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Evolutionary divergence in Philonema (Nematoda; Philometridae) parasites of B.C. salmonids Clease, Derek Fraser 1990

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EVOLUTIONARY DIVERGENCE IN PHILONEMA  (NEMATODA;  PHILOMETRIDAE) PARASITES OF B.C. SALMONIDS  by DEREK FRASER CLEASE  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIERMENTS FOR THE DEGREE OF MASTER OF SCIENCE  i n t h e Department of Zoology  We accept t h i s t h e s i s as conforming t o t h e required standard  THE UNIVERSITY OF BRITISH COLUMBIA September, 1990  © C o p y r i g h t by Derek F r a s e r C l e a s e 1990  In  presenting  degree  at the  this  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make it  agree that permission for extensive  scholarly purposes may be her  for  It  is  granted  by the  understood  that  head of copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department The University of British Columbia Vancouver, Canada  DE-6 (2/88)  II  ABSTRACT  Philonema  (Nematoda; P h i l o m e t r i d a e ) from anadromous  h o s t s , sockeye salmon  (Oncorhynchus n e r k a ) , and non-  anadromous h o s t s , rainbow t r o u t  (Oncorhynchus mykiss) , were  s t u d i e d i n o r d e r t o determine i f h o s t s w i t h d i f f e r e n t h i s t o r i e s were i n f e c t e d w i t h t h e same o r d i f f e r e n t of  Philonema.  life  species  Worms from t h e two h o s t s p e c i e s were  morphologically indistinguishable.  However, e l e c t r o p h o r e t i c  b a n d i n g p a t t e r n s produced by r e s t r i c t i o n enzyme d i g e s t i o n o f DNA e x t r a c t e d from Philonema demonstrated t h e p r e s e n c e o f two g e n e t i c t y p e s c o r r e s p o n d i n g t o t h e two h o s t s p e c i e s . T h i s s u p p o r t s t h e i d e a t h a t a t l e a s t two s p e c i e s o f Philonema a r e endemic i n B r i t i s h Columbia. o n c o r h y n c h i Kuitunen-Ekbaum,  Philonema  1933 i s a p a r a s i t e o f sockeye  salmon which undergo a l o n g ocean m i g r a t i o n b e f o r e r e t u r n i n g to  f r e s h w a t e r t o spawn, w h i l e P^ agubernaculum  Simon, 1936 i s a p a r a s i t e o f rainbow t r o u t  Simon and  (and o t h e r  salmonids) which l i v e i n l a k e s . Kokanee (0j_ n e r k a k e n n e r l y i ) , a non-anadromous o f f s h o o t of  sockeye, were i n f e c t e d w i t h t h e same worm as sockeye  p r o b a b l y because t h e two h o s t s have s i m i l a r l i f e  histories.  S t e e l h e a d s m o l t s , anadromous 0^ m y k i s s , c o n t a i n e d worms i d e n t i f i e d as P^ agubernaculum.  T h i s l i k e l y r e p r e s e n t e d an  a c c i d e n t a l i n f e c t i o n because s t e e l h e a d do not u s u a l l y c o n t a c t Philonema.  Ill  Philonema were examined, from v a r i o u s l o c a l i t i e s i n B.C. Philonema agubernaculum showed p o p u l a t i o n d i v e r g e n c e corresponding t o the d i f f e r e n t geographic l o c a l i t i e s w h i c h i t was c o l l e c t e d .  from  This l i k e l y r e f l e c t s the i s o l a t i o n  of t h e s e p a r a s i t e p o p u l a t i o n s i n unconnected watersheds. Philonema o n c o r h y n c h i showed polymorphisms many o f t h e p o p u l a t i o n s .  spread throughout  The l a c k o f p o p u l a t i o n d i v e r g e n c e  p r o b a b l y r e s u l t s from gene f l o w between p a r a s i t e p o p u l a t i o n s brought about by wandering h o s t s .  IV  TABLE OF CONTENTS  Page Abstract  I  L i s t of Figures  V  L i s t o f Tables Acknowledgements  VI VII  Introduction  1  M a t e r i a l s and Methods  5  Sample C o l l e c t i o n  5  E x t r a c t i o n o f DNA  8  DNA E l e c t r o p h o r e s i s  10  Southern T r a n s f e r  11  Hybridization  12  Microscopy  13  Results  14  Morphology o f Worms  14  Philonema from 0^ mykiss  14  Philonema from CK_ n e r k a  19  R e s t r i c t i o n Endonuclease D i g e s t i o n  23  H y b r i d i z a t i o n w i t h pBx2 Probe  52  Cladogram  55  Discussion  58  References  76  V  LIST OF FIGURES  Page 1.  Collection localities  f o r Philonema  2.  Philonema male  (post, end) from 0^ mykiss  18  3.  Philonema male  (ant. end) from 0^ mykiss  18  4.  Philonema female (ant. end) from 0^ mykiss  18  5.  Philonema male  (post, end) from 0^ nerka  22  6.  Philonema male  (ant. end) from 0^ nerka  22  7.  Philonema female (ant. end) from 0^ nerka  22  8.  Eco R l DNA d i g e s t f o r Philonema spp.  22  9.  Hae I I I DNA d i g e s t  27  10.  H i n f I DNA d i g e s t f o r Philonema spp.  29  11.  Hpa I DNA d i g e s t  f o r Philonema spp.  31  12.  Xba I DNA d i g e s t  f o r Philonema spp.  33  13  Xho I DNA d i g e s t f o r Philonema spp.  35  14.  Hpa I DNA d i g e s t  38  15.  Bam HI DNA d i g e s t f o r worms form 0^ mykiss  40  16.  H i n f I DNA d i g e s t f o r worms from 0^ mykiss  42  17.  Hae I I I DNA d i g e s t  44  18.  Bam HI DNA d i g e s t  f o r worms from 0^ nerka  47  19.  H i n f I DNA d i g e s t f o r worms from 0^ nerka  49  20.  Hpa I DNA d i g e s t  51  21.  Bands r e s u l t i n g from h y b r i d i z t i o n w i t h pBx2  54  22.  Cladogram o f Philonema spp. p o p u l a t i o n s  57  f o r Philonema spp.  f o r worms from 0^ mykiss  f o r worms from 0^ mykiss  f o r worms from 0^ nerka  7  VI  LIST OF TABLES  Page I.  P r e v a l a n c e and i n t e n s i t y o f Philonema  spp  i n 0j_ mykiss/ 0. n e r k a and 0. n e r k a k e n n e r l y i  15  VII  ACKNOWLEDGEMENT S  The a u t h o r would l i k e t o thank Dr. M a r t i n Adamson f o r s u g g e s t i n g and s u p e r v i s i n g t h i s study and f o r h e l p i n r e v i s i n g t h i s manuscript.  The a u t h o r would a l s o l i k e t o  thank Dr. Leo M a r g o l i s o f t h e P a c i f i c B i o l o g i c a l  Station,  f o r o r g a n i z i n g s e v e r a l sockeye c o l l e c t i o n s and f o r h i s interest i n this project.  Thanks a r e extended t o Dr. Don  Moerman f o r h i s a s s i t a n c e i n t e a c h i n g t h e a u t h o r t e c h n i q u e s i n v o l v e d w i t h DNA e x t r a c t i o n and s o u t h e r n b l o t t i n g . The a u t h o r would l i k e t o thank Mr. S t u B a r n e t s o n o f t h e F u l t o n R i v e r Salmon P r o j e c t , f o r h i s a s s i s t a n c e i n t h e c o l l e c t i o n o f f i s h on s e v e r a l o c c a s s i o n s .  The a u t h o r i s  g r a t e f u l t o Mrs. Karen Beckenbach, i n Dr. John Webster's l a b a t Simon F r a s e r U n i v e r s i t y , f o r p r o b i n g s o u t h e r n b l o t s i n t h i s study.  Thanks a r e extended t o Bob C a r v e t h f o r h i s h e l p  i n r e v i s i n g t h i s manuscript. t o Stewart "the C h i e f " collections.  F i n a l l y , thanks a r e extended  Noble f o r h i s a s s i s t a n c e i n f i e l d  1 INTRODUCTION  Philonema spp. ( D r a c u n c u l o i d e a ; P h i l o m e t r i d a e ) a r e nematode p a r a s i t e s i n t h e body c a v i t y o f salmonids (Salmoniformes;  Salmonidae).  Mature o v o v i v i p a r o u s  females  l e a v e t h e host by t h e r e p r o d u c t i v e system and r e l e a s e l a r v a e when t h e y c o n t a c t f r e s h w a t e r .  Larvae, i f eaten by a  s u i t a b l e copepod i n t e r m e d i a t e h o s t , develop t o become i n f e c t i v e t o the f i n a l host.  T r a n s m i s s i o n o c c u r s when  i n f e c t e d copepods a r e i n g e s t e d by a salmonid h o s t .  Larvae  l e a v e t h e copepod, p e n e t r a t e through t h e h o s t s gut, and e n t e r t h e swimbladder ( P l a t z e r and Adams, 1967).  Larvae  then l e a v e t h e swimbladder and e n t e r t h e coelom where they w i l l develop  into adults.  I n t h e coelom worms mate,  after  which males w i l l d i e . The genus c o n t a i n s n i n e nominal s p e c i e s ; t h r e e were d e s c r i b e d from N o r t h America 1933; Simon and Simon, 1936; R i c h a r d s o n ,  the f i r s t  (Kuitunen-Ekbaum, 1937) and t h e  r e m a i n i n g s i x were d e s c r i b e d from A s i a ( F u j i t a , 1939; F u j i t a , 1940; Bauer, 1946; F u k u i , 1961; Rumyantsev, 1965). The n i n e nominal s p e c i e s o f Philonema a r e m o r p h o l o g i c a l l y difficult  t o d i s t i n g u i s h s i n c e females become l i t t l e more  t h a n bags f i l l e d w i t h l a r v a e and m o r p h o l o g i c a l  characters  used f o r i d e n t i f i c a t i o n show a h i g h degree o f o v e r l a p between s p e c i e s .  P o s i t i v e i d e n t i f i c a t i o n of species i s  o f t e n n o t p o s s i b l e because worms can not be r e l i a b l y  2  diagnosed from t h e i r morphology.  Therefore,  s p e c i e s i s not c l e a r s i n c e many s p e c i e s may synonyms f o r p r e v i o u s l y d e s c r i b e d m a t e r i a l Akhemerov, 1955,  P l a t z e r , 1964,  the number of represent (Baylis,  1948,  Vismanius et a l . , 1987).  A  method independent of morphology i s t h e r e f o r e needed to r e s o l v e taxonomic problems w i t h i n the genus Philonema. Curran e t . a l .  (1985) suggested a molecular  technique  which uses r e s t r i c t i o n fragment l e n g t h d i f f e r e n c e s i n r e p e t i t i v e DNA  sequences t o i d e n t i f y nematode s p e c i e s  e a s i l y separated by morphological  characters.  Extracted  i s c l e a v e d with r e s t r i c t i o n enzymes which recognize sequences and DNA  fragments are separated  electrophoresis.  R e p e t i t i v e DNA  h i g h l y r e p e t i t i v e genes  technique  DNA  specific  u s i n g agarose g e l  sequences, corresponding  (e.g. ribosomal  to  and h i s t o n e genes),  can then be v i s u a l i z e d by s t a i n i n g with ethidium The  not  i s r a p i d , r e l a t i v e l y inexpensive  bromide.  and may  have  an advantage over p r o t e i n e l e c t r o p h o r e s i s s i n c e p r o t e i n s are f r e q u e n t l y i n f l u e n c e d by environmental and  ontogenetic  f a c t o r s making t h e i r p a t t e r n s more d i f f i c u l t to i n t e r p r e t (Curran e t . a l . , Two  1985).  