when food i s abundant and other c o n d i t i o n s are optimal f o r s u r v i v a l . Seasonal changes i n termperature, photo-p e r i o d , moisture, food, and chemicals may serve as t r i g g e r s f o r the t i m i n g of the a s s o c i a t e d p h y s i o l o g i c a l changes." The i n t e r - r e l a t i o n s h i p between hormones of the f i s h and growth and r e p r o d u c t i o n of P. oncorhynchi i s not c l e a r , but e x t e n s i v e work has been done on the p i t u i t a r y and r e p r o d u c t i v e c y c l e of f i s h ( P i c k f o r d and A t z , 1957). R e c e n t l y Hoar (1965b) reviewed the endocrine r e g u l a t i o n of r e p r o d u c t i o n i n the f i s h . He notes t h a t the p i t u i t a r y i s necessary f o r maturation of gonads i n a l l v e r t e b r a t e s above the Cyclostomes. T h e r e f o r e , i t may be s a i d t h a t the maturation of P^ oncorhynchi i s d i r e c t l y or i n d i r e c t l y dependent on the p i t u i t a r y hormones of f i s h or with some k i n d s of feedback mechanism of gonadal and gonado-t r o p i c hormones. I t has been demonstrated i n t h i s study t h a t the p i t u i t a r y hormone e f f e c t s the maturation of P^ oncorhynchi. The e f f e c t of p i t u i t a r y on p a r a s i t e s may be e i t h e r by t h e i r e f f e c t on the host or by d i r e c t e f f e c t on the worms. The e f f e c t of 1 7 - b - e s t r a d i o l on P_^  oncorhynchi needs more experimental work. 1 7 - b - e s t r a d i o l has been r e p o r t e d i n the o v a r i e s of the Oncorhynchus nerka (Hisaw, 1963). The blood estrogens of both male and female A t l a n t i c Salmon, Salmo s a l a r show a f i v e t o s i x f o l d increase at the time of spawning and 62 reach f i v e t o seven mg. /100 ml. blood (Cedar et a l , 1961). The c o n c e n t r a t i o n of 1 7 - b - e s t r a d i o l i n P a c i f i c Salmon i s not known, though i t i s b e l i e v e d that there i s an i n c r e a s e i n spawning P a c i f i c Salmon as i n A t l a n t i c Salmon. I t i s unknown i f maturation of P. oncorhynchi i n a d u l t salmon i s e f f e c t e d by 1 7 - b - e s t r a d i o l . Implanted S t i l b e s t r o l had no apparent e f f e c t on the m a t u r i t y of P_^  oncorhynchi i n both male and female prespawning salmon, but had suppressed the normal p r o g r e s s i v e growth of t e s t e s . But o v a r i e s are u n a f f e c t e d . The l a r g e doses of S t i l b e s t r o l given t o r e p r o d u c i n g sockeye may have suppressed the normal c o - o r d i n a t i n g function of the p i t u i t a r y and gonadal hormones (or s u p p r e s s i o n of gonodotropic hormones) which have r e g r e s s e d both the gonads of f i s h and the m a t u r i t y of worms. What other hormones c o u l d be i n v o l v e d that produce maturation of P^ _ oncorhynchi i n a d u l t salmon? A review of the l i t e r a t u r e c o n cerning changes i n d i f f e r e n t hormone l e v e l s of spawning salmon might answer t h i s q u e s t i o n . P a c i f i c salmon (Genus Oncorhynchus) e x h i b i t e d both h i s t o l o g i c a l and p h y s i o l o g i c a l changes d u r i n g t h e i r maturation and spawning p e r i o d (Robertson N and<£al 1961 a, b) . There was a marked h y p e r p l a s i a of the adrenal c o r t i c a l t i s s u e present i n the s e x u a l l y mature f i s h (Robertson and Wexler 1959), and a g r e a t l y i n c r e a s e d c o n c e n t r a t i o n of 1 7 - h y d r o x y c o r t i c o s t e r o i d s i n spawn-i n g salmon's bfood (Hane and Robertson, 1959). The changes i n 63 c o n c e n t r a t i o n of 17-OHCS from the sea t o the spawning grounds re p r e s e n t e d an i n c r e a s e of as much as seven f o l d . (The average i n spawning females was 79 ^ug pe r c e n t , i n males 31 /ug p e r c e n t ) . Robertson and Wexler (1960) and I d l e r et a l (1959) a l s o found a sharp i n c r e a s e i n the c o r t i s a l c o n c e n t r a t i o n i n sockeye plasma d u r i n g a b r i e f p e r i o d immediately p r e c e e d i n g spawning and d u r i n g completion of the spawning a c t . Female plasma co n t a i n e d double the c o n c e n t r a t i o n of c o r t i s o n e and C o r t i s o l i n male plasma, 41 ^ug and 26 ^ug/lOO ml. r e s p e c t i v e l y as compared with 22 /ug and 11 /ug/100 ml. I d l e r e t a l (1959) suggested t h a t the extremely h i g h l e v e l s of c o r t i s o n e and C o r t i s o l r e g u l a t e mineral metabolism i n salmon, at l e a s t d u r i n g the r i v e r m i g r a t i o n . There are osmotic and met a b o l i c changes i n f i s h when they e n t e r an e s t u a r y or f r e s h water from the sea, and C o r t i s o l and c o r t i s o n e r e g u l a t e t h i s change. Does Philonema oncorhynchi a l s o face a s i m i l a r osmotic and me t a b o l i c change along with the f i s h ? Is there any c o r r e l a t i o n between sharp i n c r e a s e of 17-OHCS i n the blood of f i s h and the r a p i d development of l a r v a e of Philonema oncorhynchi i n the p e r i t o n e a l c a v i t y of the host d u r i n g the m i g r a t i o n of salmon? R o t h s c h i l d (1965b) showed t h a t the most e f f e c t i v e hormones c o n t r o l l i n g maturation i n the r a b b i t f l e a are the c o r t i c o s t e r o i d s , produced by the adren a l glands under the c o n t r o l of the a d r e n o c o r t i c o t r o p i c hormones which are s e c r e t e d i n t u r n by the a n t e r i o r lobe of the p i t u i t -a ry. 64 There i s no experimental evidence, as y e t , t o i n d i c a t e whether gonadal hormone or hormones from the adrenal organ p l a y a r o l e i n the m a t u r i t y of Philonema oncorhynchi, or whether any of these e x e r t an e f f e c t i n d i r e c t l y through the host's metabol-ism. So f a r , i t i s known t h a t salmon p i t u i t a r y e x t r a c t has a s t i m u l a t i n g e f f e c t on the m a t u r i t y of P^ _ oncorhynchi, but no more than t h i s i s known. I t i s obvious that much more e x p e r i -mental work i s t o be done before s p e c i f i c hormones r e q u i r e d f o r the m a t u r i t y of P^ oncorhynchi i n anadromous sockeye salmon are known. 65 SECTION IV T r a n s p l a n t a t i o n of Worm. INTRODUCTION A second s e r i e s of experiments watte designed t o t e s t the h y p o t h e s i s t h a t Philonema oncorhynchi i s dependent on hormones of sockeye salmon f o r p r o d u c t i o n of l a r v a e . Worms of d i f f e r e n t ages were t r a n s p l a n t e d t o other hosts of v a r y i n g age and s p e c i e s . The purpose of the experiments was t o gain f u r t h e r i n f o r m a t i o n on the dependence of P_._ oncorhynchi on host hormones f o r the development of i t s l a r v a e and t o determine whether a s i n g l e s t i m u l u s or s u s t a i n e d s t i m u l a t i o n i s r e q u i r e d . The experiments had a d d i t i o n a l advantages of a c q u i r i n g informa-t i o n as t o the s u r v i v a l and growth of worm i n a l t e r e d s i t e s of d i f f e r e n t r e c i p i e n t h o s t s . MATERIALS AND METHODS Worms used f o r T r a n s p l a n t a t i o n a) A d u l t P_. oncorhynchi not yet g r a v i d , c o l l e c t e d from sockeye salmon at Sweltzer Creek f i s h t r a p , C u l t u s Lake one t o two months p r i o r t o spawning. b) T h i r d stage l a r v a e of P. oncorhynchi from the swimbladder of sockeye smolts from C u l t u s Lake. c) F o u r t h stage l a r v a e of P^ oncorhynchi from the body c a v i t y of a p r e c o c i o u s male sockeye salmon from C u l t u s Lake. 66 R e c i p i e n t Hosts a) Rainbow t r o u t two years of age, from Sun V a l l e y