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Observations on the pathology of saprolegniasis of Pacific salmon and on the identity of the fungi associated… Neish, Gordon Arthur 1976

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OBSERVATIONS ON THE PATHOLOGY OF SAPROLEGNIASIS OF PACIFIC SALMON AND ON THE IDENTITY OF THE FUNGI ASSOCIATED WITH THIS DISEASE by GORDON ARTHUR NEISH B.Sc., A c a d i a U n i v e r s i t y , 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Botany We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1976 © Gordon Arthur Neishy 1976. In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of Brit ish Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Depa rtment The University of Brit ish Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date OcicoW \S 1 <Q i i . . ABSTRACT The o b j e c t i v e s o f t h i s s t u d y were to i s o l a t e and c h a r a c t e r -i z e the f u n g i a s s o c i a t e d w i t h s a p r o l e g n i a s i s o f P a c i f i c salmon and to determine the c o n d i t i o n s which a l l o w the i n f e c t i o n s to become i n i t i a t e d . The f u n g i i s o l a t e d from the salmon were e i t h e r s t e r i l e S a p r o l e g n i a s p e c i e s , or S a p r o l e g n i a s p e c i e s w i t h a f f i n i t i e s t o the S. d i c l i n a - S . p a r a s i t i c a complex. Two m o r p h o l o g i c a l l y and p h y s i o l o g i c a l l y d i s t i n c t s t r a i n s were r e c o g n i z e d i n t h i s l a t t e r group. D i f f i c u l t i e s a s s o c i a t e d w i t h the i d e n t i f i c a t i o n o f these and o t h e r S a p r o l e g n i a i s o l a t e s f o c u s s e d a t t e n t i o n on the i n a d e -quacy o f e x i s t i n g s p e c i e s concepts and a l s o showed t h a t more a t t e n t i o n s h o u l d be d i r e c t e d toward the e f f e c t o f n u t r i t i o n a l and e n v i r o n m e n t a l f a c t o r s on the morphology and growth of these f u n g i . A s t u d y o f the DNA base c o m p o s i t i o n s of e l e v e n i s o l a t e s b e l o n g i n g to the genus S a p r o l e g n i a sensu s t r i c t o showed t h a t , c o n t r a r y to e x p e c t a t i o n s based on the l i t e r a t u r e , t h i s c h a r a c t e r c o u l d not be used to d i s t i n g u i s h groups a t the i n f r a g e n e r i c l e v e l , b u t the r e s u l t s d i d suggest t h a t i s o l a t e s i n c l u d e d i n the genus may have r e l a t i v e l y homogeneous genomes. The e x i s t e n c e o f a s a t e l l i t e DNA was c o n f i r m e d and was found i n a l l i s o l a t e s ex-amined. T h i s s a t e l l i t e DNA s e p a r a t e s S a p r o l e g n i a s p e c i e s from a l l o t h e r Oomycetes wh i c h have been s i m i l a r l y examined. An argument i s p r e s e n t e d f a v o u r i n g the r e j e c t i o n o f the name S a p r o l e g n i a p a r a s i t i c a Coker as a nomen ambiguum and i t i s shown t h a t a l l e x i s t i n g oogonium p r o d u c i n g i s o l a t e s i n c l u d e d i n t h i s s p e c i e s can be l e g i t i m a t e l y c o n s i d e r e d to be S a p r o l e g n i a  d i c l i n a Humphrey. I n f e c t i o n e x p e r i m e n t s , and o b s e r v a t i o n s on the h i s t o - and gross p a t h o l o g y of s a p r o l e g n i a s i s , when c o n s i d e r e d i n the l i g h t o f modern concepts c o n c e r n i n g the n a t u r e o f i n f e c t i o u s d i s e a s e s and e x i s t i n g knowledge o f the p h y s i o l o g y o f P a c i f i c salmon, suggest t h a t t h e r e i s a d i r e c t l i n k between i n c r e a s e d plasma c o r t i c o s t e r o i d l e v e l s i n the f i s h and t h e i r s u s c e p t i b i l i t y t o s a p r o l e g n i a s i s and o t h e r i n f e c t i o n s caused by n o r m a l l y non-patho-g e n i c organisms. I t i s h y p o t h e s i z e d t h a t n a t u r a l i n c r e a s e s i n the plasma c o r t i c o s t e r o i d l e v e l s of the salmon, e i t h e r a l o n e , or i n c o n j u n c t i o n w i t h f u r t h e r s t r e s s - i n d u c e d i n c r e a s e s , c r e a t e a s i t u a t i o n where n a t u r a l immunity and the a b i l i t y to r e p a i r t i s s u e damage a r e g r e a t l y i m p a i r e d . T h i s c o m b i n a t i o n o f f a c t o r s a l l o w s an i n f e c t i o n to be i n i t i a t e d and, once e s t a b l i s h e d , i t becomes p r o g r e s s i v e l y worse, and u l t i m a t e l y t e r m i n a l , a t a r a t e w h i c h can be d i r e c t l y c o r r e l a t e d w i t h i n c r e a s i n g t e m p e r a t u r e . i v TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i LIST OF ILLUSTRATIONS v i i ACKNOWLEDGEMENTS i x HISTORICAL REVIEW AND GENERAL INTRODUCTION 1 SAPROLEGNIASIS IN THE PACIFIC NORTHWEST -A BRIEF REVIEW 29 OBSERVATIONS ON THE TAXONOMY AND PHYSIOLOGY OF ISOLATES BELONGING TO THE GENUS SAPROLEGNIA  SENSU STRICTO WITH EMPHASIS ON THOSE INCLUDED IN THE SAPROLEGNIA DICLINA-SAPROLEGNIA PARA-SITICA COMPLEX 34 PART I - I s o l a t i o n and c h a r a c t e r i z a t i o n of the Saprolegnia i s o l a t e s obtained from P a c i f i c salmon 34 D e s c r i p t i o n of the study area 34 I s o l a t i o n of b a c t e r i a - f r e e , s i n g l e spore i s o l a t e s 40 F i e l d observations 46 C h a r a c t e r i z a t i o n and i d e n t i f i c a t i o n of i s o l a t e s 52 E f f e c t of temperature on vegetat i v e growth PART I I - Nuclear and s a t e l l i t e DNA base com-p o s i t i o n and the taxonomy of Saprolegnia 74 In t r o d u c t i o n 74 D e s c r i p t i o n of i s o l a t e s 80 DNA i s o l a t i o n and buoyant density determination 95 Results and d i s c u s s i o n 98 PART I I I - General taxonomic d i s c u s s i o n w i t h emphasis on the Saprolegnia d i c l i n a -Saprolegnia p a r a s i t i c a complex IQ3 V TABLE OF CONTENTS (Continued) Page OBSERVATIONS ON THE PATHOLOGY OF SAPROLEGNIASIS OF PACIFIC SALMON 122 PART I - F i e l d o b s e r v a t i o n s o n . a d u l t salmon 122 PART I I - H i s t o p a t h o l o g i c a l o b s e r v a t i o n s on a d u l t salmon 1 3 5 Methods and m a t e r i a l s 135 R e s u l t s and d i s c u s s i o n 140 PART I I I - E x p e r i m e n t a l i n f e c t i o n of young P a c i f i c salmon and p l a t y f i s h 151 I n t r o d u c t i o n 151 Methods and m a t e r i a l s 156 PART IV - A g e n e r a l d i s c u s s i o n c o n c e r n i n g the r e l a t i o n s h i p between s t r e s s and sapro-l e g n i a s i s o f P a c i f i c salmon 182 LITERATURE CITED 193 v i LIST OF TABLES Table , Page I Nomenclatural status of some i s o l a t e s of Achlya and Saprolegnia obtained from f i s h or f i s h eggs p r i o r to 1923 7 II Species of Oomycetes reported as p a r a s i t e s of f i s h 19 I I I Temperature records f o r Robertson Creek (21 December 1970-20 December 1971) 37 IV C o l l e c t i o n data f o r Saprolegnia i s o l a t e s obtained from salmonid f i s h at Robertson Creek, 1973 41 V Some c h a r a c t e r i s t i c s of the s i n g l e spore Saprolegnia i s o l a t e s derived from the c u l t u r e s obtained from salmonids at Robertson Creek, 1973 56 VI Oogonium production by some Category C c u l t u r e s 63 VII R a d i a l growth rates (mm/day) a f t e r the i n i t i a l l a g phase of some Saprolegnia i s o l a t e s growing at d i f f e r e n t temperatures i n corn meal agar 72 VI I I Buoyant d e n s i t i e s and base compositions of Saprolegnia species from the l i t e r a t u r e 79 IX I d e n t i t y of the Saprolegnia i s o l a t e s used f o r the DNA buoyant density determinations .... 81 X CsCl buoyant de n s i t y and % GC of the DNA of the Saprolegnia i s o l a t e s 100 XI Records of oospore diameters f o r the Saproleg-n i a d i c l i n a - S a p r o l e g n i a p a r a s i t i c a complex .... 117 XII Species of t e l e o s t f i s h s u c c e s s f u l l y i n f e c t e d experimentally by saprolegnian f u n g i 156 XI I I Summary of the r e s u l t s of the 16-26 February 1975 i n f e c t i o n experiments w i t h young coho salmon igg v i i LIST OF ILLUSTRATIONS F i g u r e Page 1 Mean monthly temperatures o f R o b e r t s o n Creek (December, 1970-December, 1971) 38 2 R a d i a l growth r a t e s (mm/day) a f t e r the i n i t i a l l a g phase of some S a p r o l e g n i a i s o l a t e s growing a t d i f f e r e n t tempera-t u r e s i n c o r n meal agar 73 3 M i c r o d e n s i t o m e t e r t r a c i n g s o f the cesium c h l o r i d e e q u i l i b r i u m g r a d i e n t s of the S a p r o l e g n i a i s o l a t e s ' DNA IQ2 4 H i s t o r i c a l development o f the t e r m i n o l o g y r e l a t e d t o oospore types i n the S a p r o l e g n i a c e a e 112 P l a t e I 32 I I 36 H I 48 IV 51 V 59 VI 61 V I I 66 V I I I 84 IX 90 X 92 XI 116 X I I 124 X I I I 126 XIV 128 XV 134 XVI 138 XVII 142 X V I I I 144 v i i i LIST OF ILLUSTRATIONS (Continued) P l a t e Page XIX 146 XX 148 XXI 164 XXII 174 X X I I I 177 XXIV 181 i x ACKNOWLEDGEMENTS Dr. G. C. Hughes s u g g e s t e d t h a t I c a r r y out a study o f s a p r o l e g n i a s i s o f P a c i f i c salmon and s u p p o r t e d and encouraged me t h r o u g h o u t t h i s s t u d y . To him go my s i n c e r e t h a n k s . T h i s work would not have been p o s s i b l e w i t h o u t the coopera-t i o n of the Department of the Environment, F i s h e r i e s and Marine S e r v i c e . I would p a r t i c u l a r l y l i k e t o thank Mr. K a r l P e t e r s e n , manager of the R o b e r t s o n Creek H a t c h e r y , Dr. G. R. B e l l and Mr. G. E. H o s k i n s , P a c i f i c B i o l o g i c a l S t a t i o n , Nanaimo, B. C., and Dr. E. M. Donaldson, West Vancouver L a b o r a t o r y , West Van-c o u v e r , B. C. f o r t h e i r generous a s s i s t a n c e and a d v i c e . Dr. B. R. Green encouraged and s u p p o r t e d my i n c u r s i o n i n t o the r e a l m o f m o l e c u l a r b i o l o g y and, i n c o n n e c t i o n w i t h t h i s s t u d y , I owe thanks to numerous o t h e r i n d i v i d u a l s who s u p p l i e d a s s i s t a n c e , i n f o r m a t i o n and c u l t u r e s . In p a r t i c u l a r , I would l i k e t o thank Mrs. 0. V o l k o f f and Dr. U. Padmanabhan of t h i s de-partment, Dr. M. W. D i c k , U n i v e r s i t y o f Reading, E n g l a n d , Dr. I . B. Heath, York U n i v e r s i t y , Downsview, O n t a r i o , Dr. R a l p h Emerson, U n i v e r s i t y o f C a l i f o r n i a , B e r k e l e y , and Dr. E. S. Ben-eke, M i c h i g a n S t a t e U n i v e r s i t y , E a s t L a n s i n g . I would a l s o l i k e t o thank Dr. J a c k Maze f o r s u p p l y i n g ad-v i c e and l a b o r a t o r y f a c i l i t i e s f o r the h i s t o p a t h o l o g y work, Mr. N. O t t e n f o r s u g g e s t i n g the p o t e n t i a l u s e f u l n e s s o f c a r b e n i -c i l l i n , Dr. Z. Kabata f o r t r a n s l a t i n g some R u s s i a n p a p e r s , Mr. R i c h a r d Hebda f o r t r a n s l a t i n g a P o l i s h paper, and M i s s J o s e -p h i n e Chow f o r h e l p w i t h the p r e p a r a t i o n of the p l a t e s . F i n a l -X l y , I would l i k e to thank the members of my research committee for help with the manuscript and the i n t e r l i b r a r y loan s t a f f at Woodward Library for their painstaking e f f o r t s to fi n d obscure publications. 1 HISTORICAL REVIEW AND GENERAL INTRODUCTION Oomycetes t h a t i n f e c t f i s h produce an e a s i l y r e c o g n i z e d c o t t o n y mycelium on the s u r f a c e o f the a f f e c t e d a n i m a l . As suc h , these i n f e c t i o n s have p r o b a b l y been r e c o g n i z e d s i n c e a n t i q u i t y by p e o p l e s who have depended on f r e s h w a t e r f i s h f o r f o o d . Pub-l i s h e d r e p o r t s from Europe d e s c r i b i n g t h e s e i n f e c t i o n s date from a t l e a s t the m i d - e i g h t e e n t h c e n t u r y (Aderon, 1748, c i t e d by A i n s w o r t h , 1958; S p a l l a n z a n i , 1777, c i t e d by Ramsbottom, 1916; Shrank, 1789, c i t e d by Humphrey, 1893), and f o r the n e x t c e n t u r y i n t e r e s t e d o b s e r v e r s c o n t i n u e d t o d i s c u s s the i d e n t i t y o f these f u n g i and t h e i r e f f e c t on f r e s h w a t e r a n i m a l s (Hannover, 1839, 1842, c i t e d by Buchwald, 1971; Bennet, 1842; G o o d s i r , 1842; Areshqug, 1844; Unger, 1844; R o b i n , 1853; B e r k e l e y , 1864). I n 1877 Oomycetes were, f o r the f i r s t time i n European l i t e r a t u r e , r e p o r t e d to be a s s o c i a t e d w i t h an e p i z o o t i c which came to be c a l l e d the 'salmon d i s e a s e ' . T h i s d i s e a s e was f i r s t o b s e r v e d i n the Tweed, Eden, and o t h e r r i v e r s near the E n g l i s h -S c o t t i s h b o r d e r , b u t l a t e r s p r e a d t o o t h e r r i v e r s i n Great B r i t a i n and became a sour c e o f g r e a t c o n c e r n . In 1879, a com-m i s s i o n o f i n q u i r y was s e t up by the Rt. Hon. R i c h a r d A s s h e t o n Cross t o determine " t h e causes of such d i s e a s e , and the s t e p s which i t may be e x p e d i e n t t o take to p r e v e n t i t s p r o p a g a t i o n i n the r i v e r s i n whi c h i t has a l r e a d y appeared, and i t s e x t e n s i o n to o t h e r r i v e r s " ( B u c k l a n d e t al_. , 1880) . The Commissioners, Frank B u c k l a n d , Spencer W a l p o l e , and 2 A r c h i b a l d Young, interviewed a l l those who f e l t they had a con-t r i b u t i o n to make to the i n q u i r y and the r e s u l t s were presented to both Houses of Parliament i n 1880 (Buckland et aJ_. , 1880) . The Commissioners were unable to reach any conclusions about the causes, spread, or treatment of the disease and p r o p h e t i c a l l y suggested that "increased observations by n a t u r a l i s t s , micro-s c o p i s t s , and other s c i e n t i f i c persons, prolonged over many seasons, may p o s s i b l y be necessary i n order to enable us to a r r i v e at a complete knowledge of the cause of the recent out-break of Saprolegnia, and the remedies which are a p p l i c a b l e to t h i s disease." The Commissioners f e l t that the cause "must un-doubtedly have a r i s e n from one of two circumstances. E i t h e r (1) the fungus i t s e l f must have been r e c e n t l y s t i m u l a t e d i n t o e x c e p t i o n a l a c t i v i t y , or (2) the f i s h must have been rendered e x c e p t i o n a l l y s u s c e p t i b l e to i t s a t t a c k s . " Research and debate over these two points continued f o r the next two decades. Some observers f e l t that the fungus was p r i m a r i l y r e s p o n s i b l e f o r the disease ( S t i r l i n g , 1879-80; Huxley, 1882a, 1882b), whereas others f e l t t hat the fungus was merely the consequence of a disease brought on by various predisposing f a c t o r s . There was a d i v e r -s i t y of opinion as to what the predisposing f a c t o r s might be, as i s shown by the summary prepared by Frank Buckland (Buckland et a l . , 1880) concerning the "causes to which the salmon d i s -ease and i t s spread have been a t t r i b u t e d " i n which he used the f o l l o w i n g headings: 3 "1. P o l l u t i o n s . "2. Lowness of water caused by excessive land drainage. "3. E f f e c t s of f r o s t and snow on the water. "4. Overstocking or overcrowding of f i s h . "5. O b s t r u c t i o n by w e i r s . "6. Dead f i s h l e f t i n the r i v e r s . "7. Wounds from f i s h f i g h t i n g on the spawning beds and i n j u r i e s by anglers. "8. Transport of fungus by b i r d s . " I t was p o p u l a r l y assumed that the fungus a s s o c i a t e d w i t h the 1877-1881 e p i z o o t i c of B r i t i s h salmon was Saprolegnia f e r a x , and the terms Saprolegnia and Saprolegnia ferax o f t e n seem to have been used interchangeably. The l i t e r a t u r e of the e r a , however, provides no c l e a r evidence that the fungus" growing on the f i s h was, i n f a c t , S. f e r a x . For example, Huxley (1882a) wrote: " I t has been seen that the fungus which grows on diseased salmon i s unquestionably a species of the genus Saprolegnia; and i t i s commonly i d e n t i f i e d w i t h S. f e r a x . But t h i s i d e n t i f i c a t i o n has rested upon very slender grounds. I t i s p r a c t i c a l l y almost impossible to determine the species of a Saprolegnia u n t i l the characters of i t s oosporangia and of i t s a n t h e r i d i a ( i f i t have any) have been a c c u r a t e l y made out. At present not only are we without any s u f f i c i e n t account of these organs i n the salmon Saprolegnia, but i t i s c e r t a i n that they are, at most seasons, extremely r a r e . Mr. S t i r l i n g speaks of having observed only four i n the course of a l l h i s i n v e s t i g a t i o n s ; and not a s i n g l e specimen :has presented i t s e l f i n the considerable number of diseased salmon ...which have come under our observation during the l a s t four months." For the sake of accuracy, i t should be noted that l a t e r i n the same re p o r t Huxley (1882a) wrote that he had solved t h i s problem and i d e n t i f i e d the p a r a s i t e . He wrote: "In the t h i r d week of A p r i l , however, oosporangia and a n t h e r i d i a , i n a l l respects s i m i l a r to those of the 1monoica' form of Saprolegnia f e r a x , made t h e i r 4 appearance i n a c o p i o u s growth o f the fungus on a f l y , w h i c h was i n f e c t e d on the 24th o f March from a c u l t u r e on b l a d d e r , which was a g a i n d e r i v e d from a f l y i n f e c t e d d i r e c t l y from a N o r t h Esk salmon on the 14 of March. " I t may be s a f e l y c o n c l u d e d , t h e r e f o r e , t h a t the salmon fungus i s not a p a r a s i t e p e c u l i a r to t h a t f i s h , b u t t h a t i t i s a form o f the S a p r o l e g n i a f e r a x , w h i c h , so f a r as our o b s e r v a t i o n s go...remains d e v o i d of o o s p o r a n g i a so l o n g as i t i n f e s t s the f i s h , and tends to p e r s i s t i n t h i s c o n d i t i o n f o r a l o n g t i m e , even when i t i s c u l t i v a t e d on those m a t t e r s upon which the S a p r o l e g n i a more u s u a l l y s u b s i s t s . " T h i s s t a t e m e n t was i n f l u e n t i a l i n promoting the p r o p o s a l t h a t the 'salmon fungus' was S_. f e r a x . T h e r e f o r e , i t must be p o i n t e d out t h a t H u x l e y ' s c o n c l u s i o n , based on s e r i a l t r a n s f e r s o f c u l t u r e s w h i c h were not demonstrably u n i f u n g a l , and which were m a i n t a i n e d w i t h o u t any apparent attempt to use s t e r i l e t e c h n i q u e , can s c a r c e l y be r e g a r d e d as ' s a f e ' . I must agree w i t h Large (1940) t h a t "when i t came t o f i n d i n g the p a r t o f the fungus i n the c a u s a t i o n o f the d i s e a s e , he [Huxley] was out of h i s d e p t h " and t h a t Huxley " s h o u l d never have rubbed d e a l b l u e -b o t t l e s on mouldy salmon from the Tweed. I t was not i n c h a r a c -t e r , i t was i n f r a d i g n i t a t e m , and i t was s c a r c e l y i n h i s l i n e . " A l e x a n d e r E d i n g t o n , a l e c t u r e r i n b a c t e r i o l o g y a t the Univ-e r s i t y o f E d i n b u r g h , was not c o n v i n c e d t h a t the 'salmon d i s e a s e ' fungus was c o r r e c t l y i d e n t i f i e d a f t e r h i s i n v e s t i g a t i o n s and, as h i s r e p o r t to the F i s h e r y Board f o r S c o t l a n d ( E d i n g t o n , 1889) i s one o f the more s c h o l a r l y n i n e t e e n t h c e n t u r y papers concerned w i t h the 'salmon d i s e a s e ' , I t h i n k i t i s s a f e to agree w i t h Coker's c o n c l u s i o n t h a t the f u n g i i n f e c t i n g f i s h were " f o r a l o n g time c a r e l e s s l y spoken of as JS. f e r a x " (Coker, 1923) . L i t t l e i n t e r e s t was shown thr o u g h o u t most o f the i n v e s t i -5 gations as to whether organisms other than Saprolegnia species might be a s s o c i a t e d w i t h the 'salmon disease'. The science of b a c t e r i o l o g y was i n i t s i n f a n c y , v i r u s e s were unknown, and these f a c t s , along w i t h the f a c t that the fungus was such an obvious component of the disease, probably account f o r t h i s l a c k of i n t e r e s t . In view of t h i s , i t i s i n t e r e s t i n g to note that as e a r l y as 1880, J . Rutherford observed that although he had not "been able to trace the roots of the fungus beyond the s k i n that covers the s c a l e s " (Rutherford, 1881), when he made a cut through the l e s i o n he saw "an inflamed, unhealthy-looking stratum of muscle below the s k i n " (Rutherford, 1881) and he found that sec-t i o n s of t h i s muscle "when placed under the microscope were seen to be l i t e r a l l y one mass of l i f e - - that l i f e being a species of B a c t e r i a " (Rutherford, 1881). Dr. M. C. Cooke, one of the l e a d i n g mycologists of the day, was not impressed by Rutherford's suggestion. He wrote (Cooke, 1880) : "...these B a c t e r i a are...more probably the r e s u l t of a c e r t a i n d i s i n t e g r a t i o n of the substance of the f l e s h caused by the mycelium of Saprolegnia, than a p r e l i m i n a r y d e p ravity of the f l e s h inducing the sub-sequent development of the fungus...I f e a r that we must accept the force of evidence which goes to show that the Saprolegnia appears to be the great destruc-t i v e agent i n t h i s disease. I t may be t r u e . . . that the c o n s t i t u t i o n of the f i s h i s i n a low c o n d i t i o n , that i t i s d e b i l i t a t e d , and powerless to r e s i s t the fungoid a t t a c k s ; and that t h i s c o n d i t i o n may be the r e s u l t of various secondary causes; but the theory that B a c t e r i a i n the f i s h i s the primary cause, though i t may be a new suggestion, can s c a r c e l y be accepted as a true one." Cooke's opinion d i d not p r e v a i l however, and i n 1903 Ruth-e r f o r d was apparently v i n d i c a t e d by J . Hume Patterson whose 6 c a r e f u l l y e x e c u t e d s t u d y e n t i t l e d "On the Cause of Salmon D i s -ease: A B a c t e r i o l o g i c a l I n v e s t i g a t i o n " (Hume P a t t e r s o n , 1903) seemed to demonstrate c o n c l u s i v e l y t h a t the d i s e a s e was caused by a m o t i l e , gram-negative b a c i l l u s t h a t Hume P a t t e r s o n named B a c i l l u s Salmonis P e s t i s . Hume P a t t e r s o n c o n c l u d e d t h a t the b a c i l l u s was a wound p a r a s i t e , t h a t i t c o u l d be t r a n s m i t t e d from dead f i s h to l i v i n g f i s h , t h a t s almonids were more prone to i n -f e c t i o n than: o t h e r f i s h , and t h a t the b a c i l l u s c o u l d grow w e l l i n c o l d w a t e r and seawater. As a p r o p h y l a c t i c measure, he r e -commended t h a t a l l dead f i s h be removed from the water and burned. Hume P a t t e r s o n ' s s t u d y , i n an h i s t o r i c a l s ense, marks the end o f the i n v e s t i g a t i o n s i n i t i a t e d by the 1877 e p i z o o t i c , and was g e n e r a l l y a c c e p t e d as the s o l u t i o n to the problem o f the cause o f the 'salmon d i s e a s e ' (Drew, 1909; S m i t h , 1912; Rushton, 1925) . The l a t e n i n e t e e n t h and e a r l y t w e n t i e t h c e n t u r y was an e r a of c o n s i d e r a b l e taxonomic development f o r the S a p r o l e g n i a c e a e ( D i c k , 1973) and, because taxonomic concepts had not s t a b i l i z e d , an e r a o f c o n s i d e r a b l e taxonomic and n o m e n c l a t u r a l c o n f u s i o n . F u n g i i s o l a t e d from f i s h and f i s h eggs were no e x c e p t i o n t o t h i s r u l e , and d u r i n g t h i s p e r i o d a number o f s p e c i e s were d e s c r i b e d , or i s o l a t e s named, which have s u b s e q u e n t l y been s e t a s i d e as e x c l u d e d o r d o u b t f u l t a x a , or have been reduced to synonomy i n r e c e n t monographs (Table I ) . I n a d d i t i o n , problems c e n t r e d around the c a r e l e s s use o f the name S a p r o l e g n i a f e r a x were com-pounded by the f a c t t h a t t h e r e was an o v e r l a p p i n g e q u i v o c a l use TABLE I Nomenclatural status of .some .isolates of Achlya and Saj from f i s h or f i s h eggs p r i o r to 1923. Species Achlya h o f e r i Harz I s o l a t e d from mirror carp Reference Harz (1906) Achlya n o w i c k i i Racib'orski carp R a c i b o r s k i (1886) Achlya p r o l i f e r a C. G. Nees Achlya racemosa var. s t e l l i g e r a Cornu Achlya r a d i o s a M a u r i z i o Saprolegnia esocina M a u r i z i o g o l d f i s h , Japanese f i g h t i n g f i s h , Oryzias l a t i p e s , Gusai-chi t r o u t eggs eggs of American brook t r o u t l i v i n g pike Sawada (1912, 1919), c i t e d by Wolf (1939) Humphrey (1893) Maurizio (1899) Maurizio (1896) Saprolegnia f l o c -cosa Mau r i z i o Saprolegnia para-doxa M a u r i z i o eggs of American brook t r o u t eggs of sea tr o u t Saprolegnia hypogyna var. Coregoni Coregonus sp Mauriz i o egg Maurizio (1899) Maurizio (1899) Maurizio (1899) i a obtained Present status A taxon of doubtful a f f i n i -t i e s (Johnson, 1956). Also see d i s c u s s i o n of A. t r e -leaseana by Howard et a l . (1970) . Excluded taxon (Johnson, 1956). A l s o see d i s c u s s i o n of A. treleaseana by Howard et a l . (1970). May be Achlya f l a g e l l a t a Coker (Johnson" 1956) . Synonym of Achlya c o l o r a t a Pringsheim (Johnson^ 1956) . S t i l l thought to be a v a l i d species (Johnson, 1956). Synonym of Saprolegnia ferax (Gruith.) Thuret (Seymour, 1970). A l s o a synonym of S. ferax (Seymour, 1970). ~ Al s o a synonym of S. ferax (Seymour, 1970). Synonym of Saprolegnia hypo-gyna (Pringsheim) de Bary (Seymour, 19 70). 8 o f the name A c h l y a p r o l i f e r a . T h i s s p e c i e s was d e s c r i b e d by C. G. Nees von Esenbeck i n 1823 (see Johnson [1956a} f o r a more complete d i s c u s s i o n ) , but l a t e r , i n 1851, P r i n g s h e i m a p p l i e d the same name to an i s o l a t e w hich i s now c o n s i d e r e d to be S a p r o l e g -n i a f e r a x (Seymour, 1970). Hence, t h e r e i s the d u a l problem t h a t some organisms r e f e r r e d to as ;S. f e r a x may n o t have been t h a t s p e c i e s (and, i n the case o f the 'salmon d i s e a s e f u n g u s ' , almost c e r t a i n l y was not t h a t s p e c i e s ) whereas o t h e r i s o l a t e s , r e f e r r e d to as A. p r o l i f e r a , may i n d e e d have been S. f e r a x . The l a t e n i n e t e e n t h and e a r l y t w e n t i e t h c e n t u r y was a l s o a p e r i o d o f r a p i d development f o r a q u a c u l t u r e f a c i l i t i e s i n Europe and N o r t h A m e r i c a , and i t soon became apparent to workers i n A u s t r i a ( F i e g i g e r , 1903), Canada ( H a r r i s o n , 1918; Huntsman, 1918), France ( V a l e r y - M a y e t , 1885; V i n c e n t , 1908; G r i f f o n § Maublanc, 1911), Germany (Benecke, 1886; M a u r i z i o , 1895, 1896, 1897a, 1897b, 1899), P o l a n d ( W a l e n t o w i c z , 1885; R a c i b o r s k i , 1886), and the U n i t e d S t a t e s (Ryder, 1881, 1883; Humphrey, 1893; H e n s h a l l , 1898) t h a t s a p r o l e g n i a n f u n g i posed a t h r e a t to a v a r i e t y o f f r e s h w a t e r f i s h a t a l l phases of t h e i r l i f e c y c l e . The o b s e r v a t i o n s o f these workers were augmented by o t h e r s who d e s c r i b e d i n f e c t i o n s i n a q u a r i a ( C l a r k , 1874; C l i n t o n , 1894) and i n n a t u r a l w a t e r s ( B l a n c , 1887; S c h n e t z l e r , 1888; Hardy, 1910; J o h n s t o n , 1917). Work on oomycete i n f e c t i o n s 1 around the t u r n o f the c e n t u r y 1The term ' s a p r o l e g n i a s i s ' , as used i n t h i s t h e s i s , r e f e r s to i n -f e c t i o n s o f a n i m a l s caused by f u n g i i n c l u d e d i n the S a p r o l e g -n i a c e a e . The more g e n e r a l term, 'oomycete i n f e c t i o n ' i s used when o t h e r Oomycetes (e.g. Pythium spp., L e p t o m i t u s lacteus) are not e x c l u d e d from the d i s c u s s i o n . 9 was concerned not only w i t h e p i d e m i o l o g i c a l aspects of these i n -f e c t i o n s , but al s o w i t h the means of prevention and treatment. Some authors (Ryder, 1883; Vincent, 1908; Rosenburg, 1908) recog-n i z e d , as Holder (1908) put i t , that "the p o i n t . . . r e l a t i n g to fungus a f f e c t i n g species of f i s h under c u l t i v a t i o n i s that the Chinese method of materia medica should be adopted-- namely, not the cure but to keep w e l l . " These authors emphasized the impor-tance of well-designed f a c i l i t i e s , c a r e f u l management and s t r i c t hygiene. At the same time, there was also i n c r e a s i n g i n t e r e s t i n the use of various chemicals f o r the prevention and treatment of oomycete i n f e c t i o n s . This i n t e r e s t has continued unabated to the present time and, consequently, there i s a voluminous l i t e r -ature devoted to chemotherapy of these i n f e c t i o n s . Since t h i s t h e s i s i s concerned w i t h the b i o l o g y of oomycete i n f e c t i o n s and not w i t h t h e i r treatment, I w i l l r e f e r to t h i s l i t e r a t u r e only when i t serves t h i s end. The recent reviews by Herman (1970, 1972), Hoffman § Meyer (1974), and Nelson (1974) provide r e f e r -ences and u s e f u l i n f o r m a t i o n f o r those i n t e r e s t e d i n chemothera-py of oomycete i n f e c t i o n s . In 1923, W. C. Coker published h i s c l a s s i c a l monograph "The Saprolegniaceae w i t h notes on other water molds" i n which he described s e v e r a l new species of Saprolegnia. One of these s p e c i e s , Saprolegnia paras i t i c a , subsequently came to be consid-ered v i r t u a l l y synonymous wi t h the 'fungus disease' of f i s h , i n much the same way that Saprolegnia ferax had been considered to be the ' f i s h fungus' f o r the preceding h a l f century. There are 10 s e v e r a l problems a s s o c i a t e d w i t h the name S a p r o l e g n i a p a r a s i t i c a . These problems w i l l be d i s c u s s e d o n l y b r i e f l y h e r e , because i n a l a t e r s e c t i o n o f t h i s t h e s i s I w i l l p r e s e n t a d e t a i l e d c r i t i -c a l r e v i e w o f the taxonomy and nomenclature o f S. p a r a s i t i c a . Coker (1923) d e s c r i b e d S. p a r a s i t i c a t o i n c l u d e n o n - f r u i t i n g i s o l a t e s of S a p r o l e g n i a o b t a i n e d from f i s h and f i s h eggs, thus making i t the o n l y s p e c i e s i n the genus i n whi c h s p e c i e s i d e n t i -f i c a t i o n was based on h a b i t a t and on the morphology o f the a s e x u a l r e p r o d u c t i v e s t r u c t u r e s r a t h e r than on the morphology o f i t s s e x u a l r e p r o d u c t i v e s t r u c t u r e s . L a t e r , Kanouse (1932) des-c r i b e d the s e x u a l r e p r o d u c t i v e s t r u c t u r e s o f some S a p r o l e g n i a i s o l a t e s o b t a i n e d from f i s h and f i s h eggs w h i c h she assumed were, i p s o f a c t o , £5. p a r a s i t i c a . Coker (Coker § Mathews, 1937) a c c e p t -ed Kanouse's emended d e s c r i p t i o n , but some subsequent a u t h o r s c o n t i n u e d to use the name f o r n o n - f r u i t i n g S a p r o l e g n i a i s o l a t e s o b t a i n e d from f i s h or f i s h eggs, o f t e n w i t h o u t making any appar-ent e f f o r t t o induce and observe oogonium f o r m a t i o n . B e s i d e s Kanouse's (1932) paper on S. p a r a s i t i c a , t h e r e were s e v e r a l o t h e r s i g n i f i c a n t papers concerned w i t h s a p r o l e g n i a s i s p u b l i s h e d i n the n i n e t e e n - t h i r t i e s i n c l u d i n g a major r e v i e w by Shereshevskaya (1932), a s t u d y of the water mould p o p u l a t i o n o f a f i s h h a t c h e r y by Monsma (1937) , and a s t u d y o f t h e taxonomy and p a t h o l o g y o f the f u n g i c a u s i n g s a p r o l e g n i a s i s i n the n o r t h -e a s t e r n U n i t e d S t a t e s ( T i f f n e y , 1939a, 1939b). S h e r e s h e v s k a y a 1 s (1932) r e v i e w i s the f i r s t major R u s s i a n language p u b l i c a t i o n o f which I am aware t h a t d e a l s w i t h sapro-11 l e g n i a s i s . I n t h i s paper the a u t h o r d e s c r i b e s i n f e c t i o n s o f f i s h and f i s h eggs and methods of p r e v e n t i o n and t r e a t m e n t . Shereshevskaya c o n c l u d e d t h a t s a p r o l e g n i a s i s o f f i s h was compar-a b l e t o t u b e r c u l o s i s of man; t h a t i s , a d i s e a s e caused by an omnipresent m i c r o o r g a n i s m which i n f e c t s d e b i l i t a t e d or s t r e s s e d i n d i v i d u a l s . S hereshevskaya emphasized the importance o f a v o i d -i n g s t r e s s f u l c o n d i t i o n s , p a r t i c u l a r l y when d e a l i n g w i t h m a t u r i n g s a l m o n i d s . Monsma's (1937) s t u d y o f the water mould p o p u l a t i o n o f a f i s h h a t c h e r y i n M i c h i g a n i s one o f the f i r s t a t tempts to d e t e r -mine the i d e n t i t y o f the s p e c i e s i s o l a t e d from f i s h and f i s h eggs r e l a t i v e t o the t o t a l w ater mould p o p u l a t i o n o f a p a r t i c u -l a r l o c a l i t y . More r e c e n t l y , s i m i l a r s t u d i e s have been c a r r i e d out i n Japan (Hoshina e_t a l . , 1960), The USSR (Domashova, 1971; Dudka § F l o r i n s k a y a , 1971), and England ( W i l l o u g h b y , 1969a, 1970, 1971). S t u d i e s such as these p r o v i d e e v i d e n c e which h e l p s to determine whether t h e r e are ' p a r a s i t i c ' s t r a i n s or s p e c i e s w h i c h , f o r one r e a s o n o r a n o t h e r , have a c o m p e t i t i v e advantage over o t h e r components o f the water mould p o p u l a t i o n i n terms o f u t i l i z i n g f i s h as n u t r i e n t s o u r c e s . Monsma found s e v e r a l s p e c i e s o f A c h l y a and S a p r o l e g n i a . He r e p o r t e d t h a t p a r a s i t i c a was the s p e c i e s most f r e q u e n t l y i s o l a t e d , b u t ob s e r v e d oogonium f o r m a t i o n i n o n l y 10 o f the 34 i s o l a t e s a s s i g n e d t h i s name. I n f e c t i o n e x periments u s i n g " b l u e g i l l " f r y (Monsma c a l l e d t hese f i s h Lepomis p a l l i d u s , b u t I can f i n d no r e f e r e n c e to t h i s name i n B a i l e y e_t al_. [1970] , 12 or i n o t h e r s o u r c e s . The f i s h were p r o b a b l y Lepomis m a c r o c h i r u s R a f i n e s q u e . ) a n d "small-mouthed b l a c k b a s s " ( M i c r o p t e r u s d o l o m i e u i Lacepede) f r y were n e g a t i v e , b u t experiments w i t h "Lake M i c h i g a n s h i n e r s " (no s c i e n t i f i c name g i v e n , but they were p r o b a b l y Notro-•gis. a t h e r i n o i d e s R a f i n e s q u e ) were s u c c e s s f u l . However, s i n c e c o n t r o l as w e l l as e x p e r i m e n t a l f i s h were i n f e c t e d , i t i s d i f f i -c u l t , „as Monsma p o i n t e d o u t , t o i n t e r p r e t the d a t a . N e v e r t h e l e s s , Monsma f e l t t h a t the m o r t a l i t y r a t e s showed t h a t S^ . p a r a s i t i c a was the most v i r u l e n t s p e c i e s , and t h a t i m p o r t a n t f a c t o r s pre-d i s p o s i n g the f i s h t o i n f e c t i o n were i n j u r y and the age of the f i s h . T i f f n e y (1939a) i s o l a t e d and i d e n t i f i e d f u n g i o b t a i n e d from a v a r i e t y o f f i s h as w e l l as a few amphibians and a t u r t l e . T i f f n e y took c a r e to i s o l a t e the f u n g i from l i v i n g a n i m a l s . T h i s i s a prudent p r e c a u t i o n because t h e r e i s a h i g h p r o b a b i l i t y t h a t the f u n g i i s o l a t e d i n c u l t u r e from l i v i n g f i s h are the ones a c t u a l l y growing as p a r a s i t e s on the f i s h , whereas dead f i s h (as w i l l be shown l a t e r ) can become c o l o n i z e d by a v a r i e t y o f sapro-b i c f u n g i which are not found on l i v i n g a n i m a l s and have no p a r a s i t i c r o l e . T h i s p r e c a u t i o n was not obser v e d by O'Bier (1960) who m a i n t a i n e d t h a t i t was p o s s i b l e t h a t s a p r o p h y t e s c o u l d be i s o l a t e d from l i v i n g a n i m a l s as w e l l , and t h a t p a t h o g e n i c i t y c o u l d be e s t a b l i s h e d by c a r r y i n g out i n f e c t i o n e x p e r i m e n t s . T h i s argument has some s e r i o u s f l a w s . F i r s t , w h i l e i t may be t r u e t h a t s a p r o b i c f u n g i might be i s o l a t e d from a l i v i n g f i s h , the p r o b a b i l i t y t h a t t h i s w i l l happen i s much reduced r e l a t i v e t o the p r o b a b i l i t y o f i s o l a t i n g s a p r o b i c f u n g i from a dead f i s h . 