s p e c i e s of Philonema have been r e p o r t e d from  B r i t i s h Columbia. 1933,  Philonema oncorhynchi  Kuitunen-Ekbaum,  i s a p a r a s i t e of anadromous sockeye salmon  (Oncorhynchus nerka) and P_;_ agubernaculum Simon and 1936  Simon,  supposedly i n f e c t s non-anadromous lake d w e l l i n g  salmonids.  Many lakes c o n t a i n sockeye and non-anadromous  salmonids i n f e c t e d with Philonema spp.,  therefore  B.C.  3  r e p r e s e n t s an i d e a l l o c a t i o n f o r c o l l e c t i o n i d e n t i f i c a t i o n of m a t e r i a l . c o l l e c t e d from sockeye  and  In t h i s study Philonema  was  salmon and rainbow t r o u t  (Oncorhynchus mykiss), a non-anadromous salmonid known t o c a r r y Philonema,  i n the same l a k e  (Babine Lake) and the  technique d e s c r i b e d by Curran et a l . (1985) was determine two  used t o  i f worms from these two hosts r e p r e s e n t e d one  s p e c i e s of  or  Philonema.  An i n t r i g u i n g s i t u a t i o n a l s o e x i s t e d i n the f a c t that kokanee  (0^ nerka k e n n e r l y i ) , a non-anadromous freshwater  o f f s h o o t of sockeye, from Babine Lake were i n f e c t e d with an u n i d e n t i f i e d s p e c i e s of Philonema.  Conversely, s t e e l h e a d  t r o u t , anadromous O^ mykiss, were a l s o i n f e c t e d with an u n i d e n t i f i e d s p e c i e s of Furthermore,  Philonema.  Philonema samples i n both 0^ mykiss and  nerka were c o l l e c t e d from v a r i o u s B.C. same technique was  used t o determine  0.  l o c a l i t i e s and the  i f worms from  g e o g r a p h i c a l l y separate areas had undergone p o p u l a t i o n divergence.  The  freshwater f i s h fauna of B.C.  represents a  recent r e c o l o n i z a t i o n , s i n c e much of the p r o v i n c e g l a c i a t e d 14,000 years ago.  was  During g l a c i a l r e t r e a t ,  r e i n v a d e d from areas of r e f u g i a i n the n o r t h and (McPhail and Lindsey, 198 6).  fish  south  Since t h i s time many  watersheds have become i s o l a t e d , r e s t r i c t i n g gene flow i n f i s h and t h e i r p a r a s i t e s .  Sockeye, on the other hand, are  not r e s t r i c t e d t o freshwater. may  T h e i r anadromous behaviour  take them many miles o f f shore and mix them w i t h other  4  sockeye p o p u l a t i o n s ; but t h e y show a s t r o n g homing tendency when r e t u r n i n g t o t h e i r n a t a l n u r s e r y l a k e s and spawning streams.  T h i s homing b e h a v i o u r  may r e s t r i c t gene f l o w i n  Philonema because i t i s t r a n s m i t t e d i n n u r s e r y  lakes.  5  MATERIALS AND METHODS  Sample C o l l e c t i o n  Prespawning sockeye salmon, kokanee, and rainbow and s t e e l h e a d t r o u t were examined from v a r i o u s B r i t i s h localities  ( f i g 1.).  Sockeye were c o l l e c t e d from R i v e r s  I n l e t , S p r o a t R i v e r , P i e r r e Creek, Henderson Lake and Creek.  Fulton River.  Lake, C u l t u s  Kokanee were c o l l e c t e d from P i e r r e  Rainbow t r o u t were c o l l e c t e d from F u l t o n R i v e r and  Pennask Lake; O'Connor. examined  Columbia  S t e e l h e a d s m o l t s were c o l l e c t e d from Lake  F i s h were c o l l e c t e d by s e i n e o r d i p n e t and i n t h e f i e l d o r packed on i c e and s h i p p e d t o t h e  lab f o r examination. F i s h were d i s s e c t e d by making a m i d - v e n t r a l i n c i s i o n from anus t o p e c t o r a l g i r d l e .  Philonema spp. were found  l y i n g f r e e t h r o u g h o u t t h e coelom, e n t a n g l e d i n p y l o r i c caeca and w i t h i n gonads o f some f e m a l e s .  U s i n g a bent probe,  worms were c a r e f u l l y removed t o p r e v e n t c u t i c l e r u p t u r e , p l a c e d i n d i v i d u a l l y i n 1.2 ml Nalgene c r y o v i a l s , and i m m e d i a t e l y f r o z e n a t -196°C i n l i q u i d n i t r o g e n f o r transport. for storage.  L a t e r , worms were t r a n s f e r r e d t o a -70°C f r e e z e r  6  F i g u r e 1.  Collection localities  f o r Philonema spp.  from v a r i o u s salmonid h o s t s i n B r i t i s h  Columbia.  Pl-sockeye, R i v e r s I n l e t , P2-sockeye, Sproat R i v e r , P3-sockeye, P i e r r e Creek kokanee, P i e r r e Creek  (Babine Lake), P4-  (Babine Lake), P5-sockeye,  Henderson Lake, P6-sockeye, C u l t u s Lake, P7s t e e l h e a d smolts, Lake O'Connor, P8-rainbow Fulton River  (Babine Lake), P9-rainbow  trout,  trout,  Pennask Lake, PlO-sockeye, F u l t o n R i v e r (Babine Lake).  7  8  A d u l t female Philonema spp. used f o r l i g h t microscopy were f i x e d i n c o l d 95% e t h a n o l t o prevent 1964), and t r a n s f e r r e d t o 70% ethanol  bursting (Platzer,  f o r storage.  Adult  males were f i x e d i n the same manner, or i n hot g l y c e r i n e alcohol  (5% g l y c e r i n e , 70% e t h a n o l ) .  E x t r a c t i o n o f DNA  Only female Philonema spp. were used f o r DNA e x t r a c t i o n because o f t h e i r l a r g e r s i z e .  V i a l s c o n t a i n i n g worms were  removed from the -70°C f r e e z e r and p l a c e d i n a small thermos of l i q u i d n i t r o g e n .  I n d i v i d u a l f r o z e n worms were  t r a n s f e r r e d t o 1.5 ml Eppendorfs and ground under nitrogen using a disposable p e s t l e . p r o t e i n a s e K b u f f e r [100 mM T r i s  liquid  A 0.7 ml a l i q u o t o f  (hydroxymethyl) methylamine  ( T r i s ) pH 8.0, 50 mM e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d (EDTA), 1% sodium dodecyl  s u l f a t e (SDS), 200 mM NaCl] was added t o  each Eppendorf, f o l l o w e d by approximately K.  Eppendorfs were incubated  i n a dry bath f o r 16-20 hours  at 65°C t o maximize d i g e s t i o n . times,  2 mg o f P r o t e i n a s e  DNA was e x t r a c t e d  three  a l l o w i n g at l e a s t 0.5 hours between e x t r a c t i o n s .  The  aqueous l a y e r was c o l l e c t e d by s p i n n i n g Eppendorfs f o r 2-3 minutes and t r a n s f e r r i n g the v i s c o u s supernatent Eppendorf u s i n g a wide-mouth p l a s t i c p i p e t t e .  to a clean  Extractions  proceeded as f o l l o w s : 1.  equal volume r e d i s t i l l e d phenol s a t u r a t e d with TE (10 mM T r i s pH 7.5, 1 mM EDTA)  9  2.  equal volume 1 phenol:1 sevag  (24 chloroform:1 isoamyl  alcohol). 3.  equal volume sevag. F o l l o w i n g e x t r a c t i o n , 3/5 volume of 20%  p o l y e t h y l e n e g l y c o l 8000 i n 2.5 M NaCl was p r e c i p i t a t e d overnight at 4°C.  added and DNA  was  Eppendorfs were spun i n a  m i c r o c e n t r i f u g e f o r 15 minutes t o p e l l e t DNA.  The  DNA  p e l l e t was washed twice w i t h c o l d 70% e t h a n o l and vacuum d r i e d f o r 5-10  minutes b e f o r e b e i n g resuspended i n 200 u l of  TE.  DNA  Electrophoresis  E x t r a c t e d DNA  was examined by running a l l samples  m i n i g e l s at 100 v o l t s f o r about 20 minutes. q u a n t i t y o f Hind I I I cut lambda DNA  was  on  A known  i n c l u d e d as a s i z e  standard and samples approximately 20 kb c o n t a i n i n g 20 ug or more o f DNA  were s e l e c t e d f o r d i g e s t i o n w i t h r e s t r i c t i o n  endonucleases. DNA  was  collection  sampled from at l e a s t 5 worms from each  site.  ( s u p p l i e d by BRL) Eco R l , Hae  The f o l l o w i n g r e s t r i c t i o n enzymes were used t o d i g e s t each sample;  I I I , Hind I I I , H i n f I , Hpa I , Msp  I and Xho I .  DNA  (supplied with  enzyme by the vendor), 1.0 u l bovine serum albumin  (2 mg/ml), 10 u l RNAse A  HI,  I , Pst I , Xba  D i g e s t i o n r e a c t i o n s i n v o l v e d 2-8 ug of  suspended i n 18 u l of TE, 2.5 u l 10X b u f f e r the  Bam  (10 mg/ml) and 2.5 u l of  (BSA)  10  r e s t r i c t i o n endonuclease.  The r e a c t i o n m i x t u r e was  i n c u b a t e d a t 37°C, i n a water b a t h , f o r 1-5 h o u r s , depending on a c t i v i t y o f enzymes used.  R e a c t i o n s were h a l t e d by  a d d i n g 0 . 5 M EDTA t o a f i n a l c o n c e n t r a t i o n o f 10 mM (Maniatis e t a l . , 1982). (15% f i c o l l ,  L o a d i n g b u f f e r w i t h marker dye  0.25% bromophenol  b l u e , 0.25% x y l e n e c y a n o l )  was added t o a f i n a l c o n c e n t r a t i o n o f 3%. DNA samples  were l o a d e d i n t o . 0 . 7 % agarose g e l  c o n t a i n i n g 2 . 5 mg/ml e t h i d i u m bromide f o r DNA s t a i n i n g . Lambda H i n d I I I was used as a s i z e s t a n d a r d t o c a l c u l a t e r e s t r i c t i o n fragment s i z e s .  G e l s were r u n i n a h o r i z o n t a l  g e l box, c o n t a i n i n g TBE b u f f e r  (0.089 M T r i s ,  0.089 M b o r i c  a c i d , 0.002 M EDTA), a t 30-40 v o l t s f o r 18-20 hours t o s e p a r a t e DNA r e s t r i c t i o n fragments.  G e l s were  destained  f o r 0 . 5 hours i n d i s t i l l e d water and photographed u s i n g a UV i l l u m i n a t o r box and Kodak P o l a r o i d camera. film  Kodak t y p e 55  ( p o s i t i v e / n e g a t i v e ) was used and r e p e t i t i v e DNA b a n d i n g  p a t t e r n s were t r a c e d from n e g a t i v e s . Initially,  r e s t r i c t i o n d i g e s t s o f s i n g l e samples from  a l l c o l l e c t i o n a r e a s were r u n t o g e t h e r .  Later,  repetitive  samples from v a r i o u s s i t e s d i g e s t e d w i t h s e l e c t e d enzymes were r u n .  Southern T r a n s f e r  Photographed g e l s were p l a c e d i n 0.25 M H C l f o r 10-15 minutes t o d e p u r i n a t e DNA and were t h e n soaked i n t r a n s f e r  11  buffer  (0.02 M NaOH a n d 1 M ammonium a c e t a t e ) f o r 5-10  minutes  p r i o r t o p r e p a r a t i o n of southern t r a n s f e r  Transfer apparatus and  consisted of a glass  a Plexiglass plate  reservoir  apparatus. for buffer  over the r e s e r v o i r t o support  Two p i e c e s o f Whatman 3 MM  paper  transfer buffer  G e l s were p l a c e d on w i c k s  piece was  as w i c k s .  