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Effects of parasitic copepod, Salmincola californiensis (Dana, 1852) on juvenile sockeye salmon, Oncorhynchus… Pawaputanon, Kamonporn 1980

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EFFECTS OF PARASITIC COPEPOD, Salmincola  californiensis  (Dana^1852)  ON JUVENILE SOCKEYE SALMON, Onoovhynchus  nerka  (Walbaum).  by KAMONPORN PAWAPUTANON B.Sc, K a s e t s a r t U n i v e r s i t y , T h a i l a n d , 1964 M . S c , Auburn U n i v e r s i t y , Auburn, Alabama, 1972  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  i n 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  A p r i l , 1980.  © Kamonporn Pawaputanon, 1980  E-6  In presenting t h i s thesis in p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the Library 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 study. I further agree that permission f o r extensive copying of t h i s thesis f o r s c h o l a r l y purposes may be granted by the Head of my Department or by his representatives.  It i s understood that copying or p u b l i c a t i o n  of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission.  Department of  ZOT>\Q  The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5  Date  BP  75-51 1 E  ABSTRACT  Sockeye salmon p a r a s i t i z e d w i t h Salmincola  calif'ovniensis"were  compared e x p e r i m e n t a l l y w i t h u n p a r a s i t i z e d f i s h of the same age  to d e t e r -  mine: (1) i t s e f f e c t s on growth; (2) e f f e c t s on p a r a s i t i z e d f i s h under some environmental its  s t r e s s e s and  f i s h h o s t . I t was  found  s i t e s p e r f i s h can reduce a p e r i o d o f 112 DPI. group was  (3) h e m a t o l o g i c a l e f f e c t s of t h i s p a r a s i t e on t h a t average  the weight  i n f e c t i o n l e v e l s of 31.25  of the f i s h h o s t by almost  para-  34 % w i t h i n  The r a t e o f i n c r e a s e i n l e n g t h of the i n f e c t e d  slower than t h a t o f the n o n - i n f e c t e d f i s h group though no  t i c a l l y s i g n i f i c a n t d i f f e r e n c e developed  fish statis-  d u r i n g the e x p e r i m e n t a l p e r i o d .  The p a r a s i t i z e d f i s h were found to develop anemia, e x p r e s s e d by r e d u c t i o n i n r e d c e l l c o u n t s , hemoglobin c o n c e n t r a t i o n s and values.  the  hematocrit  T h i s anemic c o n d i t i o n i s a t t r i b u t e d to h e m o d i l u t i o n o f the b l o o d ,  r e s u l t i n g from damage to the g i l l and s k i n e p i t h e l i a , and, to an osmotic imbalance  i n turn, leading  between the water and t h e i n t e r n a l f l u i d s .  addition,  the p r o g r e s s i v e r e d u c t i o n o f the r e d c e l l s  b l o o d may  be a r e s u l t of the a b s o r p t i o n of p a r a s i t e m e t a b o l i c s e c r e t i o n s  through t h e g i l l s o r t h e b u l l a .  i n the  In  circulating  Such a b s o r p t i o n seems l i k e l y because o f  the observed v a r i a t i o n s o f the c e l l s  i n t h e l e u c o c y t i c system and  the  s i g n i f i c a n t i n c r e a s e i n lymphocytes,  n e u t r o p h i l s and " g r a n u l o c y t e  cells"  i n r e l a t i o n to i n f e c t i o n time.  Furthermore,  the b l o o d of the  f i s h c l o t t e d f a s t e r than t h a t of the n o n - i n f e c t e d f i s h . of  i n f e c t i o n a marked i n c r e a s e was  a l s o observed  infected  During  the c o u r s e  i n the number o f  thrombocytes. P a r a s i t i z e d f i s h were l e s s a b l e to cope w i t h environmental A water temperature  stresses.  of 21°C was found to be the median l e t h a l temperature o  i n f e c t e d f i s h . The swimming a b i l i t y of i n f e c t e d f i s h was a l s o  reduced.  The p a r a s i t i z e d f i s h reached 50% f a t i g u e when they swam i n water of a v e l o c i t y of 65 cm/sec f o r o n l y 250 n i n .  The chance of s u r v i v a l f o r the  i n f e c t e d f i s h i n t h i s h i g h water v e l o c i t y i s only 6.6% over the p e r i o d of  600 min.  The a b i l i t y o f the i n f e c t e d f i s h to t r a n s f e r from f r e s h water  to  s a l t water was a l s o a f f e c t e d .  M o r t a l i t y of the i n f e c t e d f i s h i n c r e a s e d  d u r i n g t h i s t r a n s i t i o n and these f i s h , as i n d i c a t e d by the s a l i n i t y ence t e s t , a l s o avoided high s a l i n i t y , been ready to m i g r a t e .  prefer-  suggesting that they may n o t have  The c r i t i c a l p e r i o d o f i n f e c t i o n where marked  d i f f e r e n c e s were found i n a l l the parameters  was that p e r i o d when the  p a r a s i t e s reached maximum s i z e and a second i n f e c t i o n took p l a c e w i t h copepodids hatched from the o r i g i n a l  group.  TABLE OF CONTENTS  ABSTRACT  i i  LIST OF TABLES LIST OF FIGURES LIST OF PLATES GENERAL INTRODUCTION  1  GENERAL MATERIALS AND METHODS  5  1.  Experimental F i s h  6  2. P a r a s i t e Source 3. I n f e c t i o n Techniques 3.1. P a r a s i t e Numbers 3.2. Parasite-Days SECTION I  • • • • •  IMPACT ON. GROWTH AND WEIGHT  1. I n t r o d u c t i o n 2. M a t e r i a l s and Methods 2.1. Experimental Design 2.2. Experimental F i s h 2.3. Growth Study 2.4. Data A n a l y s i s 3. R e s u l t s 4. D i s c u s s i o n SECTION I I  1 . Introduction 2.. M a t e r i a l s and Methods 2.1. Experimental F i s h 2.2. Experimental Procedure 3. R e s u l t s 4. D i s c u s s i o n SECTION I I I 1. 2.  14 •  IMPACT ON HOST'S TEMPERATURE TOLERANCE  6 6 7 10  15 17 17 17 18 18 19 22 29 30 31 31 31 32 40  IMPACT ON HOST'S SWIMMING ABILITY  Introduction M a t e r i a l s and Methods  44 ^5 46  iv  2.1. Experimental- F i s h 2.2. Experimental Procedures 2.2.1. C r i t i c a l V e l o c i t y 2.2.2. F i x e d V e l o c i t y 3. R e s u l t s 4. D i s c u s s i o n SECTION IV  46 47 47 48 49 58  IMPACT ON HOST'S SALINITY TOLERANCE  1. I n t r o d u c t i o n '2. M a t e r i a l s and Methods 2.1. Experimental F i s h 2.2. Experimental Procedures 2.2.1. S a l i n i t y Tolerance Test 2.2.2. S a l i n i t y P r e f e r e n c e Test 3. R e s u l t s 3.1. S a l i n i t y Tolerance Test 3.2. S a l i n i t y Preference Test 4. D i s c u s s i o n SECTION V  IMPACT ON BLOOD  63 64 66 66 66 67 67 68 68 69 74 78  1 . Introduction 2. M a t e r i a l s and Methods 2.1. Experimental Design 2.2. Experimental F i s h 2.3. Blood Sampling Procedure 2.4. S t a i n i n g Technique 2.5. Hematological Determinations 2.5.1. Hemoglobin C o n c e n t r a t i o n 2.5.2. Hematocrit Value 2.5.3. E r y t h r o c y t e Osmotic F r a g i l i t y Test 2.5.4. C l o t t i n g Time 2.5.5. T o t a l Blood C e l l Counts 2.5.6. Red Blood C e l l Count 2.5.7. Red C e l l Corpuscular Value 2.5.8. D i f f e r e n t i a l C e l l Count 2.5.9. S t a t i s t i c a l A n a l y s i s of Data 3. R e s u l t s 3.1. Hemoglobin C o n c e n t r a t i o n 3.2. Hematocrit Value 3.4. C l o t t i n g Time 3.5. T o t a l Blood C e l l Count 3.6. Red Blood C e l l Count 3.7. Red Blood Corpuscular Values 3.8. White Blood C e l l Counts 3.9. D i f f e r e n t i a t i o n of Blood C e l l s 3.9.1. D i f f e r e n t i a l C e l l D e s c r i p t i o n s 3.9.1.1. E r y t h r o c y t i c S e r i e s 3.9.1.2. L e u c o c y t i c S e r i e s 3.9.1.3. Thrombocytic S e r i e s 3.9.2. D i f f e r e n t i a l C e l l Counts 3.9.2.1. E r y t h r o c y t i c S e r i e s  v  79 81 81 81 81 82 85 85 86 86 88 88 89 90 90 91 91 91 95 95 105 105 111 111 111 111 111 120 122 122 122  3.9.2.2. L e u c o c y t i c S e r i e s 3.9.2.3. Thrombocytic S e r i e s 4. D i s c u s s i o n  123 144 153  GENERAL DISCUSSION  161  REFERENCES  1  7  vi  0  LIST OF TABLES  Table  Page  I  Lengths, weights  II  V a r i a t i o n of d i s s o l v e d 0  III  Percent m o r t a l i t y and cumulative m o r t a l i t y  39  IV  R e l a t i o n of swimming speed  50  V  Mean f a t i g u e time, percent f i s h fish  and c o e f f i c i e n t of c o n d i t i o n 2  factors  25  and pH l e v e l s  33  to the percent f a t i g u e d f a t i g u e d and  s i z e of the  host  55  VI  V a r i a t i o n i n the number of p a r a s i t e s  59  VII  Percent cumulative m o r t a l i t y  72  VIII  S a l i n i t y preference test  73 .  IX  Hemoglobin c o n c e n t r a t i o n g / d l  94  X  Hematocrit  98  XI  Percent NaCl and percent hemolysis  XII  Percent blood c l o t t e d  XIII  T o t a l blood c e l l ,  XIV  C a l c u l a t e d MCHC, MCV  XV  Ranges, means and S.D.  XVI  Percentage  XVII  Mean and  value  (%) i n osmotic  test  101 104  red blood c e l l  and white blood c e l l  and MCH  counts  106 112  of l e u c o c y t i c c e l l s  and number of-immature red c e l l s  S.D.  fragility  of l e u c o c y t i c c e l l  vii  counts  119 138 141  LIST OF FIGURES  Figure 1  Page The r e l a t i o n s h i p s of p a r a s i t e numbers, p a r a s i t e - d a y s and time  9  2  Life  c y c l e of Salminoota  3  Length and weight i n r e l a t i o n to time post i n f e c t i o n  21  4  Condition factor  24  5  pH i n experimental a q u a r i a  36  6  Dissolved 0  36  7  Percent m o r t a l i t y and percent cumulative m o r t a l i t y of  2  californiensis  12  (K) d u r i n g 112 DPI  in.experimental aquaria  experimental f i s h  38  8  C r i t i c a l v e l o c i t y of experimental f i s h  52  9  R e l a t i o n s h i p s between f a t i g u e times and % f i s h f a t i g u e d ....  54  10  R e l a t i o n s h i p s between f a t i g u e time and p a r a s i t e days  56  11  Percent cumulative m o r t a l i t y i n r e l a t i o n to s a l i n i t y  71  12  Blood Sampling  84  13  R e l a t i o n s h i p s between hemoglobin and DPI  93  14  R e l a t i o n s h i p s between hematocrit value and DPI  97  15  E r y t h r o c y t e osmotic  16  Blood C l o t t i n g Time  17  R e l a t i o n s h i p s between red blood c e l l counts and DPI  108  18  Regression c o e f f i c i e n t s of red blood c e l l counts  110  19  Mean c o r p u s c u l a r volume (MCV)  114  Technique  f r a g i l i t y of experimental  fish  ,  viii  100 102  Figure  Page  20  Mean c o r p u s c u l a r hemoglobin (MCH)  114  21  Mean c o r p u s c u l a r hemoglobin c o n c e n t r a t i o n (MCHC)  115  22  Regression c o e f f i c i e n t s of white b l o o d c e l l counts  118  23  Regression c o e f f i c i e n t s of immature red c e l l counts  140  24  Regression c o e f f i c i e n t s of l e u c o c y t i c  143  25  Regression c o e f f i c i e n t s of lymphocyte counts  146  26  Regression c o e f f i c i e n t s of n e u t r o p h i l counts  148  27  Regression c o e f f i c i e n t s of counts of "granulocyte c e l l s "  28  Regression c o e f f i c i e n t s of thrombocyte counts  152  29  Host-parasite-environment  167  c e l l counts  relationships  ix  .... 150  LIST OF PLATES  Plate  Page  I  Immature and mature red c e l l s  125  II  Mature r e d c e l l s and lymphocytes  127  III  Small lymphocyte, l a r g e lymphocyte, monocyte, and n e u t r o p h i l s  macrophage 129  IV  Neutrophils  131  V  "Granulocyte c e l l s "  133  VI  Immature and mature  VII  Mature thrombocytes  thrombocytes  135 137  x  ACKNOWLEDGEMENTS  I would l i k e to express my s i n c e r e g r a t i t u d e to my  supervisor,  Dr. J.R. Adams, who guided the r e s e a r c h , p a t i e n t l y e d i t e d the manuscript and provided  i n s p i r a t i o n , encouragement and moral support.  Without h i s  enthusiasm t h i s study would not have been p o s s i b l e . I am a l s o indebted D.J.  Randall who served  to Drs. A.B. Acton, Z. Kabata, L. M a r g o l i s and  on my t h e s i s committee.  f i t e d from t h e i r c r i t i c i s m s .  In p a r t i c u l a r , I am g r a t e f u l f o r the  encouragement and moral support I would a l s o l i k e  The t h e s i s g r e a t l y bene-  of Drs.  Z. Kabata and L. M a r g o l i s .  to acknowledge the many people at the P a c i f i c  B i o l o g i c a l S t a t i o n and P a c i f i c Environmental I n s t i t u t e who s u p p l i e d the f i s h and f a c i l i t i e s  f o r the r e s e a r c h .  I appreciated  their  invaluable  a s s i s t a n c e and I am extremely g r a t e f u l f o r t h e i r f r i e n d s h i p . I have been very f o r t u n a t e  to have continued  a s s i s t a n c e from my  c o l l e a g u e s , Mr. L.R. R u s s e l l and Mr. G.S. Norman. Appreciation preparing  i s a l s o extended to Mr. J . J . K e n d a l l f o r h i s help i n  t h i s manuscript.  F i n a n c i a l support Development Agency.  was provided  by the Canadian I n t e r n a t i o n a l  1  In n a t u r a l water, sockeye salmon are found to c a r r y p a r a s i t e s of many k i n d s : Protozoa, Myxosporida, Monogenea, Trematoda, Cestoidea, Acanthocephala, H i r u d i n o i d e a , A c a r i n a and Copepoda (Margolis and A r t h u r , 1979).  Among the p a r a s i t i c copepods, of which there are more than  1,000  species known from f i s h e s throughout the world (Kabata, 1970), at l e a s t eight species of those copepods have been found on sockeye salmon. ( Margol i s and A r t h u r , 1979). Kabata (1969), r e v i s i n g the genus Salmincola,  noted that P a c i f i c  salmon are p a r a s i t i z e d by only one species of t h i s genus, 5. (Dana, 1852).  californiensis  Kabata and Cousens (1977), who studied the h o s t - p a r a s i t e  r e l a t i o n s h i p between sockeye salmon and t h i s species of copepod, demons t r a t e d the i n j u r i o u s e f f e c t s r e s u l t i n g from the presence and a c t i v i t y of  this parasite.  They noted that the s e v e r i t y of i t s e f f e c t s depends  upon the i n f e c t i o n s i t e .  In j u v e n i l e sockeye salmon, the m a j o r i t y of the  p a r a s i t e s are found i n the b r a n c h i a l c a v i t y , mainly on the b r a n c h i a l r i m w i t h some on the inner w a l l of the operculum.  A few are attached to the  g i l l f i l a m e n t s and almost a l l of these are immature.  The g i l l s are found  to have the strongest t i s s u e response to t h i s p a r a s i t e . of  the g i l l e p i t h e l i u m leads to p a r t i a l or complete f u s i o n of adjacent  filaments. for  The p r o l i f e r a t i o n  E p i t h e l i a l c e l l s become t h i c k e r , rendering them n o n - f u n c t i o n a l  gas and ion exchange.  The e f f e c t i s not r e s t r i c t e d to the e p i t h e l i a l  c e l l s but can extend to the hard s k e l e t a l t i s s u e s of the f i s h host.  The  bone may become translucent and seemingly c r y s t a l l i n e , l o s i n g i t s l a m e l l a r structure.  E v e n t u a l l y , the s t r u c t u r e of the bone i s destroyed.  Normally  2  the e f f e c t s of t h i s p a r a s i t e are s u b l e t h a l .  Nevertheless, i t may be expected  that t h i s p a r a s i t e i s r e s p o n s i b l e f o r more than simple mechanical damage. Kabata (1970) reviewed the consequences of p a r a s i t i c C r u s t a c e a on f i s h e s f i n d i n g that they have c e r t a i n g e n e r a l i z e d e f f e c t s on the host. These i n c l u d e : e f f e c t s on weight and f a t content, growth, metabolism, a l t e r a t i o n s i n the blood c h a r a c t e r i s t i c s , reproduction and behavior.  The  presence of t h i s p a r a s i t e can a l s o lead to secondary i n f e c t i o n and d e l e t e r i o u s e f f e c t s on the f i s h e r y .  j  The l i t e r a t u r e contains several references to the l o s s of weight and growth brought about by copepod i n f e c t i o n .  They create the impression  that almost a l l copepod p a r a s i t e s when present i n s u f f i c i e n t numbers can produce these losses (Mann, 1953; Kabata, 1958; and Reichenbach-Klinke et a l . , 1968).  Kabata (1958) a l s o found that the f a t content of the l i v e r  of haddock, severely p a r a s i t i z e d w i t h Lernaeoceva, 50%.  dropped by approximately  Mann (1953) concluded from h i s observations that the p a r a s i t e exerts  more influence over the weight of the f i s h than over i t s growth i n length. During the course of i n f e c t i o n , t o x i c secretions from the p a r a s i t e can exert a great deal of e f f e c t on the f i s h host. disturbances are always found. Argulus  Among them, metabolic  K o l l a t s c h (1959) has shown that a s i n g l e  l a r v a i s capable of k i l l i n g Hyphessobryoon  to four days.  Considering the e f f e c t  flammeus  w i t h i n three  of p a r a s i t e s on blood c h a r a c t e r -  i s t i c s , a blood-feeding p a r a s i t e can be expected to exert a s i g n i f i c a n t influence on the composition and volume of the blood.  However, i t has  been shown that non-blood feeding p a r a s i t e s can a l s o produce d e f i n i t e changes i n the blood of t h e i r hosts, f o r example, f i s h s u f f e r i n g from a severe i n f e c t i o n w i t h Caligus 1955).  Furthermore, Levnaea  have e x h i b i t e d signs of anemia (Goregyad, cypvlnacea.  which attaches to the f l a n k of  3 f i s h , causes a p o s i t i v e l y increase i n the number of both monocytes and polymorphonuclear granulocytes  (Bauer, 1959).  P e r s i s t e n t l o s s of blood,  r e s u l t i n g from the p a r a s i t i c i n f e c t i o n , has been found t o a f f e c t the reproductive capacity of the f i s h host (Kabata, 1958).  Behavioral  abnormalities  were a l s o observed i n many cases as i n f i s h e s i n f e c t e d w i t h AvguVus ( K o l l a t s c h , 1959).  