T r o a t Farm, Coquitlam, B.C. b) Rainbow t r o u t , 2| years of age, from the above source. c) S t e e l h e a d , the anadromous v a r i e t y of g a i r d n e r i , which were at l e a s t two years of age when used. d) Sockeye salmon (Oncorhynchus n e r k a ) . Approximately 2\ year o l d sockeye of Shuswap Lake were obtained as smolt i n 1964. e) Gold f i s h ( C a r a s s i u s c a r a s s i u s ) were obtained from a comm-e r c i a l pet shop. f ) Frogs,, (Rana p j p i e n s ) were s u p p l i e d by Vancouver B i o l o g i c a l S u p p l i e r s , V i c t o r i a . T r a n s p l a n t a t i o n of a d u l t Worms i n t o F i s h The f i s h were a n a e s t h e t i s e d with MS "222 (0.1 g m . / l i t . water). The immobilized f i s h were p l a c e d on t h e i r backs i n a cloth, h o l d e r , which was a d j u s t e d a c c o r d i n g t o the s i z e of the f i s h . D uring the o p e r a t i o n , the g i l l s were i r r i g a t e d through the -mouth with a 1:10,000 s o l u t i o n of MS 222 by means of a pump r e c y c l i n g system, p r o v i d i n g approximately 10 l i t r e s per minute. F i s h c o u l d be maintained a l i v e on the o p e r a t i n g t r a y f o r as lo n g as necess a r y . An i n c i s i o n of about \ - 1 i n c h was made i n the mid v e n t r a l l i n e , a v o i d i n g the p e l v i c bones of the f i s h . A r e t r a c t o r was used t o widen the i n c i s i o n . . Required numbers of Philonema were t r a n s p l a n t e d i n t o the body c a v i t y i n Ringer's s o l u t i o n . The worms were p l a c e d i n s i d e , e i t h e r p o s t e r i o r or a n t e r i o r t o 67 the i n c i s i o n with the h e l p of a smooth g l a s s rod. Great care had t o be taken i n p l a c i n g the female worms i n s i d e as they were very f r a g i l e . Sutures were made with " m e a s u r o l l " b l a c k s i l i c o n e t r e a t e d s u r g i c a l thread very c l o s e t o each other t o make the wound w a t e r - t i g h t . Recovery of f i s h was brought about by s w i t c h i n g from a n a e s t h e t i c t o d e c h l o r i n a t e d water. Both anaes-t h e t i c and d e c h l o r i n a t e d water was aerated and maintained at a constant temperature. T r a n s p l a n t a t i o n of l a r v a l worms i n t o f i s h . The f i s h were a n a e s t h e t i z e d i n MS 222 (0.1 g m . / l i t . water) . The immobilized f i s h was s l i p p e d v e n t r a l s i d e up i n t o the trough with a foam rubber l i n i n g and the head was r a i s e d with a foam rubber p i l l o w . The f i s h was kept secure with rubber bands. The f i s h was then f e d n a t u r a l l y i n f e c t e d d e f l a t e d swimbladders of sockeye smolts from C u l t u s Lake through ja smooth g l a s s tube i n s e r t e d i n t o the stomach. Free l a r v a e from the swimbladder i n a minimal amount of Ringer's s o l u t i o n were s q u i r t e d i n t o the stomach of f i s h through a p o l y e t h y l e n e stomach tube. T r a n s p l a n t a t i o n of A d u l t and L a r v a l Worms i n t o Frogs Operations on f r o g s were made on a d i s s e c t i n g t r a y under e t h e r a n a e s t h e s i a . The limbs were t i e d t o the o p e r a t i n g t r a y and an i n c i s i o n of 1 i n c h was made on the v e n t r a l s i d e , a v o i d i n g the v e n t r a l abdominal v e i n s . The s k i n and body w a l l were p u l l e d out with a r e t r a c t o r and worms were p l a c e d i n the abdominal c a v i t y of the f r o g with the he l p of a p a i r of f o r c e p s . 68 Larvae were t r a n s p l a n t e d i n the abdominal c a v i t y i n Ringer's s o l u t i o n with a medical p i p e t t e . S u t u r i n g was c a r r i e d out i n the same manner as i n the f i s h . Frogs were maintained i n a wooden box with a l i g h t spray of water, i n a c o n t r o l l e d environment room at 10°C. RESULTS T r a n s p l a n t a t i o n of a d u l t Philonema oncorhynchi from matured  sockeye salmon i n t o immature rainbow t r o u t . (Table IX.) Two t o f o u r non-gravid female and three t o f i v e a d u l t male P_;_ oncorhynchi were t r a n s p l a n t e d i n t o the abdominal c a v i t y of each of 37 f i s h . T r a n s p l a n t e d t r o u t d i e d between 2 and 114 days. No female P^ oncorhynchi were recovered from the female f i s h which d i e d a f t e r the second day, with the e x c e p t i o n of one f i s h i n which female worms s u r v i v e d f o r 114 days. The female P. oncorhynchi s u r v i v e d f o r nine days i n male f i s h . A-lthough Male worms s u r v i v e d i n both male and female f i s h f o r a longer p e r i o d . The two female worms t h a t s u r v i v e d i n immature female f i s h f o r 114 days d i d not show any development of the u t e r i n e c o n t e n t s , whereas i n the normal spawning host a l l female P^ _ oncorhynchi had reached the f i n a l stage of m a t u r i t y . Male F\ oncorhynchi recovered from the t r a n s p l a n t e d host were as a c t i v e as those from the normal sockeye h o s t . The d i f f -erence i n the l o n g e v i t y of P_„ oncorhynchi i n the male and female r e c i p i e n t h osts was i n s i g n i f i c a n t . 6 9 I t was observed d u r i n g the autopsy without except-i o n t h a t both male and female Philonema oncorhynchi moved toward the a n t e r i o r p o r t i o n of the abdominal c a v i t y , e n t a n g l i n g around the l i v e r and the p y l o r i c caecae. Male worms were found to be more a c t i v e than female worms i n both the c o n t r o l and r e c i p i e n t h o s t s . Female worms were seen to come out of the p e r i t o n e a l c a v i t y through the i n c i s i o n s . P a t h o l o g i c a l changes were seen i n r e c i p i e n t hosts 77 days a f t e r t r a n s p l a n t a t i o n . V i s c e r a l adhesion was recorded a r b i t r a r i l y from + t o +++. Three k i n d s of c y s t s were found i n the abdominal c a v i t y of t r a n s p l a n t e d t r o u t : a) C y s t s of s o l i d t i s s u e i n which the worms c o u l d h a r d l y be i d e n t i f i e d . b) Female worms encased i n a f i b r o u s t i s s u e and attached e i t h e r on the l i v e r or the body w a l l . c) S o f t , p a r t l y d i s i n t e g r a t e d t i s s u e of female worms. T r a n s p l a n t a t i o n of T h i r d Stage Larvae of Philoneifla oncorhynchi  i n t o the stomach of s t e e l h e a d , rainbow t r o u t , and sockeye  salmon (Figure XII and Table X.) The r a t e of development of P_^  oncorhynchi i n the r e c i p i e n t h o s t s , sockeye salmon, rainbow t r o u t , s t e e l h e a d t r o u t , and the c o n t r o l sockeye ( n a t u r a l l y i n f e c t e d i n the l a k e ) , were of the same p a t t e r n ( F i g u r e XII and Table X . ) . Although a l l the t r a n s p l a n t e d f i s h were i n f e c t e d with t h i r d stage l a r v a e , great v a r i a b i l i t y i n growth was observed i n any s i n g l e group of f i s h . T h i r d , f o u r t h , and sub-adult P. 70 F i g . XII Development of Philonema oncorhynchi i n d i f f e r e n t t r a n s p l a n t e d salmonids. 