o f Hybond-N  cut to size  Whatman 3 MM  hybridization  rolling  p a p e r were c u t t o s i z e  placed  (Amersham)  Two p i e c e s o f  and p l a c e d o v e r t h e  them o u t w i t h a l o n g g l a s s  apparatus.  and a  A i r b u b b l e s were removed a t e a c h  Handi-Wrap, w i t h a window the  into the  t r a n s f e r membrane  and p l a c e d over t h e g e l .  t r a n s f e r membrane. by  were i n s e r t e d  around  A weighted  over the g e l .  DNA  gels.  rod.  stage  A piece of  t h e g e l , was u s e d t o c o v e r  stack o f Scott paper t r a n s f e r was  towels  was  allowed to proceed  overnight. Following transfer, in  membranes were removed a n d  0.4 M NaOH f o r 20 m i n u t e s  t o d e n a t u r e DNA  1988).  Membranes  were r i n s e d w i t h 2X SSC  citrate  a n d 0.3 M N a C l ) a n d a i r d r i e d  minutes  t o c r o s s - l i n k e d DNA.  room t e m p e r a t u r e  until  (Broad e t a l . ,  (0.02 M  f o r 1 hour.  were w r a p p e d i n Handi-Wrap a n d e x p o s e d Membranes  soaked  t o UV l i g h t  sodium Membranes for 3  were t h e n s t o r e d a t  used.  Hybridization  Membranes which  were p r o b e d w i t h a c l u s t e r  i n c l u d e d t h e 5.8,  18s a n d 28s genes,  o f r i b o s o m a l genes and a  12  nontranscribed  region.  The r i b o s o m a l c l u s t e r  (pBx 2) was a  7.2 k b E c o R l f r a g m e n t i s o l a t e d f r o m t h e p i n e w o o d Bursaphelenchus x y l o p h i l u s i n P u c 19.  The p r o b e  Simon F r a s e r  (Nematoda; T y l e n c h i d a ) a n d  ( s u p p l i e d by Dr. J . Webster's  University)  e t a l . , 1982) u s i n g  contained  400 n g o f p r o b e DNA  was s e t up a s f o l l o w s : [500 mM  cloned  l a b at  was l a b e l l e d by n i c k t r a n s l a t i o n  (Maniatis  (NTB)  nematode  3 2  P  ATP.  The  reaction  i n a f i n a l v o l u m e o f 50 u l a n d  5 u l 10X n i c k t r a n s l a t i o n b u f f e r  T r i s pH 7.5, 100 mM magnesium s u l f a t e , 10  dithiothreitrol  mM  (DTT), 500 ug/ml B S A ] , 37 u l d i s t i l l e d  w a t e r , 2 U l -ATP  (10 mM GTP,  10 mM  d i l u t e d DNAse I [ d i l u t e s t o c k  TTP, 10 mM C T P ) , 1 U l  DNAse I (1 mg/ml i n 50 mM  pH 7.5, 10 mM MgS04, 1 mM DTT,  Tris  5 0 % g l y c e r o l ) t o 1/10000 i n  IX N T B ] , 1 u l DNA p o l y m e r a s e I ( a p p r o x i m a t e l y 2-4 u n i t s ) , 2 u l pBx 2  (400 n g ) , 2 u l  3 2  P  ATP  (20 u C i ) .  The  reaction  m i x t u r e was i n c u b a t e d a t 12-15°C f o r 2 h o u r s a n d was using (to  5 u l o f 0.2 M EDTA a n d 2% SDS.  remove f r e e n u c l e o t i d e s )  column with  constructed  glass  NaCl),  were p r e h y b r i d i z e d  f r e e z e r bags c o n t a i n i n g  solution  (G25 f i n e )  spin  t i p p l u g g e d a t one e n d  wool.  Membranes sealed  The p r o b e was p u r i f i e d  i n a Sephadex  from a 1 ml p i p e t  halted  [5X SSPE  100 m l o f p r e h y b r i d i z a t i o n  (5 mM EDTA, 0.06 M NaH2P04-H20,  0.3% SDS, 2.5X D e n h a r t s  0.05% p o l y v i n l y p y r r o l i d o n e were d r a i n e d  f o r 2 h o u r s a t 62°C i n  ( 0 . 0 5 % BSA,  0.7  M  0.05% f i c o l l  4 0 ) ] . Bags c o n t a i n i n g  400,  membranes  and 4 ml o f h y b r i d i z a t i o n s o l u t i o n ( p u r i f i e d  p r o b e , 5X SSPE, 0.3% SDS, 2.5X D e n h a r t s ) p e r 100 c m  2  of  13  t r a n s f e r membrane was added t o each bag.  Membranes were  h y b r i d i z e d at 62 °C overnight, washed once f o r 5 minutes and t h r e e times f o r 10 minutes each i n 2X SSPE and 0.2%  SDS.  Washed membranes were b l o t t e d dry, wrapped w i t h Saran Wrap and p l a c e d i n a d e v e l o p i n g c a s e t t e w i t h a s i n g l e i n t e n s i f y i n g screen and X-ray f i l m . a -70"C  C a s e t t e s were p l a c e d i n  f r e e z e r and f i l m was exposed f o r 5 days.  Microscopy  A d u l t male and female Philonema spp. from Babine Lake and Pennask Lake were examined  and drawn u s i n g a Nikon  microscope equipped w i t h drawing tube. c a l c u l a t e d u s i n g a d i g i t i z i n g pad and software from Sonnet-Gap.  Measurements were  (Hi-pad Texas Instruments)  14  RESULTS  Morphology o f Worms  Philonema s p . from 0^ mykiss  A d u l t male and female Philonema sp. were found i n prespawning rainbow t r o u t from F u l t o n R i v e r and t h e Pennask Lake egg c o l l e c t i o n s i t e . no p a t h o l o g y .  Worm burdens  Worms a p p a r e n t l y caused l i t t l e o r (# o f worms p e r host) i n t r o u t  were g e n e r a l l y l o w e r t h a n t h o s e i n sockeye  (Table 1) and  many t r o u t c o n t a i n e d no a d u l t worms ( f i s h were n o t examined for  larvae). S t e e l h e a d s m o l t s from Lake O'Connor were i n f e c t e d w i t h  mature Philonema s p . and worm burdens were o f t e n h i g h . Worms caused s e v e r e p a t h o l o g i c a l e f f e c t s such as g r e a t l y d i s t e n d e d , f l u i d f i l l e d b e l l i e s and t h i n abdominal w a l l s . Worms were f r e q u e n t l y found p r o t r u d i n g from t h e anus. f i s h were o b s e r v e d f l o a t i n g a t t h e s u r f a c e o f n e t pens.  Dead  15  Table I  HOST  LOCALITY  # EXAMINED  # INFECTED* INTENSITY (PREVALENCE)  sockeye  Rivers In.  10  10 (100%)  +++  sockeye  Sproat Rv.  14  14 (100%)  ++  sockeye  P i e r r e Ck.  10  10 (100%)  ++  kokanee  P i e r r e Ck.  16  16 (100%)  +  sockeye  Henderson  12  12 (100%)  ++  sockeye  C u l t u s Lk.  18  15 (83%)**  ++  steelhead  0 Connor  13  13 (100%)  +++  rainbow  F u l t o n Rv.  17  6  rainbow sockeye  1  (35%)  +  Pennask Lk. 23  11 (48%)  +  F u l t o n Rv.  11 (100%)  ++  11  # i n f e c t e d r e f e r s t o a d u l t worms only,  f i s h were not  examined f o r l a r v a e . C u l t u s lake f i s h were c o l l e c t e d on two occasions, i n October, 1987 and again second o c c a s i o n  i n November,  1988.  once  On the  f i s h were c o l l e c t e d from L y n d e l l beach  and some may have spawned and r e l e a s e d worms; may be an underestimate o f p r e v a l e n c e .  thus 83%  16  Description: Length 14.64 mm (234  Male  Maximum width 286 |im  Oesophagus c o n s i s t i n g o f a n t e r i o r muscular  407 (Xm (345 t o 488 |im) long,  glandular  ( f i g s . 2 and 3 ) :  (12.28 t o 18.68 mm).  t o 371 Lim) .  portion  (3 specimens)  portion,  into intestine.  and a p o s t e r i o r  1000 (im (891 t o 1175 fim) long,  emptying  r i n g 226 ^im (185 t o 278 Lim) from  Nerve  a n t e r i o r end, near midpoint o f muscular oesophagus. l a r g e t e s t i s b e g i n n i n g 1.11 mm  (1.06 t o 1.16 mm)  a n t e r i o r end, near o e s o p h a g o - i n t e s t i n a l posteriorly to cloacal junction  junction,  Single  from extending  j u s t a n t e r i o r t o anus.  Slender, arcuate s p i c u l e s equal and s i m i l a r , 315 |im (299 t o 333 (310  |im) l o n g .  Gubernaculum absent.  t o 402 Lim) long,  T a i l c o n i c a l , 345 |im  with s i x t o t e n p a i r s o f p a p i l l a e .  F i v e t o e i g h t p a i r s o f p r e a n a l p a p i l l a e were a l s o Female  (1 specimen)  width 772.7 Lim. portion  ( f i g . 4 ) : Length 59.7 mm.  Maximum  Oesophagus c o n s i s t i n g o f a n t e r i o r muscular  474.3 (im long,  1067.6 Lim long,  observed.  and p o s t e r i o r g l a n d u l a r  portion,  j o i n i n g a f l a t t e n e d , empty i n t e s t i n e .  Nerve  r i n g e n c i r c l i n g muscular oesophagus near i t s middle, 241.4 Lim from a n t e r i o r end o f worm. ends o f worm.  Anterior  Small ovary present at both  ovary 703.5 (im from a n t e r i o r end o f  worm and p o s t e r i o r ovary 550.5 Lim from t i p o f t a i l .  Ovaries  emptying i n t o l a r g e uterus f i l l i n g most o f worms body. Uterus c o n t a i n i n g latter  stages o f b l a s t u l a t i o n (no f i r s t  observed). t o have  many small embryos appearing t o be i n  N e i t h e r anus nor v u l v a  atrophied.  stage  larvae  observed and both assumed  17  F i g u r e s 2-4.  Philonema sp. from CK_ m y k i s s .  P o s t e r i o r end o f male. 4.  3.  A n t e r i o r end o f female.  2.  A n t e r i o r end o f male.  18  19  Philonema sp. from 0^ nerka  A d u l t male and female Philonema sp. were found i n prespawning  sockeye and kokanee from a l l sample s i t e s .  Worm  burdens were o f t e n h i g h (Table 1) but p a t h o l o g i c a l  effects  were not observed w i t h the e x c e p t i o n o f o c c a s i o n a l  thin  walled, f l u i d f i l l e d c y s t s  ( c o n t a i n i n g dead males and/or  female worms) on t h e l i v e r or body w a l l .  Philonema sp.  c o l l e c t e d from 0^ nerka tended t o be l a r g e r than those from 0. mykiss but otherwise worms were m o r p h o l o g i c a l l y indistinguishable. Description: Length  29.1 mm  (274 t o 320 Um). portion,  Male  (2 specimens)  (27.24 t o 30.95 mm).  ( f i g s . 5 and 6 ) : Maximum width 297 um  Oesophagus c o n s i s t i n g o f a n t e r i o r muscular  649 um (637 t o 660 um) long, and p o s t e r i o r  g l a n d u l a r p o r t i o n 1824 um (1723 t o 1925 um) long, into intestine.  emptying  Nerve r i n g 325 um (311 t o 339 um) from  a n t e r i o r end o f worm, e n c i r c l i n g musculo-oesophagus midpoint. 4.04 mm)  S i n g l e l a r g e t e s t i s b e g i n n i n g 2.55 mm  near i t s  (1.06 t o  from a n t e r i o r end and extending p o s t e r i o r l y t o t h e  c l o a c a l j u n c t i o n j u s t a n t e r i o r t o anus. s p i c u l e s equal and s i m i l a r , Gubernaculum absent.  Slender, arcuate  339 um long i n one o f specimens.  Conical t a i l ,  505 um long i n one  specimen, w i t h s i x p a i r s o f p o s t a n a l p a p i l l a e . of p r e a n a l p a p i l l a e were a l s o observed.  Five pairs  20  Female to  (2 specimens)  130.62 mm).  (fig.  7 ) : Length 89.79 mm  (48.96  Maximum width 725 um (495 t o 955 um).  