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Some aspects of the development of Philonema (Nematoda: Dracunculoidea) in Cyclops bicuspidatus Claus Ko, Ronald Chun Chung 1966

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SOME A S P E C T S OF THE D E V E L O P M E N T OF PHILONEMA (NEmTODA:DRACUNCULOIDEA)  IN C^L^OPS B I C U S P I D A T U S  CLAUS  by  RONALD CHUN CHUNG KO B.Sc,  U n i v e r s i t y of Manitoba, 1964  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  In the Department of Zoology  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 August, 1966  In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree a t the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and studyo  I f u r t h e r agree that permission-, f o r extensive  copying of t h i s  t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s representatives.  I t i s understood that copying  or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission.  Department of  ~^o\_  The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada  ABSTRACT  Development o f Philonema oncorhynchi and  Philonema  agubernaculum i n Cyclops b i c u s p i d a t u s were f o l l o w e d and compared by e x p e r i m e n t a l l y i n f e c t i n g the copepods w i t h l a r v a e obtained from g r a v i d female worms i n spawning salmon and trout.  Larvae were d i s s e c t e d from copepods f o r examination  a t an i n t e r v a l o f two to three days. undergo two moults i n the copepods. described.  They were found to Three l a r v a l stages a r e  A l l the l a r v a l stages o f the two s p e c i e s o f  Philonema a r e m o r p h o l o g i c a l l y i d e n t i c a l .  The r a t e s o f  development o f the l a r v a e were observed to be d i r e c t l y "  o  p r o p o r t i o n a l to temperatures between 4 to 15 C. the range was c a l c u l a t e d as 4.  The Q  for  The r a t e s o f development a t  10° were the same i n the two s p e c i e s .  The e f f e c t s o f  temperature on l a r v a l development a r e d i s c u s s e d .  An attempt  i s made to c o r r e l a t e the r a t e s o f development o f Philonema l a r v a e i n the copepods under l a b o r a t o r y c o n d i t i o n s and the i n f e c t i o n o f sockeye salmon i n Cultus Lake, B r i t i s h Columbia.  iii TABLE OF CONTENTS Page INTRODUCTION  1  1  MATERIALS AND METHODS Sources o f Philonema l a r v a e .  6  C o l l e c t i o n and maintenance o f copepods.  7  I n f e c t i o n o f copepods.  8  E x t r a c t i o n - a n d examination o f copepods.  8  F i x i n g , mounting, s t a i n i n g and m o r p h o l o g i c a l study of larvae  ^  RESULTS 1.  Some g e n e r a l o b s e r v a t i o n s on the i n f e c t i o n and maintenance o f copepods.  2.  M o r p h o l o g i c a l d e s c r i p t i o n o f developmental stages o f Philonema.  ^  a  F i r s t - s t a g e l a r v a from g r a v i d female worms.  18  b  F i r s t - s t a g e l a r v a from Cyclops b i c u s p i d a t u s .  20  c  Second-stage l a r v a .  22  d  Third-stage larva.  26  3.  Manner o f m o u l t i n g .  30  4.  E f f e c t o f temperature on r a t e o f development.  32  5.  E f f e c t o f temperature on growth.  34  iv Page  DISCUSSION Morphology.  40  Moulting.  47  E f f e c t s o f temperature on development.  48  C o r r e l a t i o n o f the r a t e s o f development o f Philonema l a r v a e i n the copepods under l a b o r a t o r y c o n d i t i o n s and the i n f e c t i o n o f salmon i n ^ C u l t u s Lake. SUMMARY  61  BIBLIOGRAPHY  63  LIST OF ABBREVIATIONS USED IN TABLES AND FIGURES  70  ERRATA  Abstract  l i n e 11  change " t o " to "and" between o o 4 and .15 C.  P. 1  last.line  Salvelinus for Salvelinius.  P. 2  para. 3 l i n e 5  tributary for tributory.  P. 3  para. 1 l i n e 6  tributary for tributory.  P. 8  para. 3 l i n e 1  bicuspidatus f o r bicupidatus.  P. 11  para. 3 l i n e 3  haematoxylin  P. 14  para. 1 l i n e 6  had f o r have.  P. 15  Frequency d i s t r i b u t i o n o f s i z e s should be read to the l e f t o f the s o l i d base l i n e f o r the t r o u t s t r a i n and the r i g h t o f the dash l i n e f o r the salmon s t r a i n .  P. 16  para. 1 l i n e 3  died f o r dies.  P. 18  para. 2 l i n e 11  conspicuous  f o r consipicuous.  P. 22  last line  conspicuous  f o r consipicuous.  P. 3 2  para. 2 l i n e 6  show f o r shows.  P. 40  para. 2 l i n e 5  show f o r shows.  P. 42  para 4 l i n e 6  analagous  P. 48  para. 3 l a s t l i n e  add " t o " a f t e r  P.. 54  para. 1 l i n e 3  "of" f o r " t o " the f i r s t young  f o r haemotoxylin.  f o r analogous. "are". instar.  LIST OF TABLES Page I II III  Measurements o f l a r v a l stages.  24  P o s i t i o n s of g e n i t a l primordia of larvae.  72  (a)  P r o p o r t i o n o f l a r v a l stages present a t d i f f e r e n t time i n t e r v a l s a t 1 5 C (salmon s t r a i n ) .  76  The number o f m o u l t i n g F i r s t - s t a g e l a r v a e r e c o v e r e d 6 days a f t e r i n f e c t i o n a t 1 5 ° C (salmon s t r a i n ) .  77  The number o f moulting Second-stage l a r v a e r e c o v e r e d 1 7 - 1 9 days a f t e r i n f e c t i o n a t 15°C (salmon s t r a i n ) .  78  (d)  P r o p o r t i o n o f l a r v a l stages present a t d i f f e r e n t time i n t e r v a l s a t 1 0 ° C .  79  (e)  The number o f moulting F i r s t - s t a g e l a r v a e r e c o v e r e d 13 day days a f t e r i n f e c t i o n a t 1 0 ° C (salmon s t r a i n ) .  80  The number o f m o u l t i n g Second-stage . r e c o v e r e d 3 0 , 3 1 days a f t e r i n f e c t i o n a t 1 0 ° c (salmon s t r a i n ) .  81  P r o p o r t i o n o f l a r v a l stages present a t d i f f e r e n t time i n t e r v a l s a t 4 ° C (salmon strain)  82  The number o f m o u l t i n g F i r s t - s t a g e l a r v a e r e c o v e r e d 2 3 - 2 5 days a f t e r i n f e c t i o n a t 4°C (salmon s t r a i n ) .  83  The number o f m o u l t i n g Second-stage r e c o v e r e d 7 4 , 7 8 days a f t e r i n f e c t i o n a t 4 ° C (salmon s t r a i n ) .  84  (b)  (c)  (f)  (g)  (h)  (i)  vi Page (j)  (k)  (1)  IV  P r o p o r t i o n o f l a r v a l stages present a t d i f f e r e n t time i n t e r v a l s a t 10 C (trout s t r a i n ) .  85  The number o f moulting F i r s t - s t a g e r e c o v e r e d 1 2 , 1 5 days a f t e r i n f e c t i o n a t 10 C (trout s t r a i n ) .  86  The number o f moulting Second-stage r e c o v e r e d 3 0 - 3 4 days a f t e r i n f e c t i o n a t 10 c ( t r o u t s t r a i n ) .  87  Summary o f the r a t e s o f moulting temperatures.  at different  The s i z e s o f l a r v a e c o l l e c t e d a t d i f f e r e n t intervals.  time  Two-ways a n a l y s i s o f v a r i a n c e on the e f f e c t o f temperature on the growth o f l a r v a e (salmon strain). (a)  S i z e s o f l a r v a e a t 15°c  91  (b)  S i z e s o f l a r v a e a t 10°c  92  (c)  S i z e s o f l a r v a e a t 4°c  93  (d)  C e l l sum t a b l e  94  (e)  A n a l y s i s of variance  table  95  Two-ways a n a l y s i s o f v a r i a n c e on the s i z e s o f salmon and t r o u t s t r a i n s Philonema l a r v a e from o •— Cyclops maintained i n 10 c . (a)  S i z e s o f l a r v a e ( t r o u t s t r a i n ) from the copepods maintained i n 10°c  96  (b)  C e l l sums t a b l e  97  (c)  A n a l y s i s of variance  table  98  LIST OF FIGURES Figure 1  2  Page Chromosomes o f hypodermal c e l l s o f m o u l t i n g F i r s t - s t a g e larvae. (a)  Philonema oncorhynchi  (b)  Philonema  8  9  10  13  agubernaculum  Comparison of the s i z e s o f l a r v a e between salmon and t r o u t s t r a i n from Cyclops b i c u s p i d a t u s m a i n t a i n e d i n 10 C.  15  (a) (b)  L a t e r a l view o f F i r s t - s t a g e l a r v a e D o r s a l view of the a n t e r i o r e x t r e m i t y o f a m o u l t i n g F i r s t - s t a g e l a r v a showing the u n i c e l l u l a r gland.  17  (a) (b)  L a t e r a l view o f Second-stage l a r v a V e n t r a l view o f the p o s t e r i o r e x t r e m i t y of the Second-stage l a r v a a t a n a l r e g i o n .  19  (a) (b)  L a t e r a l view o f T h i r d - s t a g e l a r v a . L a t e r a l view o f the r e c t a l and a n a l r e g i o n s o f E a r l y Third-sta'ge l a r v a .  21  O e s o p h a g e a l - i n t e s t i n a l v a l v e s o f (a) F i r s t stage l a r v a (b) Second-stage (c) T h i r d - s t a g e 7  vii  (a), (b) and (c) showing the g e n i t a l primordium of F i r s t , Second and T h i r d - s t a g e l a r v a respectively (d) E x c r e t o r y b l a d d e r of First-stage larva. The d i s t r i b u t i o n o o f g e n i t a l primordium i n the l a r v a l stages.  23  27  29  The d i s t r i b u t i o n o f l a r v a l stages (salmon s t r a i n ) c o l l e c t e d a t d i f f e r e n t time i n t e r v a l s from Cyclops maintained i n 15 C.  31  (a) The d i s t r i b u t i o n o f l a r v a l stages (salmon s t r a i n ) c o l l e c t e d a t d i f f e r e n t time i n t e r v a l s a t 10°c.  33  (b) The d i s t r i b u t i o n o f l a r v a l stages ( t r o u t s t r a i n ) c o l l e c t e d a t d i f f e r e n t time i n t e r v a l s a t 10°c. The d i s t r i b u t i o n o f l a r v a l stages (salmon s t r a i n ) c o l l e c t e d a t d i f f e r e n t time i n t e r v a l s from the copepods maintained i n 4 c. Comparison o f the range o f s i z e s o f the l a r v a l stages o b t a i n e d from copepods maintained i n 15°c, 10°C and 4°C. Comparison  o f the growth of l a r v a e r e c o v e r e d  frgm Cyclogs b i c u s p i d a t u s maintained i n 15 c, 10 c and 4 C.  ACKNOWLEDGMENT  The  author wishes to express h i s s i n c e r e  to Dr. J . R. Adams f o r suggesting The.author i s very much indebted h i s many t h o u g h t - s t i m u l a t i n g  gratitude  and s u p e r v i s i n g t h i s  study.  t o Dr. G.G. Gibson f o r  suggestions and f o r h i s  a s s i s t a n c e i n many ways throughout the e a r l y phase o f the research.  The p a t i e n t t e c h n i c a l a s s i s t a n c e o f Miss Wendy Woo  i s g r a t e f u l l y acknowledged. Mr.  Thanks i s a l s o due to  A.K.M. B a s h i r u l l a h f o r h i s a s s i s t a n c e i n f i e l d c o l l e c t i o n s S i n c e r e g r a t i t u d e i s e s p e c i a l l y extended to the  author's s i s t e r , Mrs. M a r i l y n Y i p , without whose encouragement and sound advice, been f i n i s h e d .  t h i s study would never have  INTRODUCTION  Two s p e c i e s o f the genus Philonema (Nematoda: Dracunculoidea) have been r e p o r t e d i n North America. These nematodes a r e p a r a s i t e s o f the body c a v i t i e s o f salmonoids. Kuitunen-Ekbaum salmon, Oncorhynchus  (1933) found t h a t a d u l t  sockeye  nerka, from E n g l i s h Bay, B r i t i s h  Columbia  were h e a v i l y i n f e c t e d w i t h nematodes b e l o n g i n g to the superfamily Dracunculoidea. as Philonema o n c o r h y n c h i .  She d e s c r i b e d these d r a c u n c u l o i d s  In the same year, Smedley (1933)  d e s c r i b e d the same s p e c i e s o f helminth from sockeye caught i n C u l t u s Lake, B r i t i s h Columbia. Adams  salmon  L a t e r , Bangham and  (1954) r e p o r t e d P. oncorhynchi i n s e v e r a l s p e c i e s o f  salmonoids throughout the f r e s h w a t e r s of B r i t i s h  Columbia.  T h i s nematode a l s o has been r e p o r t e d from A l a s k a , the S t a t e of Washington,  and the N o r t h P a c i f i c Ocean as f a r west as the  Okhotsk Sea (Margolis,  1963).  Simon and Simon (1936) d e s c r i b e d agubernaculum  Philonema  which they d i s t i n g u i s h e d from P. oncorhynchi on the  b a s i s o f s m a l l e r s i z e and d i f f e r e n t r a t i o o f the l e n g t h o f a n t e r i o r to p o s t e r i o r oesophagus.  This p a r a s i t e was found i n  the body c a v i t y and muscle o f the abdominal w a l l o f Prospium williamsoni  ( G i r a r d ) , Salmo s h a s t a (Jordan) and  Salvelinius fontinalis  ( M i t c h e l l ) from the waters o f Wyoming  2 National Forest.  Bangham (1951) r e p o r t e d the s p e c i e s from  Snake R i v e r , Wyoming, which i s a p a r t o f the Columbia  River  system. However, Bay l i s  (1947) c o n s i d e r e d P_. oncorhynchi  to be i d e n t i c a l w i t h P. agubernaculum.  He suggested t h a t the  d i f f e r e n c e i n s i z e between the two s p e c i e s might be due t o the i n f l u e n c e o f d i f f e r e n t hosts or the degree o f m a t u r i t y . Platzer  (1964) compared P_. oncorhynchi w i t h Philonema  from Kootenay Lake, B r i t i s h Columbia.  The l a t t e r were presumed  to be P. agubernaculum because o f t h e i r occurrence i n one o f the type hosts from a r i v e r system which, l i k e the type locality,  i s a t r i b u t o r y o f the Columbia.  P l a t z e r a l s o examined  the type specimens o f P. agubernaculum from the U.S. N a t i o n a l Museum.  He concluded t h a t there i s no s i g n i f i c a n t m o r p h o l o g i c a l  d i f f e r e n c e between the mature a d u l t s o f the two s p e c i e s . Meyer  (1958,1960) and V i k (1964) attempted  to s o l v e  the l i f e h i s t o r y o f P. agubernaculum by e x p e r i m e n t a l l y i n f e c t i n g Cyclops sp. w i t h l a r v a e from g r a v i d female worms. Meyer was a b l e t o keep the i n f e c t e d copepods a l i v e f o r one month b e f o r e f e e d i n g them to h a t c h e r y - r e a r e d f i n g e r l i n g , Salmo s a l a r .  No worm, however, c o u l d be found i n the f i s h when  they were a u t o p s i e d s e v e r a l months l a t e r .  