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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 the Department of Zoology We accept t h i s t h e s i s as conforming to the req u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1990 © Copyright by Derek Fraser Clease 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) II ABSTRACT Philonema (Nematoda; Philometridae) from anadromous hosts, sockeye salmon (Oncorhynchus nerka), and non-anadromous hosts, rainbow t r o u t (Oncorhynchus mykiss) , were st u d i e d i n order t o determine i f hosts w i t h d i f f e r e n t l i f e h i s t o r i e s were i n f e c t e d w i t h the same or d i f f e r e n t species of Philonema. Worms from the two host species were morp 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 . However, e l e c t r o p h o r e t i c banding patterns produced by r e s t r i c t i o n enzyme d i g e s t i o n of DNA e x t r a c t e d from Philonema demonstrated the presence of two genetic types corresponding t o the two host species. This supports the idea t h a t at l e a s t two species of Philonema are endemic i n B r i t i s h Columbia. Philonema  oncorhynchi Kuitunen-Ekbaum, 1933 i s a p a r a s i t e of sockeye salmon which undergo a long ocean mi g r a t i o n before r e t u r n i n g to freshwater t o spawn, while P^ agubernaculum Simon and Simon, 1936 i s a p a r a s i t e of rainbow t r o u t (and other salmonids) which l i v e i n l a k e s . Kokanee (0j_ nerka k e n n e r l y i ) , a non-anadromous offshoot of sockeye, were i n f e c t e d w i t h the same worm as sockeye probably because the two hosts have s i m i l a r l i f e h i s t o r i e s . Steelhead smolts, anadromous 0^ mykiss, contained worms i d e n t i f i e d as P^ agubernaculum. This l i k e l y represented an a c c i d e n t a l i n f e c t i o n because steelhead do not u s u a l l y contact Philonema. I l l 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 pop u l a t i o n divergence corresponding t o the d i f f e r e n t geographic l o c a l i t i e s from which i t was c o l l e c t e d . 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 these p a r a s i t e populations i n unconnected watersheds. Philonema oncorhynchi showed polymorphisms spread throughout many of the p o p u l a t i o n s . The l a c k of p o p u l a t i o n divergence probably r e s u l t s from gene flow between p a r a s i t e populations brought about by wandering hosts. IV TABLE OF CONTENTS Page Ab s t r a c t I L i s t of Figures V L i s t of Tables VI Acknowledgements VII In t r o d u c t i o n 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 of DNA 8 DNA E l e c t r o p h o r e s i s 10 Southern Transfer 11 H y b r i d i z a t i o n 12 Microscopy 13 Results 14 Morphology of Worms 14 Philonema from 0^ mykiss 14 Philonema from CK_ nerka 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 Di s c u s s i o n 58 References 7 6 V LIST OF FIGURES Page 1. C o l l e c t i o n l o c a l i t i e s for Philonema 7 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 Rl DNA digest for Philonema spp. 22 9. Hae III DNA digest for Philonema spp. 27 10. Hinf I DNA digest for Philonema spp. 2 9 11. Hpa I DNA digest for Philonema spp. 31 12. Xba I DNA digest for Philonema spp. 33 13 Xho I DNA digest for Philonema spp. 35 14. Hpa I DNA digest for worms from 0^ mykiss 38 15. Bam HI DNA digest for worms form 0^ mykiss 4 0 16. Hinf I DNA digest for worms from 0^ mykiss 42 17. Hae III DNA digest for worms from 0^ mykiss 44 18. Bam HI DNA digest for worms from 0^ nerka 47 19. Hinf I DNA digest for worms from 0^ nerka 4 9 20. Hpa I DNA digest for worms from 0^ nerka 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 with pBx2 54 22. Cladogram of Philonema spp. populations 57 VI LIST OF TABLES Page I. Prevalance and i n t e n s i t y of Philonema spp i n 0j_ mykiss/ 0. nerka and 0. nerka k e n n e r l y i 15 VII ACKNOWLEDGEMENT S The author would l i k e to thank Dr. Ma r t i n Adamson f o r suggesting and s u p e r v i s i n g t h i s study and f o r help i n r e v i s i n g t h i s manuscript. The author would a l s o l i k e to thank Dr. Leo Margolis of the P a c i f i c B i o l o g i c a l S t a t i o n , 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 i n t e r e s t i n t h i s p r o j e c t . Thanks are extended t o Dr. Don Moerman f o r h i s a s s i t a n c e i n teaching the author techniques i n v o l v e d w i t h DNA e x t r a c t i o n and southern b l o t t i n g . The author would l i k e t o thank Mr. Stu Barnetson of the Fu 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 the c o l l e c t i o n of f i s h on s e v e r a l occassions. The author i s g r a t e f u l t o Mrs. Karen Beckenbach, i n Dr. John Webster's l a b at Simon Fraser U n i v e r s i t y , f o r probing southern b l o t s i n t h i s study. Thanks are extended t o Bob Carveth f o r h i s help i n r e v i s i n g t h i s manuscript. F i n a l l y , thanks are extended to Stewart "the Chie f " Noble f o r h i s a s s i s t a n c e i n f i e l d c o l l e c t i o n s . 1 INTRODUCTION Philonema spp. (Dracunculoidea; Philometridae) are nematode p a r a s i t e s i n the body c a v i t y of salmonids (Salmoniformes; Salmonidae). Mature ovoviviparous females leave the host by the reproductive system and re l e a s e larvae when they contact freshwater. Larvae, i f eaten by a s u i t a b l e copepod intermediate host, develop t o become i n f e c t i v e t o the f i n a l host. Transmission occurs when i n f e c t e d copepods are ingested by a salmonid host. Larvae leave the copepod, penetrate through the hosts gut, and enter the swimbladder ( P l a t z e r and Adams, 1967). Larvae then leave the swimbladder and enter the coelom where they w i l l develop i n t o a d u l t s . In the coelom worms mate, a f t e r which males w i l l d i e . The genus contains nine nominal spe c i e s ; the f i r s t three were described from North America (Kuitunen-Ekbaum, 1933; Simon and Simon, 1936; Richardson, 1937) and the remaining s i x were described from A s i a ( F u j i t a , 1939; F u j i t a , 1940; Bauer, 1946; Fukui, 1961; Rumyantsev, 1965). The nine nominal species of Philonema are morphologically d i f f i c u l t 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 than bags f i l l e d w i t h l a r v a e and morphological characters used f o r i d e n t i f i c a t i o n show a high degree of overlap between species. 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 of t e n not 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, the number of species i s not clear since many species may represent synonyms for previously described material (Baylis, 1948, Akhemerov, 1955, Platzer, 1964, Vismanius et a l . , 1987). A method independent of morphology i s therefore needed to resolve taxonomic problems within 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 length differences i n r e p e t i t i v e DNA sequences to i d e n t i f y nematode species not e a s i l y separated by morphological characters. Extracted DNA i s cleaved with r e s t r i c t i o n enzymes which recognize s p e c i f i c sequences and DNA fragments are separated using agarose gel electrophoresis. Repetitive DNA sequences, corresponding to highly r e p e t i t i v e genes (e.g. ribosomal and histone genes), can then be v i s u a l i z e d by staining with ethidium bromide. The technique i s rapid, r e l a t i v e l y inexpensive and may have an advantage over protein electrophoresis since proteins are frequently influenced by environmental and ontogenetic factors making t h e i r patterns more d i f f i c u l t to interpret (Curran e t . a l . , 1985). Two species of Philonema have been reported from B r i t i s h Columbia. Philonema oncorhynchi Kuitunen-Ekbaum, 1933, i s a parasite of anadromous sockeye salmon (Oncorhynchus nerka) and P_;_ agubernaculum Simon and Simon, 1936 supposedly i n f e c t s non-anadromous lake dwelling salmonids. Many lakes contain sockeye and non-anadromous salmonids infected with Philonema spp., therefore B.C. 3 represents an i d e a l location for c o l l e c t i o n and i d e n t i f i c a t i o n of material. In t h i s study Philonema was c o l l e c t e d from sockeye salmon and rainbow trout (Oncorhynchus mykiss), a non-anadromous salmonid known to carry Philonema, i n the same lake (Babine Lake) and the technique described by Curran et a l . (1985) was used to determine i f worms from these two hosts represented one or two species of Philonema. An i n t r i g u i n g situation also existed i n the fact that kokanee (0^ nerka kennerlyi), a non-anadromous freshwater offshoot of sockeye, from Babine Lake were infected with an u n i d e n t i f i e d species of Philonema. Conversely, steelhead trout, anadromous O^ mykiss, were also infected with an u n i d e n t i f i e d species of Philonema. Furthermore, Philonema samples i n both 0^ mykiss and 0.  