13 Second, w h i l e an e x p e r i m e n t a l i n f e c t i o n shows t h a t the i s o l a t e used can i n f e c t the s p e c i e s o f f i s h used under a p a r t i c u l a r s e t o f e x p e r i m e n t a l c o n d i t i o n s , f a i l u r e to o b t a i n an i n f e c t i o n does not prove t h a t the i s o l a t e i s n o t , under any c i r c u m s t a n c e s , a p a r a s i t e . P a r a s i t i s m i s a complex, dynamic p r o c e s s i n v o l v i n g the h o s t , the p a r a s i t e , and t h e i r e n v i ronments, and any o f these c o u l d be a l t e r e d i n a v a r i e t y o f ways to g i v e m i s l e a d i n g r e s u l t s . T i f f n e y (1939a) o b t a i n e d 128 i s o l a t e s . Of t h e s e , 122 were ' s t e r i l e ' S a p r o l e g n i a spp. w h i c h T i f f n e y c a l l e d S a p r o l e g n i a p a r a s i t i c a , but a t the same time he proposed t h a t S. p a r a s i t i c a "may i n r e a l i t y be composed of forms of heterogeneous o r i g i n w h i c h , i f they c o u l d be i n d u c e d t o f r u i t , might be found t o b e l o n g to s e v e r a l d i f f e r e n t s p e c i e s w i t h i n t h e genus S a p r o l e g n i a " and t h a t S. paras i t i c a s h o u l d be r e g a r d e d as a " s p e c i e s o f c o n v e n i e n c e " . The r e m a i n i n g s i x i s o l a t e s c o n s i s t e d o f two i s o l a t e s o f S a p r o l e g -n i a f e r a x , two i s o l a t e s of A c h l y a f l a g e l l a t a , one s t e r i l e A c h l y a sp., and one i s o l a t e of D i c t y u c h u s sp. Of t h e s e , o n l y one i s o -l a t e (A. f l a g e l l a t a ) was o b t a i n e d from a f i s h ( L e b i s t e s r e t i c u -l a t u s ( P e t e r s ) ) . The o t h e r i s o l a t e s were o b t a i n e d from the amphibians and the t u r t l e . T i f f n e y (1939b) a l s o c a r r i e d out i n f e c t i o n experiments u s i n g a s u b c u l t u r e of Coker's type c u l t u r e o f S^ . p a r a s i t i c a . ( T i f f n e y [1939a, 1939b] appear to be the o n l y r e f e r e n c e s i n the l i t e r a -t u r e r e f e r r i n g t o Coker's type c u l t u r e . A c c o r d i n g to T i f f n e y [1939a] the c u l t u r e was d e p o s i t e d by Coker i n the C e n t r a a l b u r e a u voor S c h i m m e l c u l t u r e s , B a a r n , b u t i t has a p p a r e n t l y s u b s e q u e n t l y been l o s t . ) U s i n g zoospore s u s p e n s i o n s o b t a i n e d from t h i s ' 14 c u l t u r e , T i f f n e y r e p o r t e d t h a t he was s u c c e s s f u l i n o b t a i n i n g i n f e c t i o n s i n f i f t e e n out of s i x t e e n s p e c i e s o f f i s h used f o r the e x p e r i m e n t s . No o t h e r subsequent s t u d y has been done to show i n f e c t i o n of such a wide v a r i e t y of f i s h u s i n g a s i n g l e i s o l a t e ( T a b le X I I ) a l t h o u g h , a s . T i f f n e y p o i n t s o u t , t h e r e i s a chance t h a t t h a t p a r t i c u l a r i s o l a t e d i d not n e c e s s a r i l y cause a l l the i n f e c t i o n s because o t h e r water moulds were not e x c l u d e d from the e x p e r i m e n t a l t a n k s . T i f f n e y ' s (1939b) st u d y i s n i c e l y complement-ed by V i s h n i a c § N i g r e l l i ' s (1957) s t u d y i n w h i c h a v a r i e t y o f f u n g i (Table I I ) were used to i n f e c t a s i n g l e s p e c i e s o f f i s h (Xiphophorus maculatus ( G u n t h e r ) ) . In 1941, D a v i s § L a z a r d e s c r i b e d S a p r o l e g n i a i n v a d e r i s (=S. f e r a x ) w h i c h they r e p o r t e d as the cause of an a l i m e n t a r y t r a c t i n f e c t i o n o f rainbow t r o u t (Salmo g a i r d n e r i R i c h a r d s o n ) f i n g e r -l i n g s . T h i s r e p o r t and A g e r s b o r g ' s (1933) r e p o r t of " i n t e s t i n a l f u n g i s i t o s i s " of brook t r o u t ( S a l v e l i n u s f o n t i n a l i s ( M i t c h i l l ) ) f i n g e r l i n g s appear to be the o n l y ones which d e s c r i b e the i n i t i a l s i t e o f a s a p r o l e g n i a n i n f e c t i o n as the gut i n s t e a d o f a s i t e somewhere (e.g. s k i n , g i l l s , n a r e s ) on the s u r f a c e o f the f i s h . There i s no r e a s o n to b e l i e v e t h a t s a p r o l e g n i a s i s i s t i s s u e s p e c i f i c , and r e c e n t s t u d i e s (Bootsma, 1973; N o l a r d - T i n t i g n e r , 1973, 1974) c l e a r l y show t h a t s a p r o l e g n i a n f u n g i are not r e s t r i c -t e d t o b e i n g p a r a s i t e s of the s k i n , g i l l s , and m u s c u l a t u r e ; n o n e t h e l e s s , the r e p o r t s by D a v i s § L a z a r (1941) and A g e r s b o r g (1933) are remarkable and need c o n f i r m a t i o n . Two n o t a b l e s t u d i e s done d u r i n g the n i n e t e e n - f i f t i e s are 15 those by Lennon (1954) and by V i s h n i a c § N i g r e l l i (1957). Lennon (1954) found t h a t L e p t o m i t u s l a c t e u s was a s s o c i a t e d w i t h wounds o f f i s h caused by sea lampreys (Petromyzon marinus L . ) . T h i s i s one o f v e r y few r e p o r t s (Table I I ) which r e c o r d L. l a c -teus as a p a r a s i t e of l i v i n g f i s h . V i s h n i a c § N i g r e l l i ' s (1957) stu d y has been c i t e d p r e v i o u s l y and most of t h e i r r e s u l t s have been i n c l u d e d i i n T a b l e I I . Ta b l e I I i s a l i s t o f oomycete s p e c i e s which have been r e -p o r t e d as p a r a s i t e s o f f i s h . I t d i f f e r s from s i m i l a r l i s t s p r e p a r e d by o t h e r a u t h o r s ( T i f f n e y , 1939b; V i s h n i a c § N i g r e l l i , 1957; S c o t t § O ' B i e r , 1962; S c o t t , 1964; McKay, 1967; N o l a r d -T i n t i g n e r , 1973, 1974; Wolke, 1975; W i l s o n , 1976) i n one or more of the f o l l o w i n g ways. F i r s t , T a ble I I i n c l u d e s o n l y r e c o r d s o f oomycete s p e c i e s which I am r e a s o n a b l y sure were i s o l a t e d from (and presumably i n f e c t i n g ) l i v i n g f i s h . I have e x c l u d e d r e c o r d s o f f u n g i i s o l a t e d from dead f i s h and f i s h eggs, s i n c e i n b o t h cases t h e r e i s a good chance t h a t the f u n g i i s o l a t e d were sap-r o b e s . Second, I have i n d i c a t e d which s p e c i e s o f f u n g i have been ob s e r v e d on n a t u r a l l y i n f e c t e d f i s h , and which have been observed o n l y i n e x p e r i m e n t a l i n f e c t i o n s . T h i r d , I have atte m p t e d to r e c o r d the names of the s p e c i e s i n accordance w i t h modern nomen-c l a t u r a l and taxonomic c o n c e p t s . F i n a l l y , I have t r e a t e d Sapro-l e g n i a paras i t i c a i n an unorthodox way. N o n - f r u i t i n g i s o l a t e s c l a s s i f i e d as S a p r o l e g n i a p a r a s i t i c a Coker i n the l i t e r a t u r e have been p l a c e d w i t h S a p r o l e g n i a s p e c i e s . Only i s o l a t e s w hich pro-duced oogonia have been p l a c e d i n S a p r o l e g n i a p a r a s i t i c a , and I V 16 have i n d i c a t e d the d i s t i n c t i o n by r e f e r r i n g to these i s o l a t e s as Saprolegnia p a r a s i t i c a Coker emend. Kanouse. In some instances, I have not included references which are u s u a l l y included i n these l i s t s because I f e e l that there i s e i t h e r i n s u f f i c i e n t i nformation given to be reasonably sure that the fungus was c o r r e c t l y i d e n t i f i e d (e.g. Blanc, 1887) or the references have been c i t e d i n c o r r e c t l y . For example, most of the p r e v i o u s l y c i t e d authors (Vishniac § N i g r e l l i , 1957; Scott $ O'Bier, 1962; Sc o t t , 1964; McKay, 1967; N o l a r d - T i n t i g e r , 1973; Wolke, 1975; Wilson, 1976) have included Saprolegnia t o r u l o s a de Bary i n t h e i r l i s t s . The i n c l u s i o n of t h i s name can be traced back to T i f f n e y (1939a) who wrote: "Although fungi p a r a s i t i c to f i s h have been recog-n i z e d since 1777...and...there have been frequent reports from many parts of the world of such para-s i t i c Saprolegniaceae, the exact i d e n t i f i c a t i o n of many of the species l i s t e d i s d o u b t f u l . This i s p a r t i c u l a r l y true of such reports as that of Huxley [1882a] wherein the organism observed as a p a r a s i t e of salmon was s t a t e d to be e i t h e r Saprolegnia  t o r u l o s a or S. f e r a x . . . . " Subsequently, s t a r t i n g w i t h V i s h n i a c § N i g r e l l i (1957), S. to r u -l o s a has been c i t e d as a p a r a s i t i c species. Even i f one ignores, fo r the moment, the f a c t s that the 'salmon disease fungus' was probably never p r o p e r l y i d e n t i f i e d by Huxley or anyone else i n the nineteenth century, the i n c l u s i o n of this name s t i l l cannot be j u s t i f i e d . Huxley (1882a), while d e s c r i b i n g some c u l t u r e s , merely noted that at one point the hyphae "assumed a j o i n t e d or beaded appearance, as i n the S. t o r u l o s a of De Bary." This can hardly be considered a formal i d e n t i f i c a t i o n e s p e c i a l l y when 17 e l s e w h e r e , i n the same a r t i c l e , Huxley (1882a) noted t h a t "both P r i n g s h e i m and De Bary agree t h a t s e v e r a l s o - c a l l e d s p e c i e s , namely, S. monoica, S. t h u r e t i , and S. t o r u l o s a are merely more or l e s s permanent v a r i e t i e s of S. ferax.'" T h i s problem i s f u r t h e r c o n f u s e d by the f a c t t h a t Howard et. a l _ . (1970) have r e d e s c r i b e d S^ . t o r u l o s a de Bary and they c o n s i d e r I s o a c h l y a ( = S a p r o l e g n i a ) m o n i l i f e r a de Bary to be a synonym o f S. t o r u l o s a and, s i n c e V i s h n i a c § N i g r e l l i (1957) have r e p o r t e d s u c c e s s f u l i n f e c t i o n of p l a t y f i s h w i t h I s o a c h l y a m o n i l i f e r a (Table I I ) , the name S a p r o l e g -n i a t o r u l o s a may now be l e g i t i m a t e l y i n c l u d e d i n a l i s t of Oomycetes r e p o r t e d as p a r a s i t e s o f f i s h . A n o t h e r e r r o r d a t i n g from T i f f n e y (1939a) i s the a s s e r t i o n t h a t C l i n t o n (1894) " i d e n t i f i e d h i s fungus as e i t h e r S^ . m i x t a or S. f e r a x . " I n f a c t , C l i n t o n d i d not do t h i s . He s t a t e s q u i t e c l e a r l y t h a t because he d i d not observe oogonia or a n t h e r i d i a , " i t was i m p o s s i b l e . . . to d e t e r m i n e e x a c t l y the s p e c i e s to which t h i s fungus b e l o n g s . " T i f f n e y (1939a) a l s o s t a t e s t h a t Hine (1878-79) r e p o r t e d t h a t A c h l y a p o l y a n d r a H i l d e b r a n d was a pa r a -s i t e . I n the pages o f Hine's a r t i c l e c i t e d by T i f f n e y (1939a) and l a t e r , by W i l s o n (1976), A. p o l y a n d r a i s not mentioned a t a l l , but l a t e r , i n the same paper, Hine notes t h a t he found A. p o l y -andra growing on a dead f r o g . T h i s h a r d l y c o n s t i t u t e s p r o o f o f p a r a s i t i s m ; n e v e r t h e l e s s , the e r r o r has been p e r p e t u a t e d i n the l i t e r a t u r e (e.g. see R e i c h e n b a c h - K l i n k e , 1973). P a r e n t h e t i c a l l y , i t s h o u l d be n o t e d t h a t Johnson (1956a) b e l i e v e s t h a t Hine's i n t e r p r e t a t i o n of A. p o l y a n d r a was i n c o r r e c t and t h a t Hine's 18 i s o l a t e was, i n f a c t , A c h l y a americana Humphrey. S c o t t (1964) r e p o r t s t h a t A. americana has been i s o l a t e d from i n f e c t e d f i s h , b ut h i s r e p o r t i s based on O'Bier's (1960) t h e s i s and O'Bier does not s t a t e whether the two i s o l a t e s of A. americana w h i c h he i s o -l a t e d from Lepomis m a c r o c h i r u s were o b t a i n e d from l i v i n g f i s h or no t . A l s o , i n o c u l a t i o n experiments w i t h t h e s e . . i s o l a t e s u s i n g wounded p l a t y f i s h were n e g a t i v e . T h e r e f o r e , an a s s e r t i o n t h a t A. americana i s a p a r a s i t e , based on the e v i d e n c e p r e s e n t e d by O'Bier (1960), S c o t t £ O'Bier (1962) or S c o t t (1964), i s of l i t t l e v a l u e and these r e f e r e n c e s have not been i n c l u d e d i n T a b l e I I . However, S c o t t £j Warren (1964) p r e s e n t much more c o n v i n c i n g e v i d e n c e r e g a r d i n g the p o t e n t i a l l y p a r a s i t i c n a t u r e o f ameri-cana i s o l a t e s and t h e r e f o r e , t h i s r e f e r e n c e i s c i t e d i n Table I I . There are o t h e r minor e r r o r s . N o l a r d - T i n t i g n e r (1974) i n -c o r r e c t l y c i t e s H o shina e t a l . (1960) as h a v i n g i n f e c t e d f i s h w i t h " A c h l y a s t e l l a t u s " , i n s t e a d o f Aphanomyces s t e l l a t u s . T i f f -ney, (1939a) has been c i t e d (e.g. W i l s o n , 1976) as h a v i n g i s o l a t e d D i c t y u c h u s monosporus from T r i t u r u s v i r i d e s c e n s . T i f f n e y (1939a) s t a t e s t h a t he d i d not observe oogonia i n t h i s i s o l a t e , and merely s u g g e s t e d , as a guess, t h a t i t might be D. monosporus. The p r e c e d i n g l i s t i n g o f e r r o r s c o u l d , perhaps w i t h some j u s t i f i c a t i o n , be d i s m i s s e d as p i c a y u n e n i t p i c k i n g . Neverthe-l e s s , I f e e l t h a t i t i s the m y c o l o g i s t ' s r e s p o n s i b i l i t y to make t h i s i n f o r m a t i o n a v a i l a b l e t o the f i s h p a t h o l o g i s t . For example, Wolke (1975) g i v e s a l i s t o f twenty-seven s p e c i e s i n the S a p r o l e g n i a l e s " o f c e r t a i n i d e n t i f i c a t i o n found i n a s s o c i a t i o n TABLE U S p e c i e s o f Oomycetes r e p o r t e d Fungi 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 c e a e A c h l y a spp. A c h l y a a m b i s e x u a l i s Raper * * A c h l y a americana Humphrey A c h l y a b i s e x u a l i s Coker § Couch A c h l y a d u b i a Coker A c h l y a f l a g e l l a t a Coker A c h l y a i n t r i c a t a Beneke * * A c h l y a k l e b s i a n a P i e t e r s * * * A c h l y a p r o l i f e r a Nees von Esenbeck * * A c h l y a racemosa H i l d e b r a n d * * A c h l y a s p a r r o w i i R e i s c h e r (= A. racemosa [Johnson, 1956]) as p a r a s i t e s o f f i s h . R e f e r e n c e s T i f f n e y (1939a); V i s h n i a c § N i g r e l l i (1957); W i l l o u g h b y (1970); Bhargava e t a l . (1971); N o l a r d - T i n t i g n e r (1973, 1974). V i s h n i a c § N i g r e l l i /1957) ( < / § $ ) ; W i l l o u g h b y (1970) ( d ) ; N o l a r d - T i n t i g n e r (1973, 1974) Co)• S c o t t $ Warren (1964). V i s h n i a c § N i g r e l l i (1957) (r/ § o) ; O' B i e r (1960); S c o t t § O ' B i e r (1962). Bhargava e t al. (1971) . T i f f n e y § Wolf (1937); T i f f n e y (1939a); Domashova (1971) . Howard et a l . (19 70) . V i s h n i a c § N i g r e l l i (1957). N o l a r d - T i n t i g n e r (1974). H o s h i n a e t a l . (1960) . V i s h n i a c § N i g r e l l i (1957). TABLE I I (Continued) S p e c i e s o f Oomycetes r e p o r t e d as p a r a s i t e s of f i s h . * R e f e r e n c e s Fungi Aphanomyces spp. **Aphanomyces l a e v i s de Bary **Aphanomyces s t e l l a t u s de Bary * * C a l y p t r a l e g n i a a c h l y o i d e s (Coker § Couch) Coker * * D i c t y u c h u s sp. * * D i c t y u c h u s monosporus L e i t g e b * * I s o a c h l y a m o n i l i f e r a de Bary (= S a p r o l e g n i a t o r u l o s a de Bary [Howard e t a l . , 1970] ) I s o a c h l y a u n i s p o r a Coker § Couch (= S a p r o l e g n i a u n i s p o r a [Seymour, 19 70]) L e p t o l e g n i a caudata de Bary * * P r o t o a c h l y a paradoxa Coker X= A c h l y a (-proto) p'aradoxa [ D i c k , 1973]) S a p r o l e g n i a spp. ( A l s o i n c l u d e s n o n - f r u i t i n g i s o l a t e s c l a s s i f i e d as S a p r o l e g n i a p a r a s i t i c a Coker or one of i t s synonyms which can be found i n Seymour [ 1970]:,.) Shanor § Saslow (1944); W i l l o u g h b y (1970) V i s h n i a c § N i g r e l l i (1957). H o s h i n a e t a l . (1960). V i s h n i a c $ N i g r e l l i (1957). N o l a r d - T i n t i g n e r (1974); T i f f n e y (1939a). N o l a r d - T i n t i g n e r (1974) . V i s h n i a c $ N i g r e l l i (1957). ° Domashova (1971). W i l l o u g h b y (1970). V i s h n i a c § N i g r e l l i (1957). H u x l e y (1882a, 1882b); C l i n t o n (1894) J o h n s t o n (1917); Coker (1923); D u f f (1930); T i f f n e y (1939a, 1939b); Chidambaram (1942); Chaudhuri e t a l . (1947); Aleem et a l . (1953); Lennon (1954); V i s h n i a c § N i g r e l l i (1957); A r a s a k i e t a l . (1958); O ' B i e r (1960); F u n g i TABLE I I (Continued) S p e c i e s of Oomycetes r e p o r t e d as p a r a s i t e s of f i s h . * R e f e r e n c e s S a p r o l e g n i a spp. (Continued) S a p r o l e g n i a sp. Type I (Some i s o l a t e s c o u l d p r o b a b l y be c l a s s i f i e d as S a p r o l e g n i a  paras i t i c a Coker emend. Kanouse.) S a p r o l e g n i a d e l i c a Coker (= S. d i c T T n a [Seymour, 1970]) S a p r o l e g n i a d i c l i n a Humphrey S a p r o l e g n i a f e r a x ( G r u i t h u i s e n ) Thuret S a p r o l e g n i a i n v a d e r i s Davis § L a z a r (= S. f e r a x [Seymour, 1970]) * * S a p r o l e g n i a megasperma Coker S a p r o l e g n i a m i x t a de Bary j- S. f e r a x [Seymour, 1970]) S a p r o l e g n i a monoica P r i n g s h e i m X= S. f e r a x [Seymour, 1970]) S c o t t c] O'Bier (1962); S c o t t § Warren (1964); Dudka (1964) ; S t u a r t ^ l E u l l e r (1968); Bhargava e t a l . (1971); Domashova (1971); N o l a r d - T i n t i g n e r (1971, 1973); Bootsma (1973); Johnson (1974). W i l l o u g h b y (1968, 1,969 , 1971, 1972). V i s h n i a c § N i g r e l l i (1957); O'Bier (1960); S c o t t §. 'O'Bier (1962); Dudka £ F l o r i n s k a y a (1971); N o l a r d - T i n t i g n e r (1973). McKay (1967); W i l l o u g h b y (1970); N o l a r d - ^ T i n t i g n e r (1970, 1973). T i f f n e y (1939a); V i s h n i a c § N i g r e l l i (1957); O'Bier ( I 9 6 0 ) ; H o s h i n a e t a l . ( I 9 6 0 ) ; S c o t t § O'Bier (1962); N o l a r d -T i n t i g n e r (1970, 1971, 1973); Bhargava e t a l . (1971). D a v i s § L a z a r (1941) . V i s h n i a c § N i g r e l l i (1957). V i s h n i a c § N i g r e l l i (1957); Dudka § F l o r i n s k a y a (1971). O'Bier (1960); S c o t t § O'Bier (1962); Domashova (1971). TABLE I I (Continued) S p e c i e s of Oomycetes r e p o r t e d as p a r a s i t e s of f i s h . F u ngi S a p r o l e g n i a p a r a s i t i c a Coker emend. Kanouse * * T h r a u s t o t h e c a c l a v a t a (de Bary) Humphrey * * T h r a u s t o t h e c a primoachlya- Coker § Couch X D i c k [1973] suggests t h a t t h i s s p e c i e s s h o u l d be p l a c e d i n the genus A c h l y a . ) L e p t o m i t a l e s L e p t o m i t a c e a e L e p t o m i t u s l a c t e u s (Roth) Agardh P e r o n o s p o r a l e s P y t h i a c e a e **Pythium sp. R e f e r e n c e s Rucker (1944) ; Hoshina e t a l . (1960); O ' Bier ( I 9 6 0 ) ; S c o t t § O 'Bier (1962); N o l a r d - T i n t i g n e r (1973) . V i s h n i a c § N i g r e l l i (1957). V i s h n i a c § N i g r e l l i (1957). Lennon (1954); W i l l o u g h b y (1970). S c o t t £ Warren (1964) *See a l s o T a b l e I and the d i s c u s s i o n i n the t e x t . T h i s l i s t does not i n c l u d e s p e c i e s i s o -l a t e d o n l y from dead f i s h or f i s h eggs, but does i n c l u d e most, i f not a l l , s p e c i e s r e p o r t -ed as n a t u r a l l y o c c u r r i n g or e x p e r i m e n t a l l y i n d uced p a r a s i t e s o f f i s h . * * S p e c i e s r e p o r t e d o n l y as e x p e r i m e n t a l l y induced p a r a s i t e s . N o t . r e p o r t e d as n a t u r a l l y oc-- c u r r i n g p a r a s i t e s . An e x c e p t i o n may be Pythium spp. b u t i t i s not c l e a r from the r e p o r t s by S c o t t § Warren (1964) or S t u a r t § F u l l e r (1968) whether the Pythium spp. they i s o l a t e d were o b t a i n e d from l i v i n g , n a t u r a l l y i n f e c t e d f i s h . * * * T h i s i s the o n l y r e c e n t r e f e r e n c e to i n f e c t i o n of f i s h by t h i s s p e c i e s . T h i s s p e c i e s was commonly r e p o r t e d to be a p a r a s i t e by e a r l i e r a u t h o r s (see d i s c u s s i o n i n t e x t ) but these r e f e r e n c e s have not been i n c l u d e d because I am e i t h e r not s u r e t h a t the i s o l a t e was o b t a i n -ed from a l i v i n g f i s h or not sure t h a t the i s o l a t e was p r o p e r l y i d e n t i f i e d . 23 w i t h n a t u r a l l y o c c u r r i n g and e x p e r i m e n t a l l y i n d u c e d d i s e a s e " i n f i s h i i By e l i m i n a t i n g synonyms, e x c l u d e d or d o u b t f u l t a x a , and e r r o n e o u s l y i n c l u d e d names, the l i s t i s reduced to n i n e t e e n s p e c i e s , but does not i n c l u d e A c h l y a a mericana, A c h l y a d u b i a , A c h l y a i n t r i c a t a , I s o a c h l y a (= S a p r o l e g n i a ) u n i s p o r a , or Sapro-l e g n i a d i c l i n a ( a l t h o u g h a synonym, S. d e l i c a , i s i n c l u d e d ) , a l l o f which have been r e p o r t e d as p a r a s i t e s o f f i s h . When the l i t e r a t u r e i s c a r e f u l l y a n a l y z e d , i t becomes abund-a n t l y c l e a r t h a t s p e c i e s b e l o n g i n g to the genus S a p r o l e g n i a sensu s t r i c t o ( D i c k , 1973) are by f a r the f u n g i most f r e q u e n t l y i s o l a t e d as n a t u r a l l y o c c u r r i n g p a r a s i t e s o f f i s h (see T a b l e I I ) . More p a r t i c u l a r l y , i t can be seen t h a t s t e r i l e ' S a p r o l e g n i a s p e c i e s , i s o l a t e s b e l o n g i n g to the S a p r o l e g n i a d i c l i n a - S a p r o l e g n i a p a r a -s i t i c a complex, and i s o l a t e s w hich can be i d e n t i f i e d as S a p r o l e g -n i a f e r a x are the ones most f r e q u e n t l y i s o l a t e d from l i v i n g f i s h . T h i s i s s c a r c e l y a r e v e l a t i o n , but the e v i d e n c e has not f r e q u e n t l y been c r i t i c a l l y e v a l u a t e d , and t h i s e x p l a i n s , i n p a r t , why I have c o n c e n t r a t e d on the taxonomy o f t h i s group of f u n g i i n t h i s t h e s i s . To put t h i s i n p e r s p e c t i v e , however, i t must be p o i n t e d out t h a t most s t u d i e s o f oomycete i n f e c t i o n s have been c a r r i e d out i n the temperate r e g i o n s o f the N o r t h e r n Hemisphere where S a p r o l e g -n i a s p e c i e s are abundant (Hughes, 1962; R o b e r t s , 1963; H u n t e r , 1975) . There have been few s t u d i e s of oomycete i n f e c t i o n s i n t r o p i c a l r e g i o n s where A c h l y a s p e c i e s appear to be much more abundant than S a p r o l e g n i a s p e c i e s (Hughes, 1962; A l a b i , 1971; N o l a r d - T i n t i g n e r , 1974). Under these c i r c u m s t a n c e s , one might 24 expect t h a t A c h l y a s p e c i e s would be the ones most f r e q u e n t l y i s o -l a t e d from d i s e a s e d f i s h , and the o n l y major s t u d y o f s a p r o l e g -n i a s i s i n t r o p i c a l r e g i o n s ( N o l a r d - T i n t i g n e r , 1974) s t r o n g l y s u p p o r t s t h i s s u p p o s i t i o n . I t s h o u l d be noted though, t h a t t h i s i s p r o b a b l y a somewhat o v e r - s i m p l i f i e d a n a l y s i s o f the r e l a t i o n between g e o g r a p h i c a l p o s i t i o n and the genera of s a p r o l e g n i a n f u n g i most commonly encountered as p a r a s i t e s o f f i s h . Johnson (1974) r e p o r t s t h a t A c h l y a s p e c i e s were more commonly i s o l a t e d than S a p r o l e g n i a s p e c i e s from "dead or d i s e a s e d f i s h " i n I c e l a n d . I n f e c t i o n s o f f i s h caused by Oomycetes o t h e r than A c h l y a s p e c i e s and S a p r o l e g n i a s p e c i e s appear to be i n f r e q u e n t , but I s u s p e c t t h a t when the t r o p i c a l r e g i o n s are more i n t e n s i v e l y s t u d -i e d , i n f e c t i o n s caused by Aphanomyces and D i c t y u c h u s s p e c i e s w i l l be more f r e q u e n t l y encountered. In 1964, an e p i z o o t i c o f salmon (Salmo s a l a r L.) was noted i n s o u t h e r n E i r e , and s u b s e q u e n t l y s p r e a d to Great B r i t a i n (Munro, 1970) and c o n t i n e n t a l Europe ( K i n k e l i n $ Turdu, 1971). A t the p r e s e n t time the d i s e a s e appears to have waned, but from 1966-1974 i t was a sour c e of c o n s i d e r a b l e c o n c e r n and, c o n s e q u e n t l y , the s u b j e c t o f much r e s e a r c h and numerous r e v i e w a r t i c l e s (Pye-f i n c h § E l s o n , 1967; Anonymous, 1968; E l s o n , 1968; C a r b e r y , 1968; Carbery § S t r i c k l a n d , 1968; S t r i c k l a n d § C a r b e r y , 1968; Munro, 1970; S t e v e n s o n , 1970; R o b e r t s , 1972; Murphy, 1973; W i l s o n , 1976). The d e t a i l e d c l i n i c a l d e s c r i p t i o n s by Huxley (1882a, 1882b), E d i n g t o n (1889), and o t h e r s , l e a v e l i t t l e r e a s o n to doubt t h a t t h i s l a t e s t e p i z o o t i c was v i r t u a l l y i d e n t i c a l to the 1877 e p i z o o t -25 i c . I n i t i a l l y c a l l e d ' I r i s h salmon d i s e a s e ' or ' c o l d water c o l u m n a r i s d i s e a s e ' , the d i s e a s e i s now u n i v e r s a l l y r e f e r r e d t o as U l c e r a t i v e Dermal N e c r o s i s (UDN) ( R o b e r t s , 1972) and i s con-s i d e r e d to be a d i s e a s e which i s d i s t i n c t from the 'normal' s a p r o l e g n i a s i s a s s o c i a t e d w i t h d e b i l i t a t e d or s e x u a l l y mature f i s h ( Roberts $ Shepherd, 1974; Bauduoy c^.Tuffery, 1973). A key f e a t u r e o f UDN i s l e s i o n s w h i c h o c c u r on the non-s c a l e d areas of the body, e s p e c i a l l y the head. The f i r s t l e s i o n s are s m a l l , o v a l , b l a n c h e d patches which f r e q u e n t l y become u l c e r -a t e d and then hemorrhagic. The cause o f these i n i t i a l l e s i o n s i s unknown. A f a v o u r e d h y p o t h e s i s (see W i l l o u g h b y [1972] f o r an i n t e r e s t i n g d i s s e n t i n g o p i n i o n ) i s t h a t they are caused by an e p i t h e l i o t r o p i c v i r u s ( R o b e r t s , 1972; O ' B r i e n , 1974), but as y e t t h e r e i s no d i r e c t e v i d e n c e to s u p p o r t t h i s h y p o t h e s i s . Whether or n o t a v i r u s i s i n v o l v e d , i t appears t h a t s t r e s s ( O ' B r i e n , 1974; R e i c h e n b a c h - K l i n k e , 1974), and p o s s i b l y the presence of s u b l e t h a l c o n c e n t r a t i o n s o f p o l l u t a n t s ( R e i c h e n b a c h - K l i n k e , 1974; Wachs, 1973), may a l s o c o n t r i b u t e to the development of the d i s e a s e . P a r e n t h e t i c a l l y , i t s h o u l d be n o t e d t h a t s i n c e sub-l e t h a l c o n c e n t r a t i o n s o f p o l l u t a n t s can i n v o k e a s t r e s s response (Donaldson § Dye, 1975), these two f a c t o r s cannot be c o n s i d e r e d to be u n r e l a t e d . The i n i t i a l l e s i o n s , whatever t h e i r cause, do not appear to cause f a t a l i t i e s p e r . s e , but a c t as f o c i f o r i n f e c t i o n by b a c t e r -i a l and f u n g a l p a r a s i t e s . E v i d e n c e s u p p o r t i n g t h i s v i e w p o i n t i s 26 based on the o b s e r v a t i o n t h a t the l e s i o n s can h e a l i f the f i s h are t r e a t e d w i t h m a l a c h i t e green to c o n t r o l the development o f the fungus (Dunne, 1970 ; R oberts e_t al_. , 1971). Hume P a t t e r s o n (1903) was c o n v i n c e d t h a t the 'salmon d i s -ease' was caused by a b a c t e r i u m - B a c i l l u s Salmonis P e s t i s . Sub-s e q u e n t l y , i t has been shown t h a t Hume P a t t e r s o n ' s B a c i l l u s  Salmonis P e s t i s was p r o b a b l y a mixed c u l t u r e of Aeromonas  l i q u e f a c i e n s and Pseudomonas f l u o r e s c e n s ( B i s s e t , 1946; C a r b e r y , 1968). These b a c t e r i a a r e a s s o c i a t e d w i t h hemorrhagic s e p t i c e m i a (Wolke, 1975). They are common i n f r e s h w a t e r and can be i s o l a t e d from h e a l t h y f i s h ( C o l l i n s , 1970). They are c o n s i d e r e d t o be common f a c u l t a t i v e f i s h pathogens, and are not now thought t o be the cause of UDN. N e v e r t h e l e s s , the h y p o t h e s i s t h a t UDN might be caused by a p a r t i c u l a r s p e c i e s o r s t r a i n o f b a c t e r i u m i s an a t -t r a c t i v e one and c e r t a i n l y one t h a t had to be i n v e s t i g a t e d . The most s e r i o u s p r o p o s a l to come out o f these i n v e s t i g a t i o n s was the s u g g e s t i o n t h a t UDN was caused by a myxobacterium and was, i n f a c t , a c o l d w a t e r form of 'columnaris d i s e a s e ' (Jensen, 1965; Brown, 1966; Brown § C o l l i n s , 1966). However, the workers who most f o r c e f u l l y propounded t h i s view l a t e r m o d i f i e d t h e i r s t a n d (Brown, 1968; C o l l i n s § Brown, 1968) and, a t the p r e s e n t t i m e , the w e i g h t o f the ev i d e n c e seems to be a g a i n s t the i d e a t h a t UDN i s caused by 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 . There i s now g e n e r a l agreement t h a t the fungus p l a y s an i m p o r t a n t r o l e i n the d i s e a s e and i t i s a l s o g e n e r a l l y conceded t h a t the development of the fungus on the l e s i o n s i s an e s s e n t i a l component o f the d i s e a s e syndrome. Roberts e_t a J . (1971) n o t e d 27 t h a t t h e i r e x p eriments i n d i c a t e d " t h e importance of d e n y i n g ac-cess to s p e c i e s o f the S a p r o l e g n i a group of f u n g i because the e s t a b l i s h m e n t o f the l a t t e r w i l l , i n most c a s e s . . . r e s u l t i n a f a t a l outcome." In another paper, Roberts (1972) a g a i n noted t h a t the i m p o r t a n t p a r t p l a y e d by S a p r o l e g n i a p a r a s i t i c a c o u l d not be over-emphasized.and s u g g e s t e d t h a t UDN s h o u l d be renamed the " U l c e r a t i v e E p i d e r m a l N e c r o s i s - S a p r o l e g n i a s i s Complex" which emphasizes the f a c t t h a t n e c r o s i s of the e p i d e r m i s and i n f e c t i o n by S a p r o l e g n i a s p e c i e s are the two dominant and c o n s i s t e n t char-a c t e r i s t i c s o f the d i s e a s e . This c o n c l u s i o n p a r t i a l l y r e v e r s e s the u s u a l t r e n d i n d e a l i n g w i t h oomycete i n f e c t i o n s o f f i s h . There i s a s t r o n g tendency ( B e l l S, H o s k i n s [1971], t o n g u e - i n - c h e e k , c a l l i t " an a r t i c l e o f f a i t h " ) among hatcherymen and f i s h p a t h o l o g i s t s to m i n i m i z e the r o l e of s a p r o l e g n i a n f u n g i i n f i s h d i s e a s e because they are con-s i d e r e d to be 'secondary i n v a d e r s ' c a p a b l e of i n f e c t i n g o n l y ' d e b i l i t a t e d ' f i s h . There i s much t r u t h i n t h i s n o t i o n b u t , i n my o p i n i o n , i t i s not the whole t r u t h and represents; an over-s i m p l i f i e d v i e w p o i n t . T h i s a s p e c t o f s a p r o l e g n i a s i s w i l l be d i s c u s s e d i n d e t a i l i n the p a t h o l o g y s e c t i o n o f t h i s t h e s i s ; however, g i v e n the assumption t h a t s a p r o l e g n i a n f u n g i are merely 'sa p r o p h y t e s ' which can o c c a s i o n a l l y f u n c t i o n as 'secondary i n v a d e r s ' , and g i v e n the f a c t t h a t a v a r i e t y of s a p r o l e g n i a n f u n -g i seem to be p o t e n t i a l p a r a s i t e s , then one might e x p e c t by d e d u c t i o n t h a t f i s h may become i n f e c t e d on a s o r t o f ' f i r s t come, f i r s t s e r v e d ' b a s i s by whatever water moulds happen to i n h a b i t 28 t h a t p a r t i c u l a r w a t e r s h e d . For most, i f not a l l , s a p r o l e g n i a n i n f e c t i o n s of f i s h , and p a r t i c u l a r l y f o r s a p r o l e g n i a n i n f e c t i o n s of s a l m o n i d s , t h i s does not appear to be the case a t a l l . W i l -loughby (1968, 1969a, 1971, 1972) has done the most work on t h i s a s p e c t and m a i n t a i n s t h a t a " u s u a l l y s e x u a l l y s t e r i l e " but " d i s t i n c t and r e c o g n i z a b l e " s t r a i n c a l l e d " S a p r o l e g n i a Type I " ( W i l l o u g h b y , 1969a) i s " t h e almost i n v a r i a b l e f u n g a l p a r a s i t e o f s a l m o n i d f i s h i r r e s p e c t i v e of t h e i r s i t u a t i o n and i n c l u d i n g such a p p a r e n t l y c o m p l i c a t e d ( v i r u s i n v o l v e d ? ) c o n d i t i o n s as U'.D.N." ( W i l l o u g h b y , 1971). T h i s w i l l be c o n s i d e r e d i n d e t a i l i n the taxonomy s e c t i o n of t h i s t h e s i s because W i l l o u g h b y ' s S a p r o l e g n i a Type I appears, i n many i m p o r t a n t r e s p e c t s , to be i d e n t i c a l to most of the i s o l a t e s w hich I have o b t a i n e d from P a c i f i c salmon (Oncorhynchus s p p . ) . 29 SAPROLEGNIASIS IN THE PACIFIC NORTHWEST2-- A BRIEF REVIEW S a p r o l e g n i a s i s i s w i d e l y r e c o g n i z e d as a major a f f l i c t i o n o f P a c i f i c salmon (Qncorhynchus spp.) when they are i n f r e s h w a t e r ( F o e r s t e r , 1968; Ivankov, 1971; Wood, 1974) and r e p o r t s d e s c r i b -i n g s a p r o l e g n i a s i s of thes e f i s h i n B r i t i s h Columbia and C a l i f o r -n i a go back almost one hundred y e a r s ( B u c k l a n d et. al_. , 1880). D e s p i t e t h i s , t h e r e have been few s t u d i e s o f the p a t h o l o g y of sap-r o l e g n i a s i s of P a c i f i c salmon o r o f the taxonomy o f the f u n g i a s s o c i a t e d w i t h these i n f e c t i o n s - - the two n o t a b l e e x c e p t i o n s b e i n g the u n p u b l i s h e d t h e s e s o f Rucker (1944) and McKay (1967). Rucker s t u d i e d s a p r o l e g n i a s i s o f chin o o k salmon (0. tshawy-t s c h a (Walbaum)), sockeye salmon (0. ne r k a (Walbaum)), and s t e e l -head t r o u t (anadromous Salmo g a i r d n e r i R i c h a r d s o n ) from the Columbia R i v e r d r a i n a g e system. He c o n c l u d e d t h a t the fungus, w h i c h he i d e n t i f i e d as S a p r o l e g n i a p a r a s i t i c a Coker, was a sec-ondary i n v a d e r w h i c h c o u l d n o t n o r m a l l y i n i t i a t e an i n f e c t i o n and t h a t the p r i m a r y cause of the i n f e c t i o n s was the myxobacter-ium, F l e x i b a c t e r c o l u m n a r i s (= B a c i l l u s c o l u m n a r i s ) . Subsequent s t u d i e s have shown t h a t c o l u m n a r i s d i s e a s e i s common i n the Columbia and F r a s e r r i v e r s (Pacha § O r d a l , 1970) and t h i s has h e l p e d t o encourage the v i e w p o i n t t h a t S a p r o l e g n i a s p e c i e s can The term ' P a c i f i c Northwest', as used h e r e , r e f e r s to Oregon, Washington and B r i t i s h Columbia from the P a c i f i c c o a s t e a s t to the Rocky Mountains. 30 cause o n l y secondary i n f e c t i o n s . McKay (1967) c a r r i e d out a s e r i e s o f i n f e c t i o n experiments a t d i f f e r e n t t emperatures u s i n g young coho salmon (0. k i s u t c h (Walbaum)) and an i s o l a t e i d e n t i f i e d as S a p r o l e g n i a d i c l i n a Humphrey. As a r e s u l t of these e x p e r i m e n t s , she c o n c l u d e d t h a t the fungus c o u l d i n i t i a t e i n f e c t i o n s and she a l s o c o n c l u d e d t h a t t h e r e was a d i r e c t c o r r e l a t i o n between i n c r e a s i n g temperature and the i n c i d e n c e and r a t e o f i n f e c t i o n . Rucker (1944) had a l s o t r i e d to i n f e c t young salmon and t r o u t and r e p o r t e d t h a t most of h i s attempts had ended i n f a i l -u r e and o n l y a few i n f e c t i o n s were o b s e r v e d , e x c e p t i n the case where the f i s h were wounded and s i m u l t a n e o u s l y c h a l l e n g e d w i t h F l e x i b a c t e r c o l u m n a r i s . These r e s u l t s c o n t r a d i c t t h o s e o b t a i n e d by McKay (1967) b u t , as w i l l be d i s c u s s e d l a t e r , t h e r e may be a r e a s o n a b l e e x p l a n a t i o n f o r t h i s c o n t r a d i c t i o n based on the r e l a -t i o n of s t r e s s to d i s e a s e r e s i s t a n c e of f i s h (Wedemeyer, 1970). Most i n v e s t i g a t i o n s o f oomycete i n f e c t i o n s i n the P a c i f i c Northwest have been concerned w i t h anadromous s a l m o n i d s , b u t some work has been done on l a c u s t r i n e s a lmonids as w e l l . B e l l § H o s k i n s (1971) have d i s c u s s e d a massive e p i z o o t i c of mountain w h i t e f i s h (Prosopium w i l l i a m s o n i ( G i r a r d ) ) i n the West Arm of Kootenay Lake i n the autumn of 1969 (see P l a t e I , F i g . 1 ) . The cause of t h i s e p i z o o t i c was not d e t e r m i n e d and can be added to a growing l i s t o f w h i t e f i s h d i s e a s e s of obscure e t i o l o g y (Roberts e t a l . , 1970a; J a n d e r , 1974). The moribund w h i t e f i s h examined by B e l l § H o s k i n s (1971) a l l had oomycete i n f e c t i o n s , but these 31 P l a t e I F i g u r e 1 - Mou n t a i n w h i t e f i s h (Prosopium w i l l j a m s o n i ( G i r a r d ) ) c o l l e c t e d from the West Arm o f Kootenay Lake on 1 September 1971 by Mr. Myles Crowly, F i s h $ W i l d l i f e Branch b i o l o g i s t a t N e l s o n . The f i s h was dead when I examined i t , but a c c o r d i n g to Mr. Crowly i t resem-b l e d the w h i t e f i s h t h a t d i e d d u r i n g the e p i z o o t i c . T h i s f i s h had t h r e e fungus i n f e c t e d l e s i o n s on the r i g h t s i d e o f i t s body, one i n the v i c i n i t y o f the p e c t o r a l f i n , and two more ( e n c i r c l e d by arrows) f a r t h e r back on the body. The l e s i o n s a re not q u i t e the same as those d e s c r i b e d by B e l l § H o s k i n s (1971) as ' t y p i c a l ' f o r i n f e c t e d w h i t e f i s h from t h i s a r e a i n 1969, and, as the f i s h examined was dead, the o b s e r v a t i o n s r e p o r t e d here are o f o n l y l i m i t e d v a l u e . re 2 - Kokanee ( l a c u s t r i n e Qncorhynchus n e r k a (Walbaum)) i n R e d f i s h Creek (49°37'N, 117 U03'W) which d r a i n s i n t o Kootenay Lake on the N o r t h s i d e of the West Arm. Note the l a r g e p a t c h of fungus i n the v i c i n i t y o f the d o r s a l f i n . T h i s i s a t y p i c a l a r e a f o r i n f e c t i o n o f P a c i f i c salmon by s a p r o l e g n i a n f u n g i . Photograph . t a k e n 2 September 1971. © 33 a u t h o r s d i d not c o n s i d e r them to be e x t e n s i v e enough to account f o r the demise of the f i s h . B e l l 8j H o s k i n s (1971) a l s o d i s c u s s e d oomycete i n f e c t i o n s of a d u l t rainbow t r o u t i n the Lardeau R i v e r near G e r r a r d , B r i t i s h Columbia and I have seen a d u l t kokanee ( l a c u s t r i n e p. nerka) w i t h oomycete i n f e c t i o n s i n nearby R e d f i s h Creek ( P l a t e I , F i g . 2 ) . Oomycete i n f e c t i o n s o f a d u l t salmonids are n o t , i n f a c t , u n u s u a l and do not u s u a l l y arouse any c o n c e r n u n l e s s they are a s s o c i a t e d w i t h a s i g n i f i c a n t amount of prespawn-i n g m o r t a l i t y . As w i l l be d i s c u s s e d i n the p a t h o l o g y s e c t i o n of t h i s t h e s i s , t h e r e i s v e r y l i k e l y a d i r e c t r e l a t i o n s h i p between s a p r o l e g n i a s i s and the e n d o c r i n e p h y s i o l o g y o f m a t u r i n g s a l m o n i d s , w hich i n t u r n i s a l s o r e l a t e d to the r o l e o f s t r e s s i n p r e c i p i -t a t i n g s a p r o l e g n i a n i n f e c t i o n s . A p a r t from work on the c o m m e r c i a l l y v a l u a b l e s a l m o n i d s , l i t t l e has been p u b l i s h e d on s a p r o l e g n i a s i s of o t h e r f i s h s p e c i e s i n t h i s a r e a . L e s t e r (1974) r e p o r t e d the i s o l a t i o n of S a p r o l e g -n i a paras i t i c a from s t i c k l e b a c k s (Gas t e r o s teus a c u l e a t u s L.) a t the U n i v e r s i t y of B r i t i s h Columbia, and oomycete i n f e c t i o n s o f o t h e r s p e c i e s of f i s h m a i n t a i n e d i n the f r e s h w a t e r h o l d i n g f a c i l i t i e s a t t h i s i n s t i t u t i o n have a l s o been observed. Whenever p o s s i b l e , the t e r m i n o l o g y used f o r i n t e r v a l s i n f i s h development i s based on B a l o n (1975). A c c o r d i n g to B a l o n , the a d u l t p e r i o d "commences w i t h the f i r s t m a t u r a t i o n of gametes and i s c h a r a c t e r i z e d by a spawning r u n . . . " 34 OBSERVATIONS ON THE TAXONOMY AND PHYSIOLOGY OF ISOLATES BELONGING TO THE GENUS SAPROLEGNIA SENSU STRTCTO WITH EMPHASIS ON THOSE INCLUDED IN THE SAPROLEGNIA DICLINA-SAPROLEGNIA PARASITICA COMPLEX PART I - ISOLATION AND CHARACTERIZATION OF THE SAPROLEGNIA ISOLATES OBTAINED FROM PACIFIC SALMON DESCRIPTION OF THE STUDY AREA The f u n g i d e s c r i b e d i n t h i s p a r t o f the t h e s i s were a l l ob-t a i n e d from P a c i f i c salmon o r s t e e l h e a d t r o u t (Table IV) a t the Ro b e r t s o n Creek H a t c h e r y (49°20'N, 124°59'W) on Vancouver I s l a n d R o b e rtson Creek, a secondary o u t l e t o f Great C e n t r a l Lake ( P l a t e I I ) , i s an a r t i f i c i a l spawning c h a n n e l w h i c h was c o n s t r u c t e d by 1961. Lucas (1960) d i s c u s s e s the r e l e v a n t h i s t o r y o f the a r e a and d e s c r i b e s the s t r u c t u r e o f the spawning c h a n n e l i n d e t a i l ( a l s o , see Bardach e_t_ a l . , 1972). A major problem hampering the suc c e s s o f t h i s spawning c h a n n e l has been e x c e s s i v e prespawning m o r t a l i t y of a d u l t f i s h w hich u s u a l l y have o b v i o u s , and o f t e n e x t e n s i v e , oomycete i n f e c t i o n s . These f a c t s , combined w i t h the f a c t t h a t R o b e r t s o n Creek may become ( f o r a salmon stream) q u i t e warm i n the summer ( F i g u r e 1, T a b l e I I I ) , l e d McKay (1967) to i n v e s t i g a t e the r e l a t i o n between temperature and the i n c i d e n c e o f s a p r o l e g n i a s i s . The water f l o w i n g i n t o R o b e r t s o n Creek comes from the s u r -f a c e o f Great C e n t r a l Lake w h i c h , i n the summer, reaches a 35 P l a t e I I A e r i a l view o f R o b e r t s o n Creek (49°20'N, 124°59'W). The arrows i n d i c a t e the p o s i t i o n s o f the i n l e t i n Boot Lagoon and the o u t l e t i n the Stamp R i v e r . The photographs were t a k e n i n J u l y , 1968 and t h e r e f o r e do not show the r e c e n t l y b u i l t h a t c h e r y s t r u c t u r e s . S c a l e 1:20,000. (B. C. Government A i r Photos.) 