The i n t r o d u c t i o n of secondary i n f e c t i o n as an a f t e r -  math to the presence of crustacean p a r a s i t e s has been widely accepted and i s o f t e n r e f e r r e d to i n the l i t e r a t u r e ( N i g r e l l i , 1950; Schaperclaus, 1954). F i n a l l y , these p a r a s i t e s induce negative repercussions ery.  Heavy i n f e c t i o n by Levnaea  cypvinacea  k i l l e d 18 tons of carp i n a  s i n g l e pond i n Ohio w i t h i n two weeks (Tidd, 1933).  Savage (1935) a l s o  reported that severe i n f e c t i o n of speckled t r o u t (Salvelinus w i t h Salmincola  edwardsi  and the United States.  i n the f i s h -  frontinalis)  r e s u l t e d i n t h e i r death i n hatcheries i n Canada A f t e r c a r e f u l c o n s i d e r a t i o n of a number of e a r l i e r  works,however, i t i s obvious that there have been many d i f f i c u l t i e s i n accurately assessing the general e f f e c t s of the p a r a s i t e s on t h e i r hosts. The main d i f f i c u l t y l i e s i n our l a c k of knowledge regarding the normal c o n d i t i o n of f i s h .  A l s o , most i n v e s t i g a t o r s determined the e f f e c t s of  the p a r a s i t e from a small sample s i z e , which was p a r t i c u l a r l y true i n case of f i e l d s t u d i e s .  I n many instances an i n v e s t i g a t i o n of the e f f e c t s of a  s i n g l e p a r a s i t e species on the host was undertaken without f i r s t performing an examination t o determine i f other species were a l s o present on the host.  These oversights p o s s i b l y l e d t o the c o n t r a d i c t o r y information usu-  a l l y obtained.  For example, Kabata (1958) i s convinced that  Lernaeocera  causes d e f i n i t e and considerable l o s s of weight i n i n f e c t e d f i s h ; however, Sherman and Wise (1961) found no such e f f e c t on the weight of Gadus morhua. Contradictory f i n d i n g s are very common i n hematological  s t u d i e s ; Layman  4 (1957) mentions an i n c r e a s e i n the number of n e u t r o p h i l s i n the blood of the tench lus  {Tinea  sieboldi,  tinea)  and  the dace (Leuciscus  but Reichenback-Klinke  leueisaus)  i n f e c t e d with  et a l . (1968) d i d not f i n d any  ence between the blood c h a r a c t e r i s t i c s of p a r a s i t e f r e e Coregonus and C. fera  and the blood of f i s h c a r r y i n g l a r g e numbers of  In l i g h t of p r e v i o u s r e s e a r c h , does Salmineola, to an area of v i t a l  importance  host?  t h e r e f o r e , designed  A study was,  aola-sockeye ability  differlavaretus  Ergasilus.  which a t t a c h e s  itself  have any g e n e r a l i z e d n e g a t i v e e f f e c t s on i t s to c l a r i f y  some aspects of the  salmon h o s t - p a r a s i t e r e l a t i o n s h i p by comparing  to t o l e r a t e changes i n temperature  formance and  Ergasi-  and  salinity,  (1) growth,  Salmin(2)  (3) swimming p e r -  (4) h e m a t o l o g i c a l c h a r a c t e r i s t i c s of i n f e c t e d versus  non-  i n f e c t e d f i s h under c o n t r o l l e d c o n d i t i o n s . These parameters were s e l e c t e d f o r the f o l l o w i n g reasons: growth i s one of the f a c t o r s f r e q u e n t l y used s i t e ' s e f f e c t on the host  (Schaperclaus, 1954)  i s t i c s have been used as e x c e l l e n t many f i e l d s of study Cardwell and  Smith,  (Cope, 1961; 1971).  to determine  the f i s h e s ' a b i l i t y  and h e m a t o l o g i c a l c h a r a c t e r -  i n d i c a t o r s of p h y s i o l o g i c a l responses Slicher,  Furthermore,  1961;  sockeye  t i o n to the sea must f a c e major environmental reduce  the s e v e r i t y of a p a r a -  to withstand  B a l l and S l i c h e r ,  1962  salmon d u r i n g t h e i r  changes.  in and  migra-  T h i s p a r a s i t e may  the changes they must face i n n a t u r e .  5.  GENERAL MATERIALS AND METHODS  6  In t h i s s e c t i o n , m a t e r i a l s and methods common to a l l p a r t s of the study are d e s c r i b e d .  M a t e r i a l s and methods s p e c i f i c to i n d i v i d u a l  iments w i l l be o u t l i n e d i n the a p p r o p r i a t e 1.  exper-  sections.  Experimental F i s h Young sockeye salmon, Oncovhynchus  nerka  (Walbaum), were o b t a i n e d  from the f e d e r a l government hatchery at Rosewall Creek, Vancouver where they had been r e a r e d from f r y .  Island,  The f i s h were t r a n s f e r r e d to the  P a c i f i c B i o l o g i c a l . S t a t i o n i n Nanaimo, B r i t i s h Columbia, kept i n an aerated h o l d i n g tank at ambient  temperature and f e d once a day to s a t i a t i o n w i t h  Oregon Moist P e l l e t s (3/32"). 2.  Parasite  Source  In 1972 oaliforniensis  sockeye salmon (Dana,  smolts i n f e c t e d w i t h the copepod  Salmincola  1852) were brought to the P a c i f i c B i o l o g i c a l  from C u l t u s Lake, B r i t i s h Columbia f o r the purpose of l i f e c y c l e  Station  studies.  Copepods from that experiment, which had been maintained at the P a c i f i c B i o l o g i c a l S t a t i o n i n Nanaimo, were used as the source of i n f e c t i o n f o r the present 3.  studies.  I n f e c t i o n Techniques Egg sacs (with eggs at the pigment  female fish.  Salmincola  stage) were c o l l e c t e d from a d u l t  f o r the purpose of i n f e c t i n g the stock of p a r a s i t i z e d  These eggs were placed i n a d i s h of f r e s h water and r e f r i g e r a t e d i n  a shaker bath at 12°C f o r two to four days or u n t i l hatched.  the copepod  The copepodids were r e l e a s e d i n t o the tank of the f i s h  as the i n f e c t e d f i s h  groups.  larva selected  7 The experimental f i s h , d i v i d e d i n t o three groups according to the s p e c i f i c t e s t s i n which they were to be used, were i n f e c t e d on three d i f f e r ent  schedules. The f i r s t group (about 250 f i s h ) , which was t o be used f o r  growth and hematological s t u d i e s , was exposed to copepod larvae three days p r i o r to the experiment.  The second group (about 150 f i s h ) , intended f o r  use i n temperature tolerance and swimming a b i l i t y t e s t s , was exposed t o the  larvae seven weeks p r i o r to the commencement of the t e s t s i n temperature  tolerance.  F i s h remaining from t h i s experiment were used i n the swimming  a b i l i t y t e s t conducted approximately f i v e months a f t e r i n i t i a l exposure to the p a r a s i t e s .  The t h i r d group (about 100 f i s h ) , used i n the s a l i n i t y t e s t ,  was i n f e c t e d three months p r i o r to that experiment. 3.1  P a r a s i t e Numbers . The p a r a s i t i z e d f i s h which were used f o r growth and hematological  studies were a l s o used i n e v a l u a t i n g the l e v e l of i n f e c t i o n . A f t e r the blood was sampled and the length and weight of the f i s h were recorded, the number of p a r a s i t e s i n the buccal c a v i t y and on the s k i n was recorded f o r each f i s h . The mean numbers of p a r a s i t e s obtained a t each sampling p e r i o d are shown i n Figure 1.  The l i n e of best f i t ( s o l i d l i n e ) was drawn to show the v a r i a t i o n  i n number of p a r a s i t e s during the experiment.  I t was found that during the  3-41 Day Post I n f e c t i o n (DPI) p e r i o d the number of copepods was 22.6 ± 3.2 per  fish.  At 41 DPI the m a j o r i t y of copepods were found w i t h egg sacs but  a few f i s h were found to be i n f e c t e d w i t h copepods of the chalimus stages, i n d i c a t i n g that a second i n f e c t i o n from the f i r s t generation had occurred. The p a r a s i t e count f o r each f i s h increased very sharply w i t h the occurrence of a new generation of p a r a s i t e s ( i n d i c a t e d by the arrow w i t h open c i r c l e i n Figure 1). At the chalimus stage, the p a r a s i t e attaches to the g i l l filament by f r o n t a l f i l a m e n t s which do not provide a f i r m attachment.  Figure  1  The r e l a t i o n s h i p s of p a r a s i t e numbers, p a r a s i t e - d a y s and time S o l i d l i n e i s the l i n e of best f i t showing the v a r i a t i o n i n number of p a r a s i t e s . J  i n d i c a t e s f i n d i n g of p a r a s i t e s  I *  i n d i c a t e s that copepods found on f i s h up to t h i s p o i n t were adult  of chalimus stage  i n d i c a t e s s t a r t of second i n f e c t i o n i n d i c a t e s a high count a t t r i b u t a b l e to new generation of i n f e c t i o n  PARASITE  6  DAYS  10 This f a c t  i n combination with the f u r t h e r f a c t  that male p a r a s i t e s d i e a f t e r  mating can e x p l a i n the noted tendency f o r the number of p a r a s i t e s per f i s h to d e c l i n e .  From 42 DPI to the end of the experiment  (112 DPI), the mean  number of the p a r a s i t e s was found to be 31.25 ± 2.96 per f i s h . 3.2  Parasite-Days The  damage caused by the copepods i s p r o p o r t i o n a l not only to the  number of p a r a s i t e s present  but a l s o to the l e n g t h of time they have i n -  f e c t e d the f i s h  (Kabata and Cousens, 1977).  Since  increases u n t i l  i t reaches a f u l l y mature stage,  the s i z e of the p a r a s i t e  i t i s more accurate to  measure the degree of i n f e c t i o n by using p a r a s i t e number i n combination with " p a r a s i t e - d a y s "  r a t h e r than alone.  This l a t t e r measure  (parasite-days)  i n d i c a t e s the days that copepods of a given age are found on the f i s h  host  (Figure 2) and i s i n t e r p o l a t e d from the p a r a s i t e number by means of the formula i l l u s t r a t e d below:  T o t a l p a r a s i t e - d a y s / f i s h = (P .xD ,)+(P _xD .)+ s1 s1 s2 s2 where P_ = number of p a r a s i t e s a t a given  stage  D_ = number of days that a copepod at a given p a r a s i t i z i n g the f i s h The  parasite-days  (P xD ) sn sn  stage has been  (Figure 2)  were p l o t t e d a g a i n s t  time and a r e g r e s s i o n  line  was drawn (y = 12.4 + .36 x ) . A high p o s i t i v e c o r r e l a t i o n was noted between parasite-days  and time.  A comparison of the l i n e s , r e p r e s e n t i n g  numbers ( s o l i d l i n e ) , which w i l l be c a l l e d  parasite  " l e v e l of i n f e c t i o n " and p a r a s i t e -  days (dotted l i n e ) "degree of i n f e c t i o n , " i n F i g u r e  1 leads  to the observa-  t i o n that the l a t t e r i n d i c a t e s the r e l a t i o n between the s e v e r i t y of i n f e c t i o n and time (DPI) more a c c u r a t e l y  than does the former.  The degree  11  Figure 2  L i f e c y c l e of Salnrincola  oaliforniensvs  T h i s diagram i s adapted from Kabata and Cousens (1973). Time i n b r a c k e t s denotes d u r a t i o n of stages.- Since males d i e a f t e r mating only a d u l t females are normally found on the f i s h .  ADULT (  5  d a y s )  t  ADULT WITH EGG SACS  ADULT ( 14 days )  ( 32- 45 days )  I  CHALIMUS IV (3 days)  CHALIMUS III (2 days )  O"  CHALIMUSIV ( 4days )  CHALIMUS II ( ,1 days )  •  t  CHALIMUS I ( 1 days)  t  COPE POD  EGG AT PIGMENT STAGE  CHALIMUSIII ( 2 days )  of i n f e c t i o n was throughout  the  found to be constant at the r a t e of 7.5 experiment.  parasite-days/day  SECTION I IMPACT ON GROWTH AND WEIGHT  15  1.  Introduction Many s t u d i e s have repeatedly  of f i s h may  l e a d to growth r e t a r d a t i o n , weight l o s s , and  dition factor. with Liguta  demonstrated that p a r a s i t i c i n f e c t i o n  P i t t and  intestinatvs  Huggins (1959) a t t r i b u t e d a stunted  than n o n - p a r a s i t i z e d  to Postodiplostomum  minimum i n f e c t i o n .  i s s i g n i f i c a n t l y l e s s than that of n o n - p a r a s i t i z e d  Gasteosteous  aculeatus  with Schistocephalus  The  solidus  salvelini  (Walkey and Meakins, 1970).  fish.  other  Impairment of  percent  under weight.  Caligus  macavovi  p a r a s i t e s on growth, weight and observed by  parasitism  Scott  (1909) and  interest in  condition factors.  Kabata (1958) i n Mann (1953) a l s o  p a r a s i t i z e d with the same species were f i v e to Hotta  saura  sieboldi  evidence p o i n t s  and  Reichenbach-Klinke et a l .  lowers the c o n d i t i o n f a c t o r of  i n c r e a s i n g l y to the c o n c l u s i o n  p a r a s i t e s exert an adverse e f f e c t on the growth, weight and f a c t o r of t h e i r f i s h host.  ten  (1962) observed the r e t a r d a t i o n . o f growth by  i n Cololabis  s t a t e d that Ergasilus The  continue to s t i m u l a t e  gadoids i n f e c t e d with Lernaeocera.  found that Mevlangus  Uninfected  (Boyce, 1979). •  s t u d i e s of many r e s e a r c h e r s  Reduction i n weight was and  Lopukhina  were found to grow more r a p i d l y than those p a r a s i t i z e d  the e f f e c t of crustacean  whitings  crappie  Triaenophovus  growth i n j u v e n i l e sockeye salmon i s c l e a r l y the consequence of with Eubothrium  fish.  c o n d i t i o n i n a sample of b l a c k  (1961) found that the l e n g t h of rainbow t r o u t p a r a s i t i z e d with avassus  i n con-  Grumdan (1957) found that y e l l o w perch p a r a s i t i z e d are markedly smaller  (Pomox-is nigvomaoulatus)  a reduction  However, there  that  (1968)  Coregonus. several  condition  are c o n t r a d i c t i o n s which appear  16 i n the l i t e r a t u r e i n d i c a t i n g that some p a r a s i t e s do not have any discernable e f f e c t s . o n t h e i r f i s h hosts.  For example, Sproston and H a r t l e y (1941)  observed no d i f f e r e n c e s between the c o n d i t i o n f a c t o r s of Gadus i n f e c t e d w i t h Lernaeooeva  branahialis  and those non-infected.  mevlangus K l e i n et a l .  (1969) d i d not f i n d any s i g n i f i c a n t e f f e c t s among rainbow t r o u t i n f e c t e d by Crepidostomwn  farionis.  Tosthodiptostomum  Lewis and Nickum (1964) a l s o found no e f f e c t of  minimum on the b l u e g i l l .  R u s s e l l (1977)  found only a  s l i g h t decrease i n the growth r a t e of t r o u t corresponding to an increase i n i n f e c t i o n w i t h Truttaedaonitis even found that CatZa catla to gain weight.  truttae.  S u r p r i s i n g l y , Rao et a l . , (1972)  f i n g e r l i n g s , i n f e c t e d with black grub, seemed  I n t e r p r e t a t i o n s from these studies are complicated  f a c t that i n some cases, researchers  by the  f a i l e d t o measure e i t h e r the l e v e l of  i n f e c t i o n or the r e l a t i o n s h i p s between the extent of i n j u r i e s and d i f f e r e n t l e v e l s of i n f e c t i o n .  In some s t u d i e s , the data were obtained from the f i e l d  i n u n c o n t r o l l e d circumstances or the samples were too s m a l l . In comparing the e f f e c t s of d i f f e r e n t p a r a s i t e s on f i s h hosts, the l e v e l of i n f e c t i o n must be taken i n t o c o n s i d e r a t i o n .  The e f f e c t s of the  p a r a s i t e on the f i s h vary i n response t o the number and/or s i z e of p a r a s i t e s with which p a r t i c u l a r hosts are i n f e c t e d (Scott, 1929; Gross, 1935 and M i l l e r , 1945). the ated  host;  L i g h t i n f e c t i o n s may not produce any detectable e f f e c t s on  high i n f e c t i o n s may create s t r e s s on the h o s t and give an exagger  c o n d i t i o n . Furthermore, the c o n t r a - i n d i c a t i o n s may be due to d i f f e r e n c e s  among I p a r a s i t e species, f i s h species, f i s h age an environmental c o n d i t i o n s . The attempt to evaluate the e f f e c t s of Salmincota  on growth and  weight of j u v e n i l e sockeye salmon i n t h i s experiment should y i e l d r e l i a b l e r e s u l t s as both i n f e c t e d and non-infected  f i s h of the same age and s i z e  were maintained under c o n t r o l l e d experimental  conditions.  17 2.  M a t e r i a l s and Methods  2.1  Experimental Design Two  experiments were conducted to i n v e s t i g a t e the e f f e c t s of the p a r a -  s i t e on growth and b l o o d c h a r a c t e r i s t i c s . e f f e c t between 14 and  The f i r s t  112 days post i n f e c t i o n  (DPI).  experiment measured the F i s h were sampled f o r  growth and h e m a t o l o g i c a l d e t e r m i n a t i o n s at approximately three day for  intervals  the f i r s t month, 14 day i n t e r v a l s f o r the second month and a l s o at the  end of the experiment day.  (112 DPI).  Twelve f i s h were sampled on each sampling  A f t e r the i n i t i a t i o n of t h i s experiment, a q u e s t i o n arose about the  p o s s i b l e occurrence of e f f e c t s b e f o r e 14 DPI.  The second experiment  t h e r e f o r e a s s i g n e d f o r the p e r i o d of three to t h i r t y e i g h t DPI,  was  sample  being taken every three days. 2.2  Experimental F i s h Fourteen days b e f o r e the f i r s t  the  experiment was  s t a r t e d , f i s h from  h o l d i n g tank were t r a n s f e r r e d i n t o two experimental tanks.  s i z e s e l e c t e d i n order to keep t h i s parameter c o n s t a n t . fifty the  f i s h were used i n each experimental tank.  c o n t r o l and the other f o r the i n f e c t e d group.  One  They were  One hundred  tank was  and  assigned f o r  F i s h a s s i g n e d to the i n -  f e c t e d group were exposed to l a r g e numbers of copepod l a r v a e (approximately 30,000 l a r v a e ) by the method d e s c r i b e d  i n the s e c t i o n of t h i s paper  enti-  t l e d General M a t e r i a l s and Methods. Water s u p p l i e d to these tanks was water at ambient for  maintained at 9° ± 2°C by mixing  temperature w i t h heated water.  