nerka were c o l l e c t e d from various B.C. l o c a l i t i e s and the same technique was used to determine i f worms from geographically separate areas had undergone population divergence. The freshwater f i s h fauna of B.C. represents a recent recolonization, since much of the province was glaciated 14,000 years ago. During g l a c i a l retreat, f i s h reinvaded from areas of refugia i n the north and south (McPhail and Lindsey, 198 6). Since t h i s time many watersheds have become isolated , 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 parasites. Sockeye, on the other hand, are not r e s t r i c t e d to freshwater. Their anadromous behaviour may take them many miles o f f shore and mix them with other 4 sockeye p o p u l a t i o n s ; but they show a strong homing tendency when r e t u r n i n g t o t h e i r n a t a l nursery lakes and spawning streams. This homing behaviour may r e s t r i c t gene flow i n Philonema because i t i s t r a n s m i t t e d i n nursery l a k e s . 5 MATERIALS AND METHODS Sample C o l l e c t i o n Prespawning sockeye salmon, kokanee, and rainbow and steelhead t r o u t were examined from v a r i o u s B r i t i s h Columbia l o c a l i t i e s ( 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 , Sproat R i v e r , P i e r r e Creek, Henderson Lake, Cultus Lake and F u l t o n R i v e r . Kokanee were c o l l e c t e d from P i e r r e Creek. 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; Steelhead smolts were c o l l e c t e d from Lake O'Connor. F i s h were c o l l e c t e d by seine or d i p net and examined i n the f i e l d or packed on i c e and shipped t o the lab f o r examination. F i s h were d i s s e c t e d by making a mid-ventral 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 throughout the coelom, entangled i n p y l o r i c caeca and w i t h i n gonads of some females. Using a bent probe, worms were c a r e f u l l y removed t o prevent c u t i c l e rupture, placed 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 immediately frozen at -196°C i n l i q u i d n i t r o g e n f o r t r a n s p o r t . L a t e r , worms were t r a n s f e r r e d t o a -70°C fr e e z e r f o r storage. 6 Figure 1. C o l l e c t i o n l o c a l i t i e s for Philonema spp. from various salmonid hosts i n B r i t i s h Columbia. Pl-sockeye, Rivers Inlet, P2-sockeye, Sproat River, P3-sockeye, Pierre Creek (Babine Lake), P4-kokanee, Pierre Creek (Babine Lake), P5-sockeye, Henderson Lake, P6-sockeye, Cultus Lake, P7-steelhead smolts, Lake O'Connor, P8-rainbow trout, Fulton River (Babine Lake), P9-rainbow trout, Pennask Lake, PlO-sockeye, Fulton River (Babine Lake). 7 8 Adult female Philonema spp. used for l i g h t microscopy were fix e d i n cold 95% ethanol to prevent bursting (Platzer, 1964), and transferred to 70% ethanol for storage. Adult males were fixed i n the same manner, or i n hot glycerine alcohol (5% glycerine, 70% ethanol). Extraction of DNA Only female Philonema spp. were used for DNA extraction because of t h e i r larger s i z e . V i a l s containing worms were removed from the -70°C freezer and placed i n a small thermos of l i q u i d nitrogen. Individual frozen worms were transferred to 1.5 ml Eppendorfs and ground under l i q u i d nitrogen using a disposable pestle. A 0.7 ml aliquot of proteinase K buffer [100 mM T r i s (hydroxymethyl) methylamine (Tris) pH 8.0, 50 mM ethylenediaminetetraacetic acid (EDTA), 1% sodium dodecyl sulfate (SDS), 200 mM NaCl] was added to each Eppendorf, followed by approximately 2 mg of Proteinase K. Eppendorfs were incubated i n a dry bath for 16-20 hours at 65°C to maximize digestion. DNA was extracted three times, allowing at least 0.5 hours between extractions. The aqueous layer was co l l e c t e d by spinning Eppendorfs for 2-3 minutes and t r a n s f e r r i n g the viscous supernatent to a clean Eppendorf using a wide-mouth p l a s t i c pipette. Extractions proceeded as follows: 1. equal volume r e d i s t i l l e d phenol saturated 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. Following extraction, 3/5 volume of 20% polyethyleneglycol 8000 i n 2.5 M NaCl was added and DNA was pr e c i p i t a t e d overnight at 4°C. Eppendorfs were spun i n a microcentrifuge for 15 minutes to p e l l e t DNA. The DNA p e l l e t was washed twice with cold 70% ethanol and vacuum dried for 5-10 minutes before being resuspended i n 200 u l of TE. DNA Electrophoresis Extracted DNA was examined by running a l l samples on minigels at 100 v o l t s for about 20 minutes. A known quantity of Hind I I I cut lambda DNA was included as a size standard and samples approximately 20 kb containing 20 ug or more of DNA were selected for digestion with r e s t r i c t i o n endonucleases. DNA was sampled from at least 5 worms from each c o l l e c t i o n s i t e . The following r e s t r i c t i o n enzymes (supplied by BRL) were used to digest each sample; Bam HI, Eco Rl, Hae I I I , Hind I I I , Hinf I, Hpa I, Msp I, Pst I, Xba I and Xho I . Digestion reactions involved 2-8 ug of DNA suspended i n 18 u l of TE, 2.5 u l 10X buffer (supplied with the enzyme by the vendor), 1.0 u l bovine serum albumin (BSA) (2 mg/ml), 10 u l RNAse A (10 mg/ml) and 2.5 u l of 10 r e s t r i c t i o n endonuclease. The r e a c t i o n mixture was incubated at 37°C, i n a water bath, f o r 1-5 hours, depending on a c t i v i t y of enzymes used. Reactions were h a l t e d by adding 0.5 M EDTA t o a f i n a l c o ncentration of 10 mM (Maniatis et a l . , 1982) . Loading b u f f e r w i t h marker dye (15% f i c o l l , 0.25% bromophenol blue, 0.25% xylene cyanol) was added t o a f i n a l c o ncentration of 3%. DNA samples were loaded into. 0 . 7 % agarose g e l co n t a i n i n g 2 .5 mg/ml ethidium bromide f o r DNA s t a i n i n g . Lambda Hind I I I was used as a s i z e standard 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 . Gels were run 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), at 30-40 v o l t s f o r 18-20 hours t o separate DNA r e s t r i c t i o n fragments. Gels were destained f o r 0.5 hours i n d i s t i l l e d water and photographed using 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. Kodak type 55 f i l m ( 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 banding patterns were t r a c e d from negatives. I n i t i a l l y , r e s t r i c t i o n d i g e s t s of s i n g l e samples from a l l c o l l e c t i o n areas were run together. L a t e r , r e p e t i t i v e samples from v a r i o u s s i t e s d igested w i t h s e l e c t e d enzymes were run. Southern Transfer Photographed g e l s were placed i n 0.25 M HCl f o r 10-15 minutes t o depurinate DNA and were then soaked i n t r a n s f e r 11 b u f f e r (0.02 M NaOH and 1 M ammonium acetate) 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 apparatus. T r a n s f e r apparatus c o n s i s t e d o f a g l a s s r e s e r v o i r f o r b u f f e r and a P l e x i g l a s s p l a t e over the r e s e r v o i r t o support g e l s . Two p i e c e s of Whatman 3 MM paper were i n s e r t e d i n t o the t r a n s f e r b u f f e r as wicks. Gels were p l a c e d on wicks and a p i e c e of Hybond-N h y b r i d i z a t i o n t r a n s f e r membrane (Amersham) was cut t o s i z e and p l a c