36 TABLE I I I Temperature r e c o r d s f o r Robertson Creek (21 December 1970 - 20 December 1971).* Month Mean High Temp. (C) Mean Low Temp. (C) Range (C) December, 1970 4.4 4.1 3.3-4.7 (21-31) J a n u a r y , 19 71 3.9 3.6 2.8-4.2 F e b r u a r y , 1971 4.2 3.8 3.0-4.4 March, 1971 4.5 4.1 3.0-5.0 A p r i l , 1971 7.0 6.6 5.0-9.4 May, 1971 12.1 11.6 9.2-14.2 June, 1971 14.0 13.6 10.8-15.3 J u l y , 1971 17.6 16.7 13.3-21.1 August, 19 71 19.1 18.3 16.1-21.9 September, 1971 16.5 15.9 14.2-18.3 October, 1971 13.0 12.5 9.2-15.3 November, 1971 7.9 7.7 6.7-9.4 December, 1971 5.1 4.9 3.6-6.7 (1-20) *Based on c o n t i n u o u s temperature r e c o r d s made by Mr. J a c k Baker who k i n d l y l o a n e d me the c h a r t s f o r a n a l y s i s . 38 2 OP D J F M A M J J A S O N D 1970 1971 M o n t h F i g u r e 1 - Mean monthly temperatures o f R o b e r t s o n Creek (December, .1970 - December, 1971). 39 temperature o f 20 C (Parsons e_t a l . , 1972) . P r i o r to f l o w i n g i n t o R o b e r t s o n Creek, the water f i r s t passes through the r e l a t i v e -l y b r oad and s h a l l o w Boot Lagoon ( P l a t e I I ) and may become warm-ed f u r t h e r ; c o n s e q u e n t l y , i t i s not u n u s u a l f o r R o b e r t s o n Creek t o have temp e r a t u r e s around 22 C f o r p a r t of the summer (Table I I I ) . As a g e n e r a l r u l e , i t i s not d e s i r a b l e to keep salmon i n water w i t h a temperature much i n excess of 15 C (Bardach et. a l . , 1972; B r e t t e t a l . , 1969) and i n 1971, f o r example, R o b e r t s o n Creek exceeded t h i s temperature f o r almost a q u a r t e r (221) of the y e a r . In a d d i t i o n t o t e m p e r a t u r e , I have c a r r i e d out l e s s e x t e n -s i v e a n a l y s i s of 0 2 , pH, C 0 2 , and a l k a l i n i t y i n Ro b e r t s o n Creek. The r e s u l t s a re c o n s i s t e n t w i t h what would be ex p e c t e d of a w e l l - a e r a t e d , o l i g o t r o p h i c stream i n t h i s a r e a . The water i s always s a t u r a t e d w i t h oxygen and c o n s e q u e n t l y v a r i e s o n l y as the temperature v a r i e s , and i s i n excess o f 8.0 ppm a t a l l t i m e s . The pH remains r e l a t i v e l y c o n s t a n t around the s l i g h t l y a l k a l i n e s i d e of n e u t r a l i t y , C 0 2 i s <:< 5 ppm (the l i m i t of r e s o l u t i o n of the t e s t employed), and a l k a l i n i t y i s about 17 ppm CaC0 3 per g a l l o n . In 1972, a h a t c h e r y was b u i l t near the o u t l e t o f Ro b e r t s o n Creek by the Department o f the Environment, F i s h e r i e s and Marine S e r v i c e . T h i s h a t c h e r y , which a t p r e s e n t i s j u s t r e a c h i n g f u l l p r o d u c t i o n o f about f i v e m i l l i o n eggs, i s p r i m a r i l y concerned w i t h the c u l t u r e of chinook salmon, w i t h a l e s s e r emphasis on the p r o d u c t i o n o f coho salmon and s t e e l h e a d t r o u t . One r e a s o n 40 f o r c o n c e n t r a t i n g on the p r o d u c t i o n of chinook salmon i n p r e f e r -ence to o t h e r s p e c i e s , i s t h a t they can be r e l e a s e d a f t e r a f r e s h w a t e r n u r s e r y p e r i o d of about 90 days and c o n s e q u e n t l y , w i l l u s u a l l y a v o i d the warmest months o f the y e a r . The main spawning c h a n n e l has been more or l e s s n e g l e c t e d f o r s e v e r a l y e a r s , and i s not b e i n g m a i n t a i n e d . ISOLATION OF BACTERIA-FREE, SINGLE SPORE ISOLATES Immediately a f t e r the f i s h was k i l l e d o r , i n the case of dead f i s h , soon a f t e r c o l l e c t i o n , mycelium was removed from the f i s h w i t h s t e r i l e tweezers and p l a t e d d i r e c t l y onto Y p S s - t e l l u r -i t e agar ( D i f c o Emerson's YpSs agar w i t h 0.1 g per l i t e r p o t a s -sium t e l l u r i t e [K^TeOa] added as a b a c t e r i o s t a t i c agent [ W i l l o u g h -by, 1 9 7 0 ] ) . The c o l l e c t i o n d a t a are g i v e n i n T a b l e IV. U s u a l l y the f u n g i were s u b c u l t u r e d once more onto Y p S s - t e l l u r i t e agar b e f o r e b e i n g t r a n s f e r r e d to p l a i n YpSs agar f o r maintenance. T h i s t e c h n i q u e e i t h e r e l i m i n a t e d the b a c t e r i a c o m p l e t e l y from the c u l t u r e s , o r , a t l e a s t reduced the problem to a manageable l e v e l so t h a t the c o n t a m i n a t e d c u l t u r e s c o u l d be m a i n t a i n e d i n a r e f r i g e r a t o r (but not a t room temperature) w i t h o u t b e i n g over-grown by t h e i r b a c t e r i a l c o n t a m i n a n t s . There was no problem w i t h f u n g a l c o n t a m i n a n t s . S i n g l e spore i s o l a t e s were o b t a i n e d from t h e s e c u l t u r e s as f o l l o w s : B l o c k s of YpSs agar c o n t a i n i n g mycelium were t r a n s f e r r e d to p e t r i d i s h e s c o n t a i n i n g s t e r i l e d i s t i l l e d water and i n c u b a t e d f o r a day a t 20 C to induce z o o s p o r o g e n e s i s . The zoospores were TABLE IV C o l l e c t i o n d a t a f o r S a p r o l e g n i a i s o l a t e s o b t a i n e d from s a l m o n i d f i s h a t R o bertson Creek, 1973. I s o l a t e Date of Sex o f I n t e r v a l o f C o n d i t i o n o f F i s h D e s i g n a t i o n C o l l e c t i o n S p e c i e s of F i s h F i s h Ontogeny J u s t P r i o r to C o l l e c t i o n GAN 0 10 Aug. s. g a i r d n e r i 1 - f r y 5 dead GAN 1 12 Aug. 0. n e r k a 2 a d u l t dead < 24hr GAN 2 16 Aug. i t ? i t dead < 24 h'. GAN 3 11 II 9 i t a l i v e GAN 4 11 9 i t t i GAN 5 11 Aug. i t ? II a l i v e / m o r i b u n d GAN 6 14 Aug. s. g a i r d n e r i f r y a l i v e / a c t i v e GAN 7 15 Aug. i t - i t dead GAN 8 - t t i t GAN 9 II 0. t s h a w y t s c h a 3 - t i a l i v e GAN 10 i t t i - II dead GAN 14 04 Oct. 0. nerka c/ a d u l t a l i v e / a c t i v e / e r r a t i c GAN 16 05 Oct. 0. k i s u t c h 4 9 t t a l i v e / a c t i v e GAN 17 11 0. nerka 9 i t i t GAN 18 II 9 t t 11 GAN 19 06 Oct. 11 1 1 GAN 20 II GAN 21 11 9 II 11 GAN 22 i t 4 t t t t GAN 23 18 Oct. 9 II t t GAN 24 i t 2 t t t t TABLE IV (Continued) C o l l e c t i o n d a t a f o r S a p r o l e g n i a i s o l a t e s o b t a i n e d from s a l m o n i d f i s h a t R o b e r t s o n Creek, 1973. I s o l a t e D e s i g n a t i o n Date o f Sex of I n t e r v a l o f C o l l e c t i o n S p e c i e s of F i s h F i s h Ontogeny C o n d i t i o n o f F i s h J u s t P r i o r to C o l l e c t i o n GAN 2 5 GAN 26 GAN 27 GAN 2 8 GAN 29 GAN 30 GAN 31 GAN 3 2 GAN 33 19 Oct t t t t 20 Oct, 25 Oct, 26 Oct, 27 Oct, 0. nerk a 0. tshawytscha 0. n e r k a 4 4 4 4 4 4 4 a d u l t a l i v e / a c t i v e ( j a c k ) ( j a c k ) ( j a c k ) a l i v e / m o r i b u n d a l i v e / a c t i v e Notes: 1Salmo g a i r d n e r i R i c h a r d s o n ( s t e e l h e a d t r o u t ) . 2 0 n c o r h y n c h u s nerka (Walbaum) (sockeye salmon). 3 0 n c o r h y n c h u s tshawytscha (Walbaum) (chinook salmon). ^Oncorhynchus k i s u t c h (Walbaum) (coho salmon). 5The term ' f r y ' i s not one o f the terms approved by B a l o n (1975). As used here to the young of the year b e i n g r a i s e d i n the h a t c h e r y p r i o r to r e l e a s e . I t i s i t r e f e r s w h i c h ones f i t Balon's u n c e r t a i n d e f i n i t i o n of an a l e v i n and which would have been d e f i n e d as s m o l t s 43 then drawn o f f i n a s m a l l volume o f water w i t h a P a s t e u r p i p e t t e and s p r e a d over the s u r f a c e o f a c o r n meal agar p l a t e . A f t e r a few hours i n c u b a t i o n , i n d i v i d u a l g e r m i n a t i n g spores were removed from the p l a t e w i t h a m i c r o s p a t u l a and i n o c u l a t e d onto a f r e s h YpSs agar p l a t e . The n e x t s t e p was to de t e r m i n e e x a c t l y which i s o l a t e s were co n t a m i n a t e d by b a c t e r i a . A r e l a t i v e l y s i m p l e way to do t h i s i s to i n o c u l a t e the c u l t u r e s i n t o a b r o t h d e s i g n e d to encourage the growth o f b a c t e r i a , grow them i n a shaker i n c u b a t o r , and examine them p e r i o d i c a l l y f o r t u r b i d i t y . I f no contaminants are p r e s e n t , the b r o t h w i l l r emain c l e a r . The t e c h n i q u e I used was as f o l l o w s : A b l o c k o f YpSs agar c o n t a i n i n g mycelium was i n o c u l a t e d i n -to a f l a s k c o n t a i n i n g 50 ml TYE b r o t h ( t r y p t o n e 10 g, y e a s t e x t r a c t 5 g, and NaCl 5 g per l i t e r o f d i s t i l l e d w a ter w i t h the pH a d j u s t e d to 7.3 ± 0.1 w i t h KOH p r i o r to a u t o c l a v i n g ) and then p l a c e d i n a r e c i p r o c a t i n g i n c u b a t o r - s h a k e r m a i n t a i n e d a t 21 ± 1 C and o p e r a t i n g a t about 60 o s c i l l a t i o n s per minute w i t h a 5-cm s t r o k e . T h i s p r o v i d e s a g e n t l e r o c k i n g motion which mixes and a e r a t e s the c u l t u r e . R e s u l t s were u s u a l l y o b t a i n e d w i t h i n 24 h, but the c u l t u r e s s h o u l d be l e f t a t l e a s t 72 h i n case the i n i t i a l b a c t e r i a l i n o c u l u m was low. The r e s u l t s o f t h i s work showed t h a t s e v e n t e e n o f my t h i r t y s i n g l e spore i s o l a t e s were f r e e o f b a c t e r i a , and the r e m a i n i n g t h i r t e e n were co n t a m i n a t e d . I t i s sometimes d i f f i c u l t to o b t a i n b a c t e r i a - f r e e c u l t u r e s 44 o f S a p r o l e g n i a s p e c i e s . The b a c t e r i a t h a t o ccur i n f r e s h w a t e r and on f i s h are u s u a l l y v a r i o u s gram-negative rods ( B e l l e t a l . , 1971; B u l l o c k , 1971; C o l l i n s , 1970) which are o f t e n m o t i l e and can c o e x i s t w i t h S a p r o l e g n i a s p e c i e s under a wide range of c u l -t u r a l c o n d i t i o n s . C o n s e q u e n t l y , a v a r i e t y o f methods have been d e v i s e d to c o n t r o l or e l i m i n a t e b a c t e r i a i n c u l t u r e s o f these f u n g i (see N e i s h , 1975a). The u s u a l p r a c t i c e these days i s to use v a r i o u s a n t i b a c t e r -i a l agents to f r e e f u n g i from b a c t e r i a . W i t h r e g a r d to S a p r o l e g -n i a s p e c i e s , the two most w i d e l y used are p o t a s s i u m t e l l u r i t e , used as p r e v i o u s l y d e s c r i b e d , and p e n i c i l l i n G p l u s s t r e p t o m y c i n s u l f a t e w h i c h are i n c o r p o r a t e d i n t o agar media a t a c o n c e n t r a t i o n o f 0.5 g per l i t e r each when the agar i s as c o o l as p o s s i b l e but s t i l l m o l t e n . I d e c i d e d to t e s t a n o ther a n t i b i o t i c , c a r b e n i c i l l i n , as a p o s s i b l e a l t e r n a t i v e to the p e n i c i l l i n - s t r e p t o m y c i n c o m b i n a t i o n . C a r b e n i c i l l i n ( d i s o d i u m a - c a r b o x y b e n z y l p e n i c i l l i n , marketed under the t r a d e names 'Pyopen' [ A y e r s t , Beecham] and 'Geopen' [ P f i z e r ] ) has been i n c l i n i c a l use s i n c e about 1967 and i s used to t r e a t p y o g e n i c Pseudomonas a e r u g i n o s a i n f e c t i o n s . I t has a p p a r e n t l y not been used p r e v i o u s l y f o r o b t a i n i n g b a c t e r i a - f r e e f u n g a l c u l t u r e s . I t i s a s e m i s y n t h e t i c p e n i c i l l i n w i t h a w i d e r gram-n e g a t i v e spectrum than p e n i c i l l i n G ( K u c e r s , 1972) but the same mode of a c t i o n ( i . e . i n h i b i t i o n of b a c t e r i a l c e l l w a l l s y n t h e s i s ) . C a r b e n i c i l l i n i s somewhat l e s s s t a b l e than p e n i c i l l i n G and s h ares the same t e n d e n c i e s to be heat and a c i d l a b i l e i n s o l u -45 t i o n . T h e r e f o r e I d e c i d e d a g a i n s t the u s u a l method o f i n c o r p o r -a t i n g the a n t i b i o t i c i n t o warm agar and d e v i s e d the f o l l o w i n g method from the one used to check the c u l t u r e s f o r the presence of b a c t e r i a . From the edge o f one of the con t a m i n a t e d c o l o n i e s a c t i v e l y growing i n YpSs agar, a s m a l l b l o c k o f mycelium was removed and c a r e f u l l y p l a c e d i n t o a 250-ml beaker c o n t a i n i n g 50 ml of c o o l TYE b r o t h . The c a r b e n i c i l l i n , d i s s o l v e d i n s t e r i l e d i s t i l l e d w ater and d i l u t e d to t h e - d e s i r e d c o n c e n t r a t i o n , was added to the b r o t h j u s t b e f o r e or a f t e r the a d d i t i o n o f the b l o c k o f mycelium. S i n c e c a r b e n i c i l l i n comes i n a v i a l as a r e a d i l y s o l u b l e s t e r i l e powder, the most c o n v e n i e n t way to d i s s o l v e , d i l u t e , and d e l i v e r i t , was w i t h a s y r i n g e . The b r o t h p l u s a n t i b i o t i c was then p l a c e d i n a r e c i p r o c a t i n g s h a k e r - i n c u b a t o r and i n c u b a t e d i n the manner d e s c r i b e d p r e v i o u s l y . A f t e r about 24 h, i f the experiment was s u c c e s s f u l , the c u l t u r e was f r e e of b a c t e r i a and the mycelium was removed from the b e a k e r , p l a t e d out on YpSs agar and a l l o w e d t o grow i n t o the agar. S u b c u l t u r e s were d e r i v e d from t h i s p l a t e and were t e s t e d a g a i n , u s i n g the p r e v i o u s l y d e s c r i b e d method, to be sure t h a t the b a c t e r i a l c o n t a m i n a t i o n had been e l i m i n a t e d . The c o n c e n t r a t i o n o f a n t i b i o t i c n e c e s s a r y f o r a s u c c e s s f u l t r i a l was, o f c o u r s e , d e t e r m i n e d by expe r i m e n t . I found t h a t f o r 8 o f the 13 c u l t u r e s , c a . 2 mg/ml o f c a r b e n i c i l l i n was s u f f i c i e n t whereas f o r two o t h e r s c a . 4 mg/ml was n e c e s s a r y . The r e m a i n i n g t h r e e c u l t u r e s d i d not respond to c a . 6.5 mg/ml o f c a r b e n i c i l l i n 46 but d i d respond to c a . 4000 u n i t s / m l o f p o t a s s i u m p e n i c i l l i n G p l u s c a . 1 mg/ml of s t r e p t o m y c i n s u l f a t e (the l o w e s t c o n c e n t r a -t i o n u s e d ) . The method used f o r the p e n i c i l l i n G p l u s s t r e p t o -mycin c o m b i n a t i o n was the same as t h a t used f o r the c a r b e n i c i l -l i n , e x c e p t t h a t the a n t i b i o t i c s had to be f i l t e r s t e r i l i z e d p r i o r to use. FIELD OBSERVATIONS In a d d i t i o n t o o b t a i n i n g c u l t u r e s o f f u n g i from the f i s h , I a l s o examined water mounts o f mycelium removed d i r e c t l y from the l e s i o n s . T h i s mycelium was found t o be p r o d u c i n g numerous t e r -m i n a l s p o r a n g i a ( P l a t e I I I , F i g . 1) and u s u a l l y the f u n g i c o u l d be p r e s u m p t i v e l y i d e n t i f i e d as S a p r o l e g n i a s p e c i e s by o b s e r v i n g i n t e r n a l p r o l i f e r a t i o n or p o s i t i v e l y i d e n t i f i e d as S a p r o l e g n i a s p e c i e s by o b s e r v i n g zoospore r e l e a s e . Two a t y p i c a l types o f s p o r a n g i a were not e d : (1) s p o r a n g i a w i t h l a t e r a l e x i t p a p i l l a e ( P l a t e I I I , F i g s . 1-3) and (2) a p l a n o i d s p o r a n g i a ( P l a t e I I I , F i g . 4 ) . Zoospore r e l e a s e from a l a t e r a l e x i t p a p i l l a was ob-s e r v e d o n l y once ( P l a t e I I I , F i g s . 1, 2) and i n t h i s case zoo-spore r e l e a s e was the normal s a p r o l e g n o i d t y p e . Aleem e_t a l . (1953) have a l s o o b s e r v e d l a t e r a l e x i t p a p i l l a e i n m a t e r i a l o b t a i n e d from f i s h . A p l a n o i d s p o r a n g i a have been obser v e d by o t h e r workers who have examined f u n g i removed d i r e c t l y from f i s h ( S m i t h , 1878; Aleem e t a l . , 1953; McKay, 1967) and they may a l s o be found when s a p r o l e g n i a c e o u s f u n g i are grown under o t h e r c u l -t u r a l c o n d i t i o n s (Coker, 1923; W i l l o u g h b y , 1968; Seymour, 1970). 47 P l a t e I I I Mycelium removed d i r e c t l y from i n f e c t e d salmon. F i g u r e 1 - Z o o s p o r a n g i a . Arrow i n d i c a t e s a zoosporangium r e l e a s i n g z o o s p o r e s . Bar r e p r e s e n t s 100 ym. F i g u r e 2 - Zoosporangium i n d i c a t e d by the arrow i n F i g . 1 showing r e l e a s e of zoospores through a l a t e r a l e x i t p a p i l l a ( a r r o w ) . Bar r e p r e s e n t s 50 ym. F i g u r e 3 - Zoosporangium w i t h a l a t e r a l e x i t p a p i l l a ( a r r o w ) . Bar r e p r e s e n t s 50 ym. F i g u r e 4 - A p l a n o i d zoosporangium showing _in s i t u g e r m i n a t i o n o f z o o s p o r e s . Bar r e p r e s e n t s 50 ym. 49 I never observed oogonia i n any of the mycelium obtained d i r e c t l y from the f i s h . McKay (1967) reported f i n d i n g oogonia by d i r e c t observation of mycelium from salmon i n one of her c o l l e c t i o n s from Robertson Creek, so i t appears that oogonia may be produced by the f u n g i when they are growing on the f i s h , but t h i s i s probably a very rare event. Zoosporangia t y p i c a l of the genus Achlya (Pl a t e IV, F i g . 3) were observed only once on the corpse of a steelhead t r o u t f r y (Fi s h GAN 0, Table IV) which had been dead f o r an indeterminate period. This agrees w i t h observations by Chong (1973) who also found Achlya spp. only on dead f i s h i n t h i s l o c a l i t y . I am con-vinced that only Saprolegnia species i n f e c t salmon at Robertson Creek, and that Achlya species cannot, or at l e a s t do not, i n -f e c t these animals. During August 1973, I c a r r i e d out an experiment to assess the abundance and i d e n t i t y of saprolegniaceous spores i n the water of Robertson Creek. The experiment was done i n concrete ponds adjacent to the creek and r e c e i v i n g water from the creek. At the time, the ponds contained salmon and t r o u t f r y which were being held i n the ponds i n an unsuccessful attempt to observe n a t u r a l i n f e c t i o n of wounded or unwounded f i s h . Other concerns prevented me from doing much wit h t h i s spore abundance e x p e r i -ment, and I would not have bothered to mention i t except that the technique developed, while simple and inexpensive, appeared to be an e f f e c t i v e way to gain both q u a n t i t a t i v e and q u a l i t a t i v e i n f o r m a t i o n about a saprolegnian p o p u l a t i o n . 50 P l a t e IV Figure 1 - Arrow i n d i c a t e s a standard 9-cm p l a s t i c p e t r i d i s h c o n t a i n i n g YpSs agar and suspended i n a concrete pond to c o l l e c t zoospores. Figure 2 - Photomicrograph of the s u r f a c e of a p e t r i d i s h a f t e r being t r e a t e d i n the manner shown i n F i g . 1. The arrows i n d i c a t e germinating saprolegniaceous zoo-spores. Bar represents 100 ym. Figure 3 - Zoosporangium of A c h l y a sp. obtained from a dead Salmo g a i r d n e r i f r y ( F i s h GAN 0, Table IV). Bar r e p r e s e n t s 100 ym. 51 52 The t e c h n i q u e c o n s i s t e d o f submerging p e t r i d i s h e s c o n t a i n -i n g YpSs agar i n t o the ponds, h o r i z o n t a l to the s u r f a c e of the pond ( P l a t e IV, F i g . 1 ) . The p l a t e s were l e f t i n the ponds f o r s i x hours (the water temperature d u r i n g t h i s e xperiment was 22 C) and then they were removed, d r a i n e d , and t r a n s p o r t e d back to a nearby l a b o r a t o r y . When the p l a t e s were obse r v e d w i t h a mi c r o -scope, i t was r e a d i l y apparent t h a t s a p r o l e g n i a n spores had s e t t l e d onto the s u r f a c e o f the p l a t e and c o u l d be e a s i l y d i s c e r -ned ( P l a t e IV, F i g . 2 ) . S i n g l e spores c o u l d be removed w i t h a m i c r o s p a t u l a and t r a n s f e r r e d to f r e s h agar, thus e s t a b l i s h i n g u n i f u n g a l c u l t u r e s which c o u l d t h e n be t r a n s f e r r e d to hemp seeds and i d e n t i f i e d . CHARACTERIZATION AND IDENTIFICATION OF ISOLATES The r o u t i n e method f o r examining the morphology o f the s i n g l e spore i s o l a t e s was to p l a c e a b l o c k of YpSs agar c o n t a i n -i n g mycelium i n t o a s t a n d a r d 9-cm p l a s t i c p e t r i d i s h c o n t a i n i n g s t e r i l e d i s t i l l e d w a ter and a u t o c l a v e d hemp seeds (Cannabis  s a t i v a L . ) . The hemp seeds were u s u a l l y s p l i t , b ut l a t e r I found t h a t i t was advantageous to use whole hemp seeds w i t h a pu n c t u r e h o l e ( T i f f n e y , 1939a) s i n c e t h i s method seems to en-hance the p r o b a b i l i t y o f oogonium p r o d u c t i o n o c c u r r i n g f o r some i s o l a t e s . Other v a r i a t i o n s i n c u l t u r e t e c h n i q u e i n c l u d e d the use o f f i l t e r s t e r i l i z e d pond water i n p l a c e o f a u t o c l a v e d d i s -t i l l e d w a t e r , and the use o f a u t o c l a v e d salmon s k i n and muscle t i s s u e i n p l a c e o f the hemp seeds. 53 From p r e v i o u s work of my own and t h a t o f o t h e r s (Rucker, 1944; McKay, 1967; Chong, 1973) I had every r e a s o n to s u s p e c t t h a t the c u l t u r e s I i s o l a t e d from the salmon would b e l o n g to the S a p r o l e g n i a d i c 1 i na-S ap r o1e g n i a par as i t i c a complex. However, my p r e v i o u s work had shown t h a t temperatures around 20 C, w h i l e s u i t a b l e f o r i n d u c i n g z o o s p o r o g e n e s i s , were i n i m i c a l to the de-velopment of o o g o n i a , which must be observed b e f o r e the i n f r a -g e n e r i c d e l i m i t a t i o n of an i s o l a t e can be attempted. T h i s t r a i t i s not u n u s u a l f o r S a p r o l e g n i a s p e c i e s , e s p e c i a l l y those t h a t b e l o n g to the S. d i c l i n a - S . paras i t i c a complex. Coker (1923) n o t e d t h a t S_. d i c l i n a " i s one o f our commonest s p e c i e s , but i s o f t e n d i f f i c u l t to de t e r m i n e w i t h c e r t a i n t y because o f the i n f r e -quency o f s e x u a l r e p r o d u c t i o n a t room t e m p e r a t u r e . W h i l e the oogonia are not r a r e under o r d i n a r y c o n d i t i o n s they are not n e a r l y so a b u n d a n t l y formed as i n o t h e r s p e c i e s , and sometimes many c u l t u r e s w i l l be made b e f o r e they are found. However, s e x u a l r e p r o d u c t i o n can be g r e a t l y i n c r e a s e d by lower tempera-t u r e , and can n e a r l y always be o b t a i n e d i n an i c e box." Kanouse (1932) i n c u b a t e d her c u l t u r e s of S_. p a r a s i t i c a a t 10 C, O'Bier (1960) n o t e d t h a t 10 C "was more s a t i s f a c t o r y f o r i s o l a t e s of S a p r o l e g n i a " , and W i l l o u g h b y (1971) r e p o r t e d t h a t n i n e out of t e n S a p r o l e g n i a Type I i s o l a t e s o b t a i n e d from s a l m o n i d f i s h "produced a t l e a s t some oogonia a t low temperatures of 5°-10°C, i r r e s p e c t i v e of the l i g h t regime, w h i l e a t room temperature none were produced." I t s h o u l d be noted b e f o r e p r o c e e d i n g , however, t h a t when 54 one r e f e r s to temperatures around- 10 C as b e i n g low,, t h i s i s an a n t h r o p o c e n t r i c b i a s and i s not n e c e s s a r i l y d e s c r i p t i v e of the normal environment o f the organism. I n a temperate r e g i o n l i k e s o u t h e r n B r i t i s h Columbia, 10 C cannot be c o n s i d e r e d a low or un u s u a l t e m p e r a t u r e . A n a l y s i s o f the 1971 temperature d a t a (see Tab l e I I I , F i g . 1) f o r R o b e r t s o n Creek, f o r example, shows t h a t f o r more than h a l f the y e a r (52%) the temperature of the water was 10 C or l o w e r , and f o r almost h a l f the year (461) the temper-a t u r e of the water was 10 ± 5 C. On the o t h e r hand, the tempera-t u r e of the water was between 15 C-20 C f o r o n l y 19% of the ye a r and was a t , or i n excess o f , 20 C f o r o n l y 3% o f the y e a r . These d a t a c l e a r l y show t h a t f o r the fungus, temperatures i n excess of 20 C are u n u s u a l l y warm whereas temperatures i n the range of 10 C are q u i t e normal. When the s i n g l e spore i s o l a t e s were o b t a i n e d , they were i n -o c u l a t e d i n t o hemp seed and water c u l t u r e s and i n c u b a t e d a t 14 C to induce z o o s p o r o g e n e s i s . The f a c t t h a t some o f the c u l t u r e s were s t i l l c o n t a m i n a t e d by b a c t e r i a made no d i f f e r e n c e to these r e s u l t s . U s i n g t h i s more o r l e s s s t a n d a r d t e c h n i q u e , I was a b l e t o c o n f i r m the i d e n t i t y of a l l but f o u r o f the i s o l a t e s as Sapro-l e g n i a s p e c i e s by o b s e r v i n g zoospore r e l e a s e . The f o u r c u l t u r e s (GAN 0, GAN 1, GAN 17, GAN 5) t h a t I c o u l d n o t i d e n t i f y were a l l b a c t e r i a - f r e e and produced z o o s p o r a n g i a v e r y s p a r s e l y on hemp seeds; hence, i t was d i f f i c u l t t o observe zoospore r e l e a s e . T h i s problem was s o l v e d by s u b s t i t u t i n g s m a l l p i e c e s o f a u t o c l a v e d salmon s k i n and muscle t i s s u e f o r the hemp seeds. When grown on 55 t h i s s u b s t r a t e a t 21 C, a l l f o u r i s o l a t e s produced numerous zoo-s p o r a n g i a w h i c h r e l e a s e d t h e i r zoospores i n a s a p r o l e g n o i d manner. Thus I was f i n a l l y a b l e to c o n f i r m t h a t a l l the s i n g l e spore i s o l a t e s were S a p r o l e g n i a s p e c i e s w h i c h i s what my f i e l d obser-v a t i o n s had l e d me to e x p e c t . The t h i r t y s i n g l e spore i s o l a t e s d e r i v e d from the c u l t u r e s o b t a i n e d from t h i r t y d i f f e r e n t f i s h (Table IV) were e v e n t u a l l y grouped i n t o f o u r c a t e g o r i e s . Some of the key c h a r a c t e r i s t i c s of these c a t e g o r i e s based on zoosporangium p r o d u c t i o n and oogon-ium p r o d u c t i o n are shown i n Table V. The i s o l a t e s i n c l u d e d i n C a t e g o r i e s A and D d i d not produce oogonia o r , i n the case of the Category A i s o l a t e , produced oogonia so r a r e l y t h a t no o b s e r v a t i o n s c o u l d be made r e g a r d i n g t h e i r i n f r a g e n e r i c a f f i n i -t i e s . The i s o l a t e s i n c l u d e d i n Cate g o r y B are q u i t e d i s t i n c t from those i n c l u d e d i n Category C and can be r e a d i l y d i s t i n g u i s h -ed from the Cate g o r y C i s o l a t e s on the b a s i s o f any one of the f o l l o w i n g c h a r a c t e r s : zoospore p r o d u c t i o n , gemma p r o d u c t i o n , p r o d u c t i o n of s e x u a l r e p r o d u c t i v e s t r u c t u r e s , morphology of s e x u a l r e p r o d u c t i v e c u l t u r e s , g r o s s appearance o f c u l t u r e s grow-i n g i n YpSs a g a r , and growth r a t e . A d e t a i l e d d i s c u s s i o n and comparison of the c h a r a c t e r i s t i c s o f the Cate g o r y B and the Category C i s o l a t e s f o l l o w s . The t h r e e Category B i s o l a t e s are so s i m i l a r w i t h r e g a r d to the c h a r a c t e r i s t i c s examined, I have l i t t l e doubt t h a t they are i d e n t i c a l . Even when growing i n YpSs agar they can be s e p a r a t e d from the o t h e r c u l t u r e s because they produce much more a e r i a l mycelium w h i c h , i n an o l d e r c u l t u r e , becomes app r e s s e d to the TABLE V Cat e g o r y Some c h a r a c t e r i s t i c s of the s i n g l e spore S a p r o l e g n i a i s o l a t e s d e r i v e d from the c u l t u r e s o b t a i n e d from salmonids a t R o b e r t s o n Creek, 1973. Zoosporangium P r o d u c t i o n hemp seeds salmon t i s s u e (14±1 C) (21±1 C) Oogonium P r o d u c t i o n hemp seeds (10±1 C ) * Number of I s o l a t e s (n=30) I s o l a t e s A B poor poor good good good D good one oogonium observed, c o n s i s t e n t , abundant. s p o r a d i c , not abundant. none observed. 1 3 12 14 GAN 5. GAN 0, GAN 1, GAN 17. GAN 6, GAN 8, GAN 18-GAN 20, GAN 23, GAN 25-GAN 27, GAN 31-GAN 33. GAN 2-GAN 4, GAN 7, GAN 9-GAN 16, GAN 21, GAN 22, GAN 24, GAN 28-GAN 30. *Oogonium p r o d u c t i o n f o r the Category A i s o l a t e and one Ca t e g o r y C i s o l a t e (GAN 18) was obs e r v e d i n c u l t u r e s i n c u b a t e d a t 12 C. Oogonium p r o d u c t i o n f o r two o t h e r C a t e g o r y C i s o l a t e s (GAN 6, GAN 31) was observed i n c u l t u r e s i n c u b a t e d a t 14 C. **For f u r t h e r i n f o r m a t i o n r e g a r d i n g the source of these i s o l a t e s , r e f e r t o T a b l e IV. 57 under s u r f a c e of the p e t r i d i s h l i d . When grown on s p l i t hemp seeds i n s t e r i l e d i s t i l l e d water at about 20 C, the Category B i s o l a t e s produce a few gemmae, but no s e x u a l r e p r o d u c t i v e s t r u c t u r e s ( P l a t e V, F i g . 1 ) . When they are grown a t 10 C, they produce masses o f gemmae, oo g o n i a and a n t h e r i d i a ( P l a t e V, F i g . 2 ) . The o o g o n i a l w a l l s o f these i s o -l a t e s are t h i n and o c c a s i o n a l l y p i t t e d ( P l a t e V, F i g . 4; P l a t e V I , F i g . 5 ) . The oospheres are u s u a l l y 24-29 ym i n diameter and, a l t h o u g h a few a p p a r e n t l y mature c e n t r i c t o s u b c e n t r i c oospores were observed ( P l a t e V I , F i g s . 3, 4, 6 ) , the v a s t m a j o r i t y of the oospheres a b o r t ( P l a t e V I , F i g . 6 ) . .The a n t h e r i d i a are d i c l i n o u s i n o r i g i n and the a n t h e r i d i a l c e l l s are g e n e r a l l y c l a v a t e or t u b u l a r s t r u c t u r e s ( P l a t e V, F i g . 3; P l a t e V I , F i g . 2) t y p i c a l f o r the genus (Seymour, 1970). F e r t i l i z a t i o n tubes were o c c a s i o n -a l l y o bserved ( P l a t e V, F i g . 3; P l a t e V I , F i g . 1 ) . These t h r e e i s o l a t e s cannot be a s s i g n e d c o n f i d e n t l y t o any p r e s e n t l y r e c o g n i z e d s p e c i e s . I n s e v e r a l r e s p e c t s h o w e v e r t h e y are q u i t e r e m i n i s c e n t o f S a p r o l e g n i a k a u f f m a n i a n a P i e t e r s ( P i e t e r s , 1915a; Seymour, 1970) which Seymour (1970) c l a s s i f i e s ( c o r r e c t l y , i n my o p i n i o n ) as an i m p e r f e c t l y known or d o u b t f u l t a x o n . Some p o i n t s of s i m i l a r i t y t h a t the Category B i s o l a t e s share w i t h S^ . k a u f fmaniana (see Seymour, 1970) are the smooth, t h i n , o c c a s i o n a l l y p i t t e d o o g o n i a l w a l l s , the d i c l i n o u s a n t h e r i -d i a , and (most i m p o r t a n t ) the somewhat l a r g e r than average ( f o r the genus) oospheres which f a i l t o develop a con s p i c u o u s o o p l a s t . I must emphasize, however, t h a t I am not i d e n t i f y i n g these i s o -58 P l a t e V F i g u r e 1 - Four week o l d hemp seed c u l t u r e o f GAN 17 m a i n t a i n e d a t 20 t 1 C i n the dark. Note the complete absence of o o g o n i a . Bar r e p r e s e n t s 250 ym. F i g u r e 2 - Four week o l d hemp seed c u l t u r e of GAN 17 m a i n t a i n e d a t 9.5-12 C i n the dark. Note the numerous oogonia and compare w i t h F i g . 1. Bar r e p r e s e n t s 250 ym. F i g u r e 3 - Oogonium from a f o u r week o l d c u l t u r e of GAN 17 m a i n t a i n e d a t 9.5-12 C i n c o n s t a n t l i g h t . Arrow i n d i c a t e s a p r o b a b l e f e r t i l i z a t i o n tube. Bar r e p r e -s e n t s 20 ym. F i g u r e 4 - Oogonium from a f o u r week o l d c u l t u r e o f GAN 17 m a i n t a i n e d a t 9.5-12 C i n c o n s t a n t l i g h t . Arrows i n d i c a t e p i t s i n the o o g o n i a l w a l l . Bar r e p r e s e n t s 20 ym. 60 P l a t e VI F i g u r e 1 - Oogonium from a 40 day o l d hemp seed c u l t u r e o f GAN 0 m a i n t a i n e d a t 10 C i n the dark. Arrow i n d i c a t e s a f e r t i l i z a t i o n tube. Bar r e p r e s e n t s 10 ym. F i g u r e 2 - Oogonium from a 40 day o l d hemp seed c u l t u r e o f GAN 1 m a i n t a i n e d a t 10 C i n the dark. Note the d i c l i n o u s a n t h e r i d i a and the absence o f mature oos p o r e s . Bar r e p r e s e n t s 20 ym. F i g u r e 3 - More or l e s s c e n t r i c oospore from a 40 day o l d hemp seed c u l t u r e o f GAN 1 m a i n t a i n e d a t 10 C i n the dark. Bar r e p r e s e n t s 10 ym. F i g u r e 4 - Somewhat s u b c e n t r i c oospore from a 40 day o l d hemp seed c u l t u r e o f GAN 1 m a i n t a i n e d a t 10 C i n the dark. Bar r e p r e s e n t s 10 ym. F i g u r e 5 - Oogonium from a 40 day o l d hemp seed c u l t u r e o f GAN 1 m a i n t a i n e d a t 10 C i n the dark. Arrows i n d i c a t e p i t s i n the o o g o n i a l w a l l . Note the absence o f mature oosp o r e s . Bar r e p r e s e n t s 20 ym. F i g u r e 6 - Oogonium from a 40 day o l d hemp seed c u l t u r e o f GAN 1 m a i n t a i n e d a t 10 C i n the dark. Note the h i g h r a t e o f oosphere a b o r t i o n . Arrow i n d i c a t e s a c e n t r i c oospore. Bar r e p r e s e n t s 20 ym. 62 l a t e s as S. k a u f f m a n i a n a , but am mer e l y n o t i n g a s i m i l a r i t y . The Category B i s o l a t e s u n d o u b t e d l y b e l o n g to the S. d i c l i n a - S . p a r a s i t i c a complex. The i s o l a t e s i n c l u d e d i n Category C produce v e r y few gemmae. Oogonium p r o d u c t i o n i s s p o r a d i c (see T a b l e VI) and v e r y few oog-o n i a are produced. F u r t h e r m o r e , i n o r d e r to get any oogonium p r o d u c t i o n a t a l l , i t i s u s u a l l y n e c e s s a r y t o i n c u b a t e the c u l t u r e s a t 10 C f o r about f o u r months. I t i s a l s o advantageous to use whole hemp seeds w h i c h have been p u n c t u r e d by a ne e d l e i n s t e a d o f the u s u a l hemp seed h a l v e s . Under these c o n d i t i o n s , i f o ogonia are produced, they are produced i n a c l u s t e r near the pu n c t u r e h o l e . D u r i n g the l o n g i n c u b a t i o n p e r i o d , some o f the c u l t u r e s became c o n t a m i n a t e d by hyphomycetes or b a c t e r i a (Table V I ) . These r e s u l t s were not d i s c a r d e d because i t i s r e a s o n a b l e t o assume t h a t these contaminants were n ot accompanied by s a p r o l e g -n i a n c o n t a m i n a n t s . A l s o , the d a t a o b t a i n e d from the co n t a m i n a t e d c u l t u r e s were c o n s i s t e n t w i t h the d a t a o b t a i n e d from c u l t u r e s i n which no contaminants were no t e d . W i l l o u g h b y (1968) had a s i m i l a r problem w i t h c o n t a m i n a t i o n of h i s c u l t u r e s , and suggested t h a t the pr e s e n c e o f these contaminants might h e l p induce oogon-ium f o r m a t i o n . The r e s u l t s o f the work shown i n T a b l e VI do not c o n f i r m or deny t h i s o b s e r v a t i o n , but o t h e r work w i t h c u l t u r e s a s s o c i a t e d w i t h the i n f e c t i o n e x p eriments tend t o c o n f i r m t h i s i d e a (see P l a t e X X I I I ) . These o b s e r v a t i o n s w i l l be d e s c r i b e d l a t e r . TABLE VI Oogonium p r o d u c t i o n by some Category C i s o l a t e s . Experiment 1* Experiment 2* I n c u b a t i o n P e r i o d : 121-124 days I n c u b a t i o n P e r i o d : 134 days I s o l a t e s P r o d u c i n g Oogonia I s o l a t e s P r o d u c i n g Oogonia 1. GAN 8** GAN 8 2. GAN 19 _._ _. 3. GAN 20** 4- ----- GAN 23 5. GAN 25** -_- -. 6. GAN 26** GAN 26 7'• ----- GAN 2 7 8. GAN 32 -_. * D u p l i c a t e p l a t e s each c o n t a i n i n g a s i n g l e p u n c t u r e d hemp seed i n d i s t i l l e d w a t e r . Inoculum c o n s i s t e d o f a b l o c k o f YpSs agar c o n t a i n i n g mycelium. C u l t u r e s main-t a i n e d a t 10 + 1 C i n the dark. C u l t u r e s i n which hyphomycete or b a c t e r i a l contaminants were n o t e d . 64 The f o l l o w i n g d e s c r i p t i o n o f the Category C l i s o l a t e s i s based p r i m a r i l y on o b s e r v a t i o n s made on the c u l t u r e s shown i n Ta b l e V I . From o t h e r c u l t u r e work, i t appears t h a t GAN 18 cannot be d i s t i n g u i s h e d from these c u l t u r e s , and l e s s e x t e n s i v e o b s e r v a t i o n s on GAN 33 do n o t , a t l e a s t , e x c l u d e i t from t h i s group. As I made no d e t a i l e d o b s e r v a t i o n s on the oogonia of GAN 6 and GAN 31, i n c l u s i o n o f these two i s o l a t e s i n Category C i s c o n j e c t u r a l . The o ogonia o f the Categ o r y C i s o l a t e s are t h i n - w a l l e d and u n p i t t e d ( P l a t e V I I , F i g s . 1, 3, 6, 7 ) . U s u a l l y few oospheres mature ( P l a t e V I I , F i g s . 1-3, 7 ) . The few apparent oospores ( P l a t e V I I , F i g s . 3, 5-7) are g e n e r a l l y 18-22 ym i n di a m e t e r and c e n t r i c to s u b c e n t r i c . The a n t h e r i d i a l branches are s l e n d e r and a p p a r e n t l y d i c l i n o u s i n o r i g i n ( P l a t e V I I , F i g s . 2-4, 7 ) . The a n t h e r i d i a l c e l l s are u s u a l l y not s w o l l e n or o n l y s l i g h t l y s w o l l e n and are l a t e r a l l y a p p r e s s e d ( P l a t e V I I , F i g s . 2-4); A n t h e r i d i a were not observ e d on a l l oogonia. These i s o l a t e s appear to have a f f i n i t i e s w i t h the S_. d i c -l i n a - S . p a r a s i t i c a complex and have s e v e r a l s i g n i f i c a n t f e a t u r e s i n common w i t h S_. paras i t i c a Coker emend. Kanouse as d e s c r i b e d by Kanouse (1932) and O'Bier (1960). I f the Categ o r y C i s o l a t e s were to be a s s i g n e d t o any p r e s e n t l y r e c o g n i z e d s p e c i e s , i t would be t h i s one; however, the s m a l l number o f oogonia produced, combined w i t h the low r a t e o f oosphere m a t u r a t i o n and the d i f f i -c u l t i e s a s s o c i a t e d w i t h making a c c u r a t e o b s e r v a t i o n s on the a n t h e r i d i a l c e l l s and a n t h e r i d i a l o r i g i n , makes the c o n f i d e n t 65 P l a t e V I I F i g u r e 1 - C l u s t e r o f oogonia from a 125 day o l d hemp seed c u l t u r e o f GAN 26 m a i n t a i n e d a t 10 C i n the dark. Bar r e p r e -s e n t s 50 ym. F i g u r e 2 - Oogonium from a 125 day o l d hemp seed c u l t u r e o f GAN 26 m a i n t a i n e d a t 10 C i n the dark. Note the s l e n d e r , a p p a r e n t l y d i c l i n o u s a n t h e r i d i a s u r r o u n d i n g the oogon-ium. Bar r e p r e s e n t s 20 ym. F i g u r e 3 - Oogonium from a 122 day o l d hemp seed c u l t u r e o f GAN 8 m a i n t a i n e d a t 10 C i n the dark. The arrow i n d i c a t e s a s m a l l , o n l y s l i g h t l y s w o l l e n a n t h e r i d i a l c e l l . Bar r e p r e s e n t s 10 ym. F i g u r e 4 - Oogonium from a 122 day o l d hemp seed c u l t u r e o f GAN 20 m a i n t a i n e d a t 10 C i n the dark. Note the s l e n d e r , a p p a r e n t l y d i c l i n o u s a n t h e r i d i a s u r r o u n d i n g the oogon-ium. Bar r e p r e s e n t s 20 ym. F i g u r e 5 - More or l e s s s u b c e n t r i c oospore from a 125 day o l d hemp seed c u l t u r e of GAN 26. Bar r e p r e s e n t s 10 ym. F i g u r e 6 - Oogonium from a 125 day o l d hemp seed c u l t u r e of GAN 26 m a i n t a i n e d a t 10 C i n the dark. Note the t h i n , u n p i t t e d o o g o n i a l w a l l . T h i s oogonium has more a p p a r e n t l y mature oospores than were u s u a l l y seen i n oogonia produced by Categ o r y C c u l t u r e s . Bar r e p r e -s e n t s 20 ym. F i g u r e 7 - S m a l l oogonium from a 122 day o l d hemp seed c u l t u r e o f GAN 20 m a i n t a i n e d a t 10 C i n the dark. Note the a n t h e r i d i u m , the t h i n o o g o n i a l w a l l , and the lower oospore w i t h a p o o r l y d i f f e r e n t i a t e d o o p l a s t . Bar r e p r e s e n t s 10 ym. 67 a s s i g n a t i o n o f these i s o l a t e s t o any p r e s e n t l y r e c o g n i z e d s p e c i e s premature. Of much more i n t e r e s t i s the f a c t t h a t the Category C i s o l a t e s appear to have a s t r i k i n g resemblance to W i l l o u g h b y ' s S a p r o l e g n i a Type I i s o l a t e s w hich were o b t a i n e d from d i s e a s e d s a l m o n i d s i n England ( W i l l o u g h b y , 1968, 1969a, 1971; W i l s o n , 1976). P o i n t s o f s i m i l a r i t y i n c l u d e (1) oogonium p r o d u c t i o n o n l y a t 'low' temperatures ( c a . 