Oesophagus c o n s i s t i n g o f a n t e r i o r g l a n d u l a r p o r t i o n 1752 um (1580 t o 1923 um) long, j o i n i n g f l a t t e n e d , empty i n t e s t i n e . Nerve r i n g 286 um (227 t o 345 um) from a n t e r i o r end, e n c i r c l i n g muscular oesophagus near i t s middle. Reproductive system c o n s i s t i n g o f s m a l l a n t e r i o r ovary, 67 6 um (550 t o 802 um) from a n t e r i o r end, and small p o s t e r i o r ovary, 760 um (720 t o 799 um) from p o s t e r i o r e x t r e m i t y . Ovaries emptying  i n t o l a r g e u t e r u s f i l l i n g much o f body.  Uterus c o n t a i n i n g s i n g l e c e l l s , e a r l y g a s t r u l a e , but no f i r s t  e a r l y cleavage embryos and  stage l a r v a e were observed.  V u l v a and anus not observed and both were assumed t o have atrophied.  21  Figures 5-7.  Philonema sp. from  P o s t e r i o r end o f male. 7.  6.  A n t e r i o r end o f f e m a l e .  nerka.  5.  A n t e r i o r end o f  male.  22  23  R e s t r i c t i o n Endonuclease D i g e s t i o n  Banding p a t t e r n s produced by r e s t r i c t i o n  endonuclease  d i g e s t i o n c l e a r l y separated Philonema sp. i n t o two  groups  c o r r e s p o n d i n g t o the two host s p e c i e s from which they were collected  ( f i g s . 8-13).  steelhead trout  Thus, worms from rainbow  (0^ mykiss) had s i m i l a r banding p a t t e r n s and  c o u l d be d i s t i n g u i s h e d from those i n sockeye kokanee  and  (CK nerka k e n n e r l y i ) .  Of 10  (O^ nerka)  and  restriction  endonucleases used, only 3 r e v e a l e d bands shared by the two groups;  Hae  I I I and Hpa  and 11), and Xho  I produced 0.4  I a 3.2 kb band  kb bands  ( f i g . 13).  (figs. 9  24  F i g u r e 8.  Eco R l d i g e s t i o n of Philonema spp.(P) and C.  elegans  (N2) DNA.  Philonema spp. are from the  f o l l o w i n g h o s t s and l o c a l i t i e s ;  Pl-sockeye,  R i v e r s I n l e t , P2-sockeye, Sproat R i v e r , sockeye, P i e r r e Creek, P4-kokanee,  P i e r r e Creek,  P5-sockeye, Henderson Lake, P6-sockeye, Lake, P7-steelhead, Lake O'Connor,  River.  Cultus  P8-rainbow  t r o u t , F u l t o n R i v e r , P9-rainbow t r o u t , Lake, PlO-sockeye, F u l t o n  P3-  Pennask  25  o a CO CL  0_ CL  DC O o LU  CO  0.  in  CL  CO  CL  S!  I I I I II i n  s  u » • » 8  2.  si  i  SS no  5  26  F i g u r e 9.  Hae  I I I d i g e s t i o n o f Philonema spp. (P) and  C. elegans (N2) DNA.  Philonema spp. are from the  f o l l o w i n g h o s t s and l o c a l i t i e s ;  Pl-sockeye,  R i v e r s I n l e t , P2-sockeye, Sproat R i v e r , sockeye, P i e r r e Creek, P4-kokanee,  P i e r r e Creek,  P5-sockeye, Henderson Lake, P6-sockeye, Lake, P7-steelhead, Lake O'Connor,  River.  Cultus  P8-rainbow  t r o u t , F u l t o n R i v e r , P9-rainbow t r o u t , Lake, PlO-sockeye, F u l t o n  P3-  Pennask  27  o a. 00  o.  I I  Q. CO 0.  o. 0_  0_ CM Q.  0_  I  C\J  III I II f S3 S  S S  28  gure 10.  H i n f I d i g e s t i o n o f Philonema spp.(P) and  C. elegans (N2) DNA.  Philonema spp. are from the  f o l l o w i n g h o s t s and l o c a l i t i e s ;  Pl-sockeye,  R i v e r s I n l e t , P2-sockeye, Sproat R i v e r , sockeye, P i e r r e Creek, P4-kokanee,  P i e r r e Creek,  P5-sockeye, Henderson Lake, P6-sockeye, Lake, P7-steelhead, Lake O'Connor,  Cultus  P8-rainbow  t r o u t , F u l t o n R i v e r , P9-rainbow t r o u t , Lake, PlO-sockeye, F u l t o n R i v e r .  P3-  Pennask  29  II I II I II I II I I  II  II  I III fl 1 1 5 ^  8  •»  OB  *  »*i  5S n  o  30  F i g u r e 11. elegans  Hpa I d i g e s t i o n o f Philonema spp. (P) and C. (N2) DNA.  Philonema spp. are from the  f o l l o w i n g h o s t s and l o c a l i t i e s ;  Pl-sockeye,  . R i v e r s I n l e t , P2-sockeye, Sproat R i v e r , sockeye, P i e r r e Creek, P4-kokanee,  P i e r r e Creek,  P5-sockeye, Henderson Lake, P6-sockeye, Lake, P7-steelhead, Lake O'Connor,  Cultus  P8-rainbow  t r o u t , F u l t o n R i v e r , P9-rainbow t r o u t , Lake, PlO-sockeye, F u l t o n R i v e r .  P3-  Pennask  31  II  III III III  I 11 I II S II I  SS  32  F i g u r e 12. elegans  Xba I d i g e s t i o n o f Philonema spp.(P) and C. (N2) DNA.  Philonema spp. are from the  f o l l o w i n g h o s t s and l o c a l i t i e s ;  Pl-sockeye,  R i v e r s I n l e t , P2-sockeye, Sproat R i v e r , sockeye, P i e r r e Creek, P4-kokanee,  P i e r r e Creek,  P5-sockeye, Henderson Lake, P6-sockeye, Lake, P7-steelhead, Lake O'Connor,  River.  Cultus  P8-rainbow  t r o u t , F u l t o n R i v e r , P9-rainbow t r o u t , Lake, PlO-sockeye, F u l t o n  P3-  Pennask  33  l l l l  o a.  IIII  a. a.  IIII  a.  llll  CO  CO  a.  _Q  X  in  a. a.  CO  a. CM  a.  CM  z  llll  II  £ Is s  ss  R ai.ss.3i.  n o  s  cc p  34  Figure  13.  elegans  Xho I d i g e s t i o n o f Philonema spp.(P) and C. (N2) DNA.  Philonema spp. are from the  f o l l o w i n g h o s t s and l o c a l i t i e s ;  Pl-sockeye,  R i v e r s I n l e t , P2-sockeye, Sproat R i v e r , sockeye, P i e r r e Creek, P4-kokanee,  P i e r r e Creek,  P5-sockeye, Henderson Lake, P6-sockeye, Lake, P7-steelhead, Lake O'Connor,  Cultus  P8-rainbow  t r o u t , F u l t o n R i v e r , P9-rainbow t r o u t , Lake, PlO-sockeye, F u l t o n R i v e r .  P3-  Pennask  35  36  Banding p a t t e r n s w i t h i n groups showed l i t t l e f o r most enzymes ( f i g s .  variation  14-17), but Philonema p o p u l a t i o n s  from O^ mykiss d i f f e r e d with r e s p e c t t o p a t t e r n s produced by Bam HI, Hae I I I and H i n f I . to  different collection  These d i f f e r e n c e s  localities.  Bam HI d i g e s t i o n produced a present  corresponded  2.1 kb band t h a t was  i n F u l t o n R i v e r and Pennask Lake worms and absent i n  Lake O'Connor worms ( f i g . 15). Two bands, 3.1 and 2.8 kb, produced by Hinf I d i g e s t i o n were present Pennask p o p u l a t i o n s and absent a 2.6 kb present  i n F u l t o n and  i n Lake O'Connor worms, while  i n Lake O'Connor worms was absent  from  F u l t o n and Pennask worms ( f i g . 16). Banding p a t t e r n s produced by Hae I I I d i g e s t i o n d i s t i n g u i s h e d Philonema from t h e 3 O^ mykiss p o p u l a t i o n s ( f i g . 17). Lake O'Connor worms had 2 bands, 3.5 kb and 1.1 kb, not present  i n e i t h e r t h e F u l t o n or Pennask p o p u l a t i o n s .  F u l t o n R i v e r worms had a 2.4 kb band not present  i n the  other two p o p u l a t i o n s , and l a c k e d 2.8 kb band which was present  i n the other two.  Banding p a t t e r n s produced by the enzymes Eco R l , Hpa I ( f i g . 14) and Xho I were i d e n t i c a l f o r a l l 0^ mykiss collection  localities.  37  F i g u r e 14. mykiss.  Hpa I DNA  d i g e s t o f Philonema t a k e n from  Philonema sp. a r e from t h e f o l l o w i n g  h o s t s and l o c a l i t i e s ;  P 7 - s t e e l h e a d , Lake  O'Connor, P8-rainbow t r o u t , F u l t o n R i v e r , P9rainbow t r o u t , Pennask Lake.  3 8  0.  o  CL CL CL CO CL CO  CC  Q_  0_ co  0. CO CL  rv. CL  CL  CL  CL  I II I 5 fI J3 £,»  I  f oI P>  pj pj  S  39  F i g u r e 15.  Bam  0. mykiss.  HI DNA  d i g e s t o f Philonema taken from  Philonema sp. are from the f o l l o w i n g  hosts and l o c a l i t i e s ;  P7-steelhead, Lake  O'Connor, P8-rainbow t r o u t , F u l t o n R i v e r , rainbow t r o u t , Pennask Lake.  P9-  The 2.1 kb band i s  shared by the F u l t o n and Pennask p o p u l a t i o n s but i s not present i n the Lake O'Connor p o p u l a t i o n .  40  41  F i g u r e 16.  H i n f I DNA  0. mykiss. hosts and  d i g e s t of Philonema taken from  Philonema sp. are from the f o l l o w i n g localities;  P7-steelhead,  Lake  O'Connor, P8-rainbow t r o u t , F u l t o n R i v e r , rainbow t r o u t , Pennask Lake.  The  3.1  P9-  and 2.8  kb  bands are shared by the F u l t o n and Pennask p o p u l a t i o n s but are not present O'Connor p o p u l a t i o n . has  a 2.6  The  Lake O'Connor p o p u l a t i o n  kb band not present  populations.  i n the Lake  i n the other  two  42  CL  o. co  CO  CL  co  CL  oo CL  rr CL  CL  CL  CL  j II I'I  I I I f Zi * •  K  2.  S2.  5  *  i  S  n o csi est  43  F i g u r e 17.  Hae I I I DNA  0. mykiss.  d i g e s t o f Philonema taken from  Philonema sp. are from the f o l l o w i n g  hosts and l o c a l i t i e s ;  P7-steelhead, Lake  O'Connor, P8-rainbow t r o u t , F u l t o n R i v e r , P9rainbow t r o u t , Pennask Lake. Pennask p o p u l a t i o n s  The F u l t o n and  share the 3.6 kb band which  Lake O'Connor worms l a c k .  The Lake O'Connor  p o p u l a t i o n has two bands, 3.5 and 1.1 kb, not seen i n the other two p o p u l a t i o n s .  The 2.8 kb band i s  shared by the Pennask and Lake O'Connor populations  and the F u l t o n R i v e r p o p u l a t i o n has a  2.4 kb band not seen i n the other  populations.  44  cn  o. 0-  co  CL  co  CD 03  00 £L 00  CL  CL  CL  CL  Cw  3 3  I II I • II OS  tfi V  JC JC C5 © pJ CNl  33  45  Philonema p o p u l a t i o n s  from 0^ nerka showed banding  p a t t e r n d i f f e r e n c e s f o r t h e enzymes Bam HI, H i n f I and Hpa I, but these d i f f e r e n c e s were not r e s t r i c t e d t o any population  (figs.  18-20).  Bam HI d i g e s t i o n produced a 2.6 kb band t h a t was present  i n C u l t u s Lake worms, most Sproat Lake worms, one  worm from Henderson  Lake, one worm from P i e r r e Creek, and i n  much f a i n t e r form i n worms from F u l t o n R i v e r bands,  ( f i g . 18). Two  1.0 and 0.9 kb, produced by H i n f I d i g e s t i o n were  polymorphic.  In C u l t u s Lake worms, most Sproat R i v e r worms,  one worm from Henderson Lake and one worm from P i e r r e Creek the 0.9 kb band was more prominent.  In t h e remainder o f t h e  samples  from Henderson Lake, P i e r r e Creek, Sproat R i v e r and  samples  from R i v e r s I n l e t , t h e 1.0 kb band i s o f g r e a t e r  intensity.  However, i n samples from F u l t o n R i v e r these  bands were n e a r l y equal i n i n t e n s i t y .  Finally,  band produced by Hpa I d i g e s t i o n was present  a 1.9 kb  i n Cultus Lake  worms, most Sproat R i v e r worms and one worm from P i e r r e Creek  ( f i g . 