V i k f e d l a r v a e to  3 Cyclops s c u t i f e r b u t the copepods d i e d b e f o r e any development o f the l a r v a c o u l d take p l a c e . The Platzer  l i f e h i s t o r y o f P. oncorhynchi was s o l v e d by  (1964).  He found t h a t Cyclops b i c u s p i d a t u s serves as  an'intermediate host f o r the p a r a s i t e .  F i r s t - s t a g e larvae  were o b t a i n e d from g r a v i d female worms taken from the coelomic c a v i t y o f spawning salmon, Oncorhynchus nerka, caught  i n C u l t u s Lake, B r i t i s h Columbia.  The l a r v a e were  Introduced i n t o c u l t u r e s o f the copepods which became i n f e c t e d by i n g e s t i n g the l a r v a e . haemocoel.  The l a r v a e moulted twice i n the  I n f e c t i v e t h i r d - s t a g e l a r v a e were r e c o v e r e d  Cyclops seventy and seventeen  from  days a f t e r i n f e c t i o n when the  copepods were maintained a t 8°C and 12°C r e s p e c t i v e l y . I n f e c t e d copepods were l a t e r f e d to h a t c h e r y - r e a r e d fingerlings.  sockeye  T h i r d - s t a g e l a r v a e were o b t a i n e d from the  p e r i t o n e a l t i s s u e s and t u n i c a a d v e n t i t i a o f the swim-bladder 4 to 10 days a f t e r i n f e c t i n g the f i s h . fish,  In n a t u r a l l y  infected  f o u r t h - s t a g e l a r v a e were found i n the p e r i t o n e a l  tissue  when the f i s h were 26 months o l d , and i n the body c a v i t y when the f i s h were 3 2 months o l d .  The worms became mature  o n l y i n salmon r e t u r n i n g t o freshwater to spawn i n . t h e 4th year. Although  there i s no m o r p h o l o g i c a l  difference  between a d u l t Philonema from the freshwater host i n Kootenay  Lake and the anadromous sockeye o f Cultus Lake, there i s evidence t h a t the l i f e c y c l e s i n these two s i t u a t i o n s a r e different..  Bashirullah  (unpublished data)  found mature  P_. agubernaculum i n the body c a v i t i e s .of rainbow t r o u t , Salmo g a i r d n e r i i , caught i n May i n the Lardeau R i v e r which is  a t r i b u t o r y o f Kootenay Lake.  O b v i o u s l y these worms had  developed to m a t u r i t y i n l e s s than 12 months. t h a t there a r e two d i s t i n c t  This suggests  " b i o l o g i c a l s p e c i e s " o f Philonema  d i f f e r i n g i n developmental ra.te, one adapted to r e s i d e n t freshwater hosts, the other to anadromous hosts w i t h a four year spawning  cycle.  A s i m i l a r c o n c l u s i o n was reached by Akhmerov  (1955)  who r e s t u d i e d the s e v e r a l s p e c i e s o f d r a c u n c u l o i d s d e s c r i b e d from salmonoid f i s h e s o f the U.S.S.R. morphology, Simon,  On the b a s i s o f  he synonymized Philonema agubernaculum  1936, Philonema e l o n g a t a F u j i t a ,  Simon and  1940, Coregonema  s i b i r i c a Bauer, 1946, and Philonema•oncorhynchi Kuitunen-Ekabaum, 1933.  But he suggested t h a t the ecology o f P. oncorhynchi may  be d i f f e r e n t from t h a t o f ]?. agubernaculum because the b i o l o g y of  t h e i r hosts i s d i f f e r e n t . The f o r e g o i n g b r i e f r e v i e w g i v e s a g e n e r a l p i c t u r e  of the  the. l i f e h i s t o r y o f Philonema.  But a number o f aspects o f  development o f the p a r a s i t e i n the copepod need  clarification.  Platzer  (1964) d i d not make any  m o r p h o l o g i c a l study o f the developmental P_. oncorhynchi i n the c r u s t a c e a n host. a n a l y s i s o f the e f f e c t s o f temperature  detailed  stages of There was  no  critical  on development.  The  m o r p h o l o g i c a l development of P_. agubernaculum i n the i n t e r m e d i a t e host a l s o needs to be  studied.  T h e r e f o r e , the p r e s e n t study was d e t a i l e d i n f o r m a t i o n on the developmental  undertaken  to o b t a i n  stages of  P_. oncorhynchi i n the copepod host# to e s t a b l i s h the moulting i n t e r v a l s and the r a t e of development to temperatures finally,  and  to determine e x p e r i m e n t a l l y whether P. agubernaculum  from f r o u t d i f f e r s i n these f e a t u r e s from P. oncorhynchi i n salmon.  6 MATERIALS AND  The problem was i n f e c t i n g copepods larvae.  METHODS  approached by e x p e r i m e n t a l l y  c o l l e c t e d from C u l t u s Lake w i t h Philonema  Copepods i n f e c t e d w i t h sockeye s t r a i n  were maintained i n i n c u b a t o r s a t 4 ° c , infected with trout strain at 1 0 ° c .  Development was  established.  T h i s was  10°C and 15°C; those  (P. agubernaculum) were kept o n l y f o l l o w e d by e x t r a c t i n g and examining  l a r v a e a t l e a s t three times a week. the r a t e s o f development  (P_. oncorhynchi)  A c r i t e r i o n f o r comparing  a t the d i f f e r e n t temperatures  was  the i n t e r v a l r e q u i r e d f o r 50% o f the  l a r v a e i n a sample to show evidence o f the f i r s t or second moult, as evidenced by l o o s e n i n g o f the c u t i c l e .  Sources o f Philonema  larvae.  I n f e c t i v e Philonema oncorhynchi l a r v a e were o b t a i n e d from g r a v i d female worms f r e s h l y r e c o v e r e d from the body c a v i t y of i n f e c t e d prespawning  sockeye salmon caught a t Sweltzer Creek,  C u l t u s Lake i n November.  Philonema agubernaculum  l a r v a e were  o b t a i n e d i n a - s i m i l a r manner from rainbow t r o u t caught i n May i n Trout Lake, B r i t i s h  Columbia.  The worms were p l a c e d i n d e c h l o r i n a t e d water where they b u r s t , r e l e a s i n g the f i r s t - s t a g e l a r v a e .  The broken  p i e c e s of the females were removed by f i l t e r i n g through a  7 f i n e mesh screen.  The  l a r v a e were t r a n s f e r r e d to a 100ml  graduated c y l i n d e r to which lake water was o f inoculum.  One  ml of inoculum was  p e t r i d i s h r u l e d i n squares.  The  added making 100ml  then p i p e t t e d to a  number., of l a r v a e i n the d i s h  were counted u s i n g a hand-counter.  T h i s was  done under an  Olympus d i s s e c t i n g scope, thus g i v i n g a rough e s t i m a t i o n the c o n c e n t r a t i o n o f l a r v a e i n the  of  inoculum.  C o l l e c t i o n and Maintenance of Copepods. Plankton  was  c o l l e c t e d from d i f f e r e n t areas i n  C u l t u s Lake by means o f a #10 by a row-boat. The  plankton  quarters  b o l t i n g s i l k plankton  net towed  Both h o r i z o n t a l and v e r t i c a l tows were made.  was  emptied i n t o two  f u l l of lake water.  An  3-galIon l i v e r ice-pack was  cans,  three  added to each  can to keep the water c o o l .  S i x g a l l o n s o f lake water were  a l s o t r a n s p o r t e d back to the  laboratory.  On  a r r i v a l a t the l a b o r a t o r y , the s m a l l and  p l a n k t e r s were separated f i r s t through a b r a s s  immediately by  s i e v e of 1 mm  #12  bolting silk.  was  maintained a t a constant  flow.  The  plankton  the water  pore s i z e , then a p i e c e of  During f i l t r a t i o n ,  the water i n the  funnel  l e v e l by r e g u l a t i n g the r a t e of  so separated  i n 3 - g a l l o n museum j a r s .  siphoning  large  was  stored overnight at 1 0 ° c  8 One  hundred random samplings o f copepods were made  by a f i n e p i p e t t e .  They were t r a n s f e r r e d w i t h a drop o f  water t o a 3 x 2" c o n c a v i t y s l i d e f o r i d e n t i f i c a t i o n .  Half of  the sample was d i s s e c t e d to determine whether they were n a t u r a l l y i n f e c t e d w i t h Philonema.  I n f e c t i o n o f copepods. Approximately one thousand copepods were i n t o each o f s e v e r a l 4" finger-bowls p i e c e o f #12 b o l t i n g s i l k .  by f i l t e r i n g  concentrated through a  Philonema l a r v a e were added to  produce a c o n c e n t r a t i o n o f three l a r v a e per copepod. P l a t z e r ' s methods (1964), the finger-bowls for  Following  were k e p t a t 10°C  24 hours b e f o r e the c u l t u r e s were t r a n s f e r r e d t o 8"  stacking dishes. per d i s h .  The c o n c e n t r a t i o n o f copepodswas about 250  Each d i s h , h a l f - f u l l o f water, was covered w i t h a  p i e c e o f g l a s s t o reduce e v a p o r a t i o n . dishes was maintained Using  The water l e v e l i n the  by adding lake water from time to time.  the above method, a d u l t Cyclops  both males and females,  bicupidatus,  were found to be 3 0 to 40% i n f e c t e d  w i t h an average o f two l a r v a e each.  L a t e r experiments showed  t h a t the percentage o f i n f e c t i o n c o u l d be i n c r e a s e d t o 80 to 90% without  a l t e r i n g much the o v e r a l l degree of i n f e c t i o n .  This was done by a l l o w i n g the copepods to remain i n the  9 f i n g e r - b o w l s f o r 48 i n s t e a d of 24 hours b e f o r e b e i n g t r a n s f e r r e d to the 8" s t a c k i n g d i s h e s .  A longer exposure to  a more c o n c e n t r a t e d q u a n t i t y o f l a r v a e provides a b e t t e r chance o f b e i n g  infected.  Roughly 12,000 copepods were i n f e c t e d i n three d i f f e r e n t groups. batch;  About 8,000 were i n f e c t e d i n the f i r s t  2,000 s i x days afterwards; another  a f t e r the f i r s t  2,000 f i f t e e n days  infection.  Copepods i n f e c t e d w i t h Philonema oncorhynchi were maintained  larvae  a t 15°C and 10°C i n c o n t r o l l e d - e n v i r o n m e n t  rooms and a t 4°c i n a r e f r i g e r a t e d i n c u b a t o r .  Two c u l t u r e  dishes were t r a n s f e r r e d from 10°c t o 15°C and 4°C r e s p e c t i v e l y when 50% o f the l a r v a e from the copepods were found  undergoing  the f i r s t moult. Copepods i n f e c t e d w i t h Philonema agubernaculum l a r v a e were maintained  a t 10°C i n the c o n t r o l l e d - e n v i r o n m e n t  room. Copepods were f e d weekly by adding h a y - i n f u s i o n to each d i s h .  5 ml o f  The i n f u s i o n was prepared by  s t e e p i n g c l o v e r - h a y i n hot water and s e t t i n g i t a s i d e to c o o l . The  i n f u s i o n was used o n l y a f t e r i t was one week o l d when the  p o p u l a t i o n o f algae, b a c t e r i a and protozoans considerable s i z e .  had reached a  I t was d i s c a r d e d three weeks a f t e r  10 p r e p a r a t i o n because by t h i s time i t was algae.  u s u a l l y too f u l l  Copepods were observed to i n g e s t the c i l i a t e s  algae may  a l s o have served  of  but  as food as green-pigmented granules  were found i n the i n t e s t i n e o f copepods from the c u l t u r e .  E x t r a c t i o n and  examination of l a r v a e from copepods.  Samples of copepods were examined three to four a week.  The  c u l t u r e d i s h was  under a cool-white  times  p l a c e d on a p i e c e of b l a c k c l o t h  f l o u r e s c e n t lamp.  The  copepods were  t r a n s f e r r e d by a f i n e p i p e t t e to a Syracuse d i s h w i t h two of c h i l l e d Ringer's s o l u t i o n f o r cold-blooded  ml  invertebrates.  From t h i s Syracuse d i s h they were f i n a l l y t r a n s f e r r e d to a w e l l - s l i d e when they were d i s s e c t e d under an Olympus d i s s e c t i n g scope w i t h two sticks.  NO.000 i n s e c t pins mounted i n a p p l i c a t o r  .Extracted  l a r v a e were examined w i t h a L e i t z compound  microscope u s i n g a 10X  o c u l a r and  45X  objective.  F i x i n g , mounting, s t a i n i n g and m o r p h o l o g i c a l study of  larvae.  A f t e r examining the l a r v a e w i t h a compound microscope, they were f i x e d and (1:1.25) and  preserved  stored i n small  in formalin-acetic acid vials.  Goodey's l a c t o p h e n o l - c o t t o n Goodey, 1949)  was  modified  blue  ( F r a n k l i n and  f o r s t a i n i n g the l a r v a e i n the  11 f o l l o w i n g manner:-  Larvae were t r a n s f e r r e d from the v i a l s  to a s l i d e by a dropper.  They were caused to adhere t o the  s u r f a c e by p r e s s i n g w i t h a human e y e l a s h glued to the end o f an a p p l i c a t o r s t i c k .  E x c e s s i v e f l u i d was withdrawn b e f o r e  the a d d i t i o n o f a drop o f 0.0004% l a c t o p h e n o l - c o t t o n b l u e . Glass-wool to  f i l a m e n t s were arranged  on four s i d e s of the drop  serve as a support upon which an 18 mm  square  NO.O  c o v e r s l i p was g e n t l y l a i d w i t h a p a i r o f f i n e f o r c e p s . c o v e r s l i p was f i n a l l y Cotton-blue nuclear s t a i n .  s e a l e d w i t h Gurr's  The  glyceel.  was used s o l e l y because i t i s a good  Besides c o t t o n - b l u e , methylene-blue,  haemotoxylin-eosin,  f a s t - g r e e n and mixtures  c o t t o n - b l u e were a l s o t r i e d f o r s t a i n i n g . (0.12% i n 70% a l c o h o l ) i s a poor nucleus  o f f a s t - g r e e n and Fast-green  stain.  s t a i n e d the o u t l i n e o f s t r u c t u r e s c l e a r l y .  However, i t  I t was more  permanent than c o t t o n - b l u e which faded away i n one to two months  time. Measurements o f the l a r v a e were made on a L e i t z  compound /'.microscope w i t h an o c u l a r micrometer.  L i v i n g larvae  as w e l l as s t a i n e d specimens were s t u d i e d to t r a c e the d e t a i l s of m o r p h o l o g i c a l  development.  A s e r i e s o f diagrams was drawn  w i t h the a i d o f a L e i t z m i c r o p r o j e c t o r .  