5-12 C ) ; (2) poor oogonium p r o d u c t i o n ; (3) t h i n , u n p i t t e d o o g o n i a l w a l l s ; (4) poor oospore p r o d u c t i o n combined w i t h an apparent h i g h r a t e o f oosphere a b o r t i o n ; (5) r e l a t i v e l y s m a l l , g e n e r a l l y s u b c e n t r i c o o s p o r e s ; (6) a n t h e r i -d i a of p r o b a b l e d i c l i n o u s o r i g i n ; and (7) s l e n d e r a n t h e r i d i a , which may co v e r the e n t i r e s u r f a c e o f the oogonium. I d i d not observe z o o s p o r a n g i a o f the type t h a t W i l l o u g h b y (1969a) d e s c r i b -es as t y p i c a l f o r S a p r o l e g n i a Type I ; n o n e t h e l e s s , the s t r i k i n g p h y s i o l o g i c a l and m o r p h o l o g i c a l s i m i l a r i t y o f the Category C i s o l a t e s t o the S a p r o l e g n i a Type I i s o l a t e s , combined w i t h the f a c t t h a t they were i s o l a t e d as presumed p a r a s i t e s of e s s e n t i a l -l y the same h o s t (I b e l i e v e i t i s s a f e t o assume t h a t Salmo and Oncorhynchus s p e c i e s are c l o s e l y r e l a t e d ) , argues s t r o n g l y i n f a v o u r o f the n o t i o n t h a t t h e r e i s a p h y s i o l o g i c a l l y and mor-p h o l o g i c a l l y d i s t i n c t s t r a i n of S a p r o l e g n i a w h i c h , f o r reasons not y e t known, has a p r e d i l e c t i o n toward b e i n g a p a r a s i t e o f s a l m o n i d s . I t would be most i n t e r e s t i n g to compare W i l l o u g h b y ' s S a p r o l e g n i a Type I w i t h my Category C i s o l a t e s u s i n g the s e r o -taxonomic t e c h n i q u e s d e s c r i b e d by P e d u z z i (1975) to see how w e l l these f u n g i compare w i t h each o t h e r r e l a t i v e t o o t h e r f u n g i 68 i n c l u d e d i n the S^ . d i c l i n a - S . p a r a s i t i c a complex. Even when such r e f i n e m e n t s as i n c u b a t i n g the c u l t u r e s a t 10 C and u s i n g p u n c t u r e d hemp seeds had been worked o u t , oogon-ium p r o d u c t i o n i n most of my i s o l a t e s was s t i l l poor or n o n e x i s t -ent (Table V, Table V I ) . C o n s e q u e n t l y , I t r i e d s e v e r a l o t h e r experiments to induce oogonium f o r m a t i o n i n these i s o l a t e s , none of which was p a r t i c u l a r l y s u c c e s s f u l . F i r s t , when s a p r o l e g n i a c e o u s f u n g i do not produce o o g o n i a , a n a t u r a l assumption i s t h a t they may be h e t e r o t h a l l i c . There-f o r e I put i s o l a t e s t o g e t h e r i n v a r i o u s c o m b i n a t i o n s i n d i s t i l l e d water w i t h hemp seeds a t the same time as Experiment I i n Ta b l e VI was s e t up. Oogonia were observed o n l y i n c o m b i n a t i o n s con-t a i n i n g i s o l a t e s which s u b s e q u e n t l y produced oogonia i n s i n g l e s p o r e c u l t u r e s ; c o n s e q u e n t l y , no e v i d e n c e o f h e t e r o t h a l l i s m c o u l d be found. Other attempts i n c l u d e d u s i n g salmon t i s s u e i n p l a c e of hemp seed s , and f i l t e r s t e r i l i z e d pond water or 1.21 g l u c o s e p l u s 0.1% peptone b r o t h (Kanouse, 1932) i n p l a c e o f s t e r i l e d i s t i l l e d w a t e r . Only the Category B c u l t u r e s and one Category C c u l t u r e (GAN 26) produced oogonia on the a u t o c l a v e d salmon t i s s u e . The c u l t u r e s produced o n l y mycelium i n the hemp seed g l u c o s e - p e p t o n e b r o t h c u l t u r e s . Two Categ o r y D c u l t u r e s (GAN 3, GAN 24) and one Category C c u l t u r e (GAN 18) were i n c l u d e d ( a l o n g w i t h o t h e r i s o l a t e s t o be d e s c r i b e d l a t e r ) i n the pond water experiment. The b a s i c i m p r e s s i o n I got from t h i s e x p e r i -ment was t h a t t h e pond water might have a p o s i t i v e q u a n t i t a t i v e e f f e c t over the d i s t i l l e d water c u l t u r e s i n terms of enhancing oogonium p r o d u c t i o n i n i s o l a t e s a l r e a d y known to produce oogonia 69 i n d i s t i l l e d w a t e r , b u t the pond water had no apparent q u a l i t a -t i v e e f f e c t i n terms o f i n d u c i n g oogonium p r o d u c t i o n i n i s o l a t e s w h ich were r e f r a c t o r y i n the o t h e r e x p e r i m e n t s . EFFECT OF TEMPERATURE ON VEGETATIVE GROWTH U n t i l now, I have d i s c u s s e d temperature m a i n l y w i t h r e g a r d to those temperatures which were c o n v e n i e n t or n e c e s s a r y f o r i n d u c i n g p r o d u c t i o n o f the a s e x u a l and s e x u a l r e p r o d u c t i v e s t r u c t u r e s w h i c h must be s t u d i e d i n o r d e r to i d e n t i f y an i s o l a t e . T h i s s e c t i o n i s concerned w i t h the e f f e c t o f temperature on v e g e t a t i v e growth. There have been s e v e r a l s t u d i e s done on the v e g e t a t i v e growth of S a p r o l e g n i a i s o l a t e s (e.g. P o w e l l e t al _ . , 1972 ; Chong, 1973) and, as a g e n e r a l r u l e , i t can be s t a t e d t h a t they are mesothermic f u n g i which can grow i n a temperature range of >0 C to <35 C, and t h e i r growth r a t e w i l l i n c r e a s e as the tem-p e r a t u r e i n c r e a s e s . The temperature d a t a f o r R o b e r t s o n Creek (Table I I I ) show t h a t my i s o l a t e s would never n o r m a l l y be c h a l l e n g e d by the extremes of the temperature range, and i t i s p r o b a b l y s a f e t o assume ( i g n o r i n g the r e l a t i v e l y t h i n l a y e r o f i c e t h a t may form on these w a t e r s i n w i n t e r ) t h a t organisms l i v i n g i n the l a k e s and streams o f Vancouver I s l a n d would v e r y r a r e l y have t o t o l e r a t e t emperatures o u t s i d e a range o f 2-23 C. T h e r e f o r e , from t h e s e temperature d a t a and the l i t e r a t u r e , i t i s r e a s o n a b l e t o h y p o t h e s i z e t h a t my i s o l a t e s can grow thr o u g h o u t the y e a r i n a l o c a t i o n l i k e R o b e r t s o n Creek, and need never 70 e n t e r a 'dormant' or ' o v e r w i n t e r i n g ' p e r i o d . T h i s s u p p o s i t i o n i s s u p p o r t e d by the o b s e r v a t i o n t h a t f i s h eggs 'or f i s h may be i n f e c t e d by s a p r o l e g n i a n f u n g i t hroughout the y e a r . N o n e t h e l e s s , t h i s does not mean t h a t temperature has no e f f e c t on the patho-g e n i c i t y o f these f u n g i and, s i n c e e m p i r i c a l e v i d e n c e i s more u s e f u l than a g e n e r a l d e d u c t i o n , t h i s s t u d y was i n i t i a t e d i n o r d e r to v e r i f y and, to a c e r t a i n e x t e n t , q u a n t i f y the e f f e c t o f temperature on v e g e t a t i v e growth o f some o f my i s o l a t e s . An unexpected r e s u l t was the d i s c o v e r y t h a t these d a t a appear to have some taxonomic v a l u e as w e l l . The growth r a t e was determined from measurements of the r a d i a l i n c r e a s e of a c o l o n y growing i n an agar medium. T h i s method was used i n s t e a d o f the more a c c u r a t e d e t e r m i n a t i o n of the d r y w e i g h t o f mycelium grown i n a l i q u i d medium because i t was an e a s i e r p r o c e d u r e to c a r r y o u t , i t p r o v i d e d d a t a of ade-quate r e s o l u t i o n f o r my p u r p o s e s , and i t most n e a r l y mimicked the way i n w h i c h the mycelium u s u a l l y grows on an i n f e c t e d f i s h . D i f c o c o r n meal agar (CMA) was the medium of c h o i c e f o r these o b s e r v a t i o n s because i t i s f r e q u e n t l y used f o r maintenance o f water mould c u l t u r e s , i s t r a n s p a r e n t , and s u p p o r t s l i t t l e a e r i a l growth of mycelium. The i s o l a t e s chosen f o r the st u d y i n c l u d e d a Cate g o r y B i s o l a t e (GAN 1 7 ) , a Category C i s o l a t e (GAN 1 8 ) , and a Category D i s o l a t e (GAN 2 4 ) . The i s o l a t e s were grown a t 3 ± 1 C, 7 ± 0 . 5 C, 14 ± 1 C and 21 ± 1 C. These temperatures were chosen t o span the range of temperatures n o r m a l l y e n c o u n t e r e d by these organisms i n n a t u r e . 71 I n o c u l a c o n s i s t e d o f CMA p l u g s 8 mm i n d i a m e t e r c u t from the edge of a c t i v e l y growing c o l o n i e s . These were t r a n s f e r r e d to the c e n t r e o f 9 X 1.5 cm p l a s t i c p e t r i d i s h e s c o n t a i n i n g 25 ml CMA each. Four r e p l i c a t e s were p l a t e d out f o r each temperature and the growth r a t e f o r each r e p l i c a t e was d e t e r m i n e d by making two measurements of c o l o n y d i a m e t e r a t r i g h t a n g l e s to each o t h e r a t i n t e r v a l s o f 12, 24, or 48 h, depending on the i n c u b a t i o n t emperature and the s t a g e o f c o l o n y development. G e n e r a l l y , f o r a g i v e n temperature and t i m e , the v a r i a t i o n o f measurement w i t h i n r e p l i c a t e s was the same as the v a r i a t i o n between r e p l i c a t e s . The maximum v a r i a t i o n e n c o u n t e r e d was 3 mm, but u s u a l l y i t was l e s s . The e r r o r o f measurement was ± 1 mm. A f t e r an i n i t i a l l a g phase, which v a r i e d from l e s s than 24 h a t 21 C to about 96 h a t 3 C, the growth r a t e o f each i s o -l a t e was c o n s t a n t a t a g i v e n t e m p e r a t u r e . These growth r a t e s are shown i n Table V I I and F i g u r e 2. GAN 18 and GAN 24 had the same growth r a t e a t each temperature and c o u l d not be d i s t i n g u i s h -ed from one another on t h i s b a s i s . T h i s tends to c o n f i r m my s u s p i c i o n t h a t many, and perhaps a l l , o f the c u l t u r e s i n C a t e g o r i e s C and D are v i r t u a l l y i d e n t i c a l . The growth r a t e of GAN 17 a t each t e m p e r a t u r e , on the o t h e r hand, was c o n s i s t e n t l y 35 ± 51 f a s t e r than the o t h e r two i s o l a t e s . For a l l the i s o l a t e s , as a g e n e r a l r u l e , the growth r a t e d o u b l e d as the temperature d o u b l e d , but t h i s r e l a t i o n s h i p was more a c c u r a t e between 3 and 14 C than between 14 and 21 C, s i n c e the a c t u a l r a d i a l growth r a t e s r e c o r d e d a t 21 C were 871 of the e x p e c t e d r a t e f o r GAN 18 and GAN 24, and 89 ± 3% of the e x p e c t e d r a t e f o r GAN 17. TABLE V I I R a d i a l growth r a t e s (mm/day) a f t e r the i n i t i a l l a g phase o f some S a p r o l e g n i a i s o l a t e s growing a t d i f f e r e n t temperatures i n c o r n meal agar. Temperature (C) GAN 17 GAN 18 GAN 24 ATCG 26116* 3±1 7±0.5 10±1 14±1 21±1 3.5 7 2.5 5 2.5 5 13 18 10 13 10 13 2.5 6 8.5 11 17 *ATCC = American Type C u l t u r e C o l l e c t i o n , R o c k v i l l e , M a r y l a n d . 73 Temperature (Celsius) Figure 2 - Radial growth rates (mm/day) after the i n i t i a l lag phase of some Saprolegnia isolates growing at d i f f e r e n t tempera-tures i n corn meal agar. 74 PART I I - NUCLEAR AND SATELLITE DNA BASE COMPOSITION AND THE TAXONOMY OF SAPROLEGNIA INTRODUCTION I have shown t h a t most of the i s o l a t e s w hich I o b t a i n e d from the s a l m o n i d s a t R o b e r t s o n Creek e i t h e r d i d not produce o o g o n i a , o r produced them s p o r a d i c a l l y i n s m a l l numbers o n l y a f t e r a l o n g i n c u b a t i o n p e r i o d . F u r t h e r , I have a l s o shown t h a t f a i l u r e to observe s e x u a l 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 i s o l a t e s o b t a i n e d from f i s h i s a common e x p e r i e n c e . The f a i l u r e o f these organisms to produce oogonia i n the l a b o r a t o r y , as I have a l r e a d y s u g g e s t e d , may be a r e s u l t o f the t e c h n i q u e s used to i s o l a t e and i d e n t i f y these f u n g i , e s p e c i a l l y w i t h r e g a r d to the h i g h l y a r t i -f i c i a l environment p r o v i d e d by a x e n i c c u l t u r e t e c h n i q u e s . None-t h e l e s s , i t i s a l s o p o s s i b l e t h a t these o r g a n i s m s , i n n a t u r e , may i n d e e d be s t e r i l e or v i r t u a l l y s t e r i l e . I f t h i s i s t r u e , t h e r e i s an e c o n o m i c a l l y and e c o l o g i c a l l y i m p o r t a n t component o f the water mould p o p u l a t i o n which ca n n o t , and may never be, i d e n t i f i e d i n accordance w i t h c l a s s i c a l morphotaxonomic s p e c i e s c o n c e p t s . T h i s a l o n e , i n my o p i n i o n , i s j u s t i f i c a t i o n enough f o r p u r s u i n g n o v e l approaches to the taxonomy of the genus S a p r o l e g n i a so t h a t the a f f i n i t i e s o f d i f f e r e n t i s o l a t e s can be a s s e s s e d w i t h o u t n e c e s s a r i l y h a v i n g to observe s e x u a l r e p r o d u c -t i v e s t r u c t u r e s . However, even i n cases where c o n s i s t e n t and abundant oogonium p r o d u c t i o n can be o b t a i n e d (as i n the case o f the Category B i s o l a t e s , f o r example), s p e c i e s concepts and 75 s p e c i e s a f f i n i t i e s can s t i l l be vague and c o n t r o v e r s i a l . To d i g r e s s b r i e f l y , I t h i n k i t i s q u i t e p o s s i b l e t h a t the a b i l i t y of a S a p r o l e g n i a i s o l a t e to produce v i a b l e oospores may not be i m p o r t a n t f o r the suc c e s s or s u r v i v a l of the organism. G e n e r a l l y s p e a k i n g , p r o d u c t i o n o f v i a b l e oospores p r o v i d e s two major advantages f o r an oomycete: i t a l l o w s the g e n e t i c recom-b i n a t i o n i n h e r e n t i n s e x u a l r e p r o d u c t i o n , and i t r e s u l t s i n the p r o d u c t i o n of a spore which can w i t h s t a n d e n v i r o n m e n t a l extremes not t o l e r a t e d by the v e g e t a t i v e hyphae or a s e x u a l r e p r o d u c t i v e s t r u c t u r e s (e.g. zoospore c y s t s , gemmae), and which can remain dormant f o r extended p e r i o d s p r i o r t o g e r m i n a t i o n . Assuming t h a t the a b i l i t y t o produce these spores has been l o s t by some o f the S a p r o l e g n i a s p e c i e s t h a t i n f e c t f i s h , does t h i s n e c e s s a r i l y put these f u n g i a t a c o m p e t i t i v e d i s a d v a n t a g e , r e l a t i v e to o t h e r w ater moulds, i n the environment i n wh i c h they l i v e ? A p p a r e n t l y n o t , and the reasons f o r t h i s may be as f o l l o w s . F i r s t , a l l known s p e c i e s o f S a p r o l e g n i a are h o m o t h a l l i c ; hence, the degree o f g e n e t i c r e c o m b i n a t i o n p r o v i d e d by s e x u a l r e p r o d u c t i o n w i l l p r o b a b l y be r e l a t i v e l y l i m i t e d . Second, s i n c e these organisms appear to have s u c c e s s f u l l y adapted to a r e l a -t i v e l y s t a b l e environment, t h e r e i s p r o b a b l y l i t t l e s e l e c t i v e p r e s s u r e f a v o u r i n g g e n e t i c d i v e r s i t y anyway. These f u n g i are ca p a b l e of v e g e t a t i v e growth thr o u g h o u t the y e a r , and can a l s o reproduce a s e x u a l l y t h r o u g h o u t most, and perhaps a l l , of the y e a r as w e l l . T h e r e f o r e , g i v e n an adequate foo d s u p p l y , t h e r e i s p r o b a b l y no o c c a s i o n when oospore p r o d u c t i o n i s n e c e s s a r y to 76 ensure s u r v i v a l o f the s p e c i e s . B a l a n c e d a g a i n s t t h i s p o t e n t i a l -l y n o n e x i s t e n t s e l e c t i o n i n f a v o u r o f S a p r o l e g n i a s p e c i e s c a p a b l e o f p r o d u c i n g v i a b l e o o s p o r e s , i s the f a c t t h a t s e x u a l r e p r o d u c -t i o n i n Oomycetes, as i n o t h e r organisms t h a t r e p r o d u c e s e x u a l l y , i s a complex p r o c e s s . There are p r o b a b l y many m u t a t i o n s t h a t c o u l d o c c u r w h i c h would i n t e r f e r e w i t h the p r o d u c t i o n of v i a b l e o o s p o r e s , but would n ot o t h e r w i s e i n t e r f e r e w i t h the f u n c t i o n i n g of an organism w h i c h can s t i l l r e p r o d u c e s u c c e s s f u l l y by a s e x u a l means. Meanwhile, o t h e r m u t u a t i o n s c o u l d have o c c u r r e d w h i c h , f o r some r e a s o n , gave the s e organisms a c o m p e t i t i v e advantage over o t h e r Oomycetes and a l l o w e d them ' f i r s t c h o i c e ' , so to speak, i n terms of e x p l o i t i n g the r i c h and abundant food s u p p l y p r o v i d e d by the f i s h t h a t they i n f e c t . The genus S a p r o l e g n i a sensu s t r i c t o ( D i c k , 1973) c o n t a i n s t w e l v e (or t h i r t e e n , i f the r e c e n t l y d e s c r i b e d S. i r r e g u l a r i s Johnson § Seymour [1975] i s i n c l u d e d ) 'morphospecies' (Scudder, 1974) s e p a r a t e d from each o t h e r ( e x c e p t i n g S^ . i r r e g u l a r i s ) on the b a s i s o f f i v e key c h a r a c t e r s (Seymour, 1970): (1) pr e s e n c e or absence o f a n t h e r i d i a ; (2) a n t h e r i d i a l o r i g i n ; (3) presence or absence o f p i t t i n g on the o o g o n i a l w a l l ; (4) presence o f c e n t r i c or s u b c e n t r i c o o s p o r e s ; and, (5) oospore d i a m e t e r . The w e i g h t g i v e n each of these c h a r a c t e r s v a r i e s f o r the d i f f e r e n t s p e c i e s , and t h i s has r e s u l t e d i n b o t h b r o a d l y and n a r r o w l y d e f i n e d s p e c i e s w h i c h may be s e p a r a t e d from one another by v a r i a t i o n of o n l y one c h a r a c t e r . For example, S^ . f e r a x ( G r u i t h . ) T h u r e t i s s e p a r a t e d from S. hypogyna ( P r i n g s h e i m ) de 77 Bary on the b a s i s o f the type o f a n t h e r i d i a produced (Seymour, 1970). The n a r r o w l y d e f i n e d S. hypogyna produces no a n t h e r i d i a l branches and o n l y hypogynous a n t h e r i d i a l c e l l s . The b r o a d l y de-f i n e d S. f e r a x o f t e n produces no a n t h e r i d i a a t a l l , b u t t h i s s p e c i e s may a l s o i n c l u d e i s o l a t e s t h a t produce m o n o c l i n o u s , androgynous, and even d i c l i n o u s a n t h e r i d i a i n v a r y i n g p r o p o r t i o n s . I n o t h e r r e s p e c t s the morphology of these two s p e c i e s i s v e r y s i m i l a r . S. d i c l i n a and S_. p a r a s i t i c a , as p r e s e n t l y d e f i n e d (Seymour, 1970), a r e a l s o v e r y s i m i l a r w i t h s e p a r a t i o n o f the two s p e c i e s b e i n g p r i m a r i l y based on a s m a l l d i f f e r e n c e i n the mean oospore s i z e and the predominant type o f oospore produced. These and o t h e r a s p e c t s of the morphology of the S_. d i c l i n a - S . p a r a s i t i c a complex w i l l be d i s c u s s e d i n d e t a i l l a t e r . Because of the problems a s s o c i a t e d w i t h a c c u m u l a t i o n o f c y t o g e n e t i c i n f o r m a t i o n f o r the genus ( D i c k , 1972; D i c k § Win-T i n , 1973), the e x t e n t to w h i c h these m o r p h o l o g i c a l v a r i a t i o n s r e f l e c t g e n e t i c r e l a t i o n s h i p s i s unknown. T h e r e f o r e any one or a l l o f t h e f o l l o w i n g t h r e e p r o p o s i t i o n s may be v a l i d : (1) the m o r p h o l o g i c a l v a r i a t i o n s used to d i s t i n g u i s h s p e c i e s o f Sapro-l e g n i a r e p r e s e n t minor g e n e t i c e x p r e s s i o n s o f a b a s i c a l l y homo-geneous genome; (2) i s o l a t e s of s i m i l a r morphology have genomes t h a t are o t h e r w i s e q u i t e d i v e r g e n t ; (3) v a r i a t i o n s i n morphology are i n d i c a t i v e o f p o p u l a t i o n s w i t h s u f f i c i e n t l y d i v e r g e n t gene-t i c i n f o r m a t i o n to j u s t i f y a s s i g n i n g them to d i f f e r e n t t a x a . The l a t t e r two p r o p o s i t i o n s can be t e s t e d by d e t e r m i n i n g the d e o x y r i b o n u c l e i c a c i d base c o m p o s i t i o n o f v a r i o u s i s o l a t e s 78 a s s i g n e d t o the genus. T h i s p r o v i d e s o b j e c t i v e , r e p r o d u c i b l e i n f o r m a t i o n about a r e l a t i v e l y s t a b l e and c o n s e r v a t i v e c h a r a c t e r and can be used to demonstrate a l a c k o f r e l a t i o n between i s o -l a t e s . I t cannot be used to i n d i c a t e the e x t e n t t o which the i s o l a t e s are r e l a t e d because organisms may have v a s t l y d i f f e r e n t genomes and s t i l l have the same DNA base c o m p o s i t i o n . (For f u r t h e r i n f o r m a t i o n on the a p p l i c a t i o n o f t h i s c h a r a c t e r to the taxonomy o f f u n g i see Meyer § P h a f f , 1970; S t o r c k § A l e x o p o u l o s , 1970; Dupont § H e d r i c k , 1971; Green § D i c k , 1972; M a r t i n i e t a l . , 1972; de B e r t o l d i e t a l . , 1973; S t o r c k , 1974 and the papers c i t e d by these a u t h o r s . ) P r i o r to the i n i t i a t i o n of t h i s s t u d y , p r e v i o u s work ( S t o r c k § A l w x o p o u l o s , 1970; Green § D i c k , 1972; C l a r k - W a l k e r § Gl e a s o n , 1973) sug g e s t e d (Table V I I I ) t h a t S a p r o l e g n i a s p e c i e s might have DNA base c o m p o s i t i o n s s u f f i c i e n t l y d i v e r g e n t to make t h i s a u s e f u l c h a r a c t e r f o r s e p a r a t i n g s p e c i e s . I n a d d i t i o n , the i s o l a t e s examined by Green § D i c k (1972) and C l a r k - W a l k e r § Gleason (1973) c o n t a i n e d a s a t e l l i t e DNA which I thought might be a u s e f u l taxonomic i n d i c a t o r - - e s p e c i a l l y f o r d e l i m i t i n g i s o l a t e s i n the S_. d i c l i n a - S . p a r a s i t i c a complex. T h i s r e a s o n -i n g was based on the f a c t t h a t a l l the S a p r o l e g n i a i s o l a t e s examined by Green § D i c k (Table V I I I ) were i n c l u d e d i n t h i s complex, and i t was a l s o p o s s i b l e t h a t the i s o l a t e examined by C l a r k - W a l k e r § Gleason (1973) (Emerson's S a p r o l e g n i a sp. 47-15a = ATCC 26116) a l s o belonged t o t h i s complex and t h a t t h i s c o u l d be shown by growing i t a t low t e m p e r a t u r e s . S t o r c k § Alexopou-TABLE V I I I Buoyant d e n s i t i e s and base c o m p o s i t i o n s of S a p r o l e g n i a s p e c i e s from the l i t e r a t u r e S p e c i e s S. d i c l i n a I I S. f e r a x £5. hypogyna S^ . p a r a s i t i c a t i S. s p e c i e s I s o l a t e * APCC 203a APCC 203d ESB ESB ESB ATCC 26116 Buoyant D e n s i t y i n C s C l , g/cm3 Main Band S a t e l l i t e 1.717 1. 718 1. 718 1.717 1.705-1.708 1. 708 1. 710 1. 707 % GC 58, 46-49 59, 49 49.5, ---55.5, --59, 51 60.5, --58, 48 Reference Green $ D i c k (1972). S t o r c k § A l e x o p o u l o s (1970) 11 Green § D i c k (1972). S t o r c k § A l e x o p o u l o s (1970) C l a r k - W a l k e r § Gleason (1973). * A b b r e v i a t i o n s : APCC, A q u a t i c Phycomycete.Culture C o l l e c t i o n , Department of Botany, The Univ-e r s i t y o f Reading, Reading, England RG6 2AS; ESB, E. S. Beneke, Department o f Botany and P l a n t P a t h o l o g y , M i c h i g a n S t a t e U n i v e r s i t y , E a s t L a n s i n g , M i c h i g a n 48824; ATCC, American Type C u l t u r e C o l l e c t i o n , R o c k v i l l e , M a r y l a n d 20852. 80 l o s (1970) , on the o t h e r hand, r e p o r t e d no s a t e l l i t e DNA i n any o f the S a p r o l e g n i a i s o l a t e s they examined, i n c l u d i n g one i d e n t i -f i e d as S. p a r a s i t i c a . However, i n t h i s case i t was p o s s i b l e t h a t the s a t e l l i t e had been mi s s e d or the i s o l a t e had been i d e n t i f i e d i n c o r r e c t l y . DESCRIPTION OF ISOLATES I used e l e v e n i s o l a t e s o f S a p r o l e g n i a ( T a b l e I X , P l a t e V I I I ) f o r t h i s s t u d y , i n c l u d i n g i s o l a t e s a s s i g n e d to the f o l l o w i n g s p e c i e s : S_. d i c l i n a Humphrey, S_. hypogyna ( P r i n g s h e i m ) de B a r y , S. p a r a s i t i c a Coker, and S. f e r a x ( G r u i t h . ) T h u r e t . S a p r o l e g n i a i s o l a t e s w hich can be i d e n t i f i e d as S_. f e r a x or a s s i g n e d to the S_. d i c l i n a - S . p a r a s i t i c a complex a r e , i n g e n e r a l , the most commonly enc o u n t e r e d r e p r e s e n t a t i v e s o f the genus (see Seymour, 1970) and they are a l s o f r e q u e n t l y i s o l a t e d as p a r a s i t e s of f i s h ( T a ble I I ) ; moreover, S_. f e r a x i s the n o m e n c l a t u r a l type o f the genus and s h o u l d be w e l l r e p r e s e n t e d i n any taxonomic s t u d y of the genus. S_. hypogyna i s not a p a r t i c u l a r l y commonly i s o l a t e d s p e c i e s , but i t does have the d i s t i n c t i o n o f b e i n g a n a r r o w l y d e f i n e d t a x o n , u n l i k e S_. f e r a x or the f u n g i i n c l u d e d i n the S_. d i c l i n a - S . paras i t i c a complex. A l s o S_. hypogyna was used by S t o r c k § A l e x o p o u l o s (1970); hence, I used a l l the s p e c i e s used i n the p r e v i o u s s t u d i e s so t h a t the r e s u l t s c o u l d be compared. In a d d i t i o n to b e i n g r e a s o n a b l y r e p r e s e n t a t i v e o f the mor-p h o l o g i c a l v a r i a t i o n i n the genus S a p r o l e g n i a sensu s t r i c t o , the I d e n t i t y o f the S a p r o l e g n i a i s o l a t e s S p e c i e s I s o l a t e * 1. S. d i c l i n a - S . p a r a s i t i c a APCC 204e complex 2. " ATCC 22284 3. " CBS 282.38 4. 11 GAN 17 5. S. s p e c i e s GAN 18 6. ** GAN 24 7. S. f e r a x IBH 8. 9. i t I I APCC 204a APCC 204b TABLE IX :ed f o r the DNA buoyant d e n s i t y d e t e r m i n a t i o n s . O r i g i n M a r i o n Lake, B r i t i s h Columbia, s i t e 27. C h r i s t i a n s b u r g , V i r g i n i a from I c t a l u r u s p u n c t a t u s , England. Robertson Creek, B r i t i s h Columbia (124° 59'W,49°20*N) from Oncorhynchus n e r k a . L i t e r a t u r e R e f e r e n c e s D i c k (1971) O'Bier (1960); S c o t t § O'Bier (1962); P o w e l l e t a l . (1972). CO U n i v e r s i t y o f Leeds, England from aquarium water. Bedgebury, E n g l a n d , s i t e BQ4. Lake D i s t r i c t , England. Manton e_t a_l_. (1951, 1952); Gay § Greenwood (1966) ; Heath § Green-wood (1968, 1970a, 1970b, 1970c, 1971); Heath e t a l . (1970); Gay e t a l . TT971); O l s o n $ HeatK (1971) ; Heath (1970 , 1974, 1975a, 1975b, 1976); B e c k e t t e t a l . (1974). D i c k (1963). TABLE IX (Continued) I d e n t i t y of the S a p r o l e g n i a i s o l a t e s used f o r the DNA buoyant d e n s i t y d e t e r m i n a t i o n s . S p e c i e s I s o l a t e O r i g i n L i t e r a t u r e R e f e rences 10. S. f e r a x ATCC 26116 U n i v e r s i t y of C a l i f o r n i a , (RE 47-15a) B e r k e l e y from pond wate r . Unestam § Gleason (1968) ; Gleason e t a l . (1970) ; C l a r k - W a l k e r § Gleason (1973); G l e a s o n (1973a, 1973b, 1974) ; N e i s h (1975b). 11. S. hypogyna APCC 206a S. E. England CO t o * A b b r e v i a t i o n s : APCC, A q u a t i c Phycomycete C u l t u r e C o l l e c t i o n , Department o f Botany, The U n i v e r s i t y of Reading, Reading, E n g l a n d RG6 2AS. A l l APCC c u l t u r e s s e n t c o u r t e s y of Dr. M. W. D i c k , U n i v e r s i t y of Reading; ATCC, American Type C u l t u r e C o l l e c t i o n , R o c k v i l l e , M a r y l a n d , U.S.A. 20852; CBS, C e n t r a a l b u r e a u voor S c h i m m e l c u l t u r e s , Baarn, The N e t h e r l a n d s ; GAN, G. A. N e i s h , Department o f Botany, U n i v e r s i t y of B r i t i s h Columbia, Vancouver, Canada V6T 1W5; IBH, Dr. I . B. Heath, Department of B i o l o g y , York U n i v e r s i t y , Downsview, O n t a r i o , Canada M3J 1P3. T h i s i s o l a t e i s a l s o d e p o s i t e d i n APCC as No. 204g; RE, Dr. R a l p h Emerson, Department of Botany, U n i v e r s i t y of C a l i f o r n i a , B e r k e l e y , USA, 94 720 . 83 P l a t e V I I I P hotomicrographs o f u n d i s t u r b e d f i v e day o l d hemp seed c u l t u r e s o f the i s o l a t e s used f o r th& DNA buoyant d e n s i t y d e t e r -m i n a t i o n s showing some a s p e c t s o f the p h y s i o l o g i c a l and morpho-l o g i c a l v a r i a t i o n among these i s o l a t e s . The c u l t u r e s were m a i n t a i n e d a t 21 ± 1 C i n the dark. F i g u r e 1 - S . ? d i c l i n a (APCC 204e) showing o o g o n i a . F i g u r e 2 - S. p a r a s i t i c a (ATCC 22284) showing two o f the few l a r g e gemmae observ e d i n t h i s c u l t u r e . Oogonia were never o b s e r v e d i n t h i s i s o l a t e . F i g u r e 3 - S. d i c l i n a (CBS 282.38) showing some o f the numerous l a r g e gemmae seen i n t h i s c u l t u r e . T h i s i s o l a t e p r o-duced no oogonia a t 21 C but would do so a f t e r p r o l o n g e d i n c u b a t i o n a t lower t e m p e r a t u r e s . F i g u r e 4 - S . ? d i c l i n a (GAN 17) showing some gemmae. Swimming zoospores were a l s o seen i n t h i s c u l t u r e . A s p e c t s of the morphology and p h y s i o l o g y of t h i s C ategory B i s o l a t e have been d e s c r i b e d p r e v i o u s l y . F i g u r e 5 - S. s p e c i e s (GAN 24) showing s e v e r a l z o o s p o r a n g i a , most of which a re empty. Note the cymose zoospor-a n g i a l r e n e w a l . A s p e c t s o f the morphology and p h y s i o l o g y o f t h i s C a t e g o r y D i s o l a t e have been d e s c r i b e d p r e v i o u s l y . F i g u r e 6 - S. s p e c i e s (GAN 18) showing numerous z o o s p o r a n g i a and zoospore c y s t s . A s p e c t s o f the morphology and p h y s i o l o g y o f t h i s C ategory C i s o l a t e have been d e s c r i b e d p r e v i o u s l y . F i g u r e 7 - S_. f e r a x (IBH) showing z o o s p o r a n g i a . Oogonia were never seen i n t h i s i s o l a t e r e g a r d l e s s o f c u l t u r e c o n d i t i o n s . F i g u r e 8 - S_. f e r a x (APCC 204a) showing oogonia. T h i s i s o l a t e produces masses o f oogonia a t 21 C. F i g u r e 9 - S. f e r a x (APCC 204b) showing o o g o n i a . T h i s i s o l a t e a l s o produces masses o f oogonia a t 21 C. F i g u r e 10- S_. f e r a x (ATCC 26116) showing gemmae and one of the few oogonia produced by t h i s i s o l a t e a t 21 C. F i g u r e 11- S_. hypogyna (APCC 206a) showing the numerous oogonia produced by t h i s i s o l a t e a t 21 C. 85 i s o l a t e s used came from g e o g r a p h i c a l l y d i v e r s e l o c a t i o n s (Table I X ) , showed a r e l a t i v e l y wide range of p h y s i o l o g i c a l v a r i a t i o n (e.g. see P l a t e V I I I ) , and had been i n c u l t u r e f o r v a r y i n g p e r i o d s - ^ the o l d e s t i s o l a t e b e i n g S_. d i c l i n a (CBS 282.38) w h i c h had been i n c u l t u r e f o r more than 35 y e a r s when the DNA was i s o l a t e d from i t . The i d e n t i t y o f t h r e e i s o l a t e s (GAN 24, ATCC 22284, IBH) used i n t h i s s t u d y c o u l d n ot be c o n f i r m e d because oogonium pro-d u c t i o n was never ob s e r v e d . GAN 24, a Category D i s o l a t e , has been d i s c u s s e d p r e v i o u s l y . S. p a r a s i t i c a (ATCC 22284) was r e -p o r t e d by O'Bier (1960) to have produced o o g o n i a . The o n l y o t h e r subsequent s t u d y o f t h i s organism ( P o w e l l et. a J . , 1972) d i d n o t d e a l w i t h oogonium p r o d u c t i o n , and d e s p i t e v a r i o u s a t t e m p t s , I was never a b l e to observe oogonium p r o d u c t i o n i n t h i s i s o l a t e . I s o l a t e IBH was f i r s t d e s c r i b e d and i d e n t i f i e d as S. f e r a x by Manton e_t al_. (1951). T h e i r d e s c r i p t i o n o f the morphology o f t h i s i s o l a t e , and .the means by which oogonium pro-d u c t i o n was o b t a i n e d , i s q u i t e vague. I s o l a t e IBH has subse-q u e n t l y been used i n a number of u l t r a s t r u c t u r a l i n v e s t i g a t i o n s (see l i t e r a t u r e r e f e r e n c e s i n Table I X ) , but oogonium p r o d u c t i o n has never been obser v e d ( I . B. Heath, p e r s o n a l c o m m u n i c a t i o n ) , and I have a l s o been u n a b l e to induce oogonium p r o d u c t i o n i n t h i s i s o l a t e . T h e r e f o r e , a l t h o u g h I have e l e c t e d to r e t a i n t h i s i s o l a t e i n the s p e c i e s i n which i t was o r i g i n a l l y p l a c e d , i t must be n o t e d t h a t the i d e n t i f i c a t i o n o f t h i s i s o l a t e as S. f e r -ax cannot be c o n f i r m e d as b e i n g c o r r e c t . 86 To observe oogonium p r o d u c t i o n i n S. d i c l i n a (CBS 282.38), I found i t n e c e s s a r y to i n c u b a t e the c u l t u r e s f o r p r o l o n g e d p e r i o d s a t lower t e m p e r a t u r e s - - a c h a r a c t e r i s t i c w h i c h , as I have mentioned p r e v i o u s l y , appears to be t y p i c a l of many i s o -l a t e s i n the S^ . d i c l i n a - S . p a r a s i t i c a complex. Good r e s u l t s were o b t a i n e d by growing the i s o l a t e on hemp seed h a l v e s i n f i l t e r s t e r i l i z e d pond water a t 10 C f o r about two months. Beyond c o n f i r m i n g t h e i r i d e n t i t y , few f u r t h e r s t u d i e s were done w i t h two S. f e r a x i s o l a t e s (APCC 204a and APCC 204b) and the IS. hypogyna i s o l a t e (APCC 206a) , a l l of which produced oogonia a b u n d a n t l y on hemp seeds i n d i s t i l l e d water a t 21 C ( P l a t e VIII, F i g s . 8, 9, 11). A somewhat more d e t a i l e d s t u d y o f S. p a r a s i t i c a (APCC 204e) which a l s o produced oogonia a b u n d a n t l y a t 21 C "\ ( P l a t e VIII, F i g . 1 ) , w i l l be d i s c u s s e d l a t e r . Two o t h e r i s o -l a t e s -- GAN 17, a C a t e g o r y B i s o l a t e , and GAN 18, a Category C i s o l a t e - - have been d i s c u s s e d p r e v i o u s l y . A d e t a i l e d s t u d y was c a r r i e d out to a s s e s s the e f f e c t o f temperature on the growth and morphology o f Emerson's S a p r o l e g -n i a sp. 47-15a (ATCC 26116). As mentioned p r e v i o u s l y , t h i s i s o l a t e has had i t s DNA buoyant d e n s i t y d e t e r m i n e d i n another s t u d y ( C l a r k - W a l k e r § G l e a s o n , 1973), and i t has a l s o been the s u b j e c t of v a r i o u s p h y s i o l o g i c a l and u l t r a s t r u c t u r a l s t u d i e s (Unestam § G l e a s o n , 1968; Gleason e_t al_. , 1970; G l e a s o n , 1973a, 1973b, 1974). N e v e r t h e l e s s , p r i o r to t h i s s t u d y , t h e r e was no d e s c r i p t i o n of the morphology o f t h i s i s o l a t e , nor any account of i t s growth r a t e on a r o u t i n e agar medium. As e x p e c t e d , I 87 found t h a t temperature has a marked e f f e c t on the growth and mor-phology of t h i s i s o l a t e . An unexpected r e s u l t was the d i s c o v e r y t h a t the i s o l a t e most c l o s e l y resembles S_. f e r a x , not S. d i c l i n a . Most of the t e c h n i q u e s used to s t u d y t h i s i s o l a t e have been d e s c r i b e d p r e v i o u s l y . The morphology o f the i s o l a t e was examined by growing i t on hemp seed h a l v e s i n d i s t i l l e d water a t 21 ± 1 C, 14 ± 1 C, 10 ± 1 C, or 7 ± 0.5 C. The l i n e a r growth r a t e a t these t e m p e r a t u r e s , and a l s o a t 3 ± 1 C, on CMA was d e t e r m i n e d u s i n g the t e c h n i q u e s d e s c r i b e d on page 71. As a r u l e , the c u l t u r e s were m a i n t a i n e d i n the dark except d u r i n g p e r i o d s of o b s e r v a t i o n . However, exposure to l i g h t was not n e c e s s a r i l y r i g i d l y c o n t r o l l e d f o r e v e r y c u l t u r e , so I car-., r i e d out experiments to determine whether c o n t i n u o u s exposure to a r e l a t i v e l y low l i g h t i n t e n s i t y (344-388 l u x ) i n the same o r d e r of magnitude as t h a t n o r m a l l y e n c o u n t e r e d i n the l a b o r a t o r y , had any s i g n i f i c a n t e f f e c t on the morphology or growth o f the i s o -l a t e , s i n c e v a r i o u s workers ( K r a u s e , 1960; S z a n i s z l o , 1965; Lee c] S c o t t , 1967) have shown t h a t l i g h t may a f f e c t the morphology and growth o f S a p r o l e g n i a i s o l a t e s . The experiments were c a r r i e d out a t 10 ± 1 C and the methods used were s i m i l a r to those des-c r i b e d p r e v i o u s l y , e x cept t h a t the c u l t u r e s were exposed to con-t i n u o u s l i g h t p r o v i d e d by Westinghouse 24T12 c o o l - w h i t e f l u o r -e s c e n t l i g h t t u b e s . The r e s u l t s o f t h i s work i n d i c a t e d t h a t l i g h t o f the type and i n t e n s i t y u s e d , had no s i g n i f i c a n t e f f e c t on the growth r a t e o r morphology of ATCC 26116. S i m i l a r work w i t h GAN 17, a C a t e g o r y B i s o l a t e , a l s o showed t h a t exposure to 88 c o n t i n u o u s l i g h t o f the type and i n t e n s i t y d e s c r i b e d , had no e f f e c t on the morphology o f , t h i s i s o l a t e e i t h e r (see P l a t e V, F i g s . 3, 4 ) . The e f f e c t of l i g h t on the growth r a t e of GAN 17 was not examined. A d e s c r i p t i o n o f S_. f e r a x (ATCC 26116) f o l l o w s . For ATCC 26116, z o o s p o r a n g i a and gemmae are most e a s i l y ob-s e r v e d a t 21 C, but oogonia a re v e r y s p a r s e ( P l a t e IX, F i g . 7) and I have not observ e d a n t h e r i d i a a t t h i s t e m p e r a t u r e . A t 14 C or l e s s , o o g o n i a are v e r y abundant ( P l a t e IX, F i g . 6) and gemmae are c o n c o m i t a n t l y s p a r s e . A n t h e r i d i a can be observ e d a t 14 C as w e l l , but a r e most e a s i l y found a t 7 C. Th e - z o o s p o r a n g i a a re o f t e n i r r e g u l a r and p o l y m o r p h i c ( P l a t e X, F i g s . 1, 2) and may be t e r m i n a l or i n t e r c a l a r y , but t y p i c a l ( f o r the genus) more or l e s s c y l i n d r i c a l , t e r m i n a l z o o s p o r a n g i a w i t h a p i c a l e x i t p a p i l l a e a r e common ( P l a t e I X , F i g s . 4, 5; P l a t e X, F i g s . 3, 4 ) . Zoospore r e l e a s e i s t y p i c a l l y s a p r o l e g n o i d , but i t i s not uncommon to see zoospores e n c y s t e d w i t h i n a zoo-sporangium ( P l a t e IX, F i g . 5 ) . I n t e r n a l p r o l i f e r a t i o n can be observe d ( P l a t e I X , F i g s . 4, 5 ) . The zoospore c y s t s are u s u a l l y 12-14 ym i n d i a m e t e r . The gemmae are a l s o t e r m i n a l or i n t e r c a l -a r y and are v e r y v a r i a b l e i n s i z e and shape ( P l a t e IX, F i g . 7 ) . The oogonia can be q u i t e v a r i a b l e i n shape and may be t e r m i n a l or i n t e r c a l a r y , b u t are t y p i c a l l y t e r m i n a l w i t h a s p h e r i c a l o r p y r i f o r m shape ( P l a t e IX, F i g . 6; P l a t e X, F i g . 9 ) . The o o g o n i a l w a l l i s smooth and co v e r e d w i t h numerous co n s p i c u o u s p i t s ( P l a t e IX, F i g . 2; P l a t e X, F i g . 9 ) . T y p i c a l o o g o nia are about 45-90 ym 89 P l a t e IX O b s e r v a t i o n s on the morphology and p h y s i o l o g y o f ATCC 26116. F i g u r e s 1 and 2 - Oogonia from a 14 C c u l t u r e . Note the c e n t r i c oospores i n F i g . 1 and the prominent p i t s (arrows) on the o o g o n i a l w a l l i n F i g . 