e d over the g e l . Two p i e c e s of Whatman 3 MM paper were cut t o s i z e and p l a c e d over the t r a n s f e r membrane. A i r bubbles were removed at each stage by r o l l i n g them out with a long g l a s s rod. A p i e c e of Handi-Wrap, wi t h a window around the g e l , was used t o cover the apparatus. A weighted stack of Scott paper towels was p l a c e d over the g e l . DNA t r a n s f e r was allowed t o proceed o v e r n i g h t . F o l l o w i n g t r a n s f e r , membranes were removed and soaked i n 0.4 M NaOH f o r 20 minutes t o denature DNA (Broad et a l . , 1988). Membranes were r i n s e d with 2X SSC (0.02 M sodium c i t r a t e and 0.3 M NaCl) and a i r d r i e d f o r 1 hour. Membranes were wrapped i n Handi-Wrap and exposed t o UV l i g h t f o r 3 minutes t o c r o s s - l i n k e d DNA. Membranes were then s t o r e d at room temperature u n t i l used. H y b r i d i z a t i o n Membranes were probed w i t h a c l u s t e r of ribosomal genes which i n c l u d e d the 5.8, 18s and 28s genes, and a 12 n o n t r a n s c r i b e d r e g i o n . The r i b o s o m a l c l u s t e r (pBx 2) was a 7.2 kb Eco R l fragment i s o l a t e d from t h e pinewood nematode Bur s a p h e l e n c h u s x y l o p h i l u s (Nematoda; T y l e n c h i d a ) and c l o n e d i n Puc 19. The probe ( s u p p l i e d by Dr. J . Webster's l a b a t Simon F r a s e r U n i v e r s i t y ) 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 ( M a n i a t i s e t a l . , 1982) u s i n g 3 2 P ATP. The r e a c t i o n c o n t a i n e d 400 ng o f probe DNA i n a f i n a l volume o f 50 u l and was s e t up as f o l l o w s : 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 (NTB) [500 mM T r i s pH 7.5, 100 mM magnesium s u l f a t e , 10 mM d i t h i o t h r e i t r o l (DTT), 500 ug/ml BSA], 37 u l d i s t i l l e d w a ter, 2 Ul -ATP (10 mM GTP, 10 mM TTP, 10 mM CTP), 1 Ul d i l u t e d DNAse I [ d i l u t e s t o c k DNAse I (1 mg/ml i n 50 mM T r i s pH 7.5, 10 mM MgS04, 1 mM DTT, 50% g l y c e r o l ) t o 1/10000 i n IX NTB], 1 u l DNA polymerase 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 uCi) . 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 12-15°C f o r 2 hours and was h a l t e d u s i n g 5 u l o f 0.2 M EDTA and 2% SDS. The probe was p u r i f i e d (to remove f r e e n u c l e o t i d e s ) i n a Sephadex (G25 f i n e ) s p i n column c o n s t r u c t e d from a 1 ml p i p e t t i p p l u g g e d a t one end w i t h g l a s s w o o l . Membranes were p r e h y b r i d i z e d f o r 2 hours a t 62°C i n s e a l e d f r e e z e r bags c o n t a i n i n g 100 ml o f p r e h y b r i d i z a t i o n s o l u t i o n [5X SSPE (5 mM EDTA, 0.06 M NaH2P04-H20, 0.7 M N a C l ) , 0.3% SDS, 2.5X Denharts (0.05% BSA, 0.05% f i c o l l 400, 0.05% p o l y v i n l y p y r r o l i d o n e 4 0 ) ] . Bags c o n t a i n i n g membranes were d r a i n e d 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 probe, 5X SSPE, 0.3% SDS, 2.5X Denharts) p e r 100 cm 2 o f 13 transfer membrane was added to each bag. Membranes were hybridized at 62 °C overnight, washed once for 5 minutes and three times for 10 minutes each i n 2X SSPE and 0.2% SDS. Washed membranes were blotted dry, wrapped with Saran Wrap and placed i n a developing casette with a single i n t e n s i f y i n g screen and X-ray f i l m . Casettes were placed i n a -70"C freezer and f i l m was exposed for 5 days. Microscopy Adult male and female Philonema spp. from Babine Lake and Pennask Lake were examined and drawn using a Nikon microscope equipped with drawing tube. Measurements were calculated using a d i g i t i z i n g pad (Hi-pad Texas Instruments) and software from Sonnet-Gap. 14 RESULTS Morphology of Worms Philonema sp. from 0^ mykiss Adult 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 the Pennask Lake egg c o l l e c t i o n s i t e . Worms apparently caused l i t t l e or no pathology. Worm burdens (# of worms per host) i n t r o u t were g e n e r a l l y lower than those i n sockeye (Table 1) and many t r o u t contained no adult worms ( f i s h were not examined f o r l a r v a e ) . Steelhead smolts from Lake O'Connor were i n f e c t e d w i t h mature Philonema sp. and worm burdens were often high. Worms caused severe 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 distended, 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 the anus. Dead f i s h were observed f l o a t i n g at the surface of net pens. 15 Table I HOST LOCALITY # EXAMINED # INFECTED* INTENSITY (PREVALENCE) sockeye Rivers In. 10 10 (100%) +++ sockeye Sproat Rv. 14 14 (100%) ++ sockeye Pierre Ck. 10 10 (100%) ++ kokanee Pierre Ck. 16 16 (100%) + sockeye Henderson 12 12 (100%) ++ sockeye Cultus Lk. 18 15 (83%)** ++ steelhead 0 1 Connor 13 13 (100%) +++ rainbow Fulton Rv. 17 6 (35%) + rainbow Pennask Lk. 23 11 (48%) + sockeye Fulton Rv. 11 11 (100%) ++ # infected refers to adult worms only, f i s h were not examined for larvae. Cultus lake f i s h were co l l e c t e d on two occasions, once i n October, 1987 and again i n November, 1988. On the second occasion f i s h were c o l l e c t e d from Lyndell beach and some may have spawned and released worms; thus 83% may be an underestimate of prevalence. 16 Description: Male (3 specimens) ( f i g s . 2 and 3): Length 14.64 mm (12.28 to 18.68 mm). Maximum width 286 |im (234 to 371 Lim) . Oesophagus consisting of anterior muscular portion 407 (Xm (345 to 488 |im) long, and a posterior glandular portion, 1000 (im (891 to 1175 fim) long, emptying into i n t e s t i n e . Nerve ri n g 226 i^m (185 to 278 Lim) from anterior end, near midpoint of muscular oesophagus. Single large t e s t i s beginning 1.11 mm (1.06 to 1.16 mm) from anterior end, near oesophago-intestinal junction, extending p o s t e r i o r l y to cloacal junction just anterior to anus. Slender, arcuate spicules equal and similar, 315 |im (299 to 333 |im) long. Gubernaculum absent. T a i l conical, 345 |im (310 to 402 Lim) long, with six to ten pairs of p a p i l l a e . Five to eight pairs of preanal p a p i l l a e were also observed. Female (1 specimen) ( f i g . 4): Length 59.7 mm. Maximum width 772.7 Lim. Oesophagus consisting of anterior muscular portion 474.3 (im long, and posterior glandular portion, 1067.6 Lim long, joining a flattened, empty i n t e s t i n e . Nerve ring e n c i r c l i n g muscular oesophagus near i t s middle, 241.4 Lim from anterior end of worm. Small ovary present at both ends of worm. Anterior ovary 703.5 (im from anterior end of worm and posterior ovary 550.5 Lim from t i p of t a i l . Ovaries emptying into large uterus f i l l i n g most of worms body. Uterus containing many small embryos appearing to be i n l a t t e r stages of b l a s t u l a t i o n (no f i r s t stage larvae observed). Neither anus nor vulva observed and both assumed to have atrophied. 17 Figures 2-4. Philonema sp. from CK_ mykiss. 2. P o s t e r i o r end of male. 3. A n t e r i o r end of male. 4. A n t e r i o r end of female. 18 19 Philonema sp. from 0^ nerka Adult 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 often high (Table 1) but pathological e f f e c t s were not observed with the exception of occasional t h i n walled, f l u i d f i l l e d cysts (containing dead males and/or female worms) on the l i v e r or body wall. Philonema sp. col l e c t e d from 0^ nerka tended to be larger than those from 0. mykiss but otherwise worms were morphologically indistinguishable. Description: Male (2 specimens) ( f i g s . 5 and 6): Length 29.1 mm (27.24 to 30.95 mm). Maximum width 297 um (274 to 320 Um). Oesophagus consisting of anterior muscular portion, 649 um (637 to 660 um) long, and posterior glandular portion 1824 um (1723 to 1925 um) long, emptying into i n t e s t i n e . Nerve ring 325 um (311 to 339 um) from anterior end of worm, e n c i r c l i n g musculo-oesophagus near i t s midpoint. Single large t e s t i s beginning 2.55 mm (1.06 to 4.04 mm) from anterior end and extending p o s t e r i o r l y to the cloaca l junction just anterior to anus. Slender, arcuate spicules equal and similar, 339 um long i n one of specimens. Gubernaculum absent. Conical t a i l , 505 um long i n one specimen, with six pairs of postanal p a p i l l a e . Five pairs of preanal p a p i l l a e were also observed. 