12 RESULTS  (1)  Some g e n e r a l o b s e r v a t i o n s  on the i n f e c t i o n and  maintenance o f copepods. (a)  Some Philonema l a r v a e from g r a v i d female worms  s u r v i v e d as long as twenty-five (b)  days i n lake water a t 10°C.  No i n f e c t i o n was found i n n a u p l i i , or i n f i r s t ,  second and t h i r d copepodid stages.  However, f o u r t h and  f i f t h copepodid stages were s u s c e p t i b l e to i n f e c t i o n . i s s i m i l a r to the r e s u l t s o f Watson and P r i c e w i t h the coracidium (c)  (1960) working  o f the cestode, Triaenophorus  Cyclops i n g e s t e d  This  crassus.  free-swimming l a r v a e i n the  water and more p a s s i v e ones i n the bottom o f the c u l t u r e dish. • S i m i l a r observations  were made by L e i p e r  (1907) on the  guinea worm, Dracunculus medinensis and by Li(1935) on Procamallanus f u l v i d r a c o n i s . (d)  D i s s e c t i o n o f i n f e c t e d copepods a t h a l f - h o u r l y  i n t e r v a l s f o l l o w i n g i n f e c t i o n showed t h a t 5 t o 5% hours a r e r e q u i r e d f o r the l a r v a e to penetrate  the i n t e s t i n e and reach  the body c a v i t y . (e)  The n u t r i t i o n a l s t a t e o f l i g h t l y i n f e c t e d  (1 to 2 larvae) and h e a v i l y i n f e c t e d (5 or over) Cyclops seemed to be normal.  O i l d r o p l e t s were present  i n both  cases.  13  F i g u r e 1.  Chromosomes o f hypodermal c e l l s o f moulting First-stage . (a) (b)  larvae.  Philonema oncorhynchi P. agubernaculum  M a g n i f i c a t i o n - 1000X  14 Moorthy  (1938) r e p o r t e d t h a t Dracunculus medinensis  i n f e c t i o n had a d e t e r r e n t e f f e c t on the r e p r o d u c t i v e of Mesocyclops sp.  activity  But i n the present experiments, young  copepods w i t h 5 t o 7 l a r v a e were found t o c a r r y egg-sacs some time a f t e r i n f e c t i o n .  N a u p l i i and copepodid  stages  u s u a l l y appeared i n c u l t u r e s t h a t have been maintained f o r three to f o u r weeks. Reduction  i n a g i l i t y , was the o n l y obvious  d e t r i m e n t a l e f f e c t on h e a v i l y i n f e c t e d copepods.  Heavily  i n f e c t e d copepods u s u a l l y moved s l u g g i s h l y and tended to remain motionless periods.  on the bottom o f the c u l t u r e d i s h f o r long  No copepod i n f e c t e d w i t h t e n o r more l a r v a e c o u l d  be found two t o three weeks a f t e r i n f e c t i o n , whereas  those  i n f e c t e d w i t h one to two. l a r v a e s u r v i v e d f o r a much longer p e r i o d o f time.  S l u g g i s h movement may a d v e r s e l y  affect  s u r v i v a l because such animals are l i k e l y t o be e l i m i n a t e d by competition  i n the long: run.  No d e f i n i t e c o n c l u s i o n can be made on the e f f e c t o f heavy i n f e c t i o n on the development o f l a r v a e . l a r v a e o f d i f f e r e n t stages were r e c o v e r e d  In some cases,  from the same copepod,  whereas i n o t h e r s , a l l the l a r v a e from a copepod were the same stage.  Often,  t h i r d - s t a g e l a r v a e c o u l d be found along w i t h the  p r o c e r c o i d s o f Proteocephalus under n a t u r a l c o n d i t i o n s .  sp. which the copepods a c q u i r e d  15  F i g . 2.  Comparison o f the s i z e s of l a r v a e between salmon and t r o u t s t r a i n s from Cyclops b i c u s p i d a t u s maintained i n 10°C. (Sample size=20'for each stage) j  1  Range o f s i z e o f t r o u t s t r a i n .  I  1  Range of s i z e o f salmon s t r a i n .  '—[  Frequency d i s t r i b u t i o n o f s i z e .  . »f~'  F = First-stage S = Second-stage T = Third-stage  120011  1100"  1000}  900|  2  800jf  N  700  600  500  400S  s STAGE  16 (f)  The i n f e c t i v e t h i r d - s t a g e o f Philonema  appeared unable to l e a v e i t s c y c l o p o i d i n t e r m e d i a t e host even a f t e r the l a t t e r d i e s .  This i s d i f f e r e n t from the p r o c e r c o i d s  of the cestode, Spirometra mansonoides which can themselves from i n t a c t copepods  (Mueller,  liberate  1959).  A f t e r twenty copepods were k i l l e d by a n o x i a t i o n as a r e s u l t o f withdrawing a l a r g e q u a n t i t y o f water,  the l a r v a e  i n s i d e were observed w i t h a compound microscope f o r 12 hours. They moved a l o n g the haemocoel,  i n t o the antennae,  appendages of c a u d a l rami, attempting to leave the h o s t s . c o u l d make i t s way (g)  None  out.  The m o r t a l i t y o f i n f e c t e d copepod c u l t u r e s  was  more than 50% a t 10°C and 15°C, three to f o u r months a f t e r At 4 ° c ,  infection. 3 00 copepods  (2)  the m o r t a l i t y was  30 to 40%.  Some 200 to  remained a l i v e a t 4°C f o r more than e i g h t months.  M o r p h o l o g i c a l d e s c r i p t i o n o f developmental stages o f Philonema sp. from Cyclops b i c u s p i d a t u s . Larvae of Philonema from b o t h :trout and salmon were  found to undergo stages, there was  two moults i n Cyclops b i c u s p i d a t u s .  no m o r p h o l o g i c a l d i f f e r e n c e between the  t r o u t and salmon s t r a i n s . identical,  In a l l  Chromosome counts were a l s o  5 p a i r s per nucleus (Figure 1).  A comparison o f  (a)  L a t e r a l view of F i r s t - s t a g e  larva.  (b)  D o r s a l view o f the a n t e r i o r  extremity  of a moulting F i r s t - s t a g e l a r v a showing the u n i c e l l u l a r g l a n d .  For legend see p. 70.  the s i z e s of the v a r i o u s stages between the two presented  i n F i g u r e 2.  A two-ways a n a l y s i s of  strains i s variance  (Table VII) comparing the lengths o f the d i f f e r e n t stages s t r a i n s a t 1 0 ° c (sampled from d i f f e r e n t  these two  of  periods  w i t h i n each stage) shows t h a t a t the 99% i n t e r v a l o f confidence  there i s no s i g n i f i c a n t d i f f e r e n c e .  F i r s t - s t a g e l a r v a e from g r a v i d female worms. (Figures 3, 6a,  7a, 8, Table  I)  Body l e n g t h 490.3/u (416.3 - 527.3 yu) ; body w i d t h a t r e g i o n of nerve r i n g 15.9 13.6  - 18.5yu.  - 24.6/U; body width a t anus  C u t i c l e t h i c k , expanding a t d o r s a l b u c c a l  r e g i o n . to form c o n i c a l d e n t i c l e . Buccal c a v i t y short. c e p h a l i c end.  Amphids not d i s c e r n i b l e .  Nerve r i n g s i t u a t e d 44 to 61.8 yu from  E x c r e t o r y bladder  (Figure 7a)  22.8  - 23 yu  long, 4 yu wide ending p o s t e r i o r l y a t o e s o p h a g e a l - i n t e s t i n a l j u n c t i o n on v e n t r a l s i d e of pseudocoel; cells.  Comparatively  round nucleus,  5.13  c o n s i s t i n g of f o u r  large excretory c e l l with - 6 M  long, l o c a t e d s l i g h t l y  p o s t e r i o r to a l a r g e and c o n s i p i c i o u s nucleus oesophagus.  conspicious  i n muscular  E x c r e t o r y duct narrow, 26.69 - 27 M  long,  1 yu wide, l e a d i n g a n t e r i o r l y from e x c r e t o r y bladder t e r m i n a t i n g p o s t e r i o r to nerve r i n g ,  58.9  extremity.  - 126.7 yu long,  Muscular oesophagus 98.4  and  - 82.7yu from a n t e r i o r  19  F i g . 4.  (a)  L a t e r a l view o f Second-stage l a r v a .  (b)  Ventral  view o f the p o s t e r i o r  extremity  of Second-stage l a r v a a t a n a l r e g i o n .  _GN  EP  («•)  20 7.38 - 11.1/u wide, immediately behind b u c c a l c a v i t y ; expanding g r a d u a l l y p o s t e r i o r t o nerve r i n g . terminating i n crescent-like c e l l s ,  f o u r d o r s a l and two  v e n t r a l , each w i t h c o n s p i c i o u s nucleus w a l l e d i n t e s t i n e 88.6 - 13 5.3/u  Oesophagus  (Figure 6a).  Thick  long, c o n s i s t i n g o f ten to  twelve c e l l s ; w i d e s t r e g i o n 12.3 - 14.8/u; narrowest a t i n t e s t i n a l - r e c t a l r e g i o n , 6.2 - 1 2 . 3 ^ .  Lumen narrow, ending  anterior to i n t e s t i n a l - r e c t a l junction.  Genital  composed o f four c e l l s  primordium  (Figures 7, 8 and Table I I ) , 18.5 -  20.9 yu long, s i t u a t e d v e n t r a l l y 39 - 52.4% o f body l e n g t h from a n t e r i o r e x t r e m i t y ; a d j a c e n t t o i n t e s t i n a l - r e c t a l junction.  T h i r d c e l l from a n t e r i o r  largest,  diameter, w i t h prominent round n u c l e u s . long, 9.8 - 12.3 /u wide.  13.5 - 14.8/u i n  Rectum 49.2 - 71.3/U  Two l a r g e prominent n u c l e i ,  each  10 /u i n diameter, l o c a t e d near i n t e s t i n a l - r e c t a l j u n c t i o n , one oneeach  s i d e o f narrow lumen.  R e c t a l lumen expanding to form  a heart-shaped chamber b e f o r e opening to e x t e r i o r . and r e c t a l lumens n o t connected. near a n a l r e g i o n .  Intestinal  P o o r l y developed r e c t a l g l a n d  Phasmids n o t seen.  T a i l 58.9 - 82.7/u  long, f i l i f o r m and t a p e r i n g towards e x t r e m i t y .  F i r s t - s t a g e l a r v a e from Cyclops b i c u s p i d a t u s . F i r s t - s t a g e l a r v a e from the copepods resemble from female worms except f o r the f o l l o w i n g minor  those  differences:-  21  Fig.  5.  (a)  L a t e r a l view o f T h i r d - s t a g e  (b)  L a t e r a l view of the r e c t a l and a n a l regions of E a r l y Third-stage  larva.  larva.  22 T o t a l body l e n g t h longer, U n i c e l l u l a r gland  11 - 12 /a long, 6 - 7 m  l o c a t e d d o r s a l to b u c c a l c a v i t y . abundant chromatin g r a n u l e s .  - 44.2%  wide  (Figure 3),  Nucleus of gland round, w i t h  I n t e s t i n a l and r e c t a l lumens  connected i n l a r v a e from haemocoel. s i t u a t e d 30.3  5 7 9 . 6 ( 4 9 9 . 5 - 638.5yu) .  G e n i t a l primordium  o f body l e n g t h from a n t e r i o r extremity,  immediately a n t e r i o r to i n t e s t i n a l - r e c t a l j u n c t i o n .  Second stage.  (Figures 4, 6b,  7b,  8,  Table  Shape stumpy, w i t h b l u n t head and l e n g t h 492  - 809.3 yu;body width a t anus 12.3  D o r s a l d e n t i c l e absent. c a v i t y short. extremity.-  - 30/u  Body  - 19.7 yu. Buccal  - 74 p. from a n t e r i o r  G a n g l i o n i c n u c l e i concentrated 28.5  tail.  Amphids not d i s c e r n i b l e .  Nerve r i n g s i t u a t e d 59.4  E x c r e t o r y bladder  I)  around nerve r i n g .  long, 5 - 6/u wide; p o s t e r i o r  e x t r e m i t y ending a t a n t e r i o r p o r t i o n of g l a n d u l a r oesophagus. E x c r e t o r y duct 34.2  - 35.4/u long, opening p o s t e r i o r to nerve  r i n g , 74 - 87.3/u from a n t e r i o r end.  Oesophagus 196.8  359.2yu long, p o o r l y demarcated i n t o a n t e r i o r muscular  and  p o s t e r i o r g l a n d u l a r r e g i o n s ; w i d t h behind b u c c a l c a v i t y 4.9  - 6.2yu.  d o r s a l and  two  O e s o p h a g e a l - i n t e s t i n a l v a l v e present; with v e n t r a l nuclei. (Figure 6b) .  four  Hypodermal n u c l e i  round and c o n s p i c i o u s , showing m i t o t i c d i v i s i o n .  Intestine  Oesophageal-intestinal (a)  First-stage  (b)  Second-stage  (c)  Third-stage  valves  larva. larva. larva.  TABLE I .  Measurements o f l a r v a l stages  (in/u) (salmon s t r a i n )  (sample s i z e - 20 f o r each stage) Stage  First From female worms  Early  second  Second  Early  third*  Third  From copepods  49 0.3 579.6 564.2 (416.3-527.3) (499.5-638.3) (492-666)  732.3 876.2 (625.4-809.3) (788-964.3)  992. 2 (915.8-1037 .9  NRG  53 . 2 (47.1-61.8)  59 . 7 (49.5-65.5)  64.77 (59.4-74.1)  79. 5 (70.4-90)  91.3 (81.5-99.9)  LMO  105. 2 (98.4-126.7)  123 (103.3-159.9)  190.1 (137.7-273)  2 06.64 (150.1-350. 6)  321.1 (198-386.2)  476.9 (350-532.8)  TL  LGO  61 (53.2-65.5)  240.3 (102.6-267.9)  294 (196.8-359)  WMO  6.8 (7.38-11.1)  7 (6.2-8.6)  6.47 (6.2-6.8)  5. 5 (4.9-6.2)  5.5 (4.9-6.5)  WBNR  18.45 (15.9-24.6)  18. 5 (17.2-18.45)  17.22 (15.99-18.5)  17.22 (14.8-18.5)  13.53 (11.1-14.8)  13.53 (11.1-14.8)  WBA  13.63 (15.3-18.5)  14 (13.5-15.4)  15.2 (13.5-20.9)  14.76 (12.3-19.7)  12.8 (12.3-16)  12.3 (11.1-12.3)  118. 7 (88.6-135.3)  113.3 (79.9-159.9)  166.6 (129-258.3)  259 (209-314.9)  196.8 13 2.4 (153.8-258.3) (104.6-205. 4)  LI  4.92  TABLE I. (Continued)  * First  Stage  E a r l y second  * Second  Early  third  Third  From female worms  From copepods  Wlw  •13.63 (12.3-14.8)  13.4 (12-14.8)  11. 5 (10.8-12.3)  11.7 (9.8-15.3)  9.1 (8.6-11.1)  8.61 (7.38-9.84)  LR  55.35 (49.2-71.3)  56.8 (43.1-59.8)  51. 5 (36.9-67.6)  59.4 (51.7-68.9)  46.74 (43.1-55.4)  46.74 (36.9-61.5)  WR  9.64 (9.84-12.3)  9.9 (7.38-11.4)  9.84 (7.38-11.1)  10.3 (9.84-12.3)  7.38 (6.2-9.84)  7.9 (7.38-8.61)  107.2 (73.8-123)  119.9 (86.1-135.3)  121.5 (98.4-129.2)  130.8 (98.4-131.4)  18.45 (17.2-20.9)  18.45 (17.2-20.9)  DAT  286.5 221. 2 (190.7-270.6) (233.7-337)  LGP  18.45 (18.45-20.9)  18.45 (18.45-20.9)  14.5 (12.3-17)  18.45 (17.2-19.7)  GPR  43.7% (39-52.4)  38.4% (30.3-44. 2)  43 .9% (32.3-58.2)  51.3% (27.3-65.9)  EXP^  71.84 (58.9-82.7)  78.8 (74-82.7)  * soon a f t e r  86 ' (74-87.3)  86 (74-87.3)  47% (43. 2- 50.5) 63%(61. 3- 65.9) 100 (100-114)  100 (100-114)  larvae cast off o l dc u t i c l e .  * s i x specimens f o r each stage. to  26 209  - 314-9 yu long, c o n s i s t i n g of ten to twelve c e l l s ;  r e g i o n 9.8 6.2  - 13.5ya; narrowest a t i n t e s t i n a l - r e c t a l j u n c t i o n ,  - 8.6 yu;  lumen wide; i n t e s t i n a l w a l l s t h i n .  primordium 17.2 cells;  7b,  8).  51.7  - 19.7/U l o n g , < c l o s e l y  s i t u a t e d 27.3  extremity,  to 65.9%  Genital  packed w i t h e i g h t  of body length from a n t e r i o r  u s u a l l y p o s t e r i o r to middle o f i n t e s t i n e (Figures  Rectum swollen;  - 68.9/U long; 9.8  with  t h i c k w a l l s and narrow lumen;  - 12.3yu wide;  i n t e s t i n a l - r e c t a l junction.  