The tanks were equipped  water r e c i r c u l a t i o n and a e r a t i o n . The second experiment was experiment.  I t was  conducted one month a f t e r  of  the f i r s t  of  the f i s h to the copepod l a r v a e .  the i n i t i a t i o n  commenced on the t h i r d day a f t e r  exposure  The average number of p a r a s i t e s counted  18 during the f i r s t period (3-38) of the experiment was 22.6 ± 3.2 per f i s h . The f i s h i n both experiments  were fed w i t h Oregon Moist P e l l e t s  w i t h 3% body weight per day (recommended by B r e t t et a l . , 1969) f o r the optimum growth at a temperature of 10°C).  The amount of food was readjusted  every two weeks according to weight changes i n both groups of f i s h , while maintaining 3% body weight per day. 2.3  l! I'  Growth Stujiy A f t e r the f i s h were sampled at random f o r hematological studies (as i  described i n Section V ) , the length of each was measured t o the nearest m i l l i m e t e r and (G),  t h e i r weights, to the nearest gram.  S p e c i f i c growth r a t e  c o e f f i c i e n t of c o n d i t i o n (K) and absolute growth r a t e were computed  to determine whether a r e l a t i o n s h i p e x i s t e d between the growth of the f i s h and the presence of the p a r a s i t e using the formula: a)  S p e c i f i c growth r a t e (G) = l o g YT - l o g Yt X 100 T - t Where YT = f i n a l s i z e at time T Yt = i n i t i a l s i z e a t time t  b) C o e f f i c i e n t of c o n d i t i o n (K) = W L  X 100 3  Where W and L = weight (g) and length (cm) c)  Absolute growth r a t e = __ X 100 dt Where dW = weight d i f f e r e n c e between i n i t i a l and f i n a l measurements dt = the period between i n i t i a l and f i n a l measurement.  2.4  Data A n a l y s i s The mean lengths and weights of the c o n t r o l and i n f e c t e d f i s h groups  were compared using the t - t e s t .  Statistically  assessed at a confidence l e v e l of P = 0.05.  s i g n i f i c a n t d i f f e r e n c e s were  Regression c o e f f i c i e n t s were  19 compared using a n a l y s i s of v a r i a n c e . .Because there was a time overlap between the two experiments, from 14-38 DPI, the r e s u l t s obtained during t h i s period f o r both c o n t r o l and i n f e c t e d f i s h groups were compared ( t - t e s t , P = 0.05) and no s i g n i f i c a n t d i f f e r e n c e s were found.  For convenience of a n a l y s i s and d i s c u s s i o n , the  data from the two experiment experiment covering 3-112 3.  were pooled and r e p r e s i n t e d as a s i n g l e  DPI.  Results When the mean wet weights and lengths of i n f e c t e d and non-infected  f i s h groups were compared, a s i g n i f i c a n t d i f f e r e n c e i n weight was found although there was no s i g n i f i c a n t d i f f e r e n c e i n length.  The r e g r e s s i o n  l i n e s were drawn to show the r e g r e s s i o n c o e f f i c i e n t s of both weight and length of both groups of f i s h (Figure 3 ) . Mean weights of both i n f e c t e d and c o n t r o l f i s h samples during the period of 17-32 DPI were higher than those sampled during the p e r i o d of 41-56 DPI which can be a t t r i b u t e d not to growth, but to the f a c t that l a r g e r f i s h were sampled. The d i f f e r e n c e i n weight becomes marked a f t e r 71 DPI (Figure 3, top graph).  At the end of the experiment, the mean weight of the i n f e c t e d f i s h  group was 18.46 ± 6.24 g which was s i g n i f i c a n t l y lower than that of the c o n t r o l f i s h group, 28.34 ± 2.74  ( Table I )  The percentage gain i n weight  was computed ( H a s k e l l , 1959), the r e s u l t s showing that the non-infected f i s h group gained 25%, while the i n f e c t e d f i s h group l o s t 23.21%. I t was found that the p a r a s i t e Salminoola  reduced the p o t e n t i a l  weight of the f i s h host, j u v e n i l e sockeye salmon, by almost 34.86% at the end of the experiment (112 DPI), when they were i n f e c t e d at a l e v e l of 31.25 p a r a s i t e s per f i s h and the degree of i n f e c t i o n increased at the r a t e  20  Figure 3  Length and weight i n r e l a t i o n to time post  infection  Comparison of l e n g t h and weight between f i s h p a r a s i t i z e d with the p a r a s i t i c copepod, Salnrincola, and n o n - p a r a s i t i z e d f i s h d u r i n g the p e r i o d of 112 DPI. Open u n i t s and broken l i n e s i n d i c a t e n o n - p a r a s i t i z e d f i s h ; c l o s e d u n i t s and s o l i d l i n e s i n d i c a t e p a r a s i t i z e d fish.  21  22 of 7.5 parasite-days/day . Due  over the p e r i o d of 112  DPI.  to a slow growth r a t e and a short experimental  period, specific  growth rates (G) were found i n v a l i d as a measure to compare the growth of the i n f e c t e d and non-infected f i s h groups. c o e f f i c i e n t of c o n d i t i o n (K) was used. i n f e c t e d f i s h were compared 0.05).  Mean Ks f o r both i n f e c t e d and  non-  and found to be s i g n i f i c a n t l y d i f f e r e n t (P =  The r e g r e s s i o n l i n e was  drawn to show a r e l a t i o n between K and time  of these two groups of f i s h (Figure 4). (r = -.46)  Instead the c o n d i t i o n f a c t o r or  The negative c o r r e l a t i o n of K  f o r the i n f e c t e d f i s h groups i l l u s t r a t e s a b a s i c trend toward  decreasing K w i t h i n c r e a s i n g DPI. found quite constant  Ks of the non-infected f i s h group were  (Figure 4, Table I) since both weight and  length  increased i n the same p r o p o r t i o n throughout the experiment. 4.  Discussion The r e s u l t s obtained from t h i s experiment c l e a r l y i n d i c a t e that the  i n f e c t i o n of j u v e n i l e sockeye salmon w i t h i n i t i a l numbers of 22.6 ± Salminaola  3.2  per f i s h leads to a marked decrease (by almost 34.86%) i n the  weight of the f i s h host during the p e r i o d of 112 DPI. 3 and Table I  The r e s u l t s i n Figure  i n d i c a t e that both i n f e c t e d and c o n t r o l f i s h continue to grow  although there appeared to be a s l i g h t decrease i n the r a t e of growth i n length among i n f e c t e d f i s h when compared w i t h the c o n t r o l group.  However,  t h i s d i f f e r e n c e could not be substantiated i n a s t a t i s t i c a l l y s i g n i f i c a n t manner. A f t e r 56 days, a negative r e l a t i o n s h i p between weight and time observed.  This suggests that the r e d u c t i o n i n the weight of j u v e n i l e  sockeye salmon depends not only on the p a r a s i t e number but a l s o on the number of days the p a r a s i t e s are found on the f i s h host.  The degree of  i n f e c t i o n (measured from the r e g r e s s i o n l i n e i n Figure 1) increased  was  23  Figure 4  Condition factor  (K) during  112  DPI  R e g r e s s i o n l i n e s and r e g r e s s i o n c o e f f i c i e n t s of c o n d i t i o n f a c t o r (K) of i n f e c t e d and n o n - i n f e c t e d f i s h groups. Open squares and broken l i n e s i n d i c a t e n o n - i n f e c t e d f i s h ; c l o s e d squares and s o l i d l i n e s , i n f e c t e d f i s h .  CONDITION  FACTOR  (K)  TABLE I Lengths, weights and c o e f f i c i e n t s of c o n d i t i o n f o r i n f e c t e d and non-infected f i s h groups during the period of 112 DPI. Mean lengths and weights f o r each sampling period based on 12 f i s h . Time y  d a  Means length mm  s  Means weight g  C o e f f i c i e n t of c o n d i t i o n (K)  Infected  Control  Infected  3  145.8 + 6.18  145.7 + 9.44  24.04 + 4.35  22.59 + 4.47  .73  .73  6  +  +  +  3.94  23.90 + 4.02  .73  .76  146.4  5.59  146.4  9.47  22.80  Control  Infected  Control  10  147.8 + 3.19  145.8 + 4.43  19.69 + 2.68  23.10 + 3.39  .61  .74  13  152.5  +  145.1 + 3.58  23.88 + 3.22  25.47 ± 2.20  .67  .83  17  147.6 + 4.83  145.5  +  1.75  .67  .72  23  145.5 + 9.10  148.1 + 3.82  27.11 + 2.65  .77  .83  28  143.3  +  +  4.14  23.86 + 4.03  .85  .76  32  154.5 ± 1 .33  153.0 + 1.34  30.18 + 5.59  32.39 + 5.57  .81  .90  38  153.0  +  154.3 + 4.33  28.01 + 5.20  26.98 + 2.30  .78  .73  41  143.6 + 1.43  149.2  +  +  3.69  .74  .82  56  144.8 + 1.48  152.8 + 7.83  24.30 + 1.99  27.74 + 5.18  .80  .79  70  153.0  +  +  2.09  19.92 + 2.21  27.38 + 2.09  .60  .72  84  149.3 + 4.86  151.6 + 5.59  19.62 + 3.73  24.56 + 4.75  .58  .77  112  151.7 + 4.96  152.9 + 2.74  18.46 + 6.24  28.34 + 2.74  .55  .79  6.55  5.11 8.63  4.06  146.9  156.0  +  +  3.46 4.31  6.90  21.62  +  2.00  23.68 + 3.98 25.03  22.10  +  3.74  21.98  26.00  26 c o n s t a n t l y throughout t h i s experiment at 7.5 p a r a s i t e - d a y s / d a y .  Each  7.5  p a r a s i t e - d a y s i n c r e a s e corresponded w i t h a l o s s i n weight of almost .45 %. Corresponding to the time a t which an e a s i l y observable d i f f e r e n c e between the weight of experimental and c o n t r o l groups of f i s h was noted (about 56 DPI) there was (Figure 1) i n each f i s h . from the i n i t i a l  a dramatic i n c r e a s e i n the number of p a r a s i t e s T h i s was  a t t r i b u t a b l e to the h a t c h i n g of  i n f e c t i o n and t h e i r  attachment.  copepods  T h i s i n c r e a s e may  not  be p r o p o r t i o n a l to the noted r e d u c t i o n of the r e s p i r a t o r y area caused by the  a d u l t copepods.  f i l a m e n t s , they may  Since they attached themselves d i r e c t l y to the g i l l have i n t e r f e r e d with normal r e s p i r a t o r y  The a b i l i t y of the f i s h to convert food i n t o growth  function. i s generally  i n d i c a t e d by the " c o n d i t i o n f a c t o r , " a measure of the r e l a t i o n s h i p between length and weight.  A comparison of the i n f e c t e d and c o n t r o l f i s h groups i n  t h i s experiment i n d i c a t e s a decrease i n the c o n d i t i o n f a c t o r of the former. In the present case, however, the decrease i n the c o n d i t i o n f a c t o r may a t t r i b u t e d not to a decreased a b i l i t y  to convert food i n t o growth but to a  decrease i n the amount of food consumed by the i n f e c t e d f i s h . amount of unconsumed food was  be  A greater  found at the bottom of the tank of the i n -  f e c t e d f i s h than at the bottom of the tank of the n o n - i n f e c t e d group. Optimum growth of the f i s h depends upon t h e i r t a k i n g enough food to p r o v i d e for  b a s a l metabolism, to r e p l a c e broken down t i s s u e s and to supply normal  t i s s u e growth as w e l l as energy r e q u i r e d presence and a c i t i v t y normal  i n v a r i o u s other a c t i v i t i e s .  of the p a r a s i t e s may  The  thus i n t e r f e r e w i t h t h e i r  growth. Food i n t a k e , however, i s not the only f a c t o r of importance a f f e c t -  ing In  the growth and weight of the f i s h . the present i n s t a n c e , t h i s  0  2  uptake must a l s o be mentioned.  is a p a r t i c u l a r l y relevant factor  since  causes d i r e c t damage to the g i l l s .  Salmincola  d i c a t e that t h i s p a r a s i t e i s found i n the g i l l salmon where at an e a r l y stage  i t attaches  Kabata and  Cousens (19.77) i n -  c a v i t y of j u v e n i l e sockeye  itself  to the g i l l  filament,  moving as an a d u l t to a s i t e u s u a l l y on the b r a n c h i a l r i m or the inner side of the operculum where i t becomes more f i r m l y a f f i x e d .  The presence  a c t i v i t y of t h i s p a r a s i t e and  the pressure  the g i l l  leads to atrophy  disappearance of the d i s t a l p o r t i o n s of  filaments.  The  and  eventual  adjacent  These r e s e a r c h e r s  damage caused by  i n f e c t e d with 22.6  1977). of 0  2  f u r t h e r i n d i c a t e that the extent of  Salminaola  20% of the g i l l  which i n c r e a s e d to 31.25  s u r f a c e area was  in  initially  per f i s h during  destroyed  the  i t i s possible  (Kabata and  Cousens,  Losses of t h i s magnitude can a l s o be expected to a f f e c t the amount uptake.  Since normal l e v e l s of 0_  the balance.  must be maintained  i n t e r f e r e n c e with  those  Moreover, a r e d u c t i o n i n the g i l l  .to meet the r e levels  ( E l l i o t and Russert,  1949).  not o b t a i n s u f f i c i e n t  0.  The  age  will  s u r f a c e area  a g r e a t e r requirement f o r an i n c r e a s e i n the b a s a l metabolic I t i s v i r t u a l l y c e r t a i n that these  results  rate fish did  2  of the f i s h host i s a f u r t h e r f a c t o r i n f l u e n c i n g the  s e v e r i t y of the e f f e c t s of p a r a s i t i z a t i o n  in fish.  noted that the r a t e of growth of i n f e c t e d f i s h was  P i t t and  Grumdan (1957)  slowed to such an  extent during the p e r i o d of p a r a s i t i z a t i o n that the f i s h become Since  gill  size.  With i n f e c t i o n s of t h i s extent  quirement of b a s a l metabolism, any upset  these  them n o n f u n c t i o n a l f o r  experiment j u v e n i l e sockeye salmon were  second p a r t of the i n f e c t i o n . that at l e a s t  rendering  depends on t h e i r number and  SatnrincoZa  In the present  filaments  p a r t s of the m i s s i n g f i l a m e n t s are a f f e c t e d by  t i s s u e r e a c t i o n s , becoming t h i c k , and respiration.  i t e x e r t s on  and  the p a r a s i t e s continue  stunted.  to exert an e f f e c t throughout the l i f e  of  28 t h e i r hosts subsequent  to i n f e c t i o n , f i s h with the l o n g e s t h i s t o r y of i n -  f e c t i o n showed the g r e a t e s t r e t a r d a t i o n of growth. and Grumdan was  confirmed by the present experiment.  i n c r e a s e i n the degree of  T h i s o b s e r v a t i o n of P i t t However, the dramatic  of i n f e c t i o n i n the present study p o i n t s to the  the p a r a s i t e r a t h e r than to the age of the f i s h as the primary  on the r e t a r d a t i o n of f i s h  influence  growth.  The r e s u l t s of t h i s experiment californiensis  age  clearly  i n d i c a t e that  has a more pronounced e f f e c t on the weight  f a c t o r than on the l e n g t h of sockey  and  Salmincola  condition  salmon, f o r the p e r i o d of 112 DPI.  This  f i n d i n g confirms some p r e v i o u s s t u d i e s with p a r a s i t i c C r u s t a c e a such as those of Schaperclaus  and of Reichenbach-Klinke  silus C.  (1954) on tench,  feva  i n f e c t e d with  Ergasilus  Tinea  tinea  et a l . (1968) on sieboldi.  p a r a s i t i z e d with Covegonus  lavaretus  Erga-  and  SECTION I I IMPACT ON HOST'S TEMPERATURE TOLERANCE  30  1.  Introduction Among those environmental f a c t o r s which cause s t r e s s i n f i s h r a p i d  or extensive temperature change i s one of the most severe i n i t s e f f e c t on f i s h physiology.  Increasing water temperature above the optimum leads to  the production of such adverse e f f e c t s as r e s t r i c t e d swimming performance ( B r e t t , 1964), i n t e r f e r e n c e w i t h normal feeding h a b i t s ( P h i l l i p s et a l . , 1960), increased standard metabolism ( B r e t t , 1964), and l i p i d composition (Lewis,1962). Kinne and Kinne (1962) a l s o found that the a c t i v i t y l e v e l of the f i s h was accelerated as water temperature was r a i s e d . None of the changes which a f f e c t water q u a l i t y and can a f f e c t f r e s h water f i s h e r i e s adversely  i s g e n e r a l l y b e l i e v e d to be more common and more  s i g n i f i c a n t than reduction of d i s s o l v e d 0 .  Doudoroff and Shumway (1967)  2  c l a i m that salmonid f i s h are among the most s u s c e p t i b l e to reduction of 0_. Patterns of v a r i a t i o n i n the r e s i s t a n c e of f i s h to 0  2  l a t e d with water temperature are a l s o h i g h l y v a r i a b l e .  deficiencies correB r e t t (1956) found  that salmonids have a maximum upper l e t h a l temperature b a r e l y exceeding 25°C.  He a l s o found that the zone of tolerance can be extended i f the  temperature change i s s u f f i c i e n t l y  gradual.  Water temperature influences the r a t e of a l l metabolic of f i s h so that t h e i r need f o r 0 temperature of t h e i r medium.  2  can vary g r e a t l y w i t h a change i n the  Normally, most f i s h have the a b i l i t y to  compensate f o r a reduction of d i s s o l v e d 0 adjustments.  Randall  processes  2  by making various p h y s i o l o g i c a l  (1970) found increases i n breathing r a t e and ampli-  tude and a l s o i n the v e n t i l a t i o n - p e r f u s i o n r a t i o as a r e s u l t of reduction  31 of  the 0  2  l e v e l of water.  compensation  Ellis  (1937) a l s o r e p o r t e d that  o c c u r r e d i n g o l d f i s h , y e l l o w perch and other s p e c i e s of  at an oxygen c o n c e n t r a t i o n only a l i t t l e below 5 mg/1 was between 20 and 25°C.  fish  when the. temperature  These compensatory mechanisms r e q u i r e  expenditures and the l o n g e r the f i s h energy  respiratory  energy  i s i n the s t r e s s f u l c o n d i t i o n , the more  i s required. T h i s experiment  attempted  to f i n d out whether p a r a s i t i s m w i t h  a f f e c t s the a b i l i t y of j u v e n i l e sockeye  mincola  ses i n water temperature. atures and t h e i r a b i l i t y  salmon to withstand  Sal-  increa-  The f i s h were subjected to d i f f e r e n t water temperto cope w i t h each p a r t i c u l a r water temperature  was  evaluated i n terms of m o r t a l i t y . 2.  