3 5 0 2 5 0 150 2 6cJ z 5 5 I i -O 5 0 LU _1 Z < LU 2 4 5 4 0 -E 35 0 ! to 3 0 2 25 LU 1 20 15 10 5 la l i Is Is i i Control-1 Control -2 SOCKEYE E H Is p | i Is i Is Is Is I l i 1 L D L A R V . IN SWIMBLADDER HI1 1T» STAGE L A R V . IN COELOM i O" WORMS IN C O E L O M J WORMS IN C O E L O M TIME IN DAYS STEELHEAD PAIN BOW S O C K E Y E 71 oncorhynchi were found i n a s i n g l e t r a n s p l a n t e d f i s h . Two s i z e s of l a r v a e were found i n the swimbladders of a l l groups of f i s h with the e x c e p t i o n of the Shuswap Lake sockeye where the i n f e c t i o n was 55 days o l d . One a d u l t female oncorhynchi was found i n the body c a v i t y of a male rainbow t r o u t a f t e r 232 days of t r a n s p l a n t a t i o n . In a l l groups of f i s h , male worms were g e n e r a l l y more p r e v a l e n t i n the body c a v i t y than female worms. T h i r d stage l a r v a e were recovered from the swimbladders of rainbow t r o u t when they were a u t o p s i e d at 16, 24, 36 and 92 hours a f t e r b e i n g f e d with the n a t u r a l l y i n f e c t e d swimbladders of sockeye salmon, but none were recovered at 5 and 10 hours. The u n d i g e s t e d swimbladder and l i v e l a r v a e were found i n the stomach of the r e c i p i e n t host not l a t e r than 10 hours. T r a n s p l a n t a t i o n of t h i r d stage Larvae of Philonema oncorhynchi  i n t o the body c a v i t y of t r o u t Approximately 35 l a r v a e from the swimbladders of sockeye smoltj were t r a n s p l a n t e d i n t o the body c a v i t y of each of f i v e t r o u t (Salmo g a i r d n e r i i ) . F i s h were examined a f t e r 7 hours, 40 hours, 8 days, 13 days, and 14 days, and 0, 5, 6, 4, and 11 l a r v a e r e s p e c t i v e l y were recovered from the swim-bladder of the r e c i p i e n t h o s t s . Dead l a r v a e were found i n the v i s c e r a l mass of the f i s h . .72 T r a n s p l a n t a t i o n of f o u r t h stage l a r v a e i n t o the stomach and  body c a v i t y of t r o u t a) Approximately 30 l a r v a e were s q u i r t e d i n t o the stomach of each of the f i v e t r o u t . F i s h were s a c r i f i c e d at 24 hour i n t e r v a l s ; no l a r v a e were found i n the swimbladders of the r e c i p i e n t h o s t s . Dead l a r v a e were found i n the stomachs of those f i s h which were examined at 24 and 48 hours. b) Approximately 30 f o u r t h stage l a r v a e were t r a n s p l a n t e d i n t o the body c a v i t y of each of the f i v e t r o u t . F i s h were examined 1, 3, 5, 50,.and 223 days a f t e r t r a n s p l a n t a t i o n . No l a r v a e were found i n the swimbladder of any of the f i s h with the e x c e p t i o n of the f o u r t h f i s h i n which one l a t e t h i r d stage l a r v a was found. Larvae l i v e d i n the body c a v i t y of a l l f i s h . The l a r v a e i n the l a s t f i s h examined had an average l e n g t h of 11.78 mm. T r a n s f e r of i n f e c t i o n by p r e d a t i o n Two h a t c h e r y - r e a r e d u n i n f e c t e d rainbow t r o u t of 24.0 cm. l o n g were s t a r v e d f o r two days i n two separate tanks. One n a t u r a l l y i n f e c t e d sockeye smolt of 8.5 cm. l o n g was t r a n s f e r r e d i n t o each tank. Both t r o u t chased and captured the smolt from the a n t e r i o r end w i t h i n 10 minutes. F i s h were a u t o p s i e d at 19 and 32 hours; 53 and 22 l a r v a e r e s p e c t i v e l y were recovered from the swimbladders. Dead and l i v e l a r v a e were a l s o found i n the stomachs of both f i s h . 73 T r a n s p l a n t a t i o n of A d u l t and Fourth stage l a r v a e of Philonema  oncorhynchi i n t o F r ogs, Rana p i p i e n s a) A l l t r a n s p l a n t e d a d u l t female P^ _ oncorhynchi were found to be dead and encysted when the f r o g s were au t o p s i e d a f t e r 13, 16, and 18 days. Only one a d u l t male worm was recovered alive.- a f t e r 13 days of t r a n s p l a n t a t i o n , (Table X I a ) . V i s c e r a l adhesion was apparent i n a l l r e c i p i e n t f r o g s . Cyst formation o c c u r r e d around the a d u l t female worms. The female worms were encased i n t h i n brownish f i b r o u s ( t i s s u e and the c y s t s were e i t h e r a t t a c h e d t o the body w a l l or t o the l i v e r . Worms i n f r o g #5 were found e n c a p s u l a t e d i n one l a r g e c y s t . No c y s t was found i n f r o g #4, but loo s e decomposed worms were found i n the body c a v i t y . b) Fourth stage l a r v a e s u r v i v e d i n the body c a v i t y of t r a n s -p l a n t e d frogs f o r 18 days when the l a s t f r o g was s a c r i f i c e d , (Table X l b ) . No v i s c e r a l adhesion or any other p a t h o l o g i c a l changes were observed i n the r e c i p i e n t abnormal h o s t . T r a n s p l a n t a t i o n of Fourth Stage Larvae i n t o the Abdominal  C a v i t y of Gold f i s h Ten worms were t r a n s p l a n t e d i n t o each of 6 f i s h . F i s h were au t o p s i e d 5, 10, 47, 67, 75, and 80 days a f t e r t r a n s p l a n t a t i o n . F i v e (2 dead), 6, and 2 l a r v a e were recovered from the body c a v i t y of the f i r s t 3 f i s h respective-l y . No l a r v a e were found i n any f i s h a f t e r 47 days of t r a n s -p l a n t a t i o n . No v i s c e r a l adhesions or any other p a t h o l o g i c a l changes were observed. 74 DISCUSSION Philonema oncorhynchi i s p h y s i c a l l y and p h y s i o l o -g i c a l l y adapted f o r r e s i d e n c e i n anadromous sockeye f o r more than three y e a r s . During t h i s p e r i o d P^ _ oncorhynchi grows along with the sockeye salmon. I t i s found that maturation of(  P. oncorhynchi i s c o r r e l a t e d with the maturation of the h o s t . When a d u l t P^ oncorhynchi were t r a n s p l a n t e d i n t o s e x u a l l y immature t r o u t and abnormal f r o g h o s t s , the female worms f a i l e d t o e s t a b l i s h themselves i n the abdominal c a v i t y of the r e c i p i e n t h osts and d i e d w i t h i n a s h o r t p e r i o d . V i c t o r (1958) t r a n s p l a n t e d bovine S e t a r i a d i g i t a t a i n t o the p e r i t o n e a l c a v i t y of a r a b b i t and a male and female goat. None of the animals showed any m i c r o f i l a r i a e i n the blood and none of the a d u l t s were r e c o v e r e d . In the present study two a d u l t female oncorhynchi s u r v i v e d f o r 114 days and males g e n e r a l l y s u r v i v e d f o r a l o n g -er p e r i o d . The female worms are very s l u g g i s h which i s probably due t o t h e i r l a r g e s i z e . The s u r v i v a l of male worms may be due t o t h e i r a c t i v e m o t i l i t y which enables them t o escape the d e f e n s i v e mechanism of the h o s t . Although female P. oncorhynchi s u r v i v e d f o r 114 days they d i d not become g r a v i d i n immature t r o u t . In a s i m i l a r p e r i o d a l l female worms i n spawning sockeye would have become g r a v i d . T h i s suggests t h a t a d u l t P_._ oncorhynchi i s p h y s i o l o -g i c a l l y s p e c i f i c t o the donor sockeye host f o r m a t u r i t y and t h e r e f o r e w i l l not mature i n immature r e c i p i e n t t r o u t . I t 75 a l s o be p o s s i b l e t h a t l a r v a l p r o d u c t i o n of P_. oncorhynchi i s dependent on continued s t i m u l a t i o n of some e s s e n t i a l hormones of the matured host which are m i s s i n g i n the immature host. Larka e t a l (1963) t r a n s p l a n t e d male and female S e t a r i a s p e c i e s i n t o r a b b i t s . M i c r o f i l a r i a e of c e r v i were present i n the r a b b i t but showed no p e r i o d i c i t y . A l i c a t a and Noda (1960) found t h a t Philophthalmus from c h i c k s reached m a t u r i t y and produced eggs i n h e t e r o t r a n s p l a n t e d eyes of r a t s at about the same time as i n c h i c k s . T h i r d stage l a r v a e of oncorhynchi migrate i n t o the swimbladder when t r a n s p l a n t e d i n t o e i t h e r the stomach or body c a v i t y of t r o u t and s u r v i v e d as l o n g as the f i s h d i d . The r a t e of growth of l a r v a e i n the r e c i p i e n t host i s com-parable t o those i n donor h o s t s . Fourth stage l a r