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Saprolegnia diclina Humphrey as a parasite of the solmonid, Oncorhynchus kisutch. McKay, Diana Louise 1967

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SAPROLEGNIA DICLINA HUMPHREY AS A PARASITE OF THE SALMONID, ONCORHYNCHUS KISUTCH by DIANA LOUISE McKAY A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e Department o f BOTANY We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA November 1967 In presenting th is thesis in pa r t ia l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i sh Columbia, I agree that the Library sha l l make i t f ree ly ava i lab le for reference and Study. I further agree that permission for extensive copying of this thesis for scholar ly purposes may be granted by the Head of my Department or by h.i)s representat ives. It is understood that copying or publ icat ion of th is thesis for f inanc ia l gain shal l not be allowed without my wri t ten permission. The Univers i ty of B r i t i sh Corumbia Vancouver 8, Canada Department i i ABSTRACT Studies of Saprolegnia infections of f i s h i n B r i t i s h Columbia were made to determine disease causing agents and i n f e c t i o n conditions. Saprolegnia diclina. Humphrey was the most frequently observed para-s i t e . This fungus reproduced sexually both on f i s h tissue and hemp seed cultures. No d e f i n i t e i s o l a t i o n s of 3. parasitica Coker viere made although some non-sexually reproducing isolates of a Saprolegnia sp. were found. The v a l i d i t y of the species, S. p a r a s i t i c a , has been examined and questioned on the basis of present i d e n t i f i c a t i o n characteristics. Infection studies using S. d i c l i n a as the parasite and f i n g e r l i n g coho (Oncorhynchus kisutch) as.the host indicated a d i s t i n c t correlation between temperature and in f e c t i o n . At normal cool temperatures, e.g., o o 8 C, no in f e c t i o n occurred; at 9 C or above, some inf e c t i o n resulted. o Above 9 C, the rate of in f e c t i o n increased as temperature increased. Temperature was also associated with the time at which i n f e c t i o n occurred o o after inoculation. At 18 C, in f e c t i o n began e a r l i e r than at 13 C, Heat-shock treatment tended to reduce the temperature-time effect causing o o i n i t i a l i n f e c t i o n at 13 and at 18 C to occur almost simultaneously. Cold-shock treatment resulted i n some inf e c t i o n . Such treatment, however, did not produce the same immediate i n f e c t i o n as heat-shock. Hi s t o l o g i c a l studies demonstrated the inf e c t i o n t o be concentrated i n the host epidermis with fungal hyphae at s i t e s of heaviest i n f e c t i o n extending through the dermis and into underlying mascle tissues. i v TABLE OF CONTENTS ABSTRACT i i ACKNOWLEDGEMENT i i i TABLE OF CONTENTS i v LIST OF TABLES, PLATES AND FIGURES • INTRODUCTION AND HISTORICAL RESUME 1 MATERIALS AND METHODS 7 Col l e c t i o n and culture 7 I d e n t i f i c a t i o n 10 Infection studies: 12 Experiments demonstrating the effect of temperature increase 12 Experiments demonstrating the effect of temperature shock 1U H i s t o l o g i c a l techniques 15 RESULTS 16 Collection and i d e n t i f i c a t i o n 16 Culture techniques IS Infection studies 21 Effect of temperature increase 21 Effect of temperature shock 25 Observations on fungus development and host response 31 His t o l o g i c a l studies - 36 V TABLE OF CONTENTS (Cont'd.) DISCUSSION AO SUMMARY 54-LITERATURE CITED 56 APPENDIX 61 v i LIST OF TABLES, PLATES AND FIGURES TABLES 1 S a p r o l e g n i a c e a e r e p o r t e d a s p a r a s i t e s o f a q u a t i c a n i m a l s •£ a 2 C o l l e c t i o n d a t a and i d e n t i f i c a t i o n o f f u n g i i s o l a t e d 17 3 C o m p a r i s o n o f t h e morphology o f S a p r o l e g n i a p a r a s i t i c a C o ker and S. d i c l i n a Humphrey 43 U MSM Medium 62 5 S t o c k s o l u t i o n s f o r MSM medium 63 PLATES 1 A p p a r a t u s f o r f i s h i n f e c t i o n s t u d i e s 13 2 S a p r o l e g n i a d i c l i n a , H u m p h r e y - morphology on f i s h t i s s u e 19 3 S a p r o l e g n i a d i c l i n a Humphrey - morphology on hemp seed c u l t u r e . . 20 A Coho f i n g e r l i n g s i n f e c t e d w i t h S a p r o l e g n i a d i c l i n a 35 5 S e c t i o n s o f coho t o compare h e a l t h y t i s s u e s w i t h t i s s u e s i n f e c t e d w i t h S a p r o l e g n i a d i c l i n a 37 6 S e c t i o n s t h r o u g h f i s h t i s s u e i n f e c t e d w i t h S a p r o l e g n i a d i c l i n a . . 38 FIGURES 1 E x p e r i m e n t a l t a n k s 13 2 A q u a r i a s e t i n t a n k s d u r i n g i n f e c t i o n s t u d y 13 3 Zoosporangium w i t h e n c y s t e d p r i m a r y z o o s p o r e s 19 4 A p l a n o i d zoosporangium w i t h g e r m i n a t i n g z o o s p o r e s 19 5 Gemmae 19 6 Gemma f u n c t i o n i n g a s zoosporangium 19 7 Oogonia and o o s p o r e s . 19 v i i LIST OF TABLES, PLATES AND FIGURES (Cont'd.) FIGURES (Cont'd.) 8 Zoosporangium releasing motile, primary zoospores 20 9 Empty zoosporangium . 20 10 Oogonia, diclinous antheridia, and oospores 20 11 Mature oospore . 20 12 Effect of temperature increase on in f e c t i o n of coho with Saprolegnia d i c l i n a during 18 day periods 22 13 Effect of temperature increase on in f e c t i o n of coho with Saprolegnia d i c l i n a . . . . 23 L4 Effect of temperature increase on time of i n f e c t i o n of coho with Saprolegnia d i c l i n a during 18 day period 24-15 Effect of warm temperature shock on in f e c t i o n of coho with Saprolegnia d i c l i n a during l 6 day periods 26 16 Effect of warm temperature shock on in f e c t i o n of coho with Saprolegnia d i c l i n a during 16 day period 27 17 Effect of warm temperature shock on time of i n f e c t i o n of coho with Saprolegnia d i c l i n a during 16 day period 28 18 Effect of warm and cold temperature shock on in f e c t i o n of coho with Saprolegnia d i c l i n a during 16 day period 29 19 Effect of warm and cold temperature shock on time of inf e c t i o n of coho with Saprolegnia d i c l i n a 30 20 Infection s i t e frequencies 32 21 Summary of sp e c i f i c i n f e c t i o n s i t e s 33 22 Dorsal i n f e c t i o n 35 23 Dorsal i n f e c t i o n extending to caudal and ventral regions 35 24. Ventral i n f e c t i o n with o r i g i n at base of anal f i n 35 25 Infection with o r i g i n * at base of caudal and dorsal f i n s 35 v i i i LIST OF TABLES, PLATES AND FIGURES (Cont'd.) FIGURES (Cont'd.) 26 S e c t i o n t h r o u g h h e a l t h y t i s s u e 37 27 S e c t i o n t h r o u g h t i s s u e i n f e c t e d w i t h S a p r o l e g n i a d i c l i n a 37 28 Heavy e p i d e r m a l and d e r m a l i n f e c t i o n 38 29 Deep i n f e c t i o n showing f u n g a l hyphae p e n e t r a t i n g w e l l i n t o m uscle t i s s u e s 38 i i i ACKN0WLEDGEMET3T My s i n c e r e a p p r e c i a t i o n i s e x t e n d e d t o t h e f o l l o w i n g p e r s o n s f o r a s s i s t a n c e d u r i n g t h i s s t u d y : Dr. G.C. Hughes under whose d i r e c t i o n and s u p p o r t t h i s s t u d y was c a r r i e d o u t . Mr. A.R. Maurer ( R e s e a r c h S c i e n t i s t , Canada Department o f A g r i c u l t u r e ) , who a c t e d a s c o n s u l t a n t i n many phases o f t h e s t u d y . Dr. R . J . B a n d o n i , T. B i s a l p u t r a , D. M c P h a i l , and T.G. N o r t h c o t e o f t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a f o r v a l u a b l e a d v i c e and a s s i s t a n c e . D r. G. B e l l and Dr. T. E v e l y n o f t h e F i s h e r i e s R e s e a r c h B o a r d , Nanaimo, B r i t i s h C o l u m b i a , f o r t h e i r h e l p f u l s u g g e s t i o n s and c o - o p e r a t i o n . M e s s r s . J . B a k e r , F. F r a s e r , R. K e a r n s , and L. Sharman f o r v a l u a b l e a s s i s t a n c e i n t h e f i e l d and i n t h e l a b o r a t o r y . Mr. S. Borden f o r a s s i s t a n c e w i t h t h e s t a t i s t i c s . T h i s s t u d y was con d u c t e d d u r i n g t e n u r e o f a N a t i o n a l R e s e a r c h C o u n c i l S c h o l a r s h i p . F o r a d d i t i o n a l f i n a n c i a l s u p p o r t f o r c e r t a i n e q u i p -ment and f i e l d e x p e n s e s , I am i n d e b t e d t o a F i s h e r i e s R e s e a r c h Board U n i v e r s i t i e s G r a n t awarded t o Dr. G.C. Hughes, U n i v e r s i t y o f B r i t i s h C o l u m b i a . INTRODUCTION AND HISTORICAL RESUME Saprolegniaceous infections of f i s h and other aquatic animals have attracted the interest of biologists since Spallanzani's f i r s t report i n 1777 of "molds" growing on minnows and leeches (cf. Ramsbottom, 1916). Most investigations have, however, aimed at fungus control with l i t t l e attempt to identify the fungi involved or to c l a r i f y the conditions under-lying infection. Fisheries biologists, on the whole, have shown l i t t l e concern for fungus disease as most consider i t a secondary infection attacking only diseased or dying f i s h . Furthermore, Saprolegnia and other Phycomyeetous parasites of f i s h and f i s h eggs are relatively easily controlled by removal of infected organisms or by standard chemical treat-ments (Hoffman, 1963). These fungus infections, however, have at times aroused serious concern. High mortality rates among f i s h , especially Salmonids, suffering from fungus infection were early reported i n England and Scotland (Huxley, 1882 a, b, c; Murrary, 1885; Clinton, 1893; Patterson, 1903) and i n the United States (St. George and Barron de l a Valette, 1884; Koltz, 1883; Valery-Mayet, 1885; Rutter, 1904; Rosenberg, 1908). Whereas such early reports were mainly observational, some of the more recent studies have aimed at identification of fungi and hosts with attempts to c l a r i f y the host-parasite relationship (Kanouse, 1932; Tiffney, 1937, 1939 a, b; Shanor and Saslow, 1944-5 Vishniac and N i g r e l l i , 1957; Wolf, 1958; Scott and O'Bier, 1962; Egusa, 1963, 1965, a, b). One of the most complete historical resumes of " f i s h mold" (Rucker, 1944) indicates that, although the literature on such fungus diseases i s voluminous, our understanding of the problem i s limited. This paradox i s par t i a l l y explained by the fact that so many different - 2 -groups of people have studied these diseases. Botanists, zoologists, fisheries biologists, pathologists, and bacteriologists have a l l examined the problem from their own various points of view. The result has been many contradictory reports and many incomplete or invalid descriptions. With the exception of the many papers on control measures, the literature covers three general topics: hosts attacked, identification of the fungi involved, and etiology of the diseases themselves. Reports of fungi on almost a l l groups of Teleostei, as well as several members of the Amphibia, indicate an extremely wide host range (Tiffney, 1939 a). It has also been shown, however, that certain species are more susceptible to fungus attack than others (Tiffney, 1939 a). During Tiffney's experi-ments, attempts were made to infect several species of 10 different families of f i s h with the fungus, Saprolegnia parasitica Coker. Members of the Siluridae, Catostomidae, and Salmonidae were among the most sus-ceptible to fungus attack. Only Aguilla chrysvpa of the Anguillidae resisted infection. Identification of " f i s h molds" has been complicated by several factors - failure of certain isolates