M a t e r i a l s and Methods  2.1  Experimental F i s h F i s h used f o r t h i s experiment  came from the stock of f i s h  i n the General M a t e r i a l s and Methods.  mentioned  I n f e c t i o n techniques were d e s c r i b e d  in the same s e c t i o n . 2.2  Experimental One  i n t o two  Procedure  hundred  experimental f i s h and  100 c o n t r o l f i s h were t r a n s f e r r e d  separate tanks c o n t a i n i n g water of the same temperature.  temperature  of the two  tanks was  g r a d u a l l y a d j u s t e d by the a d d i t i o n of  heated water at a r a t e of 1°C f o r each two d a y - i n t e r v a l , u n t i l 9°C. to  The water  i t reached  The f i s h were a c c l i m a t e d to these c o n d i t i o n s f o r one week, and f e d  s a t i a t i o n with Oregon Moist P e l l e t s .  three days p r i o r to and throughout  However, they were s t a r v e d f o r  the course of the experiment  i n order  to reduce the amount of waste products, the experimental a q u a r i a having a e r a t i o n but no water r e c i r c u l a t i o n .  32 Ten f i s h were sampled  to determine t h e i r average weight and l e n g t h  and a l s o the l e v e l and degree of i n f e c t i o n .  Ten c o n t r o l and ten i n f e c t e d  f i s h were t r a n s f e r r e d i n t o each experimental aquarium.  Four a q u a r i a were  used, each c o n t a i n i n g 20 g a l l o n s of temperature c o n t r o l l e d water. temperature i n each aquarium was e s t a b l i s h e d a t 9°C. of  The 0  2  The water  content and pH  the water were measured i n each aquarium b e f o r e the f i s h were introduced  and throughout the experiment  (Table I I ) . A f t e r the f i s h were t r a n s f e r r e d  i n t o the a q u a r i a , they were l e f t three days.  to a c c l i m a t e to t h e i r new environment f o r  During these three days, waste products from the bottom of the  a q u a r i a were siphoned out u s i n g a small p l a s t i c  tube and water was r e p l a c e d .  A f t e r three days a c c l i m a t i o n at 9°C, the f i s h were subjected to gradual i n c r e a s e s i n water temperatures u n t i l course o f the experiment, the dead f i s h the of  i t reached 23°C.  During the  ( a l s o those f a i l i n g to respond t o  touch of a g l a s s rod) were removed.  The number of dead f i s h and l e v e l  i n f e c t i o n were r e c o r d e d each day.  3.  Results The average l e n g t h and weight of ten randomly  sampled  f i s h were  15.1 ± 0.92 cm and 27.8 ± 4.6 g f o r the i n f e c t e d f i s h groups and 15.2 ± 0.89 cm and 31.4 ± 5.3 g f o r the c o n t r o l . f i s h were s l i g h t l y no s t a t i s t i c a l l y  The l e v e l of i n f e c t i o n was found to be 27.2 ± 7.9 per  (mainly a d u l t s w i t h egg sacs) and degree of i n f e c t i o n was- 589.3 ±  28.1 p a r a s i t e - d a y s . but  h e a v i e r than those i n the i n f e c t e d group but there were  s i g n i f i c a n t d i f f e r e n c e s i n length and weight between these  two groups of f i s h . fish  A c c o r d i n g to the data, c o n t r o l  Reduction of the g i l l  s u r f a c e area was not measured  e r o s i o n of the f i l a m e n t s was observed. Even with three days s t a r v a t i o n p r i o r to the experiment, the f i s h  r e l e a s e d some waste products i n t o the a q u a r i a , causing an i n c r e a s e i n the  TABLE I I  V a r i a t i o n of d i s s o l v e d 0  2  and pH l e v e l s during the experiment  (1 month period) as the  temperature increased from 9° to 23°C.  Acclimated temperatures °C  1 1  0  2  ppm  pH  12:  12.25  11.40  7.0  8.1  1.3  17  21  23*  23  10.5  10.9  8.5  8.15  7.45  7.43  7.5  6.0  -  -  5.9  8.15  23  * Temperature remained constant at 23°C u n t i l the end of the experiment  Co Co  pH (Figure 5) from 7.02 to 8.10 during the second day (Table I I ) .  As the  waste products were removed and the water r e p l a c e d , the pH declined-and reached the o r i g i n a l l e v e l w i t h i n 12 days. the  The pH g r a d u a l l y declined a f t e r  12th day due t o an increase i n d i s s o l v e d G0  r e s p i r a t i o n and p o s s i b l y f i s h e x c r e t i o n ) .  2  (the by-product of f i s h  However, a t the end of the  experiment, the pH dropped to 5.9, (Figure 5) s l i g h t l y below the range recommended f o r optimum f i s h h e a l t h by E.P.A. (1973), although t h i s should not cause any concern since both i n f e c t e d and c o n t r o l f i s h groups were kept under the same c o n d i t i o n s . The t o t a l amount of d i s s o l v e d 0  2  i n the experimental aquaria de-  c l i n e d as the water temperature increased (Figure 6) though i t remained higher than the amount of d i s s o l v e d 0  2  i n normal f r e s h water  as a r e s u l t of the a e r a t i o n of the experimenta  aquaria.  (Whipple,1914)  Measurement made  on the 21st day of the experiment showed that the l e v e l of d i s s o l v e d 0 i n 2  the experimental aquaria had f a l l e n below the l e v e l of d i s s o l v e d 0 i n 2  normal f r e s h water at the same temperature.  By the end of the experiment  i t had dropped t o 7.2 ppm. No m o r t a l i t y was found during the course of water temperature changes from 9 to 15°C.  On the 14th day, when the water temperature was  17°C, some of the i n f e c t e d f i s h showed signs of s t r e s s ; they struggled and before l o s i n g t h e i r balance, swam w i t h a great deal of a c t i v i t y .  Initial  m o r t a l i t y of i n f e c t e d f i s h occurred on the 15th day when water temperature was 17°C  while i n the non-infected f i s h group, i t occurred on the 18th  day (Figure 7, Table I I I ) .  When the water temperature was increased to  21°C, severe s t r e s s appeared i n both i n f e c t e d and non-infected f i s h groups and the m o r t a l i t y rate showed a dramatic increase reaching 25% i n the i n fected f i s h group and 12.5% among the non-infected f i s h . The cumulative  35  Figure 5  pH i n experimental  aquaria  V a r i a t i o n of pH during a 1 month p e r i o d i n the aquaria  experimental  a = l e v e l of pH recommended f o r optimum h e a l t h of f i s h (E.P.A., 1973)  Figure 6  Dissolved 0  2  i n experimental  aquaria  V a r i a t i o n of the amount of d i s s o l v e d 0 i n the experimental aquaria with temperature i n c r e a s e d from 9 to 23°C. 2  The c l o s e d squares represent c o n c e n t r a t i o n of 0 measured in the experimental aquarium at the time i n d i c a t e d . Open squares represent c o n c e n t r a t i o n of d i s s o l v e d 0 i n f r e s h water (derived from Whipple, 1914). 2  2  36  1  1  1  3  15  17  19~  TEMPERATURE 2  6  10  14 TIME IN  Qj t t V I l O—•  I  11 1  2  — i  J.  13  i  6  15  —  1  10  O,  IN  ~_3  23  18  22  26  30  DAYS  IN E X P E R I M E N T A L  SOIUIIIITT Of  21 (°C)  AQUARIUM  FRESH  1  17 TEMPERATURE 1  14 TIME IN DAYS  WATER  1  19 (°C)  I  21  I  23  1  1  1  18  22  26  I  23  i  30  37  Figure 7  Percent m o r t a l i t y and percent cumulative m o r t a l i t y of experimental fish V a r i a t i o n of the m o r t a l i t y of i n f e c t e d and n o n - i n f e c t e d f i s h corresponding to temperature i n c r e a s e s ' f r o m 9 to 23°C. The arrow denotes that from the i n d i c a t e d p o i n t , the water remained at 23°C u n t i l the end of the experiment.  8C  TABLE I I I  Percent m o r t a l i t y and cumulative m o r t a l i t y of the c o n t r o l and i n f e c t e d d u r i n g the course of changing temperature from 9 to 23°C  Control Fish Day  Temperature °C  Infected  Mortality Percent Cumulative  fish  Fish  Mortality Percent Cumulat:  0-14  9-17  0  -  0  -  15  17  0  -  7.5  7.5  18  19  2.5  2.5  5.0  12.5  20  20  0  2.5  2.5  15.0  22  21  7.5  10.0  22.5  37.5  24  22  12.5  22.5  25.0  62.5  26  23  7.5  30.0  5.0  67.5  28  23  2.5  32.5  15.0  82.5  30  23  0  32.5  2.5  85.0  Co  40 m o r t a l i t y reached 50% among i n f e c t e d f i s h as compared to 22 5% i n non-infected f i s h at. the 24th day of the experiment.  A gradual reduction i n m o r t a l i t y  rate occurred i n both f i s h groups (Figure 7, top graph).  At the end of the  experiment, the cumulative m o r t a l i t y among non-infected f i s h was 32.5% w h i l e i n the i n f e c t e d f i s h group, i t went up to 85%. A m o r t a l i t y r a t e of 62.5% was found among f i s h i n f e c t e d w i t h an average of 27  Salmincola  or 589 p a r a s i t e - d a y s per f i s h  when  the water  temperature was increased to 22°C on the 24th day of the experiment. This was almost 30% higher than the m o r t a l i t y found i n the n o n - i n f e c t e d group at the same temperature and time.  The % m o r t a l i t y of both groups of f i s h  was found t o be highest during t h i s p e r i o d .  A f t e r the 24th day of the  experiment, m o r t a l i t y tended to d e c l i n e f o r both groups.  This occurrence  can be a t t r i b u t e d to a c c l i m a t i o n t o the constant temperature of 23°C assigned f o r t h i s experiment.  Only 22.5% of the non-infected group died  at the temperature found t o be the median l e t h a l temperature f o r the i n fected f i s h group.  Even at the end of the experiment when the water  temperature was 23°C only 32.5% m o r t a l i t y was observed among the noni n f e c t e d group. 4.  Discussion The above r e s u l t s i n d i c a t e that  Salminoota  reduces the a b i l i t y of  j u v e n i l e sockeye salmon to withstand increases i n temperature.  In healthy  sockeye salmon, m o r t a l i t y occurs i f the water temperature exceeds 25°C but the m o r t a l i t y rate depends mainly on the a c c l i m a t i o n time.  The zone of  tolerance can be extended i f the a c c l i m a t i o n time f o r a p a r t i c u l a r temperacture i s s u f f i c i e n t l y extended ( B r e t t , 1956). However, the exact cause of death of f i s h exposed t o h i g h water temperatures i s not known with c e r t a i n t y .  A l l a n s o n and Noble (1964) agreed  41 with s e v e r a l previous researchers factor. Ca  that osmotic s t r e s s may be the primary  I t has been shown by many workers that the a d d i t i o n of Na , Mg and +  ions increased the r e s i s t a n c e of f i s h t o high temperature.  and Dunn (1967) found that a sample of s a l t marsh  Garrbusia  affinis  +  Also Strawn was more  r e s i s t a n t to heat death than was a sample from f r e s h water. Increasing osmotic s t r e s s as a r e s u l t of i n c r e a s i n g water temperature would lead to the expenditure of a considerable amount of metabolic energy i n order that the f i s h might osmoregulate and maintain homeostasis. Thus the metabolic  a c t i v i t i e s of the f i s h are h i g h l y dependent upon the i o n i c  c o n d i t i o n s of the body f l u i d . increase t h e i r metabolic increase i n 0  2  When the water temperature increases, the fish  a c t i v i t y and t h i s increase i n turn demands an  consumption.  Beamish (1964) demonstrated that a l l species  of f i s h show a c o n t i n u a l increase i n 0  2  consumption w i t h i n c r e a s i n g temper-  ature . Temperature a f f e c t s both the amount of 0  2  that can be d i s s o l v e d  i n water and the amount f i s h r e q u i r e f o r metabolism. atures increase 0 dissolve.  consumption but decrease the amount of 0  2  Generally the rate of 0  2  2  2  the water can  consumption tends to increase more than  two-fold with a 10°C temperature r i s e . standard r a t e of 0  Higher water temper-  B r e t t (1964) has shown that the  consumption of sockeye salmon r i s e s by a f a c t o r of  almost f i v e when the temperature increases from 5 to 14°C and a l s o that i t increases f a i r l y r a p i d l y above 15°C to 220 mg/kg/hr at 25°C. The manner i n which temperature influences the s u r v i v a l of f i s h can now be c l e a r l y understood.  The m o r t a l i t y of non-infected  f i s h observed  i n the present experiment increased l i n e a r l y w i t h temperature increase. Within the 30 day period of the experiment, i t increased from 2.5% to 32% with temperature increase from 9° to 23°C.  Data from Graham (1949) and  42 Burdick et a l . (1954) a l s o suggest, a s i m i l a r r e l a t i o n s h i p between m o r t a l i t y r a t e and temperature. Among the i n f e c t e d f i s h group a l i n e a r c i a t e d with i n c r e a s i n g temperature was r a t e of i n c r e a s e i n m o r t a l i t y was n o n - i n f e c t e d f i s h group. was at  i n c r e a s e i n m o r t a l i t y asso-  a l s o observed.  Furthermore, the  g r e a t e r f o r t h i s group than that of the  The m o r t a l i t y l e v e l among the i n f e c t e d f i s h  group  found to be almost twice as h i g h than that among the n o n - i n f e c t e d  group  a water temperature of 23°C.  Even more s i g n i f i c a n t ,  the m o r t a l i t y  occurred e a r l i e r among t h i s group on the 15th day of the experiment when the 0  2  of  water temperature was  only  i n the experimental tank was 0  2  17°C.  11 ppm,  2  about 2.3 ppm h i g h e r than the l e v e l  that has been shown to l i m i t a c t i v e metabolism.  i n d i c a t e s that damage to the g i l l 0  At t h i s time the amount of d i s s o l v e d  This r e s u l t  clearly  r e s p i r a t o r y s u r f a c e l i m i t s the amount of  uptake to a l e v e l below that r e q u i r e d f o r b a s a l metabolism. The osmotic s t r e s s i s not the only s t r e s s the f i s h e x p e r i e n c e s as  a r e s u l t of i n c r e a s i n g water temperature.  The very i n t e r f e r e n c e w i t h the  r e s p i r a t o r y mechanism which the p a r a s i t e causes to the f i s h , earlier  i n s e c t i o n I, demands a h i g h e r l e v e l of 0  i f b a s a l metabolism i s to be maintained. called  2  consumption  Compensatory  i n t o p l a y to i n c r e a s e the amount of 0  2  mechanisms must be  2  changes which are  too severe by means of t h e i r r e s p i r a t o r y compensatory  there being any n o t i c e a b l e e f f e c t on m o r t a l i t y l e v e l s Privolnev,  than normal  d e l i v e r y to the t i s s u e s .  Normally f i s h can cope w i t h temperature and 0 not  discussed  mechanisms without  (Ellis,  1937;  1954; and Holeton and-Randall, 1967).  U n f o r t u n a t e l y , t h i s p a r a s i t e causes d i r e c t damage to the g i l l described i n d e t a i l  (as  i n the p r e v i o u s s e c t i o n ) and a l s o i n t e r f e r e s w i t h the  o p e r c u l a r movement and prevents complete c l o s u r e of the operculum'.  The  43 p o s s i b i l i t y of the i n f e c t e d f i s h  delivering  requirements of the t i s s u e i s thereby g r e a t l y death  are the usual r e s u l t s .  0  2  adequately f o r the  reduced;  t i s s u e anoxia and  SECTION I I I IMPACT ON HOST'S SWIMMING ABILITY  45  1.  Introduction Swimming performance, a s i g n i f i c a n t parameter i n e v a l u a t i n g  s u r v i v a l , has  been widely s t u d i e d i n r e l a t i o n to a number of  variables including f i s h dition  (Bainbridge,  temperature, 0 , C0 2  1963;  Brett,  1964,  1962; and  2  1967).  s i z e , body form (Bainbridge, Thomas, 1964)  and  solution toxicity  1958)  fish  important and  p h y s i c a l con-  such environmental c o n d i t i o n s (Fry and  Haet, 1948;  as  Davis et al..,  Many d e s c r i p t i o n s of the v a r i o u s components which  c h a r a c t e r i z e swimming movements i n salmonids a l s o e x i s t (Bainbridge,  1960;  Brett,  Jones  1958,  1964,  1967;  Fry and  Cox,  1970;  B r e t t and  Glass,  1973  and  et a l . , 1974). Owing to i t s importance and s i b l e information incorporate Fox  to the l a r g e volume of r e a d i l y a c c e s -  of f i s h swimming a b i l i t y ,  some r e s e a r c h e r s  swimming t e s t s i n t h e i r s t u d i e s of host p a r a s i t e r e l a t i o n s h i p s .  (1965) and  B u t l e r and Millemann (1971) used c r i t i c a l and  swimming t e s t s to show that t r o u t and  f a t i g u e d f a s t e r than c o n t r o l f i s h .  Schistocephalus  aculeatus.  Smith and M a r g o l i s  (1970) s t u d i e d  the swimming a b i l i t y terms of d i s t a n c e weighing up non-infected  to 50%  affects  the  fixed velocity  salmon i n f e c t e d with l a r g e numbers of  trematode metacercariae a t t a i n e d lower maximum s u s t a i n e d and  have begun to  Lester  swimming speeds  (1971) a l s o suggested  Gasterosteus.  swimming performance of  the e f f e c t s of  Eubothrium  that  salvelini  on  of sockeye salmon by measuring t h e i r performance i n  t r a v e l l e d and of the wet  found that sockeye smolt with p a r a s i t e s  weight of the fish, swam only  smolts of s i m i l a r s i z e .  This r e s u l t was  2/3  as f a r as  l a t e r confirmed  by  46 Boyce (1979), who found that the i n f e c t e d f i s h had s i g n i f i c a n t l y lower c r i t i c a l swimming speeds than d i d the c o n t r o l f i s h .  However, K l e i n et a l . (1969)  could not f i n d any e f f e c t s of an i n t e s t i n a l f l u k e , on the stamina of rainbow t r o u t .  Crepidostomum  farionis,  The e f f e c t on the swimming a b i l i t y which  R u s s e l l (1977) observed on the same f i s h i n f e c t e d w i t h the nematode, Truttaedacnitis  truttae,  was not s t a t i s t i c a l l y s i g n i f i c a n t .  I t i s not u n l i k e l y that during t h e i r migration to sea, and even during t h e i r residence i n l a k e s , sockeye salmon must sometimes swim against high water v e l o c i t i e s .  