two  large n u c l e i at  R e c t a l g l a n d w e l l developed,  comprising  twelve c e l l s c i r c u m s c r i b i n g a n a l r e g i o n .  not  T a i l 86 - 13 5.3 / 1 long, t e r m i n a t i n g  seen.  widest  a cap formed by The  thin  Phasmids  typically in  cuticle.  average s i z e  (564/i) of r e c e n t l y moulted  second-stage l a r v a e i s smaller  than t h a t of the  first-stage  l a r v a e from the copepods.  Third-stage/(Figures  5a,  6c,  7c, 8,  Table  I)  Body f i l i f o r m i n shape, w i t h c o n i c a l head and tail.  Body length, 992/u (915.8 - 1037.9 yu) ; body width a t  r e g i o n of nerve r i n g 11.1 12.3yu.  tapering  - 14.8yu; body width a t anus 11 -  C u t i c l e t h i c k , forming four l i p - l i k e s t r u c t u r e s a t  buccal region.  Amphids not d i s c e r n i b l e .  Nerve r i n g 81.5  - 99.9/u from a n t e r i o r extremity.  n u c l e i concentrated  around nerve r i n g .  Buccal c a v i t y absent. Ganglionic  Muscular oesophagus  27  F i g . 7.  (a), (b) and  (c) showing the g e n i t a l  primordium o f F i r s t , larva respectively; of F i r s t - s t a g e  Second and T h i r d - s t a g e (d)  larva.  E x c r e t o r y bladder  lb)  10/*-  EC  28 slender, 150 - 350.6/u long, 4. 9 2 yu wide. oesophagus w e l l developed, 350  - 532.8/u long; widening  g r a d u a l l y to f i l l . ' p s e u d o c o e l completely oesophageal j u n c t i o n .  Glandular  at i n t e s t i n a l -  Oesophageal-intestinal valve w e l l  developed; a prominent s t r u c t u r e w i t h four d o r s a l and v e n t r a l n u c l e i , p r o t r u d i n g i n t o lumen of i n t e s t i n e E x c r e t o r y bladder  20 - 22/u  (Fig. 6c).  long, 4yu wide; ending p o s t e r i o r l y  not f a r from muscular-glandular  oesophageal j u n c t i o n .  E x c r e t o r y duct 60 - 62.7yu long; e x c r e t o r y pore, 100 from a n t e r i o r e x t r e m i t y .  I n t e s t i n e narrow, 104.6  long; a n t e r i o r width 7.4  - 9.8/u; p o s t e r i o r width 6.2  ( F i g s . 7c, 8)  packed c e l l s ;  17.2  - 50.5%  114yu  - 7.4/u; Genital  - 20.9 yu long; ten c l o s e l y  l o c a t e d i n some specimens adjacent  oesophagus, 43.2  -  - 205.4yu  f o u r t e e n to s i x t e e n i n t e s t i n a l c e l l s ; , lumen narrow. primordium  two  to g l a n d u l a r  of body l e n g t h from a n t e r i o r extremity  i n g l a n d u l a r oesophageal r e g i o n ; i n other specimens a t a n t e r i o r p o r t i o n o f i n t e s t i n e , 61.3 extremity. walled,  Rectum 36.9  - 65.9%  from a n t e r i o r  -,61.5/u long; a n t e r i o r r e g i o n , t h i c k  lumen narrow; p o s t e r i o r r e g i o n , a f i n e tube l e a d i n g to  exterior.  R e c t a l gland w e l l developed, w i t h f o u r t e e n to  s i x t e e n c e l l s surrounding Phasmids not seen.  p o s t e r i o r t u b u l a r p o r t i o n of rectum.  T a i l 98.4  - 131.4 yu long.  Cuticle at  e x t r e m i t y of t a i l expanding d o r s a l l y to form a s h o r t p r o t r u s i o n (Fig.  5).  29  F i g . 8.  The d i s t r i b u t i o n o f g e n i t a l i n the l a r v a l stages. F = First-stage. S = Second-stage. T = Third-stage.  primordium  801  O  06 70  I Z  S X > o  601  ••  of®  • •  © 50F  40f-  30L  • • • • i  STAGE  J  1—  30 R e c e n t l y moulted t h i r d - s t a g e l a r v a e d i f f e r from the above d e s c r i p t i o n of t h i r d - s t a g e i n the f o l l o w i n g ways:-  (3)  (1)  Small p i e c e of sheath a t t a c h e d  (2)  Body l e n g t h s h o r t e r , 876.2 yu. (788 - 964.3 /u).  (3)  Glandular  (4)  I n t e s t i n e longer,  (5)  R e c t a l gland,  Manner of  oesophagus s h o r t e r , 198 153.8  tail.  - 386.2/a;..  - 258.3/u.  not so w e l l developed  ( F i g . 5b).  moulting.  Philonema l a r v a e undergo two bicuspidatus.  to  The  events of moulting  moults i n Cyclops  r  were observed to take  p l a c e i n the f o l l o w i n g sequence:(i)  Wrinkling of c u t i c l e ,  thus i n d i c a t i n g a s h o r t e n i n g  of  the body. • (ii) (iii)  Formation o f new  c u t i c l e , most e v i d e n t a t t a i l  Loosening of o l d c u t i c l e .  During the f i r s t moult,  l o o s e n i n g o f the o l d c u t i c l e was  apparent f i r s t a t  p o s t e r i o r extremity.  In the second moult,  corresponding  was  process  region,  the  the  f i r s t e v i d e n t a t the a n t e r i o r  extremity. (iv)  Shedding of o l d c u t i c l e .  In the l a t e r phase of  moulting,  a sheath e n c l o s i n g the a n t e r i o r end breaks o f f i n the form of a cap a n t e r i o r to the e x c r e t o r y pore r e g i o n .  31  F i g . 9.  The d i s t r i b u t i o n o f l a r v a l stages (salmon s t r a i n ) c o l l e c t e d a t d i f f e r e n t time i n t e r v a l s Cyclops m a i n t a i n e d i n 15°C. First-stage.  ma  Moulting  First-stage.  Second-stage. M o u l t i n g Second-stage. Third-stage. * sample s i z e o f the i n t e r v a l .  from  DAYS  AFTER  INFECTION  (A sheath i n form of a cap was  found a t t a c h i n g i n s i d e - o u t  to the head of the l a r v a by the l i n i n g of the oesophagus.) Larvae w r i g g l e f r e e of the remaining sheath s t i l l a t t a c h e d to the p o s t e r i o r  (4)  p o r t i o n of the body.  E f f e c t o f temperature  on r a t e of development.  The frequency d i s t r i b u t i o n of v a r i o u s l a r v a l stages o b t a i n e d from copepods maintained a t 15°C, different intervals  i s presented i n F i g . 9,  (For raw data see Table I I I ) due mainly to the d i f f e r e n c e  i n the p o p u l a t i o n from one s p e c i f i c stage u s u a l l y  extended  10, 11  respectively.  Sample s i z e s are not uniform, i n infections  The data shows t h a t a t a l l temperatures,  weeks.  10°C and 4°C a t  among copepods.  complete  transition  stage to another i s slow.  extends  A  over a p e r i o d of three to f o u r  (For example, a t 15°C, m o u l t i n g f i r s t - s t a g e l a r v a e from 5 t o 21 days a f t e r  infection.)  The frequency d i s t r i b u t i o n of l a r v a l stages of salmon and t r o u t 10°c  i s also  s t r a i n s o b t a i n e d from copepods maintained a t  shown i n F i g . 10a, b.  d i s t r i b u t i o n between the two unequal  sample s i z e .  distribution  The s l i g h t d i f f e r e n c e  s t r a i n s may  be caused by  N e v e r t h e l e s s , the o v e r a l l  i s more or l e s s the same.  the  frequency  in  33  Fig.  10.  (a)  The d i s t r i b u t i o n o f l a r v a l stages (salmon s t r a i n )  c o l l e c t e d at d i f f e r e n t  time i n t e r v a l s a t 10°C.  Legend - see F i g . 9  100 45  I—B—I  90-  • -a a a a •  80-  19  •  •  D  a a • a a • a a  70-  •  •  •  • •an a c •  29  •  •  60-  D  B  •  a a u • • u a a c • • u a a a • • a ci • a  102 31  _ 50|-  •z. U  •  • ac u ac a a • aa •  &  K 40 30-  ••o • c • n •c • •c • ac • ac • •c  20-  1  10r-  .•• t El " • E . • El E  1-7  8-14  15-21  22-28  29-35  DAYS AFTER 5MFECT8CN  36-42  50-114  34 The e f f e c t o f temperature on the r a t e o f moulting i s summarized i n Table IV.  The c r i t e r i o n f o r comparison i s  the time r e q u i r e d f o r 50% o f the sample to show s i g n s o f moulting.  (5)  E f f e c t o f temperature on growth. F i g u r e 12 compares the range and frequency-  d i s t r i b u t i o n o f s i z e s of the three l a r v a l stages a t three different  temperatures. A two-ways a n a l y s i s o f v a r i a n c e was performed on the  s i z e s o f the three l a r v a l stages of each stage)  (covering d i f f e r e n t  periods  from 15°C, 10°C and 4°C (Table V I . T o t a l  sample s i z e = 180). A t the 99% c o n f i d e n c e i n t e r v a l , i t i s found  that  ( i ) there i s no s i g n i f i c a n t d i f f e r e n c e between the  lengths o f the same stage from the three temperatures,  different  ( i i ) the response to temperature ( i n t e r a c t i o n )  i s the same i n a l l stages.  «  F i n a l l y , F i g u r e 13 (Table V) summarizes the p a t t e r n of growth o f Philonema l a r v a e (salmon s t r a i n ) i n d i f f e r e n t temperatures.  35  10  (b)  The d i s t r i b u t i o n o f l a r v a l stages ( t r o u t  strain)  c o l l e c t e d a t d i f f e r e n t time i n t e r v a l s a t 1 0 ° c .  Legend - see F i g . 9.  90  '26 111  Li  e a a a a B B U a a B • • 0 B a•a  103_  •  D  •  c  a a• c  o • Bn  a a D a• c  •Dc •  •  •  C  Q  D  •  D  C  • E B  a •  cK  •  1r 8-14  15-21  D  •  •  E3 R  D  3  B  a  C D C B  in Q D •  [aQ B •  aD 0  fa •  L  22-28  DAYS AFTER INFECTION  29-35  36-42  B  43-49  36  TABLE IV.  Temperature  Summary of the r a t e s o f moulting a t d i f f e r e n t temperatures  Strain  Time a t which 50% of the samples c o l l e c t e d are moulting F i r s t Stage l a r v a e  Time a t which 50% of the samples c o l l e c t e d are moulting SecondStage l a r v a e  15°C  Salmon  6 days a f t e r infection  17 to 19 days a f t e r infection  10°C  Salmon  13 days a f t e r infection  3 0 to 31 days a f t e r infection  o o 10 C-15 C  Salmon  13 days a f t e r infection  12 days a f t e r change  10°C-4°C  Salmon  13 days a f t e r infection  47 days a f t e r change  10°C  Trout  12 to 15 days after infection  3 0 to 34 days after infection  4°C  Salmon  23 to 25 days after infection  74 to 78 days after infection  (For more i n f o r m a t i o n  see Table I I I )  37  Fig.  11.  The d i s t r i b u t i o n o f l a r v a l stages (salmon s t r a i n ) c o l l e c t e d a t d i f f e r e n t time i n t e r v a l s  from  Cyclops b i c u s p i d a t u s maintained i n 4°C.  Legend - see F i g . 9  100  7-14  15-21  22-28  29-35 36-42 43-49  50-56  57-63  DAYS AFTER  64-70  INFECTION  71-77  78-84  38  Fig.  12.  Comparison o f the range of s i z e s ' o f the l a r v a l stages o b t a i n e d from copepods maintained i n 15°C  /  10°C, and 4°C.  (Sample s i z e = 2 0 f o r each stage) I  [  Frequency d i s t r i b u t i o n of F i r s t - s t a g e Frequency d i s t r i b u t i o n of Second-stage Frequency d i s t r i b u t i o n of T h i r d - s t a g e  1100-  1000-  900-  800-  •» 700-  9 •» « ff  600  500  10  TEMPERATURE *C  15  39  Fig.  13.  Comparison o f the growth o f l a r v a e recovered from Cyclops b i c u s p i d a t u s maintained i n ., ,. _o ,6 , „o . .15 C, 10 C and 4 C.  M = Moulting  40 DISCUSSION Morphology: During the process o f dvelopment, prominent m o r p h o l o g i c a l changes i n the l a r v a e o f Philonema take p l a c e mainly  d u r i n g moulting.  Only i n c r e a s e i n s i z e and changes  i n p r o p o r t i o n o f d i f f e r e n t s t r u c t u r e s occur w i t h i n each stage. D i f f e r e n c e i n s i z e i s a major f e a t u r e f o r d i s t i n g u i s h i n g the l a r v a l stages.  The t h i r d - s t a g e l a r v a i s  almost twice as long as the f i r s t 490.34/u). third.  (mean lengths,  992.2 v s .  The second-stage i s wider b u t s h o r t e r than the  Data shows t h a t there i s an upper asymptote f o r the  growth o f t h i r d - s t a g e l a r v a e .  (Figure 13. Table V)  the mean l e n g t h o f the l a r v a recovered i s almost the same as t h a t o b t a i n e d over 1 mm i n both cases.  A t 4°C  96 days a f t e r i n f e c t i o n  64 days l a t e r ,  Moreover, b e s i d e s  slightly  the c e s s a t i o n o f  growth, i t appears t h a t the t h i r d - s t a g e l a r v a may not be i n g e s t i n g food.  This observation  green-pigmented p a r t i c l e s such larvae. in  i s made on the b a s i s  that  a r e absent i n the i n t e s t i n e o f  Green-pigmented p a r t i c l e s not only are present  the i n t e s t i n e s o f the f i r s t and second-stage l a r v a e but  the i n t e n s i t y o f these p a r t i c l e s i n the i n t e s t i n e changes w i t h the i n t e n s i t y o f a l g a l growth i n the c u l t u r e dishes where  41 i n f e c t e d copepods are kept.  T h i s suggests  t h a t the l a r v a e  may be f e e d i n g on the i n t e s t i n a l contents o f the copepods. The method by which they achieve t h i s i s not known. These phenomena p a r t i a l l y i l l u s t r a t e one o f the f e a t u r e s o f Rogers' h y p o t h e s i s .  In t h i s hypothesis, Rogers  s t a t e s t h a t i n p a r a s i t i c s p e c i e s , the formation o f the i n f e c t i v e stage i s u s u a l l y preceded by a moult o r p a r t i a l . moult.  Whether the l i f e c y c l e i s d i r e c t or, i n d i r e c t , the  i n f e c t i v e stage i s a " r e s t i n g " stage i n which the normal processes o f development a r e suspended i n Philonema, the t h i r d - s t a g e l a r v a ,  (Rogers,  1962).  Here,-,  l i k e t h a t o f most  nematodes, r e p r e s e n t s the " r e s t i n g " stage i n the l i f e  cycle.  Body forms a l s o vary c o n s i d e r a b l y between the three l a r v a l stages as a r e s u l t o f a d a p t a t i o n .  The t a i l o f the  f i r s t - s t a g e l a r v a , which i s long and f i l i f o r m serves two purposes.  The l e n g t h o f the t a i l a l l o w s i t to be whipped  around, c o i l e d and u n c o i l e d a c t i v e l y , d e v i c e to keep a f l o a t .  Observations  thus f u n c t i o n i n g as a under the microscope show  t h a t a f t e r b e i n g kept iri the lake water f o r a f o r t n i g h t , l a r v a e show l e s s vigorous movements and they g r a d u a l l y s i n k to  the bottom o f the d i s h .  