to reproduce sexually (a primary requirement for valid identification); incomplete descriptions; and inadequate knowledge of potential species v a r i a b i l i t y of the involved fungi. Probably the greatest confusion arises from the fact that the fungus most frequently observed as a f i s h parasite, S. parasitica, seldom produces the sexual structures necessary for positive identification. As i s shown in Table 1, a number of species from several genera of aquatic Phycomycetes have been reported as parasites of f i s h and other TABLE 1 SAPROLEGNIACEAE REPORTED AS PARASITES OF AQUATIC ANIMALS Fungus Reference Achlya ambisexualis J.R. Raper, o* and g A. biaexualis Coker & A. Couch, of and p A. colorata Pringsheim (as A. racemosa var stelligera oornu) A. flagellata Coker 1 A. nowickii Raciborski A. polyandra Hildebrand A. prollfera C.G. Nees A. klebsiana Pieters 2 A. sparrovii Reischer Achlya sp. Aphanomyces laevis de Bary Ap. astaci Schikora A_>. daphniae Prowse Ap. hydatinae Valkanov Ap. ovide struens Gicklhorn Calyptralegnia achlyoides (Coker & Couch) Coker Dictyuchus monosporus Leitgeb Leptolegnia baltica Hohnk & V a l l i n L. caudata de Bary L. marina Atkins Frotoachlya paradoxa Coker 3 Saprolegnia delica Coker S. diclina Humphrey S. ferax (Gruith) Thuret 4 S. invaderls Davis & Lazar S. megasperma Coker 4 S. monoica Pringsheim 4 S. mixta de Bary S. parasitica Coker 5 S. torulosa de Bary Thraustotheca elavata (de Bary) Humphrey T. primoachlya Coker & Couch Vishniac & N i g r e l l i , 1957 Vishniac & N i g r e l l i , 1957 Humphrey, 1893 Tiffney & Wolf, 1937 Walentowicz, 1885 (see Tiffney, 1939 a) Hine, 1878 (see Tiffney, 1939 b) Schnetzler, 1887 (see Tiffney, 1939 b) Vishniac & N i g r e l l i , 1957 Vishniac & N i g r e l l i , 1957 Vishniac & N i g r e l l i , 1957 • Shanor & Saslow, 1944 Prowse, 1954 Prowse, 1954 Valkanov, 1931 Prowse, 1954 Vishniac & N i g r e l l i , 1957 Tiffney, 1939 b Hohnk & V a l l i n , 1953 Coker, 1923 Atkins, 1954 Vishniac & N i g r e l l i , 1957 Vishniac & N i g r e l l i , 1957 Rioux & Achard, 1956 Smith, 1878 (see Tiffney, 1939 b) Davis & Lazar, 1940 Vishniac & N i g r e l l i , 1957 Walentowicz, 1885 (see Tiffney, 1939 a, b) Clinton, 1894 (see Tiffney, 1939 b) Coker, 1923 Huxley, 1882 a Vishniac & N i g r e l l i , 1957 Vishniac & N i g r e l l i , 1957 1 Excluded taxon (Johnson, 1956). 2 Reduced to synonomy with A. racemosa (Johnson, 1956). 3 Reduced to synonomy with S. diclina (Seymour, 1966). 4 Reduced to synonomy with S. ferax (Seymour. 1966). 5 Taxon of doubtful a f f i n i t i e s (Seymour, 1966). - 3 -aquatic animals. In fact, Vishniac and Nigrelli (1957), using the Mexican platyfish, Xlphophorus maculstus. as their host, found that under various experimental conditons, 15 of the 17 species of Saprolegniaceae which they used in their study were potential parasites. Descriptions of these fungus diseases and their effects on fish are extremely inconsistent. Fungi have been reported to develop only on pre-viously injured, lesioned, or otherwise damaged tissues (Plehn, 1924); at other times, they have been reported on apparently healthy tissues (Huxley, 1882 a). Some fish reacted by vigorously rubbing infected areas (Huxley, 1882 a; Stirling, 1880; Tiffney, 1939 a); others showed no direct response to fungal growth and appeared unaware of its development. Contradictions, however, are most striking in discussions of the conditions leading to infection of fish with fungus. Such fungus diseases have been attributed to overcrowding, pollution, and reduction of water supply (Brook, 1879); to a rise in temperature (Cummins, 1954; Scott and O'Bier, 1962); to a nutritional deficiency (Jewell, Schneberger and Ross, 1933); to mechanical injury (O'Donnell, 1941; Hoffman, 1949); and to pre-disposing bacterial infections (Hardy, 1911; Davis, 1923; Rueker, 1944; Egusa, 1965 a, b). For almost every suggested condition promoting infec-tion, a contradictory argument may be found. In contrast to Cummins' and Scott's views supporting a rise in temperature as the necessary condition, Davis (1923) f ^ l t low temperaturesW5re necessary for infection. Murray (1885) felt temperature was of l i t t l e importance except in extremes. Rucker (1944)» opposing the theory of mechanical injury, cited several instances (Edington, 1889; Heacox, 1941) when deliberate mechanical injury failed to result in fungus attack. His theory of a primary bacterial - 4 -infection, however, finds a contradiction in the studies of Vishniac and N i g r e l l i (1957). These investigators found no evidence of bacterial i n -fection i n thin sections of infected tissues. In a l l the confusing array of arguments, the only consistency seems to be the general feeling that Saprolegniaceous fungi w i l l not attack healthy f i s h l i v i n g under normal conditions. Most reports suggest the fungus i s a secondary consequence of some other primary, pathogenic con-dition or agent. The cause of death once fungus infection i s established has also been a debatable point. Tiffney (1939 a) suggested three possible causes: mechanical destruction of tissues, production of toxins, and ionic upset due to surface tissue destruction. His earlier work (1936) indicated the presence of heat stable exotoxins and endotoxins. Rucker (1944)* on the other hand, was unable to find positive evidence of any toxic material produced by the fungus. Occasionally, reports of death from suffocation caused by fungus on g i l l tissues appear (Brook, 1879). These, however, are rare and most investigators, e.g., Rucker (1944) and Tiffney (1936), indicate that soffocation had no part i n the cause of death of the f i s h examined. From the above discussion, i t i s evident that the problem of Sapro-legniaceous fungus infections of f i s h demands much further experimental study. The problem becomes even more acute when we consider some of the economic factors involved. In Japan, serious outbreaks of such a fungus disease have threatened the eel-farming industry for the past 10 years. The disease, most serious i n April and May, has caused severe losses i n - 5 -pond-reared eel populations (Egusa, 1963, 1965 a, b). Egusa's experi-mental studies on infected eels suggest a primary bacterial infection. Many aspects of the disease, however, are s t i l l poorly understood and further studies are presently being carried out. In the western United States, Rucker (1944) carried out an extensive study i n the Columbia River system of chinook (0. tshawytscha), sockeye (0. nerka), and steelhead (Salmo gairdnerii gairdnerii). Serious fungus outbreaks had been en-countered among these f i s h during transplanting operations prior to construction of the Grand Coulee Dam. Similar problems have recently prompted some concern in Western Canada. With the development of several large scale, a r t i f i c i a l spawn-ing channels (MacKinnon, 196l), fungus infections have become increasingly troublesome. An example of such an area i s the Robertson Creek Spawning Channel near Port Alberni, British Columbia. Here f i s h are held i n down-stream spawning channels constructed to provide the optimum conditions for spawning Pacific salmon — mainly coho, pink (0. gorbuscha). and sockeye — and for rearing their young (Lucas, I960). Fungus infections among many of the f i s h are often rampant with high concurrent prespawning mortality rates. In 1965, for instance, losses of both coho and sockeye prespawners exceeded lp% (Kearns, 1965). Such mortalities are not uncommon among prespawning salmon. Royal and Seymour (1940) noted a 57% mortality among planted sockeye salmon of the Pugett Sound area which they attributed to "...fungus growth on the head and especially on the snout and eyes." At Robertson Creek, the f i s h are often badly bruised from continual - 6 -attempts to move past gates to natural upstream spawning beds. Many f i s h are heavily infected with fungus. The white mycelial mats of fungus often extend over 50% of the body surface. In some years, however, mechanical injury has not appeared serious enough to account for prespawning mortal-i t i e s approaching 50% and a bacterial infection was suspected. Although some f i s h had lesions characteristic of Chondrococcus columnaris infec-tions, no serious build up of the disease could be established from later bacteriological studies. As mortality rates were often higher at:;temperatures around or above 13°C, i t was f e l t they may have been temperature associated (Kearns, 1965). In the following study, an attempt has been made to test the hypo-thesis that temperature may be a c r i t i c a l factor i n predisposing f i s h , directly or indirectly, to infection by Saprolegniaceous fungi. The fungus infecting f i s h at Robertson Creek was isolated, identified as S. diclina Humphrey, and used i n experimental infection studies on fingerling coho. Considerable time was spent i n developing techniques for obtaining single-spore, bacteria-free cultures prior to identification and experimental studies. Fungi from f i s h and f i s h eggs collected at several other l o c a l -i t i e s i n British Columbia were also isolated and identified. - 7 -MATERIALS AND METHODS Collection and Culture Fungi were collected in the field from living and dead fish and from fish eggs by transferring hyphae directly to Petri dishes of potato-dextrose agar (Difco). On several occasions, fungi were removed directly from fish tissues for immediate microscopic examination. Repeated transfers using several techniques were carried out to obtain bacteria-free cultures. The following techniques were used with varying degrees of success: 1. Raper's glass ring method (Raper, 1937). 2. Agar wells: Small cubes of agar were removed from the centre of medium in Petri dishes and placed on the agar surface adjacent to the well. Fungus was inoculated at the bottom of the well and hyphal tips subsequently transferred from the surface of the agar cube. 3. Rose bengal: 35 mgA and 17 mg/1 were added to plates of various types of agar media. 4-. Potassium tellurite (Willoughby, 1962): Fungi were grown on potato-dextrose agar containing 100 mg/1 . potassium tellurite. A second treatment was used in which fungi were grown in 125 ml. Erhlenmeyer flasks containing - 8 -liquid medium - .025$ maltose and .025$ peptone in distilled, carbon-filtered water (Kanouse, 1932). To prepare flasks, 150 mg maltose and 150 mg peptone were autoclaved in 49 ml. water. 1 ml. potassium tellurite solutions were later added to each flask of cooled medium bringing total medium per flask to 50 ml. A replicated series of flasks was prepared containing concentrations of potassium tellurite ranging at small intervals from 1.0$ to 0.002$. Two discs of fungus inoculum were placed in each flask. Inoculum discs were cut with a #3 cork borer from bacteria contaminated cultures growing on potato-dextrose agar. Flasks were examined over a five day period for growth of fungi and bacteria. Bacteria-free cultures remained clear; con-taminated ones turned murky. 5. Aetidione: 500 rag/1 was added to potato-dextrose or to corn meal agar (B-B-L), autoclaved, and poured into Petri dishes. 6. Chloramphenicol: 50 mg/1 was added to the medium. Medium was prepared as in treatment 5. 7. Penicillin-G: Liquid antibiotic was added in one of two concentrations (190 mg/1 and 125 mg/l) to autoclaved potato-dextrose agar. The medium was poured into Petri dishes. - 9 -8. Penieillin-G and streptomycin-sulfate: Antibiotics (190 mg/1 penicillin-G and 100 mg/1 strepto-mycinsulfate) were added i n the dry form to autoclaved potato-dextrose agar. The medium was poured into Petri dishes. 