2. Bars r e p r e s e n t 20 ym. F i g u r e 3 - Oogonium w i t h an obvious d i c l i n o u s a n t h e r i d i u m . a 7 C c u l t u r e . Bar r e p r e s e n t s 20 ym. From F i g u r e 4 - Immature zoosporangium or a gemma p r o l i f e r a t i n g w i t h -i n an empty zoosporangium. Arrow i n d i c a t e s the former e x i t p a p i l l a . From a 14 C c u l t u r e . Bar r e -p r e s e n t s 100 ym. F i g u r e 5 - I n t e r n a l p r o l i f e r a t i o n . Arrow i n d i c a t e s the former e x i t p a p i l l a . I n t h i s c a s e , the zoospores i n the p r o l i f e r a t i n g zoosporangium had a p p a r e n t l y e n c y s t e d w i t h i n the sporangium. From a 21 C c u l t u r e . Bar r e p r e s e n t s 100 ym. F i g u r e s 6 and 7 - Comparison of 16 day o l d c u l t u r e s m a i n t a i n e d at 10 C ( F i g . 6) and 21 C ( F i g . 7 ) . Note the abund-ance of oogonia and absence o f gemmae i n F i g . 6 and the s c a r c i t y of oogonia (arrow i n d i c a t e s one) and abundance of gemmae i n F i g . 7. Bars r e p r e s e n t 250 ym. 91 P l a t e X O b s e r v a t i o n s on the morphology and p h y s i o l o g y o f ATCC 26116. F i g u r e s 1-3 - Empty z o o s p o r a n g i a . Note the v a r i a b i l i t y o f s i z e and shape. Arrows i n d i c a t e e x i t p a p i l l a e . From a 21 C c u l t u r e . Bars r e p r e s e n t 100 urn. F i g u r e 4 - Zoosporangium shown i n F i g . 3 p r i o r to zoospore r e -l e a s e . Arrow i n d i c a t e s e x i t p a p i l l a . Bar r e p r e -s e n t s 100 ym. F i g u r e 5 F i g u r e 6 F i g u r e 7 F i g u r e 9 S u b c e n t r i c oospores r e p r e s e n t s 10 ym. From a 14 C c u l t u r e . Bar Oogonium c o m p l e t e l y c o v e r e d w i t h a n t h e r i d i a . a 7 C c u l t u r e . Bar r e p r e s e n t s 20 ym. From Oogonium w i t h c l a v a t e a n t h e r i d i a l c e l l s and an ap-p a r e n t f e r t i l i z a t i o n tube ( a r r o w ) . From a 10 C c u l t u r e m a i n t a i n e d i n c o n s t a n t l i g h t . Bar r e p r e s e n t s 20 ym. F i g u r e 8 - P o r t i o n of an oogonium showing two oospores which have a d i s t o r t e d shape because of the c o n s t r a i n t s o f the o o g o n i a l w a l l . . From a 7 C c u l t u r e . Bar r e p r e s e n t s 20 ym. Group of oogonia from a 14 C c u l t u r e . Arrows i n d i -c a t e two o f s e v e r a l p i t s v i s i b l e i n t h i s f i g u r e . Bar r e p r e s e n t s 100 ym. 93 i n d i a m e t e r and c o n t a i n about 6-20 o o s p o r e s , but s m a l l e r or l a r g e r oogonia c o n t a i n i n g l e s s e r or g r e a t e r numbers of oospores are common ( P l a t e IX, F i g . 6; P l a t e X, F i g . 9 ) . The oospheres mature and become t y p i c a l l y c e n t r i c ( P l a t e IX, F i g . 1; P l a t e X, F i g . 9) but o c c a s i o n a l l y s u b c e n t r i c ( P l a t e X, F i g . 5) o o s p o r e s , which are u s u a l l y 21-26 ym i n d i a m e t e r . The oospores are always s p h e r i c a l u n l e s s t h e i r shape i s a l t e r e d by the c o n s t r a i n t s o f the o o g o n i a l w a l l ( P l a t e X, F i g . 8 ) . The a n t h e r i d i a are s p a r s e and d i c l i n o u s i n o r i g i n ( P l a t e IX, F i g . 3 ) . The a n t h e r i d i a l c e l l s are l a t e r a l l y a p p r e s s e d and may be c l a v a t e ( P l a t e X, F i g . 7) or t u b u l a r ( P l a t e X, F i g . 6) and are o f t e n branched. At 7 C i t i s not uncommon to see oogonia c o m p l e t e l y c o v e r e d w i t h an-t h e r i d i a w hich form a complex t u b u l a r mass ( P l a t e X, F i g . 6 ) . F e r t i l i z a t i o n tubes were r a r e l y and d u b i o u s l y o b s e r v e d ( P l a t e X, F i g . 7 ) . The key m o r p h o l o g i c a l f e a t u r e s o f ATCC 26116 which p l a c e i t c l o s e s t t o £5. f e r a x are i t s p r e d o m i n a n t l y c e n t r i c o o s p o r e s , c o n s p i c u o u s l y p i t t e d o o g o n i a l w a l l , and g e n e r a l absence of an-t h e r i d i a ( P l a t e IX, F i g s . 1, 2; P l a t e X, F i g . 9 ) . The o c c a s i o n -a l s u b c e n t r i c oospores and the d i c l i n o u s o r i g i n o f the a n t h e r i -d i a do not e x c l u d e the i s o l a t e from Seymour's (1970) expanded concept o f the s p e c i e s . A f t e r an i n i t i a l l a g phase, which v a r i e d from l e s s than 24 h a t 21 C to about 96 h a t 3 C, the growth r a t e o f ATCC 26116 on CMA was -- l i k e t h a t o f GAN 17, GAN 18 and GAN 24 -c o n s t a n t f o r a g i v e n t e m p e r a t u r e . These growth r a t e s are g i v e n 94 i n T a ble V I I and F i g u r e 3 and show t h a t w i t h a d o u b l i n g of the te m p e r a t u r e , t h e r e i s a d o u b l i n g o f growth r a t e w i t h i n the temp-e r a t u r e range s t u d i e d . T h i s p a t t e r n i s s i m i l a r to t h a t encount-e r e d i n the o t h e r i s o l a t e s (Table V I I , F i g u r e 3 ) . ATCC 26116 i s not unique among S. f e r a x i s o l a t e s i n h a v i n g poor oogonium p r o d u c t i o n a t 'room t e m p e r a t u r e ' . I t has been known s i n c e P i e t e r s ' (1915b, 1915c) s t u d i e s t h a t temperature can have a marked e f f e c t on the m o r p h o l o g i c a l development o f S. f e r a x i s o l a t e s . P i e t e r s (1915b) found t h a t temperatures around 15 C were b e s t f o r o b t a i n i n g c e r t a i n o o g o n i a l p r o d u c t i o n i n an i s o l a t e of S^ . f e r a x c o l l e c t e d i n H e i d e l b e r g , Germany, and he a l s o found ( P i e t e r s , 1915c) t h a t f o r some c u l t u r e s o f S. f e r a x c o l l e c t e d i n Ann A r b o r , M i c h i g a n , low temperatures (12-15 C) enhanced a n t h e r -i d i a l p r o d u c t i o n . Lee § S c o t t (1967) found t h a t t h e i r i s o l a t e of S. f e r a x would n ot produce oogonia a t 25 C but d i d so abund-a n t l y a t 15 C. These r e s u l t s are i n agreement w i t h those o b t a i n e d f o r ATCC 26116. However, t h i s p a t t e r n i s by no means u n i v e r s a l f o r S^ . f e r a x i s o l a t e s . I have a l r e a d y shown t h a t two of the S. f e r a x i s o l a t e s used i n t h i s s t u d y (APCC 204a $ APCC 204b) c o u l d b o t h form oogonia i n abundance a t 21 C and Krause (1960) has r e p o r t e d t h a t an S>. f e r a x t h a t he s t u d i e d c o u l d form oogonia a t 27 C. There a r e a p p a r e n t l y no r e p o r t s o f f e r a x i s o l a t e s t h a t are c a p a b l e o f f o r m i n g oogonia a t temperatures i n excess o f 20 C but are unable to do so a t lower t e m p e r a t u r e s , p r o v i d e d s u f f i c i e n t time i s a l l o w e d t o compensate f o r the s l o w e r development o f the i s o l a t e s a t the lower t e m p e r a t u r e s . Thus the 95 d a t a a v a i l a b l e to date i n d i c a t e t h a t t h e r e i s a v a r i a b l e upper l i m i t c o n d u c i v e to p r o l i f i c o o g o n i a l development f o r S_v f e r a x i s o l a t e s and t h a t t e m p e r a t u r e s i n excess o f t h i s l i m i t w i l l cause the i s o l a t e to tend to form gemmae i n s t e a d o f oogonia. A n o t h e r i n t e r e s t i n g c h a r a c t e r i s t i c o f ATCC 26116 i s i t s a b i l i t y t o produce o o g o n i a i n agar media. I o b s e r v e d oogonium f o r m a t i o n i n b o t h CMA and YpSs agar. T h i s a b i l i t y i s s h a r e d by the two o t h e r i d e n t i f i a b l e S. f e r a x i s o l a t e s used i n t h i s s t u d y (APCC 204a § APCC 204b) b o t h o f which c o u l d produce oogonia i n YpSs agar. ( T h e i r a b i l i t y to produce oogonia i n CMA was not checked.) Coker (1923) n o t e d t h a t S^ . f e r a x " i s our o n l y Sapro-l e g n i a t h a t forms oogonia i n t h i s medium [ c o r n meal agar] except S. monoica [= S.~ f e r a x (Seymour, 1 9 7 0 ) ] . . . . " Presumably t h i s c h a r a c t e r i s t i c i s i n some way r e l a t e d to the a b i l i t y o f these f u n g i to produce oogonia w i t h o u t a n t h e r i d i a ( p a r t h e n o g e n e t i c a l l y ) , but to my knowledge, the n a t u r e o f t h i s phenomenon has never been i n v e s t i g a t e d . None of the o t h e r S a p r o l e g n i a i s o l a t e s used i n t h i s s t u d y produced oogonia i n agar media, even though some of them, as noted p r e v i o u s l y , produce oogonia a b u n d a n t l y on hemp seeds i n water under s u i t a b l e c o n d i t i o n s . DNA ISOLATION AND BUOYANT DENSITY DETERMINATION The method used f o r the i s o l a t i o n o f the DNA was adapted from Green § D i c k (1972) and Marmur (1961). S i n c e ATCC 26116 has had i t s DNA buoyant d e n s i t y d e t e r m i n e d u s i n g somewhat d i f -f e r e n t methods i n a c o m p l e t e l y independent s t u d y ( C l a r k - W a l k e r 96 § G l e a s o n , 1973), i t was used to check t h i s method. The c u l t u r e s used f o r the s t u d y were r o u t i n e l y m a i n t a i n e d on YpSs agar and were t e s t e d f o r the presence of b a c t e r i a l con-t a m i n a t i o n by growing them i n TYE b r o t h . T h i s p r o c e d u r e i s d e s c r i b e d on page 43. To h e l p r u l e out the p o s s i b i l i t y o f v i r a l o r i g i n o f a s a t e l -l i t e DNA band, two of the i s o l a t e s (GAN 17 § GAN 18). were examin-ed u s i n g t e c h n i q u e s s i m i l a r to those of V o l k o f f e_t a l . (1972) . S i n c e a n o t h e r member o f the S a p r o l e g n i a c e a e , Aphanomyces a s t a c i S c h i k o r a , may c o n t a i n m y c o p l a s m a - l i k e s t r u c t u r e s (Heath § Unestam, 1974), t h i s p o s s i b i l i t y was t e s t e d by i s o l a t i n g DNA from i s o l a t e IBH which has been e x t e n s i v e l y examined by e l e c t r o n m i c r o s c o p y (see l i t e r a t u r e r e f e r e n c e s i n T a b l e IX) and i n which no mycoplas-m a - l i k e s t r u c t u r e s have ever been d e t e c t e d ( I . B. Heath, p e r s o n a l communication). To o b t a i n mycelium, a s m a l l b l o c k of i n o c u l u m was removed from a s t o c k c u l t u r e m a i n t a i n e d on YpSs agar and p l a c e d i n one l i t e r o f b r o t h c o n t a i n i n g 10 g g l u c o s e , 5 g peptone, and 5 g y e a s t e x t r a c t . These c u l t u r e s were grown a t 21 ± 1 C i n a r e c i -p r o c a t i n g i n c u b a t o r shaker s e t a t about 60 o s c i l l a t i o n s per minute w i t h a 5-cm s t r o k e f o r 5-9 days. The y i e l d s o b t a i n e d ranged from about 4-25 g wet w e i g h t of mycelium depending on the i s o l a t e used and the i n c u b a t i o n t i m e . The mycelium was h a r v e s t e d by s u c t i o n f i l t r a t i o n , washed i n d i s t i l l e d w a t e r , f r o z e n i n l i q u i d N2 and ground t o a f i n e powder w i t h a mortar and p e s t l e . The f r o z e n powder was suspended i n a. 97 b u f f e r (1 M N a C l , 0.1 M d i e t h y l d i t h i o c a r b a m a t e , 0.1 M t r i s , 0.005 M EDTA, pH 9.0 ± 0.1) and to t h i s s u s p e n s i o n was added an e q u a l volume of l i q u i d p h e n o l - b u f f e r m i x t u r e (9:1, v/v) and then 1 ml o f 201 (w/v) sodium d o d e c y l s u l f a t e (SDS) was added f o r each 10 ml o f the t o t a l s u s p e n s i o n . The s u s p e n s i o n was s t i r r e d f o r 30 min a t room t e m p e r a t u r e , the phases were s e p a r a t e d by c e n t r i -f u g a t i o n , and the aqueous phase d e p r o t e i n i z e d once w i t h p h e n o l and once w i t h c h l o r o f o r m - i s o a m y l a l c o h o l (24:1, v/v) (Marmur, 1961) . Two volumes o f c o l d 95% e t h a n o l were added to the aqueous s u p e r n a t a n t and l e f t o v e r n i g h t a t 4 C to ensure complete p r e c i p i -t a t i o n o f the n u c l e i c a c i d s . The p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n , d i s s o l v e d i n 9 ml of 0.1 X SSC, and made up to SSC (0.15 M N a C l , 0.015 M sodium c i t r a t e ) by the a d d i t i o n o f 10 X SSC. The s o l u t i o n was then i n c u b a t e d a t 37 C w i t h 100 u n i t s / m l T x - r i b o n u c l e a s e , 50 yg/ml p a n c r e a t i c r i b o n u c l e a s e , and 50 yg/ml a-amylase f o r 30 min, more enzymes added, and the i n c u b a t i o n c o n t i n u e d f o r another 30 min. T h i s was f o l l o w e d by two t r e a t m e n t s w i t h p r e -d i g e s t e d pronase a t 45 C, and another d e p r o t e i n i z a t i o n w i t h c h l o r o f o r m - i s o a m y l a l c o h o l . The DNA was p r e c i p i t a t e d w i t h 95% e t h a n o l , d i s s o l v e d i n 0.1 SSC and p r e c i p i t a t e d w i t h i s o p r o p a n o l -a c e t a t e EDTA (Marmur, 1961) o v e r n i g h t a t 4 C. T h i s p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n , washed w i t h e t h a n o l , d i s s o l v e d i n 0.1 X SSC, made up to SSC and s t o r e d f r o z e n . U l t r a v i o l e t a b s o r p t i o n s p e c t r a ( A m a x = 257 rim; 260/280 r a t i o s 1.80-1.96) and d i p h e n y l a m i n e assays ( B u r t o n , 1968) were 98 used to e s t i m a t e the p u r i t y and c o n c e n t r a t i o n o f the DNA p r e p a r a -t i o n s . Y i e l d s o b t a i n e d were on the o r d e r o f 1-3 mg per 100 g wet w e i g h t o f mycelium. DNA base c o m p o s i t i o n s were det e r m i n e d from the buoyant d e n s i t y i n C s C l assuming a v a l u e o f 1.731 g/cm 3 f o r M i c r o c o c c u s  l y s o d e i k t i c u s DNA ( S c h i l d k r a u t e t a l . , 1962). A t l e a s t two d e t e r m i n a t i o n s were made f o r each sample. The r e s u l t s are a c c u r -a t e to ± 0.001 g/cm 3 or ± 1% GC. There was no s i g n i f i c a n t d i f -f e r e n c e i n the r e s u l t s o b t a i n e d from runs made more than e i g h t months a p a r t . RESULTS AND DISCUSSION The main DNA component o f a l l the i s o l a t e s (Table X, F i g u r e 3) has a buoyant d e n s i t y of 1.717 ± 0.001 g/cm 3 (58 ± 1% GC) whi c h i s w i t h i n the range o f e x p e r i m e n t a l e r r o r . These r e s u l t s (see T a b l e V I I I ) agree w e l l w i t h those o b t a i n e d by Green § D i c k (1972) f o r t h r e e i s o l a t e s b e l o n g i n g to the :S. d i c l i n a - S . p a r a s i t i c a complex, and by C l a r k - W a l k e r § Gleason (1973) f o r S. f e r a x (ATCC 26116). These r e s u l t s are a l s o i n r e a s o n a b l e agreement w i t h those o b t a i n e d by S t o r c k § A l e x o p o u l o s (1970) f o r J3. hypogyna and S^ . p a r a s i t i c a (59 v e r s u s 55.5 and 60.5% GC r e s p e c t i v e l y ) . However, t h e i r r e p o r t of 49.5% GC f o r S. f e r a x i s 9.5% lower than my v a l u e f o r f o u r d i f f e r e n t i s o l a t e s o f t h a t s p e c i e s and, i n f a c t , c o i n c i d e s w i t h the p o s i t i o n o f the s a t e l -l i t e component. T h i s c o u l d n o t be due to e x p e r i m e n t a l e r r o r i n d e t e r m i n a t i o n o f the base c o m p o s i t i o n , s i n c e they used the same 99 buoyant d e n s i t y method. U n f o r t u n a t e l y , t h i s problem must remain u n r e s o l v e d s i n c e the i s o l a t e i n q u e s t i o n i s no l o n g e r e x t a n t (E. S. Beneke, p e r s o n a l communication). T h e r e f o r e , e x c l u d i n g one q u e s t i o n a b l e e x c e p t i o n , the v a r i a -t i o n of base c o m p o s i t i o n in. the genus S a p r o l e g n i a sensu s t r i c t o appears to be w i t h i n a t l e a s t 5% GC (55.5-60.51 GC) and i s p r o b a b l y w i t h i n 3% GC (57-591 GC). C l e a r l y , a n a l y s i s of DNA base c o m p o s i t i o n cannot be used f o r s e p a r a t i n g s p e c i e s o f S a p r o l e g n i a i n accordance w i t h c l a s s i c a l m o r p h o l o g i c a l s p e c i e s c o n c e p t s ; however, the d a t a do suggest t h a t S a p r o l e g n i a may be a r e l a t i v e l y homogeneous taxon (a c o n c l u s i o n s u p p o r t e d by the r e c e n t s e r o t a x -onomic st u d y by P e d u z z i [1975]) and t h a t any i s o l a t e whose base c o m p o s i t i o n i s not between 55.5 and 60.51 s h o u l d be e x c l u d e d from the genus. F u r t h e r c l a r i f i c a t i o n o f the i n f r a g e n e r i c r e -l a t i o n s h i p s of S a p r o l e g n i a s p e c i e s may have to be based on a n a l y -s i s o f the DNA-DNA homologies between d i f f e r e n t i s o l a t e s . A l l the i s o l a t e s examined have a more or l e s s w e l l - d e f i n e d s a t e l l i t e DNA w i t h a buoyant d e n s i t y o f 1.706-1.709 g/cm 3 (47-50% GC). The d a t a are g i v e n i n Ta b l e X and F i g u r e 3. T h i s s a t e l l i t e DNA was a l s o found i n t h r e e i s o l a t e s o f the S. d i c l i n a - S . p a r a -s i t i c a complex by Green § D i c k (1972), who showed t h a t i t was d e o x y r i b o n u c l e a s e s e n s i t i v e and not due to c o n t a m i n a t i o n o f c u l t u r e s by b a c t e r i a or o t h e r f u n g i . The s a t e l l i t e was a l s o r e -p o r t e d by C l a r k - W a l k e r $ Gleason (1973) i n S. f e r a x (ATCC 26116). They showed t h a t the s a t e l l i t e was not m i t o c h o n d r i a l DNA, which had a much lower buoyant d e n s i t y o f 1.685 g/cm 3. No v i r u s - l i k e TABLE X C s C l buoyant d e n s i t y and % GC o f the DNA o f the S a p r o l e g n i a i s o l a t e s . * Buoyant D e n s i t y , g/cm 3** % GC S p e c i e s I s o l a t e Main Band S a t e l l i t e Main Band S a t e l l i t e 1. S. d i c l i n a - S . para-s i t i c a complex APCC 204e 1. 718 1.708 59 49 2. 11 ATCC 22284 1. 717 1.707 58 48 3. tt CBS 282.38 1. 718 1. 708 59 49 4. i t GAN 17 1.717 1. 708 58 49 5. S. s p e c i e s GAN 24 1. 718 1. 707 59 48 6. tt GAN 18 1. 717 1. 707 58 48 7. S. f e r a x IBH 1. 718 1.707 59 48 8. tt APCC 204a 1. 718 1. 707 59 48 9. I I APCC 204b 1. 718 1.708 59 49 10. t i ATCC 26116 1. 718 1.708 59 49 11. S. hypogyna APCC 206a 1. 718 1. 706 59 47 Provenance and i d e n t i f i c a t i o n g i v e n i n Table IX. The v a l u e s g i v e n are the mean of two s e p a r a t e d e t e r m i n a t i o n s . The range o f buoyant d e n s i t i e s f o r the main band was 1.716-1.718 g/cm 3; f o r the s a t e l l i t e , 1.706-1.709 g/cm 3. 101 F i g u r e 3 M i c r o d e n s i t o m e t e r t r a c i n g s o f the cesium c h l o r i d e e q u i l i b r i u m g r a d i e n t s of the S a p r o l e g n i a i s o l a t e s ' DNA. The numbers r e p r e s e n t buoyant d e n s i t i e s i n g/cm 3. The peak on the r i g h t i s r e f e r e n c e M i c r o c o c c u s l y s o d e i k t i c u s DNA (p = 1.731 g/cm 3). The l i n e s i n the f i g u r e a p p r o x i -mate the c o r r e c t buoyant d e n s i t y ; f o r p r e c i s e v a l u e s r e f e r t o T a b l e X. 102 S. diclina-S. parasitica complex S. ferax APCC 204e ATCC 22284 CBS 282.38 GAN 17 S. sp. GAN 24 11.718 GANIS-^o/i 1.73? IBH APCC 204a-APCC 204b ATCC 26116 S. hypogyna APCC 206a ;1.718 1.707i 1731 103 p a r t i c l e s were d e t e c t e d i n GAN 17 and GAN 18, b o t h of which have d i s t i n c t s a t e l l i t e bands. A mycoplasmal o r i g i n i n u n l i k e l y be-cause i s o l a t e IBH has a w e l l - s e p a r a t e d s a t e l l i t e peak, and no m y c o p l a s m a - l i k e b o d i e s have ever been d e t e c t e d i n i t . I t i s t h e r e f o r e most l i k e l y t h a t the s a t e l l i t e DNA i s of n u c l e a r o r i g i n . N u c l e a r s a t e l l i t e DNA has been found i n v a r i o u s e u k a r y o t e s (Walker, 1971; I n g l e e_t a_l. , 1973) but they have not been common-l y r e p o r t e d f o r f u n g i ( S t o r c k , 1974) and have not been found i n any o t h e r Oomycete. The amount of s a t e l l i t e DNA i n the S a p r o l e g n i a i s o l a t e s v a r i e d from about 201 o f the t o t a l DNA e x t r a c t e d ( F i g u r e 3, GAN 18) to about 5% ( F i g u r e 3, APCC 204b). As y e t t h e r e i s no e x p l a n a t i o n f o r t h i s phenomenon. I t may be a r t e f a c t , r e p r e s e n t r e a l d i f f e r e n c e s i n the genome (Gibson § H e w i t t , 1972), or be r e l a t e d to the p h y s i o l o g i c a l s t a t e o f the organism a t the time of h a r v e s t . For example, i f the s a t e l l i t e DNA were c o d i n g f o r r i b o s o m a l RNA, the v a r i a t i o n i n amount c o u l d r e p r e s e n t a m p l i f i -c a t i o n o f the rRNA genes ( I n g l e e t a_l. , 1973) . When the n a t u r e of the s a t e l l i t e component i s b e t t e r u n d e r s t o o d , i t may prove to be o f taxonomic v a l u e a t the g e n e r i c l e v e l . PART I I I - GENERAL TAXONOMIC DISCUSSION WITH EMPHASIS ON THE SAPROLEGNIA DICLINA-SAPROLEGNIA PARASITICA COMPLEX Up to now I have d i s c u s s e d the S^ . d i c l i n a - S . p a r a s i t i c a complex w i t h o u t r e a l l y d e f i n i n g i t . T h i s group, as I d e f i n e i t , c o n s i s t s of those S a p r o l e g n i a i s o l a t e s w i t h p r e d o m i n a n t l y d i c l i n -104 ous a n t h e r i d i a and t h i n , u n p i t t e d or i n c o n s p i c u o u s l y p i t t e d (com-pa r e d to S_. f e r a x , f o r example) o o g o n i a l w a l l s . The complex i n c l u d e s S. d i c l i n a Humphrey, S. kau f f m a n i a n a P i e t e r s , and S_. p a r a s i t i c a Coker serisu Seymour (1970). When Coker (1923) d e s c r i b e d S.. p a r a s i t i c a , he d e f i e d the v e n e r a b l e taxonomic t r a d i t i o n c o n c e r n i n g the n e c e s s i t y o f observ-i n g o o g o n i a b e f o r e a s a p r o l e g n i a c e o u s i s o l a t e c o u l d be d e s c r i b e d or i d e n t i f i e d . Coker e s s e n t i a l l y d e f i n e d S_. p a r a s i t i c a on the b a s i s o f the s u b s t r a t u m ( f i s h or f i s h eggs) from which i t was o b t a i n e d , and the f a c t t h a t i t d i d not produce oogonia under normal c u l t u r e c o n d i t i o n s . Thus, when Kanouse (1932) i s o l a t e d some S a p r o l e g n i a spp. from f i s h and f i s h eggs, she assumed t h a t they were, i p s o f a c t o , S_. paras i t i c a . She found oogonia i n some of her c u l t u r e s , n o t e d t h a t they were d i s t i n c t from the oogonia of f u n g i b e l o n g i n g to the S_. f e r a x group, and went on to d e s c r i b e S_. p a r a s i t i c a as a m o r p h o l o g i c a l l y and p h y s i o l o g i c a l l y d i s t i n c t e n t i t y w i t h o u t ever n o t i n g the obvi o u s s i m i l a r i t y o f her i s o l a t e s to S_. d i c l i n a . I t i s i m p o r t a n t to note t h a t Kanouse's d e s c r i p -t i o n o f 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 was based n ot on Coker's type c u l t u r e (which i s , as n o t e d p r e v i o u s l y , a p p a r e n t l y no l o n g e r e x t a n t ) , b u t p r i m a r i l y on s i n g l e spore i s o l a t e s d e r i v e d from a c u l t u r e o b t a i n e d from w h i t e f i s h eggs which had come from a h a t c h e r y i n M i c h i g a n . Kanouse (1932) a l s o c a r r i e d out s e v e r a l n u t r i t i o n a l e x p e r i -ments wh i c h a p p a r e n t l y c o n v i n c e d both Coker (Coker § Mathews, 1937) and T i f f n e y (1939a) t h a t S. p a r a s i t i c a would not produce 105 oogonia e x c e p t when i t was grown on s p e c i a l media. T h i s i s not t r u e . I t i s q u i t e c l e a r t h a t Kanouse obse r v e d oogonia i n c u l -t u r e s grown on hemp seeds i n d i s t i l l e d w a t e r . I n f a c t Kanouse's d e s c r i p t i o n o f S. p a r a s i t i c a was based p r e d o m i n a n t l y on i t s appearance i n the hemp seed c u l t u r e s which she s a i d " i n many .re s p e c t s ... appear more normal.'! The main d i a g n o s t i c f e a t u r e s o f S. p a r a s i t i c a Coker emend. Kanouse when grown on hemp seeds (as i s the u s u a l case nowadays) were i t s t h i n , u n p i t t e d o o g o n i a l w a l l s , d i c l i n o u s a n t h e r i d i a , and s m a l l (18-22 ym) , s u b c e n t r i c (sensu Coker, 1923) o o s p o r e s . Coker (Coker § Mathews, 1937) i n c o r p o r a t e d Kanouse's d e s c r i p t i o n i n t o h i s l a t e r d e s c r i p t i o n o f p a r a s i t i c a ; t h e r e f o r e , i t may be assumed t h a t he a c c e p t e d Kanouse's emended d e s c r i p t i o n . Hence, the modern concept o f S a p r o l e g n i a p a r a s i t i c a dates from 1932, or a t l e a s t from no l a t e r t han 1937, and a f t e r t h i s date a u t h o r s s h o u l d not have used the name f o r i s o l a t e s t h a t d i d not produce oogonia. U n f o r t u n a t e l y , t h i s d i d not happen, and numerous a u t h o r s (e.g. T i f f n e y , 1939a, 1939b; Bangham § Hunter, 1939; F i s h § Hanavan, 1948; Burrows, 1949; Sparrow, 1952; Lennon, 1954; Johnson, 1956b, 1974; Hoshina § Ookubo, 1956; D a y a l , 1958; Wolf, 1958; Beneke $ S c h m i t t , 1961; S c h m i t t $ Beneke, 1962; Lee, 1962; G o p a l a k r i s h n a n , 1965, 1968; Bhargava e_t a l . , 1971; C l i n e § P o s t , 1972; V o l z § Beneke, 1972) c o n t i n u e d t o use the name, S_. p a r a s i t i c a , i n i t s o r i g i n a l sense or a t . l e a s t i t was not made c l e a r t h a t i t was not used i n i t s o r i g i n a l sense. 106 O ' Bier (1960) was among the f i r s t to emphasize the u n d e s i r a b i l i -t y o f t h i s p r a c t i c e . He w r o t e : "The use of a s e x u a l c h a r a c t e r s and the p a r a s i t i c h a b i t as the d i a g n o s t i c f e a t u r e s o f t h i s s p e c i e s have made the t axon the ' c a t c h - a l l ' f o r a l l non-f r u i t i n g i s o l a t e s o f S a p r o l e g n i a even though r e p r o -d u c t i v e s t r u c t u r e s have been d e s c r i b e d f o r the s p e c i e s ... "The a u t h o r b e l i e v e s t h a t the e a s i e s t and l e a s t hazardous s o l u t i o n to t h i s problem f o r the p r e s e n t i s to f o l l o w the p r o c e d u r e used i n t h i s paper and i n c l u d e i n p a r a s i t i c a o n l y those f u n g i t h a t have e x h i b i t e d s e x u a l i t y and possess the d i a g n o s t i c f e a t u r e s o f the t a x o n . " E a r l i e r i n t h i s t h e s i s I have shown t h a t some S a p r o l e g n i a i s o l a t e s may f a i l t o produce the s t r u c t u r e s n e c e s s a r y f o r i d e n t i -f i c a t i o n when grown under what might be c a l l e d 'normal' c u l t u r e c o n d i t i o n s (e.g. growing them on hemp seeds i n s t e r i l e d i s t i l l e d w a t er a t 20 C ) , but are q u i t e c a p a b l e of p r o d u c i n g these s t r u c -t u r e s f o l l o w i n g the a p p r o p r i a t e m o d i f i c a t i o n (e.g. changing the i n c u b a t i o n t e m p e r a t u r e , p r o v i d i n g a d i f f e r e n t n u t r i e n t s o u r c e ) o f the c u l t u r e c o n d i t i o n s . F u r t h e r m o r e , I have shown t h a t t h i s c h a r a c t e r i s t i c i s not l i m i t e d to a s i n g l e s p e c i e s or c l e a r l y d e f i n e d group w i t h i n the genus s i n c e i t a p p l i e s to the supposed-l y d i s p a r a t e 'S. d i c l i n a group' and ' S_. f e r a x group'. T h e r e f o r e , d e f i n i n g a s p e c i e s of S a p r o l e g n i a u s i n g a n e g a t i v e c r i t e r i o n , such as f a i l u r e o f the organism to produce o o g o n i a , i s c l e a r l y an u n a c c e p t a b l e p r a c t i c e . In t h i s r e g a r d , i t i s i n t e r e s t i n g to note the f o l l o w i n g comment by T i f f n e y (1939a): "One of the w r i t e r ' s forms, w h i c h . . . appeared to be S a p r o l e g n i a p a r a s i t i c a f a i l e d to f r u i t i n the same s a l t s o l u t i o n s d e s c r i b e d by Kanouse, but d i d 107 f r u i t f i n a l l y when grown on hemp seed, and was i d e n t i f i e d as S. f e r a x . Both forms i n t h e i r a s e x u a l s t a g e s agreed w i t h . C o k e r ' s d e s c r i p t i o n o f S. paras i t i c a and bo t h upon f r u i t i n g e x h i b i t e d the c h a r a c t e r i s t i c s o f two e n t i r e l y d i f f e r e n t s p e c i e s . I t seems...that these cases are i n d i c a -t i v e o f the p o s s i b i l i t y of a c u r i o u s s i t u a t i o n i n S_. p a r a s i t i c a , namely, t h a t t h i s ' s p e c i e s ' , d e f i n e d on a s e x u a l c h a r a c t e r s , may i n r e a l i t y be composed of forms o f heterogeneous o r i g i n w h i c h , i f they c o u l d be in d u c e d t o f r u i t , might be found to b e l o n g t o s e v e r a l d i f f e r e n t s p e c i e s w i t h i n the genus S a p r o l e g n i a . " L a t e r , i n the same paper, T i f f n e y (1939a) s u g g e s t s t h a t S_. p a r a -s i t i c a s h o u l d be t r e a t e d as a " s p e c i e s o f c o n v e n i e n c e " , a se n t i m e n t a p p a r e n t l y s u p p o r t e d by Wolf (1958) who su g g e s t s t h a t " f o r p r a c t i c a l purposes fungus i n f e s t a t i o n of eggs can be p r e -s u m p t i v e l y a s c r i b e d t o S_. p a r a s i t i c a . " C l e a r l y t h e n , the name S a p r o l e g n i a paras i t i c a can be, and has been, used i n two q u i t e d i f f e r e n t senses. F i r s t , the name can be used as a c o n v e n i e n t r e p o s i t o r y f o r any S a p r o l e g n i a i s o -l a t e w hich appears t o be unable t o produce oogonia and has been' o b t a i n e d from f i s h or f i s h eggs. F u r t h e r m o r e , t h i s i d e n t i f i c a -t i o n can be made w i t h o u t making any s e r i o u s attempt t o c u l t u r e t h e s e i s o l a t e s under c o n d i t i o n s which might encourage oogonium f o r m a t i o n . U s i n g these c r i t e r i a , a l l the i s o l a t e s w hich I o b t a i n e d from the salmon c o u l d have been c a l l e d S a p r o l e g n i a  p a r a s i t i c a Coker. On the o t h e r hand, the name S a p r o l e g n i a p a r a -s i t i c a can a l s o r e f e r t o those S a p r o l e g n i a i s o l a t e s w i t h t h i n o o g o n i a l w a l l s , d i c l i n o u s a n t h e r i d i a and s u b c e n t r i c o o s p o r e s , r e g a r d l e s s of the source o f the i s o l a t e s . An example of t h i s type i s APCC 204e (Table I X ) . T h i s i s o l a t e was o b t a i n e d n o t from 108 f i s h or f i s h eggs, but f r o m sediments from M a r i o n Lake, B. C. ( D i c k , 1971); n e v e r t h e l e s s , i n a p e r s o n a l communication, Dr. M. W. D i c k has su g g e s t e d t h a t t h i s i s o l a t e has a f f i n i t i e s c l o s e r to S. p a r a s i t i c a than S. d i c l i n a . T h i s o p i n i o n i s presumably based on the f a c t t h a t APCC 204e has p r e d o m i n a n t l y s u b c e n t r i c oospores ( P l a t e X I , F i g . 1 ) , and the presence of s u b c e n t r i c oospores i s c o n s i d e r e d to be a key c h a r a c t e r s e p a r a t i n g S_. pa r a -s i t i c a from S_. d i c l i n a (Seymour, 1970). In my o p i n i o n , the use o f the name S a p r o l e g n i a p a r a s i t i c a i n these d i f f e r e n t senses has l e d to c o n f u s i o n and i n a c c u r a c y , has, i n many c a s e s , p r o v i d e d a f a c i l e s o l u t i o n t o what i s , i n r e a l i t y , a d i f f i c u l t taxonomic problem, and h a s , o c c a s i o n a l l y , l e n t an aura o f f a l s e r e s p e c t a b i l i t y t o p o o r l y s t u d i e d , and p o s s i b l y i n c o r r e c t l y i d e n t i f i e d , S a p r o l e g n i a i s o l a t e s . As a p a r t i a l s o l u t i o n t o t h i s problem, I propose t h a t the name Sapro-l e g n i a p a r a s i t i c a Coker s h o u l d be r e j e c t e d as a nomen ambiguum i n accordance w i t h A r t i c l e 69 o f the I n t e r n a t i o n a l Code o f B o t a n i c a l Nomenclature ( S t a f l e u ejt al_. , 1972) whi c h s t a t e s t h a t "a name must be r e j e c t e d i f i t i s used i n d i f f e r e n t senses and so has become a l o n g - p e r s i s t a n t s o u r c e o f e r r o r . " Acceptance of t h i s p r o p o s a l l e a v e s the problem o f what to do w i t h the i s o l a t e s t h a t do produce oogonia and have been i d e n t i f i e d as S_. p a r a s i t i -ca . I b e l i e v e t h a t these i s o l a t e s ( i . e . , S. p a r a s i t i c a Coker emend. Kanouse) can be c o n s i d e r e d to be synonyms of S a p r o l e g n i a d i c l i n a Humphrey. My reasons f o r t h i s o p i n i o n w i l l be g i v e n i n the f o l l o w i n g d i s c u s s i o n . 109 Monsma (1937) was a p p a r e n t l y one of the f i r s t t o note t h a t S. p a r a s i t i c a Coker emend. Kanouse belongs to the " d i c l i n a group" of the genus. O'Bier (1960) a l s o n o t e d the s i m i l a r i t y o f S. p a r a -s i t i c a t o S. d i c l i n a and S. k a u f f m a n i a n a , but m a i n t a i n e d t h a t i t was s e p a r a t e from S^ . d i c l i n a because i t had no p i t s on the oogon-i a l w a l l and t h a t i t was s e p a r a t e from k a u f fmaniana because i t had s m a l l e r o o s p o r e s . Seymour (1970), who has w r i t t e n the f i r s t major monograph of the genus s i n c e Coker q Mathews (1937), a l s o r e c o g n i z e d the a f f i n i t i e s o f S_. p a r a s i t i c a to S^ . d i c l i n a and S^ k a u f fmaniana, but s t i l l e l e c t e d t o m a i n t a i n S^ . p a r a s i t i c a as a s e p a r a t e s p e c i e s . S i n c e I b e l i e v e , c o n t r a r y to Seymour (1970), t h a t no u s e f u l purpose i s s e r v e d by m a i n t a i n i n g S>. p a r a s i t i c a as a s e p a r a t e s p e c i e s , I propose t o examine Seymour's d i s c u s s i o n of S_. p a r a s i t -i c a i n some d e t a i l . I n t h i s c o n t e x t , i t must be n o t e d t h a t Seymour's d i s c u s s i o n of J3. paras i t i c a c o n t a i n s a f a c t u a l e r r o r and i s not i n t r i n s i c a l l y l o g i c a l . Seymour (1970) s t a t e s : " I o b s e r v e d oogonia and a n t h e r i d i a o n l y i n the Utah and V i r g i n i a i s o l a t e s . These two i s o l a t e s do, however, have c h a r a c t e r i s t i c s almost i d e n t i c a l w i t h those o f S a p r o l e g n i a Kauffmaniana: (1) s p a r s e and i n f r e q u e n t p r o d u c t i o n o f s e x u a l s t r u c t u r e s , (2) s i z e and shape of o o g o n i a , (3) u n p i t t e d o o g o n i a l w a l l s , (4) number and o r i g i n o f a n t h e r i d i a l b r a n c h e s , and (5) oospore number. Inasmuch as oospore s t r u c t u r e was n e i t h e r d e s c r i b e d nor i l l u s t r a t e d by P i e t e r s [1915c] the o n l y c o n t r a s t i n g c h a r a c t e r between S^ . Kauf fmaniana and JS. p a r a s i t i c a appears to be the l a r g e r oospores i n the former. I n the o r i g i n a l d i a g n o s i s , P i e t e r s [1915c] r e p o r t e d the oospores to average about 30 y i n d i a m e t e r , as compared to 23 y (Coker, 1923) [ T h i s i s an e r r o r . Coker r e p o r t e d oospore d i a m e t e r s f o r S. paras i t i c a o n l y once, and i n t h a t case (Coker § Mathews, 1937), he merely c o p i e d the d a t a from Kanouse (1932). I t appears t h a t Seymour got t h i s v a l u e from O'Bier ( I 9 6 0 ) . ] and 18-110 22 y (Kanouse, 1932) f o r S. p a r a s i t i c a . The oospores of the V i r g i n i a i s o l a t e ranged from 22 t o 33 y, w i t h an average d i a m e t e r o f 28 y; the Utah i s o l a t e e x h i b i t -ed a range o f 18-28 y , a v e r a g i n g 24 y i n d i a m e t e r . " L a c k i n g c o n c l u s i v e e v i d e n c e to the c o n t r a r y , Sapro-l e g n i a p a r a s i t i c a , sensu Kanouse, i s p r e s e n t l y c o n s i d -e red a v a l i d s p e c i e s . I f e e l j u s t i f i e d i n r e s t r i c t i n g the name to o n l y those i s o l a t e s w hich c o n s i s t e n t l y m a n i f e s t these two f e a t u r e s : s m a l l (ave. 23 y ) , sub-c e n t r i c o o s p o r e s . " T h i s q u o t a t i o n c l e a r l y demonstrates Seymour's l a c k o f i n t r i n -s i c l o g i c i n d e c i d i n g on the taxonomic d i s p o s i t i o n of S_. p a r a s i t - i c a . He obser v e d oogonia i n o n l y two i s o l a t e s . I n one, the average oospore d i a m e t e r was 24 ym, i n the o t h e r , 28 ym. He then goes on t o r e s t r i c t the s p e c i e s t o i s o l a t e s w i t h an average oospore d i a m e t e r o f 23 ym (a d e c i s i o n based, a t l e a s t i n p a r t , on an i n c o r r e c t l y c i t e d a u t h o r i t y ) and c o n s e q u e n t l y e l i m i n a t e s the o n l y two i s o l a t e s i n wh i c h he observed oogonium f o r m a t i o n . T h e r e f o r e , by h i s own d e f i n i t i o n , Seymour (1970) d i d not see S_. paras i t i c a ; n e v e r t h e l e s s , he c o n c l u d e s t h a t i t i s d i s t i n c t from not o n l y S_. k a u f f m a n i a n a but a l s o S. d i c l i n a . For example, i n h i s d i s c u s s i o n of S_. d i c l i n a , Seymour (1970) w r i t e s : "The r e l a t i o n s h i p between S a p r o l e g n i a d i c l i n a and S. p a r a s i t i c a i s not y e t f u l l y understood -! The two s p e c i e s o c c a s i o n a l l y share some o f the same c h a r a c -t e r i s t i c s : (1) u n p i t t e d o o g o n i a l w a l l s , (2) d e l a y i n the f o r m a t i o n o f s e x u a l r e p r o d u c t i v e s t r u c t u r e s , (3) a n t h e r i d i a l b ranch o r i g i n , and (4) oospore t y p e . D e s p i t e t h i s i n t e r g r a d a t i o n , a l l o f the c o l l e c t i o n s examined were c l e a r l y r e f e r r a b l e t o e i t h e r one or the o t h e r o f the two t a x a . " As mentioned p r e v i o u s l y , the ' c e n t r i c ' oospores o f S. d i c -l i n a are c o n s i d e r e d to be a key c h a r a c t e r i s t i c f o r s e p a r a t i n g t h i s s p e c i e s from S. p a r a s i t i c a w h i c h produces ' s u b c e n t r i c ' o o s p o r e s . Indeed, i t i s c l e a r from Humphrey's (1893) d e s c r i p -I l l t i o n of S. d i c l i n a t h a t he c o n s i d e r e d t h i s s p e c i e s to have 'cen-t r i c ' oospores and i t i s e q u a l l y c l e a r from Kanouse's (1932) d e s c r i p t i o n o f S. p a r a s i t i c a t h a t the oospores o f her i s o l a t e s were ' s u b c e n t r i c ' . The p o i n t t h a t ' s c o n s i s t e n t l y i g n o r e d , however, i s t h a t ' s u b c e n t r i c ' oospores were d e f i n e d i n 1923, t h i r t y y e a r s a f t e r Humphrey d e s c r i b e d S^ . d i c l i n a and Humphrey (1893) makes i t p e r f e c t l y c l e a r , b o t h i n w r i t i n g and i n h i s f i g u r e s , t h a t h i s concept o f the ' c e n t r i c ' oospore (see F i g u r e 4 ) i n c l u d e s b o t h the ' c e n t r i c ' and the ' s u b c e n t r i c ' oospore as d e f i n e d by Coker (1923). T h e r e f o r e , Humphrey's concept of d i c l i n a c l e a r l y i n c l u d e s forms t h a t may have e i t h e r ' c e n t r i c ' or ' s u b c e n t r i c ' oospores sensu Coker (1923), Johnson (1956a), D i c k (1969a), Seymour (1970) and Howard (1971). F u r t h e r m o r e , t h e r e i s no e v i d e n c e t o suggest t h a t Humphrey's r e c o g n i t i o n o f o n l y two oospore t y p e s - - c e n t r i c and e c c e n t r i c - - i s , a t l e a s t f o r the S a p r o l e g n i a c e a e , f u n d a m e n t a l l y wrong. Both the c e n t r i c and sub-c e n t r i c oospore have e s s e n t i a l l y the same s t r u c t u r e ( D i c k , 1969a; Howard, 1971) , the d i f f e r e n c e between them b e i n g based merely on the p o s i t i o n of the o o p l a s t r e l a t i v e t o . t h e oospore w a l l . In my o p i n i o n , much c o n f u s i o n would be e l i m i n a t e d and no demonstrable harm would be done i f , a t l e a s t f o r the genus S a p r o l e g n i a sensu  s t r i c t o , we went back to Humphrey's d e f i n i t i o n of oospore t y p e s . A c c e p t a n c e of t h i s v i e w p o i n t means t h a t f o r the t w e l v e or t h i r -t e e n s p e c i e s t h a t can be i n c l u d e d on the genus S a p r o l e g n i a sensu  s t r i c t o , S^ . a n i s o s p o r a de Bary would be d e f i n e d as h a v i n g eccen-t r i c o o s p o r e s , and the r e m a i n i n g s p e c i e s would be d e f i n e d as Figure 4_ H i s t o r i c a l development of the terminology r e l a t e d to oospore types i n the Saprolegniaceae A. B. CENTRIC C. CENTRIC CENTRIC SUBCENTRIC SUBCENTRIC SUBCENTRIC TYPE I TYPE I I 1 TYPE I I I 2 EXCENTRIC ECCENTRIC ECCENTRIC Humphrey (1893) Coker (1923) Johnson (1956) D CENTRIC SUBCENTRIC SUBCENTRIC TYPE I TYPE I I ECCENTRIC SUBECCENTRIC Seymour (1970) Howard (1971) x T h i s type i s not n e c e s s a r i l y i n c l u d e d i n Humphrey's d e f i n i t i o n but i t i s not excluded e i t h e r . 