v a e from the body c a v i t y continued t o l i v e i n the abdominal c a v i t y of t r a n s p l a n t e d t r o u t and developed as i n the sockeye host (donor), but they were unable t o i n f e c t the f i s h when i m p l a n t e d , i n t o the stomach. In other words, the f o u r t h stage l a r v a e have l o s t t h e i r c a p a c i t y t o migrate back t o t h e i r o r i g i n a l s i t e . I t i s not ve r y s u r p r i s i n g t o observe t h a t f o u r t h stage l a r v a e have s u b s i s t e d i n the p e r i t o n e a l c a v i t y of gold f i s h f o r 47 days and i n f r o g s f o r 18 days. Larvae would probably have l i v e d l onger i n f r o g s i f the h o s t s had s u r v i v e d . G e n e r a l l y , salmonids serve as ho s t s f o r Philonema so f a r there are no r e p o r t s of Philonema i n hosts other than 76 salmonids with the e x c e p t i o n of s t i c k l e b a c k s which have been r e p o r t e d from the U.S.S.R. by Spassky e t a l i n 1961. The author has t r i e d t o i n f e c t g o l d f i s h with t h i r d stage l a r v a e but f a i l e d t o do so. T h i s may be due t o e i t h e r host s p e c i f i c i t y or because of the pneumatic duct of g o l d f i s h i s c l o s e d . There-f o r e the s u r v i v a l of f o u r t h stage l a r v a e i n the p e r i t o n e a l c a v i t i e s of g o l d f i s h and f r o g s suggest that the p e r i t o n e a l c a v i t i e s of these abdnormal hosts can meet some e s s e n t i a l n u t r i -t i o n a l and other p h y s i c o - c h e m i c a l requirements of l a r v a e . French (1965)c^iMargolis (personal communication) observed v i s c e r a l adhesions i n high sea salmon. Prespawning male and p r e c o c i o u s male sockeye e x h i b i t e d v i s c e r a l adhesions ( P l a t z e r , 1964). The author a l s o observed v i s c e r a l adhesions i n s i m i l a r f i s h e s . Richardson (1937), Needham (1965), and Pippy (1966) observed p a t h o g e n i c i t y and v i s c e r a l adhesions i n d i f f e r e n t s p e c i e s of t r o u t i n f e c t e d with Philonema  agubernaculum but the author f a i l e d to observe any patho-g e n i c i t y i n the t r o u t of t h i s study. Encystment of female P. oncorhynchi i n the abdominal c a v i t i e s of t r o u t and f r o g s i s probably due to host r e a c t i o n . I n c o m p a t a b i l i t y between host ( f r o g ) and implanted nematodes i n the body c a v i t y r e s u l t e d i n death of the p a r a s i t e s i n a much s h o r t e r p e r i o d than i n t r o u t . In response t o implanted a d u l t Philonema, the host probably r e a c t s around the p a r a s i t e and e n c a p s u l a t e s i t . Male worms mostly escape encystment probably because of t h e i r a c t i v e movement. I t i s e a s i e r f o r 77 the host t o develop e p i t h e l i a l outgrowths around immobile or i n a c t i v e p a r a s i t e s than a c t i v e male worms. Male worms may d i f f e r from the a d u l t females i n some important b i o l o g i c a l way other than s i z e and a c t i v i t y . Adhesions and decomposition of female worms i n f r o g s appear s i m i l a r t o t h a t r e p o r t e d by Goodchi (1954), where he found " p a r a s i t e s became immeshed i n a stroma of phagocytes and f i b r e s " . The form a t i o n of c y s t s i n the r e c i p -i e n t t r o u t and f r o g may be due t o the same reason argued by Fawcett e t a l (1947) who suggested t h a t the host responses are c o r r e l a t e d with the bulk of f o r e i g n implant. B a c t e r i a i n t r o -duced with the worms may c o n t r i b u t e t o the prompt, e f f i c i e n t p h a g o c y t o s i s and e a r l y demise of p a r a s i t e . On the other hand, Reichkenbach-Kline (1955) suggests t h a t the " c y s t f o r m a t i o n " does not f o l l o w as a response t o mechanical factory. I t on l y appears as a r e a c t i o n t o a s p e c i f i c chemical s t i m u l u s s u f f e r e d by f i s h t i s s u e s i n the presence of p a r a s i t e s . " Philoneiqa can be t r a n s f e r r e d from one host t o another by f e e d i n g the s m a l l i n f e c t e d f i s h t o l a r g e c a r n i v o r o u s Salmonidae. T h i r d stage l a r v a e of oncorhynchi are found i n the swimbladder of rainbow t r o u t 19 hours a f t e r a meal of n a t u r a l l y i n f e c t e d sockeye smolt. T h i s suggests t h a t sockeye smolt or other i n f e c t e d f i s h can act as p a r a t e n i c h o s t ^ t o Philonema. T h i s would probably h e l p t o e x p l a i n the i n f e c t i o n s of P. agubernaculum i n l a r g e non-anadromous t r o u t which do not feed on pl a n k t o n . 78 "Host s p e c i f i c i t y i s w e l l r e c o g n i z e d i n P a r a s i t o l o g y but l e s s w ell r e c o g n i z e d i s an organ s p e c i f i c i t y which oper-ates i n p a r a s i t i c o r i e n t a t i o n t o and maintenance i n s p e c i f i c organ of the h o s t / 8 (Goodchild, 1954). P„ oncorhynchi has shown i n the present s t u d i e s both host and organ s p e c i f i c i t y . Host s p e c i f i c i t y has been d i s c u s s e d i n s e c t i o n I I . T h i r d stage l a r v a e e i t h e r from d e f l a t e d swimbladder or from swimbladder of i n t a c t f i s h migrated from the stomach i n t o the swimbladder of the r e c i p i e n t host when t r a n s p l a n t e d . When t r a n s p l a n t e d i n t o the body c a v i t y , the t h i r d stage l a r v a e migrated i n t o the swimbladder but the f o u r t h stage l a r v a e l i v e d i n the body c a v i t y of r e c i p i e n t h osts without m i g r a t i n g i n t o the swim-b l a d d e r . I t suggests that t h i r d and f o u r t h stage l a r v a e of P. oncorhynchi are very much s p e c i f i c t o t h e i r s i t e and host. Summary T r a n s p l a n t e d a d u l t P_._ oncorhynchi d i d not become g r a v i d i n immature r e c i p i e n t host d u r i n g the 114 days of the experiment. A d u l t worms d i e d i n a s h o r t e r p e r i o d when t r a n s p l a n t e d i n abnormal host ( f r o g ) . T h i r d stage l a r v a e had migrated i n t o the t i s s u e of swimbladder when t r a n s p l a n t e d i n t o the stomach and the coelom of f i s h . F o u rth stage l a r v a e had l i v e d i n the coelom but were f l u s h e d out from the stomach. 79 SECTION V. Development of Philonema oncorhynchi i n n a t u r a l l y i n f e c t e d sockeye of R i v e r s I n l e t . INTRODUCTION The t h i r d l i n e of evidence b e a r i n g on q u e s t i o n of hormonal r e g u l a t i o n of l a r v a l p r o d u c t i o n i n I\_ oncorhynchi i s t o be found i n c e r t a i n n a t u r a l p o p u l a t i o n s of sockeye salmon which d i f f e r i n the l e n g t h of t h e i r r e p r o d u c t i v e c y c l e . In the commercial salmon f i s h e r y at R i v e r s I n l e t , B r i t i s h Columbia, a d u l t sockeye salmon u s u a l l y r e t u r n t o Owikeno Lake t o spawn i n t h e i r 4th or.5th year, a f t e r 2 or 3 years of ocean r e s i d e n c e . A few p r e c o c i o u s males r e t u r n t o spawn i n t h e i r 3rd year and a v e r y few f i s h r e t u r n i n t h e i r 6th year. P. oncorhynchi i s known t o occur i n t h i s p o p u l a t i o n . The R i v e r s I n l e t f i s h can t h e r e f o r e be used t o t e s t the h y p o t h e s i s of hormonal dependence of P^ oncorhynchi on i t s h o s t . I f the worm i s dependent on the host hormonal s t i m u l u s t o produce i t s l a r v a e , then a l l age groups.in the R i v e r s I n l e t f i s h p o p u l a t i o n should c o n t a i n maturing worms as the f i s h r e t u r n to the spawning grounds. On the other hand, i f the