20 Female (2 specimens) ( f i g . 7): Length 89.79 mm (48.96 to 130.62 mm). Maximum width 725 um (495 to 955 um). Oesophagus consisting of anterior glandular portion 1752 um (1580 to 1923 um) long, joining flattened, empty i n t e s t i n e . Nerve rin g 286 um (227 to 345 um) from anterior end, e n c i r c l i n g muscular oesophagus near i t s middle. Reproductive system consisting of small anterior ovary, 67 6 um (550 to 802 um) from anterior end, and small posterior ovary, 760 um (720 to 799 um) from posterior extremity. Ovaries emptying into large uterus f i l l i n g much of body. Uterus containing single c e l l s , early cleavage embryos and early gastrulae, but no f i r s t stage larvae were observed. Vulva and anus not observed and both were assumed to have atrophied. 21 F i g u r e s 5 - 7 . Philonema sp. from n e r k a . 5 . P o s t e r i o r end o f male. 6 . A n t e r i o r end o f male. 7 . A n t e r i o r end o f female. 22 23 R e s t r i c t i o n Endonuclease Digestion Banding patterns produced by r e s t r i c t i o n endonuclease digestion c l e a r l y separated Philonema sp. into two groups corresponding to the two host species from which they were co l l e c t e d ( f i g s . 8-13). Thus, worms from rainbow and steelhead trout (0^ mykiss) had si m i l a r banding patterns and could be distinguished from those i n sockeye (O^ nerka) and kokanee (CK nerka kennerlyi). Of 10 r e s t r i c t i o n endonucleases used, only 3 revealed bands shared by the two groups; Hae III and Hpa I produced 0.4 kb bands (fi g s . 9 and 11), and Xho I a 3.2 kb band ( f i g . 13). 24 Figure 8. Eco Rl digestion of Philonema spp.(P) and C.  elegans (N2) DNA. Philonema spp. are from the following hosts and l o c a l i t i e s ; Pl-sockeye, Rivers Inlet, P2-sockeye, Sproat River, P3-sockeye, Pierre Creek, P4-kokanee, Pierre Creek, P5-sockeye, Henderson Lake, P6-sockeye, Cultus Lake, P7-steelhead, Lake O'Connor, P8-rainbow trout, Fulton River, P9-rainbow trout, Pennask Lake, PlO-sockeye, Fulton River. 25 o a CO CL 0_ CL DC O o LU CO 0. in CL CO CL S! I I I I II s i n u » • » 8 2. si i SS no 5 26 Figure 9. Hae III digestion of Philonema spp. (P) and C. elegans (N2) DNA. Philonema spp. are from the following hosts and l o c a l i t i e s ; Pl-sockeye, Rivers Inlet, P2-sockeye, Sproat River, P3-sockeye, Pierre Creek, P4-kokanee, Pierre Creek, P5-sockeye, Henderson Lake, P6-sockeye, Cultus Lake, P7-steelhead, Lake O'Connor, P8-rainbow trout, Fulton River, P9-rainbow trout, Pennask Lake, PlO-sockeye, Fulton River. 27 o a. 00 o. Q. I I CO 0. o. 0_ 0_ CM Q. 0_ C\J f S3 S I III I II S S 28 gure 10. Hinf I digestion of Philonema spp.(P) and C. elegans (N2) DNA. Philonema spp. are from the following hosts and l o c a l i t i e s ; Pl-sockeye, Rivers Inlet, P2-sockeye, Sproat River, P3-sockeye, Pierre Creek, P4-kokanee, Pierre Creek, P5-sockeye, Henderson Lake, P6-sockeye, Cultus Lake, P7-steelhead, Lake O'Connor, P8-rainbow trout, Fulton River, P9-rainbow trout, Pennask Lake, PlO-sockeye, Fulton River. 29 I I II I II I II I II I I I III II fl 1 1 5 ^ • » OB * 8 » * i 5 S n o 30 Figure 11. Hpa I digestion of Philonema spp. (P) and C.  elegans (N2) DNA. Philonema spp. are from the following hosts and l o c a l i t i e s ; Pl-sockeye, . Rivers Inlet, P2-sockeye, Sproat River, P3-sockeye, Pierre Creek, P4-kokanee, Pierre Creek, P5-sockeye, Henderson Lake, P6-sockeye, Cultus Lake, P7-steelhead, Lake O'Connor, P8-rainbow trout, Fulton River, P9-rainbow trout, Pennask Lake, PlO-sockeye, Fulton River. 31 I I S I I I III III III I 11 I II SS 32 Figure 12. Xba I digestion of Philonema spp.(P) and C.  elegans (N2) DNA. Philonema spp. are from the following hosts and l o c a l i t i e s ; Pl-sockeye, Rivers Inlet, P2-sockeye, Sproat River, P3-sockeye, Pierre Creek, P4-kokanee, Pierre Creek, P5-sockeye, Henderson Lake, P6-sockeye, Cultus Lake, P7-steelhead, Lake O'Connor, P8-rainbow trout, Fulton River, P9-rainbow trout, Pennask Lake, PlO-sockeye, Fulton River. 33 o a. l l l l a. CO a. a. I I I I I I I I l l l l _Q X CO a. in a. a. CO a. CM a. CM z l l l l II £ I s s R ai.ss.3i. s s n o s cc p 34 Figure 13. Xho I digestion of Philonema spp.(P) and C.  elegans (N2) DNA. Philonema spp. are from the following hosts and l o c a l i t i e s ; Pl-sockeye, Rivers Inlet, P2-sockeye, Sproat River, P3-sockeye, Pierre Creek, P4-kokanee, Pierre Creek, P5-sockeye, Henderson Lake, P6-sockeye, Cultus Lake, P7-steelhead, Lake O'Connor, P8-rainbow trout, Fulton River, P9-rainbow trout, Pennask Lake, PlO-sockeye, Fulton River. 35 36 Banding patterns within groups showed l i t t l e v a r i a t i o n for most enzymes (f i g s . 14-17), but Philonema populations from O^ mykiss d i f f e r e d with respect to patterns produced by Bam HI, Hae III and Hinf I. These differences corresponded to d i f f e r e n t c o l l e c t i o n l o c a l i t i e s . Bam HI digestion produced a 2.1 kb band that was present i n Fulton River 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 digestion were present i n Fulton and Pennask populations and absent i n Lake O'Connor worms, while a 2.6 kb present i n Lake O'Connor worms was absent from Fulton and Pennask worms ( f i g . 16). Banding patterns produced by Hae III digestion distinguished Philonema from the 3 O^ mykiss populations ( f i g . 17). Lake O'Connor worms had 2 bands, 3.5 kb and 1.1 kb, not present i n either the Fulton or Pennask populations. Fulton River worms had a 2.4 kb band not present i n the other two populations, and lacked 2.8 kb band which was present i n the other two. Banding patterns produced by the enzymes Eco Rl, Hpa I ( f i g . 14) and Xho I were i d e n t i c a l for a l l 0^ mykiss c o l l e c t i o n l o c a l i t i e s . 37 Figure 14. Hpa I DNA d i g e s t of Philonema taken from mykiss. 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 , P9-rainbow t r o u t , Pennask Lake. 3 8 CC Q_ 0. o CL CL CL CO CL CO 0_ c o 0. CO CL rv. CL CL CL CL I II I 5 f I I J3 £,» f I P> o pj pj S 39 Figure 15. Bam HI DNA digest of Philonema taken from 0. mykiss. Philonema sp. are from the following hosts and l o c a l i t i e s ; P7-steelhead, Lake O'Connor, P8-rainbow trout, Fulton River, P9-rainbow trout, Pennask Lake. The 2.1 kb band i s shared by the Fulton and Pennask populations but i s not present i n the Lake O'Connor population. 40 41 Figure 16. Hinf I DNA digest of Philonema taken from 0. mykiss. Philonema sp. are from the following hosts and l o c a l i t i e s ; P7-steelhead, Lake O'Connor, P8-rainbow trout, Fulton River, P9-rainbow trout, Pennask Lake. The 3.1 and 2.8 kb bands are shared by the Fulton and Pennask populations but are not present i n the Lake O'Connor population. The Lake O'Connor population has a 2.6 kb band not present i n the other two populations. 42 CL o. co CO CL co CL oo CL CL CL CL CL rr j II I I I I f Zi * • * K 2. S2. i 'I 5 S n o csi est 43 Figure 17. Hae III DNA digest of Philonema taken from 0. mykiss. Philonema sp. are from the following hosts and l o c a l i t i e s ; P7-steelhead, Lake O'Connor, P8-rainbow trout, Fulton River, P9-rainbow trout, Pennask Lake. The Fulton and Pennask populations share the 3.6 kb band which Lake O'Connor worms lack. The Lake O'Connor population has two bands, 3.5 and 1.1 kb, not seen i n the other two populations. The 2.8 kb band i s shared by the Pennask and Lake O'Connor populations and the Fulton River population has a 2.4 kb band not seen i n the other populations. 44 CD 03 cn o. 0-co CL co 00 £L 00 CL CL CL CL Cw 3 3 I II I • II 3 3 OS tfi V JC JC C5 © pJ CNl 45 Philonema populations from 0^ nerka showed banding pattern differences for the enzymes Bam HI, Hinf I and Hpa I, but these differences were not r e s t r i c t e d to any population ( f i g s . 18-20). Bam HI digestion produced a 2.6 kb band that was present i n Cultus Lake worms, most Sproat Lake worms, one worm from Henderson Lake, one worm from Pierre Creek, and i n much f a i n t e r form i n worms from Fulton River ( f i g . 