On the other hand, the whipping  movement may a c t as a gesture which a t t r a c t s the a t t e n t i o n o f  42 copepods l e a d i n g t o i n g e s t i o n . made by L i (1935) and L e i p e r The  Comparable suggestions  (1907) f o r other  were  species.  second-stage i s s h o r t and stumpy, due to the  shedding o f the long f i r s t - s t a g e t a i l . permit an e x t e n s i v e  The t h i n c u t i c l e may  i n c r e a s e i n l e n g t h d u r i n g the second  moult. The  head o f the t h i r d - s t a g e i s c o n i c a l while i t s  body i s f i l i f o r m .  Since i t has t o migrate from the i n t e s t i n e  o f the f i s h to the t u n i c a a d v e n t i t i a o f the swim-bladder a f t e r b e i n g i n g e s t e d by the f i s h host  ( P l a t z e r and Adams, manuscript  i n p r e s s ) , t h i s type o f body form may f a c i l i t a t e The  penetration.  c o n i c a l head r e t u r n s to a rounded form f o l l o w i n g the  t h i r d moult i n the f i s h .  This was shown by a moulting  t h i r d - s t a g e l a r v a recovered  from an i n v i t r o c u l t u r e  experiment c a r r i e d out by the author. Only the f i r s t - s t a g e l a r v a i s equipped w i t h the dorsal denticle.  After infection,  these l a r v a e have to migrate  from the i n t e s t i n e o f the copepod to the body c a v i t y where development t o i n f e c t i v e stage o c c u r s .  These f a c t s suggest  the p o s s i b l e f u n c t i o n o f t h i s p a r t i c u l a r s t r u c t u r e .  I t may  be analogous t o the hooks o f the oncospheres o f cestodes which a c t as a p e n e t r a t i v e d e v i c e  (Thomas, 193 7).  The  f o l l o w i n g o b s e r v a t i o n made by L i (193 5) confirms the presumption:  "When i n g e s t e d , Procama1lanus f u l v i d r a c o n i s  43 l a r v a e moved f r e e l y along  the whole d i g e s t i v e t r a c t .  The  l a r v a c o i l e d i t s e l f d o r s a l l y as f a r as i t c o u l d go and then w i t h a sudden s p r i n g i n g a c t i o n , the a n t e r i o r p o r t i o n was s t r e t c h e d and the d o r s a l spine s t r u c k on the i n t e s t i n a l wall of cyclops."  Working w i t h Dracunculus medinensis which  a l s o possesses a d o r s a l d e n t i c l e , Isaev  (1934) r e p o r t e d  t h a t i n the i n t e s t i n e o f copepod, the l a r v a assumed the form o f an elongate r i n g w i t h the ends meeting. straightened  i t s c e p h a l i c end and pressed  intestinal wall.  Then the l a r v a i t a g a i n s t the  A s i m i l a r behaviour was observed i n  Philonema. A large l i g h t staining c e l l i s located d o r s a l l y a t the c e p h a l i c e x t r e m i t y from copepods.  o f the f i r s t - s t a g e l a r v a e  Though no connection  the d o r s a l d e n t i c l e i s v i s i b l e , in  extracted  between t h i s c e l l ..and  i t s l o c a t i o n and i t s absence  subsequent l a r v a l stages l e a d to the b e l i e f t h a t i t may  be a u n i c e l l u l a r gland r e s p o n s i b l e a s s o c i a t e d w i t h the p e n e t r a t i o n  forhistolytic  secretion  i n t o the body c a v i t y o f the  copepod. I f the assumption i s c o r r e c t , one should a l s o expect to f i n d the same s t r u c t u r e i n f i r s t - s t a g e l a r v a e from g r a v i d female worms.  But an examination o f two hundred l a r v a e  y i e l d e d negative  results.  T h i s may be due to the f a c t  that  44 a l l the  l a r v a e examined were mounted l a t e r a l l y  (because  u s u a l l y l a r v a e r e c o v e r e d from female worms were c o i l e d ) .  The  u n i c e l l u l a r gland which i s s i t u a t e d d o r s a l l y , t h e r e f o r e , c o u l d not be The  detected. oesophageal-intestinal  v a l v e o f second  and  t h i r d - s t a g e l a r v a e i s a t h i c k muscular s t r u c t u r e w i t h f i v e to s i x n u c l e i .  I t i s s i m i l a r i n appearance to those o f  l a r v a e of S t r o n g y l o i d e s  the  s t e r c o r a l i s , Necator americanus  and Ancylostoma canium, a l l of which have s i x to e i g h t n u c l e i (Nicholas, side and i n both  1956).  two  The  n u c l e i , three  to four on the  dorsal  on the v e n t r a l s i d e , are always very prominent  stages. The  e x c r e t o r y b l a d d e r s of d i f f e r e n t stages d i f f e r  only i n s i z e .  The number of c e l l s remains constant.  average lengths  of the b l a d d e r and  excretory  The  duct measure  50yU i n the f i r s t - s t a g e , 63 yU i n the second and  80 yu i n the  third. The  g e n i t a l primordium i n c r e a s e s  i n number of  cells  but not i n s i z e (mean 18.54yu) as the l a r v a grows from the f i r s t to the t h i r d - s t a g e . first-stage,  F i g u r e 8 shows t h a t i n the  the g e n i t a l primordium i s g e n e r a l l y  located  between 39 to 42% of body length from the a n t e r i o r  extremity.  In the second-stage, i t s p o s i t i o n v a r i e s c o n s i d e r a b l y ,  from  45 43  to 66% whereas i n the t h i r d - s t a g e ,  two d i s t i n c t i v e  p o s i t i o n s o f g e n i t a l p r i m o r d i a appear. from 43  to 51%,  the other from 61 to  There are two  One  group ranges  66%.  possible interpretations for  bimodal d i s t r i b u t i o n of g e n i t a l p r i m o r d i a i n the First,  the d i f f e r e n c e i n p o s i t i o n s may  dimorphism.  In nematodes, the  be a form of  e s p e c i a l l y the g e n i t a l pores, are always l o c a t e d  evidence to Philonema, the are s i t u a t e d between 43 The  sexual organs,  anterior  Applying t h i s  l a r v a e whose g e n i t a l p r i m o r d i a  to 51% must be  the f u t u r e  females.  group whose organs are s i t u a t e d between 61 to 66%  the males.  This supposition  also explains  wide range of p o s i t i o n s may  are  satisfactorily  the d i s t r i b u t i o n of g e n i t a l p r i m o r d i a i n the The  third-stage.  female r e p r o d u c t i v e  to the c o r r e s p o n d i n g organs i n males.  the  second-stage.  be an i n d i c a t i o n of  the  m i g r a t i o n o f g e n i t a l p r i m o r d i a i n t h i s stage. This i n t e r p r e t a t i o n i s confirmed by  the r e s u l t s of  Schwartz and A l i c a t a (1934) working w i t h Necator americanus larvae.  They observed t h a t the g e n i t a l p r i m o r d i a of s i x  days o l d l a r v a e from the l o c a t e d i n the  l a s t t h i r d of the i n t e s t i n e , whereas i n  younger t h i r d - s t a g e intestine.  lungs of i n f e c t e d guinea pigs were  they were a n t e r i o r to the middle of  Larvae w i t h a more p o s t e r i o r g e n i t a l primordium  were c o n s i d e r e d fourth-stage,  to be males because i n female larvae o f  the a n t e r i o r and  p o s t e r i o r o v a r i e s branch from  a p o i n t corresponding to the more c e n t r a l p o s i t i o n of  the  g e n i t a l primordium. Nichols  (19 56)  i n A s c a r i s lumbricoides t h i r d - s t a g e larvae.  a l s o mentioned that sexual was  dimorphism  f i r s t e v i d e n t i n middle  Males are d i s t i n g u i s h e d from females i n  t h a t they have s i x c e l l s i n the r e c t a l gland i n s t e a d of three.  No  corresponding o b s e r v a t i o n  c o u l d be made i n  Philonema because the number of c e l l s i n the r e c t a l gland i s u s u a l l y not w e l l  defined.  Secondly, the d i f f e r e n c e i n p o s i t i o n i n g e n i t a l primordium may larvae.  be  Yoshida  due  to the presence of two  (1966) r e p o r t e d  i n g e n i t a l primordium i s one  species  of  that d i f f e r e n c e i n p o s i t i o n  of the d i a g n o s t i c f e a t u r e s  d i s t i n g u i s h between the f o u r t h - s t a g e duodenale and Necator americanus.  to  l a r v a e of I Ane.ylostoma  This suggests  the  p o s s i b i l i t y t h a t the d i f f e r e n c e i n p o s i t i o n i n g e n i t a l primordium i n d i c a t e s t h a t t r o u t and Philonema occur c o n c u r r e n t l y the two  salmon s t r a i n s of  i n the same l a k e .  s t r a i n s r e a l l y e x i s t concurrently  and  However, i f could-be  d i s t i n g u i s h e d i n t h i s manner, there must be a l s o d i s t i n c t i v e groups of g e n i t a l p r i m o r d i a  two  i n second-stage l a r v a e .  47 But no such o b s e r v a t i o n c o u l d be made.  Moreover, the  p o s i t i o n s o f the g e n i t a l organs a r e the same i n the a d u l t s of  the two s t r a i n s o f Philonema. Therefore, the f i r s t  i n t e r p r e t a t i o n seems to provide  a b e t t e r e x p l a n a t i o n f o r the bimodal d i s t r i b u t i o n o f g e n i t a l primordia.  Moulting: Little Lee  i s known about m o u l t i n g  i n nematodes.  (1965) d e f i n e d moulting as the formation o f a new  cuticle,  the l o o s e n i n g o f o l d c u t i c l e and the r u p t u r e and  e c d y s i s o f the o l d c u t i c l e w i t h the ensuing escape o f the larva.  T h e • s i g n i f i c a n c e o f moulting  not c l e a r .  i n the l i f e c y c l e i s  I t may be a mechanism o f growth r e l a t e d to the  p r o p e r t i e s o f the c u t i c l e or i t may be concerned r i d o f nitrogenous waste m a t e r i a l s .  Rogers  with g e t t i n g  (1962)  questioned the v a l i d i t y o f the l a t t e r hypothesis  (1) because  he has demonstrated t h a t ammonia and urea can be e x c r e t e d even through (2)  the a d u l t c u t i c l e o f A s c a r i s lumbricoides and  because nitrogenous wastes are e x c r e t e d by l a r v a e  without d i f f i c u l t y  (Weinstein and Haskins,  1955).  In Philonema, moulting probably f u n c t i o n s p a r t l y as a p h y s i o l o g i c a l a d a p t a t i o n t o a new environment.  The  assumption i s based on the f o l l o w i n g o b s e r v a t i o n s which  48 i n d i c a t e t h a t the p h y s i o l o g y o f the l a r v a l stages i s different.  F i r s t - s t a g e l a r v a e from g r a v i d female worms  can s u r v i v e i n lake water whereas the second-stage from copepods b u r s t s i n the same medium.  A f t e r the second moult,  l a r v a e , now b e i n g the i n f e c t i v e stage, can a g a i n w i t h s t a n d an a q u a t i c medium.  So one may s p e c u l a t e t h a t some  p h y s i o l o g i c a l changes  r e l a t e d to the p r o p e r t i e s of the c u t i c l e  have o c c u r r e d d u r i n g the second moult as a p r e p a r a t i o n f o r e n t e r i n g the forthcoming new environment.  E f f e c t s o f temperature on development. Information c o n c e r n i n g the e f f e c t s o f temperature on the development  o f p a r a s i t e s i s scanty.  Most o f the  a v a i l a b l e l i t e r a t u r e deals only with s u p e r f i c i a l observations.  Attempts were seldom made to c o r r e l a t e the  p h y s i o l o g i c a l i n t e r a c t i o n s between the host and i t s p a r a s i t e s , i n response t o ambient  temperature  changes.  As i n a l l other animals, there a r e l i m i t s o f t o l e r a n c e t o temperature f o r each p a r a s i t e .  Fluctuation of  temperature w i t h i n the l i m i t s w i l l e i t h e r decrease o r i n c r e a s e the r a t e o f metabolism.  In g e n e r a l , the e f f e c t s o f  temperature changes on p a r a s i t e s a r e : -  49 i)  Shorten the d u r a t i o n of egg i n c u b a t i o n . A temperature  i n c r e a s e to c e r t a i n  limits  a c c e l e r a t e s f i s s i o n ; a decrease, a g a i n to a c e r t a i n extent, slows down the p r o c e s s .  Maximal and minimal  l i m i t s are  c h a r a c t e r i s t i c f o r g i v e n s p e c i e s , e.g. f o r the b r a n c h i u r a n o crustacean, A r g u l u s f o l i a c e u s , i t must not be l e s s than 10 C; f o r the p a r a s i t i c copepod, Lernae c y p r i n a c e a , not l e s s than 14°c  (Bauer,  1959).  A t room temperature  (22°C) the eggs of A s c a r i s  lumbricoides take about 28 days to develop i n t o l a r v a e ; a t 31°C they r e q u i r e o n l y 14 days ii)  Shorten the l e n g t h of postembryonic  second-stage  (Fairbarn,  1955).  development.  A t 7.5°C, 21 to 28 days are r e q u i r e d f o r the l a r v a e of Dochmoides stenocephala  (Ancylostomidae)  m a t u r i t y i n n u t r i e n t agar.  A t 27°C, they became mature i n  51 hours iii)  to reach  (Gibbs and Gibbs, 19 59).  Increase oxygen  consumption.  The t h i r d - s t a g e l a r v a e of the nematode, N i p p o s t r o n g y l u s b r a s i l i e n s i s , have a h i g h r a t e of oxygen consumption (Wilson,  a f t e r b e i n g s u b j e c t e d to a temperature  1965).  rise  50 iv)  Increase the a c t i v i t y o f i n v a s i v e phases. Free-swimming l a r v a e o f the trematode, D i c l y b o t h r i u m  armaturn are a c t i v e f o r f i v e to s i x hours a t 13°C; a t 24°C, they are a c t i v e f o r one and a h a l f hour  (Bauer, 1959).  The  frequency o f c o n t r a c t i o n o f Bucephalus elegans c e r c a r i a e i n c r e a s e s from 0 - 102/minute when the temperature i s o i n c r e a s e d from 0 to 28 C. v)  Change the s i z e of l a r v a e . T h i s a s p e c t of temperature i n f l u e n c e i s not w e l l  documented.  C i l i o s p o r e s o f the c i l i a t e ,  I c t h y o p h t h i r i u s sp.  formed a t 20 to 22°C have an average l e n g t h o f 30/u; 7 t o 8°C average 50yu (Bauer, 1959). Vogt  those a t  However, a c c o r d i n g to  (1938), the p r o c e r c o i d s o f the cestode,- Triaenophorus  nodulosus, a t t a i n e d , a f t e r seventeen days i n Cyclops sp. the l e n g t h o f 283yu a t 4°C; 326 u a t 8.5°C; 345 / i a t 14.5°C. Gibbs and Gibbs  (19 59) a l s o observed t h a t a t 7.5°C the average  l e n g t h o f the f r e e - l i v i n g stage o f the nematode, Dochmoides stenocephala, was vi)  532yu as compared to 608yu a t 25°C.  