occurrence of l a r v i g e r o u s worms i n the 4 year o l d spawning sockeye of C u l t u s Lake ( S e c t i o n I) may be (merely) the r e s u l t of a c o i n c i d e n c e between a 4 year c y c l e i n the f i s h and a 3 year developmental p a t t e r n i n the worm, 80 only one age c l a s s of the R i v e r s I n l e t p o p u l a t i o n should c o n t a i n the g r a v i d Philonema. V a r i o u s age c l a s s e s of the R i v e r s I n l e t salmon popula-t i o n were examined t o determine the s t a t e of development of worms they harboured. MATERIALS AND METHODS Four year o l d sockeye salmon, Oncorhynchus nerka were captured from the North P a c i f i c Ocean between l a t i t u d e 48' 02" and l o n g i t u d e 39' 56 H i n A p r i l , 1965; from the mouth of R i v e r s I n l e t i n the commercial f i s h i n g area number 9 of the Department of F i s h e r i e s , Government of Canada, i n August, 1965 and spawn-i n g f i s h from the Owikeno Lake i n October, 1965 0 P r e c o c i o u s sockeye and 5^ sockeye were c o l l e c t e d i n August, 1965 from Owikeno Lake and commercial f i s h i n g area number 9 r e s p e c t i v e l y . A l l the f i s h were kept f r o z e n . A l l the f i s h were aged and i d e n t i f i e d as having been of R i v e r s I n l e t o r i g i n by d i s t i n g u i s h i n g scale c h a r a c t e r i s t i c s by the members of M e r i s t i c L a b o r a t o r y , B i o l o g i c a l S t a t i o n of the F i s h e r i e s Research Board of Canada, Nanaimo, B r i t i s h Columbia. In the present study, the G i l b e r t (1913) system of age d e s i g n a t i o n i s used, the l a r g e type of A r a b i c f i g u r e i n d i -c a t e s t o t a l age when caught and the s u b s c r i p t the year of l i f e i n which i t migrated from the f r e s h water. 81 C o l l e c t i o n and Measurements of A d u l t Philonema oncorhynchi Frozen f i s h were l e f t over n i g h t at room temperature and a u t o p s i e d with a m i d - v e n t r a l i n c i s i o n from the base of the p e c t o r a l f i n s t o the anus. Complete v i s c e r a was p l a c e d i n a bowl of Ringer's s o l u t i o n . The swimbladder and p e r i t o n e a l membrane were separated and mounted on a 25 x 13 x 3 mm. p l e x i s l i d e . The t i s s u e was spread on the p l e x i s l i d e with needles. Another p i e c e of s l i d e of the same s i z e was p l a c e d on the teased out swimbladder and screwed down on both ends t o keep the t i s s u e pressed. The p l a t e was examined f o r l a r v a e under a b i n o c u l a r d i s s e c t i n g microscope. The v i s c e r a was teased apart i n Ringer's s o l u t i o n and the adult worms were separated out. Ringer's s o l u t i o n with contents was poured i n t o a c y l i n d e r and allowed t o s e t t l e . When s e t t l e d , the supernatent s o l u t i o n was poured out and a l l the sediment washed twice t o remove the dark c o l o u r f o r e a s i e r examination under the microscope. A f t e r , washing, the contents were poured i n t o a p a r t i t i o n e d p l e x i g l a s s p e t r i d i s h and examined f o r s m a l l Philonema. A l l the worms were presetted i n 80% e t h a n o l and s t o r e d i n a v i a l . A l l the a d u l t worms were c l e a r e d i n l a c t o p h e n o l f o r 2 t o 5 days beforeimeasurement. The t o t a l l e n g t h of the male and immature Philonema were measured by the method used f o r l a r g e l a r v a e as d e s c r i b e d i n S e c t i o n I. Measurements of the t o t a l l e n g t h of the a d u l t worms were c a r r i e d out on the m i l l i m e t e r s c a l e mounted on a p l e x i s l i d e board under a b i n o c u l a r microscope. 82 RESULTS (Tables X I I , X I I I , and XIV. F i g u r e s X I I I , XIV, XV) The a d u l t sockeye salmon of R i v e r s I n l e t o r i g i n spawns i n Owikeno Lake, B r i t i s h Columbia d u r i n g the p e r i o d , September t o e a r l y December i n t h e i r f o u r t h or f i f t h year, a f t e r two or three years of ocean r e s i d e n c e . The average f o r k l e n g t h , body weights, gonad weights and m a t u r i t y index of the f i s h captured i n A p r i l , ,1965 at l a t -i t u d e 48' 02" and l o n g i t u d e 39' 56" was 44.4 cm., 1002.8 gm., 1.49 gm. and 0.14 r e s p e c t i v e l y f o r male f i s h and 48.0 cm., 1170.0 gm., 9.45 gnu , and 0.81 r e s p e c t i v e l y f o r female f i s h . Three males and one female were found immature and the r e s t 'maturing' at the time of capture a c c o r d i n g t o the m a t u r i t y i n d i e : e s (Godfrey 1961 and I s h i d a e t a l 1958). These f i s h were approximately f i v e t o s i x months before the entrance i n t o streams and s i x to nine months before spawning. F i s h of fou r and f i v e years of age captured i n August, 1965 from the o u t l e t of R i v e r s I n l e t were d e f i n i t e l y mature and only three t o fou r months before the spawning. The f o r k l e n g t h , body weight and gonad weights of 52 sockeye were higher than 4g (Table X I I . ) . 4 2 sockeye of October, 1965 were captured from Owikeno Lake p r i o r t o spawning. They showed decreased body weight but i n c r e a s e d weight of ovary. The mean weight of t e s t e s of f i s h i n c r e a s e d from 1.49 gm, when caught i n A p r i l , 1965 t o 73.4 gm. i n October, 1965, and the ovary from 9.45 gm. t o 327.3 gm. (Figure X I I I ) . 83 F i g . X I I I R e l a t i o n s h i p between the degree of maturation of sockeye salmon of R i v e r s I n l e t and the time of c a t c h . 20 TIME OF CATCH 84 The p r e c o c i o u s sockeye were found mature i n August, 1965 i n t h e i r t h i r d year, having mean t e s t e s weight of 20.6 gm. and m a t u r i t y index of 3.63. An estimate of growth i n s i z e of male and female P. oncorhynchi from both sexes of f i s h were obtained s e p a r a t e l y by c a l c u l a t i n g a s e r i e s of r e g r e s s i o n s a g a i n s t the age and time the f i s h were cap t u r e d . The growth i n d i s t a n c e of nerve r i n g from a n t e r i o r end and t o t a l oesophagus of d i f f e r e n t sexes of worms ob t a i n e d from the d i f f e r e n t sexes of the f i s h at d i f f e r e n t times of the year and d i f f e r e n t age composition of sockeye (Table XV). The p r o g r e s s i v e growth i n the s i z e of Philonema  oncorhynchi are s i g n i f i c a n t i n both male and female f i s h of the same age, ( i . e . 4g) when captured i n A p r i l , August, and October, 1965, but not s i g n i f i c a n t when compared a g a i n s t d i f f e r e n t age groups of f i s h ( i . e . 