18). Two bands, 1.0 and 0.9 kb, produced by Hinf I digestion were polymorphic. In Cultus Lake worms, most Sproat River worms, one worm from Henderson Lake and one worm from Pierre Creek the 0.9 kb band was more prominent. In the remainder of the samples from Henderson Lake, Pierre Creek, Sproat River and samples from Rivers Inlet, the 1.0 kb band i s of greater i n t e n s i t y . However, i n samples from Fulton River these bands were nearly equal i n in t e n s i t y . F i n a l l y , a 1.9 kb band produced by Hpa I digestion was present i n Cultus Lake worms, most Sproat River worms and one worm from Pierre Creek ( f i g . 20) (the Henderson Lake and Fulton River populations were not examined). Banding patterns produced by the enzymes Eco Rl, Hae III, Xho I and Xba were i d e n t i c a l for a l l O^ nerka c o l l e c t i o n l o c a l i t i e s . 46 Figure 18. Bam HI DNA digest of Philonema from 0. nerka. Philonema sp. are from the following hosts and l o c a l i t i e s ; Pl-sockeye, Rivers Inlet, P2-sockeye, Sproat River, P3-sockeye, Pierre Creek, P4-kokanee, Pierre Creek, P5-sockeye, Henderson Lake, P6-sockeye, Cultus Lake, PlO-sockeye, Fulton River. The 2.6 kb band corresponds to pBx 2, a cl u s t e r of ribosomal genes i s o l a t e d from B.  xylophilus. 48 Figure 19. Hinf I DNA digest of Philonema from 0. nerka. Philonema sp. are from the following hosts and l o c a l i t i e s ; Pl-sockeye, Rivers Inlet, P2-sockeye, Sproat River, P3-sockeye, Pierre Creek, P4-kokanee, Pierre Creek, P5-sockeye, Henderson Lake, P6-sockeye, Cultus Lake, PlO-sockeye, Fulton River. 4 9 50 Figure 20. Hpa I DNA d i g e s t of Philonema from 0. nerka. 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 ; P2-sockeye, Sproat R i v e r , P4-kokanee, P i e r r e Creek, P5-sockeye, Henderson Lake, P6-sockeye, Cultus Lake. 51 CO to CL a CO a. in o. CL in o. in a 2 2 CM Q. 2! I II I ^ I I I cv 2i SE- 2. II II n © s CO p. 52 Hybridization with pBx 2 Probe Southern blots taken from gels were probed with pBx 2 from Bursaphelenchus xylophilus (Nematoda; Tylenchida). Bands r e s u l t i n g from hybridization with the pBx 2 probe are summarized i n f i g . 21. Banding patterns produced by the enzymes Hae III, Hpa I, Hinf I and Xho I were i d e n t i c a l for Philonema from 0^ mykiss and 0^ nerka. Southern blots from r e s t r i c t i o n digests with other enzymes were not available since heavy background contamination made bands impossible to discern. The 3.2 kb pBx 2 fragment produced by Xho I digestion may correspond to a band of sim i l a r size seen i n f i g . 13, Xho I digestion of extracted Philonema DNA. Hae III digestion produced 4 pBx 2 fragments; the 1.4 kb fragment i s sim i l a r i n size to a band seen i n Hae III digestion of DNA from worms i n mykiss ( f i g s . 9 and 17) but i t has no corresponding band i n worms from (X^ nerka. Si m i l a r l y , the 2.1 kb pBx 2 fragment produced by Hpa I digestion i s similar i n size to a band seen i n Hpa I digestion of worms from 0^ mykiss ( f i g s . 11 and 14) but a sim i l a r band i s absent i n worms from 0. nerka. 53 Figure 21. Summary of 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 the pBx 2 probe, a c l u s t e r of ribosomal genes i s o l a t e d from x y l o p h i l u s . T -Philonema sp. from CJ^ mykiss, S - Philonema sp. from 0. nerka. 0 - N/A 55 Cladogram The most parsimonious arrangement of shared bands described from f i g s . 8-20 i s indicated by a cladogram ( f i g . 22). The cladogram indicates the two groups of Philonema corresponding to the host species from which they were removed ( i . e . . PI, P2, P3, P4, P5, P6 and P10 are from 0.  nerka and P7, P8 and P9 are from 0^ mykiss). Worms from 0.  nerka resulted i n an unresolved polychotomy for a l l populations. Worms from 0^ mykiss indicated Fulton River (P8) and Pennask (P9) populations were more clos e l y related to one another than either was to Lake O'Connor (P7) worms. 56 Figure 22. Cladogram representing hypothetical relationships of 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 following hosts and l o c a l i t i e s ; P l -sockeye, Rivers Inlet, P2-sockeye, Sproat River, P3-sockeye, Pierre Creek, P4-kokanee, Pierre Creek, P5-sockeye, Henderson Lake, P6-sockeye, Cultus Lake, P7-steelhead, Lake O'Connor, PS-rainbow, Fulton River, P9-rainbow, Pennask Lake, PlO-sockeye, Fulton River. 57 O. mykiss O. nerka H*mc(Ukt) 58 DISCUSSION The genus Philonema contains nine nominal species i n anadromous and non-anadromous salmonids d i s t r i b u t e d throughout the Northern hemisphere. Three species of Philonema have been described from North America, including the type P_^  oncorhynchi Kuitunen-Ekbaum 1933. Philonema  oncorhynchi was o r i g i n a l l y described from prespawning Oncorhynchus nerka captured i n English Bay, Vancouver, B r i t i s h Columbia (Kuitunen-Ekbaum, 1933). The species has subsequently been recorded i n 0^ nerka from various l o c a l i t i e s along coastal B r i t i s h Columbia, Vancouver Island and Alaska (Bangham and Adams, 1954, Margolis, 1963, Pennel et. a l . , 1973, Bailey and Margolis, 1987), Akhmerov (1955) reported P^ oncorhynchi i n 0^ nerka from Kamchatka. Philonema oncorhynchi has been found i n the lower Fraser River drainage (Cultus Lake) but i s apparently absent from the upper Fraser (Bailey and Margolis, 1987). Margolis (pers. comm.) indicated that of 75 adult sockeye examined from the lower Columbia only three infected f i s h were found and a t o t a l of 4 P_^  oncorhynchi worms were recovered; no worms were found i n 48 smolts examined from lakes i n the Columbia drainage. It seems l i k e l y that Philonema  oncorhynchi i s not present i n the Columbia River and reported infections may represent wandering f i s h since 59 c o l l e c t i o n data from t h i s study suggests prevalence of P.  oncorhynchi approaches 100% i n endemic areas. Philonema agubernaculum Simon and Simon, 1936 was the second species of Philonema to be described from Prosopium  williamsoni, Salvelinus f o n t i n a l i s and Salmo shasta (=Oncorhynchus mykiss) from Green River Lakes, M i l l creek, and Freemont Lake i n the Wyoming National Forest. It has since been reported 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 , S.  namaycush, S• alpinus, Coregonus clupeaformis and Prosopium  cylindracium from coastal Labrador (Hicks and T h r e l f a l l , 1973), from S^ f o n t i n a l i s and landlocked Salmo salar from Maine (Meyer, 1954), and from S^ alpinus from the Koukdiuak River, B a f f i n Island (Dick and Belosevic, 1981). A t h i r d North American species, P^ s a l v e l i n i Richardson, 1937, was described from S^ f o n t i n a l i s i n Lake Edward, Quebec. The remaining species of Philonema were described from Asia. Philonema s i b i r i c a (Bauer, 1946) Rumyantsev, 1965, o r i g i n a l l y described from Coregonus albula, was redescribed i n the same host from Lake Kuita, U.S.S.R. Rumyantsev (1965) suggested that P^ s i b i r i c a might be a synonym of P.  agubernaculum. Two species of Philonema were described from Japan: 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^ elongata F u j i t a , 1940 i n Oncorhynchus kawamurae from Lake Tazawa. F i n a l l y , three species were described from Kamchatka, U.S.S.R.; P. kondai F u j i t a , 1939 i n Oncorhynchus keta, P. tenuicauda F u j i t a , 60 1939 i n CJ^  nerka and s a l v e l i n i F u j i t a , 1939 i n Salvelinus  leucomaenis. F u j i t a was not aware that s a l v e l i n i had been preoccupied by Richardson's (1937) species. The species can be divided into two groups based on host biology: P_j_ oncorhynchi, P. kondai, P^ tenuicauda and P. elongata occur i n anadromous salmonids of the genus Oncorhynchus. These salmonids t y p i c a l l y undergo long ocean migrations 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 non-anadromous salmonids belonging to diverse genera (Coregonus, Oncorhynchus, Prosopium, Salmo and Salvelinus) which usually breed several times during t h e i r l i f e . Of the anadromous Oncorhynchus, 0. nerka i s most commonly infected with Philonema. The few reports of Philonema i n other anadromous Oncorhynchus may well represent cross-infections from sockeye. The reason for t h i s r e s t r i c t i o n presumably has to do with the fact that sockeye remain for at least a year i n nursery lakes. This i s presumably the s i t e of transmission for Philonema. The copepod Cyclops bicuspidatus i s an abundant plankton i n many B.C. lakes (Foerster, 1968) and i s an important food source for planktivorous salmonids. Platzer and Adams (1966) demonstrated that C_^  bicuspidatus i s a suitable intermediate host for Philonema. This may explain why salmonids that l i v e i n streams and ri v e r s , and do not have access to the abundant lake plankton, are rarely infected with Philonema. 