Increase the developmental r a t e of l a r v a l phases i n the i n t e r m e d i a t e host. A number o f examples  DeGuisti  are a v a i l a b l e i n t h i s r e s p e c t .  (1949) found t h a t the acanthocephalan,  Leptorhynchoides thecatus ^ r e q u i r e d a t l e a s t two months to  51 develop-, t o the i n f e c t i v e stage, i n H y a l l e l a a z t e c a maintained a t 13°C.  Conversely, f u l l development was o  a t t a i n e d i n 30 t o 32 days i f the amphipods  were kept a t 25 C.  S i m i l a r o b s e r v a t i o n s have been made i n many trematodes (Vogel, 1934; Stadun, 1952; D i n n i k , 1964; Olson, 1966).  McCoy (1928) demonstrated t h a t C e r c a r i a hamata  changed i n t o m e t a c e r c a r i a e i n " s u n - f i s h " , Eupotomus sp. a f t e r o o two t o three weeks a t 25 C and a f t e r s i x weeks a t 14 C. In  o r i b a t i d mites, the development o f c y s t i c e r c o i d s  of  the cestode, Monococoestus americanus, was a c c e l e r a t e d .  At  o o 15 C, development was completed i n 81 days; a t 20 C, 52 o  days; 25 C, 45 days  (Freeman, 1952).  In t h e i r work w i t h  Hymenolepis diminuta, Voge and Turner (1956) noted t h a t l a r v a l development c o u l d be accomplished between o  15°C (15 days  r e q u i r e d ) and 37 C (5 days r e q u i r e d ) and t h a t temperature below t h i s range was u n s a t i s f a c t o r y .  Temperature higher than  37°C was l e t h a l f o r the p a r a s i t e , though n o t always f o r the host.  Thus the t o l e r a n c e span o f the p a r a s i t e and i t s host  do n o t n e c e s s a r i l y  coincide.  Dracunculus medinensis, a nematode r e l a t e d to Philonema, undergoes i t s f i r s t moult between seventh day and the second moult between  the f i f t h and  the e i g h t h and t w e l t h  day a f t e r i n f e c t i o n o f Cyclops i n hot weather  (90 to 102°F)  52 the c o r r e s p o n d i n g moults take p l a c e r e s p e c t i v e l y between the e i g h t h and  t w e l t h day  s i x t e e n t h day  and between the t h i r t e e n t h and  a f t e r i n f e c t i o n (Moorthy, 1938).  Cystopsis  a c i p e n s e r i s l a r v a e become i n v a s i v e 14 - 15 days a f t e r o i n f e c t i o n , i n amphipods maintained a t 18 to 20 C but a f t e r 22 days a t 8°C  ( J a n i c k i and Rasin,  1929).  only  Wuchereria  b a n c r o f t i a t t a i n s t h i r d - s t a g e i n mosquitoes a t 54.4°F a t 3 0 - 43°F development e i t h e r f a i l s a f t e r reaching  first-stage  (Hu,  to occur or the  1934).  Gibson  larvae die  (1965)  n o t i c e d t h a t t h i r d - s t a g e l a r v a e o f M i c r o f i l a r i a sp. B c o u l d be recovered k e p t a t 74°F.  On  i n 6.5  days i f i n f e c t e d b l a c k f l i e s were  the other  hand, a t 52 - 68°F,  t h i r d - s t a g e were found eleven  few  days a f t e r i n f e c t i o n .  From the f o r e g o i n g b r i e f review, one  generalization  can be made on the e f f e c t s of temperature on the development of p a r a s i t e s .  Within  the l i m i t s of t o l e r a n c e , an  increase  i n ambient temperature, under i n v i v o or i n v i t r o will  a c c e l e r a t e the r a t e f u n c t i o n o f p a r a s i t e s .  i n temperature w i l l In order  have the o p p o s i t e  conditions, A  decrease  effect.  to o b t a i n a b e t t e r p i c t u r e of the e f f e c t s  o f temperature on the development of l a r v a l stages under in vivo conditions,  i t i s a l s o necessary to understand  d i r e c t e f f e c t s o f temperature on the host  itself.  the  53 Coker  (1933,  1934) r e v e a l e d t h a t there was an  i n v e r s e c o r r e l a t i o n between the s i z e o f copepods and the environmental temperature.  The average s i z e of female Cyclops  v e r n a l i s which were maintained a t 28 - 3 0°C was found t o be 1.11 mm.  In c o n t r a s t , the value was 1.62 mm f o r those k e p t  a t 8°C. A s i m i l a r c o r r e l a t i o n e x i s t e d i n Cyclops s e r r u l a t u s and Cyclops v i r i d i s .  But the v a l i d i t y of t h i s  i s q u e s t i o n e d on the b a s i s t h a t  investigation  (1) no s t a t i s t i c a l  evaluation  of data was made, and (2) the samples were n o t d i v i d e d  into  d i f f e r e n t age groups. A c c o r d i n g t o Manfredi  (1923), the e n t i r e  developmental c y c l e o f Cyclops b i c u s p i d a t u s l a s t e d about 1 - lh months a t 13 - 1 5 ° c and about t h r e e weeks a t 20 - 27°C. Ewers  (1936) s t a t e d t h a t i n c r e a s i n g temperature would speed  up the r a t e o f development  i n most Cyclops u n t i l a c e r t a i n  optimum was reached f o r each s p e c i e s and f u r t h e r i n c r e a s e would slow down development.  A c c o r d i n g to Rylov  (1948), the  eggs o f Cyclops v i r i d i s develop i n two to three days a t 22 - 23°C b u t a t low temperatures d u r i n g w i n t e r , the process w i l l take ten to f i f t e e n days. s p e c i e s w i l l complete  A t an optimum temperature,  the e n t i r e metamorphosis i n o n l y three  to four weeks; a t low temperatures, the process l a s t s s e v e r a l months.  this  54 As e a r l y as 1929, working w i t h the Daphnia  cladoceran,  l o n g i s p i n a . Brown demonstrated that the length of  generation  from the b e g i n n i n g to the f i r s t young i n s t a r t o  the end o f f i r s t a d u l t i n s t a r was  a f f e c t e d by  temperature.  o o A t 20 C, the time r e q u i r e d i s 187 days; a t 25 C, the p e r i o d is  138 days.  The Q  f o r the range i s 1.31.  1 Q  Oxygen consumption o f the marine copepod,  Calanus  o sp. i s d i r e c t l y p r o p o r t i o n a l to temperature.  A t 5 C, the  oxygen used i n ml/1000 copepods/hour  at 1 0 ° c this  value  i s r a i s e d to 0.38; Briefly,  a t 15°C, i t i s 0.61  the above i n f o r m a t i o n  r a t e f u n c t i o n o f s m a l l crustaceans, temperature  i s 0.26;  (Marshall,  1935).  r e f l e c t s t h a t the  l i k e copepods,  i s also  dependent.  , Coming back to Philonema,  i f the i n t e r v a l of time  from i n f e c t i o n to m o u l t i n g (on a 50% sample b a s i s ) a t one p a r t i c u l a r temperature i s d i v i d e d by t h a t of a temperature For  1.7,  (Table I V ) , we have the f o l l o w i n g v a l u e s : -  the f i r s t moult,  4°c);  3.8, 1.6  higher  4.1  2.1  (15°C v s . 10°C);  (15°C v s . 4 ° C ) .  (15°C v s . 10°C);  2.9,  (2) 2.5  1.6,  1.9  (1)  (10°C v s .  F o r the second moult, (10°C vs. 4°C); 4.1,4.2  (15°C vs. 4 ° C ) . If  i g n o r i n g the s l i g h t d e v i a t i o n s which may be  to the l a c k o f u n i f o r m i t y o f sample s i z e s , these i n d i c a t e t h a t an i n c r e a s e o f 5°C a c c e l e r a t e s  values  the r a t e o f  due  m o u l t i n g almost two times; an i n c r e a s e o f 1 1 C shortens the i n t e r v a l r e q u i r e d by as much as four times.  That i s t o say,  the response to temperature i s the same i n f i r s t and secondstage l a r v a e and the  f o r the r a t e o f moulting f o r  Philonema i s 4. T h i s was confirmed by the experiment o f t r a n s f e r r i n g one d i s h from 1 0 to 1 5 ° C and 4 ° C a f t e r 5 0 % o f the l a r v a e r e c o v e r e d were found t o be m o u l t i n g f i r s t - s t a g e .  The one  t r a n s f e r r e d t o 1 5 ° C was found undergoing the second moult twelve days a f t e r change; the one p l a c e d a t 4 ° C , 4 7 days a f t e r change, f o u r times as long. Normally, Qio  v a l u e s a s s o c i a t e d w i t h thermochemical  r e a c t i o n s range from 2 t o 3 .  T h e r e f o r e , the Qio  obtained i s r e l a t i v e l y high.  (But Vernberg and Vernberg  (1965)  value o f 4  working w i t h the r e d i a e o f the trematode, Himasthla  q u i s s e t e n s i s , demonstrated t h a t the Q-^Q was 9.2 f o r the m e t a b o l i c r a t e o f the warm a c c l i m a t e d forms between 12 and 18°C).  With such a h i g h Q  1Q  v a l u e and the f a c t s t h a t the'  r a t e f u n c t i o n s o f p a r a s i t e s and copepods a r e temperature dependent,  i t may be j u s t i f i e d to argue t h a t the e f f e c t s o f  temperature on the r a t e o f development  o f Philonema i n i t s  i n t e r m e d i a t e host are t w o ^ f o l d , a d i r e c t and an i n d i r e c t Any i n c r e a s e i n ambient  temperature may i n f l u e n c e the  effect.  56 metabolism o f the p a r a s i t e d i r e c t l y .  This change i n  temperature a l s o a c c e l e r a t e s the metabolism o f the crustacean host.  Since many p h y s i o l o g i c a l requirements o f a p a r a s i t e  are e n t i r e l y host dependent, an i n c r e a s e i n metabolism o f the host w i l l i n d i r e c t l y a f f e c t the metabolism o f a p a r a s i t e . Thus, the i n t e r a c t i o n o f the d i r e c t and i n d i r e c t e f f e c t s on the r a t e f u n c t i o n s o f the p a r a s i t e i s r e s p o n s i b l e f o r producing  a high Q Bauer  value.  (1959) concluded t h a t there i s a  c h a r a c t e r i s t i c optimal Species  temperature t y p i c a l f o r a given  species.  o f the same genus, even p a r a s i t i z i n g the same host,  are o f t e n c h a r a c t e r i z e d by d i f f e r e n t temperature optima. Voge and Turner  (1956) found t h a t the r a t e s o f development o f  d i f f e r e n t cestode l a r v a e v a r i e d c o n s i d e r a b l y w i t h i n the same species o f intermediate  host when kept a t the same temperature.  Because the r a t e s o f moulting  as w e l l as the morphology o f  t r o u t and salmon s t r a i n s Philonema l a r v a e a r e a l i k e , i t i s tempting to s p e c u l a t e  t h a t Philonema from t r o u t and salmon  belong to the same s p e c i e s .  However, i t may not be  j u s t i f i a b l e to e s t a b l i s h such an i d e n t i t y merely based on the evidence o f m o r p h o l o g i c a l  resemblance and the s i m i l a r i t y  of the p a t t e r n o f response to temperature between the l a r v a l forms.  57 There i s no e f f e c t o f temperature on the s i z e s o f the l a r v a e .  Larvae r e c o v e r e d from 4°C appear to be l a r g e r than  those from 15°C or 10°C (Figure 16, Table I I I ) .  But two-ways  a n a l y s i s o f v a r i a n c e comparing the. .lengths o f the three :  d i f f e r e n t stages from 15°C, 10°C and 4 ° c  gave  highly  i n s i g n i f i c a n t F v a l u e s a t the 99% confidence i n t e r v a l (Table V I ) .  T h i s i s c o n t r a d i c t o r y t o some o f the o b s e r v a t i o n s  concerning the e f f e c t s o f temperature on the s i z e o f l a r v a l stages which are summarized  i n the p r e v i o u s review.  The  probable e x p l a n a t i o n i s t h a t , as compared to the temperature ranges o f Vogt  (1933) and Gibbs and Gibbs  (1959) ( 8 t o 22°C;  7.5 t o 25°C), the 4 t o 15°C range adopted i n the p r e s e n t experiment i s q u i t e narrow.  A wider temperature range may  have a more c r u c i a l e f f e c t on growth thus r e s u l t i n g i n a prominent change o f s i z e .  C o r r e l a t i o n o f the r a t e s o f development  o f Philonema i n  Cyclops under l a b o r a t o r y c o n d i t i o n s and the i n f e c t i o n o f Sockeye Salmon i n C u l t u s Lake. F i n a l l y , a p p l y i n g the i n f o r m a t i o n o f the r a t e s o f development  o f Philonema i n Cyclops b i c u s p i d a t u s under  l a b o r a t o r y c o n d i t i o n s , an attempt i s made to e l u c i d a t e the i n f e c t i o n o f sockeye salmon i n C u l t u s Lake.  58 Every year i n C u l t u s Lake, spawning of sockeye salmon u s u a l l y s t a r t s i n mid November and l a s t s t i l l December  ( F o e r s t e r , 1925).  mid  A c c o r d i n g to P l a t z e r and Adams  (manuscript i n p r e s s ) , as the f i s h spawns female Philonema worms pass out w i t h the eggs and f i r s t - s t a g e l a r v a e are r e l e a s e d when the females b u r s t i n the l a k e . Cyclops b i c u s p i d a t u s , which a r e c o n c e n t r a t e d a t a depth o f 25 to 30 meters the  ( F o e r s t e r , 1934)  a t t h i s time o f  year, become i n f e c t e d by i n g e s t i n g the l a r v a e .  The  average temperatures a t 25 to 3 0 meters between the b e g i n n i n g of. November and the end of December are c a l c u l a t e d as 6.7°C and 6.3°C (from R i c k e r s 1  between 1934 and 1936). of  (1937) temperature data taken From December onwards, the temperature  the lake g r a d u a l l y drops.  In mid January, the average  temperature a t a l l depths i s 5.8°C. development  I t has been shown t h a t  to i n f e c t i v e t h i r d - s t a g e under  laboratory  c o n d i t i o n s c o u l d be completed one month a f t e r i n f e c t i o n a t 10°C; two and h a l f months a t 4°C.  T h e r e f o r e , w i t h the above  temperature c o n d i t i o n s i n the lake, by mid January, two months a f t e r i n f e c t i o n , there are some l a r v a e ready f o r i n f e c t i n g young f i s h . A c c o r d i n g to Wolf  (1905), Cyclops b i c u s p i d a t u s i s  p o l y c y c l i c i . e . capable o f having more than two g e n e r a t i o n s  d u r i n g the year.  