20) (the Henderson Lake and F u l t o n R i v e r  p o p u l a t i o n s were not examined). Banding p a t t e r n s produced by t h e enzymes Eco R l , Hae I I I , Xho I and Xba were i d e n t i c a l f o r a l l O^ nerka collection  localities.  46  F i g u r e 18. nerka.  Bam HI DNA  d i g e s t o f Philonema from 0.  Philonema sp. are from the f o l l o w i n g h o s t s  and l o c a l i t i e s ;  Pl-sockeye, R i v e r s I n l e t ,  P2-  sockeye, Sproat R i v e r , P3-sockeye, P i e r r e Creek, P4-kokanee, P i e r r e Creek, P5-sockeye,  Henderson  Lake, P6-sockeye, C u l t u s Lake, PlO-sockeye, F u l t o n River.  The 2.6 kb band corresponds t o pBx 2, a  c l u s t e r o f ribosomal genes xylophilus.  i s o l a t e d from B.  48  F i g u r e 19. nerka.  H i n f I DNA  d i g e s t of Philonema from 0.  Philonema sp. are from the f o l l o w i n g hosts  and l o c a l i t i e s ;  Pl-sockeye, R i v e r s I n l e t ,  P2-  sockeye, Sproat R i v e r , P3-sockeye, P i e r r e Creek, P4-kokanee, P i e r r e Creek, P5-sockeye,  Henderson  Lake, P6-sockeye, C u l t u s Lake, PlO-sockeye, F u l t o n River.  4 9  50  F i g u r e 20. nerka.  Hpa I DNA d i g e s t o f Philonema from 0. Philonema sp. a r e from t h e f o l l o w i n g h o s t s  and l o c a l i t i e s ;  P2-sockeye, Sproat R i v e r , P4-  kokanee, P i e r r e Creek, P5-sockeye, Henderson Lake, P6-sockeye, C u l t u s Lake.  51  to  CL  a CO  a. in o. CL  CO  in  o. in a  2  2 CM Q.  2!  I II I ^ II I cv  2i  SE-  2.  II In I©  s CO  p.  52  Hybridization  w i t h pBx 2 Probe  Southern b l o t s taken from g e l s were probed w i t h pBx 2 from Bursaphelenchus x y l o p h i l u s  (Nematoda;  Tylenchida).  Bands r e s u l t i n g from h y b r i d i z a t i o n w i t h the pBx 2 probe are summarized i n f i g . 21.  Banding p a t t e r n s produced by the  enzymes Hae I I I , Hpa I, H i n f I and Xho I were i d e n t i c a l f o r Philonema  from 0^ mykiss and 0^ nerka.  Southern b l o t s  from  r e s t r i c t i o n d i g e s t s w i t h other enzymes were not a v a i l a b l e s i n c e heavy background contamination made bands i m p o s s i b l e to d i s c e r n .  The 3.2 kb pBx 2 fragment produced by Xho I  d i g e s t i o n may correspond t o a band o f s i m i l a r s i z e seen i n fig.  13, Xho I d i g e s t i o n o f e x t r a c t e d  Philonema DNA.  I I I d i g e s t i o n produced 4 pBx 2 fragments; fragment  the 1.4 kb  i s s i m i l a r i n s i z e t o a band seen i n Hae I I I  d i g e s t i o n o f DNA from worms i n but  Hae  mykiss  ( f i g s . 9 and 17)  i t has no corresponding band i n worms from (X^ nerka.  S i m i l a r l y , t h e 2.1 kb pBx 2 fragment produced by Hpa I digestion  i s s i m i l a r i n s i z e t o a band seen i n Hpa I  d i g e s t i o n o f worms from 0^ mykiss  ( f i g s . 11 and 14) but a  s i m i l a r band i s absent i n worms from 0. nerka.  53  F i g u r e 21.  Summary o f a l l bands r e s u l t i n g from  h y b r i d i z a t i o n w i t h t h e pBx 2 probe, a c l u s t e r o f r i b o s o m a l genes i s o l a t e d from  xylophilus.  T -  Philonema sp. from CJ^ m y k i s s , S - Philonema sp. from 0. n e r k a .  0  -  N/A  55  Cladogram  The most parsimonious arrangement  o f shared bands  d e s c r i b e d from f i g s . 8-20 i s i n d i c a t e d by a cladogram ( f i g . 22).  The cladogram i n d i c a t e s t h e two groups o f Philonema  c o r r e s p o n d i n g t o t h e host s p e c i e s from which they were removed  ( i . e . . P I , P2, P3, P4, P5, P6 and P10 are from 0.  nerka and P7, P8 and P9 are from 0^ mykiss). nerka r e s u l t e d i n an u n r e s o l v e d polychotomy populations.  for a l l  Worms from 0^ mykiss i n d i c a t e d F u l t o n R i v e r  (P8) and Pennask to  Worms from 0.  (P9) p o p u l a t i o n s were more c l o s e l y  related  one another than e i t h e r was t o Lake O'Connor (P7) worms.  56  F i g u r e 22.  Cladogram r e p r e s e n t i n g  hypothetical  r e l a t i o n s h i p s o f Philonema spp. samples based on r e p e t i t i v e bands from f i g s .  8-20.  Philonema spp.  are from the f o l l o w i n g hosts and l o c a l i t i e s ;  Pl-  sockeye, R i v e r s I n l e t , P2-sockeye, Sproat R i v e r , P3-sockeye, P i e r r e Creek, P4-kokanee,  Pierre  Creek, P5-sockeye, Henderson Lake, P6-sockeye, C u l t u s Lake, P7-steelhead, Lake O'Connor, PSrainbow, F u l t o n R i v e r , P9-rainbow, PlO-sockeye, F u l t o n  River.  Pennask  Lake,  57  O. mykiss O. nerka  H*mc(Ukt)  58  DISCUSSION  The genus Philonema c o n t a i n s nine nominal s p e c i e s i n anadromous and non-anadromous salmonids d i s t r i b u t e d throughout t h e Northern hemisphere.  Three s p e c i e s o f  Philonema have been d e s c r i b e d from North America, the  type P_^ oncorhynchi Kuitunen-Ekbaum 1933.  oncorhynchi was o r i g i n a l l y Oncorhynchus British  d e s c r i b e d from  including  Philonema  prespawning  nerka captured i n E n g l i s h Bay, Vancouver,  Columbia  (Kuitunen-Ekbaum,  1933).  The s p e c i e s has  subsequently been recorded i n 0^ nerka from v a r i o u s localities  along coastal B r i t i s h  Columbia, Vancouver  Island  and A l a s k a (Bangham and Adams, 1954, M a r g o l i s , 1963, Pennel et.  a l . , 1973, B a i l e y and M a r g o l i s , 1987), Akhmerov  (1955)  r e p o r t e d P^ oncorhynchi i n 0^ nerka from Kamchatka. Philonema oncorhynchi has been found i n t h e lower F r a s e r R i v e r drainage (Cultus Lake) but i s a p p a r e n t l y absent from the  upper F r a s e r  (Bailey and M a r g o l i s , 1987).  Margolis  (pers. comm.) i n d i c a t e d t h a t o f 75 a d u l t sockeye examined from t h e lower Columbia only t h r e e i n f e c t e d f i s h were found and a t o t a l  o f 4 P_^ oncorhynchi worms were recovered; no  worms were found i n 48 smolts examined from l a k e s i n the Columbia d r a i n a g e .  I t seems l i k e l y t h a t  Philonema  oncorhynchi i s not present i n t h e Columbia R i v e r and r e p o r t e d i n f e c t i o n s may r e p r e s e n t wandering  fish  since  59  collection  data from t h i s study suggests p r e v a l e n c e of P.  oncorhynchi approaches 100% i n endemic a r e a s . Philonema agubernaculum  Simon and Simon, 1936 was the  second s p e c i e s o f Philonema t o be d e s c r i b e d from Prosopium williamsoni, Salvelinus fontinalis (=Oncorhynchus and Freemont  and Salmo shasta  mykiss) from Green R i v e r Lakes, M i l l creek,  Lake i n the Wyoming N a t i o n a l F o r e s t .  s i n c e been r e p o r t e d from S^ f o n t i n a l i s  from  Newfoundland  (Sandeman and Pippy, 1967), from S^ f o n t i n a l i s , namaycush, S• a l p i n u s , Coregonus  Maine  S.  c l u p e a f o r m i s and Prosopium  c y l i n d r a c i u m from c o a s t a l Labrador 1973), from S^ f o n t i n a l i s  I t has  (Hicks and T h r e l f a l l ,  and l a n d l o c k e d Salmo s a l a r  from  (Meyer, 1954), and from S^ a l p i n u s from the Koukdiuak  River, B a f f i n  Island  (Dick and B e l o s e v i c ,  1981).  A third  North American s p e c i e s , P^ s a l v e l i n i Richardson, 1937, d e s c r i b e d from S^ f o n t i n a l i s  was  i n Lake Edward, Quebec.  The remaining s p e c i e s of Philonema were d e s c r i b e d from Asia.  Philonema s i b i r i c a (Bauer, 1946)  o r i g i n a l l y d e s c r i b e d from Coregonus  Rumyantsev,  a l b u l a , was  i n the same host from Lake K u i t a , U.S.S.R.  1965,  redescribed  Rumyantsev  (1965) suggested t h a t P^ s i b i r i c a might be a synonym of P. agubernaculum. Japan:  Two  s p e c i e s of Philonema were d e s c r i b e d from  P. ochotense F u j i t a  i n Fukui, 1961  (host and  l o c a l i t y unrecorded) and P^ e l o n g a t a F u j i t a , Oncorhynchus  kawamurae from Lake Tazawa.  1940 i n  Finally,  s p e c i e s were d e s c r i b e d from Kamchatka, U.S.S.R.; Fujita,  1939 i n Oncorhynchus  three P. kondai  keta, P. tenuicauda F u j i t a ,  60  1939  i n CJ^ nerka and  leucomaenis.  salvelini Fujita,  F u j i t a was not aware t h a t  been p r e o c c u p i e d by Richardson's The  1939 i n S a l v e l i n u s s a l v e l i n i had  (1937) s p e c i e s .  s p e c i e s can be d i v i d e d i n t o two groups based on  host b i o l o g y :  P_j_ oncorhynchi, P. kondai, P^ tenuicauda and  P. e l o n g a t a occur i n anadromous salmonids Oncorhynchus.  o f t h e genus  These salmonids t y p i c a l l y undergo l o n g ocean  m i g r a t i o n s and spawn only once i n t h e i r l i f e t i m e s .  On the  other hand, P_;_ agubernaculum, P. s a l v e l i n i Richardson 1937, P. s a l v e l i n i F u j i t a 1939 and P^ s i b i r i c a occur i n nonanadromous salmonids b e l o n g i n g t o d i v e r s e genera Oncorhynchus, Prosopium, Salmo and  (Coregonus,  S a l v e l i n u s ) which  u s u a l l y breed s e v e r a l times d u r i n g t h e i r  life.  Of t h e anadromous Oncorhynchus, 0. nerka i s most commonly i n f e c t e d with Philonema.  The few r e p o r t s o f  Philonema i n other anadromous Oncorhynchus may w e l l r e p r e s e n t c r o s s - i n f e c t i o n s from sockeye.  The reason f o r  t h i s r e s t r i c t i o n presumably has t o do with the f a c t that sockeye  remain  f o r at l e a s t a year i n nursery l a k e s .  This  i s presumably t h e s i t e o f t r a n s m i s s i o n f o r Philonema.  The  copepod Cyclops b i c u s p i d a t u s i s an abundant p l a n k t o n i n many B.C.  l a k e s (Foerster, 1968) and i s an important  f o r p l a n k t i v o r o u s salmonids. demonstrated  food source  P l a t z e r and Adams (1966)  t h a t C_^ 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 f o r Philonema. l i v e i n streams  T h i s may e x p l a i n why salmonids  and r i v e r s ,  that  and do not have access t o t h e  abundant l a k e plankton, are r a r e l y i n f e c t e d w i t h  Philonema.  61  Philonema s p e c i e s are m o r p h o l o g i c a l l y very s i m i l a r and t h i s has l e d t o taxonomic problems i n t h e genus.  