61 Philonema species are morphologically very si m i l a r and t h i s has led to taxonomic problems i n the genus. Baylis (1948) suggested P_j_ agubernaculum and P_;_ oncorhynchi were i d e n t i c a l and the smaller size of the former was attributable to host differences or degree of maturity of worms. Akhmerov (1955) considered P^ agubernaculum, P.  elongata, and P^ s i b i r i c a synonyms of P^ oncorhynchi, and a recent Russian key to the Nematoda (Vismanius e t . a l . , 1987) synonymizes a l l of Fu j i t a ' s species with P^ oncorhynchi, recognizing only one other species, P^ s i b i r i c a , i n waters of the U.S.S.R. In t h i s study, Philonema spp. from 0^ mykiss and 0.  nerka were morphologically indistinguishable. Characters such as number of caudal p a p i l l a e i n males have been used i n the diagnosis of new Philonema species (Fujita, 1939). However, v a r i a b i l i t y i n both preanal and postanal caudal p a p i l l a e was noted i n the population of Philonema from Pennask Lake. This character probably has l i t t l e value for species i d e n t i f i c a t i o n . Simon and Simon (1936) suggested that P^ agubernaculum could be d i f f e r e n t i a t e d from P^ oncorhynchi by i t s smaller size and by the r a t i o of anterior muscular oesophagus to posterior glandular oesophagus, given as 1:2.7 for P.  agubernaculum and 1:1.1 for P_^_ oncorhynchi. No difference between Philonema from 0^ mykiss or CK_ nerka was noted for oesophageal r a t i o s i n worms examined and values ranged from 62 1:2.3 to 1:3.1. Although Philonema from mykiss tended to be smaller than those from nerka, morphometric analysis by Platzer (1964) showed a high degree of overlap i n length measurements for Philonema spp. from 0^ nerka, 0. keta, 0.  mykiss, landlocked salar, Salvelinus malma, S. f o n t i n a l i s and P_j_ williamsoni, making size a questionable character for d istinguishing Philonema species. Platzer (1964) demonstrated that size of adult Philonema varied depending on host species as well as host sex, and further i l l u s t r a t e d overlap i n v a r i a t i o n of such characters as distance from anterion end to nerve ring, muscular and glandular oesophagus, number of caudal pap i l l a e , and t a i l and spicule length, and concluded that P_j_ agubernaculum was morphologically i d e n t i c a l with P_^  oncorhynchi. Bashirullah (1966) decided the two worms were d i s t i n c t species based on differences i n l i f e history. He suggested P. oncorhynchi i s a parasite of anadromous salmonids and undergoes slower development, taking 32-35 months to reach maturity. Sockeye do not return to fresh water for two or more years and Bashirullah (1966) showed that elevated host hormones p r i o r to spawning stimulated female worms to undergo t h e i r f i n a l maturation. Bashirullah (1966) suggested that P^_ agubernaculum, a parasite of non-anadromous salmonids, develops more quickly reaching maturity i n 6-12 months. He suggested that these worms matured independently of host hormones but had no 63 experimental evidence to support t h i s . Developmental d i f f e r e n c e s are not host determined because c r o s s - i n f e c t i o n experiments ( B a s h i r u l l a h , 1966) show tha t developmental r a t e was unaf f e c t e d when worms were i n d i f f e r e n t hosts. B a s h i r u l l a h (1966) f u r t h e r supported h i s t h e s i s t h a t the species 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 agubernaculum (0. mykiss from Kootenay Lake) and oncorhynchi (0. nerka from Cultus Lake) were g e o g r a p h i c a l l y separated, r a i s i n g the p o s s i b i l i t y t hat d i f f e r e n c e s r e s u l t e d from population v a r i a t i o n i n a s i n g l e , widely d i s t r i b u t e d species. This study examined r e s t r i c t i o n fragment banding patterns i n r e p e t i t i v e sequences of DNA and supports B a s h i r u l l a h 1 s i n t e r p r e t a t i o n t h a t P^ agubernaculum and P.  oncorhynchi represent d i s t i n c t species. Results 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, corresponding t o the two host species from which they were c o l l e c t e d . Banding patterns were d i f f e r e n t f o r most enzymes used. Only 3 enzymes (Hae I I I , Hpa I and Xho I) revealed shared bands. Results from h y b r i d i z a t i o n w i t h the c l u s t e r of ribosomal 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 speci e s . This may be because the pBx 2 probe represents a c l u s t e r of ribosomal genes which may be h i g h l y conserved 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 r e l a t e d species. 64 Even though oncorhynchi and agubernaculum were co l l e c t e d from s i t e s where t h e i r respective hosts were sympatric (e.g. Babine Lake), the two groups of worms s t i l l maintained t h e i r genetic identity, implying no hybridization occurs even when the opportunity presumably e x i s t s . The two Philonema groups probably represent good b i o l o g i c a l species and are hereafter referred to as P^ agubernaculum (infecting 0. mykiss) and P_^  oncorhynchi (infecting 0^ nerka) . Curran et a l . (1985) demonstrated the f e a s i b i l i t y of using r e s t r i c t i o n fragment length differences of r e p e t i t i v e DNA sequences (such as ribosomal and histone genes) as a t o o l for rapid i d e n t i f i c a t i o n of nematode species. Many nematode groups include species that are d i f f i c u l t to d i s t i n g u i s h morphologically making i d e n t i f i c a t i o n d i f f i c u l t . Using Eco Rl, Curran et. a l . (1985) demonstrated r e s t r i c t i o n fragment length differences for r e p e t i t i v e genes among selected species and populations of nematodes belonging to the genera Caenorhabditis, Heterorhabditis, Meloidogyne, Romanonmermis, Steinernema and T r i c h i n e l l a . R e s t r i c t i o n endonucleases recognize and cleave s p e c i f i c sequences of nucleotides i n double-stranded DNA. The size and number of r e s u l t i n g fragments vary depending on the number and location of cut s i t e s . 