2 T h i s type has only been noted by Johnson (1956a) who found i t i n two i s o l a t e s of Achlya treleaseana (Humphrey) Kauffman which were considered to be aberrant. 113 h a v i n g c e n t r i c o o s p o r e s . T h i s p r o p o s a l does not mean t h a t we s h o u l d cease making d e t a i l e d o b s e r v a t i o n s on oospore t y p e , but t h a t t h i s c h a r a c t e r i s t i c s h o u l d o n l y be used f o r s e p a r a t i n g i s o -l a t e s , and not f o r d e l i m i t i n g t a x a a t the i n f r a g e n e r i c l e v e l . I would s u p p o r t t h i s v i e w p o i n t even i f p a r t i c u l a r i s o l a t e s always had e x c l u s i v e l y one oospore type or the o t h e r , but t h i s i s by no means the c a s e . Even Seymour (1970), i n a r e l a t i v e l y c o n s e r v a -t i v e taxonomic t r e a t m e n t o f the genus, r e c o g n i z e s t h a t f i v e s p e c i e s i n the genus S a p r o l e g n i a sensu s t r i c t o (S. d i c l i n a Hum-phrey, S_. f e r a x ( G r u i t h . ) T h u r e t , S_. f u r c a t a M a u r i z i o , S. t e r r e s -t r i s Cookson ex Seymour, S_. u l i g i n o s a Johannes) as w e l l as the r e c e n t l y d e s c r i b e d S. i r r e g u l a r i s Johnson § Seymour can have b o t h c e n t r i c and s u b c e n t r i c o o s p o r e s , a l t h o u g h one type or the o t h e r u s u a l l y p r e d o m i n a t e s . An i n t e r e s t i n g i s o l a t e r e l e v a n t t o t h i s d i s c u s s i o n i s APCC 204e. T h i s i s o l a t e , w i t h i t s d i c l i n o u s anth-e r i d i a and t h i n o o g o n i a l w a l l w h i c h appears to be u n p i t t e d ex-cep t a t p o i n t s o f a n t h e r i d i a l a t t a c h m e n t , c l e a r l y b e l o ngs to the S_. d i c l i n a - S. p a r a s i t i c a complex and, s i n c e the oospheres mature, i t can be r e a d i l y s e p a r a t e d from the p o o r l y u n d e r s t o o d S. k a u f f -maniana. With t h i s i s o l a t e t h e n , a d e c i s i o n must be made as to whether the organism s h o u l d be c o n s i d e r e d t o be S_. d i c l i n a or S_. paras i t i c a (sensu Seymour [1970]) and t h i s d e c i s i o n has to be based on a n a l y s i s o f the oospore s i z e and t y p e . My o b s e r v a t i o n s i n d i c a t e t h a t over h a l f , and perhaps as many as t h r e e - f o u r t h s o f the oospores of t h i s i s o l a t e are s u b c e n t r i c Type I or s u b c e n t r i c Type I I ( P l a t e X I , F i g s . 1, 3, 4) w i t h the v a s t m a j o r i t y of these 114 b e i n g s u b c e n t r i c Type I . Hence, t h i s predominance of s u b c e n t r i c oospores i m m e d i a t e l y suggests t h a t the i s o l a t e has a f f i n i t i e s to paras i t i c a . However, a p p r o x i m a t e l y a q u a r t e r or more o f the oospores o f t h i s i s o l a t e appear to be c e n t r i c ( P l a t e X I , F i g s . 2, 5 ) , a c h a r a c t e r i s t i c not c o n s i d e r e d c o m p a t i b l e w i t h the p r e s e n t l y a c c e p t e d concept o f S. p a r a s i t i c a . A n o t h e r i m p o r t a n t c h a r a c t e r t h e n , i s oospore d i a m e t e r and much has been made of the v a l u e o f t h i s c h a r a c t e r as a taxonomic i n d i c a t o r ( D i c k , 1969a; Seymour, 1970). In my o p i n i o n , oospore d i a m e t e r i s of no use f o r s e p a r a t i n g s p e c i e s i n the S^ . d i c l i n a -S. p a r a s i t i c a complex. T a b l e XI g i v e s v a r i o u s oospore d i a m e t e r s r e p o r t e d i n the l i t e r a t u r e f o r d i f f e r e n t s p e c i e s which are now i n c l u d e d i n the complex and, a l t h o u g h t h e r e i s a d i s t i n c t t e n -dency f o r i s o l a t e s c l a s s i f i e d as £>. p a r a s i t i c a Coker emend. Kan-ouse to be a t the s m a l l end o f the range, i t cannot be s t a t e d ' t h a t t h e r e i s any obvi o u s d i s j u n c t i o n and, f o r t h a t m a t t e r , t h i s appears to be t r u e f o r the genus S a p r o l e g n i a sensu s t r i c t o i n g e n e r a l , w i t h the p o s s i b l e e x c e p t i o n of !S. l i t o r a l i s Coker and S. u l i g i n o s a Johannes n e i t h e r o f which s h o u l d be c o n f u s e d w i t h any member o f the d i c l i n a - S.' paras i t i c a complex. A n a l y s i s o f oospore d i a m e t e r f o r APCC 204e was based on measurement o f 116 oosp o r e s . Of t h e s e , 82% f e l l w i t h i n a range of 24-28 ym and o n l y 13% had an oospore d i a m e t e r of 23 ym or l e s s w i t h the r e m a i n i n g 5% h a v i n g an oospore d i a m e t e r of 29 ym or more. The mean o f a l l measurements was 25 ym, and the mean of the median 82% was 25.5 ym. Both v a l u e s a re c l e a r l y i n excess o f Seymour's 115 P l a t e XI Photomicrographs of oospores from a 45 day o l d c u l t u r e of APCC 204e m a i n t a i n e d a t 10 ± 1 C i n c o n s t a n t l i g h t . S c a l e r e -p r e s e n t s measurements i n ym. F i g u r e 1 - S u b c e n t r i c Type I (sensu Howard, 1971) oospore. F i g u r e s 2, 5 - C e n t r i c o o s p o r e s . F i g u r e s 3, 4 - S u b c e n t r i c Type I I (sensu Howard, 1971) oos p o r e s . 116 TABLE XT Records o f oospore diameters f o r the S a p r o l e g n i a d i c l i n a - S a p r o l e g n i a p a r a s i t i c a complex. Sp e c i e s Oospore Diameters (ym) A u t h o r i t y 1. S. c r u s t o s a v a r . I M a u r i z i o * (12-) 19. 5-22 M a u r i z i o (1899) 2. S. c r u s t o s a v a r . I I M a u r i z i o * 17- 27 i t 3. s; c r u s t o s a v a r . I l l M a u r i z i o * 22- 29 t i 4. s. d e l i c a Coker* (14.8- ) 25-27 ( -33) Coker (1923) 5 . i t 1 1 25- 28 Hoshina e t a l . (1960) 6 . t t i t 21- 23 N o l a r d - T i n t i g n e r (1973) 7. s. d i c l i n a Humphrey ca. 25 Humphrey (189 3) 8 . t l t t (20-) 23- 24 ( -26) Coker (1923) 9 . t t t t (12-) 23- 26 ( -36) Seymour (1970) 10 . t t t i 24 N o l a r d - T i n t i g n e r (1973) 11. I t t i 15- 28 McKay (1967) 12. s. k a u f f m a n i a n a P i e t e r s ca. 30 P i e t e r s (1915a) 13 . s. p a r a s i t i c a Coker emend. Kanouse 18- 22 Kanouse (1932) 14. 19- 24 ( -28-30) Monsma (1937) 15 . (18.2-) 23. 2 (-31.2) Hoshina e t a l . (1960) 16 . ave. 23 O'Bier (1960) 17. (16-) 18- 24 ( -28) Seymour (1970) 18 . » 21. 5 N o l a r d - T i n t i g n e r (1973) * S p e c i e s c o n s i d e r e d by Seymour (1970) to be synonyms o f S a p r o l e g n i a d i c l i n a Humphrey. 118 l i m i t o f 23 ym f o r the mean oospore d i a m e t e r o f S. paras i t i c a . Hence, f o r APCC 204e we are l e f t i n the unhappy p o s i t i o n o f h a v i n g a S a p r o l e g n i a i s o l a t e t h a t p r o d u c e s , i n abundance, ev e r y m o r p h o l o g i c a l s t r u c t u r e n e c e s s a r y f o r i d e n t i f i c a t i o n , y e t i t cannot be a s s i g n e d to any s p e c i e s w i t h c o n f i d e n c e a c c o r d i n g t o Seymour (1970). N e v e r t h e l e s s , t h i s i s o l a t e i_s_ u n q u e s t i o n a b l y S a p r o l e g n i a d i c l i n a Humphrey (sensu Humphrey [ 1 8 9 3 ] ) . In summary, I b e l i e v e t h a t a r e a s o n a b l e and d e f e n s i b l e s o l u -t i o n to the problems a s s o c i a t e d w i t h the taxonomy of the S_. d i c -l i n a - S . p a r a s i t i c a complex i s to f i r s t e l i m i n a t e the name Sapro-l e g n i a p a r a s i t i c a Coker as a nomen ambiguum and second, to a l l o w a f a i r l y wide range of m o r p h o l o g i c a l v a r i a t i o n f o r S a p r o l e g n i a  d i c l i n a Humphrey so i t can accommodate a l l i s o l a t e s w i t h the f e a t u r e s d e s c r i b e d p r e v i o u s l y ; t h a t i s , (1) p r e d o m i n a n t l y d i c l i n -ous a n t h e r i d i a , (2) t h i n , u n p i t t e d or i n c o n s p i c u o u s l y p i t t e d o o g o n i a l w a l l s , and (3) c e n t r i c (sensu Humphrey [1893]) oospores w i t h an average d i a m e t e r o f 25 ± 5 ym. These i s o l a t e s would then be r e g a r d e d as r e p r e s e n t a t i v e o f S a p r o l e g n i a d i c l i n a sensu l a t o and i t must be emphasized t h a t t h i s s o l u t i o n , i n my o p i n i o n , does n o t s u b v e r t Humphrey's o r i g i n a l concept of the s p e c i e s . My s o l u -t i o n t h e n , i s v e r y s i m i l a r to one o f the s o l u t i o n s proposed by Johnson § Seymour (1974) f o r the problem of i d e n t i f y i n g A c h l y a  americana Humphrey i s o l a t e s , and I agree w i t h these workers t h a t t h i s s o l u t i o n "has the advantage of a d m i t t i n g a d d i t i o n a l d a t a t h a t may come to l i g h t w i t h o u t r e q u i r i n g t h a t i n v e s t i g a t o r s a l s o 119 d e a l w i t h an accompanying c l u t t e r o f b i n o m i a l s o f dubious v a l i d i -t y . " The DNA base c o m p o s i t i o n study p r e s e n t e d i n t h i s t h e s i s , combined w i t h the s e r o t a x o n o m i c work of P e d u z z i (1975) , s u p p o r t s the i d e a t h a t the 'S^ . f e r a x group', the 'S. d i c l i n a group', and perhaps o t h e r s p e c i e s i n the genus may have a c l o s e g e n e t i c r e -l a t i o n s h i p . However, a l l known S a p r o l e g n i a i s o l a t e s a r e , f o r a l l p r a c t i c a l p u r p o s e s , r e p r o d u c t i v e l y i s o l a t e d from one another even i f they are g e n e t i c a l l y i d e n t i c a l ( i . e . , the b i o l o g i c a l s p e c i e s concept sensu Scudder [1974] i s i n a p p l i c a b l e to the genus). There i s , as y e t , no o t h e r a c c e p t a b l e e m p i r i c a l b a s i s f o r d i v i d -i n g i s o l a t e s of t h i s genus i n t o groups a t the i n f r a g e n e r i c l e v e l e x c ept by comparison of m o r p h o l o g i c a l c h a r a c t e r s and i n t h a t c a s e , s p e c i e s l i m i t s are based on h i s t o r i c a l p r e c e d e n t , o p i n i o n , and a u t h o r i t y , u s i n g c h a r a c t e r s which may, or may n o t , be i n d i c a -t i v e of g e n e t i c u n r e l a t e d n e s s a t whatever (as y e t u n d e f i n e d ) l e v e l i s c o n s i d e r e d to be s u f f i c i e n t f o r p l a c i n g i s o l a t e s i n d i f f e r e n t t a x a . Some i d e a o f the c h a o t i c n a t u r e of d e l i m i t a t i o n o f the 'morphospecies' i n the S a p r o l e g n i a c e a e can be o b t a i n e d from a r e c e n t statement by D i c k (1972) who n o t e d t h a t t h i s group c o n t a i n s n a r r o w l y d e f i n e d and e c o l o g i c a l l y c o n s t a n t t a x a , p r e c i s e -l y d e f i n e d but i n d i s t i n c t t a x a , and b r o a d l y d e f i n e d t a x a w i t h c o n s t a n t s u b u n i t s , and t h a t t h e r e f o r e , d e s c r i p t i o n of a new s p e c i e s would r e q u i r e more than the d e m o n s t r a t i o n o f d i s j u n c t i o n i n one or two c h a r a c t e r s and, c o n v e r s e l y , t h a t a range of grada-t i o n s among i s o l a t e s would not be enough to demonstrate synonomy. 120 A p p l i c a t i o n of t h e s e c r i t e r i a can l e a d to a s i t u a t i o n as D i c k (1972) admits where u s i n g e s s e n t i a l l y the same c r i t e r i a , one c o u l d on one hand e r e c t a new s p e c i e s , and on the o t h e r hand suggest t h a t o t h e r ,taxa may be c o n s p e c i f i c . T h i s i s tantamount to s t a t i n g , "a s p e c i e s i s what I say i t i s " and, by s u b s t i t u t i n g the word ' d e f i n e ' i n p l a c e of 'use' and the word ' s p e c i e s ' i n p l a c e o f 'word', i s more than v a g u e l y r e m i n i s c e n t o f Humpty Dumpty's c e l e b r a t e d comment to A l i c e i n Lewis C a r r o l l ' s Through the L o o k i n g G l a s s : " 'When I use [ d e f i n e ] a word [ s p e c i e s ] , ' Humpty Dumpty said" i n a r a t h e r s c o r n f u l t o n e , ' i t means j u s t what I_ choose i t to mean-- n e i t h e r more nor l e s s . ' " 'The q u e s t i o n i s , ' s a i d A l i c e , 'whether you can make words [ s p e c i e s ] mean so many d i f f e r e n t t h i n g s . ' "' 'The q u e s t i o n i s , ' s a i d Humpty Dumpty, 'which i s to be M a s t e r - - t h a t ' s a l l . ' " Given the e x t e n t o f our i g n o r a n c e c o n c e r n i n g the genus Sap-r o l e g n i a , i t i s j u s t as r e a s o n a b l e to assume t h a t i s o l a t e s which are m o r p h o l o g i c a l l y s i m i l a r may have l e s s g e n e t i c i n f o r m a t i o n i n common than i s o l a t e s w hich are m o r p h o l o g i c a l l y d i s s i m i l a r , as i t i s to assume t h a t converse o f t h i s s t a t e m e n t . T h i s statement can be made because s p e c i e s d e l i m i t a t i o n i s based on the morphology of s e x u a l r e p r o d u c t i v e s t r u c t u r e s which may, i n some c a s e s , be more or l e s s v e s t i g i a l , w hich are u s u a l l y not n e c e s s a r y f o r s u r -v i v a l of the o r g anism, and which p r o b a b l y r e p r e s e n t the phenotyp-i c e x p r e s s i o n of o n l y a s m a l l p a r t o f the genome. Fu r t h e r m o r e , the p r a c t i c e o f b a s i n g taxonomic d e c i s i o n s on d i s j u n c t i o n s o f s t a t i s t i c a l averages d e r i v e d from measurements of m o r p h o l o g i c a l 121 c h a r a c t e r s ( f o r example, see D i c k , 1969b; Johnson 6j Seymour, 1974), may be m i s l e a d i n g and, a t any r a t e , a v o i d s the c e n t r a l q u e s t i o n . For example, D i c k (1969a) s t a t e s t h a t " f o r most taxon-omic purposes the extreme range i s u n i m p o r t a n t and u n r e l i a b l e " and he s t r e s s e s the importance o f d e t e r m i n i n g a c c u r a t e l y , the c e n t r a l tendency. T h i s approach has v a l u e i n t h a t i t demands t h a t i s o l a t e s be examined more t h o r o u g h l y , but i t s h o u l d be r e -membered t h a t the extreme ranges g i v e us some c l u e s c o n c e r n i n g the g e n e t i c p o t e n t i a l o f the organism and, when ranges o v e r l a p , I wonder i f much m u t a t i o n i s r e q u i r e d b e f o r e d i s p a r a t e c e n t r a l t e n d e n c i e s s t a r t t o converge. For example, f o r AECC 204e, the c e n t r a l tendency i s a p p a r e n t l y toward s u b c e n t r i c Type I oospores about 25 ym i n d i a m e t e r . However, more than 10% o f the oospores examined were 23 ym i n d i a m e t e r or s m a l l e r and about a q u a r t e r of the oospores were c e n t r i c . How much g e n e t i c change i s r e q u i r -ed b e f o r e 70% i n s t e a d o f 10% of the oospores a re 23 ym or s m a l l e r and b e f o r e 75% i n s t e a d of 25% of the oospores a re c e n t r i c ? Q u e s t i o n s l i k e t h i s cannot be answered a t p r e s e n t , but s o p h i s t i -c a t e d and p o w e r f u l t o o l s (e.g.,DNA-DNA h y b r i d i z a t i o n t e c h n i q u e s ) are a v a i l a b l e and may e v e n t u a l l y enable us to answer th e s e ques-t i o n s . U l t i m a t e l y , the taxonomy of the genus S a p r o l e g n i a w i l l have to be based not o n l y on morphology, but a l s o on the e v i d e n c e o b t a i n e d from d e t a i l e d s t u d i e s o f the b i o c h e m i s t r y , p h y s i o l o g y , c y t o l o g y , and e c o l o g i c a l r o l e o f numerous i s o l a t e s which must be s t u d i e d w i t h no p r e c o n c e i v e d taxonomic b i a s . 12 2 OBSERVATIONS ON THE PATHOLOGY OF SAPROLEGNIASIS OF PACIFIC SALMON PART I - FIELD OBSERVATIONS ON ADULT SALMON Most o f the o b s e r v a t i o n s d e s c r i b e d i n t h i s s e c t i o n are based on d e t a i l e d o b s e r v a t i o n s made on twenty (10 d and 10 oJ sockeye salmon (Oncorhynchus n e r k a (Walbaum)) a t R o b e r t s o n Creek ( P l a t e I I ) d u r i n g August and O c t o b e r , 1973. These o b s e r v a t i o n s are c o r r o b o r a t e d by s i m i l a r o b s e r v a t i o n s made on coho salmon (0. k i s u t c h (Walbaum)) i n 1971 and 1973, and on c h i n o o k salmon (0. t s h a w y t s c h a (Walbaum)) i n 1973. Data c o n c e r n i n g the f i s h exam-i n e d i n d e t a i l i n 1973 are g i v e n i n T a b l e IV. L e s i o n s i n f e c t e d by s a p r o l e g n i a n f u n g i a r e u s u a l l y easy to r e c o g n i z e on salmon and a l s o make the f i s h much more con s p i c u o u s ( P l a t e X I I , P l a t e X I I I , P l a t e XIV, P l a t e XV); hence, i t i s o f t e n p o s s i b l e to make f i e l d o b s e r v a t i o n s on the appearance and behav-i o u r of i n f e c t e d f i s h under more or l e s s n a t u r a l c o n d i t i o n s . The l e s i o n s may o c c u r anywhere on the body or g i l l s o f the a n i m a l , but are most f r e q u e n t l y found on the d o r s a l s u r f a c e o f the a n i m a l and the most common s i t e s o f i n f e c t i o n are the head, the a r e a near the base o f the d o r s a l f i n , the r e g i o n o f the a d i p o s e f i n and c a u d a l p e d u n c l e , and the o t h e r f i n s . Many i n -f e c t e d f i s h , t h e r e f o r e , have a r a t h e r c h a r a c t e r i s t i c appearance ( P l a t e XIV, F i g s . 1, 2; P l a t e XV, F i g s . l [ c ] , 2) which i s appar-123 P l a t e X I I Photograph of a coho (0. k i s u t c h ) salmon t a k e n through the t r a n s p a r e n t w a l l i n the f i s h v i e w i n g room a t drop s t r u c -t u r e no. 4 ( L u c a s , 1960), September, 1971. T h i s f i s h has j u s t a minor S a p r o l e g n i a i n f e c t i o n a s s o c i a t e d w i t h an appar-ent trauma on the d o r s a l s u r f a c e of the shead. 124 125 P l a t e X I I I A group of salmon ( c o n s i s t i n g p r i m a r i l y , i f n o t com-p l e t e l y , o f 0. k i s u t c h ) w i t h S a p r o l e g n i a i n f e c t i o n s . Arrows i n d i c a t e the heads o f the i n f e c t e d i n d i v i d u a l s . Note how the i n f e c t e d areas make the f i s h much more v i s i b l e i n the w a t e r . These f i s h a r e g a t h e r e d i n a q u i e t a r e a j u s t upstream from drop s t r u c t u r e no. 4. Photograph t a k e n September, 1971. Coho salmon near drop s t r u c t u r e no. 4. Note the Sap-r o l e g n i a i n f e c t i o n a s s o c i a t e d w i t h a major trauma on the d o r s a l s u r f a c e o f the head. I n f e c t i o n s on the back and on the p e c t o r a l f i n can a l s o be ob s e r v e d . T h i s f i s h i s one of those shown i n F i g . 1 as i n d i c a t e d by the arrow g o i n g from F i g . 1 to F i g . 2. 1 2 6 127 P l a t e XIV Photograph of a sockeye salmon t a k e n through the t r a n s p a r e n t w a l l i n the f i s h v i e w i n g room at drop s t r u c t u r e no. 4, O c t o b e r , 1973. Arrows i n d i c a t e a reas i n f e c t e d by S a p r o l e g n i a on the head and i n the a r e a j u s t a n t e r i o r to the d o r s a l f i n . These are two o f the areas most commonly i n f e c t e d . Photograph of a sockeye salmon t a k e n near the t e s t flume ( L u c a s , 1960), O c t o b e r , 1973. Arrows i n d i -c a t e the major areas i n f e c t e d by S a p r o l e g n i a . Note the massive i n f e c t i o n on the head and a l s o the i n -f e c t e d areas on the p e c t o r a l , d o r s a l , a d i p o s e , and c a u d a l f i n s . 128 129 e n t l y not o n l y t y p i c a l of s a p r o l e g n i a s i s o f P a c i f i c salmon but i s a l s o t y p i c a l o f s a p r o l e g n i a s i s of A t l a n t i c salmon (Salmo s a l a r L.) as w e l l ( W i l l o u g h b y , 1969b; P y e f i n c h § E l s o n , 1967). I t i s o f t e n n o t e d t h a t t h i s c h a r a c t e r i s t i c appearance may be due to the f a c t t h a t s a p r o l e g n i a n i n f e c t i o n s are most e a s i l y i n i t i a t e d i n the u n s e a l e d areas of the integument. I t i s not u n d e r s t o o d why t h i s might be the c a s e , and i t i s not an i n f l e x i b l e r u l e , s i n c e i n f e c t i o n s may a l s o be i n i t i a t e d i n s c a l e d r e g i o n s i n the absence of any apparent trauma, a f a v o u r e d a r e a b e i n g the d o r s a l s u r f a c e between the head and the d o r s a l f i n ( P l a t e XV). A p a r t from making o b s e r v a t i o n s on f i s h w hich were a l r e a d y i n f e c t e d , I a l s o o b t a i n e d some p r e l i m i n a r y i n f o r m a t i o n on the i n i t i a t i o n and subsequent development of the f u n g a l i n f e c t i o n s on f i s h a p p a r e n t l y f r e e o f i n f e c t i o n . T h i s work was c a r r i e d out d u r i n g 10-16 August 1973 and c o n s i s t e d o f t r a n s f e r r i n g , as c a r e -f u l l y as p r a c t i c a b l e , f o u r sockeye salmon (GAN 1, GAN 2, GAN 3, GAN 4; T a b l e IV) from the b r a i l p o o l ( L ucas, 1960) to a l a r g e f i b e r g l a s s tank where i t was p o s s i b l e to make a c c u r a t e o b s e r v a -t i o n s and to see (1) i f the f i s h would become i n f e c t e d , (2) how l o n g i t would take f o r the i n f e c t i o n s to be i n i t i a t e d , (3) where the l e s i o n s would o c c u r on the f i s h , and (4) how q u i c k l y , and i n what way, the i n f e c t i o n s would p r o g r e s s once they were i n i t i a t e d . The w ater temperature d u r i n g these o b s e r v a t i o n s was 21 ± 2 C. I t seems p r o b a b l e then t h a t the f i s h were exposed to a t l e a s t f o u r e x t e r n a l s t r e s s o r s i n a d d i t i o n to the i n t e r n a l p h y s i o l o g i -c a l s t r e s s a s s o c i a t e d w i t h m a t u r a t i o n and m i g r a t i o n and the 130 s t r e s s caused by the i n f e c t i o n s t h e m s e l v e s , once i n i t i a t e d . These are (1) temperature s t r e s s , (2) s t r e s s a s s o c i a t e d w i t h the t r a n s f e r of the f i s h from the b r a i l p o o l to the t a n k , (3) s t r e s s a s s o c i a t e d w i t h confinement i n the tank and (4) s t r e s s caused by the presence o f the o b s e r v e r . A l l f o u r f i s h became i n f e c t e d by S a p r o l e g n i a s p e c i e s . How-ev e r , GAN 1, a mature male, d i e d between 24 and 42 h a f t e r b e i n g p l a c e d i n the tank and the f u n g a l i n f e c t i o n d i d not appear to be e x t e n s i v e enough to be a major f a c t o r c o n t r i b u t i n g to the death of t h i s f i s h . GAN 2, a f e m a l e , was found dead on the morning o f 16 August, 13 h a f t e r i t had l a s t been obse r v e d a l i v e . T h i s f i s h had numerous areas i n f e c t e d by S a p r o l e g n i a , but f o r the most p a r t they were s m a l l c i r c u l a r l e s i o n s s c a t t e r e d over the body, and I s u s p e c t t h a t i n t h i s case the f i s h may have had dermal u l c e r s caused by a b a c t e r i a l i n f e c t i o n , and t h a t these u l c e r s a c t e d as the f o c i o f i n f e c t i o n f o r some, and perhaps most, of the fungus i n f e c t e d l e s i o n s . The r e m a i n i n g two f i s h were c a p t u r e d a l i v e on 16 August. The l e s i o n s on one of these f i s h (GAN 4) were not y e t e x t e n s i v e and they o c c u r r e d on those areas t h a t appear to be most prone to f u n g a l i n f e c t i o n - the d o r s a l s u r f a c e of the head, the a r e a a t the base o f the d o r s a l f i n , t h e a r e a between the head and the d o r s a l f i n , the a d i p o s e f i n , and the c a u d a l f i n . The r e m a i n i n g f i s h (GAN 3) had e x t e n s i v e f u n g a l l e s i o n s , and b e s t i l l u s t r a t e s the k i n d o f d a t a t h a t may be ob-t a i n e d from t h i s type o f work. The f o l l o w i n g i s a d e t a i l e d d i s -c u s s i o n o f the i n i t i a t i o n and development of s a p r o l e g n i a s i s on 131 t h i s f i s h . GAN 3 was an a d u l t female sockeye salmon w i t h a s t a n d a r d l e n g t h of c a . 0.47 m ( P l a t e XV, F i g . l [ d ] ) . When c a p t u r e d a t the b r a i l p o o l the f i s h had an apparent a b r a s i o n i n the p r e m a x i l -l a r y a r e a ( p o s s i b l y a s s o c i a t e d w i t h the normal changes t h a t o c c u r i n t h i s r e g i o n d u r i n g m a t u r a t i o n ] and an obvious severe i n j u r y on the l e f t s i d e o f the c a u d a l p e d u n c l e . A l s o some damage to the l e f t p e c t o r a l f i n and to the c a u d a l f i n was not e d . O t h e r w i s e the f i s h appeared to be i n good c o n d i t i o n and was a l e r t and a c t i v e . Four days a f t e r GAN 3 was p l a c e d i n the tank ( P l a t e XV, F i g . l [ a ] ) , i t was obvio u s t h a t the f i s h had minor f u n g a l i n f e c t i o n s on the d o r s a l s u r f a c e of the head and i n the c a u d a l a r e a . How-e v e r , the f i s h was s t i l l a l e r t and a c t i v e as i s demonstrated by the poor q u a l i t y o f P l a t e XV, F i g . 1(a) which a t t e s t s t o the f a c t t h a t the f i s h s t a y e d as f a r away from the o b s e r v e r as i t c o u l d . The most d r a m a t i c change o c c u r r e d d u r i n g the nex t twenty-f o u r h o u r s . By t h i s time l a r g e and obvious f u n g a l l e s i o n s had dev e l o p e d on the f i s h ( P l a t e XV, F i g . l [ b ] ) . As e x p e c t e d , the i n j u r y on the c a u d a l peduncle a c t e d as a focus f o r one of the i n f e c t i o n s ; however, the two l e s i o n s on the d o r s a l s u r f a c e near the d o r s a l f i n were unexpected. A l s o , I had e x p e c t e d an i n f e c -t i o n to de v e l o p on the head, but I thought t h a t the a b r a s i o n i n the p r e m a x i l l a r y a r e a would be the f o c u s o f t h i s i n f e c t i o n . I n f a c t , the i n f e c t i o n was i n i t i a t e d f a r t h e r back on the head ( t h i s was t r u e f o r GAN 4, a l s o ) . Twenty-four hours l a t e r , GAN 3 was 132 s t i l l a l i v e ( P l a t e XV, F i g . l [ c ] ) but was o b v i o u s l y moribund. I t was v e r y l e t h a r g i c , b l i n d on one s i d e , and h overed c l o s e to the s u r f a c e of the w a t e r , i n s t e a d o f s t a y i n g c l o s e to the bottom as a h e a l t h y f i s h would. As soon as i t was photographed, i t was k i l l e d and t a k e n to the l a b o r a t o r y f o r f u r t h e r i n v e s t i g a t i o n . The major f u n g a l l e s i o n s found on GAN 3 o c c u r r e d on the head, on the d o r s a l s u r f a c e between the head and the d o r s a l f i n , a t and near the bases of the d o r s a l , a n a l , and p e l v i c f i n s , on b o t h p e c t o r a l f i n s and the c a u d a l f i n , and on the c a u d a l p e d u n c l e . There was no i n f e c t i o n o f the g i l l s , and t h i s o b s e r v a t i o n h o l d s t r u e f o r most of the a d u l t salmon which I examined a t R o b e r t s o n Creek. ( T h i s i s i n d i s t i n c t c o n t r a s t to a s i t u a t i o n where c o l u m n a r i s d i s e a s e i s common. In t h a t c a s e , i n f e c t i o n o f the g i l l s i s normal.) I n o t e d a y e l l o w i s h exudate near the v e n t o f GAN 3, but found no g r o s s a b n o r m a l i t i e s o f the i n t e r n a l organs. However, i t i s q u i t e p o s s i b l e t h a t something s i g n i f i c a n t was m i s s e d s i n c e , a t t h a t t i m e , I was q u i t e i n e x p e r i e n c e d a t t h i s type of e x a m i n a t i o n . A few nematodes were found i n the c o e l o m i c c a v i t y , but they were p r o b a b l y Philonema s p e c i e s , and these organisms are g e n e r a l l y not a s s o c i a t e d w i t h any p a t h o l o g i c a l c o n d i t i o n of sockeye salmon. As i n t i m a t e d p r e v i o u s l y , the development of s a p r o l e g n i a s i s on salmon g e n e r a l l y has a v e r y p r e d i c t a b l e e f f e c t on the behav-i o u r o f the f i s h . As the i n f e c t i o n p r o g r e s s e s , the f i s h become i n c r e a s i n g l y l e t h a r g i c , t i r e more e a s i l y , and are l e s s respon-s i v e to t h r e a t e n i n g s t i m u l i . O f t e n they become unable to swim 133 P l a t e XV O b s e r v a t i o n s on the i n i t i a t i o n and p r o g r e s s of sapro-l e g n i a s i s on a 5 sockeye salmon (GAN 3; T a b l e I V ) . F i g . 1(a) shows the appearance of the f i s h on 14 Aug-u s t , c a . 96 h a f t e r i t was p l a c e d i n the tank; F i g . 1(b) shows the f i s h 24 h l a t e r , and F i g . 1 ( c ) , 48 h l a t e r , j u s t b e f o r e c a p t u r e on 16 August. F i g . 1(d) shows the f r e s h l y k i l l e d f i s h . Arrows on F i g . 1(c) and F i g . 1(d) are a t the same l o c a t i o n . Note t h a t the i n c r e a s i n g q u a l i t y o f the photographs from F i g . 1(a) through to 1(c) are almost a photogrammetric r e c o r d of the i n c r e a s i n g l e t h a r g y of the f i s h and i t s con-c o m i t a n t l a c k o f response to the presence of an observ-e r , h e l p e d , no doubt, by the f a c t t h a t the f i s h was b l i n d on the l e f t s i d e on 16 August. See t e x t f o r f u r t h e r d i s c u s s i o n . The s c a l e r e f e r s to F i g . 1(d) o n l y . Photograph o f a 4 sockeye salmon (GAN 14; T a b l e IV) t a k e n 4 October 1973. T h i s f i s h engaged i n p e r i o d i c bouts o f f r e n z i e d a c t i v i t y f o l l o w e d by p e r i o d s o f apparent e x h a u s t i o n which a l l o w e d me to approach the f i s h c l o s e l y enough to take the photographs. T h i s f i s h demonstrates a t y p i c a l p a t t e r n o f s a p r o l e g n i a s i s o f salmon. Fungal l e s i o n s o c c u r on the d o r s a l s u r f a c e o f the head, near the base of the d o r s a l f i n , on the a d i p o s e f i n , and on the p e c t o r a l , p e l v i c , and c a u d a l f i n s . No i n f e c t i o n of the g i l l s was n o t e d , nor were any g r o s s a b n o r m a l i t i e s o f the i n t e r n a l organs n o t e d . A few nematodes ( p r o b a b l y Philonema s p e c i e s ) were found i n the c o e l o m i c c a v i t y , but t h i s i s not c o n s i d e r -ed to be symptomatic of a p a t h o l o g i c a l c o n d i t i o n . The f i s h i s c a . 0.55 m l o n g . 134 135 a g a i n s t the c u r r e n t o f the stream and t e n d to congregate i n areas where t h e r e i s l i t t l e c u r r e n t ( P l a t e X I I I , F i g . 1 ) . GAN 14 ( P l a t e XV, F i g . 2 ) , a male sockeye salmon w h i c h I examined on 4 October 1973, v a r i e d somewhat from t h i s p a t t e r n . T h i s f i s h was found i n the t e s t f l u m e , and p e r i o d i c a l l y engaged i n f r e n z i e d a c t i v i t y , jumping out o f the water and l a n d i n g on i t s s i d e , as i f i t were t r y i n g to shake o f f something. The r e a s o n f o r t h i s a t y p i c a l b e h a v i o u r was not d i s c o v e r e d . PART I I - HISTOPATHOLOGICAL OBSERVATIONS ON ADULT SALMON METHODS AND MATERIALS G e n e r a l l y the f i s h were caught i n the b r a i l p o o l or near the t e s t flume ( L u c a s , 1960) and d e a l t w i t h i m m e d i a t e l y or t r a n s -f e r r e d to l a r g e f i b e r g l a s s tanks where they c o u l d be h e l d f o r f u r t h e r o b s e r v a t i o n s and removed when c o n v e n i e n t . The f i s h were / then k i l l e d e i t h e r by a blow to the head, or by t e a r i n g the g i l l s on one s i d e and a l l o w i n g the a n i m a l to b l e e d to d e a t h . T h i s l a t t e r t e c h n i q u e i s more humane than i t sounds, and i t a v o i d e d the n e c e s s i t y o f damaging the head integument. In t h e l a b o r a t o r y , a f t e r the fungus c u l t u r e s had been p l a t e d o u t , the p o s i t i o n and e x t e n t o f the e x t e r n a l l e s i o n s were n o t e d and b l o c k s of u n i n f e c t e d and i n f e c t e d t i s s u e were removed from the f i s h (25-50 min a f t e r c a p t u r e ) and f i x e d i n 10% n e u t r a l b u f f e r e d f o r m a l i n , a p r o c e d u r e recommended by Bucke (1972). T h i s m a t e r i a l was s u b s e q u e n t l y d e h y d r a t e d i n methyl a l c o h o l , 136 c l e a r e d w i t h t o l u e n e , embedded i n P a r a p l a s t , and s e c t i o n e d a t 10 ym. G a l i g h e r § K o z l o f f (1971) was found to be an e x c e l l e n t guide f o r t h i s p a r t o f the work. As mentioned p r e v i o u s l y , the g i l l s were r a r e l y i n f e c t e d i n the a d u l t f i s h I examined, and c o n s e q u e n t l y , a l l the m a t e r i a l I o b t a i n e d d u r i n g August and O c t o b e r , 1973 was integument and muscle t i s s u e and a d i s c u s s i o n o f s a p r o l e g n i a s i s of these t i s s u e s i s the s u b j e c t o f t h i s s e c t i o n . However, i n November, 1973 I was i n v o l v e d i n the e x a m i n a t i o n o f some a d u l t coho salmon w i t h s a p r o l e g n i a s i s o f the g i l l s . These f i s h (27 November 1973, D.R. No. 228) were t r a n s p o r t e d to the P a c i f i c B i o l o g i c a l S t a t i o n , Nanaimo, B. C. f o r n e c r o p s y and l a t e r , t h r ough the c o u r t e s y o f Dr. G. R. B e l l , I r e c e i v e d some b l o c k s o f g i l l t i s s u e embedded i n p a r a f f i n w h i c h I s u b s e q u e n t l y s e c t i o n e d and s t a i n e d . The r e s u l t s o f t h i s work are shown i n P l a t e XVI. By comparing u n i n -f e c t e d t i s s u e ( P l a t e XVI, F i g . 1) w i t h i n f e c t e d t i s s u e ( P l a t e XVI, F i g . 2 ) , the e x t e n s i v e damage w i t h which the fungus i s a s s o c i a t e d may be e a s i l y d i s c e r n e d ; however, the e x t e n t to which t h i s damage may be a t t r i b u t e d to the fungus i s not known. Never-t h e l e s s , I t h i n k i t i s w o r t h w h i l e to p r e s e n t t h i s i n f o r m a t i o n because t h e r e i s l i t t l e i n f o r m a t i o n a v a i l a b l e i n the l i t e r a t u r e r e g a r d i n g the h i s t o p a t h o l o g i c a l appearance of s a p r o l e g n i a s i s o f the g i l l s . For example, E l l e r (1975) does not i l l u s t r a t e , or even r e f e r t o , s a p r o l e g n i a s i s o f the g i l l s . The two most p o p u l a r t e c h n i q u e s f o r d e m o n s t r a t i n g s a p r o l e g -n i a n . f u n g i i n f i s h t i s s u e are G r o c o t t ' s (1955) methenamine 137 P l a t e XVI S a p r o l e g n i a s i s o f the g i l l s o f an a d u l t cf coho salmon. Photomicrographs shown are Cibachrome p r i n t s o b t a i n e d from o r i g -i n a l c o l o u r t r a n s p a r e n c i e s made w i t h Kodak photomicrography f i l m 2483. P A S - l i g h t g reen; X 73. F i g u r e 1 - U n i n f e c t e d g i l l f i l a m e n t and l a m e l l a e . The two dark r e d areas on the l e f t s i d e are c a r t i l a g e . The dark green areas are masses of e r y t h r o c y t e s . F i g u r e 2 - I n f e c t e d g i l l l a m e l l a e . The p i n k areas on the r i g h t and i n the c e n t r e o f t h i s p h o t o m i c r o g r a p h are sapro-l e g n i a n hyphae. Note the massive d e s t r u c t i o n of the t i s s u e . 138 139 s i l v e r n i t r a t e method (Bucke, 1972; N o l a r d - T i n t i g n e r , 1973; Bootsma, 1973; Wolke, 1975) and the p e r i o d i c a c i d - S c h i f f s t e c h -n i q u e used i n c o n j u n c t i o n w i t h a l i g h t green c o u n t e r s t a i n (PAS-l i g h t green) (McKay, 1967; R o b e r t s e t a l . , 1969, 1970c). I used the l a t t e r t e c h n i q u e and found t h a t i t gave v e r y s a t i s f a c t o r y r e s u l t s . However, t h e r e are some minor problems a s s o c i a t e d w i t h the p r e s e n t a t i o n o f r e s u l t s o b t a i n e d by u s i n g t h i s t e c h n i q u e . F i r s t , as G a l i g h e r § K o z l o f f (1971) have p o i n t e d o u t , l i g h t green S F . y e l l o w i s h may fade r a p i d l y and the s u b s t i t u t i o n o f f a s t green FCF f o r l i g h t green (as suggested by G a l i g h e r § K o z l o f f [1971]) does not g i v e s a t i s f a c t o r y r e s u l t s . The b e s t s o l u t i o n i s to examine and photograph the m a t e r i a l as soon as p r a c t i c a b l e a f t e r i t has been s t a i n e d . In t h i s r e g a r d , I o b t a i n e d e x c e l l e n t r e -s u l t s u s i n g Kodak photomicrography c o l o r f i l m 2483. The photo-m i c r o g r a p h s were ta k e n w i t h a N i k o n M i c r o f l e x Model AFM photo-m i c r o g r a p h i c attachment w i t h the c o l o u r b a l a n c i n g f i l t e r i n p l a c e , the t u n g s t e n l i g h t s o u r c e s e t a t 8 V, and the ASA o f the f i l m s e t a t 16. The f i l m was d e v e l o p e d u s i n g Kodak p r o c e s s E-4 a c c o r d i n g to the m a n u f a c t u r e r ' s i n s t r u c t i o n s . Second, a l t h o u g h the P A S - l i g h t green t e c h n i q u e y i e l d s a t t r a c t i v e and u n e q u i v o c a l r e s u l t s i n c o l o u r ( P l a t e s XVI, XIX, and XX), i t can be d i f f i c u l t to o b t a i n s u f f i c i e n t c o n t r a s t between the f u n g a l hyphae and the background so t h a t monochrome photomicrographs can be made suc-c e s s f u l l y . T h i s problem was s o l v e d by u s i n g Kodak Wra t t e n f i l -t e r s no. 15 ( y e l l o w ) and no. 58 (green) as recommended by McGavin (1972). 140 RESULTS AND DISCUSSION The u n i n f e c t e d integument o f P a c i f i c salmon i s s i m i l a r to t h a t o f o t h e r s a l m o n i d s . ( B u l l o c k § R o b e r t s , 1975). I t c o n s i s t s o f a m u l t i c e l l u l a r s t r a t i f i e d squamous e p i t h e l i u m ( o f v a r i a b l e t h i c k n e s s , but always t h i n n e s t underneath the s c a l e s ) which con-t a i n s a v a r i a b l e number of s t r o n g l y P A S - p o s i t i v e mucous c e l l s ( P l a t e X V I I ) . The basement membrane (BM) i s r a t h e r prominent when s t a i n e d by the P A S - l i g h t green t e c h n i q u e and i s p r o b a b l y s i m i l a r to the complex BM d e s c r i b e d by Roberts e t a l . (1970b) f o r the A t l a n t i c salmon. The BM i s u s u a l l y smooth and prominant dermal r i d g e s are a b s e n t , but these were o c c a s i o n a l l y seen ( P l a t e X V I I , F i g . 2) and may r e p r e s e n t areas where wounds have h e a l e d (Roberts e t a l . , 1971). The dermis c o n s i s t s of two p a r t s ( P l a t e X V I I , F i g . 1 ) . The o u t e r l a y e r ( s t r a t u m spongiosum) c o n s i s t s of a l o o s e network o f c o l l a g e n and r e t i c u l i n f i b r e s w hich s u p p o r t the melanophores and s c a l e s , and the i n n e r l a y e r ( s t r a t u m compactum) i s m a i n l y com-posed o f dense c o l l a g e n b u n d l e s . The hypodermis i s composed o f l o o s e l y o r g a n i z e d c o n n e c t i v e t i s s u e and anchors the integument to the m u s c u l a t u r e . As the f u n g a l hyphae r a d i a t e away from the f o c u s of the i n -f e c t i o n , they f i r s t d e s t r o y the e p i d e r m i s and, as the i n f e c t i o n p r o g r e s s e s , the hyphae can s u b s e q u e n t l y p e n e t r a t e the BM, extend i n t o the dermis and, i n some cases a t l e a s t , c o n t i n u e growing i n t o the hypodermis and m u s c u l a t u r e ( P l a t e X V I I I , F i g . 1; P l a t e XIX; P l a t e XX). I n some cases the m y c e l i a l mat was a s s o c i a t e d 141 P l a t e XVII U n i n f e c t e d sockeye salmon s k i n . P A S - l i g h t green; X 84 Key f o r f i g u r e ; e - e p i d e r m i s ; sc - s c a l e ; s t s - s t r a -tum spongiosum; s t c - s t r a t u m compactum; hy - hypoder-mis; m - muscle. S k i n from an u n i n f e c t e d r e g i o n showing dermal p a p i l l a e t h a t may be a s s o c i a t e d w i t h the s i t e o f a h e a l e d wound P A S - l i g h t green; X 210. 