9. Silver-ring method (Powell & Tenny, 1964.) s To test the oligodynamic effect of silver ions on con-taminated cultures, a silver ring (3.5 mm i n diameter and 1.5 mm i n height) was placed over the depression of a Shoemaker culture slide (Clay-Adams). The slide and ring were placed in a Petri dish and autoclaved. Sub-sequent treatment followed that for Raper's glass-ring method. 10. Ultra-violet light (Blank & Tiffney, 1936): Plates of levulose-peptone agar medium were prepared and irradiated as outlined by Blank and Tiffney. The lamp used was a General Electric Germicidal, 30 watt. Irradi-ated plates were inoculated with fungal discs removed from cultures grown on potato-dextrose agar. Cultures were examined and compared with non-irradiated control plates for growth rate of fungi and presence of bacteria. 11. Low temperature: Potato-dextrose and corn meal agar cultures were incu-bated at 5°C for a two week period. Control cultures were incubated at 13°C. - 10 -For a l l of the above techniques, cultures were tested for presence of bacteria by subculturing in liquid maltose-peptone medium. Most tech-niques were tried individually, in various combinations with each other, and in series. Numbers one and two were combined with three, four, and seven respectively, and number ten with three, four, six, and seven. Single spore cultures were obtained by growing fungi on hemp or sesame seeds in distilled carbon-filtered water. Following production and release of motile spores by the fungus, some of the water containing the spores was sprayed onto plates of potato-dextrose agar. A fine spray was obtained by using a fine glass atomizer connected to a tank of compressed air. Hyphae from individual germinating spores were subcultured for identification. Stock cultures were maintained in tubes of oatmeal or corn meal agar incubated at 13°C. Cultures were transferred every six to eight weeks. Identification Identification of fungi was based on measurements made on sexually mature, hemp seed cultures. A culture of S. parasitica. C.B.S. Meurs, was obtained from the Centraal bureau voor Schimmeleultures, Baarn, Netherland, as a comparative check during identification. Average measurements were made on the basis of 25 observations. References and keys included Humphrey (1893), Coker (1923), Coker and Matthews (1937), Johnson (1956), and Seymour (1966). Several techniques were tried to induce sexual reproduction in those cultures which failed to produce oogonia and antheridia on hemp seed - 11 -cultures. The C.B.S. culture of S. parasitica, a local non-sexually reproducing culture, and a culture of S. diclina (isolate D38) were grown in flasks of dilute peptone-leucine or dilute peptone-maltose liquid medium using a technique introduced by B. Kanouse (1932). Cultures of S. parasitica and S. diclina (D38) .were treated with two steroids and with three raw oils, substances found to stimulate sexual reproduction in certain Phythlum spp. (Haskins. et a l , 1964.). Fungi were grown i n i t i a l l y on plates containing 25 ml solid MSM medium (personal communication, R.J. Bandoni, 1967). The ingredients and method of prepara-tion for MSM medium are given in the Appendix. Discs of inoculum were sub-sequently transferred to 125 ml Erhlenmeyer flasks containing 50 ml liquid MSM medium. Following 2U hours growth in these flasks, fungal discs were transferred to flasks containing a modified MSM medium prepared without the addition of dextrose and casein. Individual steroids or oils were placed in this second set of flasks. The steroids, cholesterol and si t -osterol, were dissolved in 95$ ethyl alcohol. Stock steroid solutions were prepared such that 1 ml of stock solution added to 50 ml of medium resulted in steroid concentrptions of 1, 5 or 10 ppm. The raw oils — linseed, olive, and corn o i l — were added directly to flasks with a 2 ml pipette. One, two, or three drops were added to each flask. Control flasks contained 1 ml 95$ ethyl alcohol. All cultures were examined regularly over a three week period for the presence of oogonia and antheridia. - 12 -Infection Studies  Experiments demonstrating the effect of temperature increase: Experimental f i s h tanks were set up as i s shown i n Plate 1. Three glass aquaria (9" x 6n x 7") were suspended i n each of the three larger wooden tanks. Water temperature i n each wooden tank was maintained by a thermostatically controlled heater working against a continuous supply of o cold water. Tanks I , I I , and I I I were maintained at 8, 13 and 18 C respectively i n experiments I , I I , and I I I . During experiments IV, V and VI, the temperature i n tank I ranged between 9 and 10°C. A constant 18 hour light-day was regulated by 60 watt bulbs set i n wooden hoods over each tank. Tin f o i l , covering half the surface of each aquarium, allowed f i s h to move f r e e l y between l i g h t and shade. Four l i t e r s of autoclaved, dechlorinated water were placed i n each aquarium. Aeration was supplied by indi v i d u a l air-stones. Experimental f i s h , f i n g e r l i n g coho, seined i n L i t t l e Campbell River, B r i t i s h Columbia, were a l l of the same size range (averaging 3.5 cm from snout to end of caudal peduncle) and of the same age group. Freshly cap-tured f i s h were held i n large stock tanks where they were given at least a seven day period to adjust to the laboratory conditions. Water temperature o i n stock tanks was 8 C. Prior to introduction t o experimental aquaria, f i s h were removed from stock tanks and held i n a separate container at room tem-perature to allov.a gradual increase i n water temperature to that of the ex-perimental aquaria. Four f i s h were introduced into each of the nine aquaria. Fungus inoculum was prepared from cultures of S. d i c l i n a . i s o l a t e D38. Single agar discs cut from cultures grown i n P e t r i dishes containing - 13 -PLATE I A p p a r a t u s f o r f i s h i n f e c t i o n s t u d i e s F i g u r e : (1) E x p e r i m e n t a l t a n k s . 0: o u t l e t f o r c o n t i n u o u s w a t e r s u p p l y t o m a i n t a n k ; H: hood w i t h b u i l t - i n l i g h t f i x t u r e . (2) A q u a r i a s e t i n t a n k s d u r i n g i n f e c t i o n s t u d y . T: t a n k ; A: g l a s s a q u a r i u m c o n t a i n i n g f o u r f i n g e r l i n g coho; TH: t h e r m o s t a t f o r h e a t i n g u n i t ; AH: a i r h o s e ; F: f o i l c o v e r ; I : w a t e r i n l e t . PLATE 1 - u -25 ml solid MSM Medium were transferred to 125 ml Erhlenmeyer flasks con-taining 25 ml liquid MSM medium. After 24- hours, fungal discs were trans-ferred to small Petri dishes containing 15 ml sterile, distilled carbon-filtered water. These cultures, following 12 hours incubation, were inocu-lated directly into 2 of the 3 aquaria in each tank. The third and uninoculated aquarium acted as a control. Fish introduced on day 1 were exposed to fungus on day 3 and on every third day thereafter until day 18 when the experiment terminated. During the 18 day period, fish were fed daily with frozen brine shrimp and observed closely at approximate 12 hour intervals. At each observation, water temperature, general condition and behaviour of fish, and development of fungus or other infections, were recorded. A l l dead fish were removed and preserved in 10$ formalin or in Bouin's solution. A positive infection was recorded only i f fungus was observed growing on the tissues of a living fish. For each experiment, fungus on a few fish was re-isolated and identified. A l l remaining fish at the close of an experiment were preserved in formalin and examined microscopically both externally and internally for fungus or other infec-tions. The experiment was run in triplicate. Experiments demonstrating the effect of temperature shock; The procedure followed that for the previous temperature experiments with the following changes. Four tanks each containing three aquaria were o o o o maintained at 3.5-4.5 , 9-10 , 13 , and 18 C, respectively. Water tempera-ture in the 3.5-4.5° tank was controlled by a thermostatically controlled copper, cooling coil. Fish were transferred immediately from stock tanks to experimental aquaria thus subjecting them to a sudden rather than gradual temperature change. The i n i t i a l inoculum was added to aquaria on - 15 -day one rather than on day three. Histological Techniques Several fish, immediately upon death from fungus infection, were fixed in Bouin's solution. Paraffin sections were prepared and stained by the periodic acid-Schiff technique combined with a light green counter-stain (Emmons, et al . , 1963). Other techniques used were a variant of Mallory's using haematoxylineosin with aniline crystal violet ( L i l l i e , 1954) and a cresyl fast violet stain (Puchtler and Sweat, 1964). - 16 -RESULTS Collection and Identification Table 2 summarizes collection sites, hosts and fungi isolated during this investigation. Sexual reproduction was observed on a l l cultures ex-cept DI. The most common parasitic species was S. diclina which occurred at a l l collection sites except Adams River. The Adams River fungus plus two Genesse Creek isolates, D26 and D28-G4h, keyed out as S. diclina. How-ever, oogonia and antheridia were much slower in development and much fewer in number than those of other cultures of S. diclina. The fine often wavy hyphae closely resembled that of the C.B.S. culture of S. parasitica. Further study is necessary for positive identification of these three isolates. Examination of S. diclina removed directly from fish tissue re-vealed several interesting morphological features. Whereas in hemp seed culture, the fungus produced typical Saprolegnioid sporangia with free swimming primary zoospores, on fish tissue, i t produced masses of cylin-drical, aplanoid sporangia (Plate 2, figure 4). Many such sporangia were observed with the long tubes of germinating spores projecting from a l l sides. At no time was the release of primary, free-swimming zoospores observed. On one occasion, S. diclina was found producing oogonia and antheridia on fish tissue. Plate 2 illustrates the morphology of S. diclina. isolate DAD as i t appeared directly after removal from the tissues of a live, adult coho. A l l isolates of S. diclina produced masses of oogonia, antheridia, and mature oospores in hemp seed culture. Oospores were not typically centric as described by Coker (1923) or TABLE 2: Collection data and identification of fungi isolated Collection Code No. Date Locality H03t Host Condition Identification Dl D3 D4 D51 14/6/66 27/6/66 27/6/66 15/5/67 Hatchery, U.B.C. II Tt tt It It II Sasterosteus aculeatus l i v i n g dead dead dead ? Saprolegnia sp. Salmo salar Achlya oblongata Oncorhynchus gorbuscha S. diclina Eggs. G. aculeatus S. diclina D6 12/9/66 C.B.S., Baarn, Holland — — S. parasitica D7 D41 30/9/66 1/11/66 Fisheries Research Bd., Nanaimo, B.C. 11 Eggs. 0.