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 gels because they are represented i n high numbers. Bands of equal size are assumed to be homologous, and similar banding patterns produced by a given enzyme r e f l e c t relatedness. It i s 65 u n l i k e l y that the same enzyme w i l l produce r e p e t i t i v e bands of the same size i n unrelated stretches of DNA. These assumptions are supported by empirical data. Curran et a l . (1985) noted that the number of shared bands decreased as more d i s t a n t l y related groups were compared. For example, transgeneric comparisons showed no shared bands while comparisons of closely related species revealed only a small percentage of shared bands; within species, most or a l l bands were shared. This implies that shared bands indicate recent shared ancestry, and supports use of t h i s technique i n examining population and species relationships i n morphologically indistinguishable groups. R e s t r i c t i o n fragment length differences are of l i t t l e use i n investigating relationships among d i s t a n t l y related taxa because few, i f any, shared bands occur. Worms co 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 n sockeye from the same region and are i d e n t i f i e d as oncorhynchi. This i s not surprising since kokanee represent a s e l f sustaining offshoot, derived from an anadromous sockeye population i n the lake (Scott and Crossman, 1973, Foote e t . a l . , 1989), that forgo ocean migration and remain i n the nursery lake. Kokanee, l i k e sockeye, spawn once i n t h e i r l i f e t i m e , usually i n t h e i r fourth year. Philonema oncorhynchi i s probably ca r r i e d into the kokanee population when the l a t t e r arises from the sockeye population which are or were i n the same nursery lake. Material from steelhead 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 trout/ but smolts are not usually exposed to Philonema because the freshwater stage of t h e i r l i f e cycle usually involves stream or r i v e r residence (Withler, 1966). These smolts were probably carrying worms that c i r c u l a t e i n resident salmonids. Philonema spp. are normally non-pathogenic, but severe pathological effects were noted i n steelhead smolts. Smolts l i k e l y became infected when they were placed i n the lake which presumably contains an endemic salmonid population carrying P_^  agubernaculum and pathology probably resulted because these hosts are immunologically naive. Sakanari and Moser (1990) studied pathological changes induced by pleurocercoids of the cestode Lacistorhynchus d o l l f u s i i n east and west coast striped bass (Morone s a x a t i l i s ) . They showed that these parasites induced more intense pathological changes i n east coast st r i p e d bass. They suggested that because east coast st r i p e d bass do not contact d o l l f u s i they are immunologically naive and t h i s r e s u l t s i n a stronger pathological response. On the other hand, Sakanari and Moser (1990) suggested that west coast s t r i p e d bass, introduced from the east coast, have adapted to d o l l f u s i because they have been associated with the parasite for more than 20 generations. Dracunculoids are tissue dwelling parasites of vertebrates and t h e i r cycles t y p i c a l l y involve use of copepods as intermediate hosts. This l i f e s t y l e presents a 67 problem because f i r s t stage larvae must reach the external environment to be ingested by the intermediate host. Many dracunculoids undergo a migration to subcutaneous tissues where they breach the host tegument to release t h e i r larvae. However, Philonema spp. make use of the host's reproductive system and pass to the external environment with the hosts roe during spawning (Platzer, 1964). Philonema spp. use copepods of the genus Cyclops as intermediate hosts (Vik, 1964, Bashirullah, 1966, Platzer and Adams, 1966, Ko and Adams, 1969) and larvae undergo development to the i n f e c t i v e t h i r d stage within these hosts. Since P_;_ agubernaculum and P. oncorhynchi are known to occur i n sympatry (e.g.. Babine Lake) and both species use the same intermediate host, natural cross-infections should occur, and t h i s could lead to hybridization and possible breakdown of species b a r r i e r s . Cross-infection experiments by Bashirullah (1966) demonstrate that P^ agubernaculum and P^ oncorhynchi are capable of i n f e c t i n g a variety of salmonids. A sub-adult P.  agubernaculum was recovered from an experimentally infected sockeye a f t e r 161 days, and a rainbow trout experimentally infected with P^ oncorhynchi survived 34 6 days afte r which time a fourth stage worm was recovered from the swimbladder. There i s also evidence for natural cross-infections. Migrant sockeye from Shushwap lake and Seton Lake were found to be infected with immature Philonema spp. (Bashirullah, 1966, Margolis, pers. comm.). Since P_^  oncorhynchi i s absent from the upper Fraser and Columbia Rivers these 68 infections l i k e l y represent cr o s s - i n f e c t i o n with P.  agubernaculum. While cross-infection apparently occurs, no mature P. agubernaculum was recovered from the 35 sockeye and kokanee examined in the present study, yet a l l were presumably sympatric with salmonids infected by P.  agubernaculum. Similarly, no P^ oncorhynchi were recovered from the 5 Fulton River rainbow trout known to be sympatric with sockeye harboring Philonema. Bashirullah (1966) argued that hybridization i s prevented because agubernaculum has a yearly l i f e cycle and can not survive the extended migration of sockeye. Furthermore, he believed that unlike P_;_ oncorhynchi, P.  agubernaculum develops d i r e c t l y to adulthood independent of cues from host hormones. He showed that P^ agubernaculum develops more rapidly than P^ oncorhynchi regardless of host but never recovered gravid females of P^ agubernaculum. Even afte r six months, worms were subgravid. This i s consistent with the hypothesis that P^ agubernaculum requires host hormonal cues before undergoing i t s f i n a l maturation. Circumstantial evidence suggests P.  agubernaculum i s t i e d to host hormonal changes since Platzer (1964) only recovered mature worms from reproductively mature hosts i n the wild. It seems more l i k e l y that jP^ agubernaculum and P.  oncorhynchi have similar l i f e h i s t o r i e s ; both can reproduce only when the host reproduces. Development i n the f i n a l host i s probably diphasic; worms develop to adulthood 69 d i r e c t l y but require host hormonal cues to undergo f i n a l maturation. The fact that P_^_ oncorhynchi develops more slowly than agubernaculum merely r e f l e c t s i t s adaptation to a host which breeds only once usually at four years. If i t were not true, then when agubernaculum entered hosts which may not mature for several years or hosts such as charr (genus Salvelinus) which breed every other year, worms would be at a reproductive dead-end. Furthermore, kokanee spend t h e i r entire l i v e s feeding on plankton and benthic invertebrates (Scott and Crossman, 1973) and should be exposed throughout t h e i r l i f e to Philonema spp. Therefore, at least those P_^  agubernaculum picked up a year p r i o r to spawning should survive to host spawning. Bashirullah (1966) examined 200 kokanee examined from Kootenay Lake and only one contained an adult Philonema; P. oncorhynchi i s absent i n t h i s area and the i n f e c t i o n probably represents P.  agubernaculum. Rainbow trout generally take 3-5 years to mature and may l i v e as long as 8 years (Scott and Crossman, 1973); therefore, P^ oncorhynchi acquired early should have ample time to grow and reach maturity. No cross-infections were observed, but sample size was small because only the 5 worms examined from Fulton River were known to be sympatric with P. oncorhynchi. It seems l i k e l y that successful cross-infections by Philonema spp. are prevented by a host immune response which encapsulates and destroys worms before they can mature. 