32's, 4 2 S ' a n d 5 2 S ^ captured at the same time. Growth i n d i s t a n c e of nerve r i n g and t o t a l oesophagus from the a n t e r i o r end of worms were dependent on the s i z e of worms but were u n a f f e c t e d by the age of the f i s h or worms. In other words, the d i s t a n c e t o nerve r i n g and l e n g t h of oesoph-agus i n c r e a s e when the t o t a l l e n g t h of worms i n c r e a s e s . An apparent r e l a t i o n s h i p e x i s t s between the growth r a t e of female Philonema oncorhynchi and gonad of f i s h of same age. The growth of female Philonema oncorhynchi i n s i z e was dependent i n the p r o g r e s s i v e growth of the gonad of the f i s h . G r a v i d i t y 85 of the female Philonema oncorhynchi was d i r e c t l y p r o p o r t i o n a l to the r i p e n e s s of eggs of the f i s h ( F i g u r e XIV). Random g r a v i d -i t y of female worms was observed i n male f i s h ( F i g u r e XV). V i s c e r a l adhesions were observed i n only seven of the t o t a l 168 f i s h of R i v e r s I n l e t examined. The occurrence of c y s t s i n f i s h was not dependent on the age and sex of the f i s h . I t was very much v a r i a b l e but tended to show more c y s t s per male f i s h i r r e s p e c t i v e of age and time of capture of f i s h . DISCUSSION The sharp i n c r e a s e i n gonad weight of sockeyein the present study i s i n g e n e r a l agreement with the f i n d i n g s of I s h i d a and Miyaguchi (1958) and Godfrey (1961). The gonad weight of f i s h caught i n A p r i l i s very low i n comparison t o August of the same year. The gonad weight i n c r e a s e d from 1=49 gm. i n A p r i l t o 65.4 gm. f o r male and 9.45 gm. t o 77.5 gm. f o r female f i s h . D uring t h i s s h o r t p e r i o d , P_^  oncorhynchi showed r a p i d growth which i s d i r e c t l y p r o p o r t i o n a l t o the sharp i n c r e a s e of gonad of f i s h . The maturation of female worms i s d i r e c t l y dependent on the r i p e n i n g of o v a r i e s of f i s h . C o n v e r s e l y , maturation of female P_^  oncorhynchi depends not only on m a t u r i t y of t e s t e s but a l s o on some other unknown f a c t o r ( s ) as the g r a v i d i t y of P,  oncorhynchi and r i p e n e s s of t e s t e s are not p a r a l l e l . T h i s sugg-e s t s t h a t the maturation of P^ oncorhynchi i s not dependent f u l l y on gonadal hormones but a l s o on hormone or hormones of the p i t u i t a r y and with some other mechanism of r e l a t i o n between 86 F i g . XIV R e l a t i o n s h i p between the ovary weight and the p r o d u c t i o n of l a r v a e i n Philonema oncorhynchi from sockeye salmon of R i v e r s I n l e t . 4501 0 coo OOCO CCO 000 ticab o X B b 00 03X0 O CD O OX00 OOCD oa JL JL JL EGG MOR NO EMB TAIL TAIL DEVELOPMENTAL STAGES 8? F i g . XV R e l a t i o n s h i p between the t e s t i s weight of f i s h and the p r o d u c t i o n of l a r v a e i n Philonema oncorhynchi from sockeye salmon. 0 r e p r e s e n t s the number of worms. TESTIS WEIGHT IN GRAMS _ o o o g o o o o ~l 1 1 1 I I 1 I 9 0 0 8 f < m I-o I m l o § § 8 § 8 §8§* 8 8 8 0 8 ° 9 o o o gpo 8 5| 88 p i t u i t a r y and gonad of f i s h . The dependence of l a r v a l produc-t i o n i n Philonema oncorhynchi on hormones has been d i s c u s s e d i n S e c t i o n I I I of present s t u d i e s . 42 male and female sockeye of the A p r i l c a t c h are mostly immature a c c o r d i n g to the m a t u r i t y i n d i c e s (Godfrey, 1961 and I s h i d a et a l 1958, 1961). The f i s h of A p r i l , though immature t might be the f i s h t h a t would have spawned i n the f a l l of the same year they were captured. One male and two female f i s h of the A p r i l catch probably would not spawn u n t i l one year l a t e r , c o n s i d e r i n g the very low gonad weight. No Philonema were found i n the body c a v i t y of these three f i s h . Larvae from the swimbladder would probably reach a d u l t stage i n one year's time when f i s h would a l s o reach sexual m a t u r i t y . Young Philonema  oncorhynchi i n the abdominal c a v i t y of f i s h of the A p r i l catch would probably reach the a d u l t form i n October which would be comparable to worms c o l l e c t e d from f i s h i n October, G e n e r a l l y , sockeye has a f o u r t o s i x year r e p r o d u c t i v e c y c l e , of P_^  oncorhynchi of C u l t u s Lake was shown t o be adapted t o f o u r year c y c l e of the host. Question: Can P._ oncorhynchi d e l a y t h e i r r e p r o d u c t i v e c y c l e i n f i s h which spawn i n t h e i r f i f t h year? Present s t u d i e s show f u l l y grown a d u l t P^ oncorhynchi i n §^ sockeye w/veft examined i n August. These f i s h would have spawned d u r i n g September t o e a r l y December of the same year and t h e i r worms would a l s o become g r a v i d . U s u a l l y female worms i n 5 salmon are l a r g e r i n s i z e than i n 42 f i s h when captured at the same time. Body weight and 89 and gonad weight of 5g sockeye are gr e a t e r than 4g but have reached the s i m i l a r sexual maturity,, T h i s suggests that the r e p r o d u c t i v e c y c l e of P_^  oncorhynchi f o l l o w s the r e p r o d u c t i v e c y c l e of i t s host> but i s not dependent on age of worm, (Adult P. oncorhynchi are found i n matured sockeye of ^2 a n c i ^2 age) . Larvae of Po_ oncorhynchi are not found i n the swim-bladder of spawning sockeye from C u l t u s Lake but are common i n spawning f i s h of Owikeno Lake, I t i s not p o s s i b l e t o e x p l a i n t h i s o b s e r v a t i o n at t h i s s t a g e . However, i t i s c l e a r that a l l l a r v a e i n f i s h do not suceed i n r e a c h i n g r e p r o d u c i n g stage„ perhaps due t o unknown e c o l o g i c a l or p h y s i o l o g i c a l f a c t o r s . The low i n f e c t i o n r a t e i n sockeye of the A p r i l c a t c h i s probably due t o d i f f e r e n t f o r a g i n g zones of j u v e n i l e sockeye i n the l a k e . Ruggles (1966) s t a t e d that abundance of zooplank-t e r s which are the most important food of young sockeye, i s v a r i a b l e i n seasons and a l s o i n f o u r d i f f e r e n t b a s i n s of Owikeno Lake. The most abundant z o o p l a n k t e r s i n the l a k e , with the e x c e p t i o n of northern end, are Diaptomus s p e c i e s and Cyclops  v e r n a l i s . These two types of copepods are important food f o r young sockeye i n Owikeno Lake. So f a r i t i s known that Cyclops b i c u s p i d a t u s i s a s u i t a b l e i n t e r m e d i a t e host of oncorhynchi. Meyer (1960) i n f e c t e d Diaptomus s p a t u l o c r e n a t u s , E u c y c l o p s a g i l i s , E p i s c h u r a  l a c u s t r i s , Mesocyclops edax and Cyclops s c u t i f e r with l a r v a e of Philonema^ agubernaculum. The author i n f e c t e d Mesocyclops edax 90 with l a r v a e of Philonema oncorhynchi e x p e r i m e n t a l l y but had very low r a t e of i n f e c t i o n i n comparison of Cyclops b i c u s p i d a t u s . Diaptomus oregonensis and Mesocyclops edax are common i n Babine Lake, B r i t i s h Columbia and^lalso i n f e c t a b l e by P^ oncorhynchi. T h i s o b s e r v a t i o n l e a d s t o a c o n c l u s i o n t h a t s e v e r a l s p e c i e s of copepod may serve as an i n t e r m e d i a t e host of Philonema s p e c i e s . T h e r e f o r e i t can be suggested t h a t Diaptomus s p e c i e s and Cy c l o p s  v e r n a l i s a ct as in t e r m e d i a t e hosts f o r P^ oncorhynchi i n Owikeno Lake, B r i t i s h Columbia. 91 SUMMARY AND CONCLUSIONS 1) Philonema oncorhynchi Kuitunen-Ekbaum, 1933. The growth and maturation of P^ oncorhychi are r a p i d d u r i n g the l a s t 6 months of the host's l i f e . The pr o d u c t i o n of l a r v a e i n F\_ oncorhychi i s synchronized with the r i p e n i n g of the f i s h . Larvae of the worms are r e l e a s e d when the f i s h spawn i n freshwater. The l o n g d u r a t i o n of developmental p a t t e r n of P.  oncorhynchi i s d i f f e r e n t from t h a t of the other d r a c u n c u l o i d s , i n which development i s completed i n 12 months. 2) The appearance of v i s c e r a l adhesions i s c o r r e l a t e d with the m i g r a t i o n of the l a r v a e from the swimbladder i n t o the body c a v i t y of the f i s h at the age of 23 months. The adhesions seem t o disappear with the onset of m a t u r i t y i n the f i s h and with the m i g r a t i o n of the f i s h towards the e s t u a r i e s . No adhesions are found i n the spawning f i s h . 