Baylis  (1948) suggested P_j_ agubernaculum and P_;_ oncorhynchi were i d e n t i c a l and the s m a l l e r s i z e o f the former was a t t r i b u t a b l e t o host d i f f e r e n c e s or degree o f m a t u r i t y o f worms.  Akhmerov (1955) c o n s i d e r e d P^ agubernaculum, P.  e l o n g a t a , and P^ s i b i r i c a synonyms o f P^ oncorhynchi, recent Russian key t o t h e Nematoda (Vismanius  and a  e t . a l . , 1987)  synonymizes a l l o f F u j i t a ' s s p e c i e s with P^ oncorhynchi, r e c o g n i z i n g only one other s p e c i e s , P^ s i b i r i c a ,  i n waters  o f the U.S.S.R. In t h i s study, Philonema spp. from 0^ mykiss and 0. nerka were m o r p h o l o g i c a l l y i n d i s t i n g u i s h a b l e .  Characters  such as number o f caudal p a p i l l a e i n males have been used i n the d i a g n o s i s o f new Philonema s p e c i e s ( F u j i t a ,  1939).  However, v a r i a b i l i t y i n both p r e a n a l and p o s t a n a l caudal p a p i l l a e was noted i n t h e p o p u l a t i o n o f Philonema Pennask Lake. species  from  T h i s c h a r a c t e r probably has l i t t l e v a l u e f o r  identification.  Simon and Simon (1936) suggested t h a t P^ agubernaculum c o u l d be d i f f e r e n t i a t e d from P^ oncorhynchi by i t s s m a l l e r s i z e and by t h e r a t i o o f a n t e r i o r muscular oesophagus t o p o s t e r i o r g l a n d u l a r oesophagus, g i v e n as 1:2.7 f o r P. agubernaculum and 1:1.1 f o r P_^_ oncorhynchi.  No d i f f e r e n c e  between Philonema from 0^ mykiss or CK_ nerka was noted f o r oesophageal  r a t i o s i n worms examined and v a l u e s ranged  from  62  1:2.3  t o 1:3.1.  Although Philonema  be s m a l l e r than those from by P l a t z e r  landlocked  and P_j_ w i l l i a m s o n i , for  mykiss tended to  nerka, morphometric a n a l y s i s  (1964) showed a h i g h degree of o v e r l a p i n l e n g t h  measurements f o r Philonema mykiss,  from  spp. from 0^ nerka, 0. keta, 0.  salar,  fontinalis  making s i z e a q u e s t i o n a b l e c h a r a c t e r  d i s t i n g u i s h i n g Philonema  demonstrated  S a l v e l i n u s malma, S.  species.  Platzer  t h a t s i z e of a d u l t Philonema  (1964)  varied  depending  on host s p e c i e s as w e l l as host sex, and f u r t h e r  illustrated  o v e r l a p i n v a r i a t i o n of such c h a r a c t e r s as d i s t a n c e from a n t e r i o n end t o nerve r i n g , muscular  and g l a n d u l a r  oesophagus, number of caudal p a p i l l a e , and t a i l l e n g t h , and concluded t h a t P_j_ agubernaculum  and  spicule  was  m o r p h o l o g i c a l l y i d e n t i c a l w i t h P_^ oncorhynchi. Bashirullah  (1966) d e c i d e d the two worms were d i s t i n c t  s p e c i e s based on d i f f e r e n c e s i n l i f e h i s t o r y .  He  suggested  P. oncorhynchi i s a p a r a s i t e of anadromous salmonids  and  undergoes slower development, t a k i n g 32-35 months t o reach maturity.  Sockeye do not r e t u r n t o f r e s h water f o r two  more years and B a s h i r u l l a h  (1966) showed t h a t e l e v a t e d host  hormones p r i o r t o spawning s t i m u l a t e d female worms t o undergo t h e i r f i n a l maturation.  Bashirullah  (1966)  suggested t h a t P^_ agubernaculum, a p a r a s i t e of nonanadromous salmonids, develops more q u i c k l y r e a c h i n g m a t u r i t y i n 6-12  months.  or  He suggested t h a t these worms  matured independently of host hormones but had  no  63  experimental evidence t o support t h i s .  Developmental  d i f f e r e n c e s a r e not h o s t d e t e r m i n e d because e x p e r i m e n t s ( B a s h i r u l l a h , 1966)  cross-infection  show t h a t d e v e l o p m e n t a l r a t e  was u n a f f e c t e d when worms were i n d i f f e r e n t h o s t s . Bashirullah  (1966)  f u r t h e r supported h i s t h e s i s that the  s p e c i e s were d i s t i n c t w i t h p r o t e i n e l e c t r o p h o r e s i s . However, sample s i t e s f o r Kootenay Lake) and  agubernaculum  o n c o r h y n c h i (0.  (0.  mykiss from  n e r k a from C u l t u s  Lake) were g e o g r a p h i c a l l y s e p a r a t e d , r a i s i n g t h e p o s s i b i l i t y t h a t d i f f e r e n c e s r e s u l t e d from p o p u l a t i o n v a r i a t i o n i n a single, widely d i s t r i b u t e d species. T h i s s t u d y examined r e s t r i c t i o n fragment b a n d i n g p a t t e r n s i n r e p e t i t i v e sequences o f DNA  and s u p p o r t s  B a s h i r u l l a h s i n t e r p r e t a t i o n t h a t P^ agubernaculum 1  oncorhynchi represent d i s t i n c t species.  and P.  R e s u l t s from t h i s  study demonstrate t h a t Philonema spp. f a l l i n t o two g e n e t i c a l l y d i s t i n c t groups, c o r r e s p o n d i n g t o t h e two h o s t s p e c i e s from which t h e y were c o l l e c t e d . were d i f f e r e n t f o r most enzymes used.  Banding p a t t e r n s Only 3 enzymes  I I I , Hpa I and Xho I) r e v e a l e d s h a r e d bands.  (Hae  R e s u l t s from  h y b r i d i z a t i o n w i t h t h e c l u s t e r o f r i b o s o m a l genes (pBx2) probe f a i l e d t o show any d i f f e r e n c e s between Philonema species.  T h i s may be because t h e pBx 2 probe r e p r e s e n t s a  c l u s t e r o f r i b o s o m a l genes which may be h i g h l y c o n s e r v e d and t h e r e f o r e show no d i f f e r e n c e s between c l o s e l y species.  related  64  Even though  oncorhynchi  and  agubernaculum were  c o l l e c t e d from s i t e s where t h e i r r e s p e c t i v e hosts were sympatric maintained  (e.g. Babine Lake), the two t h e i r genetic i d e n t i t y ,  groups of worms s t i l l  i m p l y i n g no h y b r i d i z a t i o n  occurs even when the opportunity presumably e x i s t s . Philonema groups probably  represent good b i o l o g i c a l  and are h e r e a f t e r r e f e r r e d t o as P^ agubernaculum 0. mykiss) and P_^ oncorhynchi  The  two  species  (infecting  ( i n f e c t i n g 0^ nerka) .  Curran et a l . (1985) demonstrated the f e a s i b i l i t y  of  u s i n g r e s t r i c t i o n fragment l e n g t h d i f f e r e n c e s of r e p e t i t i v e DNA  sequences  (such as ribosomal  and h i s t o n e genes) as a  t o o l f o r r a p i d i d e n t i f i c a t i o n of nematode s p e c i e s .  Many  nematode groups i n c l u d e species t h a t are d i f f i c u l t d i s t i n g u i s h m o r p h o l o g i c a l l y making i d e n t i f i c a t i o n  to difficult.  Using Eco R l , Curran e t . a l . (1985) demonstrated r e s t r i c t i o n fragment l e n g t h d i f f e r e n c e s f o r r e p e t i t i v e genes among s e l e c t e d s p e c i e s and p o p u l a t i o n s of nematodes belonging  to  the genera C a e n o r h a b d i t i s , H e t e r o r h a b d i t i s , Meloidogyne, Romanonmermis, Steinernema and  Trichinella.  R e s t r i c t i o n endonucleases r e c o g n i z e and c l e a v e sequences of n u c l e o t i d e s i n double-stranded  DNA.  The  and number o f r e s u l t i n g fragments vary depending on number and l o c a t i o n of cut s i t e s .  specific size  the  R e s t r i c t i o n fragments of  r e p e t i t i v e genes are v i s i b l e on agarose g e l s because they are r e p r e s e n t e d  i n high numbers.  Bands of equal s i z e  assumed t o be homologous, and s i m i l a r banding p a t t e r n s produced by a given enzyme r e f l e c t r e l a t e d n e s s .  It i s  are  65  u n l i k e l y t h a t the same enzyme w i l l produce r e p e t i t i v e bands of the same s i z e i n u n r e l a t e d s t r e t c h e s of DNA. assumptions are supported  by e m p i r i c a l data.  These Curran et a l .  (1985) noted t h a t the number of shared bands decreased more d i s t a n t l y r e l a t e d groups were compared.  as  For example,  t r a n s g e n e r i c comparisons showed no shared bands while comparisons of c l o s e l y r e l a t e d s p e c i e s r e v e a l e d only a small percentage of shared bands; bands were shared. recent shared  w i t h i n s p e c i e s , most or a l l  T h i s i m p l i e s t h a t shared bands i n d i c a t e  ancestry,  and supports  use  of t h i s  technique  i n examining p o p u l a t i o n and s p e c i e s r e l a t i o n s h i p s i n morphologically  i n d i s t i n g u i s h a b l e groups.  Restriction  fragment l e n g t h d i f f e r e n c e s are of l i t t l e use i n i n v e s t i g a t i n g r e l a t i o n s h i p s among d i s t a n t l y r e l a t e d taxa because few,  i f any,  shared bands occur.  Worms c o l l e c t e d from kokanee i n Babine Lake were i d e n t i c a l with those i d e n t i f i e d as  i n sockeye from the same r e g i o n and  oncorhynchi.  kokanee represent  T h i s i s not  s u r p r i s i n g since  a s e l f s u s t a i n i n g o f f s h o o t , d e r i v e d from  an anadromous sockeye p o p u l a t i o n i n the lake Crossman, 1973,  are  Foote e t . a l . ,  (Scott  and  1989), t h a t forgo ocean  m i g r a t i o n and remain i n the nursery  lake.  Kokanee,  like  sockeye, spawn once i n t h e i r l i f e t i m e , u s u a l l y i n t h e i r f o u r t h year.  Philonema oncorhynchi  i s probably  carried into  the kokanee p o p u l a t i o n when the l a t t e r a r i s e s from the sockeye p o p u l a t i o n which are or were i n the same nursery lake.  M a t e r i a l from s t e e l h e a d smolts from net pens i n Lake  66  O'Connor was  i d e n t i f i e d as  agubernaculum.  Steelhead are  an anadromous d e r i v i t i v e of rainbow t r o u t / but smolts not u s u a l l y exposed t o Philonema because the  are  freshwater  stage of t h e i r l i f e c y c l e u s u a l l y i n v o l v e s stream or r i v e r residence  (Withler, 1966).  These smolts were probably  c a r r y i n g worms t h a t c i r c u l a t e i n r e s i d e n t salmonids. Philonema spp. are normally non-pathogenic,  but  severe  p a t h o l o g i c a l e f f e c t s were noted i n s t e e l h e a d smolts.  