70 Bashirullah (1966) observed cysts containing Philonema i n the body cavity of sockeye from Shushwap Lake. Philonema  agubernaculum and P^ oncorhynchi have undergone molecular divergence, as evidenced by di f f e r e n t banding patterns, and t h i s divergence may be associated with antigenic changes which res u l t i n rejection when Philonema spp. enters an inappropriate host. This host immune response may prevent hybridization between the two species of Philonema by destroying one species p r i o r to maturation and mating. However, i t i s also possible that cross-infections and hybridization are prevented because worms are unable to mature i n unsuitable hosts due to physiological differences in the host species. Fish r e s t r i c t e d to is o l a t e d freshwater drainage systems often undergo genetic divergence from neighboring populations i n other watersheds because land masses and s a l t water act as ba r r i e r s to disp e r s a l . Philonema agubernaculum samples showed banding patterns s p e c i f i c to 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 there i s no gene flow between populations since t h e i r hosts are probably r e s t r i c t e d to t h e i r respective drainage basins. Pennask Lake and Fulton River populations were nearly i d e n t i c a l (distinguished by only two Hae III bands), while Lake O'Connor worms appear to have undergone greater divergence from the two mainland populations since d i f f e r e n t banding patterns were observed for three enzymes (Bam HI, Hae III and Hinf I ) . Curran et a l . (1985) demonstrated 71 banding pattern differences between species but f a i l e d to show differences between populations of Caenorhabditis  elegans. This led them to speculate that r e s t r i c t i o n fragment length differences may represent markers for populations which can no longer fre e l y interbreed and they may be associated with speciation, when genetic i s o l a t i o n allows rapid 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 pattern differences seem to r e f l e c t genetic i s o l a t i o n , there i s no reason to conclude that each population represents a d i f f e r e n t species. It seems more l i k e l y that, l i k e morphological differences, differences i n banding patterns are l i k e l y to occur among p o t e n t i a l l y interbreeding populations i n widely d i s t r i b u t e d species. Minchella et. a l . (1989) studied r e s t r i c t i o n fragment length differences i n urban and s y l v a t i c populations of T r i c h i n e l l a s p i r a l i s (Nematoda; T r i c h i n e l l i d a e ) . A l l 12 r e s t r i c t i o n enzymes used successfully separated s y l v a t i c (wild) T r i c h i n e l l a from urban (swine) T r i c h i n e l l a . Most enzymes gave similar banding patterns for s y l v a t i c populations. However, the enzyme Cla I distinguished a l l s y l v a t i c i s o l a t e s , suggesting that s y l v a t i c 1\_ s p i r a l i s i s organized into more or less d i s t i n c t populations. Most of B.C. was glaciated as recently as 14,000 years ago. Melting and subsequent retreat of g l a c i e r s resulted i n formation of large lakes and streams (Briggs, 1986), and f a c i l i t a t e d reinvasion of freshwater f i s h from areas of refugia i n the north and south. The Fraser River system was 72 p r i n c i p a l l y recolonized from the Columbia River v i a g l a c i a l lakes i n the Okanagan v a l l e y . The Skeena system has a f i s h fauna si m i l a r to the Columbia River, i n d i c a t i n g a previous connection v i a the Fraser, probably while the Skeena r i v e r was blocked and i t s t r i b u t a r i e s flowed east into the Fraser (McPhail and Lindsey, 1986). Thus, the close relationship between Pennask Lake and Fulton River P^ agubernaculum may represent t h e i r common o r i g i n v i a a Columbia River dispersal event. On the other hand P^ agubernaculum from Lake O'Connor may have diverged much e a r l i e r , following an early p o s t g l a c i a l migration 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 population which survived i n a central coast refugia during the most recent Pleistocene g l a c i a t i o n . There i s increasing evidence, mainly botanical, that an area i n the Queen Charlottes and northern Vancouver Island was unglaciated during the most recent Pleistocene g l a c i a t i o n (McPhail and Lindsey, 1986, Ogilvie, 1989). I f P^ agubernaculum from Lake O'Connor represents a population which survived i n t h i s refugia, then i t should prove to be more sim i l a r to worms from the Queen Charlottes. Philonema agubernaculum from southern Vancouver Island should be more similar to the southern mainland since i t was probably colonized from t h i s area following g l a c i a l retreat. Results from Bam HI, Hinf I and Hpa I digestion appear to indicate two genetic types of P^ oncorhynchi, however, these two types occurred within the same population (e.g. 73 Sproat River, Henderson Lake, Fulton River and Pierre Creek) and are best interpreted as polymorphisms. Sockeye salmon are anadromous and undergo an ocean migration which takes them o f f shore before they return to spawn i n fresh water. Like other salmonids, sockeye show a strong homing tendency for t h e i r nursery lake (and natal stream). If homing were perfect, i t would r e s t r i c t gene flow i n P^ oncorhynchi populations and possibly lead to geographic v a r i a t i o n because worms are transmitted i n nursery lakes. A study by Quinn et a l . ( 1987) using protein electrophoresis and parasite prevalence data suggested sockeye may stray less than 1% of the time. However, t h i s small amount of straying may be s u f f i c i e n t to prevent population divergence i n t h e i r parasites or may res u l t i n mixing i f changes do occur. Sockeye introductions may also have contributed to mixing of populations of P^ oncorhynchi. Sockeye have been transplanted into many B.C. watersheds i n recent times. If infected smolts were transplanted they could seed uninfected lakes with P^ oncorhynchi or mix with an e x i s t i n g population. Supporting evidence for t h i s hypothesis i s d i f f i c u l t to f i n d since many introductions are unrecorded and when they are, method of transplant i s often not stated. However, most f i s h are transplanted as eggs (Foerster, 1968) which would not carry 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. and should f a c i l i t a t e further studies on host and geographic 74 d i s t r i b u t i o n s of the group. Philonema agubernaculum has been reported from a wide v a r i e t y of salmonid hosts but our specimens were c o l l e c t e d from 0^ mykiss; i t remains t o be seen whether a s i n g l e species of Philonema i n f e c t s a l l non-anadromous 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 the geographic d i s t r i b u t i o n of P_j_ oncorhynchi 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 species because i t appears t o l a c k geographic 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 the geographic d i s t r i b u t i o n of P_^  agubernaculum. This species has been reported from v a r i o u s l o c a l i t i e s across North 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 divergence and those from more d i s t a n t geographic areas may show degrees of divergence t h a t argue against 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. oncorhynchi i s probably r e s t r i c t e d t o 0^ nerka. Rarely, i t has been reported from 0^ keta (Akhemerov 1955, P l a t z e r , 1964) but i t i s probably not a common p a r a s i t e of chum and other P a c i f i c salmon f o r e c o l o g i c a l reasons. Sockeye are unique i n that smolts spend 1-3 years feeding i n a nursery lake which i s the focus of the Philonema i n f e c t i o n . I f 0^ nerka i s the only n a t u r a l host f o r P.  oncorhynchi then worms should only be found w i t h i n t h i s hosts range. Reports of oncorhynchi outside the range of sockeye are l i k e l y m i s i d e n t i f i c a t i o n s . 75 P a c i f i c salmon (genus Oncorhynchus) represent a recent lineage, 2-3 m i l l i o n years old (Thomas et a l . , 1986), and sockeye diverged from t h i s lineage about 2 m i l l i o n years ago. Philonema oncorhynchi may have coevolved with sockeye from an ancestor found i n more primitive non-anadromous salmonids, i n response to a change i n the host's l i f e h i story. Philonema oncorhynchi probably arose sometime after t h i s event since the worm i s not present throughout the whole host range; i t i s absent from the Upper Fraser and Columbia Rivers. Philonema oncorhynchi may have arisen on the A s i a t i c or Alaskan coast during Pleistocene g l a c i a t i o n and colonized the B.C. coast when sockeye populations reinvaded from areas of refugia i n Beringia (McPhail and Lindsey, 1986). 76 REFERENCES Akhmerov, A.kh. 1955. Parasite fauna of the fishes of the r i v e r Kamchatka. Izvest. Tilchookeansk. N. I. Inst. Rybn. Khoz. Okeanogr., 43: 99-137. Bailey, R.E. and L. Margolis. 1987. Comparison of parasite fauna of juvenile sockeye salmon (Oncorhynchus nerka) from southern B r i t i s h Columbia and Washington State Lakes. Can. J. Zool., 65: 420-431. 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