3) Philonema agubernaculum Simon and Simon, 1936. Non-anadromous salmonoids are the normal hosts of t h i s d r a c u n c u l o i d . A l l developmental stages of P^ agubernaculum are the same as those of P^ oncorhynchi. P. agubernaculum reaches the a d u l t stage i n 182 days i n e x p e r i m e n t a l l y i n f e c t e d rainbow t r o u t and g r a v i d worms occur i n immature y e a r l i n g w i l d t r o u t . 4) The l i f e c y c l e of P^ agubernaculum i s d i f f e r e n t from t h a t of P^ oncorhynchi but i s s i m i l a r t o those of the other d r a c u n c u l o i d s . The c o n t i n u a t i o n of the l i f e c y c l e i s dependent on the discharge of the worm i n t o a lake at the time of spawning. 92 5) The d i f f e r e n c e s between the two forms are a t t r i b u -t e d t o the f o l l o w i n g : a) Length of r e p r o d u c t i v e c y c l e , b) r e p r o d u c t i v e i s o l a t i o n i n the host, c) e l e c t r o p h o r e t i c p a t t e r n s , a'ad d) l a s t l y , d i f f e r e n t b i o l o g y of h o s t s . 6) The h y p o t h e s i s , t h a t the worm i s dependent on hormonal s t i m u l u s from the host f o r s y n c h r o n i z a t i o n of rep r o d -u c t i o n , was t e s t e d e x p e r i m e n t a l l y . Larvae were present i n the u t e r i of 33 of 45 female worms recovered from 11 p i t u i t a r y -i n j e c t e d f i s h , 11 t o 47 days a f t e r treatment. In the same p e r i o d , only 2 of 55 female worms from u n t r e a t e d f i s h c o n tained l a r v a e . Treatment of the'green' f i s h with s t i l b e s t r o l i n h i b i t -ed gonadal development but i t had no apparent e f f e c t on the worm. 7) Two other l i n e s of evidence were d i s c u s s e d t o support the hypothesis of hormonal dependence of P. oncorhynchi on i t s h o s t . F i r s t l y , l a r v i g e r o u s female worms were found only i n the r i p e sockeye salmon p o p u l a t i o n of R i v e r s I n l e t , whether 3, 4 or 5 years o l d . Rapid development of the worm occurred d u r i n g the l a s t 6 months of the f i s h ' s l i f e which i s p a r a l l e l e d t o the r a p i d gonadal development. Secondly, n o n - l a r v i g e r o u s P. oncorhynchi from the green f i s h f a i l e d t o become g r a v i d when t r a n s p l a n t e d i n t o immature t r o u t . LITERATURE CITED 93 Akhmerov, A. Kh. 1955. P a r a s i t e fauna of the f i s h e s of the r i v e r Kamchatka. I z v e s t . T i lchookeansk. N. 1. I n s t . Rybn. Khoz. 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Response of gonads i n immature t r o u t (Salmo g a i r d n e r i ) t o e x t r a c t s of p i t u i t a r y glands from a d u l t salmon. Gen. Comp. E n d o c r i n o l . , 5(2):197-206. Simon, J . R. and F. Simon 1936. Philonema agubernaculum s p e c i e s nov. ( D r a c u n c u l i d a e ) , a nematode from the body c a v i t y of f i s h e s . P a r a s i t o l o g y , 28: 440-442. Smedley, E. M. 1933. Nematode p a r a s i t e s from Canadian marine and freshwater f i s h e s . C o n t r i b . Canad. B i o l , and F i s h . n.s. V o l . 8 and 14 pp. 169-179. Smyth, J . D. 1962. I n t r o d u c t i o n t o animal p a r a s i t o l o g y . The E n g l i s h U n i v e r s i t i e s Press L t d . London, E.C.I. Chapt. 10. S t r e l k o v , I. A. and Shulman 1960 . P a r a s i t e i n f e s t a t i o n of f i s h i n the p r o j e c t e d l a k e — c o n s t r u c t i o n area of the Amur R i v e r B a s i n . 10th Conf. P a r a s i t o l . 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C i t e d from Kuitunen-Ekbaum, 1937. Table Date Exam'd Smolts, May '65 12 4 2200 20 0 0 1965 June '65 13 5 1850 20 0 0 Aug. '65 15 7 450 1+ 1 5 0 0 Sept. '65 16 8 335 3+ 1 1 5 0 0 Jan. '66 20 12 1050 20+ 5 3 2 10 0 0 Apr. '66 23 15 1238 20+ 24 26 2 10 7 + June '66 25 17 64 20+ 12 37 5 4 ++ Smolts, 2 y r . 1965 May '65 24 4 & 16 650 3+ 1 4 2 7 0 0 Smolts,'63 kept i n sea May water i n Nanaimo '65 35 28 7 2 4 5 1 3 2 0 P r e c o c i o u s Oct. male '65 Nov. '65 '65 29 30 21 22 769 225 45 32 9 56 9 11 30 135 3 9 5 5 4 4 +++ ++ Prespawn- Sept. '64 40 32 15 30 59 5 0 0 i n g Oct. '64 41 33 1 5 37 162 10 1 0 Nov. '64 42 34 1 53 203 10 0 0 Dec. '65 43 35 29 137 10 0 0 o o * approximate estimate Table I I . Measurements of Philonema oncorhynchi of Sockeye of d i f f e r e n t ages from C u l t u s Lake. Mean s i z e s of Philonema oncorhynchi H i s t o r y Date and age worms of f i s h c o l l e c t e d Smolt, 1965 May 12 months 1965 Smolt, 1965 June 13 months 1965 Smolt, 1965 August 15 months 1965 Smolt, 1964 Sept. 16 months 1964 Smolt, 1965 Jan. 20 months 1966 Stages i n Stages i n swimbladder body c a v i t y 3rd 4th 4th M F 10 1.16 104.26 248.93 480.62 10 1.02 100.4 228.6 468.3 10 1 1.18 3.4 108.9 222.0 244.2 399.6 492.0 971.2 10 2 1.08 6.27 102.53 180.3 225.9 410.7 481.2 929.6 10 10 5 3 2 1.19 4.6 5.22 14.3 50. 01 104.22 152.8 195.9 222.3 259. 3 236.38 334.4 422.4 445.8 516. 4 512.0 871.7 818.5 1082.2 936. 0 F e a t u r e s measured 1. No. measured 2. Length (mm.) 3. Nerve Ring (/u) 4. Muse. Oes. (/u) 5. Gland.Oes. (/u) Table I I . Continued Smolt, 1965 A p r i l 23 months 1966 Smolt, 1965 June 25 months 1966 Smolt, 1963 A p r i l 35 months 1965 Pr e c o c i o u s Oct.-male Nov. 29-30 months 1965 Oct.-Nov. 41-42" months 1965 male a d u l t 4 0 female a d u l t Oct.-4 Nov. 41-42 2months 1965 20 1.79 124.4 306.9 544.7 20 3.32 143.3 312.7 773.6 5 20 2 8.78 18 .74 17.0 213. 7 234 .9 283.0 451. 7 566 .0 521.7 652. 1 1536 .1 1172.3 10 10 15 4 .1 9. 5 21 .7 149 .0 192. 5 256 .7 329.8 422. 2 502 .4 881 .6 992. 42 1521 .0 7 2 5 5 3 .43 4. 4 17 .83 26.52 129 .6 151. 2 238 .1 252.4 298 .1 302. 5 474 .9 531.8 823 .1 823. 4 1534 .3 1325.4 20 15 10 8 7 .3 6. 49 19 .15 146.82 179 .2 189. 7 260 .0 345.0 360 .0 365. 6 491 .6 669.5 947 .9 1022. 2 1424 .1 1648.3 20 20 28 .7 119.7 312 .1 384.4 576 .8 771.0 1654 .0 1758.4 20 20 30 .95 202.95 326 .1 364.6 617 .8 738.9 1677 .0 1737.9 103 Table I I I . I n f e c t i o n o f j i t r o u t with Philonema agubernaculm Sex D u r a t i o n of No. Worms Philonema  i n f e c t i o n Recovered 3rd 4th stage sub-adult a d u l t stage M F M F M F M 30 5 5 _ _ _ _ _ _ M 152 7 - - - 2 2 1 2 M 152 7 - - - 3 2 1 1 M 162 6 4 _ _ 1 _ 1 _ M 169 1 1 - - _ _ _ _ M 176 4 1 3 M 178 2 2 - - -M 181 2 - - - 1 - - 1 M 182 3 - - - - - 1 2 F 3 1 F 58 3 2 2 - _ _ _ _ F 118 9 - - - 3 5 1 -F 151 3 - - - - 2 1 F 151 3 - - - 2 1 - -F 160 2 - - - _ i _ i F 163 1 - - - 1 - - -F 166 1 - - - - - - 1 F 168 5 1 - - 2 1 - -F 169 1 1 - - _ _ _ _ F 173 7 2 - - - - 1 2 F 178 1 - - - - - - 1 Rate of i n f e c t i o n 65.6 percent with mean l a r v a e 2.3 Table IV. E f f e c t of 17-b E s t r a d i o l , Salmon P i t u i t a r y e x t r a c t s and S t i l b e s t r o l on Philonema s p e c i e s i n immature two year o l d Sockeye from Shuswap Lake. Treatments 17-b E s t r a d i o l No.of No. of f i s h I n f e c t e d used f i s h 20 No. of l i v e worms No. of dead and encysted female worms IF & 2M Dura t i o n of experiment 103 Remarks No e f f e c t on female worms. I n f a n t i l e gonads and no change was observed on f i s h , Salmon P i t u i t a r y 20 10 5M 94 I n f a n t i l e gonads and no change was observed on f i s h . S t i l b e s t r o l mixed food 20 12 IF 12 103 No e f f e c t on female worms. I n f a n t i l e gonads and no change was observed on f i s h , C o n t r o l 30 8 3F 8 103 No e f f e c t on female worms. I n f a n t i l e gonads and no change was observed on f i s h . Table V. E f f e c t of salmon p i t u i t a r y e x t r a c t on female Philonema  oncorhynchi recovered from the t r e a t e d f i s h .  (Time i n days) Developmental Stages* 7-12 19-22 47 T o t a l Egg 4 0 0 4 Morula 7 5 0 12 T a i l l e s s 6 4 6 16 T a i l e d 0 10 8 18 T o t a l 17 19 14 50 106 Table V I ( a ) . E f f e c t of Salmon P i t u i t a r y e x t r a c t s on Sockeye Sex D u r a t i o n Body wt. Gonad wt. Mat. Index N u p t i a l C o l o r (gm.) (gm.)  