-nerka Eggs, 0. gorbuscha dead ? dead ? S. diclina S. diclina D8, D9 D10 D34, D35 D37, D38, DUO B39 D43, DU, D45, D45a D46, D47, D49b 6/10/66 6/10/66 21/10/66 21/10/66 21/10/66 16/5/67 16/5/67 Robertson Creek, B.C. Tt It tt Tt tt IT II tt It II tt tt 0. kisutch (prespawner) 0. gorbuscha (prespawner) 0. nerka 0. kisutch 0. kisutch water samples water samples li v i n g l i v i n g dead l i v i n g dead S. diclina S. diclina S. diclina S. diclina S. diclina S. diclina Saprolegnia sp. D23, D24 9/10/66 Adams River, B.C. 0. nerka dead Saprolegnia sp. D26, D28-G4b D28,-'G3 29/9/66 29/9/66 Genesse Creek, B.C. tt tt 0. nerka (spawned) 0. nerka (spawned dead dead Saprolegnia sp. S. diclina - 18 -Johnson (1956). The ooplasm, placed slightly to one side, vas surrounded by several layers of o i l droplets, up to three on one side and five on the other (Plate 3, figure l l ) . In a l l other characteristics, the fungus coin-cided with Coker's description of S. dicl i n a (Coker, 1923). Plate 3 i l l -ustrates the morphology of S. d i c l i n a . isolate D40, in hemp seed culture. Attempts to obtain sexual reproduction i n the type culture of S. parasitica f a i l e d for a l l but one treatment. A few mature oogonia and antheridia developed i n three flasks containing one, two, or three drops of corn o i l . In a repetition of the treatment, no sexual organs developed. Culture Techniques Attempts to obtain bacteria-free cultures using any single one of the usual techniques proved unsuccessful. Superficially, cultures appeared uneontaminated; however, subculturing i n liquid medium demonstrated the presence of bacteria. Microscopic examination of hyphae submerged in agar revealed a mass of motile bacteria swarming in a narrow, l i q u i d band adjacent to hyphal walls. These bacteria proved to be gram negative rods which showed no sensitivity to p e n i c i l l i n . Treatment with potassium t e l l u r i t e indicated the bacterial contim-inant was far more tolerant to the treatment than were the fungi tested. Bacterial contaminants tolerated up to 0.08$ potassium t e l l u r i t e . Fungal growth was inhibited at a l l concentrations above .006$. Bacteria-free cultures were eventually obtained by repeated trans-fers using the glass or silver ring technique alternately with growth on irradiated medium. Even this method, however, was not consistently sue-- 19 -PLATE 2 Saprolegnia diclina Humphrey - morphology on f i s h tissue: Figure I (3) Zoosporangium with encysted primary zoospores. (4) Aplanoid zoosporangium with germinating zoospores. (5) Gemmae. (6) Gemma functioning as zoosporangium. (7) Oogonia and oospores. P L A T E 2 - 20 -PLATE 3 Saprolegnia diclina Humphrey - morphology on hemp seed culture i Figure: (8) Zoosporangium releasing motile, primary zoospores; renewal of zoosporangium by internal proliferation. Scale a (9) Empty zoosporangium; renewal by side branching. Scale a. (10) Oogonia, diclinous antheridia* and oospores. Scale a. (11) Mature oospore showing peripheral rings of o i l droplets surrounding ooplasm; position of ooplasm slightly off-center demonstrating variation from recognized centric or subcentric oospore types. Scale b. P L A T E 3 8 - 21 -eessful. Many cultures, after long periods of incubation, became contamin-ated suggesting the presence of extremely resistant bacterial spores. Infection Studies Effect of temperature Increase: Results from experiments I, II, and III indicated a distinct corre-lation between temperature and infection of coho with S. diclina. The effect of water temperature, summarized for each experiment in Figure 12, was similar in each of the three 18 day periods. Figure 13 gives combined results. Statistical analysis using a Chi-square test demonstrated a sig-nificant change in fungus mortality at the three temperature regimes at . o o the 99% confidence level. At temperatures between 8 and 9 C no infection with S. diclina occurred; one control and one experimental fish died of o o unknown causes. Some fungus infection occurred at both 13 and 18 G. In each experiment, the number of infected fish was greatest at the higher o o temperature. The difference between infection at 13 and 18 C, however, was not statistically significant. At these temperatures, no fish in the experimental tanks died of causes other than fungus infection. A few con-trol fish died — some owing to a bacterial infection causing t a i l rot and some owing to fungus infection. Fungus contamination in these aquaria probably resulted from the transfer of fungus spores from experimental aquaria by splash from air hoses or from jumping fish. Further analysis indicated a correlation with temperature and the time of infection. Figure 1U illustrates the number of fish infected on o o each day at 13 and at 18 C. Analysis of this data using the Mann-Whitney u test for non-parametric statistics (Siegel, 1956) indicates a significant - 22 -Figure: (12) Effect of temperature increase on infection of coho with Saprolegnia diclina during 18 day periods. FIGURE 12 in n U. o cc UJ m ZD FUNGUS 8 6 4 2 0 4 2 0 8 6 4 2 0 4 2 0 8 6 4 2 D 4 2 0 CONTROL FUNGUS CONTROL FUNGUS• CONTROL EXPERIMENT I ( A p r i l 5-21) I EXPERIMENT II ( A p r i l 24-May 11 i zza Ll EXPERIMENT I I I (May 16-31) F i s h h e a l t h y t h r o u g h o u t . Water temperature ( C) F i s n d i e d from funqus i n f e c t i o n , F i s h - d i e d from causes o t h e r thar fungus. Effect of temperature increase on infection of coho with Saprolegnia diclina. Chart summarizes results from experiments I, II, and III. FIGURE 13 FUNGUS a : in UJ m 2 4 2 2 2 0 18 1 6 14 1 2 1 0 8 6 4 2 • EZ3 CONTROL 1 2 1 0 8 -."6 4 2 k • E l B - 9 • F i s h h e a l t h y t h r o u g h o u t , . • • 1 3 W a t e r t e m p e r a t u r e (°C) F i s h d i e d f r o m f u n g u s i n f e c t i o n < 1B m F i s h d i e d f r o m c a u s e s o t h e r t h a n f u n g u s o - 2U -Effect of temperature increase on time of infection of coho with Saprolegnia diclina during 18 day period. F I G U R E 14 1B°C cn CP 1 c 1 • s -C CO •rl tt-<(-• U 01 XI E 3 3 4 5 6 7 8 9 1 0 1 1 12 13 14 15 16 17 '18 13°C J L -i I L (A J L 3 4 5 6 7 8 9 1 0 1 1 12 1 3 14 1 5 16 1 7 1i D a y f u n g u s a p p e a r e d on f i s h - 25 -difference at the 99% confidence level between infection times for the two temperatures. At 18°C, the majority of infections occurred within the o i f i r s t seven days while at 13 C infection occurred only during the second week. Effect of temperature shock! Figures 15, 16, 17, 18, and 19 summarize results from experiments IV, V, and VI. In two of the three experiments, fungus infection occurred at a l l three temperatures. As in experiments I, II , and III, some control f i s h died of fungus infection and some of unknown causes, possibly bacter-i a l infections. Analysis of data using a Chi-square test revealed no sig-nificant difference at the 95% confidence level between the three treat-o o ments. The overall effect at 13 and at 18 C was not significantly d i f f e r -ent from that of experiments I, I I , and III. Some change, however, occurred i n the time of infection. Figure 17 demonstrates a leveling out in the times of infection at 13° and 18°C. The Mann-Whitney u test gave no significant difference at the 95% confidence level between the two dis-tributions. Infection at 13°C began earlier after temperature shock with a marked i n i t i a l fungus attack on day five. Although not s t a t i s t i c a l l y significant, the total number of infected f i s h was slightly higher for both temperatures than that for experiments using a gradual rather than sudden temperature increase. Figures 18 and 19 give results of experiments V and VI in which a cold temperature treatment was included. A summary of infection times (Figure 19) demonstrates that while fungus infection did occur at o 3.5 - 4.5 C, i t was both lower i n frequency and later i n development than o o at the 13 or 18 C. - 2 6 -Effect of warm temperature shock on i n f e c t i o n of coho with Saprolegnia d i c l i n a during 16 day periods. FIGURE 15 FUNGUS 8 6 4 2 0 4 2 0 8 6 4 2 • 4 2 • 8 6 4 2 • 4 2 • Ll CONTROL FUNGUS CONTROL FUNGUS .. CONTROL 9-10 • F i s h h e a l t h y t h r o u g h o u t . EXPERIMENT IV (May 31-June 15) Ll EXPERIMENT V (May 1 7 - J u l y 2) I EXPERIMENT VI ( J u l y 5-20) 1 3 Water t e m p e r a t u r e ( C) Fish . , i.disd from fungus i n f e c t i o n , I B 1 w F i s h d i e d from causes o t h e r than fungus „. - 27 -E f f e c t o f warm t e m p e r a t u r e shock on i n f e c t i o n o f coho w i t h S a p r o l e g n i a d i c l i n a . C h a r t summarizes r e s u l t s f r o m e x p e r i m e n t s I V , V, and V I . FIGURE 16 FUNGUS cn •H M CD XI E • 24 22 2D 18 16 14 12 10 6 -2 -• 12 10 8 6 4 2 0 CONTROL 9-10 Water temperature ( DC) • F i s h h e a l t h y t h r o u g h o u t . F i s h d i e d from' fungus i n f e c t i o n , 13 F i s h d i e d from causes o t h e r than fungus. - 28 -Figure: (17) Effect of warm temperature shock on time of i n f e c t i o n of coho with Saprolegnia d i c l i n a during 1 6 day period. FIGURE 17 Day f u n g u s a p p e a r e d on f i s h . - 29 -Figure: (18) Effect of warm and cold temperature shock on infection of coho with Saprolegnia diclina during 1 6 day-periods. Chart summarizes results from experiments V and VI. FIGURE 18 FUNGUS • F i s h h e a l t h y t h r o u g h o u t . F i s h d i e d from fungus i n f e c t i o n . F i s h d i e d from causes o t h e r t h a n f u n g u s . - 30 -Figure: (19) Effect of warm and cold temperature shock on time of infection of coho with Saprolegnia diclina during 16 day periods. F I G U R E 19 3 2 1 0 3 2 1 • 3 2 1 0 3.5 - 4.5 C muim vm Em 9 - 10 C Y77A 77A 13° C J I L 18° c 'A JL 1 2 3 4 5 6 7 9 10 11 12 13 14 15 16 Day f u n g u s appeared on f i s h Observations on fungus development and host response: Close observation of fish during a l l experiments revealed striking variations in patterns of fungus development and host response. Fish response ranged from nervous darting about to apparent indifference to the presence of fungus. Fungus infection was at times pre-ceded by a general sluggishness of the hosts, such fish swimming close to the water surface or lying s t i l l on the bottom of the aquarium. At other times, no abnormal symptoms or patterns of behaviour preceded infection. Fungus always began as a small white tuft of hyphae protruding from fish tissues. No ulcerative sores or other eruptions preceded fungus infec-tion. At no time were any fish observed rubbing infected areas against the aquarium sides. As the fungus advanced, infected tissues became pro-gressively paralyzed and fish movement was seriously hampered. Fish eventually lay motionless on the surface or bottom of the aquarium and died shortly thereafter. The site of infection was extremely variable. The i n i t i a l infection was often on or at the base of fins, especially the dorsal, caudal, and pectoral fins. Data on infection sites are summarized in Figures 20 and 21. Analysis using a Chi-square test demonstrated non-randomness at the 99$ confidence level in the areas of body surface first attacked by fungus. For over 50$ of the 76 fish examined, fungus began on area IV, the region of the caudal peduncle and caudal fin. Little difference existed in the frequency of infection initiating on the fins or directly on the body tissues. Of the 42 records of body infections, over 50$ began on the caudal peduncle. Only four infections initiated on the gills. Although - 32 -F i g u r e : (20) I n f e c t i o n s i t e f r e q u e n c i e s based on o b s e r v a t i o n s made on 76 coho i n f e c t e d w i t h S a p r o l e g n i a d i c l i n a . 