F i s h c a r r y i n g a s i g n i f i c a n t number of p a r a s i t e s ,  p a r t i c u l a r l y p a r a s i t e s which exert a d i r e c t e f f e c t on the r e s p i r a t o r y system, w i l l s u f f e r from a decreased a b i l i t y to produce these v a r i a t i o n s i n swimming speed required during m i g r a t i o n . The present experiment was conducted t o determine whether or not the p a r a s i t i c copepod  Salminaola  i s r e s p o n s i b l e f o r any reduction i n the  stamina of sockeye salmon during the swimming at near c r i t i c a l v e l o c i t y . 2. 2.1  M a t e r i a l s and Methods Experimental F i s h The i n f e c t e d f i s h used i n t h i s experiment came from the same stock  as those described i n Section I I .  They had been i n f e c t e d f o r about f i v e  months and c a r r i e d large numbers of  S.  californiensis.  An average of 67  of the p a r a s i t e s at various stages i n t h e i r l i f e c y c l e s was counted per f i s h . Some of the experimental f i s h were randomly sampled and examined and found free of other p a r a s i t e s and kidney disease. Both c o n t r o l and i n f e c t e d f i s h were t r a n s f e r r e d from the P a c i f i c B i o l o g i c a l S t a t i o n i n Nanaimo to the U n i v e r s i t y of B r i t i s h Columbia and kept i n separate tanks equipped w i t h running water and 12-h photoperiods. The water i n these h o l d i n g tanks was 9 ± 2°C.  The f i s h were f e d once a day to  47 s a t i a t i o n w i t h Oregon Moist P e l l e t s . 2.2  Experimental Procedures The swimming performance t e s t s were performed using a open c i r c u i t  stamina chamber c o n s i s t i n g of a 12.7 cm ID p l e x i g l a s s tube, 86 cm i n length (Jones et a l . , 1974).  A s e r i e s of three 0.3 cm mesh g r i d s a t the upstream  end of the chamber introduced microturbulence i n the p l e x i g l a s s tube, r e s u l t i n g i n an e s s e n t i a l l y f l a t v e l o c i t y p r o f i l e and allowing a maximum f l o w rate of about 100 cm/sec.  At the downstream end, an e l e c t r i f i e d g r i d (5  v o l t ) stimulated the f i s h to swim.  Fresh water was added to the system  continuously and oxygen l e v e l s i n the water were maintained a t s a t u r a t i o n by the a d d i t i o n of a i r through a i r s t o n e s i n the chamber. During the experiment, water supplied f o the chamber was 8 ± 3°C. I t was c i r c u l a t e d by a v a r i a b l e speed pump which was a l s o used to increase the v e l o c i t y of the water i n the chamber. Two types of swimming performance t e s t were performed. ved c r i t i c a l v e l o c i t y and f i x e d v e l o c i t y t e s t s 2.2.1  Critical  These i n v o l -  respectively.  Velocity  Due to the l i m i t e d number of i n f e c t e d f i s h a v a i l a b l e ,  critical  v e l o c i t y t e s t s were conducted only w i t h twenty c o n t r o l f i s h w i t h the i n t e n t i o n of e s t a b l i s h i n g a base l i n e f o r the c a l c u l a t i o n of a f i x e d v e l o c i t y used i n t e s t i n g the e f f e c t of  S.  californiensis  i n f e c t i o n on the swimming  a b i l i t y of experimental f i s h . The twenty c o n t r o l f i s h were introduced, f i v e a t a t i m e , i n t o the stamina chamber, w i t h water v e l o c i t y adjusted to 10 cm/sec. The f i s h were allowed to adapt to t h i s new environment f o r about one hour p r i o r to t e s t i n g to minimize the e f f e c t of handling (Jones et a l . , 1974). The speed of the water was then increased i n increments of about 10 cm/sec. a t 60 minute  48 i n t e r v a l s as recoimuended by B r e t t (1967) u n t i l the f i s h became f a t i g u e d or exhausted and f e l l back against the e l e c t r i c g r i d . the  As they became f a t i g u e d ,  f i s h were removed and t h e i r f a t i g u e times were recorded. T o t a l length (cm)  and weight (g) of each exhausted f i s h were measured. C r i t i c a l v e l o c i t i e s ( V - c r i t . ) were determined by i n t e r p o l a t i o n as described below f o l l o w i n g Jones et a l . (1974). V-crit. = V P  where  + (V, - V ) x t F fl f  P  = penultimate water v e l o c i t y = f i n a l water v e l o c i t y  (cm/sec)  (cm/sec)  tF = time to f a t i g u e a t V_ (sec) TI = time between v e l o c i t y increments (sec). 2.2.2  Fixed V e l o c i t y This t e s t was used to determine the e f f e c t of  SaZmincota  on the  swimming a b i l i t y of the i n f e c t e d f i s h i n comparison to that of the c o n t r o l f i s h group. The t e s t was conducted a t 90% of the c a l c u l a t e d c r i t i c a l v e l o c i t y determined f o r the twenty c o n t r o l f i s h . The duration of the t e s t was 600 min ( B r e t t , 1967). Water was s u p p l i e d to the chamber a t 8 + 3 C. C o n t r o l f i s h used i n the experiment were s e l e c t e d by s i z e such that they were s i m i l a r to i n f e c t e d f i s h , thereby m i n i m i z i n g the e f f e c t of any s i z e d i f f e r e n c e on swimming a b i l i t y (Bainbridge, 1958). T h i r t y c o n t r o l and i n f e c t e d f i s h were assigned f o r t h i s t e s t . f i s h from each group were tested a t the same time.  Three  The f i s h were starved  for one day p r i o r to the experiment to minimize the amount of excreted waste, which would increase pH and NH  3  levels.  The i n t r o d u c t o r y process was the  same as i n the c r i t i c a l v e l o c i t y t e s t except that the experiment f i s h were l e f t overnight i n the chamber.  The water v e l o c i t y was increased i n s i x  49 increments, each l a s t i n g 10 min u n t i l the t e s t i n g v e l o c i t y reached 90% of the calculated c r i t i c a l velocity.  The t e s t v e l o c i t y was maintained f o r 600 min.  Fatigued f i s h were removed very c a r e f u l l y from the chamber i n order t o prevent disturbance to other f i s h . separately.  The f a t i g u e time f o r each f i s h was recorded  For those f i s h which swam throughout the 600 min t e s t i n g time,  600 min was assigned as " f a t i g u e time."  Length and weight of each f i s h were  measured and the number of p a r a s i t e s was counted f o r each of the i n f e c t e d fish. 3.  Results The c r i t i c a l v e l o c i t i e s of the 20 f i s h were computed (Table IV) and  p l o t t e d against per cent fatigued  (Figure P). F i f t y per cent of the f i s h  f a t i g u e d at the v e l o c i t y of 70 cm/sec.  The approximate 5 and.95% f a t i g u e  l e v e l s occurred a t 40 and 87 cm/sec r e s p e c t i v e l y . The v e l o c i t y used to t e s t the d i f f e r e n c e between the swimming a b i l i ty of the i n f e c t e d and c o n t r o l f i s h groups was 90% of the c r i t i c a l v e l o c i t y determined  from the 20 c o n t r o l f i s h , i . e . 65 cm/sec.  I t i s apparent from Figure 9 that no f i s h f a t i g u e d during the f i r s t 45 min at the f i x e d v e l o c i t y (65 cm/sec).  Thereafter, i n f e c t e d f i s h  s t a r t e d to f a t i g u e e a r l i e r than c o n t r o l f i s h . forced to swim a t 65 cm/sec f o r about 250 min. c o n t r o l f i s h were f a t i g u e d .  They became 50% f a t i g u e d when At t h i s time only 7% of the  The c o n t r o l f i s h d i d not reach the 50% f a t i g u e  l e v e l u n t i l they had swum longer than 500 min.  At the end of the 600 min  t e s t i n g time, 14 c o n t r o l f i s h , compared w i t h only two i n f e c t e d f i s h , were s t i l l swimming (Table V). These r e s u l t s i n d i c a t e that the i n f e c t e d f i s h have l e s s a b i l i t y t o swim a t high v e l o c i t i e s than do the c o n t r o l f i s h . The l e v e l of i n f e c t i o n was a l s o found t o a f f e c t the swimming a b i l i t y of f i s h .  Figure 10 i n d i c a t e s a negative c o r r e l a t i o n (-.73) between time t o  TABLE IV R e l a t i o n of swimming speed to the per cent fatigued f o r 20 sockeye salmon of mean s i z e 14.47+ 3.8 cm and 26.62 ± 5.15 g tested at 8 ± 3°C f o r 400 min. V e l o c i t i e s were r a i s e d by 10 cm/sec every 60 min.  Testing v e l o c i t y (cm/sec)  40  50  60  70  80  Testing time (min)  60-120  120-180  180-240  240-300  300-360  360-400  Fatigue times (min)  115  170  195  290  315  360  225 235  295 295 298 300 300  322 340 340 346 350 355  400  66.3  79.35  86.32  95.0  Mean c r i t i c a l v e l o c i t y  49.1  58.3  Grand mean c r i t i c a l v e l o c i t y I  t'r  O f  90  72.39 (n=20)  '  Percent f i s h fatigued  5  10  25  55  90  100  51  Figure 8  C r i t i c a l v e l o c i t y of experimental  fish  C r i t i c a l v e l o c i t y of j u v e n i l e sockeye salmon obtained f r o n 20 tested f i s h , t o t a l length of 14.47 + 3.8 cm and weight 26.62 +5.15 g at 8+3°C f o r 400 min i n t e r v a l .  SWIMMING  SWIMMING  VELOCITY  VELOCITY  (L/sec)  (cm/sec)  ho  53  Figure 9  Relationships  between f a t i g u e times and % f i s h  fatigued-  Comparison of the f a t i g u e times of i n f e c t e d and c o n t r o l f i s h groups a t a f i x e d v e l o c i t y (65 cm/sec) a t 8 i 2°C w i t h 600 min t e s t i n g time. The numbers i n b r a c k e t s i n d i c a t e the numbers of f i s h s t i l l swimming at the end of t e s t i n g time. Arrow i n d i c a t e s the time when 50% of f i s h were found f a t i g u e d .  TABLE V Mean f a t i g u e  times, % f i s h f a t i g u e d and s i z e of the i n f e c t e d and c o n t r o l f i s h t e s t e d at the f i x e d ' v e l o c i t y of 65.0 cm/sec a t 8 i 2°C f o r 600 min t e s t i n g time. Infected  Lgue time  Control Size  % Fatigue L(cm)  F a t i g u e time  %',• Fatigue  Size L (cm)  W(g)  W(g)  45  3.3  14.25  20.9  164  3.3  15.7  32.4  62.5  9.9  15.41  30.5  242  6.6  14.05  27.9  86.0  16.5  14.78  24.45  260  9.9  15.41  31.9  116.25  29.7  13.9  22.9  316.5  16.5  14.25  29.7  138.0  36.3  15.1  30.8  346.0  18.8  16.02  36.8  169.0  39.6  13.9  28.42  359.4  23.1  14.73  30.5  224.0  42.9  14.5  24.48  419.7  33.0  15.41  29.8  240.5  52.8  15.0  29.6  441 .5  39.6  13.95  28.3  267.5  66.0  14.2  24.1  462  46.2  15.81  36.4  279.0  72.6  14.7  23.7  513  49.5  14.97  29.2  495.0  75.9  13.8  22.6  600  52.8  14.17  27.8  531.0  82.5  13.4  24.8  571.0  85.8  14.2  24.3  600  92.4  15.4  31.29  1  l  2 infected f i s h  2 14 c o n t r o l f i s h  s t i l l swimming at the end s t i l l swimming a t the end  2  of t e s t i n g t ime. of t e s t i n g time.  Ln Ui  56  F i g u r e 10  R e l a t i o n s h i p s between f a t i g u e time and p a r a s i t e - d a y s Regression l i n e i n d i c a t e s the r e l a t i o n s h i p s between f a t i g u e time and p a r a s i t e - d a y s d u r i n g the 600 min t e s t i n g time a t f i x e d v e l o c i t y . V e r t i c a l l i n e = standard d e v i a t i o n . P o i n t s on the top of the graph i n d i c a t e the f a t i g u e times of f i s h with average i n f e c t i o n s of about 400 p a r a s i t e - d a y s .  58 f a t i g u e and p a r a s i t e - d a y s .  Fatigue  time g r a d u a l l y d e c l i n e d when the p a r a s i t e -  days i n c r e a s e d from 640 to about 1170 (Table V I ) .  I t i s i n t e r e s t i n g to note  that no f i s h f a t i g u e d d u r i n g the p e r i o d of about 300 to 500 min and that  fish  f a t i g u e d between 500 to 600 min t e s t i n g time were found to have average number of p a r a s i t e - d a y s  of 407 ± 28.  c o r r e l a t i o n between p a r a s i t e - d a y s I t can be concluded with  This i n d i c a t e d that there i s a negative  and f a t i g u e time (Figure 10).  that when j u v e n i l e sockeye salmon, p a r a s i t i z e d  f o r 640-1230 p a r a s i t e - d a y s , are f o r c e d to swim at h i g h  Salmincola  v e l o c i t y of 65 cm/sec, the chance of s u r v i v a l f o r the i n f e c t e d f i s h i s only 6.6%. 4.  Discussion The  adverse e f f e c t s exerted on the swimming a b i l i t y of j u v e n i l e  sockeye salmon by  observed i n the present  Salnrineola  that p a r a s i t i z e d f i s h have l e s s a b i l i t y do n o n - i n f e c t e d  sockeye.  experiment demonstrated  to t o l e r a t e a c t i v e swimming than  The r e s u l t s a l s o c o n f i r m that t h i s a b i l i t y i s  r e l a t e d to the l e v e l of i n f e c t i o n . Increased  l e v e l s and degrees of i n f e c t i o n i n c r e a s e the amount of  r e s p i r a t o r y area destroyed. can be taken up by the f i s h .  T h i s , i n t u r n , l i m i t s the amount of 0 The i n c r e a s e d metabolic  with a c t i v e swimming demands an i n c r e a s e i n 0 requirements f o r 0 (Brett,  1964).  2  2  uptake.  i n c r e a s e very r a p i d l y with  Whenever the amount of 0  2  Moreover, the  2  r e s p i r a t o r y area  that can be absorbed.  r e q u i r e d by the t i s s u e exceeds the 0  f i s h f a t i g u e and e v e n t u a l l y death r e s u l t s .  associated  i n c r e a s e s i n swimming speed  However, the r e d u c t i o n i n the g i l l  o b v i o u s l y causes a r e d u c t i o n i n the amount of 0  activity  2  uptake,  Demands f o r i n c r e a s e d 0  take among p a r a s i t i z e d f i s h not only come from i n c r e a s i n g a c t i v i t y swimming) but a l s o from the s t r e s s exerted  which  2  on the f i s h host by the  2  up-  (active  TABLE VI  V a r i a t i o n i n the number of p a r a s i t e s , c a l c u l a t e d as p a r a s i t e days, found i n the f a t i g u e d  Mean f a t i g u e time mm  Mean p a r a s i t e days  45.0  1139.0  62.5  1130.5  86.0  900.0  116.25  1074.5  138.0  1170.0  169.0  960.0  224.0  750.0  240.5  982.5  267.5  922.5  279.0  640.0  495.0  410.0  531.0  365.5  571.0  413.0  600.0  440.5  Grand mean  976 ± 112  407 ±  28  fish.  parasite.  L e s t e r (1971) has found a d i f f e r e n c e between 0  2  during the a c t i v e swimming of s t i c k l e b a c k s i n f e c t e d w i t h  consumption Sohistoaephalus  and non-infected ones.  solidus  Various stresses are associated w i t h the presence and a c t i v i t y of this  parasite.  F i r s t l y , most important among these i s the d i r e c t damage  caused t o the g i l l surface area.  Secondly, r e l a t i v e t o the s i t e of i n f e c -  t i o n , there w i l l be an i n t e r f e r e n c e w i t h both the r e s p i r a t o r y and locomotory mechanisms.  The locomotory mechanism can be observed much more  c l e a r l y i n sockeye f r y than i n the j u v e n i l e because i n the former, the major s i t e s of i n f e c t i o n (about 65%) are the base of the f i n (Kabata and Cousens, 1977) and the f i n i t s e l f . fin  Interference w i t h the movement of the  or damage to the f i n w i l l upset the locomotory balance of the f i s h .  T h i r d l y , osmotic balance w i l l be upset as a r e s u l t of extensive damage to the  e p i t h e l i a of the g i l l and/or s k i n .  L e s t e r and Adams (1974) suggested  that damage to the e p i t h e l i a l c e l l s of the s t i c k l e b a c k s caused by may d i s r u p t normal f u n c t i o n s of the e p i t h e l i u m such as i t s  Gyrodaotylus  p r o v i d i n g a b a r r i e r f o r i o n i c exchange and may e v e n t u a l l y lead to the death of  the f i s h .  I f t h i s i s the case  on i t s host. + as Na , Ca  Salmincola  should have a s i m i l a r e f f e c t  In cases where there i s e p i t h e l i a l damage e s s e n t i a l ions such  ++  + and Mg  tend to escape from the e p i t h e l i a l c e l l s l e a d i n g t o  a reduction i n the t o t a l c o n c e n t r a t i o n of e l e c t r o l y t e s (Lockwood, 1964) and consequently lead to the development of osmotic s t r e s s .  Under s t r e s s  c o n d i t i o n s the adrenal cortex of f i s h i s a c t i v a t e d (Fagerlund, 1967; Hoar, 1975) r e s u l t i n g i n the release of G l u c o c o r t i c o i d s which break down p r o t e i n i n t o glucose. becomes.  The more body p r o t e i n that breaks down, the weaker the f i s h  61 In cases of heavy i n f e c t i o n  (such as i n t h i s experiment), the f i s h  w i l l be subjected to the v a r i o u s s t r e s s e s mentioned e a r l i e r . the  r e d u c t i o n i n the g i l l  ing  of the compensatory mechanism  the  a b i l i t y of the f i s h  In a d d i t i o n  s u r f a c e area, combined with i n e f f e c t i v e to m a i n t a i n 0  2  function-  uptake, d r a s t i c a l l y reduces  to cope with a c t i v e swimming.  F a t i g u e among these  f i s h occurs e a r l i e r than among those without p a r a s i t e s . Among f i s h with l i g h t  infections,  i f the area of the g i l l  i s not too great and the f i s h can make use of t h e i r compensatory normal 0  2  uptake may not be upset and f a t i g u e w i l l not occur.  swimming i s r e q u i r e d the amount of 0  2  needed by the f i s h  f i s h have to i n c r e a s e the e f f e c t i v e n e s s of t h e i r 0  ity.  When a c t i v e  i n c r e a s e s and the which  Furthermore, other  occur d u r i n g long p e r i o d s of swimming i n water of h i g h v e l o c -  P h y s i o l o g i c a l responses to these s t r e s s e s may a c t i v a t e the adrenal  c o r t e x , lead to p r o t e i n breakdown too  mechanisms,  uptake mechanisms,  2  i n t u r n , leads to an i n c r e a s e i n energy e x p e n d i t u r e . s t r e s s e s may  surface  and consequently, to the f i s h  week to cope with f u r t h e r a c t i v e metabolic a c t i v i t y .  