Smolts  l i k e l y became i n f e c t e d when they were p l a c e d i n the lake which presumably c o n t a i n s an endemic salmonid p o p u l a t i o n c a r r y i n g P_^ agubernaculum and pathology probably because these hosts are immunologically n a i v e . Moser  resulted Sakanari  and  (1990) s t u d i e d p a t h o l o g i c a l changes induced by  p l e u r o c e r c o i d s of the cestode L a c i s t o r h y n c h u s d o l l f u s i i n east and west coast s t r i p e d bass  (Morone s a x a t i l i s ) .  They  showed t h a t these p a r a s i t e s induced more i n t e n s e p a t h o l o g i c a l changes i n east coast s t r i p e d bass.  They  suggested t h a t because east coast s t r i p e d bass do not contact  d o l l f u s i they are immunologically naive and  r e s u l t s i n a s t r o n g e r p a t h o l o g i c a l response. hand, Sakanari and Moser s t r i p e d bass, to  this  On the other  (1990) suggested t h a t west coast  i n t r o d u c e d from the east coast, have  adapted  d o l l f u s i because they have been a s s o c i a t e d w i t h the  p a r a s i t e f o r more than 20 g e n e r a t i o n s . D r a c u n c u l o i d s are t i s s u e d w e l l i n g p a r a s i t e s of v e r t e b r a t e s and t h e i r c y c l e s t y p i c a l l y i n v o l v e use of copepods as i n t e r m e d i a t e h o s t s .  This l i f e  s t y l e presents a  67  problem because f i r s t  stage l a r v a e must reach the e x t e r n a l  environment t o be i n g e s t e d by the i n t e r m e d i a t e h o s t .  Many  d r a c u n c u l o i d s undergo a m i g r a t i o n t o subcutaneous t i s s u e s where they breach the host tegument t o r e l e a s e t h e i r l a r v a e . However, Philonema spp. make use o f the host's r e p r o d u c t i v e system and pass t o t h e e x t e r n a l environment w i t h the hosts roe d u r i n g spawning  ( P l a t z e r , 1964).  Philonema spp. use  copepods o f the genus Cyclops as i n t e r m e d i a t e hosts (Vik, 1964,  B a s h i r u l l a h , 1966, P l a t z e r and Adams, 1966, Ko and  Adams, 1969) and l a r v a e undergo development t o the i n f e c t i v e t h i r d stage w i t h i n these h o s t s . P. oncorhynchi  Since P_;_ agubernaculum and  are known t o occur i n sympatry  (e.g.. Babine  Lake) and both s p e c i e s use the same i n t e r m e d i a t e host, n a t u r a l c r o s s - i n f e c t i o n s should occur, and t h i s c o u l d l e a d t o h y b r i d i z a t i o n and p o s s i b l e breakdown o f s p e c i e s b a r r i e r s . C r o s s - i n f e c t i o n experiments by B a s h i r u l l a h (1966) demonstrate t h a t P^ agubernaculum and P^ oncorhynchi are capable o f i n f e c t i n g a v a r i e t y o f salmonids.  A sub-adult P.  agubernaculum was recovered from an e x p e r i m e n t a l l y  infected  sockeye a f t e r 161 days, and a rainbow 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 w i t h P^ oncorhynchi  s u r v i v e d 34 6 days a f t e r which  time a f o u r t h stage worm was recovered from the swimbladder. There i s a l s o evidence Migrant  for natural cross-infections.  sockeye from Shushwap lake and Seton Lake were found  t o be i n f e c t e d w i t h immature Philonema spp. ( B a s h i r u l l a h , 1966, absent  M a r g o l i s , p e r s . comm.).  Since P_^ oncorhynchi i s  from the upper F r a s e r and Columbia R i v e r s these  68  i n f e c t i o n s l i k e l y represent c r o s s - i n f e c t i o n w i t h P. agubernaculum.  While c r o s s - i n f e c t i o n a p p a r e n t l y occurs, no  mature P. agubernaculum was  recovered from the 35  sockeye  and kokanee examined i n the present study, yet a l l were presumably sympatric with salmonids agubernaculum.  i n f e c t e d by  P.  S i m i l a r l y , no P^ oncorhynchi were recovered  from the 5 F u l t o n R i v e r rainbow t r o u t known t o be w i t h sockeye h a r b o r i n g Bashirullah prevented because  Philonema.  (1966) argued t h a t h y b r i d i z a t i o n i s agubernaculum has a y e a r l y l i f e  and can not s u r v i v e the extended m i g r a t i o n of Furthermore,  sympatric  cycle  sockeye.  he b e l i e v e d t h a t u n l i k e P_;_ oncorhynchi,  P.  agubernaculum develops d i r e c t l y t o adulthood independent cues from host hormones.  of  He showed t h a t P^ agubernaculum  develops more r a p i d l y than P^ oncorhynchi r e g a r d l e s s of host but never recovered g r a v i d females of P^ agubernaculum. Even a f t e r s i x months, worms were s u b g r a v i d .  This i s  c o n s i s t e n t w i t h the hypothesis t h a t P^ agubernaculum r e q u i r e s host hormonal cues b e f o r e undergoing  i t s final  maturation.  P.  C i r c u m s t a n t i a l evidence suggests  agubernaculum i s t i e d t o host hormonal changes s i n c e P l a t z e r (1964) only recovered mature worms from r e p r o d u c t i v e l y mature hosts i n the w i l d . It  seems more l i k e l y t h a t jP^ agubernaculum and  oncorhynchi have s i m i l a r l i f e h i s t o r i e s ; only when the host reproduces. host i s probably d i p h a s i c ;  both can  Development i n the  P. reproduce final  worms develop t o adulthood  69  d i r e c t l y but r e q u i r e host hormonal cues t o undergo f i n a l maturation.  The  slowly than  f a c t t h a t P_^_ oncorhynchi  agubernaculum merely  develops more  r e f l e c t s i t s adaptation  t o a host which breeds only once u s u a l l y at f o u r years. i t were not t r u e , then when which may charr  If  agubernaculum e n t e r e d hosts  not mature f o r s e v e r a l years or hosts such as  (genus S a l v e l i n u s ) which breed every other year, worms  would be at a r e p r o d u c t i v e dead-end.  Furthermore,  kokanee  spend t h e i r e n t i r e l i v e s f e e d i n g on p l a n k t o n and b e n t h i c invertebrates  (Scott and Crossman, 1973)  exposed throughout at l e a s t those  and should be  t h e i r l i f e t o Philonema spp.  P_^ agubernaculum p i c k e d up a year p r i o r t o  spawning should s u r v i v e t o host spawning. (1966) examined 200  Bashirullah  kokanee examined from Kootenay Lake and  only one c o n t a i n e d an a d u l t Philonema; absent  Therefore,  P. oncorhynchi i s  i n t h i s area and the i n f e c t i o n probably r e p r e s e n t s P.  agubernaculum. Rainbow t r o u t g e n e r a l l y take 3-5 may  l i v e as long as 8 years  t h e r e f o r e , P^ oncorhynchi  (Scott and Crossman, 1973);  a c q u i r e d e a r l y should have ample  time t o grow and reach m a t u r i t y . observed,  years t o mature and  but sample s i z e was  No c r o s s - i n f e c t i o n s were  s m a l l because only the 5 worms  examined from F u l t o n R i v e r were known t o be sympatric w i t h P.  oncorhynchi. It seems l i k e l y t h a t s u c c e s s f u l c r o s s - i n f e c t i o n s by  Philonema spp. are prevented by a host immune response encapsulates and d e s t r o y s worms b e f o r e they can mature.  which  70  Bashirullah  (1966) observed  c y s t s c o n t a i n i n g Philonema i n  the body c a v i t y o f sockeye from Shushwap Lake. agubernaculum and P^ oncorhynchi divergence,  Philonema  have undergone  molecular  as evidenced by d i f f e r e n t banding p a t t e r n s , and  t h i s divergence  may be a s s o c i a t e d with a n t i g e n i c changes  which r e s u l t i n r e j e c t i o n when Philonema spp. e n t e r s an i n a p p r o p r i a t e host.  This host immune response may prevent  h y b r i d i z a t i o n between the two s p e c i e s o f Philonema by d e s t r o y i n g one s p e c i e s p r i o r t o maturation  and mating.  However, i t i s a l s o p o s s i b l e t h a t c r o s s - i n f e c t i o n s and h y b r i d i z a t i o n are prevented  because worms are unable t o  mature i n u n s u i t a b l e hosts due t o p h y s i o l o g i c a l d i f f e r e n c e s i n t h e host s p e c i e s . F i s h r e s t r i c t e d t o i s o l a t e d freshwater o f t e n undergo g e n e t i c divergence  from  drainage  systems  neighboring  p o p u l a t i o n s i n other watersheds because l a n d masses and s a l t water a c t as b a r r i e r s t o d i s p e r s a l .  Philonema agubernaculum  samples showed banding p a t t e r n s s p e c i f i c t o geographic l o c a l i t i e s from which they were c o l l e c t e d probably  because  t h e r e i s no gene flow between p o p u l a t i o n s s i n c e t h e i r are probably  r e s t r i c t e d t o t h e i r r e s p e c t i v e drainage  hosts basins.  Pennask Lake and F u l t o n R i v e r p o p u l a t i o n s were n e a r l y identical  ( d i s t i n g u i s h e d by only two Hae I I I bands),  Lake O'Connor worms appear t o have undergone divergence  greater  from t h e two mainland p o p u l a t i o n s s i n c e d i f f e r e n t  banding p a t t e r n s were observed Hae  while  I I I and H i n f I ) .  Curran  f o r t h r e e enzymes  (Bam HI,  e t a l . (1985) demonstrated  71  banding p a t t e r n d i f f e r e n c e s between s p e c i e s but show d i f f e r e n c e s between p o p u l a t i o n s elegans.  of  Caenorhabditis  T h i s l e d them t o s p e c u l a t e t h a t  fragment l e n g t h d i f f e r e n c e s may  f a i l e d to  restriction  represent markers f o r  p o p u l a t i o n s which can no longer f r e e l y i n t e r b r e e d and may  be a s s o c i a t e d with s p e c i a t i o n , when g e n e t i c  they  isolation  allows r a p i d f i x a t i o n of novel genome c h a r a c t e r i s t i c s . While banding p a t t e r n d i f f e r e n c e s seem to r e f l e c t i s o l a t i o n , there i s no reason population represents  genetic  t o conclude t h a t each  a d i f f e r e n t s p e c i e s . I t seems more  l i k e l y t h a t , l i k e morphological  differences, differences in  banding p a t t e r n s are l i k e l y t o occur among p o t e n t i a l l y interbreeding populations  i n widely  distributed  species.  M i n c h e l l a e t . a l . (1989) s t u d i e d r e s t r i c t i o n  fragment  l e n g t h d i f f e r e n c e s i n urban and s y l v a t i c p o p u l a t i o n s Trichinella spiralis  (Nematoda; T r i c h i n e l l i d a e ) .  