1 F I G U R E 20 - 33 -Figure: (21) Summary of specific infection sites based on 79 observations of i n i t i a l infections of coho with Saprolegnia di c l i n a . Number of f i s h - 34 -rare, auch g i l l infections developed rapidly and were extremely destructive, k i l l i n g the f i s h in a few hours. Similar infections often began at the base of pectoral fins from which they progressed quickly to g i l l tissues. Once established the fungus spread rapidly over body tissues, k i l l -ing f i s h from 12 to 96 hours after i t s f i r s t appearance. The pattern of fungus growth varied as much as the site of infec-tion. Tissues beneath fungal mats appeared white. Close examination revealed loss of pigment i n tissues from which hyphae protruded. Some-times these unpigmented areas extended 1 or 2 mm beyond; the leading edge of the fungus forming concentric ring patterns. Within hours, hyphae erupted from these unpigmented tissues and a second unpigmented band formed outside the f i r s t . In another frequent pattern of infection, sur-rounding tissues showed no such i n i t i a l response. Fungal hyphae formed a compact white mat closely flanked by normally pigmented f i s h tissues. The extent of superficial fungal growth at the time of death varied from a small tuft protruding from one operculum to large fungus patches covering over two-thirds of the body surface. Death never occurred as a result of infection on the fins alone; some body tissues were always i n -fected. Plate 4 shows extent of fungus infection on several f i s h photo-graphed minutes after death. In several aquaria, a pattern of aggression developed among the four f i s h with the largest generally acting as the aggressor. The resulting f i n and t a i l nipping sometimes l e f t smaller f i s h with ragged fi n s , espec-i a l l y the caudal and pectoral f i n s . Although these areas were at times attacked by fungus, they were just as often free of fungus infection. The - 35 -PLATE A Coho f i n g e r l l n g s i n f e c t e d w i t h S a p r o l e g n i a ^ d i c l i n a . A r r o w s i n d i c a t e e x t e n t o f e x t e r n a l f u n g u s development a t t i m e o f f i s h d e a t h . F i g u r e : (22) D o r s a l i n f e c t i o n , x 2 L/2. (23) D o r s a l i n f e c t i o n e x t e n d i n g t o c a u d a l and v e n t r a l r e g i o n s . Note m e c h a n i c a l l y damaged c a u d a l f i n w i t h o u t f u n g u s i n f e c t i o n . X 2 1/2 (24) V e n t r a l i n f e c t i o n w i t h o r i g i n a t base o f a n a l f i n . x 2 1/2 (25) I n f e c t i o n w i t h o r i g i n a t t h e base o f c a u d a l and d o r s a l f i n s , x 6 P L A T E 4 - 36 -largest and most aggressive f i s h seldom contracted fungus. A few f i s h i n both control and experimental tanks contracted an i n -fection, probably bacterial, which caused tissue damage of the caudal peduncle. For the few f i s h involved, such regions were common sites of fungus infection. Rough post-mortem examinations of a l l f i s h — infected and uninfected — for internal fungus infections or for parasites other than fungi and bacteria were a l l negative. Histological Studies Of the three staining techniques used, only the periodic acid-Schiff method gave d i f f e r e n t i a l staining. With t h i s method, fungal hyphae appeared light purple to pink against turquoise staining epidermis and musculature and purple staining dermis and connective tissues. Sections through normal and infected tissues are shown in Plates 5 and 6 . Internal fungus infections were concentrated i n tissues immediately beneath super-f i c i a l l y infected areas. Fungus was heaviest i n the epidermis where, at times, a mesh of branching hyphae completely replaced the layer of epiderm-a l c e l l s . Whereas i n some sections hyphae were found i n the epidermis only, in others they were observed i n deeper tissues. Such hyphae penetra-ted thick, dermal tissues to ramify underlying musculature. Although fewer i n number than epidermal hyphae, these hyphae were equal i n diameter (5-Bp). Hyphae rarely penetrated more than 2 or 3 mm into underlying muscle tissues. In a few sections, however, hyphae extended through dorsal musculature into the cavity of the vertebral column. Epidermal and dermal infections re-sulted i n distinct breakdown of host c e l l s . Muscle infections, however, - 37 -PLATE 5 Sections of coho to compare healthy tissues with tissues infected with Saprolegnia d i c l i n a Figure: (26) Section through healthy t i s s u e . D = dermis; E = epidermis; M = muscle tiss u e , x 250 (27) Section through tissue infected with Saprolegnia d i c l i n a . D = dermis; E = fragmented epidermis; M = muscle t i s s u e ; F = fungal hypha. x 300. PLATE 5 - 38 -PLATE 6 S e c t i o n s t h r o u g h f i s h t i s s u e i n f e c t e d w i t h S a p r o l e g n i a d i c l i n a F i g u r e : (28) Heavy e p i d e r m a l and d e r m a l i n f e c t i o n . D = d e r m i s ; E = f r a g m e n t e d e p i d e r m i s ; F = f u n g a l hypha; M = muscle t i s s u e , x 200 (29) Deep i n f e c t i o n showing f u n g a l hyphae p e n e t r a t i n g w e l l i n t o m u s c l e t i s s u e s ; e p i d e r m i s c o m p l e t e l y d e s t r o y e d . D = d e r m i s ; F = f u n g a l hypha; M = muscle t i s s u e , x 100 PLATE 6 - 3 9 -produced l i t t l e tissue destruction beyond a narrow zone immediately adjacent to the dermis. Except for a small channel created by the hyphal filament, such tissues often appeared normal. In no sections were fungal hyphae observed within internal organs. - AO -DISCUSSION Our u n d e r s t a n d i n g o f S a p r o l e g n i a c e o u s i n f e c t i o n s among f i s h — t h e i r b i o l o g y , e f f e c t s , and o v e r a l l s i g n i f i c a n c e — i s v e r y i n a d e q u a t e . An e v a l u a -t i o n o f p r e v i o u s s t u d i e s i s c o m p l i c a t e d by many f a c t o r s . One o f t h e most s e r i o u s d i f f i c u l t i e s a r i s e s f r o m t h e use o f n o n - s t a n d a r d i z e d c u l t u r e and i d e n t i f i c a t i o n p r o c e d u r e s . Many r e f e r e n c e s t o p u r e - c u l t u r e t e c h n i q u e s a r e f o u n d i n t h e l i t e r a t u r e ( R a p e r , 1937; T i f f n e y , 1939 b; Jo h n s o n , 1956; Seymour, 1966). A l t h o u g h t h e methods us e d were r e p o r t e d s u c c e s s f u l , s i m i l a r t e c h n i q u e s u s e d i n t h i s s t u d y p r o v e d o n l y p a r t i a l l y s a t i s f a c t o r y . R e s u l t s i n d i c a t e t h a t a c o m p l e t e l y " p u r e " , b a c t e r i a - f r e e c u l t u r e o f a p a r a s i t i c w a t e r moold i s a r a r e phenomenon I The t h e o r y t h a t b a c t e r i a w i l l n o t move a l o n g hyphae w h i c h p e n e t r a t e a s o l i d , a g a r medium does n o t a p p e a r v a l i d f o r a l l c a s e s . T h i s s i t u a t i o n i s perhaps e x p l a i n e d by t h e f a c t t h a t many o f t h e a q u a t i c b a c t e r i a a r e v e r y m o t i l e and t o l e r a n t o f n e a r a n a e r o b i c c o n d i t i o n s . F u r t h e r m o r e , b o t h b a c t e r i a and f u n g i a r e known t o produce s t r o n g enzymes ( S t a n i e r , e t a l . , 1963; L i l l y and B a r n e t t , 1951), many o f w h i c h a p p e a r cap-a b l e o f l i q u i f y i n g a n a g a r g e l . The p r e s e n c e o f t h i n f i l m s o f l i q u i d a g a r s u r r o u n d i n g hyphae c u t o u t o f s o l i d c u l t u r e s d u r i n g t h i s s t u d y may be t h e r e s u l t o f d i g e s t i v e enzymes produced by t h e g r o w i n g h y p h a l t i p . V i a b l e b a c -t e r i a moved a s f r e e l y t h r o u g h t h i s zone a s t h r o u g h a l i q u i d n u t r i e n t medium. Perhaps t h e r e a s o n t h a t p r e v i o u s i n v e s t i g a t o r s were s a t i s f i e d w i t h u s u a l p u r e - c u l t u r e t e c h n i q u e s i s because o f t h e f a c t t h a t c o n t a m i n a t e d c u l t u r e s o f t e n a p p e a r p u r e . O n l y c l o s e m i c r o s c o p i c e x a m i n a t i o n o r growth on a r i c h , n u t r i e n t medium r e v e a l s p r e s e n c e o f b a c t e r i a l c o n t a m i n a n t s . - a -During this study, cultures Which i n i t i a l l y gave clear solutions in liquid medium often turned cloudy after a period of time. Such delayed evidence of contamination may have resulted from the presence of resistant bacterial spore stages. The most effective pure-culture technique during this study involved repeated transfer of cultures using combinations of known proced-ures together with periodic transfer to a nutrient-rich li q u i d medium as a test for contamination. Results obtained during these culture studies j u s t i f y some comment on the use of ultra-violet irradiated media. Whereas many cultures during this study grew successfully on such media, some did not remain viable. I t i s f e l t that, even though irradiation was not applied directly to the fungi, the resulting products were capable of preventing fungal as well as bacter-i a l growth. The technique was eventually abandoned owing to the fear of introducing mutant strains of fungi. Unless further investigations are made on the effect of such media on the fungi, this technique should prob-ably be regarded with some suspicion. The problems complicating identification of these parasitic fungi are innumerable. Countless studies, both i n the past and more recently, have done l i t t l e to c l a r i f y the confusion. Perhaps the basis of the trouble stems from the characteristics used to distinguish genera and species i n the Saprolegniaceae. Hyphal diameter and growth habit, measure-ments and structure of sexual structures, measurements and development of asexual structures, spore production and diameter — a l l are used i n the separation of species. Several, as has been shown by Salvin (19£L» 1942), are subject to a high degree of variation within a species or genus. In - 42 -many keys, e.g., Coker, 1923, extreme measurements for species often over-lap, hemp seeds form the standard medium, and identification often necessi-tates rough estimates. Seymour, in a recent monograph of the genus Saprolegnia (1966). attempted to revise the genus on the basis of growth on a chemically-defined standard medium. Although Seymour's approach to the problem has long been overdue, his choice of medium may not be entire-ly suitable. Several attempts to grow fungi isolated in this study on Seymour's MSPS medium were unsuccessful. No logical explanation has been found to account for this failure unless a faulty or contaminated chemical was used. Since the most commonly reported fungus parasite of fish is S. parasitica, a closer look at the taxonomic position of this species seems necessary. S. parasitica was originally defined purely on the basis of presence on fish tissues and lack of sexual reproductive structures (Coker, 1923). Most other characteristics were similar to many other species of Saprolegnia. When sexual reproduction was observed (Kanouse, 1932), the fungus was redescribed and retained as a distinct species. The main distinguishing points of Kanouse's description are given in Table 3. Only three characteristics differentiate her species from certain other Saprolegnia species, as, for instance, S. diclina. These are growth on fish, reduced ability to produce sexual structures, and presence of sub-centric oospores. In order to demonstrate some of the problems inherent in Kanouse's description of the species i t is necessary to consider a l l three characteristics individually. As has already been demonstrated in Table 1, many species of TABLE 3* Comparison of the morphology of Saprolegnia parasitica Coker and S. diclina Humphrey Species Zoo-spore Dia-meter Oogonium shape Oogonium size Antheridial type Oospore type Oospore Diameter Oospore per Oogonium Reference or Source S. parasitica 9-11.5 — — — — — — Coker, 1923 S. parasitica 12 pyriform, clavate, sub-spherical