Then  becoming fatigue  results. Because of the v a r i a t i o n  i n the degree of i n f e c t i o n  i t was not p o s s i b l e to p r e d i c t the exact c r i t i c a l  (Table VII)  f a t i g u e time f o r j u v e n i l e  sockeye salmon swimming i n highspeed water (65 cm/sec). However, a p o s i t i v e c o r r e l a t i o n between  the degree of i n f e c t i o n and f a t i g u e time was  Some of the l i t e r a t u r e  shows that p a r a s i t e s other than g i l l  s i t e s exert a marked e f f e c t on the swimming a b i l i t y solidus  p l e r o c e r c o i d on s t i c k l e b a c k  (Lester,  i n sockeye salmon (Smith, 1973; Boyce, 19 79); salmonids Bubophorus  (S.  gaivdnevi  aonfusus  and  0.  obtained.  kisutoh)  i n rainbow t r o u t  of f i s h :  1971); Nanophyetus  Eubothvium salminoola  paraSchtstoeeph&lus saZvelini  in  ( B u t l e r and Millemann, 1971) and (Fox, 1965).  The damage to the f i s h  62 host caused by these p a r a s i t e s i s not w e l l understood but Wood and Yasutake (1956) found evidence of marked o b s t r u c t i o n and mechanical i n j u r y to the heart v e n t r i c l e , muscle  f i b e r s , r e t i n a , kidney tubules, pancreatic t i s s u e  and g a l l bladder as the r e s u l t of N. salmincola  infection.  This may i n d i c a t e  that the damage i s r e l a t e d to the migration of the c e r c a r i a e through the t i s s u e s or t o i t s o b s t r u c t i o n of the i n t e r n a l organs. Salmincola,  a g i l l p a r a s i t e , can be used as a good example of how  p a r a s i t e s exert adverse e f f e c t s on the swimming a b i l i t y of t h e i r f i s h hosts. These e f f e c t s have never before been studied i n r e l a t i o n to g i l l p a r a s i t e s . The r e s u l t s of t h i s experiment should t h e r e f o r e , be u s e f u l as a g u i d e l i n e for future studies of the impact of g i l l p a r a s i t e s on the swimming a b i l i t y of f i s h .  SECTION IV IMPACT ON HOST'S SALINITY TOLERANCE  64  1.  Introduction When sockeye  ment, a profound  salmon move from a f r e s h water to a s a l t water e n v i r o n -  change takes p l a c e i n t h e i r p h y s i o l o g i c a l c o n d i t i o n .  The  t r a n s f o r m a t i o n process i s very complex. Hoar (1976) r e f e r s to smolt  t r a n s f o r m a t i o n as a seasonal phenomenon.  For q u i t e some time i t has been understood photoperiods  t h a t the seasonal p r o g r e s s i o n of  synchronized the s m o l t i f i c a t i o n process  (Baggerman, 1960).  The work of Zaugg and Wagner (1973), Wagner (1974) and Clarke et a l . (1978) emphasize the photoperiod as a main environmental onset of parr-smolt t r a n s f o r m a t i o n .  factor controlling  the  Beside the photoperiod, Zaugg and  McLain (1976) have demonstrated the important  i n f l u e n c e of temperature  on  the d u r a t i o n of the smolt phase i n y e a r l i n g coho salmon. In  order to t o l e r a t e environmental  changes, a s p e c t a c u l a r change  must take p l a c e p r i o r to commencing seaward m i g r a t i o n .  Parry (1966)  who  s t u d i e d the osmotic a d a p t a t i o n of the f i s h , r e f e r r e d to the p o s s i b i l i t y of a mechanism f o r osmoregulation salmon.  The  ronmental of  d e v e l o p i n g d u r i n g the j u v e n i l e l i f e  same author a l s o mentioned that the a b i l i t y  change i s a f a c u l t y which may  juvenile l i f e  though i t may  of the  to t o l e r a t e e n v i -  develop d u r i n g the e n t i r e p e r i o d  change suddenly p r i o r to the seaward  migration. In  a sea water environment, where s a l t c o n c e n t r a t i o n s are much  g r e a t e r than those w i t h i n the f i s h ' s body, the n a t u r a l tendency water to f l o w from the f i s h to i t s environment by osmosis. water content w i t h i n the f i s h , water must be imbibed  is for  To maintain  and excess  s a l t must  65 be e l i m i n a t e d .  The e x c r e t i o n and absorption of c h l o r i d e v i a the g i l l s i s a  matter of some i n t e r e s t . on the g i l l filament of  Copeland (1948) presented  evidence of a c e l l type  responsible f o r c h l o r i d e t r a n s f e r .  Fundulus  studies by the same author i n 1950 s e n s i t i v e to b l o o d - s a l t l e v e l s .  Further  suggested that t h i s c e l l i s l a b i l e and  According to the observation of Kessel  and  Beams (1962) these c e l l s e x i s t embedded i n the s t r a t i f i e d e p i t h e l i a l l a y e r . Datta Munchi (1964) found these c e l l s at the base of and among the secondary g i l l lamellae.  E l e c t r o n microscopic  studies by Threadgold and Houston (1964)  implicated a s p e c i a l e p i t h e l i a l c e l l r i c h i n mitochondria ble  f o r the secretory f u n c t i o n .  as being r e s p o n s i -  These c e l l s increased i n numbers at the p a r r -  smolt transformation but became reduced during the post-smolt  stage.  This  c e l l has been r e f e r r e d to many times as the " c h l o r i d e secretory c e l l . " P r i o r to m i g r a t i o n , the necessary transformation or r e o r g a n i z a t i o n of body f u n c t i o n must already have been i n i t i a t e d to a c e r t a i n degree. These processes seem to be very e f f i c i e n t i n j u v e n i l e sockeye salmon, which q u i c k l y grow to smolt s i z e and can adapt to sea water as (Kennedy et a l . , 1976).  underyearlings  Occasionally sockeye salmon f r y have been reported  migrating to the sea from r i v e r systems without associated lakes (Foerster, 1968). The studies of s a l i n i t y r e l a t i o n s h i p s can be traced back i n the l i t e r a t u r e f o r more than a century.  Changes i n the environmental s a l i n i t y  are marked and e a s i l y reproduced i n the l a b o r a t o r y , and a large volume of information relevant to s a l i n i t y preference of anadromous f i s h i s already available.  Therefore  t h i s experiment focussed on the question of whether  or not the p a r a s i t i c copepod ance to s a l i n i t y changes.  Salminoola  causes any reduction i n the t o l e r -  To my knowledge, no studies of the e f f e c t of  p a r a s i t e s on f i s h s a l i n i t y tolerance e x i s t at present.  66 2.  M a t e r i a l s and Methods  2.1.  Experimental F i s h The y e a r l i n g sockeye salmon used i n t h i s experiment came from Rose-  w a l l Hatchery.  They were t r a n s f e r r e d to the P a c i f i c B i o l o g i c a l S t a t i o n and  kept i n running water at ambient temperature.  Before the copepod l a r v a were  introduced, 10 f i s h were randomly s e l e c t e d and examined, and found to be free of p a r a s i t e s and b a c t e r i a l kidney disease. Then the f i s h were d i v i d e d i n t o two groups w i t h approximately 200 f i s h i n each, and were put i n t o two separate tanks. A l a r g e number of copepod l a r v a (hatched i n the laboratory) were introduced to  the f i s h chosen to be " i n f e c t e d f i s h group". Both i n f e c -  ted and c o n t r o l f i s h were kept (approximately 2 1/2 months) i n running water at ambient temperature u n t i l the experiment s t a r t e d . 2.2.  Experimental Procedures Even though the sockeye salmon were found to be able to adapt to sea  water as underyearlings (Kennedy et a l . , 1976), i n order to reduce any problems which might occur as a r e s u l t of incomplete s m o l t i f i c a t i o n , the experimental f i s h , both c o n t r o l and i n f e c t e d , were held i n tanks equipped w i t h a r t i f i c i a l l i g h t ( d i f f u s e d incandescent lamps at the top of the tanks) to c o n t r o l photoperiods. There were four sequential photoperiods (12-h, 14-h, 16-h and 17-h) e a c h " l a s t i n g two weeks.  A f t e r eight weeks, the f i s h were kept  on a long d a y l i g h t photoperiod (17-h) u n t i l the experiment was s t a r t e d at.the beginning of June.  The water i n the h o l d i n g tanks was c o n t r o l l e d at 12 ± 3°C  by mixing ambient and heated water w i t h a flow rate of 40 gal/h.  The f i s h  were fed once a day to s a t i a t i o n w i t h Oregon Moist P e l l e t s . Two types of experiments were conducted to t e s t whether the p a r a s i t i c copepod has any a d d i t i o n a l e f f e c t s on j u v e n i l e sockeye salmon while they are m i g r a t i n g : the t e s t s were a s a l i n i t y tolerance t e s t and a s a l i n i t y preference test.  67 2.2.1.  S a l i n i t y Tolerance Test This t e s t was performed to see whether  a f f e c t s the a b i l i t y  Salmineola  of sockeye salmon to withstand s a l i n i t y changes.  I t was conducted from the  beginning of June to the middle of J u l y i n e i g h t small c y l i n d r i c a l  tanks.  The f r e s h and s a l t water supplies f o r the experimental tanks came from separate pipes and were mixed i n a connecting pipe before being passed i n t o the temperature  controller.  Each pipe had i t s own valve f o r a d j u s t i n g  t  I . the amount of water r e q u i r e d f o r each experimental p e r i o d . supplied with water at a constant temperature  of  12°C.  A l l of the experimental tanks were set outdoors. of the experiment  The tanks were  At the beginning  the tanks were supplied w i t h f r e s h running water at a  constant flow r a t e of 60 gal/h from the top of the tank.  The overflow  water was drained out through the overflow p i p e , each tank therefore o b t a i n ing new water at a l l times.  Ten i n f e c t e d f i s h and ten c o n t r o l f i s h were  introduced together i n t o each of the experimental tanks and l e f t to adjust to the new environmental c o n d i t i o n s f o r one week.  By the gradual a d d i t i o n  of sea water, the s a l i n i t y was then adjusted at increments of 5%»per f i v e day i n t e r v a l s , u n t i l i t reached  100%  sea water (about 28-30%^).  The  fish  were maintained i n f u l l strength sea-water f o r 15 days. Observations on f i s h behavior as water s a l i n i t y increased were made during the f i v e hours a f t e r each increment and during a two hour period (between 1.00-3.00 pm) on the remaining four days of each f i v e day p e r i o d . Salinity  tolerance was measured by recording the time of death, the  m o r t a l i t y then being expressed i n terms of % cumulative m o r t a l i t y . 2.2.2.  S a l i n i t y Preference Test This t e s t was conducted using a method modified a f t e r Houston (1957)  i n an aquarium (18x32x18 i n . ) which was d i v i d e d i n t o two compartments of  68 equal volume by a c e n t r a l p a r t i t i o n  14 i n . h i g h .  A water " b r i d g e " 2.5 i n .  h i g h was c r e a t e d over the c e n t r a l p a r t i t i o n e n a b l i n g the f i s h to move between the compartments.  The aquarium was s e t i n a l a r g e r o v a l tank i n which  running water of constant temperature  (10°C) was kept a t 1/2 the h e i g h t of  the aquarium to i n s u r e that the water temperature constant throughout  the experiment.  i n the aquarium was kept  The s i d e s of the aquarium were covered  with b l a c k p l a s t i c to prevent d i s t u r b a n c e s and the top was covered with a g l a s s cover to a l l o w o b s e r v a t i o n of the d i s t r i b u t i o n of the f i s h . The  freshwater compartment was f i l l e d  t i t i o n with f r e s h water and the second  almost  to the top of the par-  compartment with sea water f o r the  experimental, and fresh'water f o r the c o n t r o l group.  Four r e p l i c a t i o n s were  made f o r the experimental and two f o r the c o n t r o l experiment. experiment,  five  i n f e c t e d and f i v e n o n - i n f e c t e d f i s h were put i n t o the f r e s h  water compartment and l e f t over n i g h t . to  For each  The next day water was slowly added  the bottom of the f r e s h water compartment through a g l a s s tube u n t i l a  water b r i d g e 2.5 i n . h i g h was e s t a b l i s h e d over the c e n t r a l p a r t i t i o n . the c o n t r o l experiment, with f r e s h water.  both o r i g i n a l and a l t e r n a t e compartments were  For filled  I l l u m i n a t i o n was a t the c e n t e r of the room about 20 f e e t  from the t e s t i n g aquarium; i t had no n o t i c e a b l e e f f e c t on the behavior of the  fish. The number of f i s h  i n each compartment was determined  at  30, 60, 90, 120, 180 and 240 min.  or  control,  The percentage  f o r s i x periods  of f i s h , e i t h e r  infected  i n the seawater compartment was taken as a measure of the  p r e f e r e n c e of the f i s h f o r seawater. 3. 3.1.  Results S a l i n i t y Tolerance Test Observations on the behavior of the f i s h a f t e r each p e r i o d of i n -  creasing s a l i n i t y  i n the experimental  tank i n d i c a t e d that both  i n f e c t e d and  69 non-infected  f i s h swam i n a normal f a s h i o n , w i t h no s t r u g g l i n g or f a t i g u e ,  when the s a l i n i t y was between  0-15%o.  i n f e c t e d f i s h group at t h i s time.  Only 1.5% m o r t a l i t y occurred i n the  A f t e r the s a l i n i t y was adjusted t o 20%.,  some of the i n f e c t e d f i s h appeared t o lose t h e i r e q u i l i b r i u m ; a c t i v e swimming and s t r u g g l i n g appeared o c c a s i o n a l l y .  At the end of the f i v e day period  20% water s a l i n i t y , 22.5% m o r t a l i t y was found among the i n f e c t e d f i s h o  ure 11, Table VII) as compared to only 1.5 among the non-infected  with  (Fig-  group.  The m o r t a l i t y i n the i n f e c t e d group increased r a p i d l y t o 43.5% and then to 61.5% when the s a l i n i t y increased  from 20%. to 25%. and then to the f u l l  strength of seawater (28-30%. s a l i n i t y ) .  At t h i s p o i n t , the m o r t a l i t y among  the c o n t r o l f i s h reached only 4.5%. At the end of the experiment when the s a l i n i t y was 28-30%. only 10% of the i n f e c t e d f i s h survived, as compared w i t h a s u r v i v a l rate of almost 90% among the non-infected  fish.  The r e s u l t s of t h i s experiment show that  non-parasitized  sockeye salmon have a high a b i l i t y to t o l e r a t e s a l i n i t y changes. t i o n w i t h an average of 24  Salminoola  juvenile Parasitiza-  per f i s h reduced t h i s a b i l i t y by  approximately 14%. 3.2.  S a l i n i t y Preference Test General observations on the behavior of f i s h were made, f i r s t l y , i n  the c o n t r o l experiment.  I n i t i a l l y , both i n f e c t e d and non-infected  a marked tendency to remain i n the o r i g i n a l compartments.  f i s h had  An hour a f t e r the  water bridge was formed, they began to swim back and f o r t h , the f i n a l response being approximately the same i n both groups of f i s h (Table V I I I ) . However, i n the experimental group, both i n f e c t e d and non-infected  f i s h were  r e l a t i v e l y i n a c t i v e during the p r e l i m i n a r y r e s t i n g period before the water l e v e l i n the f r e s h water compartment was increased; a few i n f e c t e d f i s h were swimming slowly c l o s e to the water surface.  When the water bridge was  70  Figure 11  Percent cumulative m o r t a l i t y i n r e l a t i o n to s a l i n i t y Per cent cumulative m o r t a l i t y of i n f e c t e d and c o n t r o l f i s h during the course of s a l i n i t y changes. The f i s h remained i n f u l l strength sea water (28-30% ) f o r 15 days. o  SALINITY 5 1  10 1  15 •  20 1  25 1  %o 1  28-30.  TABLE VII  Percent  cumulative  m o r t a l i t y of i n f e c t e d and n o n - i n f e c t e d  Time  Salinity  days  %  % Cumulative M o r t a l i t y  Mean l e n g t h cm Non-infected  f i s h during the s a l i n i t y  Infected  5  5  -  10  10  -  15  15  -  14.2  20  20  13.7  13.6  25  25  -  28-30  30  28-30  Non-infected  1  Infected  tolerance test  Mean p a r a s i t e per  fish  1.5  23.2  1.5  22.5  22.9  13.4  1.5  43.5  25.6  14.1  14.7  4.5  61 .5  24.2  35  13.2  13.5  6.0  65.5  20.7  28-30  40  14.9  14.0  9.0  88.5  29.2  28-30  45  13.3  14.0  10.5  90.0  23.4  T o t a l number of t e s t f i s h = 80 *LT 50 occurred between the s a l i n i t y 1  of 25-28%„  TABLE VIII  S a l i n i t y preference  :ing  test for  Salinity %  period  i n f e c t e d and n o n - i n f e c t e d  l  j u v e n i l e sockeye salmon at 12°C  Percentage of f i s h i n the s a l t water compartments  Percentage of f i s h i n the a l t e r n a t e f r e s h water 2  Min  FC  SC  Non-infected  Infected  Non-infected  Infected  0-30  6  25  10  10  20  10  30-60  8  22  30  25  20  10  60-90  9  20  25  15  50  20  90-120  10  19  65  40  20  50  120-180  12  18  60  25  60  40  180-240  13  17  75  25  40  60  FC = f r e s h water compartment SC = s a l t water compartment = s a l i n i t y i s measured at the center of each compartment = c o n t r o l experiment 1  2  CO  74 established,  the i n f e c t e d f i s h c r o s s e d the c e n t r a l p a r t i t i o n t o the sea water  before the c o n t r o l non-infected f i s h  f i s h d u r i n g the i n i t i a l o b s e r v a t i o n .  swam back and f o r t h across the c e n t r a l p a r t i t i o n .  sionally, darting, interrupted  Both i n f e c t e d and  perhaps r e s u l t i n g from the i n t e r f e r e n c e  Occa-  by the p a r a s i t e s ,  the normal behavior of the f i s h ; t h i s l a s t e d f o r a few minutes  b e f o r e the f i s h r e t u r n e d to the normal r e s t i n g  state.  The number of f i s h , e i t h e r i n f e c t e d or n o n - i n f e c t e d , i n the a l t e r nate s a l t water compartment and the i n t e n s i t y of the i n f e c t i o n f o r the i n f e c t e d f i s h were recorded a t the end of each t e s t i n g p e r i o d . are  shown i n Table V I I I .  