Therefore, i n mid December when the l a s t  b a t c h o f salmon a r r i v e s to spawn, young a d u l t copepods as w e l l as f o u r t h and f i f t h copepodid stages are a v a i l a b l e f o r infection.  Walter  (1922) s t a t e d t h a t a d u l t Cyclops  can l i v e f o r up to 10 t o 14 months.  viridis  He a l s o assumed t h a t  s m a l l e r C y c l o p i d a e l i v e from 4 t o 6 months w h i l e b i c y c l i c s p e c i e s l i v e f o r 10 to 12 months.  Burckhardt  (1900)  suggested t h a t C y c l o p i d a e l i v e from 8 to 14 months.  In the  l a b o r a t o r y , some Cyclops b i c u s p i d a t u s i n f e c t e d w i t h the l a r v a e o f Philonema were maintained f o r more than 8 months at 4 ° c . But one q u e s t i o n i s :  can the i n f e c t e d copepods  s u r v i v e the s e v e r i t y o f w i n t e r when the minima v a r y from 2.6 t o 5.2°C, c o n s i d e r i n g the average o f a l l depths 1937)?  Cyclops b i c u s p i d a t u s i s a P a l e a r c t i c  (Ricker,  eurythermal  copepod which has been c o l l e c t e d from lakes as c o l d as 4°C or  as warm as 27°C (Rylov, 1948).  Moreover, Roy (1932)  r e p o r t e d t h a t these copepods can s u r v i v e over w i n t e r i n the form o f r e s t i n g stage by c o v e r i n g i t s e l f w i t h a s e c r e t i o n produced by the s k i n gland.  A s i m i l a r phenomenon was a l s o  observed by B i r g e and Juday  (19 08) i n the bottom layers, o f  many North American l a k e s .  So, i t i s l i k e l y t h a t some  i n f e c t e d Cyclops can w i t h s t a n d the c o l d temperature  of winter.  In the l i g h t o f t h i s i n f o r m a t i o n ,  together  with  the f a c t t h a t Philonema l a r v a e have no means o f l e a v i n g t h e i r hosts,  i t i s h i g h l y probable t h a t copepodid  stages  and young a d u l t s i n f e c t e d i n mid December c o u l d s u r v i v e as long as f i v e to s i x months t i l l o f the year, offers age  e a r l y May.  the f r y become free-swimming i n the l a k e .  a good o p p o r t u n i t y  time This  f o r Philonema t o i n f e c t a second  c l a s s o f salmon, the f i r s t c l a s s ,  having  During t h i s  l e f t the lake i n A p r i l .  i n f e c t e d i n January,  61 SUMMARY  (1)  Philonema l a r v a e were found to undergo two moults i n Cyclops b i c u s p i d a t u s .  (2)  Three developmental stages are d e s c r i b e d .  Larvae o f  t r o u t and salmon s t r a i n s are m o r p h o l o g i c a l l y identical.  A two-ways a n a l y s i s o f v a r i a n c e gave h i g h l y  i n s i g n i f i c a n t F values when the s i z e s o f the l a r v a l stages o f the two s t r a i n s from copepods maintained a t 10 c were • (3)  compared.  The r a t e s o f development o f Philonema l a r v a e were o d i r e c t l y p r o p o r t i o n a l to temperature between 4 and 15 C.  (4)  At 1 0 ° c ,  the r a t e s o f development were the same i n  salmon and t r o u t (5)  Temperature may  strains. have d i r e c t and i n d i r e c t e f f e c t s on the  r a t e s o f development o f Philonema l a r v a e i n copepods. (6)  Q ^ Q f o r the range 4 to 1 5 ° c was found to be approximately 4.  (7)  Two-ways a n a l y s i s o f v a r i a n c e showed t h a t  (a)  the extent  o f growth i n the three stages from the three temperatures, 15°C, 10°C and 4°C were the same (b)  the three stages  have the same response ( i n t e r a c t i o n ) to temperature as f a r as growth was concerned.  62  (8)  The experimental  data i n d i c a t e t h a t i t i s p o s s i b l e f o r  one age c l a s s o f salmon i n C u l t u s Lake to a c q u i r e i n f e c t i o n s w i t h Philonema a t two d i f f e r e n t p e r i o d s o f t h e i r lake r e s i d e n c e  (May a f t e r h a t c h i n g and January to  A p r i l before migration).  63 BIBLIOGRAPHY Akhmerov, A. 1955. The p a r a s i t e fauna of Kamchatka R i v e r fishes. I z v e s t . Tikhook. No - I . I n s t . Ryon, Khoz. Okeanogr. 43: 99-13 7. Bangham, R.V. 1951. P a r a s i t e s of f i s h i n the Snake R i v e r Drainage and i n Yellowstone Lake, Wyoming. Z o o l o g i c a 36: 213-217. 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Etude sur l e development l a r v a i r e de guelgues especes du genre C y c l o p s . 1. Ann. B i o l . L a c , _12: 272-303. M a r g o l i s , L. 1963. p a r a s i t e s as i n d i c a t o r s o f the g e o g r a p h i c a l o r i g i n o f sockeye salmon, Oncorhynchus nerka (Walbaum) o c c u r r i n g i n the North P a c i f i c Ocean and a d j a c e n t seas. B u l l . 11, I n s t . North pac. F i s h . Comm.  66 M a r s h a l l , S.M. e t . a l . 1935. Oxygen consumption o f the copepod, Calanus. J . Mar. B i o l . Assn. U.K. ^0: 1-27. McCoy, O.R. 1928. L i f e - h i s t o r y s t u d i e s on trematodes from M i s s o u r i . J . P a r a s i t . 14: N.4. Meyer, M.C. 1958. S t u d i e s on Philonema agubernaculum, a d r a c u n c u l o i d nematode i n f e c t i n g salmonids. J . p a r a s i t . 44 (Supple.), 42. 1960. Notes on Philonema agubernaculum and other r e l a t e d d r a c u n c u l o i d s i n f e c t i n g salmonids. Libro Homenajeal D r . 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P a r a s i t . 42_: 363-399. Olson, R.E. 1966. Some experimental f i s h hosts o f the S t r i g i e d trematode Bolbophorus confusus and e f f e c t s of temperature on c e r c a r i a and m e t a c e r c a r i s . J . P a r a s i t . 52: 327-334. Pennak, R.W. 1963. Species i d e n t i f i c a t i o n o f freshwater C y c l o p o i d Copepoda o f U.S. Trans. Am. M i c r o s : Soc. v o l . LXXXII(4): 353-349.  67 P l a t z e r , E.G. and Adams, J.R. The l i f e - h i s t o r y o f D r a c u n c u l o i d , Philonema oncorhynchi, i n an anadromous host, Oncorhynchus nerka. Manuscript i n p r e s s . Poole, J.B. 1956. R e a c t i o n to temperature by i n f e c t i v e l a r v a e of Nematodirus f i l i c o l l i s , T r i c h o s t r o n g y l i d a e (Nematoda). Can. J . Comp. Med.-_20: 169-172. R i c k e r , W.E. 1937. P h y s i c a l and chemical f a c t o r s o f C u l t u s Lake, B r i t i s h Columbia. J . o f B i o l . B u l l . Canada, _3: 362-401. Rylov, V.M. 1948. Fauna of U.S..S.R. C r u s t a c e a . V o l . 3. Freshwater C y c l o p o i d a . (Published f o r N a t i o n a l Science Foundation Washington D.C.and the Smithsonian I n s t , by I s r a e l . ) Rogers, W.P.  Roy,  1962.  The Nature o f P a r a s i t i s m .  Academic P r e s s .  J . 1932. Copepods e t Cladoceres de l ' o u e s t de l a France. 1-224.  Smedley, E.M. 1933. Nematode p a r a s i t e s from Canadian marine •and fresh-water f i s h e s . Contr. Can. B i o l . F i s h . 8: 169-179. Standen, O.D. 1952. E x p e r i m e n t a l i n f e c t i o n of A u s t r a l o r b i s g l a b r a t u s w i t h Schistosoma mansoni. 1 I n d i v i d u a l and mass i n f e c t i o n of s n a i l s and the r e l a t i o n s h i p of i n f e c t i o n to temperature and season. An. Trop. Med. & P a r a s i t . 46: 48-53. Schacher, J.F. 19 57. C o n t r i b u t i o n to the l i f e - h i s t o r y and l a r v a l morphology o f Toxocara c a n i s . J . P a r a s i t . 43: 599-612. Schwartz, B. and A i c a t a , J . 1934. Development o f the human hookworm, Necator americanus, i n guinea p i g s . Am. J . Hyg. 2_9_ 317-328. :  Svensson, R. 1925. A m o r p h o l o g i c a l d i s t i n c t i o n between i n f e c t i v e l a r v a e o f Ancylostoma and Necator, Proc. Soc. Exp. B i o l . Med. 22: 261-262.  68 Thomas, W. 1937- Environmental r e l a t i o n s and l i f e - h i s t o r y o f the tape worm B o t h r i o c e p h a l u s r a r u s . J . P a r a s i t . 23: 133-152. Vernberg,  Vik,  W. and Vernberg, F. 1965. I n t e r r e l a t i o n s h i p s between p a r a s i t e s and t h e i r h o s t s . 1. Comparative metabolic p a t t e r n s o f thermal a c c l i m a t i o n o f l a r v a l trematodes w i t h t h a t o f t h e i r host. Comp. Biochem. P h y s i o l . 14: 557-566.  R. 1964. Notes on the l i f e h i s t o r y o f Philonema agubernaculum Simon e t Simon, 1936 (Nematoda) Can. J . Z o o l . 42: 511-512.  Voge, M. and Turner, A. 1956. E f f e c t o f temperatue on l a r v a l development o f the Cestode, Hymenolepis diminuta. Exp. P a r a s i t . _5: 580-586. Vogel, H. 1934. D i e E n t w i c k l u n s y p l u s von Opisthorchis f e l i n e u s nebst Bemerkungen uber d i e Systemat^,ik und Epidemiolgie. Z o o l o g i c a Bd. 33_, H.86.  ii  Vogt, K. 1938. E x p e r i m e n t e l l e Untersuchungen uber d i e Grunde von Masseninfektionen m i t P l e r o c e r c o i d e n des Fischbandwurms Triaenophorus n o d u l o s i s . Z t s c h r . F i s c h e r e i _36: 93-224. Walter, E. 1922. Uber d i e Lebensdauer der f r e i l e b e d e n II  Susswasser C y c l o p i d e n und andere Fragen i h r e r B i o l o g i e . Z o o l . Jahrb., S y s t . 44: 375-420. Ward and Whipple. 19 59. Freshwater John Wiley and Sons I n c .  B i o l o g y 2nd e d i t i o n .  Watson, N. and P r i c e , J . 1960. Experimental i n f e c t i o n s o f C y c l o p o i d copepods w i t h Triaenophpus crassus F o r e l and T. nodulosus. Can. J . Z o o l . 38: 345-3 56. Weinstein, P. and Haskins, W. 1955. Chemical evidence o f an e x c r e t o r y f u n c t i o n f o r the s o - c a l l e d e x c r e t o r y system of the f i l a r i f o r m l a r v a e o f Nippostrongylus muris. Exp. P a r a s i t . 4: 226-243. Wharton, R.H. 1959. A simple method o f mounting and p r e s e r v i n g f i l a r i a l l a r v a e . B u l l . W.H.O. 20: 729-730.  69 W i l l e y , A.  1923. Notes on the d i s t r i b u t i o n o f f r e e - l i v i n g Copepoda i n Canada waters. Const. Canad. B i o l . Ottawa. _1: 303-334.  Wilson, P.A. 1965. The e f f e c t o f temperature change on the oxygen uptake o f the i n f e c t i v e l a r v a e o f N i p p o s t r o n g y l u s b r a z i l i e n s i s . Exp. P a r a s i t . 17: 318-325. Wolf, E. 1965. Die F o r t p f l a n z u n g s v e r h a l t n i s s e unsurer einheimischen Copeoden. Z o o l . Jahr., Syst. _22: 101-280. Yoshida, Y. 1966. 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J . p a r a s i t . 52: 122-125.  70 LIST OF ABBREVIATIONS USED IN TABLES AND FIGURES  A  - anus  BC  - buccal cavity  DAT  - d i s t a n c e from p o s t e r i o r e x t r e m i t y to anus  EC  - excretory  EP  - e x c r e t o r y pore  EXP  - d i s t a n c e from c e p h a l i c end t o e x c r e t o r y pore  GN  - ganglionic nuclei  GO  - glandular  GP  - g e n i t a l primordium  GPR  - d i s t a n c e from c e p h a l i c end to g e n i t a l primordium  HC  - n u c l e i o f hypodermal  INT  - intestine  LGO  - length of glandular  LGP  - l e n g t h o f g e n i t a l primordium  LI  - length of i n t e s t i n e  LMO  - l e n g t h o f muscular  LR  - l e n g t h o f rectum  MO  - muscular  NINT  - nucleus o f i n t e s t i n e  NR  - nerve r i n g  NRG  - the d i s t a n c e from c e p h a l i c end to middle o f nerve r i n g  ON  - nucleus o f oesophagus  cell  oesophagus  cells  oesophagus  oesophagus  oesophagus  71  OIV  - oesophageal-intestinal valve  R  - rectum  RG  - r e c t a l gland  TL  - t o t a l l e n g t h of body  WBA  - body width a t anus  WBNR  - body width a t nerve r i n g  WIw  - g r e a t e s t width of i n t e s t i n e  Win  - width o f i n t e s t i n e b e f o r e  WR  - width of rectum  joining  rectum  72  TABLE I I .  Stage  F i r s t stage from female worms  F i r s t stage from Cyclops bicuspidatus  P o s i t i o n s of g e n i t a l primordia of larvae  Lengths o f larvae i n ya  Lengths o f g e n i t a l primordia from c e p h a l i c end i n yu  % o f body l e n g t h from cephalic  416.00  190.65  45.8  543.9  221.40  40.7  471.75  217.71  46.1  527.25  209.10  40.0  499.50  196.80  49.6  410.70  215.25  52.4  527. 5  221.55  42. 0  482.85  184.50  39.0  471.75  196.8  41. 7  527.25  209.0  40. 0  588.3  178.35  30.3  541.2  228.78  42.3  549.81  221.40  40.3  567.03  209.10  36.8  547.3 5  196.80  36.0  196.80  35.9  531.2  235.00  44.2  531.2  209.1  39.4  553.5  225.1  40.8  535.05  V  TABLE I I (Continued)  721. 5  196.8  27.3  666.0  215.3  32.3  666. 0  320.8  48.2  565.2  329.1  58.2  527.3  268.1  50.8  721. 5  432.3  59.9  527.3  240  45.5  528. 0  264  50. 0  527. 5  3 05.3  57.8  577.3  297.7  51. 5  699. 0  378.0  54.0  516. 2  323 .3  62.6  616.1  402.3  65. 2  660. 5  364.7  55.2  538.4  311.7  57.8  602.7  370.5  61.5  721.5  274.5  38.1  666. 0  374.9  56.3  699.3  409.62  58. 5  615.0  405.90  65.9  723.24  314.9  43.5  731.85  420.7  57.5  781.1  343.4  44. 0  TABLE I I . (Continued) J  Third  888.0  570.2  64.2  921.3  573.7  62.3  1054.5  645.8  61.1  915.0  652.7  71.3  888.0  428.2  48.2  893.6  639.5  71.5  1010.1  507.9  50.3  860.25  502.5  58.4  959.4  599.0  62.5  1025.8  648.2  63.2  915.6  399.6  43.6  893.5  399.8  44.7  982.4  445.3  45.3  899.1  388.5  43.2  971.25  572.4  58.9  943.5  608.8  64.5  925.75  573.15  61.9  838.05  476.0  55.5  832.5  521.7  62.5  1004.6  470.2  45.5  972.5  632.4  64.9  1043.4  526.5  50.5  953.3  455.1  47.7  1019.67  624.8  61.3  923.73  575.6  62.3  647.1  12.0  1014.2  TABLE I I (Continued  674.3  53.5  924.35  585.75  63.4  869. 2  439.0  50.5  959.4  599.0  62.5  1025.8  648.2  63.2  915.86  399.6  43.6  893. 5  399.8  44.7  982.4  445.3  45.3  899.1  388.5  43.2  1113.8  TABLE I I I . P r o p o r t i o n of l a r v a l stages p r e s e n t a t d i f f e r e n t time i n t e r v a l s . (a)  A t 15°C (salmon s t r a i n )  Time (days a f t e r infection) Total sample size  1-7  8-14  102  168  * Stages  % o f Stages present  * M - Moulting  50-M 1st 52-lst  49. 02-M 1 s t 50. 