to spherical 65-135 x 60-95 diclinous, few andro-gynous sub-centric 18-22 3-25 Kanouse, 1932 S. parasitica 9-11 clavate to pyri-form or irregu-l a r 75-850 x 20-80 diclinous sub-centric 16-28 2-40 Seymour, 1966 S. diclina spherical, oval or pyriform d 35-100 diclinous centric 20-26 1-20 Coker, 1937 S. diclina 10-12 spherical or pyriform 32-110 x 52-65 diclinous centric, occasion-a l l y sub-centric 12-36 1-28 Seymour, 1966 S. dicl i n a 10-13 spherical to oblong d 4-5-80 diclinous sub-centric 15-28 1-20 Robertson Creek, 1966 - UK Saprolegnia as well as several other genera of the Saprolegniaeeae have been reported as f i s h parasites, S. parasitica i t s e l f i s not l i m i t e d t o growth on f i s h tissue and grows f r e e l y i n culture as a saprophyte on various seeds and other media. Thus, growth habit i n no way distinguishes t h i s species from certain other members of the genus. The second distinguishing characteristic e s s e n t i a l l y involves a degree of a b i l i t y t o produce oogonia and antheridia. At present l i t t l e i s known of the factors inducing sexual reproduction i n t h i s fungus. I t has been suggested (Kanouse, 1932; Rucker, 1944) that a reduction of food supply induces sexual reproduction. This p r i n c i p l e , used f o r other species of Saprolegniaeeae by Klebs (1899), Kauffman (1908), and Pieter3 (1915), has been used to explain the f a i l u r e of oogonia and antheridia to develop on f i s h tissues. Although the principle may apply f o r 3. p a r a s i t i c a . i t does not apply f o r a l l other p a r a s i t i c f i s h molds. The fact that, during t h i s study, a small patch of fungus producing sexual structures was observed among hyphae on a heavily infected f i s h suggests more thorough examination of tissues i s necessary before concluding sexual reproduction does not occur on f i s h . The same pr i n c i p l e has been used by Kanouse to explain the development i n culture of oogonia and antheridia on S. paras i t i c a grown i n d i l u t e peptone and maltose or peptone and leucine solutions. No such structures, however, developed during the present study when the G.B.S. culture was grown i n a duplicate series of Kanouse's media. Sexual reproduction was recently observed i n three cultures grown i n l i q u i d MSM medium containing a few drops of corn o i l . The p o s s i b i l i t y of a sexual stimulant i n corn o i l was suspected. However, since a r e p e t i -t i o n of the treatment f a i l e d to duplicate r e s u l t s , i t was f e l t some other - 45 -f a c t o r , e.g., c o n t a m i n a t i o n o f g l a s s w a r e , i n d u c e d s e x u a l r e p r o d u c t i o n . O c c a s i o n a l r e p o r t s o f o o g o n i a and a n t h e r i d i a produced by S. p a r a s i t i c a a r e f o u n d i n t h e l i t e r a t u r e ( T i f f n e y , 1939 a ; R u c k e r , 1944; S c o t t , 1964; Seymour, 1966). A b r i e f c o m p a r i s o n o f t h e s e d e s c r i p t i o n s i s g i v e n i n T a b l e 3. I n a l l c a s e s , t h e most s i g n i f i c a n t , common f a c t o r was t h e l o n g p e r i o d o f t i m e p r i o r t o s e x u a l r e p r o d u c t i o n . S c o t t r e p o r t s "...extreme v a r i a b i l i t y i n t h e t i m e r e q u i r e d f o r f o r m a t i o n o f s e x u a l o r g a n s " w i t h a minimum t i m e o f o v e r f o u r weeks a f t e r i s o l a t i o n and a maximum o f w e l l o v e r a y e a r . The p r o b l e m o f d e l a y e d s e x u a l r e p r o d u c t i o n i s n o t l i m i t e d t o S. p a r a s i t i c a . T i f f n e y (1939 b) r e p o r t s a c u l t u r e o f S. f e r a x w h i c h d i d not r e v e a l i t s i d e n t i t y f o r some t i m e a f t e r i s o l a t i o n . On t h e b a s i s o f s i m i l a r a s e x u a l s t r u c t u r e s , i t had been t e n t a t i v e l y a s s i g n e d t o S. p a r a s i t i c a . I n t h e p r e s e n t s t u d y s e v e r a l c u l t u r e s o f S. d i c l i n a were s l o w t o produce o o g o n i a and a n t h e r i d i a . U n t i l more i s known o f t h e f a c t o r s i n d u c i n g s e x u a l r e p r o d u c t i o n i n S. p a r a s i t i c a and o t h e r s p e c i e s o f t h e genus, a c c u r a t e i d e n t i f i c a t i o n w i l l c o n t i n u e t o be s e r i o u s l y h i n d e r e d . The t h i r d i m p o r t a n t f e a t u r e o f S. p a r a s i t i c a — p r o d u c t i o n o f s u b c e n t r i c ^ o o s p o r e s — may a l s o have l i m i t a t i o n s a s a d i s t i n g u i s h i n g c h a r a c t e r i s t i c . The f u n g u s , S. d i c l i n a . h a s a l s o been r e p o r t e d t o o c c a -s i o n a l l y produce s u b c e n t r i c o o s p o r e s (Seymour, 1966). I s o l a t e s o f S. d i c l i n a u s e d i n t h e p r e s e n t s t u d y c o n s i s t e n t l y p r oduced suc h o o s p o r e s . I n t h e s e i s o l a t e s , two f o r m s o f s u b c e n t r i c o o s p o r e s were o b s e r v e d — o o s p o r e s w i t h one l a y e r o f o i l d r o p l e t s on one s i d e o f t h e c e n t r a l ooplasm and s e v e r a l on t h e o t h e r , and o o s p o r e s w i t h two t o t h r e e l a y e r s on one s i d e and up t o f i v e on t h e o t h e r . The f i r s t t y p e was c o n s i s t e n t w i t h Johnson's (1956) d e f i n i t i o n o f s u b c e n t r i c ; t h e s e c o n d , a s y e t n o t - 46 -described in the literature, could represent an intermediate form between centric and subcentric. That oospore type within a single species can be so unstable suggests that i t is perhaps a poor taxonomic criterion. Further investigation of potential variability of oospore type in S. diclina. S. parasitica, and perhaps in other species of the genus is necessary before using i t as a major distinguishing characteristic. Kanouse's early description emphasizes another major problem in identification of S. parasitica — the considerable capacity for variation within a single isolate. She found that "...on solid media, nearly a l l oogonia were spherical and antheridial filaments were androgynous. Many oogonia developed without antheridia. In liquid environments, oogonia were spherical, subspherical, to pyriform or clavate and many were inter-calary. The antheridial branches were always diclinous. Very few oogonia developed parthenogenetically." (Kanouse, 1932). Tiffney (1939 b) in an extensive study of the morphology of S. parasitica isolated eight separate strains on the basis of variation in measurements of asexual reproductive structures alone. A considerable overlap of measurements, however, re-sulted in the eight strains forming a continuous series. Table 3 compares pertinent characteristics of isolates of S. parasitica described in the literature. Observations on sexual structures of S. parasitica made during the present study were incomplete owing to the small number of oogonia observed and failure of oospores to fully mature. For comparison, descriptions of S. diclina from this study and from several other sources are also given. Both the high degree of variability recognized within the species S. parasitica and the basic similarity of - 47 -sexual reproductive structures with those of S. diclina became apparent. It is interesting to note that Scott (1964) reported that 14. isolates of S. parasitica differed l i t t l e from isolates of S. diclina except in the complete absence of oogonial pitting. Since S. diclina is itself "...with-out pitting except where antheridia touch" (Coker, 1923)> the difference becomes less and less clear. The above comparison has not been made to suggest S. parasitica is synonymous with S. diclina but rather to emphasize the fact that S. parasitica is an extremely ill-defined taxon, even on the basis of its sexual structures. Criticism in the past has been directed to identifica-tion on the basis of asexual structures alone. For this reason Rucker (1944-) referred to S. parasitica as the "waste-basket" for Saprolegnia: Scott (1964) called it a "catch-all" for non-fruiting, parasitic isolatesj Tiffney (1939 b) spoke of forms of "...heterogeneous origin which, i f they could be induced to fruit, might belong to several different species within the genus, Saprolegnia." The present author would also question identifi-cation on the basis of sexual organs since their particular morphology does not appear to differ significantly from that of certain other members of the genus. The presence of physiologic strains within species of Saprolegnia is suggested as a possible explanation for the variable capacity for sexual reproduction between isolates and between certain species. Furthermore, the possibility exists that certain of these strains differ in their requirements for sexual reproduction. Some of these fungi may have very narrow limits in the various environmental and nutritional factors required for the production and maturation of sexual organs. - 48 -Because of the insecure status of the taxon, S. parasitica was con-sidered a poor organism to use during experimental studies. For this reason S. diclina was used in a l l infection studies. The isolate used resembled Coker's S. diclina Humphrey for a l l but one characteristic — production of subcentric oospores. As has already been pointed out, S. diclina does occasionally produce typical subcentric oospores (Seymour, 1966). The stability of oospore type has also been questioned. For these reasons the presence of subcentric oospores rather than centric oospores was not considered a valid criterion for placing the isolate in a new species. For the purpose of this study, therefore, the fungus has been referred to as S. diclina Humphrey. The unusual morphology of S. diclina observed on fish tissues is perhaps an adaptive mechanism for spore dispersal. In fast-flowing, often turbulent streams, tiny zoospores must often be destroyed or lost before contacting a suitable substrate for germination. The production of aplanoid sporangia allows whole sporangia to act as dispersal mechanisms. These structures could feasibly be carried long distances before spores are released thu3 increasing spore distribution through time and space. The question "Is the fungus a primary or secondary parasite?" has been repeatedly asked but never completely answered in the literature. The fact that infection requires some predisposing condition is generally accepted. Several suggested conditions have already been outlined. Results from the present infection studies indicate infection is definitely correlated with some such predisposing condition. Certain facts regarding this condition have also been elucidated. Mechanical injury, although an important factor, is not the main condition leading to infection. - 49 -Treatment o f f i s h b o t h p r i o r t o and d u r i n g i n f e c t i o n s t u d i e s a t t e m p t e d t o m i n i m i z e m e c h a n i c a l i n j u r y . F i s h n e v e r t h e l e s s were s u s c e p t i b l e t o fungus a t t a c k . I t was a l s o n o t i c e d t h a t o c c a s i o n a l i n j u r i e s r e s u l t i n g f r o m a g g r e s s i v e f i n and t a i l n i p p i n g were n o t n e c e s s a r i l y s i t e s o f i n f e c t i o n . A l t h o u g h exposed t o a c o n t i n u a l s u p p l y o f f u n g u s s p o r e s , t h e s e f i s h o f t e n r e m a i n e d f r e e o f f u n g u s i n f e c t i o n t h r o u g h o u t t h e 16 o r 18 day e x p e r i m e n t s . The f a c t t h a t i n j u r y a l o n e may n o t be enough t o p r e d i s p o s e f i s h t o f u n g u s a t t a c k was s