experiment.  S i m i l a r c a l c u l a t i o n s were made f o r the c o n t r o l  The percentage of n o n - i n f e c t e d f i s h  ment g r a d u a l l y  increased.  The r e s u l t s  i n the s a l t water  At the end of the experiment  compart-  (240 min t e s t i n g  time) about 75% of the n o n - i n f e c t e d f i s h appeared i n t h i s compartment. i n f e c t e d f i s h seemed to avoid f i s h remained s u c c e s s f u l l y 4.  h i g h s a l i n i t y water.  i n the a l t e r n a t e  The  Only 25% of the i n f e c t e d  s a l t water  compartment.  Discussion Because of t h e i r w e l l  developed a b i l i t y  to osmoregulate salmonid  f i s h can move from f r e s h to s a l t water w i t h great e f f i c i e n c y . (1966) demonstrated t h i s a b i l i t y water.  The f i s h  Cpnte et a l .  i n coho salmon by immersing them i n sea  i n that experiment s u c c e s s f u l l y adapted to sharp  increases  i n osmotic c o n c e n t r a t i o n from 143 to about 400 mOsmol f o l l o w e d by a r e t u r n to near f r e s h water l e v e l s . This salmon  suggests that m o r t a l i t y  (Figure  stress.  11)  among the n o n - i n f e c t e d j u v e n i l e  i n the present experiment does not r e s u l t from osmotic  I t may, however, be a t t r i b u t e d  established  i n the present  that  to the absence of food, a c o n d i t i o n  experiment.  The d r a s t i c increase indicates  sockeye  i n the m o r t a l i t y  of t h e " i n f e c t e d  f i s h probably  t h e i r e f f i c i e n c y to t r a v e l from f r e s h to s a l t water i s  75 disrupted.  T h i s can be a t t r i b u t e d to a r e d u c t i o n i n t h e i r a b i l i t y to osmo-  r e g u l a t e as w i l l be d i s c u s s e d below. It  has been r e p o r t e d that the development of the a b i l i t y of anadromous  f i s h to osmoregulate. i n sea water depends upon a p r i o r p h y s i o l o g i c a l metamorphosis  r e f e r r e d to as the p a r r - s m o l t t r a n s f o r m a t i o n (Black, 1951; Houston,  1961; Houston and Threadgold,  1963).  Apart from environmental  changes,  r e g u l a t i o n of osmotic and i o n i c c o n c e n t r a t i o n s i n the b l o o d appears one of the most important f a c t o r s i n t h i s t r a n s f o r m a t i o n .  t o be  The mechanism  (chloride-secreting c e l l s ) involved i n this regulation l i e s p r i n c i p a l l y  within  the g i l l  abil-  ity  structure.  Any damage to the g i l l  would probably d i s r u p t that  l e a d i n g e i t h e r to a decrease i n the number o f these c e l l e s or to the  hinderance of t h e i r e f f i c i e n c y i n performing t h e i r s e c r e t o r y f u n c t i o n . thermore  such damage may prevent the e l e v a t i o n of g i l l  Fur-  Na, K s t i m u l a t e d  ATPase a c t i v i t y which has been r e l a t e d to seaward m i g r a t i o n (Baggerman, 1960; Otto and Mclnery, Severe the normal  1970; Zaugg and McLain,  1972).  i n j u r y to the e p i t h e l i a l c e l l s , g i l l s and s k i n , would upset  e p i t h e l i a l c e l l f u n c t i o n s , such as a c t i n g as an i o n b a r r i e r and  may e v e n t u a l l y lead to a decrease i n the c o n c e n t r a t i o n o f e l e c t r o l y t e s , most i m p o r t a n t l y of sodium, which p l a y a key r o l e t r a n s m i s s i o n of s t r e s s s i g n a l s from the b r a i n the case, the f i s h would be unable F i s h s i z e appears 1957).  i n the normal  (Baker,  1966) .  neuroIf this i s  to endure the s t r e s s e s .  to r e l a t e to the process of s m o l t i f i c a t i o n  (Elson,  Wagner et a l . (1969) made a very i n t e r e s t i n g o b s e r v a t i o n i n t h e i r  study of osmotic and i o n i c r e g u l a t i o n i n chinook salmon. were growing more r a p i d l y  The salmon, which  than normal, possessed r e g u l a t o r y systems which  were e i t h e r more f u n c t i o n a l with r e s p e c t to a given s a l t g r a d i e n t or capable of  being i n i t i a t e d more q u i c k l y  i n response  to changes i n environmental  76 salinity.  Hoar (1976) agreed w i t h the conclusions i n these e a r l i e r papers  that l a r g e r f i s h are more r e s i s t a n t to s a l i n i t y change and that the onset of smolt c h a r a c t e r i s t i c s i s s i z e dependent.  P a r a s i t i z e d f i s h are smaller than  non-infected f i s h of the same age, thus, the parr-smolt transformation w i l l be delayed i n these f i s h , h i n d e r i n g t h e i r a b i l i t y to t o l e r a t e s a l i n i t y changes during seaward m i g r a t i o n . Considering a l l the p o s s i b i l i t i e s mentioned above, i t seems l i k e l y that an incomplete parr-smolt transformation process may occur i n the i n fected f i s h .  Therefore, during the course of the s a l i n i t y tolerance t e s t  the i n f e c t e d f i s h might not have the a b i l i t y to osmoregulate e f f e c t i v e l y . This would have lead to an osmotic c o n c e n t r a t i o n d i f f e r e n c e between the blood and the water.  Again energy expenditure  i s demanded t o meet the  upset i n homeostasis. Severe i r r i t a t i o n to the f i s h e i t h e r from the p a r a s i t e s or from other causes tends to increase the a c t i v i t y of the f i s h .  This i s suggested  by the f a c t that during the s a l i n i t y tolerance t e s t s , dash swimming was often observed.  Again t h i s would lead to an increased 0  i n f e c t e d f i s h are normally l e s s able to meet.  2  demand, which the  The t h r e s h o l d number of  p a r a s i t e s needed to produce t h i s severe i r r i t a t i o n was not measured but the average l e v e l of 24  Salmincola  d r a s t i c a l l y increased the m o r t a l i t y r a t e  among the i n f e c t e d f i s h during a s a l i n i t y tolerance t e s t .  Until better  r e s u l t s are obtained, t h i s number can presumably be used as the number of p a r a s i t e s needed to c r i t i c a l l y l i m i t the a b i l i t y of sockeye salmon to t r a n s f e r from f r e s h water to s a l t water. An incomplete parr-smolt transformation process i n the i n f e c t e d f i s h i s confirmed by the avoidance a c t i v i t y observed among those i n f e c t e d f i s h during the s a l i n i t y preference t e s t .  This r a i s e s the i n t e r e s t i n g question  77 of what f a c t o r or f a c t o r s r e a l l y have the g r e a t e s t  impact on t h i s  T h i s q u e s t i o n would have to be c o n s i d e r e d s e p a r a t e l y future  investigation.  readiness.  and q u a n t i f i e d  in a  SECTION V IMPACT ON BLOOD  79  1.  Introduction Among important  h e a l t h , hematological tology  s t u d i e s have r e c e i v e d p a r t i c u l a r a t t e n t i o n , f i s h hema-  i s becoming an i n c r e a s i n g l y u s e f u l t o o l f o r the f i s h e r y b i o l o g i s t and  research imental  i n d i c a t o r s o f the e f f e c t s of the environment on f i s h  ichthyologist.  To  e s t a b l i s h the b a s i c f i t n e s s of f i s h f o r exper-  purposes, Cope (1961) suggested s e v e r a l observations  hematological  measurements, should  be made.  S i m i l a r l y , Johnson  suggested that with the s t a n d a r d i z a t i o n of techniques, normal values  i n c l u d i n g some (1968)  procedures, and  f o r v a r y i n g environmental c o n d i t i o n s and l a b o r a t o r y  f i s h hematology may be used f o r a v a r i e t y of s t u d i e s l a t i o n s i n e x i s t i n g and changing environmental Slicher  (1961) and B a l l  i n c l u d i n g that of popu-  conditions.  and S l i c h e r (1962) used blood  as an e x c e l l e n t  i n d i c a t o r of p h y s i o l o g i c a l responses i n e n d o c r i n o l o g i c a l s t u d i e s . (1950) has used blood  counts to evaluate  that the number of e r y t h r o c y t e s Bouck and B a l l  conditions,  Katz  the d i e t s of f i s h on the grounds  responds q u i c k l y to some d i e t a r y d e f i c i e n c i e s .  (1966) found hematology a u s e f u l t o o l f o r monitoring s t r e s s  l e v e l s i n f i s h exposed to a q u a t i c  pollution.  niques s u b s t a n t i a l l y help with d i f f e r e n t i a l  Because hematological  diagnosis,  tech-  e s p e c i a l l y i n cases  with s i m i l a r c l i n i c a l p i c t u r e s , numerous f i s h b i o l o g i s t s have employed hematological The  procedures to assess the c o n d i t i o n of the f i s h . determination  of blood  Estimates of the number of blood  parameters i n v o l v e s s e v e r a l  techniques.  c e l l s have been made by v i s u a l count  (Shaw, 1930; Hesser, 1960 and Mulcahy, 1970).  Because of the v a r i o u s  disadvantages i n t h i s approach however, modern e l e c t r o n i c methods have taken over i n human hematology and the same procedure i s now g e n e r a l l y used  80 i n f i s h hematology. o f t e n used  Along with c e l l counts, hemoglobin d e t e r m i n a t i o n i s  to analyze blood c o n d i t i o n s and  d e t e c t i n g anemia.  A l l the methods used  i t i s . t h e simplest method f o r  f o r hemoglobin determination i n human  blood have been t r i e d with the f i s h b l o o d .  At p r e s e n t , cyanmethemoglobin i s  c o n s i d e r e d to be the method of c h o i c e (Larsen and 1964;  Snieszko,  1961;  Larsen,  and Muleahy, 1970). M i c r o h e m a t o c r i t values are n e a r l y always employed as a h e m a t o l o g i c a l  index s i n c e o n l y a small amount of blood i s needed f o r the d e t e r m i n a t i o n . In a d d i t i o n to red blood c e l l c h a r a c t e r i s t i c s , white b l o o d c e l l s have been used with accuracy  i n monitoring s t r e s s response  S o i v i o and O i k a r i ,  1976  and McLeay and Gordon, 1'977), i n a s s e s s i n g the h e a l t h  of  1972  and Hickey,  fish  (Blaxhall,  ance of f i s h  (Watson et a l . , 1956  that the white blood c e l l count environmental The of  1976), and  (Swift and L l o y d ,  i n assessing disease  and C o r b e l , 1975),  i s a more r e l i a b l e  Belova  1974;  resist-  (1966)  suggested  i n d i c a t o r of u n f a v o r a b l e  c o n d i t i o n s and s t r e s s e s than other h e m a t o l o g i c a l  parameters.  r o l e of white blood c e l l types i n the h e m a t o l o g i c a l defense mechanism f i s h and  the e f f e c t s of environmental  s t r e s s e s on the outbreak  are becoming i n c r e a s i n g l y more c l e a r (Snieszko, 1974;  of d i s e a s e  C o r b e l , 1975;  Ellis  et a l . , 1976). However, e v a l u a t i o n of the nature and extent of these i n f l u ence are hampered by c o n t r a d i c t i o n s i n the terminology encountered l i t e r a t u r e . These c o n t r a d i c t i o n s can be a t t r i b u t e d mainly  to the s t a i n i n g  techniques used. However, advances i n immunology, i n e l e c t r o n and i n s t a i n i n g  techniques have suggested  i n the  microscopy  that these c o n t r a d i c t i o n s may  soon be r e s o l v e d . Terminology Formerly,  r e g a r d i n g the white blood c e l l s has become l e s s c o n f u s i n g .  i t had been g e n e r a l l y agreed  were very s i m i l a r m o r p h o l o g i c a l l y .  that lymphocytes and  Ferguson.  thrombocytes  (1976), however, has  r e c e n t l y that lymphocytes and thrombocytes are not only d i s s i m i l a r  demonstrated  81 m o r p h o l o g i c a l l y , but are a l s o not r e l a t e d developmentally.  Some c o n t r a -  d i c t i o n s i n terminology and consequently even a q u e s t i o n about of some of the c e l l s remained.  McKnight  the e x i s t e n c e  (1966) claimed that no e o s i n o p h i l s  or monocytes were found i n the f i s h he examined but L e s t e r and D a n i e l s (1976) demonstrated  the occurrence of these c e l l s , u s i n g . e l e c t r o n microscopy.  On c o n s i d e r i n g the many r e p o r t s on the occurrence of white b l o o d c e l l s and the terminology used  to d e s c r i b e them, i t seems that most workers  a s s i s t e d by the d e s c r i p t i o n s a v a i l a b l e i n the l i t e r a t u r e have independantly developed  t h e i r own  terminology.  There  d e f i n i t i o n s f o r most of the c e l l s .  i s no general agreement upon  However, because of the noted  and r a p i d i t y of response of the white blood c e l l s to any  specific  sensitivity  environmental  changes, they have r e c e i v e d much a t t e n t i o n from many r e s e a r c h e r s .  Some of  them have used f i s h b l o o d to evaluate the nature of the e f f e c t s of p a r a s i t e s and d i s e a s e s on f i s h  (Watson e t a l . ,  Mawdesley-Thomas, 1969;  Carbery,  The present experiments crustacean p a r a s i t e 2.  1970  1956;  Weinreb, 1958;  and Einszporn-Orecka,  attempted  1969;  1970).  to study the e f f e c t s of the  on the blood of sockeye  Salnrinaola,  Enomato,  salmon.  M a t e r i a l s and Methods  2.1.  Experimental  Design  T h i s experiment  has  the same experimental design as d e s c r i b e d i n  S e c t i o n I. 2.2.  Experimental F i s h The f i s h used f o r h e m a t o l o g i c a l determinations were the same group  as was 2.3.  used f o r the growth study d e s c r i b e d i n d e t a i l Blood Sampling  i n S e c t i o n I.  Procedure  F i s h were removed at random one at a time from the experimental  tanks  with a small net and p l a c e d i n a 1:10,000 s o l u t i o n of the a n e s t h e t i c MS-222 (Smith and B e l l ,  1967)  f o r f i v e minutes.  A f t e r removal  from the a n e s t h e t i c ,  82 they were b l o t t e d dry with a paper The blood was dry,  towel.  then removed by  1 ml. s y r i n g e (needle s i z e  1/2"  the f o l l o w i n g syringe method. no.  A clean,  26 gauge) i s i n s e r t e d through  the  musculature  i n the area between the d o r s a l and adipose f i n s and j u s t above  the l a t e r a l  line.  vertebrae.  The needle  In t h i s p o s i t i o n , the needle w i l l  i s then c a r e f u l l y lowered u n t i l  a o r t a which l i e s under the v e r t e b r a l column c a p i l l a r y a c t i o n i n t o the s y r i n g e . i s s h i e l d e d from  i n i t i a l l y contact i t touches  (Figure 12).  T h i s technique  the  the d o r s a l  Blood i s drawn by  ensures  that the blood  contamination.  Various blood a n t i c o a g u l a n t s have been used by other r e s e a r c h e r s : oxalate c r y s t a l oxalate  (McCay, 1929), ammonium o x a l a t e ( S c h l i c h e r ,  ( F i e l d e t a l . , 1943), sodium c i t r a t e  1970), and h e p a r i n  (Yokayama, 1947;  Hesser,  i s h i g h l y recommended f o r f i s h b l o o d . t h e r e f o r e used i n a l l blood sampling The was  first  1927), sodium  (Catton, 1951), EDTA (Mulcahy, 1960;  Summerflet, 1967).  H e p a r i n i z e d c a p i l l a r y tubes were to delay blood  drop of blood from the s y r i n g e was  clotting. discarded.  One  then smeared on each of three s l i d e s f o r the d i f f e r e n t i a l c e l l  The b l o o d l e f t blood c e l l  i n the s y r i n g e was  count,  drop count.  transferred into micropipettes for t o t a l  i n t o S a h l i p i p e t t e s f o r hemoglobin determination and  m i c r o c a p i l l a r y tubes 2.4.  Heparin  f o r hematocrit  into  determination.  S t a i n i n g Technique The  smeared s l i d e s were l e f t o v e r n i g h t  with a M o d i f i e d Wright-Giemsa s t a i n  ( a i r d r i e d ) and  then s t a i n e d  ( H a l i c o D i f f Quik Stain) as d e s c r i b e d  below. The  s l i d e s were f i r s t  dipped 'into a f i x a t i v e  68451 A) f o r 5 one-second d i p s . s l i d e s was  allowed  68451 B).  T h i s procedure  Any  solution  ( D i f f Quik no.  e x c e s s i v e f i x a t i v e s o l u t i o n on  the  to d r a i n before immersion i n t o s o l u t i o n I ( D i f f Quik was  repeated with s o l u t i o n I I ( D i f f Quik no.  no.  83  Figure  12  Blood Sampling  Technique  T h i s f i g u r e shows the needle i n s e r t e d through the muscle t i s s u e and p e n e t r a t i n g the d o r s a l a o r t a . When the needle i s i n s e r t e d i n t o the b l o o d v e s s e l , the blood w i l l be drawn through c a p i l l a r y a c t i o n .  85 68451 C).  The  s l i d e s were dipped 5 times  i n t o each one  of these two  t i o n s i n a f a s h i o n s i m i l a r to the d i p p i n g i n the f i x a t i v e  solu-  solution.  Imme-  d i a t e l y a f t e r d i p p i n g i n t o s o l u t i o n I I , the s l i d e s were r i n s e d i n d i s t i l l e d water (pH 6.8)  f o r 1 min.  and allowed  to dry.  I n c r e a s i n g the number of d i p p i n g s i n t o S o l u t i o n s I and  I I , as  recommended by the manufacturer f o r b e t t e r s t a i n i n g of e o s i n o p h i l s and basop h i l s of human b l o o d , was  t r i e d w i t h some smeared s l i d e s .  improvement i n d i f f e r e n t i a t i o n was tried  noted.  i n order to i d e n t i f y the white b l o o d Wright's  The  However, no  f o l l o w i n g s t a i n s were a l s o  cells  stain  Giemsa s t a i n Pappenheim s t a i n Peroxidase B u f f y coat was  tests.  a l s o smeared on g l a s s s l i d e s which were s t a i n e d i n  H a l i c o D i f f Quik S t a i n and a l s o i n the other s t a i n s mentioned above. 2.5.  