98-1st  74-M 1st 25-1st 69-2nd  44.05-M 1 s t 14.88-1st 41.07-2nd  15-21  22-28  201  41  14-M 1 s t 4-lst 78-M 2nd 81-2nd 24-3rd  35-63  24  2-M 2nd ll-2nd 28-3rd  24-3rd  6.97-M 1 s t 4.87-M 2nd 1.99-lst 26. 80-2nd 3.8.8-M 2nd 68. 29-3rd 40.29-2nd 11.94-3rd  100-3rd  TABLE III. (Continued)  (b)  The number o f moulting F i r s t - s t a g e a f t e r i n f e c t i o n a t 15°C (salmon  Date when copepods  infected  Date when larvae, examined Days a f t e r i n f e c t i o n when larvae examined No. o f larvae examined Stages  present  l a r v a e r e c o v e r e d 6 days  strain)  10/20/65  11/ 6/65  10/26/65  11/12/65  6  6  17  8  10-M 1 s t 7-lst  3-M 1 s t 5-lst  T o t a l No. of l a r v a e examined = 25 No. o f M o u l t i n g  1st  % of Moulting 1st  = 13 = 52  TABLE I I I (Continued)  (c)  The number of moulting second-stage l a r v a e r e c o v e r e d 17-19 days a f t e r i n f e c t i o n a t 15°C (salmon s t r a i n )  Date when copepods i n f e c t e d  11/ 6/65  10/20/65  11/12/65  11/  6/65  10/24/65  Date when larvae examined  11/24/65  11/ 7/65  11/29/65  11/25/65  11/12/65  Days a f t e r i n f e c t i o n when l a r v a e examined  18  18  17  19  19  No. o f l a r v a e examined  13  6  7  19  32  Stages present  11-M 2nd 2-2nd  6-2nd  2-M 2nd 5-2nd  13-M 2nd 6-2nd  14-M 2nd 18-2nd  T o t a l No. o f l a r v a e examined = 77 No. o f m o u l t i n g 2nd  = 40  %  = 50.6  o f m o u l t i n g 2nd  TABLE I I I (Continued) (d)  A t 10°c (salmon  Time (days after infection) Total sample size  1-7  19  strain)  8-14  102  15-21  29  4-M 1st 15-1st  56-M 1st 46-1st  20-M 1st 9-2nd  21. 05-M 1st 78. 95-1st  54.8-M 1st 45.09-lst  68. 97-M 1st 31. 03-2nd  Stages  % of Stages present  \  22-28  29-35  31  133  36-42  20  43-112  45  l i - M 1st 3-lst 74-M 2nd 43-2nd 2-3rd  1-M 1st 9-M 2nd 10-3rd  4-2nd 41-3rd  16. 13-M 1 s t 8. 27-M 1 s t 3. 23-M 2nd 2. 2 6 - l s t 55. 64-M"2nd 51. 61-2nd 29. 03-3rd 32. 33-2nd 1. 50-3rd  5-M 1st 45-M 2nd 50-3rd  8.9-2nd 91.l-3rd  5-M 1st 16-2nd 1-M 2nd 9-3rd  TABLE I I I (Continued) (e)  The number of moulting F i r s t - s t a g e l a r v a e r e c o v e r e d 13 days a f t e r i n f e c t i o n at.lO°C (salmon s t r a i n )  Date when copepods i n f e c t e d  11/ 6/65  11/12/65  Date when l a r v a e examined  11/19/65  11/25/65  Days a f t e r i n f e c t i o n when l a r v a e examined  13  13  No. o f larvae examined  33  10  Stages present  15-M 1 s t 18-lst  5-M 1st 5-lst  T o t a l No. o f l a r v a e examined = 43 No. o f l a r v a e M o u l t i n g = 23 %  o f M o u l t i n g Stage  = 53.5  TABLE I I I (Continued)  (f)  The number of moulting second-stage r e c o v e r e d 3 0, 31 days a f t e r i n f e c t i o n a t 10°C (salmon s t r a i n )  Date when copepods i n f e c t e d  11/6/65  11/21/65  11/21/65  Date when larvae examined  12/7/65  12/21/65  12/22/65  Days a f t e r i n f e c t i o n when larvae examined No. of l a r v a e examined Stages present  30  30  31  17  14  32  11-M 2nd 6-2nd  6-M 2nd 8-2nd  15-M.2nd 17-2nd  T o t a l No. o f l a r v a e examined = 63 No. o f l a r v a e m o u l t i n g = 32 %  o f l a r v a e m o u l t i n g = 50.  TABLE III(Continued) (g)  A t 4°C (salmon s t r a i n )  Time(days after infection Total sample s i z e Stages % o f Stages •present.. : Time (days after infection Total sample s i z e  Stages  1-14  18 ' A l l 1st  100-lst.  57-63  22  15-21  28  22-28  67  3-M 1 s t 25-lst  39-M 1st 28-1st  10.7-M 1st 89.3-lst  58.3-M 1st 41.8-lst •  64-70  99  2-M 1st 16-M 1st 3-lst 51-2nd 17-2nd 23.1-M 2nd 9-3rd  71-77  52 3-M 1st 22-2nd 27-M 2nd  9.1-M 1st 16.6-M 1st 5.8-M 1 s t % o f Stages 13.6-lst 51.5-2nd 42.3-2nd present 77.3-2nd 23.2-M 2nd 51.9-M 2nd 9.09-3rd  29-35  26 26-M 1 s t  100-M 1 s t .  78-84  .10  36-42  25  43-49  15  25-M 1 s t  15-M 1 s t  100-M 1 s t  100-M 1 s t  85-91  21  50-56  26 10-M 1 s t 16-2nd 38.5-M 1 s t -.61. 5-2nd  92-154  55  10-M 2nd  3-lst 10-M 2nd 8-3rd  55-3rd  100-M 2nd  14.3-lst 47.6-M 2nd 38.l-3rd  100-3rd  CO  TABLE I I I (Continued)  (h)  The number of moulting F i r s t - s t a g e r e c o v e r e d 23-25 days a f t e r i n f e c t i o n a t 4°C (salmon s t r a i n )  Date when copepods i n f e c t e d  11/ 6/65  11/12/65  11/6/65  Date when larvae examined  11/29/65  12/ 6/65  12/1/65  Days a f t e r i n f e c t i o n when larvae examined  23  24  25  No. o f l a r v a e examined  11  17  14  Stages present  5-M 1st 5-lst  10-M 1 s t 7-lst  6-M 1st 8-lst  T o t a l No. o f l a r v a e examined = 42 No. o f l a r v a e m o u l t i n g % of larvae moulting  =21 .= 50  TABLE I I I (Continued)  (i)  The number o f moulting second-stage r e c o v e r e d 74, -78 days a f t e r i n f e c t i o n a t 4°C (salmon s t r a i n )  Date when copepods i n f e c t e d Date when larvae examined  11/ 6/65  11/21/65  11/ 6/65  1/19/66  2/ 3/66  1/23/66  Days a f t e r i n f e c t i o n when larvae examined  74  74  78  No. o f larvae examined  10  23  12  6-M 2nd- 10-M 2nd 13-2nd 4-2nd  Stages present  6-M 2nd 6-2nd  T o t a l No. o f l a r v a e examined = 45 No. o f l a r v a e m o u l t i n g = 22 %  o f l a r v a e m o u l t i n g = 49  TABLE I I I (Continued) (j)  A t 10°c (Trout s t r a i n )  Time, (days after infection) T o t a l sample size  Stages  % o f stages present  .*  1-14  15-21  22-28  90  103  61  15-M 1st 75-1st  21-M 1st 6-lst 76-2nd  16.67-M 1 s t . 20.4-M 1st 83.3-lst 5.8-lst 73.7-2nd  29-35  80  36-42  43-49  111  26  13-M 1st 31-2nd 16-M 2nd l-3rd  52-M 2nd 25-2nd 4-3rd  19-M 2nd 4-2nd 88-3rd  3-M 2nd 23-3rd  21.3-M 1st 50.8-2nd 26.2-M 2nd 2.7-3rd  65-M 2nd 31.3-2nd 5-3rd  17-M 2nd 3.6-2nd 79.3-3rd  11.5-M 2nd 88.5-3rd  * due to the l a c k o f specimen, i t was i m p o s s i b l e t o f o l l o w the l a r v a l stages up to 112 days a f t e r i n f e c t i o n as i n the case of the salmon s t r a i n .  oo  TABLE I I I (Continued)  (k)  The number of moulting F i r s t - s t a g e r e c o v e r e d 12,15 days o , .. a f t e r i n f e c t i o n a t 10 C (trout s t r a i n )  5/29/65  5/29/65  6/11/65  6/14/65  Days a f t e r i n f e c t i o n when larvae examined  12  12  No. o f larvae  30  30  Date when copepods Date when larvae  infected  examined  examined  Stages present  15-M 1st 15-lst  17-M 1 s t 13-lst  T o t a l No. of larvae examined = 60 No. o f larvae moulting = 32 % o f larvae m o u l t i n g = 53.3  TABLE I I I (Continued)  (1)  The number of moulting second-stage r e c o v e r e d 3 0-34 days a f t e r i n f e c t i o n a t 10°C (trout s t r a i n )  Date when copepods i n f e c t e d  5/29/65  5/29/65  5/29/65  5/29/65  Date when larvae  6/29/65  7/ 1/65  7/ 2/65  6/28/65  examined  Days a f t e r i n f e c t i o n when larvae examined No. o f larvae examined Stages present  31  33  34  30  8  9  28  5  5-M 1st 4-lst  4-M 1st 4-lst  15-M 1 s t 13-lst  T o t a l No. o f  l a r v a e examined = 50  No. o f  l a r v a e m o u l t i n g = 26  %  o f l a r v a e m o u l t i n g = 52  2- M Is 3- l s t  TABLE V.  Temperature  Stage First  o 15 C  The s i z e s o f l a r v a e c o l l e c t e d a t d i f f e r e n t time i n t e r v a l s Sample  size  Mean l e n g t h  in  Z  1  Range i n /a  Time (days a f t e r infection)  576.3  555  580. 5  499.5  7  570.6  492  7  565.5  532.8-582.8  14  6  650.4  621.6-693.8  16  7  719.1  721.5-738.2  18  7  853.9  832.5-904.7  19  12  967.1  899.1-1061.1  22  8  975.3  932.4-1032.3  61  5  436.6  421.4-452.1  5  7  483.3  402.9-541.2  8  3  446.3  412.7-547.4  10  655.8  405.2-743.7  14 & 19  12  -593.9 8 & 9 -666  12  Second  Third  Moulting 1 st  Moulting 2 nd  TABLE V Temperature  Stage  10°C  Sample size 14  (Continued) Mean l e n g t h i n yu  Range i n yu  Time (days a f t e r infection)  542.7  501.9-588.3  545.0  504.3-574.4  11  10  555.5  473.6-660.4  16  8  590.8  544.9-721. 5  21 & 23  7  614.1  575.6-643.9  26  9  685.2  625.4-754.8  5  741.7  538.74-809.34  34  7  913.3  860.3-999.0  34  6  972.2  943.5-993.5  46  7  976.9  888  -1037.9  60  5  1009.0  993.5-1043.4  108  439.2  300.1-527.3  13  5  592.0  333.0-732.6  25  4  675.0  638.3-704.9  30  First  Second 30 & 31  Third  Moulting 1st Moulting 2nd  TABLE V (Continued) Temperature  Stage  4°C First  Second  Third  Sample size  Moulting 2nd  Range i n /a  Time (days a f t e i infection)  10  543.4  516.6-608.9  12  5  522.7  483.4-544.9  16  4  563.9  537.5-605.2  23  10  675.9  589.2-788.1  55 & 53  7  714.6  627.3-794.6  58 & 60  .9  721.3  572.7-794.6  69  15  896.7  725.7-1021.2  84  11  886.0  788.1-964.3  7  1005.7  932.3-1086.1  96  5  991.7  923.7-1004.9  13 2  1008.8  . 959.4-1029.3  160  -5 • Moulting 1st  Mean l e n g t h i n /a  87 & 89  5  465.2  413.3-595.3  25  •6  403 .1  335.8-451.4  39  4  688.2  602.7-830.3  74  5  820.6  761.4-915  82 & 81  . 91 TABLE VI.  (a)  Two-ways a n a l y s i s o f variance on the e f f e c t of temperature on the growth o f l a r v a e (salmon s t r a i n ) .  Sizes of larvae  at  o 15 C. Stages  First  Second  Third  593.85  528.90  860.25  611.50  527.25  838.05  582.75  532.80  843.60  582.75  584.25  860.25  555.00  611.50  832.50  555.00  666.00  838.05  582.75  638.25  925.78  555.00  621.60.  954.60  582.75  666.00  1004.55  555.00  572.65  960.15  582.75  732.60  960.15  638.25  732.60  899.10  555.00  721.50  899.10  611.50  738.15  1061.05  627.15  721.50  1015.65  499.50  666.00  1026.75  572.65  593.85  987.90  . 582.75  666.00  1032.30  584.25  560. 55  971.25  582.75  721.50  949.05  11592.90  12803.20  16806.18  TABLE VI (Continued)  S i z e s o f l a r v a e a t 10°C Stage Second  Third  588.3  551.04  832.50  541.2  547.3 5  860.25  512.9  544.89  860.25  549.81  552.27  838.05  526.44  575.64  888.00  536.28  578.69  976.80  501.84  643.88  965.70  567.03  666.00  971.25  549.81  611.31 .  982.35  533.82  621.74  987.90  547.35  691.85  971.25  .533.82  651.26  999.00  522.75  809.34  1010.01  585.48  781.05  1026.75  535.05  785.97  1010.10  562.11  538.74  103 7.85  531.20  793.50  1010.01  531.20  668.40 ..  999.00  553.50  625.43  915.00  574.41  669.71  949.05  10884.30  12878.06  17043.12  First  TABLE VI  c)  (Continued)  S i z e s o f l a r v a e a t 4°C  Stages First  Second  Third  520.29  510.45  882.45  530.13  622.38  865.80  608.85  589.17  876.90  516.60  634.68  854.70  552.29  567.03  876.90  523.98  664.20  870.84  544.89  651.90  940.95  483.39  664.20  970.47  571.95  699.30  953.25  537.51  666.00  908.97  541.04  627.30  964.30  605.16  645.75  976.60  569.49  694.95  918.87  553.50  725.70  1025.82  551.04  768.75  1019.67  516.60  745.58  1029.33  541.20  762.60  1004.91  542.43  794.51  1009.83  530.73  753.99  974.16  544.89  778.59  980.31  10885.94  13567.03  18904.65  TABLE VI (Continued)  (d)  C e l l sum t a b l e  Stages First Temp I  (e)  Third  11592.90  12803.20  16806.18  41202. 28  1 Q  10884.30  12878.06  17043.12  40805. 48  lll^  10885.94  13567.03  18904.65  43357. 62  33363.14  39248.29  52753.95  125365. 38  15 TempiI Temp  Second  n  Analysis  of variance  Source o f v a r i a t i o n I n s t a r SS  table  SS  df  MS  3294507.48  2  1647253.74  72894.38  2  36447.19  93466.1  2x2  23366.52  Error  8517262.8  171  49808.5  Total  11978130.8  179  Temp SS I n t e r a c t i o n SS  F  temp =  MS  36447.19 = 0.73 49808.50  temp = EMS  A t 0.01 l e v e l , f o r 2 and <* df, t a b u l a t e d F = 2.35, Therefore,  temperature has no s i g n i f i c a n t e f f e c t on the  e x t e n t o f growth o f the d i f f e r e n t  F interaction  =  I EMS  l a r v a l stages.  = 23366.52 = 0.47 49808.50  A t 0.01 l e v e l , f o r 4 and  d f , t a b u l a t e d F = 1.99  Therefore,  between temperatures and l a r v a l  the i n t e r a c t i o n  stages are h i g h l y i n s i g n i f i c a n t i . e . the response t o temperature i s the same i n the d i f f e r e n t  stages.  96 TABLE V I I .  (a)  Two-ways a n a l y s i s o f v a r i a n c e on the s i z e s o f salmon and t r o u t s t r a i n s Philonema l a r v a from Cyclops m a i n t a i n e d i n 10°C. S i z e s o f l a r v a e ( t r o u t s t r a i n ) from the copepods m a i n t a i n e d i n 10°C. Stages  First  Second  Third  489.50  602.7  865.00  489.50  666. 0  860.25  603.93  693.75  832.50  585.48  699.3 0  888.00  571.95  615.00  860.25  606.39  688.80  999.00  516.60  567.03  915.75  505.05  569.49  971.25  527.25  560. 55  943.50  582.75  607.62  921.30  492.00  754.80  999.00  533.82  721.50  943.50  582.75  701.10  1010.10  555.00  723.24  1054.50  611.50  731.85  1110.00  473.55  738.00  1054.50  582.75  544.89  1054.50  571.95  582.75  949.05  605.16  560. 55  943.50  585.48  606.39  888.00  11072.36  12935.31  19063.45  97 TABLE VII. (Continued)  (b)  C e l l sum t a b l e Stages First  Second  Third  Salmon  10884.30  12878.06  17043 . 12  40805. 48  Trout  11072.36  12935.31  19063 .45  43071. 12  219 56.66  25813.37  36106 . 57  83876. 60  (c)  Analysis  of variance  Source o f v a r i a t i o n I n s t a r SS Location  SS  I n t e r a c t i o n SS  table  SS 2675367.85  df 2  MS 1337683.9  26109.4  1  26109.40  66900.8  2x1  33450.40 34644.95  Error  3949525.15  114  Total  6727902.34  119  •c i  MS l o c a t i o n  F location = EMS  26109.4  n  ^  - — — „ „ ^ •- 0.75 34644.95  A t 0.01 l e v e l , f o r 1 and 114 d f , t a b u l a t e d F = 2.76  T h e r e f o r e , there i s no s i g n i f i c a n t d i f f e r e n c e between the l a r v a l s i z e s o f the two s t r a i n s o f Philonema.  F interaction = EMS  At  = 33450.40 = 0.96 34644.95  0.01 l e v e l , f o r 2 and 114 df, t a b u l a t e d F= 2.36  Therefore, the c a l c u l a t e d F value f o r i n t e r a c t i o n i s highly insignificant.  

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