u g g e s t e d b y R u c k e r (1944-). P r e s e n t d a t a a p p e a r t o c o n f i r m h i s o p i n i o n . On t h e wh o l e , t h e p o s s i b i l i t y o f t e m p e r a t u r e a s a p r e d i s p o s i n g a g e n t has met w i t h l i t t l e s u p p o r t ( e . g . , M u r r a y , 1885; R u c k e r , 1944). R u c k e r s t a t e s t h a t i n e a r l y a t t e m p t s t o i n o c u l a t e t r o u t and salmon f i n g e r -l i n g s w i t h S. p a r a s i t i c a " . . . r e t e n t i o n o f f i s h a t t e m p e r a t u r e s a p p r o a c h i n g t h e maximum f o r f i s h d i d not cause f u n g u s t o d e v e l o p . " He c o n c l u d e d t h a t f u n g u s d i s e a s e , among t h e salmon and t r o u t examined, f o l l o w e d a p r i m a r y b a c t e r i a l i n v a d e r , Chondrococcus c o l u m n a r ! s . The b a c t e r i a l i n f e c t i o n was a t t r i b u t e d i n p a r t t o h i g h summer t e m p e r a t u r e s . R e s u l t s f r o m t h e p r e s e n t s t u d y do n o t c o n c u r w i t h R u c k e r ' s t h e o r y t h a t a p r i m a r y b a c t e r i a l i n f e c t i o n i s t h e n e c e s s a r y p r e d i s p o s i n g a g e n t . A l t h o u g h s u c h i n f e c t i o n s a r e u n d o u b t e d l y v u l n e r a b l e s i t e s f o r s e c o n d a r y f u n g u s i n f e c t i o n s ( u n p u b l i s h e d r e p o r t , Wood, 19&5), t h e y do n o t a p p e a r t o be a n a b s o l u t e r e q u i r e m e n t f o r f u n g u s i n f e c t i o n . C a r e f u l o b s e r v a t i o n s d u r i n g t h e p r e s e n t s t u d i e s i n d i c a t e d t h a t a t h i g h e r t e m p e r a t u r e s t h e f u n g u s i n f e c t e d f i s h r e g a r d l e s s o f t h e p r e s e n c e o r absence o f o b v i o u s b a c t e r i a l i n f e c t i o n s . A t l o w e r t e m p e r a t u r e s f i s h were a l m o s t c o m p l e t e l y r e s i s t a n t - 50 -to fungus attack. As temperature increased, susceptibility to fungus i n -fection also increased. The time required for fungus to develop, on the other hand, was decreased. The temperatures used during experiments I to VI were chosen on the basis of results from t r i a l experiments and f i e l d observations at Robertson o Creek. Both indicated l i t t l e infection occurred at 8 C while relatively o serious infection began at 13 C or above. A l l temperatures used were with-in the growth range of S. parasitica (Duff, 1929; Powell, 1966) as well as that of other species of Saprolegnia (Pieters, 1915; Cotner, 1930; Perrott, I960). Rucker's failure to obtain infection at higher temperatures i s puzzling on the basis of present results. However, i t i s possible that the fungus he used had a different optimum temperature for infection or that i t belonged to a less virulent strain than the one used i n the present study. o The fact that fungus infection became relatively serious at 13 C seems to coincide with reports of serious bacterial and fungal infections at Robertson Creek at temperatures above 13 C. Wood (1965), i n a recent study of pre-spawning mortalities i n the Fraser River, suggests correlation with serious C. columnaris infections and spawning ground temperatures in o excess of 13 C. So many reports of high mortalities, due to bacteria, fungi, or both, suggest some significance should be attached to temperatures o close to 13 C. Recently, pathologists have begun to consider with some interest the fact that few diseases, except those having reached epidemic status, attack healthy individuals i n their normal environment. Certain environmental changes w i l l often upset the physiology of an organism with - 51 -the result that certain natural resistance mechanisms are broken down. It is suggested that, at temperatures at or above 13°C, salmon may lose much of their resistance to bacterial and to fungal infections. Whether or not Saprolegniaceous fungi are serious disease organisms among salmon fingerlings in nature, these facts remain clear: S. diclina, a common fungus in British Columbia waters, can and does act as a primary invader; fungus infections are generally, i f not always, lethal; infection is correlated with higher temperatures; and, finally, the temperatures at which such infections occur in the laboratory are found in certain arti-f i c i a l spawning grounds in British Columbia. Treatment with sudden temperature shock has not been reported in the literature. Present studies indicated that such treatment, while slightly altering the pattern of infection, did l i t t l e to change the over-a l l incidence of infection. A comparison of figures 13 and 16 reveals a o o striking similarity in the results obtained at 13 and at 18 C. The fact that during heat-shock treatment, the 8°C tank reached 10°C during an un-expected heat spell explains the presence of fungus infection in these tanks. Cold temperature shock did not appear to break down resistance to o fungus attack. Prolonged cold temperature treatment at U C, however, did result in some infection. Further tests are necessary before drawing con-clusions on the overall effect of such lower than normal temperatures on fish susceptibility to fungus infection. The mechanism by which Saprolegniaceous fungi k i l l fish is s t i l l poorly understood. Present studies do l i t t l e to clarify this problem. Close observations at frequent, sometimes hourly intervals, revealed a - 52 -considerable amount of variation i n the course of the disease. Whereas some f i s h died within hours of the f i r s t evidence of fungus, others re-mained alive for as long as six days. Some died with apparently minor infections; others with heavy infections covering much of the body surface. Rucker (1944.) reports a similar lack of correlation between extent of body-surface infection and time of f i s h death. On the basis of so much variation, i t i s d i f f i c u l t to assign death to any one factor. While tissue damage might account for deaths from heavy infections, i t does l i t t l e to explain deaths from very light infections extending over but a few square m i l l i -meters of body surface. Although Rucker (1944-) was unable to demonstrate production of exotoxins, the possibility of toxins cannot be discounted. The fact that on most f i s h , the leading edge of visible fungus infection was often preceded i n the. present study by loss of dark pigments in adja-cent tissues, indicates some chemical change probably occurs. This change i s perhaps brought about by substances produced by fungal hyphae as they penetrate epidermal and dermal tissues prior to eruption. Results from histological studies indicate that the fungus i s con-centrated i n superficial tissues, especially the epidermis. In a l l heavily infected regions, the layer of epidermal cells was almost completely re-placed by fungus hyphae. The dermis layer i n young 0. kisutch i s limited to tough connective tissues with l i t t l e spongy tissue. The fungus was observed penetrating directly through this tough layer and into tissues below. Whereas hyphae caused serious tissue damage in the epidermis and dermis, they had l i t t l e mechanical effect on muscle tissue. Muscle infec-tions rarely extended more than a millimeter or two from the surface. Tiffney (1939 a) has suggested superficial mechanical injury results in - 53 -ionic upset serious enough to k i l l the fish. Here, too, the problem of variation in extent of infection and death of fish presents an argument against the theory of ionic upset as the sole cause of death. The answer to the above problem, as to the many others surrounding Saprolegniaceous infections of fish and other aquatic vertebrates, may never be found in one, single factor. The considerable variation in a l l aspects of the disease — host susceptibility, fungus morphology, develop-ment, and course of infection — lead one to conclude that the disease requires much further investigation. - 54 -SUMMARY P a r a s i t i c Saprolegniaceous fungi were isolated from f i s h from several l o c a l i t i e s i n B r i t i s h Columbia, obtained i n pure culture, and i d e n t i f i e d . Saprolegnia d i c l i n a Humphrey was found i n f i v e of the seven l o c a l i t i e s sampled. Studies of S. d i c l i n a as i t appeared on f i s h tissue revealed mor-phological differences from the fungus grown i n culture. Primary zoospores were retained i n the sporangium which i t s e l f often acted as a dispersal agent. Sexual reproduction was observed on f i s h t i s s u e . The problems i n i d e n t i f i c a t i o n of many species of Saprolegnia and the status of the taxon, S. p a r a s i t i c a , have been examined and discussed i n r e l a t i o n to observations of B r i t i s h Columbia fungi and references to the l i t e r a t u r e . Experimental i n f e c t i o n studies on f i n g e r l i n g coho with S. d i c l i n a were carried out to determine the effect of temperature on in f e c t i o n rate. o o At 8 C no i n f e c t i o n occurred. Infection began at 9 C and increased with a further increase i n temperature. Infection occurred e a r l i e r at higher temperatures. Fish treated with heat-shock were infected sooner than those treated with a gradual change over the same temperature difference. Cold-shock resulted i n some infe c t i o n . However, as in f e c t i o n was delayed, i t could not be correlated d i r e c t l y with shock treatment. Close observation of infected f i s h revealed the existence of much va r i a t i o n i n the development, course, and extent of the disease. - 55 -H i s t o l o g i c a l s t u d i e s d e m o n s t r a t e d h y p h a l p e n e t r a t i o n and heavy t i s s u e damage i n t h e e p i d e r m i s , some i n t h e d e r m i s , and l i t t l e i n t h e u n d e r l y i n g m u s c l e t i s s u e s . No h y p h a l p e n e t r a t i o n was o b s e r v e d i n i n t e r n a l o r g a n s . _ $6 _ LITERATURE CITED A t k i n s , D. 1954. F u r t h e r n o t e s on a mar i n e member o f t h e S a p r o l e g n i a e e a e , L e p t o l e g n i a m a r i n a n. s p . , i n f e c t i n g c e r t a i n i n v e r t e b r a t e s , i - Mar. B i o l . A s s o c . U.K. 33:616-625. B l a n k , l . H . and W.N. T i f f n e y . 1936. The use o f u l t r a - v i o l e t i r r a d i a t e d c u l t u r e media f o r s e c u r i n g b a c t e r i a - f r e e c u l t u r e s o f S a p r o l e g n i a . M y c o l o g i a 28: 324-329. B r o o k , G. 1879. N o t e s on t h e salmon d i s e a s e i n t h e E s k and t h e Eden. T r a n s , and P r o c . B o t a n . Soc. E d i n b u r g h 12: 389. C l i n t o n , G.F. 1893. O b s e r v a t i o n s and e x p e r i m e n t s on S a p r o l e g n i a i n f e c t -i n g f i s h . B u l l . U.S. F i s h . Commission. 13s 163-172. C o k e r , W. C. 1923. The S a p r o l e g n l a c e a e w i t h n o t e s on o t h e r w a t e r m o l d s . U n i v e r s i t y o f N o r t h C a r o l i n a P r e s s , C h a p e l H i l l . 201 p. and V.D. Matthews. 1937. S a p r o l e g n i a l e s : S a p r o l e g n i a e e a e , E c t r o g e l l a c e a e , L e p t o m i t a c e a e . N. Amer. F l o r a 2: 15-67. C o t n e r , F.B. 1930. The development o f t h e z o o s p o r e s i n oomycetes a t optimum t e m p e r a t u r e s and t h e c y t o l o g y o f t h e i r a c t i v e s t a g e s . Amer. J . B o t . 17: 511-546. Cummins, R. 1954. M a l a c h i t e - g r e e n o x a l a t e used t o c o n t r o l f u n g u s on y e l l o w p i k e p e r c h eggs i n j a r h a t c h e r y o p e r a t i o n s . P r o g . F i s h  C u l t . 