142 143 P l a t e X V I I I F i g u r e 1 - I n f e c t e d sockeye salmon s k i n (Table IV; GAN 24) show-i n g complete l o s s o f the e p i d e r m i s and p e n e t r a t i o n o f the f u n g a l hyphae i n t o the muscle t i s s u e . Arrows i n d i c a t e the p o s i t i o n o f some o f the hyphae. PAS-l i g h t green; X 84. Key f o r f i g u r e : sc - s c a l e ; s t c -s t r a t u m compactum; hy - hypodermis; m - muscle. F i g u r e 2 - Hyphae (arrows) a s s o c i a t e d w i t h l e u c o c y t i c i n v a s i o n o f the hypodermis o f a sockeye salmon (Table IV; GAN 28). P A S - l i g h t green; X 210. 144 145 P l a t e XIX Photomicrographs showing i n f e c t e d sockeye salmon t i s s u e . The top f i g u r e shows s a p r o l e g n i a n hyphae i n the s t r a t u m compactum of the d e r m i s , i n the hypodermis, and i n the muscle t i s s u e o f GAN 24 (Table I V ) . The bottom f i g u r e shows s a p r o l e g n i a n hyphae i n the hypodermis and muscle t i s s u e o f GAN 20 (Table I V ) . In b o t h cases the hyphae can be r e a d i l y seen as the r e d or p i n k t u b u l a r , t h r e a d - l i k e s t r u c t u r e s seen i n c r o s s or l o n g i t u d i n a l s e c t i o n . Note the l a c k of i n f l a m m a t o r y r e s p o n s e . P A S - l i g h t green; X 184. 146 147 P l a t e XX P h o t o m i c r o g r a p h showing s a p r o l e g n i a n hyphae i n the hypoder-mis and muscles o f a c h i n o o k j a c k (Table IV; GAN 29). Arrows i n d i c a t e some o f the hyphae. P A S - l i g h t green; X 184. 148 149 w i t h dermal u l c e r s w h i c h showed l e u c o c y t i c i n f i l t r a t i o n o f the dermis and hypodermis ( P l a t e X V I I I , F i g . 2 ) . I n o t h e r c a s e s , i n -c l u d i n g the cases i n w h i c h f u n g a l p e n e t r a t i o n o f the m u s c u l a t u r e was o b s e r v e d ( P l a t e X V I I I , F i g . 1 ) , an i n f l a m m a t o r y response seemed to be v i r t u a l l y a b s e n t . Wolke (1975) has a l s o n o t e d t h a t t h e r e o f t e n seems t o be a weak i n f l a m m a t o r y response t o S a p r o l e g -n i a i n f e c t i o n s . These are p r e l i m i n a r y r e s u l t s and must be i n t e r p r e t e d cau-t i o u s l y . More work needs to be done, p a r t i c u l a r l y w i t h r e g a r d to the i n f l a m m a t o r y response and the r e l a t i o n of s a p r o l e g n i a s i s to c o n c u r r e n t i n f e c t i o n s by o t h e r organisms. As a w o r k i n g hypo-t h e s i s , I suggest t h a t those areas where the f u n g a l hyphae are a s s o c i a t e d w i t h an i n f l a m m a t o r y response may r e p r e s e n t those areas where t h e r e are c o n c u r r e n t f u n g a l and b a c t e r i a l i n f e c t i o n s , and those areas where an i n f l a m m a t o r y response i s absent may r e -p r e s e n t i n f e c t i o n s where fungus i s the o n l y pathogen p r e s e n t . There was no apparent s t r u c t u r a l d i f f e r e n c e between u n i n f e c t -ed t i s s u e o b t a i n e d from salmon w i t h s a p r o l e g n i a s i s and t i s s u e o b t a i n e d from an a p p a r e n t l y h e a l t h y salmon and f i x e d i m m e d i a t e l y a f t e r the a n i m a l was k i l l e d . However, i t i s p o s s i b l e t h a t p o s t  mortem changes may have o c c u r r e d i n the i n f e c t e d r e g i o n s d u r i n g the time l a g between the death o f the f i s h and the f i x a t i o n of the t i s s u e b l o c k s . F u r t h e r , r e c e n t s t u d i e s (G. E. H o s k i n s , p e r s o n a l communication) have shown t h a t f i s h from R o b e r t s o n Creek may a l s o be i n f e c t e d by Aeromonas s a l m o n i c i d a and Aeronomas 150 l i q u e f a c i e n s . I t i s w e l l known t h a t the dermal u l c e r s r e s u l t i n g from i n f e c t i o n by these b a c t e r i a (Wolke, 1975) can be f o c i f o r S a p r o l e g n i a i n f e c t i o n s and the r e s u l t i n g h i s t o p a t h o l o g i c a l p i c -t u r e can be d i f f i c u l t to i n t e r p r e t . N e v e r t h e l e s s , the r e s u l t s p r e s e n t e d thus f a r s u g g e s t t h a t the S a p r o l e g n i a s p e c i e s t h a t i n f e c t P a c i f i c salmon are pathogens c a p a b l e of i n v a d i n g l i v i n g t i s s u e and t h a t , i n some c a s e s , the fungus may be the p r i m a r y i n f e c t i o u s agent c a u s i n g the l e s i o n s w i t h which i t i s a s s o c i a t e d . The d a t a a l s o show t h a t s a p r o l e g n i a n i n f e c t i o n s are not t i s s u e s p e c i f i c . T h i s c o n c l u s i o n agrees w i t h r e c e n t o b s e r v a t i o n s by o t h e r workers (Bootsma, 1973; N o l a r d - T i n t i g n e r , 1971, 1973, 1974; Wolke, 1975; Dukes, 1975) and show the f o l l y o f r e f e r r i n g to s a p r o l e g n i a s i s as a 'dermatomycosis' as has been done so o f t e n i n the p a s t , p a r t i c u l a r l y by R u s s i a n a u t h o r s . To d a t e , t h e r e i s no e v i d e n c e to suggest t h a t s a p r o l e g n i a n f u n g i a r e c a p a b l e o f c a u s i n g s y s t e m i c i n f e c t i o n s , nor does i t appear l i k e l y t h a t they produce any t o x i n s (Rucker, 1944) . The damage done by these f u n g i can be d i r e c t l y r e l a t e d to the t i s s u e d e s t r o y e d i n the immediate a r e a o f the hyphae. Assuming t h a t the fungus i s the o n l y pathogen, the time of death w i l l be a f u n c t i o n o f the growth r a t e o f the f u ngus, the i n i t i a l s i t e o f the i n f e c -t i o n , the type o f t i s s u e d e s t r o y e d , and the a b i l i t y o f the i n -d i v i d u a l f i s h to w i t h s t a n d the s t r e s s o f the d i s e a s e . T h i s l a t t e r p o i n t w i l l be d i s c u s s e d i n d e t a i l i n the f i n a l s e c t i o n o f t h i s t h e s i s . 151 PART I I I - EXPERIMENTAL INFECTION OF YOUNG PACIFIC SALMON AND PLATYFISH INTRODUCTION The purpose o f the i n f e c t i o n e x p eriments i s to determine whether p a r t i c u l a r i s o l a t e s a re p o t e n t i a l pathogens. S u c c e s s f u l i n f e c t i o n e x p eriments h e l p to f u l f i l l Koch's p o s t u l a t e s and, a t the same t i m e , p r o v i d e i n s i g h t s i n t o the c o n d i t i o n s which w i l l a l l o w the i n i t i a t i o n o f an i n f e c t i o n . B e f o r e an i n f e c t i o n e xperiment u s i n g Oomycetes can be con-s i d e r e d a s u c c e s s , two c r i t e r i a must be s a t i s f i e d . F i r s t , the fungus must be o b s e r v e d growing on a l i v i n g f i s h and must be seen to i nvade l i v i n g t i s s u e . I f the fungus i s found on a dead f i s h , or does not s p r e a d beyond n e c r o t i c t i s s u e on a l i v i n g f i s h , p a r a -s i t i s m has n o t been e s t a b l i s h e d . Second, i t must be demonstrated t h a t the fungus i n f e c t i n g the f i s h i s the i s o l a t e t h a t i s b e i n g t e s t e d , and not another s t r a i n . T h i s second c r i t e r i o n may be d i f f i c u l t t o determine s i n c e i t i s i m p o s s i b l e , a t the p r e s e n t t i m e , to e a s i l y d i s t i n g u i s h between d i f f e r e n t s t r a i n s o f Sapro-l e g n i a s p e c i e s ; hence, r e i s o l a t i o n p r o c e d u r e s may not p r o v i d e d e f i n i t i v e r e s u l t s . T h i s problem can be a v o i d e d by d e s i g n i n g the experiment so t h a t i t can be shown t h a t i t i s r e a s o n a b l e to assume t h a t Oomycetes which c o u l d be c o n f u s e d w i t h the s t r a i n b e i n g t e s t e d have been e l i m i n a t e d from the e x p e r i m e n t . T h i s can be done by d i s i n f e c t i o n f o l l o w e d by s t r i c t q u a r a n t i n e ( O ' B i e r , 152 1960; S c o t t § Warren, 1964), or by s e t t i n g up s u i t a b l e c o n t r o l s so t h a t the o n l y v a r i a b l e i s the presence o f the t e s t i s o l a t e i n the e x p e r i m e n t a l tanks (McKay, 1967). As y e t , t h e r e i s no s t a n d a r d i z e d e x p e r i m e n t a l method which w i l l i n d i c a t e whether or not a g i v e n oomycete i s a p o t e n t i a l f i s h p a r a s i t e . Some au t h o r s (Hume P a t t e r s o n , 1903; Rucker, 1944; Egusa, 1965; Egusa § N i s h i k a w a , 1965) have r e p o r t e d t h a t f u n g a l i n f e c t i o n s w i l l become e s t a b l i s h e d o n l y i n c o n j u n c t i o n w i t h con-c u r r e n t b a c t e r i a l i n f e c t i o n s . Other a u t h o r s have a p p a r e n t l y ob-t a i n e d p r e s u m p t i v e p r i m a r y i n f e c t i o n s by m e r e l y e x p o s i n g the f i s h to zoospores of a s u s p e c t e d s a p r o l e g n i a n p a r a s i t e ( T i f f n e y , 1939b; H o s h i n a § Ookubo, 1956; Hoshina e t a l . , 1960; O ' B i e r , 1960; S c o t t § Warren, 1964; McKay, 1967). In most c a s e s , however, thes e and o t h e r a u t h o r s have r e p o r t e d t h a t i t was n e c e s s a r y , or a t l e a s t d e s i r a b l e , to wound the f i s h b e f o r e e x p o s i n g them to the fungus ( T i f f n e y § Wolf, 1937; T i f f n e y , 1939b; Hoshina § Ookubo, 1956; V i s h n i a c $ N i g r e l l i , 1957; O ' B i e r , 1960; Egusa, 1963; S c o t t § Warren, 1964) and, i n some s t u d i e s (Hoshina § Ookubo, 1956; N o l a r d - T i n t i g n e r , 1970, 1971, 1973, 1974), t h i s has been c a r r i e d one s t e p f u r t h e r and the wounded a r e a on the f i s h has been brou g h t i n t o d i r e c t c o n t a c t w i t h the f u n g a l mycelium. Table X I I g i v e s a l i s t of the s p e c i e s o f f i s h w hich have succumbed to e x p e r i m e n t a l l y i n d u c e d p r e s u m p t i v e p r i m a r y i n f e c -t i o n s caused by s a p r o l e g n i a n f u n g i . Even a c u r s o r y g l a n c e a t t h i s t a b l e shows t h a t most work o f t h i s type has been r e s t r i c t e d 153 to i n d i v i d u a l s b e l o n g i n g to two c l o s e l y r e l a t e d genera ( L e b i s t e s , Xiphophorus) and, e x c l u d i n g these f i s h , few s t u d i e s have been done which i n d e p e n d e n t l y c o n f i r m and extend the r e s u l t s d e s c r i b -ed i n the o r i g i n a l r e p o r t s . C o n s e q u e n t l y , l i t t l e i s known about the c o n d i t i o n s which w i l l p e r m i t the e s t a b l i s h m e n t o f experiment-a l l y i n d u c e d s a p r o l e g n i a n i n f e c t i o n s o f n o n - c y p r i n o d o n t f i s h , i n c l u d i n g f i s h w h i c h are known to succumb to n a t u r a l s a p r o l e g n i a n i n f e c t i o n s and w h i c h form the b a s i s o f v a l u a b l e commercial and s p o r t s f i s h e r i e s and, where some r e s u l t s are a v a i l a b l e , the con-c l u s i o n s r e a c h e d by d i f f e r e n t workers may be a p p a r e n t l y c o n t r a -d i c t o r y . For example, Rucker (1944) and McKay (1967) are the o n l y ones who have attempted to e x p e r i m e n t a l l y i n f e c t young on-c o r h y n c h i d s w i t h s a p r o l e g n i a n f u n g i and, as mentioned p r e v i o u s l y , these two workers reached c o n c l u s i o n s which are a p p a r e n t l y opposed. Rucker c o n c l u d e d t h a t the f i s h would not become i n f e c t e d i n the absence of 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 and McKay c o n c l u d e d t h a t the f u n g i c o u l d a c t as v i r u l e n t p r i m a r y pathogens. O b v i o u s l y , f u r t h e r work s h o u l d be done to determine the f a c t o r s i n f l u e n c i n g the s u s c e p t i b i l i t y of f i s h to s a p r o l e g n i a s i s i n o r d e r to g a i n c r i t i c a l i n s i g h t i n t o the r e l a t i o n o f s a p r o l e g n i a n f u n g i to d i s -eases of f i s h and a t the same time f i n d an e x p l a n a t i o n f o r appar-ent paradoxes l i k e the one j u s t c i t e d . I n t h i s s e c t i o n I w i l l d e s c r i b e some of my u n s u c c e s s f u l and s u c c e s s f u l attempts t o i n f e c t f i s h w i t h S a p r o l e g n i a s p e c i e s and i n the f i n a l s e c t i o n I w i l l p r e s e n t an argument f a v o u r i n g a TABLE X I I Sp e c i e s o f t e l e o s t f i s h s u c c e s s f u l l y i n f e c t e d S p e c i e s o f F i s h ANABANTIDAE Helostoma temmincki C u v i e r § V a l e n c i e n n e s ANGUILLIDAE A n g u i l l a j a p o n i c a Temminck § S c h l e g e l CATOSTOMIDAE Erim y z o n s u c e t t a (Lacepede) CENTRARCHIDAE Lepomis gibbosus (Linnaeus) ( c i t e d as Eupomitus  g i b b o s u s ) M i c r o p t e r u s s a l m o i d e s (Lacepede) Pomoxis n i g r o m a c u l a t u s (Lesueur) ( c i t e d as P. s p a r o i d e s ) CICHLIDAE T i l a p i a sp. CYPRINIDAE C a r a s s i u s a u r a t u s (Linnaeus) S e m o t i l u s a t r o m a c u l a t u s ( M i t c h i l l ) CYPRINODONTIDAE Fundulus h e t e r o c l i t u s (Linnaeus) L e b i s t e s r e t i c u l a t u s ( P e t e r s ) p e r i m e n t a l l y by s a p r o l e g n i a n f u n g i . R e f e r ences S c o t t § Warren (1964). H o s h i n a § Ookubo (1956); Hoshina e t a l . (1960) . T i f f n e y (1939b) . T i f f n e y (19 39b) . T i f f n e y (1939b) . T i f f n e y (1939b) . N o l a r d - T i n t i g n e r (1970) . T i f f n e y (1939b) . T i f f n e y (1939b) . T i f f n e y § Wolf (1937); T i f f n e y (1939b) . T i f f n e y (1939b); S c o t t § Warren (1964); N o l a r d - T i n t i g n e r (1970, 1971 , 1973, 1974) . TABLE X I I (Continued) S p e c i e s o f t e l e o s t f i s h s u c c e s s f u l l y i n f e c t e d ex Sp e c i e s o f F i s h CYPRINODONTIDAE (Cont'd) M o l l i e n e s i a l a t i p i n n a Leseur Xiphophorus h e l l e r i H e c k e l Xiphophorus maculatus (Gunther) ESOCIDAE Esox n i g e r Lesueur ( c i t e d as E. r e t i c u l a t u s ) ICTALURIDAE I c t a l u r u s nebulosus Lesueur ( c i t e d as Ameiurus  nebulosus) PE RCICHTHYIDAE Morone americana (Gmelin) PERCIDAE P e r c a f l a v e s c e n s ( M i t c h i l l ) SALMONIDAE Oncorhynchus k i s u t c h (Walbaum) Salmo g a i r d n e r i R i c h a r d s o n ( c i t e d as S. i r i d e u s ) Salmo s a l a r Linnaeus ( c i t 6 d as S. sebago) Salmo t r u t t a Linnaeus ( c i t e d as 53'. f a r i o ) t a l l y by s a p r o l e g n i a n f u n g i . R e f e r e n c e s S c o t t 8, Warren (1964) . S c o t t § Warren (1964); N o l a r d -T i n t i g n e r (1970, 1971, 1973, 1974) V i s h n i a c $ N i g r e l l i (1957); S c o t t § Warren (1964) . T i f f n e y (1939b) . T i f f n e y (1939b) . T i f f n e y (1939b) . T i f f n e y (1939b) . McKay (1967) . T i f f n e y (1939b) . T i f f n e y (1939b) . T i f f n e y (1939b) . 156 c a u s a l r e l a t i o n s h i p between the i n i t i a t i o n o f s a p r o l e g n i a s i s and c e r t a i n p h y s i o l o g i c a l changes t h a t o c c u r i n P a c i f i c salmon a t d i f f e r e n t s t a g e s o f t h e i r l i f e c y c l e . METHODS AND RESULTS In 1972 I made some attempts to o b t a i n e x p e r i m e n t a l i n f e c -t i o n s o f coho salmon a l e v i n s . These a t t e m p t s , l i k e s i m i l a r ones made by Rucker (1944), a l l f a i l e d . However, t h e r e are many l a b -o r a t o r y v a r i a b l e s which c o u l d a d v e r s e l y a f f e c t these e x p e r i m e n t s ; t h e r e f o r e , i n August 1973, I t r i e d a f i e l d e xperiment which s h o u l d have e l i m i n a t e d most of the v a r i a b l e s t h a t would v i t i a t e a l a b o r a t o r y e x p e r i m e n t . Wounded and unwounded coho salmon, chi n o o k salmon, and s t e e l h e a d t r o u t a l e v i n s were p l a c e d i n t o con-t a i n e r s w h i c h were immersed i n c o n c r e t e ponds r e c e i v i n g t h e i r w ater s u p p l y d i r e c t l y from R o b e r t s o n Creek. The p r e s ence of p o t e n t i a l l y p a r a s i t i c f u n g i w i t h s u f f i c i e n t i n o c u l u m p o t e n t i a l to i n i t i a t e an i n f e c t i o n was a s s u r e d because, a t the same t i m e , I o b s e r v e d the i n i t i a t i o n of s a p r o l e g n i a s i s i n a d u l t f i s h , and I was a l s o a b l e to c o n f i r m the presence of s a p r o l e g n i a n f u n g i i n the ponds by d i r e c t o b s e r v a t i o n ( P l a t e IV, F i g s . 1, 2 ) . The water temperature d u r i n g these o b s e r v a t i o n s was i n excess o f 19 C and, a c c o r d i n g to McKay's (1967) r e s u l t s , t h i s s h o u l d have p r o v i d e d an' o p t i m a l temperature f o r the i n i t i a t i o n o f s a p r o l e g -n i a s i s . N e v e r t h e l e s s , the f i s h d i d not become i n f e c t e d , and f u r t h e r l a b o r a t o r y e xperiments i n 1974 u s i n g coho salmon a l e v i n s and wounded p l a t y f i s h a l s o f a i l e d . 157 As a l a s t r e s o r t , to see i f i t was p o s s i b l e f o r me to o b t a i n any s u c c e s s f u l e x p e r i m e n t a l i n f e c t i o n s , I used S a p r o l e g n i a p a r a -s i t i c a (ATCC 22284) ( T h i s i s o l a t e , a c c o r d i n g to O'Bier [1960], i s a v i r u l e n t p a t h o g e n . ) i n an attempt to i n f e c t wounded p l a t y f i s h (Xiphophorus maculatus) ( T h i s s p e c i e s , a c c o r d i n g to V i s h n i a c q N i g r e l l i [1957] and S c o t t q Warren [1964] , i s q u i t e s u s c e p t i b l e to s a p r o l e g n i a s i s . ) and I d e v i s e d a t e c h n i q u e to ensure t h a t t h e r e would be an o p t i m a l o p p o r t u n i t y f o r the fungus to i n f e c t the f i s h . A d e s c r i p t i o n of t h i s e x p eriments f o l l o w s : Four p l a t y f i s h were removed from t h e i r h o l d i n g tanks and p l a c e d i n a e r a t e d d i s t i l l e d water known to be f r e e o f oomycete c o n t a m i n a t i o n . Each f i s h was then wounded on one s i d e o f the c a u d a l peduncle by s c r a p i n g i t w i t h a s c a l p e l and then a hemp seed w i t h ATCC 22284 growing on i t was sewn onto the d o r s a l s i d e of the c a u d a l p eduncle w i t h a w h i t e c o t t o n t h r e a d . As soon as t h i s p r o c e d u r e was completed, the f i s h were p l a c e d i n a s m a l l r e c t a n g u l a r g l a s s aquarium (0.15 x 0.24 x 0.15 m) c o n t a i n i n g 1.5 l i t e r s of a e r a t e d d i s t i l l e d w a t e r . The experiment was c a r -r i e d out a t room temperature which v a r i e d between 19-27 C d u r i n g the c o u r s e of the e x p e r i m e n t . A l l f i s h became moribund w i t h i n 24-36 h as e v i d e n c e d by. t h e i r b e h a v i o u r . T h i s c o n s i s t e d of l y i n g u p s i d e down on the bottom of the aquarium e x h i b i t i n g weak o p e r c u l a r movements and o n l y o c c a s i o n a l weak movement of the p e c t o r a l f i n s . The f i s h were removed p r i o r to d e a t h , k i l l e d , and examined. Upon c l o s e e x a m i n a t i o n I was a b l e to determine t h a t the f i s h had become 158 i n f e c t e d and t h a t the i n f e c t i o n c o u l d s p r e a d beyond the s i t e o f the o r i g i n a l trauma. T h i s experiment had no c o n t r o l s and i t was i m p o s s i b l e f o r me to a s c e r t a i n t o what degree the f u n g a l i n f e c t i o n , as opposed to the trauma, had c o n t r i b u t e d t o the d e a t h o f the f i s h . Never-t h e l e s s , I was a b l e to det e r m i n e t h a t the t e c h n i q u e used was p o t e n t i a l l y u s e f u l f o r o b t a i n i n g s u c c e s s f u l e x p e r i m e n t a l i n f e c -t i o n s . S u b s e q u e n t l y , I c a r r i e d out a s i m i l a r e x periment s u b s t i -t u t i n g young coho salmon f o r the p l a t y f i s h and a g a i n I o b t a i n e d s u c c e s s f u l r e s u l t s . T h i s encouraged me to make an o t h e r attempt t o i n f e c t young salmon w i t h my i s o l a t e s u s i n g the t e c h n i q u e o f a t t a c h i n g hemp seeds t o the f i s h . The f i s h used f o r t h i s work were coho salmon which had been c o n c e i v e d a r t i f i c a l l y a t the R o b e r t s o n Creek H a t c h e r y on 29 Octo-ber 1973, r a i s e d i n i n c u b a t o r t r a y s , and ponded i n l a r g e f i b e r -g l a s s tanks on 25 March 1974. I o b t a i n e d the f i s h on 27 March 1974 and a t t h a t time they had an average w e i g h t o f about 0.4 g. U s i n g a c o m b i n a t i o n o f hypothermia and h y p e r o x i a , I was a b l e to t r a n s p o r t the f i s h from R o b e r t s o n Creek to the U n i v e r s i t y o f B r i t i s h Columbia and d u r i n g March and A p r i l 1974, I made some u n s u c c e s s f u l attempts to i n f e c t these a l e v i n s . The r e m a i n i n g f i s h were h e l d i n . f i b e r g l a s s a q u a r i a and the water was changed p e r i o d i c a l l y ; however, i n June 1974, when I r e t u r n e d from a p e r i o d o f absence, I found the f i s h o b v i o u s l y d i s t r e s s e d and ex-h i b i t i n g a w h i r l i n g motion. I p e r c e i v e d t h i s to be due to poor water q u a l i t y , n o t to w h i r l i n g d i s e a s e , and when the f i s h were 159 t r a n s f e r r e d to a h o l d i n g tank w i t h a c o n s t a n t f l o w o f f r e s h w a t e r , many of the f i s h appeared to r e c o v e r c o m p l e t e l y and com-menced to f e e d w e l l , to d i g e s t t h e i r f o o d w e l l , and to e x h i b i t normal b e h a v i o u r a l r e s p o n s e s . By the time the experiments were s e t up on 16 F e b r u a r y 1975, the f i s h were almost a y e a r o l d , had been a p p a r e n t l y h e a l t h y f o r more than s i x months, and were 0.08 ± 0.01 m l o n g . U n f o r t u n a t e l y , I do not know whether they would be c o n s i d e r e d to be a l e v i n s or smolts a c c o r d i n g to B a l o n (1975) . I t was o n l y l a t e r t h a t I became aware of the p o s s i b l e importance of t h i s d i s t i n c t i o n r e l a t i v e to the s u s c e p t i b i l i t y of young salmon to s a p r o l e g n i a s i s . On 15 F e b r u a r y 1975, the water e n t e r i n g the h o l d i n g tank was c u t o f f , a f i l t e r was p l a c e d i n the t a n k , and the water was a l l o w e d to come up to room temperature to m i n i m i z e the e f f e c t s o f t emperature shock. On 16 F e b r u a r y , a l l the r e m a i n i n g young coho salmon were t r a n s f e r r e d from the h o l d i n g tank to an aquarium c o n t a i n i n g a e r a t e d d i s t i l l e d w a ter known to be f r e e of oomycete c o n t a m i n a t i o n . The purpose o f t h i s p r o c e d u r e was to reduce p o s s i b l e e x t r a n e o u s oomycete c o n t a m i n a t i o n and, a t the same t i m e , make i t e a s i e r to s e l e c t and c a t c h the f i s h pr,ior to t h e i r opera-t i o n . The f i s h were removed from t h i s d i s t i l l e d water 'washing' and h o l d i n g aquarium one a t a t i m e , the o p e r a t i o n was performed ( A n e s t h e t i c s were not n e c e s s a r y and were not employed;.), and the f i s h were then t r a n s f e r r e d to the s m a l l g l a s s e x p e r i m e n t a l a q u a r i a (0.15 x 0.24 x 0.15 m) each o f which c o n t a i n e d 2.5 l i t e r s o f d i s t i l l e d w a t e r . T h i s water was a e r a t e d w i t h aquarium a i r 160 stones and the a q u a r i a were c o v e r e d w i t h aluminum f o i l to m i n i -mize the s t r e s s imposed by human a c t i v i t y i n the v i c i n i t y o f the a q u a r i a , and to p r e v e n t the f i s h from jumping out o f the a q u a r i a . One aquarium was used f o r each o f seven experiments and f o u r f i s h were p l a c e d i n each aquarium e x c e p t f o r experiment no. 7 (see Table X I I I ) i n which two f i s h were used. The e x p e r i m e n t a l aquar-i a were k e p t i n l a r g e wooden tanks c o n t a i n i n g t h e r m o s t a t i c a l l y c o n t r o l l e d c o n t i n u o u s l y c i r c u l a t i n g r e f r i g e r a t e d w a t e r . Most experiments were c a r r i e d out a t 17 ± 2 C (see Table X I I I ) , but one was c a r r i e d out a t 7 ± 2 C t o determine the n a t u r e of the r e l a t i o n s h i p between temperature and the i n i t i a t i o n o f s a p r o l e g -n i a s i s . The f i s h were not f e d d u r i n g the experiment to m i n i m i z e as much as p o s s i b l e f o u l i n g o f the water. A d e s c r i p t i o n of the experiment f o l l o w s . T h i s i n f o r m a t i o n i s a l s o summarized i n Table X I I I . In the f o l l o w i n g d e s c r i p t i o n s , when I r e f e r t o a hemp seed or t h r e a d b e i n g sewn on to a f i s h t h i s r e f e r s t o a p r o c e d u r e where a sewing n e e d l e was used to p u n c t u r e the d o r s a l muscle mass near the a d i p o s e f i n . The n e e d l e was then used to draw a w h i t e c o t t o n t h r e a d through the wound and, f o l l o w i n g t h i s , the t h r e a d was t i e d t o g e t h e r . P l a t e s XXI and XXII show the r e s u l t of t h i s p r o c e d u r e . Care was t a k e n to a v o i d , as much as p o s s i b l e , f u r t h e r i n j u r y t o the f i s h . W i t h p r a c t i c e , t h i s p rocedure can be c a r r i e d out q u i c k l y and the f i s h can be r e s t r a i n e d s e c u r e l y w i t h o u t harm, d e s p i t e the l a c k o f a n e s t h e t i c . 161 Experiment I_ - A u t o c l a v e d hemp seeds were sewn onto the f i s h . i T h i s experiment was a c o n t r o l d e s i g n e d to determine whether the f i s h c o u l d s u r v i v e the e x p e r i m e n t a l p r o c e d u r e s and to e s t a -b l i s h whether contaminant water moulds w h i c h c o u l d be c o n f u s e d w i t h the e x p e r i m e n t a l i s o l a t e s had been e l i m i n a t e d . I f these f i s h had d i e d b e f o r e the o t h e r f i s h , or had become i n f e c t e d , t h i s would have c a s t c o n s i d e r a b l e doubt on the v a l i d i t y of the o t h e r e x p e r i m e n t s . F o r t u n a t e l y , n e i t h e r o f these events o c c u r r e d and t h i s a l l o w e d me to have some degree o f c o n f i d e n c e i n the r e s u l t s o b t a i n e d from the o t h e r e x p e r i m e n t s . Experiment I I - A u t o c l a v e d hemp seed were sewn onto the f i s h . S a p r o l e g n i a sp. (GAN 18) and a u t o c l a v e d hemp seed h a l v e s were added to the wa t e r . T h i s experiment was s e t up to e s t a b l i s h the v a l i d i t y o f the assumptions made about experiment I , the c o n t r o l ; t h a t i s , to show t h a t f u n g i i n the water c o u l d c o l o n i z e the hemp seed a t t a c h -ed to the f i s h and then proceed t o i n f e c t the f i s h . One of the f i s h used f o r t h i s experiment l o s t the hemp seed a t t a c h e d to i t and d i d not become i n f e c t e d by S a p r o l e g n i a . T h i s f i s h d i e d on the e i g h t h day o f the experiment from unknown causes. The r e -m a i n i n g t h r e e f i s h became i n f e c t e d to v a r y i n g degrees. One o f these was found dead on the t h i r d day, one was removed from the aquarium i n a moribund c o n d i t i o n on the ev e n i n g o f the f o u r t h day, and the t h i r d d i e d sometime b e f o r e 12:00 h on the s i x t h day. 162 Experiment I I I - Hemp seeds w i t h S a p r o l e g n i a sp. (GAN 18) grow-i n g on them were sewn onto the f i s h . Two days a f t e r t h i s experiment was s e t up a l l the f i s h were s t i l l a l i v e and a l l were o b v i o u s l y i n f e c t e d . On the morning o f the t h i r d day I found t h a t one f i s h had d i e d o v e r n i g h t . A n o t h e r f i s h , i n an o b v i o u s l y moribund c o n d i t i o n , was a l s o removed from the aquarium. By 17:45 h o f the same day the r e m a i n i n g two f i s h were a l s o i n a n o t i c e a b l y moribund c o n d i t i o n . One o f these f i s h d i e d by 20:00 h on the t h i r d day and the experiment was t e r m i n a t -ed. One of the f i s h from t h i s experiment i s shown i n P l a t e XXI. Experiment IV - T h i s experiment was s i m i l a r t o experiment I I I except t h a t the f i s h were m a i n t a i n e d a t 7 ± 2 C i n s t e a d o f 17 ± 2 C (see T a b l e X I I I ) . A l l the f i s h became i n f e c t e d and the i n f e c t i o n s p r o g r e s s e d i n much the same way as i n experiment I I I ; however, they p r o g r e s s -ed a t a much s l o w e r r a t e . By t h e ~ f o u r t h day ( w e l l a f t e r e x p e r i -ment I I I had been t e r m i n a t e d ) a l l the f i s h were s t i l l a l i v e and j u s t showing the f i r s t s i g n s o f i n f e c t i o n and i t was not u n t i l the morning o f the e i g h t h day t h a t two l i v i n g , but moribund, f i s h were removed from the aquarium. The r e m a i n i n g two f i s h s u r v i v e d u n t i l the e v e n i n g o f the n i n t h day when one of them d i e d . S i n c e the o t h e r f i s h was o b v i o u s l y moribund, the experiment was t e r m i n -a t e d a t t h i s t ime. One o f the f i s h from t h i s experiment i s shown i n P l a t e XXI. The obvious c o n c l u s i o n to be drawn from experiments I I I and IV i s t h a t lower temperatures do not p r e v e n t i n f e c t i o n , b u t they 163 P l a t e XXI Young coho salmon i n f e c t e d by S a p r o l e g n i a sp. (GAN 1 8 ) . The f i s h i n the top photograph i s from experiment I I I (Table X I I I ) and the f i s h i s shown on the t h i r d day o f the e x p e r i m e n t . The f i s h i n . t h e bottom photograph i s from experiment IV (Table X I I I ) and the f i s h i s shown on the e i g h t h day o f the e x p e r i -ment. Note t h a t the e x t e n t o f the i n f e c t i o n on b o t h f i s h i s about the same. A l s o b o t h f i s h have s i m i l a r hemorrhagic areas on the c a u d a l p e d u n c l e . 165 do s i g n i f i c a n t l y r e t a r d the i n i t i a t i o n and development of an i n f e c t i o n and t h e r e b y p r o l o n g the l i f e o f the i n f e c t e d f i s h . E x periment V - A w h i t e c o t t o n t h r e a d w i t h no hemp seed a t t a c h e d was sewn onto the f i s h . The f i s h were then l e f t a l o n e f o r alm o s t two days b e f o r e a u t o c l a v e d hemp seed h a l v e s and hemp seeds w i t h S a p r o l e g n i a sp. (GAN 18) growing on them were added to the water i n the aquarium. Four days a f t e r the fungus was added, a l l f o u r f i s h were s t i l l a l i v e and one was i n f e c t e d . By the sev e n t h day a f t e r the fungus had been added, t h r e e o f the f i s h had become i n f e c t e d and had d i e d . The one r e m a i n i n g f i s h l o s t the c o t t o n t h r e a d a t t a c h e d to i t and d i d not become i n f e c t e d . T h i s experiment e s t a b l i s h e d t h a t i t was not n e c e s s a r y t o have a hemp seed a t t a c h e d to the f i s h i n o r d e r f o r an i n f e c t i o n to be i n i t i a t e d . The s i t e o f the wound was the p o i n t of i n i t i a -t i o n o f i n f e c t i o n i n a l l c a s e s . Experiment VI - The f i s h were not p u r p o s e l y i n j u r e d . A u t o c l a v e d hemp seed h a l v e s and hemp seeds w i t h S a p r o l e g n i a sp. (GAN 18) growing on them were added to the aquarium. On the b a s i s o f p a s t e x p e r i e n c e , I d i d n o t expect t h i s ex-pe r i m e n t t o be s u c c e s s f u l ; hence, i t was w i t h some s u r p r i s e t h a t I n o t e d t h a t one o f the f i s h had become i n f e c t e d i n the r e g i o n o f the c a u d a l peduncle by the e i g h t h day. T h i s f i s h was found dead on the morning o f the n i n t h day. None o f the o t h e r f i s h became i n f e c t e d . 166 T h i s experiment i s as c l o s e as I ever came to r e p l i c a t i o n of McKay's (1967) r e s u l t s and i t must be noted t h a t I cannot c a t e g o r i c a l l y s t a t e t h a t the f i s h t h a t became i n f e c t e d was not wounded. I t c o u l d have been i n a d v e r t e n t l y wounded when the exper-iment was s e t up o r i t c o u l d have been i n j u r e d by the o t h e r f i s h i n the aquarium. Young coho salmon are a g g r e s s i v e f i s h and t a i l n i p p i n g i s the u s u a l way i n which dominant f i s h h a r a s s s u b o r d i n -at e f i s h . E xperiment V I I - T h i s experiment was s i m i l a r to experiment I I I except t h a t o n l y two f i s h were used and S a p r o l e g n i a sp. (GAN 17) was used i n s t e a d o f S a p r o l e g n i a sp. (GAN 1 8 ) . Both f i s h became i n f e c t e d and succumbed r a p i d l y . The ex-peri m e n t was t e r m i n a t e d a p p r o x i m a t e l y 44 h a f t e r i t was begun and, a t t h a t t i m e , b o t h f i s h were s t i l l a l i v e , but were o b v i o u s l y moribund. The 16-26 Fe b r u a r y 1975 experiments appeared to f i n a l l y e s t a b l i s h t o a r e a s o n a b l e degree of c e r t a i n t y t h a t the S a p r o l e g -n i a i s o l a t e s from R o b e r t s o n Creek were p o t e n t i a l p a r a s i t e s . The f u n g i were observed to grow on l i v i n g f i s h and i t was e s t a b l i s h -ed t h a t the mycelium c o u l d grow i n t o l i v i n g t i s s u e w e l l beyond the i n i t i a l s i t e o f the i n f e c t i o n . F u r t h e r m o r e , i t i s r e a s o n a b l e to c o n c l u d e t h a t the s a p r o l e g n i a n i n f e c t i o n was, i n most c a s e s , the major f a c t o r p r e c i p i t a t i n g the death o f the f i s h . T h i s con-c l u s i o n i s based on the f a c t t h a t none o f the experiment I f i s h d i e d which i n t u r n s u g gests t h a t the e x p e r i m e n t a l p r o c e d u r e s per 167 s e , w h i l e h a r d l y p r o v i d i n g o p t i m a l c o n d i t i o n s f o r the f i s h , were not l e t h a l . A l s o o n l y one of the twenty-two f i s h exposed to the f u n g a l i s o l a t e s d i e d b e f o r e the t e r m i n a t i o n o f the experiments w i t h o u t f i r s t s u s t a i n i n g a s a p r o l e g n i a n i n f e c t i o n . The time o f i n i t i a t i o n and subsequent development o f the i n f e c t i o n s was i n f l u e n c e d by temperature and by the p r o x i m i t y of the f u n g a l i s o l a t e t o a v u l n e r a b l e a r e a (which, i n a l l cases e x c e p t f o r the one experiment VI f i s h , was an obvious wound); hence, the f i r s t f i s h to become i n f e c t e d and d i e were those from experiments I I I and V I I . Once i n i t i a t e d , the e x t e n t o f i n f e c t i o n t o l e r a t e d by the f i s h v a r i e d . The f i s h i l l u s t r a t e d i n P l a t e XXI show more or l e s s the maximum e x t e n t of i n f e c t i o n t o l e r a t e d by these f i s h b e f o r e they became o b v i o u s l y moribund as e v i d e n c e d by the permanent l o s s o f normal e q u i l i b r i u m . The g e n e r i c i d e n t i f y of the fungus on and near the hemp seed a t t a c h e d t o the f i s h c o u l d be p r e s u m p t i v e l y e s t a b l i s h e d s h o r t l y a f t e r an experiment was t e r m i n a t e d ( P l a t e XXIV, F i g s . 6, 7) but I c o u l d n o t c a t e g o r i c a l l y prove t h a t the fungus i n f e c t i n g a f i s h was the i s o l a t e i n t r o d u c e d i n t o the aquarium. However, i n thes e and subsequent experiments w i t h s u c c e s s f u l c o n t r o l s (e.g. see P l a t e X X I I I , F i g s . 1-5), when hemp seeds w i t h f u n g i growing on them were removed from e x p e r i m e n t a l a q u a r i a and then i n c u b a t e d i n p e t r i d i s h e s and s u b s e q u e n t l y examined, I found no re a s o n to b e l i e v e t h a t these f u n g i were d i f f e r e n t from those i n t r o d u c e d i n t o the aquarium. C o n t a c t w i t h the aquarium environment d i d , TABLE X I I I Summary o f the r e s u l t s o f the 16-26 February 1975 i n f e c t i o n e x p eriments w i t h young coho salmon. I I IV. V. VI V I I Experiment A u t o c l a v e d hemp seeds sewn onto f i s h . No Sapro-l e g n i a i s o l a t e i n t r o d u c e d i n t o aquarium ( c o n t r o l e x p e r i m e n t ) . A u t o c l a v e d hemp seeds sewn onto f i s h . S a p r o l e g n i a sp. (GAN 18) added to water. (GAN 18) growing I I I . Hemp seeds w i t h S a p r o l e g n i a sp on them sewn onto f i s h . Hemp seeds w i t h S a p r o l e g n i a sp. (GAN 18) growing on them sewn onto f i s h . C o t t o n t h r e a d o n l y sewn onto f i s h . S a p r o l e g n i a sp (GAN 18) added to aquarium ca. two days l a t e r . F i s h not p u r p o s e l y wounded. S a p r o l e g n i a sp. (GAN 18) added t o water. Hemp seeds w i t h S a p r o l e g n i a sp. on them sewn onto f i s h . (GAN 17) growing Temp.(C) 17 + 2 17 + 2 17 ± 2 7 ± 2 17 ± 2 17 ± 2 17 ± 2 *See t e x t f o r f u r t h e r i n f o r m a t i o n . **The t h r e a d came o f f the f i s h t h a t was not i n f e c t e d . ***The hemp seed came o f f t h i s f i s h . Wound d i d not h e a l . No. o f F i s h 4 4 4 4 4 2 No. o f No. o f F i s h Dead F i s h from Other I n f e c t e d Causes 0 3 4 4 3** 1 2 0 1 * ft A 0 0 0 0 0 169 however, appear to enhance oogonium and gemma p r o d u c t i o n i n some i s o l a t e s ( P l a t e X X I I I , F i g s . 1-5). T h e r e f o r e , as f a r as I c o u l d d e t e r m i n e , I had f i n a l l y d i s -c o v e r e d a t e c h n i q u e whereby, w i t h r e l a t i v e ease, i t was p o s s i b l e to o b t a i n c o n s i s t e n t l y s u c c e s s f u l e x p e r i m e n t a l i n f e c t i o n s o f f i s h by s i m p l y a t t a c h i n g hemp seeds to t h e i r c a u d a l p e d u n c l e s . I n no case d u r i n g the 16-26 Feb r u a r y experiments d i d a f i s h w i t h a hemp seed a t t a c h e d to i t remain f r e e o f i n f e c t i o n when exposed to a f u n g a l i s o l a t e . A l s o , I found t h a t i t was b e t t e r to use" l a r g e r f i s h because the attachment o f the hemp seed d i d prop o r -t i o n a t e l y l e s s damage and an i n f e c t i o n , once e s t a b l i s h e d , c o u l d p r o g r e s s f a r t h e r b e f o r e the f i s h became moribund. The f i s h used f o r the nex t s e t of experiments were ch i n o o k salmon a l e v i n s (which were l a r g e r than o t h e r s a l m o n i d s p e c i e s of comparable age) o b t a i n e d from R o b e r t s o n Creek. These f i s h had been a r t i f i c i a l l y c o n c e i v e d on 20 October 1974, i n c u b a t e d i n h a t c h e r y t r a y s , and ponded on 13 March 1975. I o b t a i n e d the f i s h on 16 May 1975, brought them back t o the U n i v e r s i t y of B r i t i s h C olumbia, and p l a c e d them i n a h o l d i n g tank r e c e i v i n g a c o n s t a n t f l o w o f water. A number o f these f i s h d i e d d u r i n g the f i r s t two days, but the s u r v i v i n g f i s h a c c l i m a t e d w e l l and by 25 May c o n d i t i o n s appeared to be q u i t e s t a b l e . The f i r s t s e r i e s of experiments w i t h these young ch i n o o k salmon was s e t up on 26 May 1975 and was s i m i l a r to experiment I I o f the 16-26 Fe b r u a r y s e r i e s . The f i s h were removed from the h o l d i n g t a n k , 'washed' i n d i s t i l l e d w a t e r , and then an a u t o c l a v e d 170 hemp seed was sewn onto the c a u d a l peduncle i n the r e g i o n o f the ad i p o s e f i n . Seven a q u a r i a , each o f which c o n t a i n e d t h r e e l i t e r s o f a e r a t e d d i s t i l l e d w a t e r , were used and t h r e e f i s h , w i t h hemp seeds a t t a c h e d , were p l a c e d i n t o each one. A few hours a f t e r the f i s h were p l a c e d i n the a q u a r i a , s t e r i l e hemp seed h a l v e s were added and s i x d i f f e r e n t f u n g a l i s o l a t e s (IBH, APGC 204b, ATCC 26116, APCC 206a, APCC 204e, or CBS 282.38) were p l a c e d i n d i v i d u -a l l y i n t o one o f s i x a q u a r i a . The s e v e n t h aquarium a c t e d as a c o n t r o l . The water temperature was m a i n t a i n e d a t 14-17 C. T h i s experiment f a i l e d t o produce s u c c e s s f u l i n f e c t i o n s ; however, none o f the i s o l a t e s used had been i s o l a t e d from f i s h and they may not have been p o t e n t i a l pathogens. To t e s t t h i s p o s s i b l i t y I s e t up another s e r i e s o f experiments u s i n g the Robertson Creek c u l t u r e s . T h i s second s e r i e s o f experiments was s e t up on 14 June 1975. The t e c h n i q u e s used were s i m i l a r t o those used to s e t up experiments I I and I I I o f the 16-26 Feb r u a r y 1975 s e r i e s ( T a ble X I I I ) . However, the 14 June s e r i e s d i f f e r e d from p r e v i o u s ex-pe r i m e n t s i n t h a t a p p r o x i m a t e l y 0.15 mg per l i t e r o f m a l a c h i t e green was added to the h o l d i n g tank and the f i s h were k e p t i n t h i s s o l u t i o n f o r about 30 min b e f o r e they were removed from the h o l d i n g tank and t r a n s f e r r e d to an aquarium c o n t a i n i n g a e r a t e d d i s t i l l e d w a t e r . Three f i s h were p l a c e d i n each e x p e r i m e n t a l aquarium each o f which c o n t a i n e d about 2.5 l i t e r s o f a e r a t e d d i s t i l l e d water m a i n t a i n e d a t about 17 C. The f i s h were exposed to t h r e e S a p r o l e g n i a i s o l a t e s (GAN 17, GAN 18 and GAN 24). 