F 3 1928.0 82.0 4.25 — F 7 1701.0 62.0 3.64 — F 10 2495.0 132.5 5.3 + F 11 1587.5 129.0 8.12 , +++ F 11 2041.5 115.5 5.66 ++ F 12 1587.5 97.0 6.1 ++ F 19 2041.5 120.0 5.88 ++ F 20 2948.5 339.0 11.5 ++ F 22 2041.5 157.0 7.7 +++ F 47 1928.0 159.0 8.24 +++ F 47 1474.5 106.0 7.2 +++ F 47 2041.5 137.0 6.7 + M 10 2495.0 59.0 2.36 ++ M 22 2268.0 10.0 0.44 ++ M 47 2154.0 47.0 2.18 +++ Table V I ( b ) . C o n t r o l F 7 1928.0 76.0 3.94 F 7 1928.0 83.0 4.3 — F 15 2722.0 119.0 4.33 _ F 15 1814.5 93.0 5.12 — F 16 1361.0 60.5 4.44 — F 31 1588.0 83.0 5.22 — F 31 1996.0 165.0 8.26 _ F 32 1474.5 94.0 6.37 — F 49 1724.0 157.5 9.13 ± M 7 1474.5 21.0 1.4 M 15 2041.5 31.0 1.5 _ M 24 1928.0 35.0 1.8 _ M 31 1905.0 15.0 0.79 — M 49 2948.5 37.0 1.25 — Table V I I . E f f e c t of s t i l b e s t r o l on female Philonema oncorhynchi recovered from the t r e a t e d f i s h . Developmental (Time i n days) stages 1-9 11-18 20-23 T o t a l F i s h Egg Morula Embryo T a i l l e s s T a i l e d T o t a l 12 2 1 15 M 4 1 0 5 F 10 11 8 29 M 4 7 2 13 F 0 3 1 4 M 0 3 1 4 F - - - - M - - F M - - F 22 16 10 48 M 8 11 3 22 F 108 Table V I I I . E f f e c t of implanted s t i l b e s t r o l on t e s t e s of prespawning sockeye Days F i s h Weight (gms.) s T 3 1474 I 6 2268 B 9 1906 E 11 1361 S 11 1928 T 17 1814 R 18 1814 0 20 1474 L 23 2268 ad Weight Mat. Index (gms.) 5.5 .37 9.5 .42 5.44 .285 2.8 .208 6.5 .34 8.0 .44 9.2 .507 2.0 .135 6.5 .28 C 7 1474 21.0 1.4 0 15 2041 31.0 1.5 N 16 2041 23.5 1.15 T 24 1928 35.0 1.8 R 32 1769 22.5 1.27 0 37 907 20.5 2.27 L 37 1701 26,5 1.56 49 2948 37.0 1.25 49 2041 83.5 4.09 49 1928 50.5 2.62 Table IX. T r a n s p l a n t a t i o n of a d u l t male and female Philonema oncorhynchi i n body c a v i t y of t r o u t . Worms Length Mat. Days Worms Recovered I n t e n s i t y of En c y s t e d Sex (cm.) Index Elapsed T r a n s p l a n t e d ( a l i v e ) adhesions worms F 27.0 .061 2 5 F & 5 M 5 F & 5 M __ F 28.5 .003 3 4 F & 3 M 1 M — — M 27.4 .191 9 4 F & 4 M 2 F & 2 M — • — M 28.0 .005 10 4 F & 4 M — • — _ _ M 27.5 .247 12 4 F & 4 M 3 M — __ F 29.0 .118 20 4 F & 4 M — — . — M 25.2 .01 24 4 F & 4 M — — 1 F F 30.2 .838 32 3 F & 4 M 2 M — — F 26.5 .047 32 3 F & 4 M 1 M — --M 29.0 .117 37 3 F & 3 M 2 M — __ M 28.5 .173 48 4 F & 4 M — — — M 29.5 .092 57 4 F & 4 M — — — F 30.6 1.25 68 4 F & 4 M — — — F 29.5 .87 70 3 F & 4 M — + 1 F M 27.8 .305 77 3 F & 4 M — + — M 26.5 .19 80 3 F & 3 M — + • — F 29.5 .017 80 3 F & 3 M — -F 30.5 .03 81 3 F & 3 M — + 2 F M 30.5 .214 83 2 F & 2 M 1 M + 2 F M 31.5 .205 85 3 F & 3 M — ++ 2 F M 28.5 .004 85 3 F & 3 M — — 2 F F 30.0 .03 85 4 F & 4 M — + 2 F F 30.3 .03 85 3 F & 3 M — — 1 F F 30.5 .023 86 2 F & 3 M 1 M + F 29.4 .029 86 2 F & 2 M — — M 28.5 .02 86 3 F & 3 M 2 M + 2 F Table IX. Continued M 32.0 .01 94 2 F & 2 M 2 M — - 1 F M 31.2 .007 98 2 F & 2 M — ++ M 31.0 .002 99 2 F & 2 M 2 M _«. «---== F 28.0 .052 99 2 F & 2 M 4 M + 2 F M 29.5 .235 100 2 F & 2 M — 1 F F 29.8 .057 101 3 F & 3 M 1 M + 2 F F 29.5 .045 101 3 F & 3 M 3 M ++ 1 F F 31.4 .024 102 2 F & 2 M 1 F F 33.0 .023 106 3 F & 3 M 2 M + 1 F M 30.0 .228 110 3 F & 3 M — 2 F F 30.8 .226 114 3 F & 3 M 2 F & 2 M — I-1 o Table X. Summary of recovery of t r a n s p l a n t e d l a r v a e from salmonid f i s h . Mean S i z e o f Worms Larvae i n swimbladder 3rd 4th Philonema i n 4th stage M "B.C. No. of f i s h with F adhesions days age of f i s h No. of f i s h used perce: i n f ec Sockeye, 1.19 4.6 5.22 14.3 50.01 0 237 20' mo. 10 100 C u l t u s Lake * C o n t r o l I (10) (10) (5) (3) (2) Sockeye 1 .64 2.43 18.74 17.0 7 311 23 mo. 10 100 C u l t u s Lake C o n t r o l II (25) (7) (20) (2) S teelhead 1.35 5 .42 7 .0 20.62 0 238- 2 5 80' Trout <4) (6) (1) (2) 240 Rainbow 1.67 6.81 7.72 15.46 56.46 2 223- 2 10 100 Trout (6) (10) (12) (6) (5) 252 Sockey 1.25 0 55 2 8 100 Shuswap (10) Lake 1.37 4.07 8.38 207 2 1 100 (2) (1) (3) * Number measured Table XI. T r a n s p l a n t a t i o n of a d u l t and f o u r t h stage l a r v a e i n t o body c a v i t y of Frogs Number Sex Duration of f r o g s of f r o g s (days) Group A I M 13 2 M 16 3 M 18 4 F 18 5 F 18 Group B 6 M 11 7 M 11 8 M 13 9 F 13 10 F 18 No. of worms t r a n s p l a n t e d Recovery Cyst Adhesions 2 F & 2 M 1 l i v e M 2 F i n + 1 dead M 1 c y s t 2 F & 2 M 2 l i v e M 1 F + 1 dead F 2 F & 2 M 2 dead M 2 F ++ 2 F & 2 M 2 dead M + 2 dead F 2 F & 2 M 2 dead M a l l i n + 2 do ad F 1 c y s t 10 f o u r t h 4 dead 0 0 stage l a r v a e ft 7 dead 0 0 »» 3 l i v e 0 0 2 dead tf 5 l i v e 0 0 ft 5 l i v e 0 0 2 dead Table X I I . Summary of m e r i s t i c measurements of f i s h of d i f f e r e n t ' a g e composition of R i v e r s I n l e t . Age of 4. Average Average Average Time Sex Area l e n g t h body gonad f i s h of of of f i s h weight weight Mean No. No. caught f i s h c a t c h (cm.) (gm.) (gm.) M.I. exam'd i n f e c t e d August M Owikeno 35.2 555.6 20.6 3.63 20 17 1965 Lake A p r i l M Lat .48'02 44.4 1002.8 1.49 .14 7 7 1965 F Long.39'56 48.0 1170.0 9.45 .81 14 13 August M O u t l e t of 52.9 2113.7 65.4 3.03 12 10 1965 F R i v e r s In. 51.2 1810.7 77.5 4.13 15 15 October M Owikeno 50.4 1502.7 73.4 5.06 37 36 1965 F Lake 52.4 1793.9 327.3 18.33 38 36 August M O u t l e t of 61.5 3203.3 91.0 2.89 6 6 1965 F R i v e r s In. 60.3 3005.4 139.3 4.51 19 18 Table X I I I . Summary of occurrence of Philonema oncorhynchi i n f i s h of d i f f e r e n t age composition of R i v e r s I n l e t . Age Time Sex No. with Philonema Av. No. of of f i s h of No. v i s c e r a l Av. No. Av. No. l a r v a e i n C y s t s f i s h caught F i s h examM adhesions of F. of M. swimbladder T o t a l _ % 3 August M 20 0 1.5 .6 12.9 15 .75 2 1965 A p r i l M 7 1 1.2 1.1 5.57 14 2.0 1965 F 14 1 .63 1.08 5.1 49 3.5 August M 12 4 18.0 7.0 .75 15 1.25 1965 F 15 0 17.8 7.8 , .13 4 .26 October M 37 0 14.6 10.8 7.0 124 3.34 1965 F 38 0 16.13 9.89 2.1 73 1.92 August M 6 1 9.3 5.0 0 23 3.82 1965 F 19 0 15.4 6.3 .3 45 2.36 Table XIV. Measurements of Philonema oncorhynchi from R i v e r s I n l e t Sockeye Measurements of female Philonema Measurements of male Philonema Sex of Age of f i s h f i s h M M M M Time Mean leng t h mm. Mean Nervering /u Mean t o t a l Oe s. mm. Mean le n g t h mm. Mean N e r v e r i n g /u Mean tot; Oes. mm August 81.8 314.5 1.99 23.65 312.6 2.12 1965 (3) (3) (3) (3) (3) (3) A p r i l 5.5* 9.5* 1965 (3) (3) A p r i l 40.74 _ 20.65 _ 1965 (3) (2) August 76.77 280.6 2.08 19.23 265.8 1.96 1965 (38) (40) (39) (27) (27) (27) August 59.02 260.9 1.97 19.94 255.0 2.01 1965 (60) (68) (67) (44) (44) (41) October 111.09 326.5 2.37 21.85 289.51 2.15 1965 (112) (126) (123) (117) (117) (116) October 140.59 346.6 2.39 22.68 297.7 2.17 1965 (134) (137) (136) (110) (108) (108) August 91.0 313.5 2.25 21.81 302.4 2.182 1965 (19) (24) (23) (16) (16) (15) August 95.64 292.5 2.17 22.46 306.7 2.25 1965 (78) (83) (80) (45) (44) (42) * 4th stage en Table XV. Results of Regression of Size of Worm on age of f i s h and time caught Standard error Y Simple regression: Y = A + bx Y = siz e of worms Standard F - value error b df calculated tabulated s t a t i s t i c a l s i g n i f i c a n c e Male worms Male f i s h Age Time 13.24 12.27 24.84 4.86 -.61 3.03 3.63 .65 130 .02 21.18 3.84 3.0 NS ** Male worms female f i s h Age Time 5.23 4.28 13.01 4.16 1.83 1.94 .92 .20 173 4.1 90.5 3.84 3.0 * ** Female worms female f i s h Age 50.79 144.80 -10.31 7.34 Time 34.76 -116.28 25.22 1.54 233 1.97 267.45 3.84 3.0 NS ** Female worms Male f i s h Age Time 46.68 34.55 94.43 -62.77 -0.73 17.72 11.76 1.6 142 .003 116.29 3.84 3.0 NS ** NS - not s i g n i f i c a n t * - .05 l e v e l ** - .01 l e v e l 

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