r e s t r i c t i o n enzymes used s u c c e s s f u l l y separated (wild) T r i c h i n e l l a from urban  populations.  suggesting  Most  for sylvatic  t h a t s y l v a t i c 1\_ s p i r a l i s i s  i n t o more or l e s s d i s t i n c t  Most of B.C. ago.  sylvatic  However, the enzyme C l a I d i s t i n g u i s h e d a l l  sylvatic isolates, organized  A l l 12  (swine) T r i c h i n e l l a .  enzymes gave s i m i l a r banding p a t t e r n s  of  was  populations.  g l a c i a t e d as r e c e n t l y as 14,000 years  M e l t i n g and subsequent r e t r e a t of g l a c i e r s r e s u l t e d i n  formation  of l a r g e lakes and  streams  f a c i l i t a t e d r e i n v a s i o n of freshwater r e f u g i a i n the n o r t h and south.  (Briggs, 1986),  and  f i s h from areas  of  The F r a s e r R i v e r system  was  72  p r i n c i p a l l y r e c o l o n i z e d from the Columbia R i v e r v i a g l a c i a l l a k e s i n the Okanagan v a l l e y .  The Skeena system has a f i s h  fauna s i m i l a r t o the Columbia R i v e r , i n d i c a t i n g a previous connection  v i a the F r a s e r , probably  was blocked  while the Skeena  river  and i t s t r i b u t a r i e s flowed east i n t o the F r a s e r  (McPhail and Lindsey,  1986).  Thus, the c l o s e r e l a t i o n s h i p  between Pennask Lake and F u l t o n R i v e r P^ agubernaculum  may  represent t h e i r common o r i g i n v i a a Columbia R i v e r d i s p e r s a l event.  On the other hand P^ agubernaculum from Lake  O'Connor may  have d i v e r g e d much e a r l i e r ,  f o l l o w i n g an e a r l y  p o s t g l a c i a l m i g r a t i o n across the S t r a i t of Georgia. A l t e r n a t i v e l y , Lake O'Connor worms may  represent a  p o p u l a t i o n which s u r v i v e d i n a c e n t r a l coast r e f u g i a d u r i n g the most recent P l e i s t o c e n e g l a c i a t i o n . evidence,  There i s i n c r e a s i n g  mainly b o t a n i c a l , t h a t an area i n the Queen  C h a r l o t t e s and northern Vancouver I s l a n d was d u r i n g the most recent P l e i s t o c e n e g l a c i a t i o n Lindsey,  1986, O g i l v i e ,  Lake O'Connor r e p r e s e n t s  1989).  unglaciated (McPhail  and  I f P^ agubernaculum from  a p o p u l a t i o n which s u r v i v e d i n t h i s  r e f u g i a , then i t should prove t o be more s i m i l a r t o worms from the Queen C h a r l o t t e s .  Philonema agubernaculum from  southern  Vancouver I s l a n d should be more s i m i l a r t o the  southern  mainland s i n c e i t was probably  c o l o n i z e d from t h i s  area f o l l o w i n g g l a c i a l r e t r e a t . R e s u l t s from Bam HI, H i n f I and Hpa I d i g e s t i o n appear t o i n d i c a t e two g e n e t i c types  of P^ oncorhynchi,  however,  these two types o c c u r r e d w i t h i n the same p o p u l a t i o n (e.g.  73  Sproat R i v e r , Henderson Lake, F u l t o n R i v e r and P i e r r e Creek) and are best i n t e r p r e t e d as polymorphisms.  Sockeye salmon  are anadromous and undergo an ocean m i g r a t i o n which takes them o f f shore b e f o r e they r e t u r n t o spawn i n f r e s h water. L i k e other salmonids,  sockeye  show a s t r o n g homing  f o r t h e i r nursery l a k e (and n a t a l stream).  I f homing were  p e r f e c t , i t would r e s t r i c t gene flow i n P^  oncorhynchi  p o p u l a t i o n s and p o s s i b l y l e a d t o geographic  variation  because worms are t r a n s m i t t e d i n nursery l a k e s . Quinn et a l . ( 1987)  may  A study by  using protein electrophoresis  p a r a s i t e p r e v a l e n c e data suggested sockeye may than 1% of the time.  tendency  and  stray  less  However, t h i s s m a l l amount of s t r a y i n g  be s u f f i c i e n t t o prevent p o p u l a t i o n divergence i n t h e i r  p a r a s i t e s or may  r e s u l t i n mixing i f changes do occur.  Sockeye i n t r o d u c t i o n s may  a l s o have c o n t r i b u t e d t o  mixing of p o p u l a t i o n s of P^ oncorhynchi. t r a n s p l a n t e d i n t o many B.C.  Sockeye have been  watersheds i n recent times.  If  i n f e c t e d smolts were t r a n s p l a n t e d they c o u l d seed u n i n f e c t e d l a k e s with P^ oncorhynchi or mix with an e x i s t i n g population.  Supporting evidence f o r t h i s h y p o t h e s i s i s  d i f f i c u l t t o f i n d s i n c e many i n t r o d u c t i o n s are  unrecorded  and when they are, method of t r a n s p l a n t i s o f t e n not However, most f i s h are t r a n s p l a n t e d as eggs which would not c a r r y  (Foerster,  Philonema.  The technique used i n t h i s study allows easy i d e n t i f i c a t i o n of Philonema spp. i n B.C. f a c i l i t a t e f u r t h e r s t u d i e s on host and  stated.  and should geographic  1968)  74  d i s t r i b u t i o n s o f t h e group.  Philonema agubernaculum has  been r e p o r t e d from a wide v a r i e t y o f s a l m o n i d h o s t s but our specimens were c o l l e c t e d from 0^ m y k i s s ;  i t remains t o be  seen whether a s i n g l e s p e c i e s o f Philonema i n f e c t s a l l nonanadromous salmonids i n B.C.  I t may be r e l a t i v e l y easy t o  c h a r a c t e r i z e t h e g e o g r a p h i c d i s t r i b u t i o n o f P_j_ o n c o r h y n c h i and r e s o l v e m i s i d e n t i f i c a t i o n s f o r t h i s s p e c i e s because i t appears t o l a c k g e o g r a p h i c v a r i a t i o n .  However, i t w i l l be  more d i f f i c u l t t o c h a r a c t e r i z e t h e g e o g r a p h i c d i s t r i b u t i o n o f P_^ agubernaculum.  T h i s s p e c i e s has been r e p o r t e d from  v a r i o u s l o c a l i t i e s a c r o s s N o r t h America and may a l s o be present i n A s i a .  I s o l a t e s from B.C. show d i v e r g e n c e and  t h o s e from more d i s t a n t g e o g r a p h i c areas may show degrees o f d i v e r g e n c e t h a t argue a g a i n s t t h e i r i n c l u s i o n i n a s i n g l e species. P. o n c o r h y n c h i i s p r o b a b l y r e s t r i c t e d t o 0^ n e r k a . R a r e l y , i t has been r e p o r t e d from 0^ k e t a (Akhemerov 1955, P l a t z e r , 1964) but i t i s p r o b a b l y not a common p a r a s i t e o f chum and o t h e r P a c i f i c salmon f o r e c o l o g i c a l r e a s o n s . Sockeye a r e unique i n t h a t s m o l t s spend 1-3 y e a r s f e e d i n g i n a n u r s e r y l a k e which i s t h e f o c u s o f t h e Philonema infection.  I f 0^ n e r k a i s t h e o n l y n a t u r a l h o s t f o r P.  o n c o r h y n c h i t h e n worms s h o u l d o n l y be found w i t h i n t h i s h o s t s range.  Reports of  sockeye a r e l i k e l y  o n c o r h y n c h i o u t s i d e t h e range o f  misidentifications.  75  P a c i f i c salmon  (genus Oncorhynchus) represent a recent  l i n e a g e , 2-3 m i l l i o n years o l d (Thomas et a l . , 1986), and sockeye d i v e r g e d from t h i s l i n e a g e about ago.  Philonema oncorhynchi may  2 million  years  have coevolved w i t h  sockeye  from an ancestor found i n more p r i m i t i v e non-anadromous salmonids, history.  i n response t o a change i n the host's  life  Philonema oncorhynchi probably arose sometime  a f t e r t h i s event s i n c e the worm i s not present the whole host range; and Columbia  Rivers.  i t i s absent  throughout  from the Upper F r a s e r  Philonema oncorhynchi may  have a r i s e n  on the A s i a t i c or Alaskan coast d u r i n g P l e i s t o c e n e glaciation  and c o l o n i z e d the B.C.  coast when  sockeye  p o p u l a t i o n s reinvaded from areas of r e f u g i a i n B e r i n g i a (McPhail and Lindsey, 1986).  76 REFERENCES  Akhmerov, A.kh. 1955. P a r a s i t e fauna o f t h e f i s h e s of the r i v e r Kamchatka. I z v e s t . Tilchookeansk. N. I. I n s t . Rybn. Khoz. Okeanogr., 43: 99-137. B a i l e y , R.E. and L. M a r g o l i s . 1987. 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Biochemical g e n e t i c comparison of sockeye salmon and kokanee, the anadramous and nonanadramous forms of Oncorhynchus nerka. Can. J . F i s h . Aquat. S c i . , 46: 149-158. F u j i t a , T. 1939. On the nematoda-parasites of the p a c i f i c salmon. J . F a c u l . Agr. Hokkaido Imp. Univ., 42: 239266. F u j i t a , T. 1940. F u r t h e r notes on nematodes of salmonid f i s h e s i n Japan. Japan. J . Zool., 8: 377-394. Fukui, T. 1961. S t u d i e s on the p a r a s i t e s of salmonid f i s h e s of Japan. B u l l . Yokohama Mun. Univ. S o c , 12 (2): 1-66. Hicks, F . J . and W. T h r e l f a l l . 1973. Metazoan p a r a s i t e s of salmonids and coregonids from c o a s t a l Labrador. J. F i s h . B i o l . , 5: 399-415. Ko, R.C. and J.R. Adams. 1969. The development of Philonema oncorhynchi (Nematoda: P h i l o m e t r i d a e ) i n Cyclops b i c u s p i d a t u s i n r e l a t i o n t o temperature. Can. J . Z o o l . , 47: 307-312. Kuitunen-Ekbaum, E. 1933. 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Can., 24: 1911-1943.  79  S c o t t , W.B and E . J . Crossman. 1973. Freshwater F i s h e s of Canada. B u l l . 184, F i s h . Res. Bd. Can., 966p. Simon, J.R. and F. Simon. 1936. Philonema agubernaculum sp.nov. (dracunculidae), a nematode form 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. Thomas, W.K., R.E. W i t h l e r and A.T. Beckenbach. 1986. M i t o c h o n d r i a l DNA a n a l y s i s o f P a c i f i c salmonid evolution. Can. J . Zool., 64: 1058-1064. Vik, R. 1964. Notes on the l i f e h i s t o r y o f Philonema agubernaculum Simon et Simon, 1936 (Nematoda). Can. J . Zool., 42: 511-513. Vismanius, K.O., V.V. Lomakin, V.P. Rojtman, M.K. Semenova and Trofimenko. 1987. Nemathelminthes. Opredel. Faune U.S.S.R., 149: 199-310. W i t h l e r , I.L. 1966. V a r i a b i l i t y i n l i f e h i s t o r y c h a r a c t e r i s t i c s of s t e e l h e a d t r o u t (Salmo g a i r d n e r i ) along the P a c i f i c Coast o f North America. J . F i s h . Res. Bd. Can., 23: 365-393.  

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