171 Only one i n f e c t e d f i s h was observed from t h i s s e r i e s o f ex-pe r i m e n t s and i n t h a t case the i n f e c t i o n was not v e r y e x t e n s i v e and was p r o b a b l y not s i g n i f i c a n t as a cause o f death. T h i s i n f e c t e d f i s h was observed i n the aquarium i n whi c h hemp seeds w i t h S a p r o l e g n i a sp. (GAN 24) growing on them had been a t t a c h e d to the f i s h . The f i s h was found i n a moribund c o n d i t i o n on the f i f t h day o f the experiment and i t had a hemorrhagic a r e a on the c a u d a l p e d u n c l e . The hemp seed and s t r i n g were no l o n g e r a t t a c h -ed t o the f i s h . These two s e r i e s o f experiments w i t h young c h i n o o k salmon made i t o b v i o u s t h a t even the o s t e n s i b l y d r a s t i c p r o c e d u r e o f sewing onto the f i s h a hemp seed w i t h a p o t e n t i a l l y p a r a s i t i c S a p r o l e g n i a i s o l a t e growing on i t d i d n ot a s s u r e t h a t t h e s e a n i m a l s would become i n f e c t e d . My l a s t attempt to i n f e c t the chinook salmon was made on 4 October 1975. A t t h i s time I had o n l y f o u r f i s h l e f t . The ex-p e r i m e n t a l p r o c e d u r e s were s i m i l a r to those used f o r experiments I and I I I o f the 16-26 Fe b r u a r y 1975 s e r i e s (Table X I I I ) e x c e p t t h a t o n l y two f i s h were p l a c e d i n each aquarium and each aquarium c o n t a i n e d o n l y two l i t e r s o f a e r a t e d d i s t i l l e d w a t e r . By about 54 h a f t e r the experiment was s e t up, i t was o b v i -ous t h a t one o f the f i s h w i t h S a p r o l e g n i a sp. (GAN 18) a t t a c h e d to i t was i n f e c t e d , and about 79 h a f t e r the experiment was s e t up, the f i s h was removed a l i v e , but i n an o b v i o u s l y moribund con-d i t i o n , and k i l l e d . P l a t e XXII shows t h i s f i s h i m m e d i a t e l y a f t e r i t was k i l l e d and i t can be seen t h a t the i n f e c t i o n i s s i m i l a r 172 to the i n f e c t i o n s o f coho salmon caused by the same f u n g a l i s o -l a t e under s i m i l a r e x p e r i m e n t a l c o n d i t i o n s ( P l a t e X X I ) . The o t h e r f i s h exposed to S a p r o l e g n i a sp. (GAN 18) d i d not become i n -f e c t e d , nor d i d e i t h e r o f the c o n t r o l f i s h . T h i s e x p e r i m e n t , w h i l e h a r d l y an u n e q u i v o c a l s u c c e s s , d i d p r o v i d e a s m a l l degree o f r e p l i c a t i o n f o r the 16-26 Febr u a r y s e r i e s and d i d i n d i c a t e t h a t the p r e v i o u s f a i l u r e s were p r o b a b l y n o t due to a p o t e n t i a l l o s s o f p a t h o g e n i c i t y by the f u n g a l i s o l a t e . D u r i n g the summer of 1975, I a l s o made some attempts t o i n -f e c t g o l d f i s h ( C a r a s s i u s a u r a t u s ) and p l a t y f i s h (Xiphophorus  m a c u l a t u s ) . These a t t e m p t s , l i k e those w i t h the salmon, were g e n e r a l l y u n s u c c e s s f u l ; however, a few noteworthy o b s e r v a t i o n s were made and these are r e c o r d e d below. The experiments w i t h the g o l d f i s h were s e t up on 20 June 1975 and were s i m i l a r to experiment I I of the 16-26 Febr u a r y 1975 s e r i e s e x c e p t t h a t the a u t o c l a v e d hemp seed was a t t a c h e d to the f i s h near the base o f the d o r s a l f i n . . Four a q u a r i a were used. One, l i k e e xperiment I o f the 16-26 Febr u a r y s e r i e s , s e r v e d as a c o n t r o l and a d i f f e r e n t S a p r o l e g n i a i s o l a t e (GAN 17, GAN 18, or GAN 24) was p l a c e d i n t o each o f the o t h e r t h r e e a q u a r i a . The f u n g i grew w e l l and become e s t a b l i s h e d on the hemp seeds a t t a c h e d to the f i s h i n . a l l tanks except the c o n t r o l t a n k , but no i n f e c -t i o n s o c c u r r e d . One o f the hemp seeds which presumably had S a p r o l e g n i a sp. (GAN 18) growing on i t was removed from a f i s h a t the t e r m i n a -t i o n of the experiment and was p l a c e d i n t o a p e t r i d i s h c o n t a i n -173 P l a t e XXII Chinook salmon (0.09 m long) i n f e c t e d by..Saprolegnia sp. (GAN 18). The photograph was t a k e n i m m e d i a t e l y a f t e r the f i s h was k i l l e d . T h i s f i s h i s from the 4-9 October experiment and i s shown 79 h a f t e r the experiment was s e t up. Note t h a t t h i s f i s h has an i n f e c t i o n s i m i l a r to those shown i n P l a t e XXI. 174 175 i n g s t e r i l e d i s t i l l e d water and a s t e r i l e p u n c t u r e d hemp seed. T h i s c u l t u r e was then i n c u b a t e d i n the dark a t 10 C f o r about t h r e e months and examined. U n l i k e an a x e n i c c u l t u r e o f S a p r o l e g -n i a sp. (GAN 1 8 ) , I found t h a t t h i s c u l t u r e had produced numerous oogonia and gemmae ( P l a t e X X I I I , F i g s . 1-5). P r e v i o u s o b s e r v a -t i o n s under more c o n t r o l l e d c o n d i t i o n s had shown t h a t S a p r o l e g n i a sp. (GAN 18) was p r o b a b l y a Categ o r y C i s o l a t e , and the oogonia o b s e r v e d i n the ' g o l d f i s h aquarium c u l t u r e ' were c o n s i s t e n t w i t h t h i s c o n c l u s i o n . Assuming t h a t the ' g o l d f i s h aquarium c u l t u r e ' c o n t a i n e d o n l y the one S a p r o l e g n i a i s o l a t e (and t h i s i s a reason-a b l e a s s u m p t i o n ) , then t h e s e o b s e r v a t i o n s may be q u i t e s i g n i f i -c a n t because they suggest t h a t exposure t o the aquarium e n v i r o n -ment, and the r e s u l t a n t c o n t a c t w i t h a v a r i e t y of o t h e r m i c r o -organisms, c r e a t e d a s i t u a t i o n whereby S a p r o l e g n i a sp. (GAN 18) was i n d u c e d t o produce oogonia and gemmae i n numbers w e l l i n excess o f those o b s e r v e d i n a x e n i c o r m i l d l y c o n t a m i n a t e d c u l -t u r e s . Moreover, these o b s e r v a t i o n s do not r e p r e s e n t an i s o l a t e d c a s e . S i m i l a r o b s e r v a t i o n s were made on o t h e r o c c a s i o n s . These o b s e r v a t i o n s must be r e g a r d e d as t e n t a t i v e s u b j e c t t o c o n f i r m a -t i o n by more s t r i n g e n t l y c o n t r o l l e d e x p e r i m e n t s ; n e v e r t h e l e s s , the r e s u l t s s t r o n g l y suggest t h a t many supp o s e d l y ' s t e r i l e ' S a p r o l e g n i a i s o l a t e s may be s t e r i l e o n l y i n the l a b o r a t o r y and t h a t i n n a t u r e , i n a s s o c i a t i o n w i t h the v a r i e t y o f mic r o o r g a n i s m s w i t h w h i c h they n o r m a l l y c o e x i s t , they may produce oogonia r e g u l a r l y ( a l s o , see d i s c u s s i o n on pp. 75, 76). 176 P l a t e X X I I I F i g u r e s 1-5 - S a p r o l e g n i a sp. (GAN 18) a f t e r b e i n g removed from an aquarium c o n t a i n i n g g o l d f i s h . (See t e x t f o r f u r t h e r i n f o r m a t i o n . ) T h i s c u l t u r e produced numer-ous oogonia ( F i g s . 1, 3-5) w i t h t h i n o o g o n i a l w a l l s , s l e n d e r a n t h e r i d i a o f a p p a r e n t l y d i c l i n o u s o r i g i n ( F i g s . 3, 4 ) , some a p p a r e n t l y mature s u b c e n t r i c oospores ( F i g . 5 ) , and c h a i n s o f gemmae ( F i g . 2 ) . T h i s c u l t u r e i s v e r y r e m i n i s c e n t o f S a p r o l e g n i a  paras i t i c a Coker emend. Kanouse. For F i g s . TJ 2, the bar r e p r e s e n t s 50 ym; f o r F i g s . 3-5, the bar r e p r e s e n t s 20 ym. F i g u r e s 6,7 - Z o o s p o r a n g i a (presumably of S a p r o l e g n i a sp. [GAN 18]) from the a r e a near t h e hemp seed on one of the f i s h from experiment I I I ( F i g . 6) and on one o f the f i s h from experiment IV ( F i g . 7) from the 16-26 Febr u a r y 1975 s e r i e s o f e x p e r i m e n t s . Note the i n t e r n a l p r o l i f e r a t i o n w h i c h a l l o w s t h e s e f u n g i t o be p r e s u m p t i v e l y i d e n t i f i e d as S a p r o l e g n i a s p e c i e s . The bar r e p r e s e n t s 50 ym. 177 178 A l t h o u g h I f a i l e d to o b t a i n e x p e r i m e n t a l i n f e c t i o n o f the g o l d f i s h , I d i d i n a d v e r t e n t l y prove t h a t these f i s h were n ot i n -h e r e n t l y immune to s a p r o l e g n i a s i s . On 24 June, h a v i n g d e s p a i r e d o f ever a t t a i n i n g a s u c c e s s f u l e x p e r i m e n t a l i n f e c t i o n o f these f i s h , I p u t the r e m a i n i n g g o l d f i s h i n t o the same h o l d i n g tank as the c h i n o o k salmon. When I examined these g o l d f i s h c l o s e l y a week l a t e r , I found t h a t they had obvious s a p r o l e g n i a n i n f e c t i o n s i n the c a u d a l r e g i o n . I s u s p e c t t h a t what happened was t h a t these f i s h were c o n t i n u a l l y a t t a c k e d by the salmon i n the same way t h a t dominant salmon would a t t a c h s u b o r d i n a t e salmon and t h a t the r e s u l t a n t s t r e s s , combined w i t h the wounds i n the c a u d a l a r e a , l o w e r e d the r e s i s t a n c e o f the g o l d f i s h to the p o i n t where they were no l o n g e r immune to s a p r o l e g n i a s i s . E x periments w i t h p l a t y f i s h were more s u c c e s s f u l than those w i t h the g o l d f i s h , but they d i d not y i e l d as c o n c l u s i v e r e s u l t s as I thought they might. Only the experiment i n v o l v i n g the f i s h shown i n P l a t e XXIV w i l l be d e s c r i b e d s i n c e i t i l l u s t r a t e s an i n t e r e s t i n g phenomenon. For t h i s e x p e r i m e n t , the p l a t y f i s h were removed from t h e i r h o l d i n g tank and p l a c e d i n an aquarium c o n t a i n i n g 0.3-0.4 mg per l i t e r o f m a l a c h i t e green f o r about 60 min. They were then t r a n s -f e r r e d t o f r e s h a e r a t e d d i s t i l l e d w a t e r . The f i s h shown i n P l a t e XXIV i s one o f f o u r f i s h exposed t o S a p r o l e g n i a sp. (GAN 18). Two o f these f i s h had a u t o c l a v e d hemp seeds a t t a c h e d to t h e i r c a u d a l peduncles and two ( i n c l u d i n g the one i n P l a t e XXIV) 179 were wounded i n the r e g i o n o f the c a u d a l p e d u n c l e . A f t e r t h e i r o p e r a t i o n , these f i s h were p l a c e d i n the e x p e r i m e n t a l aquarium and then hemp seeds w i t h S a p r o l e g n i a sp. (GAN 18) growing on them were added to the wa t e r . A u t o c l a v e d hemp seed h a l v e s and excess f i s h food p r o v i d e d f u r t h e r s u b s t r a t e s f o r the f u n g a l i s o l a t e . The experiment was begun a t 16:00 h on 6 J u l y 1975. A t t h a t time the temperature i n the e x p e r i m e n t a l aquarium was between 25-26 C and between 16:00 h and 17:00 h i t was br o u g h t down to about 21 C. A t t h a t p o i n t the r e f r i g e r a t i o n u n i t was t u r n e d o f f and the temperature i n the aquarium was a l l o w e d to s l o w l y e q u i l i b r a t e w i t h ambient t e m p e r a t u r e . By 11:30 h on 7 J u l y , one o f the f i s h w i t h a hemp seed a t t a c h e d had d i e d . The o t h e r t h r e e f i s h were a l i v e and t h e r e was e x t e n s i v e f u n g a l growth i n the aquarium. A c o n t r o l aquarium was f r e e o f any obvious s a p r o l e g n i a n f u n g i . By 13:15 h on 7 J u l y , I found one o f the r e m a i n i n g t h r e e f i s h moribund. The aquarium water was v e r y murky by t h i s t i m e , so a l l t h e s e f i s h were t r a n s f e r r e d t o a c l e a n aquarium c o n t a i n i n g two l i t e r s o f d i s t i l l e d w a t e r . The moribund f i s h , w hich had a s a p r o l e g n i a n i n f e c t i o n , d i e d s h o r t l y a f t e r t h i s t r a n s f e r . T h i s l e f t two l i v i n g f i s h - one w i t h a hemp seed a t t a c h e d and the f i s h shown i n P l a t e XXIV. By t h i s time i t was apparent t h a t t h i s l a t t e r f i s h was i n f e c t e d . P l a t e XXIV shows the f i s h w h i l e s t i l l a l i v e or v e r y s h o r t l y a f t e r death a t 23:00 h on 7 J u l y . W h i l e not the r e s u l t o f a 180 P l a t e XXIV P l a t y f i s h i n f e c t e d by S a p r o l e g n i a sp. (GAN 18). T h i s f i s h i s from the 6-7 J u l y 1975 experiment and i s shown a l i v e , b u t moribund, c a . 31 h a f t e r the experiment was s e t up. T h i s f i s h was i n j u r e d i n the a r e a o f the c a u d a l peduncle and i s i n f e c t e d i n t h a t r e g i o n , but note a l s o the i n f e c t i o n s between the p e c t o r -a l and p e l v i c f i n s and on the n o s t r i l . These areas were not p u r p o s e l y i n j u r e d . 1 8 1 182 t i d y e x p e r i m e n t , I b e l i e v e t h a t i t was i n f e c t e d by S a p r o l e g n i a sp. (GAN 18). The i n t e r e s t i n g t h i n g about t h i s p a r t i c u l a r c a s e , however, i s t h a t the i n f e c t i o n s a re not j u s t a s s o c i a t e d w i t h the d e l i b e r a t e l y i n j u r e d a r e a on the c a u d a l p e d u n c l e , but a l s o o c c u r on a r e a s t h a t were not d e l i b e r a t e l y i n j u r e d , p a r t i c u l a r l y the a r e a between the p e c t o r a l and p e l v i c f i n s , and the a r e a i n the v i c i n i t y of the r i g h t n o s t r i l . I t c o u l d be argued, o f c o u r s e , t h a t these areas were i n a d v e r t e n t l y i n j u r e d ; n e v e r t h e l e s s , i t i s i n t e r e s t i n g to note t h a t the l a t t e r i n f e c t i o n resembles a type of s a p r o l e g n i a s i s known as S t a f f ' s d i s e a s e w h i c h a f f e c t s the o l f a c t o r y p i t s o f c a r p i n e a s t e r n Europe (Bauer e_t a l . , 1973). Perhaps, i n some f i s h , t h i s p a r t i c u l a r l o c a t i o n i s u n u s u a l l y v u l n e r a b l e to s a p r o l e g n i a n i n f e c t i o n s . PART IV - A GENERAL DISCUSSION CONCERNING THE RELATIONSHIP BETWEEN STRESS AND SAPROLEGNIASIS OF PACIFIC SALMON "There are many s i t u a t i o n s i n which the microbe i s a c o n s t a n t and u b i q u i t o u s component o f the environment but causes d i s e a s e o n l y when some weakening of the p a t i e n t by a n o t h e r f a c t o r a l l o w s i n f e c t i o n t o p r o c e e d u n r e s t r a i n -ed. . . T h e o r i e s o f dis-eases must account f o r the s u r p r i s i n g f a c t t h a t , i n any community, a l a r g e p e r c e n t a g e o f h e a l t h y and normal i n d i v i d -u a l s c o n t i n u a l l y h a r b o r p o t e n t i a l l y p a t h o g e n i c microbes w i t h o u t s u f f e r i n g any symptoms or l e s i o n s . " Rene Dubos .(1955) S a p r o l e g n i a s p e c i e s a re a u b i q u i t o u s and normal component of the salmon's environment and are c a p a b l e o f growth ( f o r exam-p l e , compare Tab l e s I I I and V I I ) . a n d r e p r o d u c t i o n throughout the 183 y e a r as saprobes on a wide v a r i e t y o f s u b s t r a t e s ( a l s o see d i s -c u s s i o n on pp. 69, 70. There may be some s e a s o n a l v a r i a t i o n i n inoculum p o t e n t i a l ( S u z u k i , 1960a, 1960b; S u z u k i § Hatekayama, 1961; Hughes, 1962; R o b e r t s , 1963; H u n t e r , 1975), but i t has as y e t to be demonstrated t h a t t h e r e are c e r t a i n times o f the y e a r when f u n g i known to be p a r a s i t e s o f f i s h i n a p a r t i c u l a r water-shed (Dudka, 1964; W i l l o u g h b y , 1969a, 1970, 1971) are absent from t h a t w a t e r s h e d . T h e r e f o r e , i t i s a r e a s o n a b l e , i f unproven, assumption t h a t salmon are exposed to p o t e n t i a l l y p a r a s i t i c f u n g i t h r o u g h o u t t h e i r s t a y i n f r e s h w a t e r . T h i s assumption i s s u p p o r t e d by the f a c t t h a t s a p r o l e g n i a n i n f e c t i o n s o f f i s h o r f i s h eggs may be observed a t v i r t u a l l y any time of the y e a r . However, most salmon ( w i t h the p o s s i b l e e x c e p t i o n o f s e n e s c e n t a d u l t s ) do n o t become i n f e c t e d by these f u n g i , so we can conclude t h a t the S a p r o l e g n i a s p e c i e s t h a t i n f e c t salmon are not h i g h l y v i r u l e n t , b u t are f a c u l t a t i v e , o p p o r t u n i s t i c p a r a s i t e s . T h i s i s not an o r i g i n a l c o n c l u s i o n , but too o f t e n i n the p a s t i t has been used as an argument f o r m i n i m i z i n g the importance o f s a p r o l e g n i -a s i s as a d i s e a s e o f f i s h (see d i s c u s s i o n on pp.26-28 ) , i n s t e a d of b e i n g r e c o g n i z e d as the f i r s t s t e p toward u n d e r s t a n d i n g the n a t u r e o f a complex h o s t - p a r a s i t e i n t e r a c t i o n . A l s o , i t must be emphasized t h a t many i m p o r t a n t d i s e a s e s o f f i s h , i n c l u d i n g those caused by some pseudomonads, aeromonads, m y x o b a c t e r i a , and p r o t o z o a n s , are caused by ' o p p o r t u n i s t i c ' p a r a s i t e s ( R o b e r t s o n e t a l . , 1963; Wedemeyer, 1970; S n i e s z k o , 1974), but f o r some 184 r e a s o n s a p r o l e g n i a n f u n g i have been s i n g l e d out as somehow l e s s i m p o r t a n t than t h e s e . For example, i f a f i s h has both an aero-monad i n f e c t i o n and a s a p r o l e g n i a n i n f e c t i o n , most f i s h p a t h o l o -g i s t s would r e g a r d the aeromonad as the 'primary pathogen' and the s a p r o l e g n i a n fungus as a 'secondary i n v a d e r ' . I n many cases t h i s may be the c o r r e c t i n t e r p r e t a t i o n but the danger of t h i s a t t i t u d e i s t h a t i t i g n o r e s the p o s s i b i l i t y t h a t , under c e r t a i n c i r c u m s t a n c e s , s a p r o l e g n i a n f u n g i can a p p a r e n t l y a c t as l e t h a l p r i m a r y pathogens, or as the more s e r i o u s and d e s t r u c t i v e patho-gen i n a mixed i n f e c t i o n . F o r t u n a t e l y , t h i s a t t i t u d e i s changing and f i s h p a t h o l o g i s t s are g e n e r a l l y b e g i n n i n g to adopt a more b a l a n c e d view of the r o l e o f f u n g i i n f i s h d i s e a s e s (see pp. 26,27). Wolke (1975) r e c e n t l y w r o t e : "While m y c o t i c d i s e a s e s o f f i s h are known to e x i s t . . . f a r l e s s i s known about these d i s e a s e s than i s known about d i s e a s e s o f b a c t e r i a l e t i o l o g y . T h i s l a c k of knowledge may w e l l stem from the consensus t h a t f u n g i i n f e c t i n g f i s h are secondary i n v a d e r s and though they may e v e n t u a l l y r e s u l t i n m o r t a l i t y , i t i s the u n d e r l y i n g p r i m a r y b a c t e r i a l or v i r a l d i s e a s e which must be c o n t r o l l e d and towards which major r e s e a r c h e f f o r t s have been d i r e c t e d . There can be l i t t l e ques-t i o n , however, t h a t some p i s c i n e f u n g a l d i s e a s e out-b r eaks u n a s s o c i a t e d w i t h o t h e r p a t h o g e n i c agents have been e p i z o o t i c i n p r o p o r t i o n and r e s p o n s i b l e f o r mass m o r t a l i t y . " ^ I t has o f t e n been n o t e d t h a t s a p r o l e g n i a n i n f e c t i o n s o f salmon are f r e q u e n t l y a s s o c i a t e d w i t h wounds and l e s i o n s and a l s o t h a t h a n d l i n g these f i s h may p r e d i s p o s e them to i n f e c t i o n (Wood, 1974) . The c o n c l u s i o n t h a t may be drawn from these o b s e r v a t i o n s i s t h a t t h e r e must be some d i s r u p t i o n o f the integument b e f o r e a s a p r o l e g n i a n i n f e c t i o n i s i n i t i a t e d ; t h a t i s , s a p r o l e g n i a n 185 f u n g i are 'wound p a r a s i t e s ' . However, the h o s t - p a r a s i t e i n t e r -a c t i o n i s c l e a r l y more c o m p l i c a t e d than t h i s because t h i s o b s e r v a -t i o n does not account f o r two i m p o r t a n t f a c t s . F i r s t , b o t h my f i e l d and l a b o r a t o r y s t u d i e s w i t h young salmon have shown t h a t wounding these f i s h and then e x p o s i n g them to p o t e n t i a l l y patho-g e n i c s a p r o l e g n i a n f u n g i does not a s s u r e t h a t they w i l l become i n f e c t e d . Second, my i n f e c t i o n experiment d a t a , f i e l d o b s e r v a -t i o n s , d i s c u s s i o n s w i t h f i s h e r y b i o l o g i s t s , and the l i t e r a t u r e , a l l t end to agree t h a t salmon are not e q u a l l y prone to s a p r o l e g -n i a n i n f e c t i o n s d u r i n g a l l s t a g e s o f t h e i r p o s t - e m b r y o n i c f r e s h -water l i f e h i s t o r y . As a g e n e r a l r u l e , t h e r e appear to be two s t a g e s i n the l i f e h i s t o r y o f salmon when they are p a r t i c u l a r l y prone to s a p r o l e g n i a s i s - - d u r i n g the p r o c e s s o f s e x u a l m a t u r a t i o n and d u r i n g and a f t e r the a l e v i n ( p a r r ) - s m o l t t r a n s f o r m a t i o n . Any t h e o r y c o n c e r n i n g the c o n d i t i o n s w h i c h a l l o w a s a p r o l e g n i a n i n -f e c t i o n to be i n i t i a t e d must account f o r these two o b s e r v a t i o n s . The s u p e r f i c i a l c o n c l u s i o n , of c o u r s e , i s t h a t d u r i n g these two d i f f i c u l t p e r i o d s o f p h y s i o l o g i c a l adjustment the salmon-.'s former defences and r e p a r a t i v e c a p a c i t y are so i m p a i r e d t h a t , when i n j u r e d , they a r e no l o n g e r a b l e to r e s i s t i n f e c t i o n by n o r m a l l y n o n - p a t h o g e n i c organisms. T h i s c o n c l u s i o n i s too g e n e r a l to be of much use, b u t i t does l e a d to the key q u e s t i o n . Can a mechan-ism be proposed to e x p l a i n how the p h y s i o l o g i c a l changes t h a t o c c u r i n salmon d u r i n g these two phases of t h e i r l i f e h i s t o r y p r e d i s p o s e these f i s h to i n f e c t i o n by p a r a s i t e s to which they 186 are n o r m a l l y r e s i s t a n t ? In the case o f m a t u r i n g salmon, I am c o n v i n c e d t h a t a major p a r t o f the answer t o t h i s q u e s t i o n i s con n e c t e d w i t h what i s a l r e a d y known about the r e l a t i o n of s t r e s s to d i s e a s e r e s i s t a n c e o f f i s h (Wedemeyer, 1970) and the s i m i l a r i -t y of s t r e s s - i n d u c e d m e t a b o l i c a l t e r a t i o n s t o the normal p h y s i o -l o g i c a l changes t h a t o c c u r i n m a t u r i n g salmon (Woodhead, 1975) . In the case o f young salmon the p i c t u r e i s l e s s c l e a r , but r e c e n t work by McLeay (1975) suggests t h a t the same b a s i c mechan-ism, a s s o c i a t e d i n p a r t , i n t h i s c a s e , w i t h the p h y s i o l o g i c a l changes t h a t o c c u r to ensure s u c c e s s f u l t r a n s f o r m a t i o n from f r e s h w a t e r t o s a l t w a t e r , a l s o p r e d i s p o s e s the f i s h t o s a p r o l e g -n i a n i n f e c t i o n s . My b a s i c h y p o t h e s i s i s t h a t t h e r e i s a d i r e c t l i n k between i n c r e a s e d l e v e l s of.: plasma c o r t i c o s t e r o i d s and the s u s c e p t i b i l i t y o f salmon to s a p r o l e g n i a s i s . The f o l l o w i n g d i s -c u s s i o n p r e s e n t e v i d e n c e s u p p o r t i n g t h i s h y p o t h e s i s . I t i s becoming w e l l e s t a b l i s h e d t h a t the p i t u i t a r y - i n t e r r e n -a l a x i s o f m a t u r i n g salmon i s v e r y s e n s i t i v e to n o n s p e c i f i c s t r e s s (Wedemeyer, 1970; Donadlson § Dye, 1975) and the sockeye salmon from R o b e r t s o n Creek are no e x c e p t i o n ( F a g e r l u n d , 1967). The i n c r e a s e i n c o r t i c o s t e r o i d l e v e l s as a r e s u l t o f s t r e s s l owers the r e s i s t a n c e of f i s h to i n f e c t i o n by o p p o r t u n i s t i c p a r a -s i t e s (Wedemeyer, 1970; S n i e s z k o , 1974) and may, p a r e n t h e t i c a l l y , lower the r e s i s t a n c e of A t l a n t i c salmon to UDN as w e l l (Reichen-b a c h - K l i n k e , 1974; O ' B r i e n , 1974). More to the p o i n t , t h e r e i s a l s o some e m p i r i c a l e v i d e n c e w h i c h shows a p o s i t i v e c o r r e l a t i o n between the incidence,, o f s a p r o l e g n i a s i s and i n c r e a s e d c o r t i c o -187 s t e r o i d l e v e l s ( R o b e r t s o n e t a_l. , 1963; F a g e r l u n d , 1967 ; Donald-son $ F a g e r l u n d , 1968; Roth, 1972). F u r t h e r m o r e , i t i s now q u i t e w e l l e s t a b l i s h e d t h a t t h e r e i s a marked i n c r e a s e i n plasma c o r t i c o s t e r o i d l e v e l s d u r i n g the normal m a t u r a t i o n of P a c i f i c salmon (Donaldson £j F a g e r l u n d , 1970; Woodhead, 1975). T h i s i n c r e a s e i s r e l a t e d , - i n p a r t , to c o r t i c o -s t e r o i d - r e g u l a t e d p r o t e i n c a t a b o l i s m and g l u c o n e o g e n e s i s , and i s p r o b a b l y a l s o r e l a t e d to d e g e n e r a t i v e changes t h a t o c c u r i n the t i s s u e s of the f i s h . T h i s normal i n c r e a s e i n c o r t i c o s t e r o i d l e v e l s a l o n e may h e l p to p r e c i p i t a t e i n f e c t i o n s i n some f i s h . Presumably, v a r i o u s s t r e s s o r s such as c r o w d i n g , i n j u r y , s u b o p t i -mal water t e m p e r a t u r e s , h a n d l i n g , or the presence o f s u b l e t h a l c o n c e n t r a t i o n s o f p o l l u t a n t s would cause f u r t h e r i n c r e a s e s i n c o r t i c o s t e r o i d c o n c e n t r a t i o n s w i t h the r e s u l t t h a t more f i s h would then become more s u s c e p t i b l e to i n f e c t i o n . The i n f e c t i o n i t s e l f , o f c o u r s e , i s a l s o a s t r e s s o r . The r e s u l t o f a l l t h i s c o u l d be a v i c i o u s s p i r a l w h i c h , g i v e n a s u i t a b l e s e t o f c i r c u m -s t a n c e s , c o u l d end i n the premature death o f a s i g n i f i c a n t pro-p o r t i o n of the f i s h b e l o n g i n g to a g i v e n p o p u l a t i o n . Recent work by McLeay (1975), when combined w i t h the r e s u l t s o f my i n f e c t i o n e x p e r i m e n t s , suggests t h a t the s u s c e p t i b i l i t y o f young salmon to s a p r o l e g n i a s i s i s a l s o r e l a t e d to an i n c r e a s e i n plasma c o r t i c o s t e r o i d l e v e l s . R e f e r r i n g back to by i n f e c t i o n e xperiment d a t a , i t can be seen t h a t my one r e a l l y s u c c e s s f u l s e r i e s o f i n f e c t i o n e x p eriments was c a r r i e d out u s i n g coho salmon 188 coming i n t o the second s p r i n g o f t h e i r l i f e , the time when they would n o r m a l l y (and p r o b a b l y i n t h i s case as w e l l ) undergo the a l e v i n ( p a r r ) - s m o l t t r a n s f o r m a t i o n . Wood (1974) has n o t e d t h a t " w i t h i n a week or two f o l l o w i n g the h a n d l i n g a group of f i s h , e s p e c i a l l y ' s l i v e r e d - u p ' y e a r l i n g s [ i . e . , salmon u n d e r g o i n g the a l e v i n ( p a r r ) - s m o l t t r a n s f o r m a t i o n ] , a number of the f i s h w i l l u s u a l l y show fungoused areas on the t a i l and body". A c c o r d i n g to McLeay (1975), t h i s i s a l s o the p e r i o d i n the l i f e o f the salmon when the p i t u i t a r y - i n t e r r e n a l a x i s i s a c t i v a t e d by c o l d -temperature a c c l i m a t i o n and s m o l t i f ication... McLeay merely s t a t e s t h a t " t h i s a c t i v a t i o n i s p r o b a b l y n e c e s s a r y to p r o v i d e i n c r e a s e d m e t a b o l i c energy f o r the numerous... a l t e r a t i o n s a s s o c i -a t e d w i t h t h i s [ a l e v i n ( p a r r ) - s m o l t ] t r a n s f o r m a t i o n " . I would take t h i s a b i t f u r t h e r and su g g e s t t h a t the t r a n s i t i o n from f r e s h w a t e r to s a l t w a t e r may not be p o s s i b l e p r i o r to a c t i v a t i o n o f the p i t u i t a r y - i n t e r r e n a l a x i s because t h e r e i s e v i d e n c e which shows t h a t the g l u c o c o r t i c o i d s of salmon are not o n l y concerned w i t h r e g u l a t i o n o f g l u c o n e o g e n e s i s , but a l s o have an i m p o r t a n t o s m o r e g u l a t o r y f u n c t i o n as w e l l ( O l i v e r e a u , 1962; U t i d a e t a l . , 1972; Woodhead, 1975). McLeay (1975) a l s o found t h a t the..increase i n p i t u i t a r y -i n t e r r e n a l a c t i v i t y was c o r r e l a t e d w i t h lymphopenia which may have been a r e s u l t o f the l y m p h o c y t o l y t i c a c t i o n o f the c o r t i c o -s t e r o i d s . T h i s o b s e r v a t i o n agrees c o m p l e t e l y w i t h the w e l l e s t a -b l i s h e d p o s i t i v e c o r r e l a t i o n between i n c r e a s e d plasma c o r t i c o -s t e r o i d l e v e l s and de c r e a s e d immunocompetency and p r o v i d e s a 189 l o g i c a l h y p o t h e s i s t o e x p l a i n the i n c r e a s e d s u s c e p t i b i l i t y o f the salmon to n o r m a l l y n o n p a t h o g e n i c organisms such as S a p r o l e g -n i a s p e c i e s . T h i s may a l s o e x p l a i n the a p p a r e n t l y poor i n f l a m -matory response t o s a p r o l e g n i a s i s n o t e d i n the h i s t o p a t h o l o g i c a l s t u d y . P a r e n t h e t i c a l l y , I might add t h a t t h i s c o n c e p t i o n o f s a p r o l e g n i a s i s i s c o n s i s t e n t w i t h the much l a r g e r body o f know-ledge r e l a t i n g to mycoses o f man and o t h e r mammals. I t i s now w i d e l y r e c o g n i z e d t h a t c o r t i s o n e t h e r a p y , or d i s e a s e s which i n t e r -f e r e w i t h immunocompetency, such as u n c o n t r o l l e d d i a b e t e s or l e u k e m i a , can p r e d i s p o s e a p a t i e n t to mycoses ( e . g . , phycomycosis [= z y g o m y c o s i s ] , a s p e r g i l l o s i s , c a n d i d i a s i s ) which are a l s o caused by u b i q u i t o u s and n o r m a l l y non-pathogenic f u n g i (Conant et_ a l . , 1971). A n o t h e r i m p o r t a n t (and not u n r e l a t e d ) f a c t o r to be c o n s i d e r -ed i s the a s c o r b i c a c i d m e t a b o l i s m of the f i s h . Salmon are known to have a d i e t a r y r e q u i r e m e n t f o r V i t a m i n C ( A s h l e y et. al_. , 1975). I n the case o f m a t u r i n g salmon, these r e s e r v e s become d e p l e t e d because o f i n a n i t i o n and c o n s e q u e n t l y the a b i l i t y of the f i s h t o r e p a i r t i s s u e damage i s g r e a t l y i m p a i r e d ( T r i p l e t t and C a l a p r i c e , 1974; A s h l e y e_t aj_. , 1975) a t a time i n t h e i r l i v e s when they are q u i t e l i k e l y t o s u f f e r damage to the integument. Such an e x p l a n a t i o n does not a p p l y to young salmon, of c o u r s e , u n l e s s they have been m a i n t a i n e d on a d e f i c i e n t d i e t , b u t i n t h i s r e g a r d i t s h o u l d be no t e d t h a t an i n c r e a s e i n l e v e l s o f plasma c o r t i c o -s t e r o i d s w i l l a l s o cause d e p l e t i o n o f a s c o r b i c a c i d r e s e r v e s 190 (Wedemeyer, 1969, 1970). Presumably t h e n , even i f the salmon are b e i n g f e d an adequate d i e t , under s u f f i c i e n t l y s t r e s s f u l con-d i t i o n s they c o u l d c o n c e i v a b l y s u f f e r from a de_ f a c t o a s c o r b i c a c i d d e f i c i e n c y . The c o n d i t i o n o f the mucous i s a l s o an i m p o r t a n t f a c t o r to be c o n s i d e r e d w i t h r e s p e c t to the r e l a t i o n between the i n t e g r i t y o f the integument and the s u s c e p t i b i l i t y o f the salmon to sapro-l e g n i a s i s . T h i s a s p e c t has been d i s c u s s e d by W i l l o u g h b y (1972) and W i l s o n (1976) and, w i t h r e g a r d to the p r e s e n t d i s c u s s i o n , I w i l l j u s t note two p o i n t s . F i r s t , W i l l o u g h b y (1972) has p r e s e n t -ed e v i d e n c e w h i c h suggests t h a t the mucous o f s e x u a l l y immature sal m o n i d s i s more r e s i s t a n t to i n v a s i o n by s a p r o l e g n i a n f u n g i than the mucous o f s e x u a l l y mature s a l m o n i d s . I am not aware o f any s a t i s f a c t o r y e x p l a n a t i o n f o r t h i s phenomenon. Second, t h e r e i s e v i d e n c e w h i c h suggests t h a t mucous p r o d u c t i o n i s c o n t r o l l e d by the e n d o c r i n e system. The main hormone i n v o l v e d i n t h i s ac-t i v i t y appears to be p r o l a c t i n , but t h e r e i s a l s o e v i d e n c e which does not s u p p o r t t h i s v i e w p o i n t (see r e v i e w by Lam, 1972) . P r e -sumably t h e r e might be an i n t e r a c t i o n between p r o l a c t i n and the i n t e r r e n a l c o r t i c o s t e r o i d s (see U t i d a e_t a l . , 1972 ; M e i e r , 1972) b u t , as y e t , I would n ot l i k e to s p e c u l a t e on the n a t u r e of t h i s i n t e r a c t i o n , o r i t s r e l a t i o n ( i f any) to the s u s c e p t i b i l i t y of the f i s h t o s a p r o l e g n i a s i s . In summary, I am c o n v i n c e d t h a t t h e r e i s a d i r e c t l i n k be-tween i n c r e a s e d plasma c o r t i c o s t e r o i d l e v e l s i n P a c i f i c salmon and t h e i r s u s c e p t i b i l i t y to s a p r o l e g n i a s i s . These h i g h e r l e v e l s o c c u r i n response to the p h y s i o l o g i c a l r e q u i r e m e n t s o f the salmon 191 and are p a r t i c u l a r l y pronounced a t c e r t a i n c r i t i c a l p e r i o d s i n the l i f e o f the salmon ( i . e . , d u r i n g s m o l t i f i c a t i o n and d u r i n g m i g r a t i o n and m a t u r a t i o n ) and, a t these times o f h i g h c o r t i c o -s t e r o i d l e v e l s , t h e r e may be f u r t h e r s t r e s s - i n d u c e d i n c r e a s e s i n p i t u i t a r y - i n t e r r e n a l a c t i v i t y . As plasma c o r t i c o s t e r o i d l e v e l s i n c r e a s e , the f i s h become more and more s u s c e p t i b l e t o i n f e c t i o n and, a t the same t i m e , l e s s a b l e to m a i n t a i n the i n t e g r i t y o f t h e i r integument. T h i s c o m b i n a t i o n o f f a c t o r s r e n d e r s them sus-c e p t i b l e to i n f e c t i o n by S a p r o l e g n i a s p e c i e s and o t h e r u b i q u i t o u s f a c u l t a t i v e pathogens. V a r i a t i o n i n the s t r e s s response o f d i f -f e r e n t s p e c i e s o r p o p u l a t i o n s o f f i s h t o v a r i o u s e x t e r n a l s t r e s s -o rs a t d i f f e r e n t times i n t h e i r l i v e s may w e l l e x p l a i n , to some e x t e n t , some o f the a p p a r e n t l y c o n t r a d i c t o r y r e s u l t s o b t a i n e d by i n v e s t i g a t o r s who have c a r r i e d out i n f e c t i o n e x p e r i m e n t s . C e r t a i n l y , more e m p i r i c a l e v i d e n c e i s needed to c o n f i r m t h i s h y p o t h e s i s beyond a r e a s o n a b l e doubt, but the c l o s e agreement between the s m a l l amount o f e m p i r i c a l e v i d e n c e a v a i l a b l e , and what would t h e o r e t i c a l l y be e x p e c t e d from the l i t e r a t u r e , i s so good, I have l i t t l e doubt t h a t f u r t h e r i n v e s t i g a t i o n i n t h i s a r e a would be v e r y r e w a r d i n g . I n terms o f p r a c t i c a l a p p l i c a t i o n t o f i s h e r i e s management d e c i s i o n s , the c o n c l u s i o n reached i n t h i s t h e s i s c o n c e r n i n g the n a t u r e o f the s a p r o l e g n i a n - o n c o r h y n c h i d i n t e r a c t i o n a g a i n empha-s i z e s the extreme importance of a v o i d i n g , as much as p o s s i b l e , s t r e s s f u l s i t u a t i o n s -- e s p e c i a l l y w i t h r e g a r d to sm o l t s and m a t u r i n g a d u l t s . F u r t h e r m o r e , i f my c o n c l u s i o n i s c o r r e c t , i t suggests t h a t the common p r a c t i c e o f t a g g i n g salmon s m o l t s j u s t 192 p r i o r to r e l e a s e i s done a t t h e w o r s t p o s s i b l e time w i t h r e f e r -ence to the h e a l t h o f the f i s h (see Roberts et. a l . , 1973). A l s o my c o n c l u s i o n shows why attempts to t r e a t or p r e v e n t d i s e a s e s o f f i s h by c h e m o t h e r a p e u t i c or c h e m o p r o p h y l a c t i c i n t e r v e n t i o n may, i n some s i t u a t i o n s , do more harm than good s i n c e the h a n d l i n g o f the f i s h and exposure o f the a n i m a l s to the c h e m i c a l s may i n v o k e a more se v e r e s t r e s s response than the d i s e a s e i t s e l f and t h e r e -f o r e , w i l l i n c r e a s e the p o s s i b i l i t y o f m o r t a l i t y . F i n a l l y , I would l i k e to emphasize once more the p o s s i b i l i t y t h a t S a p r o l e g n i a s p e c i e s may be a b l e to a c t as p r i m a r y pathogens. S i n c e , w i t h i n a g i v e n w a t e r s h e d , i t would not be u n u s u a l f o r s e v e r a l o p p o r t u n i s t i c p a r a s i t e s to be p r e s e n t , i t i s r e a s o n a b l e to e x p e c t t h a t the f i s h w i l l most o f t e n s u f f e r from mixed i n f e c -t i o n s ; however, the p o s s i b i l i t y t h a t S a p r o l e g n i a s p e c i e s a l o n e might i n i t i a t e i n f e c t i o n s cannot be i g n o r e d . T h i s i n t u r n sug-g e s t s t h a t i n c e r t a i n cases where a n t i b a c t e r i a l t h e r a p y or pro-p h y l a x i s i s c a r r i e d o u t , i t might a l s o be a d v i s a b l e to i n s t i t u t e a n t i m y c o t i c t h e r a p y or p r o p h y l a x i s as w e l l because many commonly used a n t i b a c t e r i a l drugs are not p a r t i c u l a r l y t o x i c to s a p r o l e g -n i a n f u n g i ( e . g . , see Amend e t a l . , 1969) and the s t r e s s i n d u c e d by the t r e a t m e n t may s e r v e to encourage the i n i t i a t i o n of sapro-l e g n i a s i s . 193 LITERATURE CITED Aderon. 1748. (not seen; c i t e d by A i n s w o r t h , 1958) A g e r s b o r g ,<? H. P. K. 1933. S a l i e n t problems i n the a r t i f i c i a l r e a r i n g of s a l m o n i d f i s h e s , w i t h s p e c i a l r e f e r e n c e t o i n t e s t i n a l f u n g i s i t o s i s and the cause of w h i t e - s p o t d i s e a s e . T r a n s. Am. F i s h . 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