16: 79-82. D a v i s , H.S. 1923. A new b a c t e r i a l d i s e a s e o f f r e s h - w a t e r f i s h e s . B u l l . U.S. B u r . F i s h . 38: 261-280. and E.C. L a z a r . 1940. A new f u n g u s d i s e a s e o f t r o u t . T r a n s . Amer. F i s h . Soc. 70: 264-271. D u f f , D.C.B. 1929. A p h y s i o l o g i c a l s t u d y o f c e r t a i n p a r a s i t i c S a p r o l e g n i a e e a e . S t u d i e s f r o m t h e B i o l o g . S t a t , o f Canada 5: 195-202. E d i n g t o n , A. 1889. On t h e S a p r o l e g n i a o f salmon d i s e a s e and a l l i e d f o r m s . R e t t . F i s h . Bd. S c o t . 7: 368-382. E g u s a , S. 1963. S t u d i e s on S a p r o l e g n i a s i s o f t h e e e l . - I . The r e s i s t a n c e o f t h e e e l t o f u n g u s i n f e c t i o n s . B u l l . J a p . Soc. S c i . F i s h . 29: 27-36. . 1965 a. The existence of a primary infectious disease i n the so-called "fungus disease" i n pond-reared eels. B u l l . Jap. S c i . F i s h . 31: 517-526. and T. Nishikawa. 1965 b. Studies of a primary infectious disease i n the so-called fungus disease of eels. B u l l . Jap. Soc. Fi s h. 31: 804.-813. Emmons, C.W., C.H. Binford, and J. P. Utz. 1963. Medical mycology. Lea and Febiger, Philadelphia. 380 p. Hardy, A.D. 1911. Association of alga and fungus i n salmon disease. Proc. Royal Soc. V i c t . 23:27. Haskins, R.H., A.P. Tulloch, and R.C. Micetich. 1964. Steroids and the stimulation of sexual reproduction of a species of Pythium. Canad. J. Microbiol. 10:187-195. Heacox, C. 1941. Handle trout with dry hands. Hunting and Fishing. Feb. (not seen). Hoffman, G.L. 1949. Iso l a t i o n of Saprolegnia and Achlya with p e n i c i l l i n and streptomycin, and attempts to infect f i s h . Prog. Fish Cult. 11:171-174-. . 1963. Parasites .of fresh water f i s h I . Fungi 1. Fungi CSaprolegnia and relatives) of f i s h and f i s h eggs. Fishery  Leaflet 564. U.S. Dept. of the In t . , F i s h and W i l d l i f e Service, Wash. D.C. 6 p. Hohnk, W. and St. V a l l i n . 1953. Epidemisches Absterben von Eurytemora verursacht durch Leptolegnia b a l t i c a nov. spec. Veroff. Inst. Meeresforsch. Bremerhaven 2:215-223. Humphrey, J.E. 1893. The Saprolegniaceae of the U.S. with notes on other species. Trans. Amer. P h i l . Soc. N.S. 17:63-148. Huxley, T.H. 1882 a. Saprolegnia i n r e l a t i o n t o salmon disease. Quart. J. Mjc. Soc. 22:311-333. . 1882 b. A contribution to the pathology of the epidemic known as salmon disease. Proc. Royal Soc. London 33: 381-389. . 1882 c. The salmon disease. Nature 25:437-440. Jewell, M.E., E. Schneberger, and J.A. Ross. 1933. The vitamin requirements for goldfish and channel cat. Trans. Amer. Fish. Soc. 63:338-347. Johnson, T.W. J r . 1956. The genus Achlya: morphology and taxonomy. Univ. of Michigan Press, Ann Arbor. 180 p. - 58 -Kanouse, B.B. 1932. A physiological and morphological study of Saprolegnia parasitica. Mycologia 24.: 431-452. Kauffman, C.H. 1908. A contribution to the physiology of the Saprolegniaeeae with special reference to the variations of the sexual organs. Annals of Bot. 22:361-387. Kearns, R.K. 1965. Summary of 1965 Robert son Creek f a l l program, unpublished report. Dept. of Fish, of Canada, Vancouver, B.C. Klebbs, G. 1899. Zur Physiologie der Fortpflanzung einiger Pilze. 2. Jahrb. f. wiss. Bat. 33:513-593. Koltz, J.P.J. 1883. The a r t i f i c i a l propagation of f i s h . Rept. U.S. Comm'r. Fish. 8:491-516. L i l l i e , R.D. 1954. Hi st opatholo gic technique and practical histo-chemistry. McGraw-Hill, Toronto. 501 p. L i l l y , V.G. and H.L. Barnett. 1951 Physiology of fungi. McGraw-Hill, New York. 464 p. Lucas, K.C. I960. The Robertson Creek Spawning Channel. Canad. Fish  Culturist 27:3-23. MacKinnon, D. 1961. Man-made spawning channels for Pacific salmon. Canad. Geographical J. July. Murray, G. 1885. Notes on the inoculation of fishes with Saprolegnia  ferax. J. Bot. 23:302-308. O'Donnell, D.J. 1941. A new method of combating fungus infections. Prog. Fish Culturist 56:18-20. Patterson, J.H. 1903. On the cause of salmon disease. Pub. Fish. Bd. Scot. 52 p. Perrott, E. I960. The ecology of some aquatic Phycomycetes. Trans. B r i t . Mycol. Soc. 43:19-30. Pieters, A.J. 1915. The relation between vegetative vigor and reproduction i n some Saprolegniaeeae. Amer. J. Bot. 2:529-576. Plehn, M. 1924. Praktikum der Fischkrankheiten. E. Schweizerbartsche Verlagsbuch-handlung, Stuttgart. 179 p. (not seen). Powell, J.R. and W.B. Tenny. 1964. Application of the oligodynamic effect to the separation of bacteria from Saprolegnia. Virginia J. Sci. 15:298. - 59 -P o w e l l , J.R., J r . 1966. Some p h y s i o l o g i c a l a s p e c t s o f growth and  r e p r o d u c t i o n i n S a p r o l e g n i a p a r a s i t i c a C o k e r . 128 p. D o c t o r a l D i s s e r t a t i o n . V i r g i n i a P o l y t e c h n i c I n s t . , B l a c k s b u r g . P rowse, G.A. 1954. Aphanomyces d a p h n i a e s p . nov. p a r a s i t i c i n D a phnia  h y a l i n a . T r a n s . B r i t . Myc. Soc. 37:22-28. P u c h t e r , H. and P. Sweat. 1964. A e r e s y l f a s t v i o l e t s t a i n f o r b a c t e r i a and f u n g i i n t i s s u e . S t a i n T e c h . 39:1-5. Ramsbottom, J . 1916. Some n o t e s on t h e h i s t o r y o f c l a s s i f i c a t i o n o f t h e Phycomycetes. T r a n s . B r i t . Myc. Soc. 5:324-350. R a p e r , J.R. 1937. A method o f f r e e i n g f u n g i f r o m b a c t e r i a l c o n t a m i n a t i o n . S c i e n c e 85:342. R i o u x , J.A. and F. A c h a r d . 1956. Entomophytose m o r t e l l e a* S a p r o l e g n i a d i c l i n a Humphrey 1892 dans un e l e v a g e d'Aedes b e r l a n d i Seguy 1921. V i e e t M i l i e u 7:326-335. ( n o t s e e n ) . R o s e n b e r g , A. 1908. E x p e r i e n c e i n a b a t i n g d i s e a s e among b r o o k t r o u t . B u l l . B u r . F i s h . 28:943-945. R o y a l , L.A. and Seymour. 1940. B u i l d i n g new salmon r u n s . Puget Sound sockeye salmon p l a n t i n g s show v a r y i n g d e g r e e s o f s u c c e s s . P r o g . F i s h  C u l t u r i s t . 52:1-7. R u c k e r , R.R; 1944. A s t u d y o f S a p r o l e g n i a i n f e c t i o n s among f i s h . 92 p. D o c t o r a l D i s s e r t a t i o n . U n i v e r s i t y o f Washi n g t o n , S e a t t l e . R u t t e r , C. 1904. N a t u r a l H i s t o r y o f t h e q u i n n a t salmon. A r e p o r t o f i n v e s t i g a t i o n s i n t h e Sacramento R. 1896-1901. B u l l . U.S. Bur. F i s h . Comm. 22:65-142. S t . George and B a r r o n de l a V a l e t t e . 1884. The enemies o f f i s h . R e p t . U.S. Comm'r. F i s h . 9:795-811. S a l v i n , S.B. 1941. C o m p a r a t i v e s t u d i e s o f t h e p r i m a r y and s e c o n d a r y z o o s p o r e s o f t h e S a p r o l e g n i a c e a e I . I n f l u e n c e o f t e m p e r a t u r e . M y c o l o g i a 33:592-600. . 1942. V a r i a t i o n o f s p e c i f i c and v a r i e t a l c h a r a c t e r i n d u c e d i n a n i s o l a t e o f B r e v i l e g n i a . M y c o l o g i a 34:38-51. S c o t t , W.W. and A.H. 0 ' B i e r . 1962. A q u a t i c f u n g i a s s o c i a t e d w i t h d i s e a s e d f i s h and f i s h eggs. P r o g . F i s h C u l t u r i s t 24:3-15. S c o t t , W.W. 1964. F u n g i a s s o c i a t e d w i t h f i s h d i s e a s e . Developments o f I n d u s t r i a l M i c r o b i o l . 5:97-148. - 60 -Seymour, R.L. 1966. A revision of the genus Saprolegnia. 106 p. Doctoral Dissertation. Virginia Polytechnic Inst., Blacksburg, Virginia. Shanor, L. and Saslow. 1944. Aphanomyces as a f i s h parasite. Mycologia 36:413-415. Siegel, S. 1956. Non-parametric statistics for the behavioral sciences. McGraw-Hill, Toronto. 312 p. Stanier, R.Y., M. Doudorff, and E.A. Adelberg, 1963. The microbial world. Prentice-Hall Inc., Englewood C l i f f s , New Jersey. 753 p. S t i r l i n g , A.B. 1880. Notes on the fungus disease affecting salmon. Rept. U.S. Comm'r Fish. 6:525-529. Tiffney, W.N. 1936. A study of species of Saprolegnia attacking f i s h . Doctoral Dissertation. Harvard University, Cambridge, Mass. and F.T. Wolf. 1937. Aehf.ya flagellata as a f i s h parasite. J. Elisha Mitchell Sci. Soc. 53:298-300. Tiffney, W.N. 1939 a. The host range of Saprolegnia parasitica. Mycologia 31:310-321. . 1939 b. The identity of certain species of the Saprolegniaoaae parasitic to f i s h . J. Elisha Mitchell Sci. Soc. 55:134-151. Vglery-Mayet. 1885. Hatching salmon eggs at Montpellier, France, and trouble with fungus. Bull. U.S. Fish. Comm. 5:272. Valkanov, A. 1931. Uber Morphologie und Systematikder rotatorien-befallenden Pilze. Arch. Protistenk. 74:5-17. (not seen) Vishniac, H.S. and R.F. N i g r e l l i , 1957. The a b i l i t y of Saprolegniaeeae to parasitize platyfish. Zoologica 42:131-134-Willoughby, L.G. 1962. The fruiting behavior and nutrition of Cladochytrium replicatum Karling. Ann. Bot. N.S. 26:13-36. Wolf, K. 1958. Fungus or Saprolegnia infestation of incubating f i s h eggs. Fishery Leaflet #460, U.S. Fish and Wildlife Service, Vol. 460. Wood, J.W. 1965. A report on f i s h disease as a possible cause of pre-spawning mortalities of Fraser River sockeye. Unpublished report for International Pacific Salmon Fisheries Commission, New Westminster. 24 p. - 61 -APPENDIX - 62 -TABLE U MSM Medium 1. Buffer: K H 2 R E 4 1.0 g/1 2. Inorganic nutrients: CaCl 2 .6H 2Q 0.005 n CoCl 2 . 6 H 20 0.002 n CUSOY4 0.0002 n Fe(N0 3)3.9H 20 0.0002 n H 3 B O 3 0.002 ti MnS0^.4H20 0.0001 n NaCl 0.01 n (NH^ ) 6 M o 7 0 2 r 7 H 2 0 0.002 H Zn304.7H20 0.0002 tt 3. Organic nutrients: Casein hydrolysate 1.5 tt Dextrose 2.0 tt Biotin 0.005 mg/1 Inositol 5.0 n Nicotinurie acid 1.0 n P-Aminobenzoic acid 0.1 n Pantothenol 0.5 n Pyridoxine-HCl 0.1 n Riboflavin 0.5 n Thiamine 0.1 v. it A . Agar (for solid media only) 20.0 g/1 5. Distilled carbon-filtered water 1.0 1 6. Dissolved KHgPO^ , casein hydrolsate, NaCl, and agar in 900 ml water in 1^ .. flask with detachable 25 ml dispenser. Using sterile pipettes, added 2 ml stock mineral solution A and 1 ml stock mineral solution B. 7. Dissolved dextrose in 100 ml. water in separate Erhlenmeyer flask. 8. Autoclaved flasks from steps 6 and 7 at 15 lbs for 15 minutes. 9. Added dextrose solution, to hot medium. 10. Using sterile pipette, added 2 ml. stock vitamin solution to slightly cooled medium. - 63 -TABLE 5 Stock Solutions for MSM Medium Stock Mineral Solution A: 1. CuSO/ 0.4398 g/1 Fe(N0 3) v9H ?0 0.7235 " MnS0,?4H20 0.203 " ZnS02.7H20 0.4398 » 2. Dissolved ingredients from step 1 i n 600 ml. d i s t i l l e d carbon-f i l t e r e d water. 3. Added enough concentrated HgSO^ to yield a clear solution. 4. Made volume up to 1 l i t e r with water. 5. Autoclaved at 15 lbs for 15 minutesj stored at 13°C. 6. Used 2 ml. stock solution per l i t e r medium. Stock Mineral Solution B: 1. CaCl 2.2H 20 0.05 g/1 CoGl 2 . 6 H 20 0.02 n H 3B0 3 0.02 » ( N H ^ . M O T O ^ . T J ^ O 0.02 N 2. Dissolved ingredients from step 1 in 100 ml. d i s t i l l e d carbon-f i l t e r e d water. 3. Autoclaved at 15 lbs for 15 minutes; stored at 13°C. 4. Used 1 ml. stock solution per l i t e r medium. Stock Vitamin Solution: 1. D-Biotin 0.001 g/1 I-Inositol 1.0 " Nicotinuric acid 0.2 n P-Aminobenzoie acid 0.02 " Pantothenol 0.1 n Pyridoxine-HCl 0.02 n Riboflavin 0.1 " Thiamine-HGl 0.02 " 2. Dissolved ingredients from step 1 i n 400 ml. 20$ ethyl alcohol. 3. Stored at room temperature. 4.. Used 2 ml stock solution per l i t e r medium. 

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