Hematological  Determinations  2.5.1. Hemoglobin C o n c e n t r a t i o n Owing to i t s e x t e n s i v e use 1961;  Larsen,  1964;  f o r hemoglobin (Hb) f o r Hb  Mulcahy, 1970) determination  d e t e r m i n a t i o n was  Drabkin's  i n f i s h hematology  (Larsen and  the Cyanmethemoglobin method was i n t h i s experiment. diluent  Sodium b i c a r b o n a t e  The  Potassium  ferricyanide  s o l u t i o n used  1.0  3  cyanide  used  solution  (NaHC0 )  Potassium  Snieszko,  (KCN)  ' (K Fe(CN)g) 3  D i s t i l l e d water to make 1000  0.05 0.2  g. g. g.  ml.  F e r r i c y a n i d e converts hemoglobin i r o n from the f e r r o u s to the s t a t e to form methemoglobin, which combines with potassium the s t a b l e cyanmethemoglobin.  cyanide  ferric  to produce  The a b s o r p t i o n values of the hemoglobin from  86 t h i s s o l u t i o n were measured w i t h a p h o t o e l e c t r i c c o l o r i m e t e r u s i n g an a b s o r p t i o n band i n the r e g i o n of 540 nm. The Hb c o n c e n t r a t i o n ( g / d l of blood) was obtained by c a l i b r a t i o n from a prepared  standard curve.  The standard curve was e s t a b l i s h e d by d i -  l u t i n g Hycel Cyanmethemoglobin Standard  (Hycel no. 117) with Hycel Cyanmethe-  moglobin Reagent to get Hb concentratons  of 5, 10, 15 and 20 g/dl and  measuring the absorbance of each d i l u t i o n a t 540 nm.  The absorbance of  each standard was then p l o t t e d a g a i n s t i t s c o n c e n t r a t i o n . The hemoglobin c o n c e n t r a t i o n of a blood sample was measured by d i l u t i n g 0.02 ml. of blood from a S a h l i p i p e t t e i n t o 5 ml. of cyanmethemoglobin reagent, mixing to stand a t room temperature themoglobin.  10 ml. c u v e t t e s c o n t a i n i n g  the s o l u t i o n w e l l and a l l o w i n g i t  f o r 5 minutes to permit the formation of cyanme-  A subsample was then t r a n s f e r r e d to a smaller c u v e t t e , i n which  the absorbance was measured a t 540 nm. and compared to the standard  curve.  T h i s gave the hemoglobin c o n c e n t r a t i o n i n g / d l . 2.5.2  Hematocrit  Value  H e p a r i n i z e d m i c r o c a p i l l a r y tubes 0.2 ± 0.02 mm. w a l l ) approximately m i c r o c e n t r i f u g e a t 3000 rpm.  (77 mm.  i n l e n g t h 1.1-1.2 mm. ID,  3/4 f i l l e d with blood, were spun i n a  F i v e tubes were used  f o r each sampled  fish.  The hematocrit values were then computed u s i n g the f o l l o w i n g formula: Hematocrit  value  (%) = ^ L  x 100  2  where L_ i s the h e i g h t of packed r e d c e l l s the t o t a l b l o o d specimen.  The grey-white  packed r e d c e l l s was i n c l u d e d i n L_.  i n mm. and L layer  2  i s the h e i g h t of  (buffy l a y e r ) above the  The mean of the 5 samples was c a l -  culated . 2.5.3.  E r y t h r o c y t e Osmotic F r a g i l i t y Test The osmotic  of the r e d c e l l s .  fragility  t e s t p r o v i d e s an i n d i c a t i o n of the f r a g i l i t y  Suspended i n a hypotonic  s o l u t i o n of sodium c h l o r i d e r e d  87 cells  take up water, s w e l l , become spheroid and, a f t e r r e a c h i n g a c r i t i c a l  volume, e v e n t u a l l y b u r s t . F i s h used f o r t h i s experiment were not the same as those d e s c r i b e d i n s e c t i o n V. the  They came from the same stock but they were i n f e c t e d w i t h  copepodid l a r v a 2 months p r i o r to the experiment.  Only 100 f i s h were  assigned to each of the experimental tanks ( c o n t r o l and i n f e c t e d f i s h were kept i n separate tanks a t the same temperature of 9 ° C ) . The average  fish  s i z e and weight a t the end of 2 months were 17.83 ± 1.97 cm, and 59.5 ± 4.3 g  f o r the c o n t r o l and 16.46 ± 2.01 cm and 55.47 ± 5.0 g  f e c t e d group.  f o r the i n -  The average number of p a r a s i t e s per f i s h was 23.9 ± 4.6 and  547 ± 48 p a r a s i t e - d a y s . Blood was sampled u s i n g the technique d e s c r i b e d e a r l i e r , although the  s y r i n g e and needle s i z e had to be changed  to 3 1/2 ml. and 25 gauge  r e s p e c t i v e l y a c c o r d i n g to f i s h s i z e and the amount of blood r e q u i r e d f o r the  test. For  the q u a n t i t a t i v e method of measuring osmotic f r a g i l i t y ,  sodium  c h l o r i d e s o l u t i o n s of 0.85, 0.75, 0.65, 0.60, 0.55, 0.50, 0.45, 0.35, 0.30, 0.20, 0.10 and 0 percent were made from 10% sodium c h l o r i d e at pH 7.4. F i v e ml. of each s o l u t i o n was t r a n s f e r r e d i n t o blood was added to each of the tubes.  13 tubes and 0.05 ml. of  The tubes were immediately mixed  and c e n t r i f u g e d a t 3000 rpm. f o r 5 minutes. The degree of hemolysis was recorded by measuring the a b s o r p t i o n values of the mixed d i l u t i o n , photometer  (further diluted  1 to 5) u s i n g a s p e c t r o -  a d j u s t e d to a wave length, of 540 nm. 7  The % hemolysis was o b t a i n e d by comparing a standard tube of d i s t i l l e d water  the experimental tube with  i n which hemolysis was 100%.  20 i n f e c t e d f i s h was compared with the b l o o d of 20 c o n t r o l  fish.  Blood from  88 2.5.4.  C l o t t i n g Time T h i s experiment was done f o l l o w i n g the osmotic f r a g i l i t y  the  remaining experimental f i s h from that t e s t  3 months).  test  using  (these f i s h had been i n f e c t e d  The average number of p a r a s i t e s was 29 ± 4.5 per f i s h , w h i l e the  average length and weight were 17.92 ± 1.73 cm and 58.91 ± 3.6 g f o r the con t r o l and 16.20 ± 2.17 cm and 53.24 ± 4.9 g f o r the i n f e c t e d group P l a i n c a p i l l a r y tubes were used i n i t i a l l y  (n = 10).  to determine c l o t t i n g  time  However, due to r a p i d b l o o d c l o t t i n g , e s p e c i a l l y of b l o o d from the i n f e c t e d fish,  the r e s u l t s from the i n i t i a l  c l o t t i n g time t e s t were deemed  unreliable  The experiment was repeated u s i n g h e p a r i n i z e d m i c r o c a p i l l a r y tubes to  p r o l o n g blood c l o t t i n g .  A f t e r b l o o d was removed from the f i s h by the  technique d e s c r i b e d e a r l i e r , l a r y tubes.  Each' tube was f i l l e d  from one f i s h was s u f f i c i e n t a stopwatch was s t a r t e d . off  i t was immediately t r a n s f e r r e d i n t o m i c r o c a p i l -  to f i l l  a t l e a s t 25 tubes.  Blood  At the same time,  A short segment of the c a p i l l a r y tubes was broken  every 2 minutes u n t i l a f i b r i n  thus denoting  to approximately 4/5 of i t s volume.  thread was seen to connect the fragments,  the end p o i n t of the experiment.  Every 2 minutes a r e c o r d  was made of those w i t h c l o t t e d blood and those where c l o t t i n g had not y e t occurred.  The c a p i l l a r y tubes of c l o t t e d b l o o d were r e c o r d e d .  100% blood  c l o t t e d was denoted by the time when a l l tubes were c l o t t e d . 2.5.5.  T o t a l Blood C e l l Counts Immediately  a f t e r sampling, b l o o d was t r a n s f e r r e d i n t o m i c r o p i p e t t e s  u s i n g 5, 10 u l m i c r o p i p e t t e s f o r each sampled f i s h . was d i l u t e d  i n a b o t t l e containing  Blood from each p i p e t t e  100 ml. of 0.9% f i l t e r e d  sodium c h l o r i d e  to make a 1:10,000 suspension. A C o u l t e r Counter was used to count the number of c e l l s suspension of sampled b l o o d .  i n a 1/2 ml.  The a p e r t u r e of the g l a s s c y l i n d e r was  100 um.  89 A c o i n c i d e n c e c o r r e c t i o n was made by r e f e r r i n g manufacturer.  to a c h a r t s u p p l i e d by the  The number of c e l l s per c u b i c m i l l i m e t e r of b l o o d was  then  calculated. 2.5.6.  Red Blood C e l l As mentioned  Count  i n the blood sampling procedure, the second, t h i r d  f o u r t h drops of blood were each smeared on one of three g l a s s s l i d e s the  " t w o - s l i d e method."  s l i d e which was  back a g a i n s t the b l o o d u n t i l i t spread by c a p i l l a r y slides.  angle of 35-40 degrees) was  The "spread s l i d e " then pushed  were a i r d r i e d and then s t a i n e d  brought  a c t i o n a l o n g the  inter-  ( s t i l l maintained at an  forward at a moderate speed so that  blood spread evenly along the other s l i d e .  next  using  T h i s method i n v o l v e d a second s l i d e h e l d at an  angle of 35-40 degrees to the c e n t r e of the f i r s t  face between the two  and  The completed smeared  slides  (using a procedure to be d e s c r i b e d i n the  section). The s t a i n e d smears were examined under o i l immersion at a magnifica-  t i o n of 1500 x.  Red blood c e l l s  (both immature and mature) and white b l o o d  c e l l s were counted and recorded from a t o t a l of 30 microscope f i e l d s f o r each s l i d e  ( f i e l d s c o n t a i n i n g no g r e a t e r than 100 c e l l s but no l e s s than 20  c e l l s were counted). The t o t a l numbers of RBC lated.  and WBC  from three s l i d e s were then c a l c u -  A c c o r d i n g to the t o t a l b l o o d c e l l counts r e c e i v e d from the C o u l t e r  Counter d e s c r i b e d e a r l i e r ,  the number of red cells/mm  3  can be  calculated  u s i n g the formula: RBC  c e l l s / mm  3  =  JL  W+R  x  K  where R » t o t a l number of red c e l l s from 3  slides  W = t o t a l number of white c e l l s from 3  slides  K = t o t a l number of c e l l s , both red and white c e l l s per mm  3  C o u l t e r Counter.  from  90  2 . 5 . 7 .  Red C e l l Corpuscular Three e r y t h r o c y t i c  Value  i n d i c e s were c a l c u l a t e d from the h e m a t o c r i t , hemo-  g l o b i n and RBC value u s i n g the f o l l o w i n g formula: i i r™mr\ Hematocrit (%) x 1 0 Mean c o r p u s c u l a r volume (MCVj = r — — • —-.—(millions/mm ——\ -,—r%) RBC count v  3  v* i u i /UPD\ Hemoglobin g/dl x 1 0 Mean c o r p u s c u l a r hemoglobin (MCH; = - . . — • 7 — r \ * RBC count ( m i l l i o n s/mm ) 6  3  = Hemoglobin g/dl x 1 0 0 Hematocrit (%)  Mean c o r p u s c u l a r hemoglobin c o n c e n t r a t i o n (MCHC) 2 . 5 . 8 .  D i f f e r e n t i a l C e l l Count All  the c e l l  the s t a i n s used  types were c l a s s i f i e d as f a r as p o s s i b l e a c c o r d i n g to  i n t h i s experiment  (1976)  , L e s t e r and D a n i e l s  (1977)  , Einszporn-Orecka  Katz  (1949).  and f o l l o w i n g the d e s c r i p t i o n G o l o v i n a  (1976),  (1973),  Lehmann and Sturenberg  Watson e t a l .  In d i f f e r e n t i a l c e l l  counts  (1963),  (1975),  Wienreb  (1958)  be  i d e n t i f i e d with t h i s  from  t o be an  salmon because more c e l l s  could  stain.  I d e n t i f i c a t i o n of c e l l s following  and  the c e l l s were d i f f e r e n t i a t e d  the smeared s l i d e s s t a i n e d with H a l i c o D i f f Quik which was found a p p r o p r i a t e s t a i n f o r the blood of sockeye  Ellis  d u r i n g c o u n t i n g was aided by a p p l y i n g the  criteria:  Immature r e d c e l l  - round  to o v a l c e l l with round  to o v a l nucleus  light  blue-grey Mature r e d c e l l  - an o v a l c e l l with o v a l n u c l e u s , l i g h t orange-brown cytoplasm  Small lymphocyte  - a small c e l l with a dense blue purple nucleus and a narrow r i m of blue cytoplasm,  Large  lymphocyte  - similar  f r a y e d margin  to the above c e l l but l a r g e r , no f r a y e d margin,  may not have an indented  nucleus  91 Neutrophil Monocyte  - l a r g e c e l l with lobed nucleus and granular <  - round c e l l w i t h e c c e n t r i c and deeply c l e f t nucleus very loose  Macrophage  cytoplasm and  chromatin  - as large as or l a r g e r than RBC,  w i t h a number of vacuoles  Thrombocyte immature  - very dense nucleus, pale cytoplasm, may  or may not form  pseudopodia mature  - a c e l l w i t h packed e c c e n t r i c oval nucleus, granules s t a i n very  2.5.9.  cytoplasmic  lightly  S t a t i s t i c a l A n a l y s i s of Data The means of each hematological parameter of the i n f e c t e d groups were  compared w i t h those of the c o n t r o l group using student's t - t e s t .  Statistic-  a l l y s i g n i f i c a n t d i f f e r e n c e s were measured at a confidence l e v e l of P =  0.05.  L i n e a r r e g r e s s i o n l i n e s were drawn and r e g r e s s i o n c o e f f i c i e n t s were compared using a n a l y s i s of variance.  The data from some of the hematological t e s t s  w i t h non-homogeneous varianc 3. 3.1.  were transformed,  using l o g 10.  Results Hemoglobin Concentration Mean hemoglobin concentration of i n f e c t e d f i s h was compared w i t h  concentration of the c o n t r o l group using a t - t e s t . concentration of Hb was  s i g n i f i c a n t l y lower (P = 0.05)  i n the c o n t r o l group (Figure 13). 0.1  to 6.90  ± 0.17  I t appeared that the i n the i n f e c t e d than  The Hb concentration dropped from 8.81  g/dl (Table IX) i n i n f e c t e d f i s h during the  ±  experimental  period of 112 days, while i n the c o n t r o l group i t remained constant w i t h i n the range of 8.60-8.86 g / d l . during the same p e r i o d of time (Figure 13). During the p e r i o d of 3-41  DPI there were v a r i a t i o n s i n the Hb  c e n t r a t i o n i n both c o n t r o l and i n f e c t e d f i s h groups but there were no  con signif-  icant d i f f e r e n c e s . A f t e r 41 DPI, the Hb concentration was g r e a t l y reduced.  92  Figure  13  R e l a t i o n s h i p s between hemoglobin and DPI Comparison of the hemoglobin c o n c e n t r a t i o n s of the blood of i n f e c t e d and c o n t r o l f i s h groups i n r e l a t i o n to i n f e c t i o n time. Each p o i n t represents mean values. V e r t i c a l l i n e represents S.D. Number on each p o i n t represents sample s i z e .  HEMOGLOBIN  o  C6  CONCENTRATION  g m / dl  TABLE IX  Hemoglobin c o n c e n t r a t i o n g/dl measured from the blood of i n f e c t e d and n o n - i n f e c t e d f i s h groups during the 3 - 112 DPI  Day Post I n f e c t i o n 10  13  17  23  28  32  38  41  56  70  84  112  mean  1.81  8.88  8.70  8.30  8.55  8.43  8.62  8.89  8.34  8.30  7.84  6.81  7.45  6.90  S.D.  .10  .11  .17  .18  .20  .12  .16  .14  .14  .09  .13  .17  .08  .17  12  12  11  24  20  22  24  24  20  20  10  9  8  8  me an  8.83  8.62  8.86  8.70  8.63  8.85  8.66  8.50  8.70  8.62  8.88  8.56  S.D.  .15  .14  .18  .20  sample size  12  20  11  10  sample size  .12  .15  .14  12  12  23  .18 24  .13  .17  .19  .15  22  24  23  23  I = I n f e c t e d f i s h group C = C o n t r o l f i s h group  8.60 .15 12  8.70 .17 11  95 These r e s u l t s c l e a r l y e f f e c t on j u v e n i l e sockeye t i o n i n the i n f e c t e d f i s h .  i n d i c a t e t h a t Satmincola  e x e r t s an adverse  salmon by causing the r e d u c t i o n of Hb c o n c e n t r a During the p e r i o d of 112 DPI, when the f i s h were  p a r a s i t i z e d with 31.25 Satmincola,  Hb c o n c e n t r a t i o n i n the i n f e c t e d  fish  dropped by 20%. 3.2.  Hematocrit  Value  No s i g n i f i c a n t d i f f e r e n c e between the blood of i n f e c t e d and c o n t r o l f i s h groups (Figure 14) was found p e r i o d of 3-41 DPI. f i s h group appeared the experiment.  i n the mean hematocrit values d u r i n g the  The r e d u c t i o n of the hematocrit value of the i n f e c t e d a f t e r 41 DPI, and g r a d u a l l y d e c l i n e d toward the end of  At 112 DPI i t had dropped to 34.51% while i n the c o n t r o l  group., i t remained a t 46.21% (Table X ) . I t i s obvious t h a t Satmincola value of i n f e c t e d j u v e n i l e  sockeye  can cause a r e d u c t i o n i n hematocrit  salmon by 22.2% d u r i n g a p e r i o d of 112  days when the f i s h were i n f e c t e d w i t h an average  of 31.25 Satmincola  per  fish. 3.3.  E r y t h r o c y t e Osmotic  Fragility  There was no s i g n i f i c a n t d i f f e r e n c e between the e r y t h r o c y t e osmotic f r a g i l i t y of the i n f e c t e d and the c o n t r o l groups (P=.05). f e c t e d and the c o n t r o l groups reached  100% hemolysis with the % NaCl a t  0.30 (Table X I ) , which i s very s i m i l a r to the osmotic human blood 3.4.  Both the i n -  f r a g i l i t y of normal  (Figure 15).  C l o t t i n g Time In p r e l i m i n a r y t e s t s i n which p l a i n c a p i l l a r y tubes were used  the blood of the c o n t r o l f i s h c l o t t e d w i t h i n 3-4 min (Figure 16). The h e p a r i n i z e d m i c r o c a p i l l a r y tubes can p r o l o n g blood c l o t t i n g from 3-4 min to almost  25 min i n the blood of the c o n t r o l f i s h .  The r e s u l t s are shown  96  Figure  14  R e l a t i o n s h i p s between hematocrit  value and DPI  V a r i a t i o n i n hematocrit values of the blood of i n f e c t e d and c o n t r o l f i s h groups d u r i n g the experimental p e r i o d of 3-112 DPI. Each p o i n t r e p r e s e n t s a mean v a l u e . V e r t i c a l l i n e r e p r e s e n t s S.D. Number on each p o i n t r e p r e s e n t s sample s i z e .  TABLE X  Hematocrit value  (%) of i n f e c t e d and c o n t r o l f i s h groups during  112 DPI  Day Post I n f e c t i o n 3  I  10  13  17  23  28  32  38  41  56  70  84  112  mean  44.4  45.3  44.0  43.9  45.0  46.5  44.5  42.5  46.0  45.2  41.7  34.2  34.1  34.5  S.D.  1.5  0.6  .8  1.7  1.0  0.7  0.8  1.3  1.1  0.9  1.6  1.5  0.9  0.8  20  20  10  9  8  8  sample size  C  6  )  2  n  n  u  2  Q  2  2  ^  n  mean  46.1  45.2  46.7  46.7  46.9  47.5  45.6  46.2  47.5  45.0  45.9  47.2  45.0  46.2  S.D.  0.8  1.3  1.1  0.7  0.6  0.2  1.8  0.6  1.4  1.8  0.7  1.2  1.9  1.8  sample s lze  u  n  n  ^  u  2  2  2  3  2  3  I = Infected f i s h  2  3  2  Q  u  ] Q  u  u  group  C = C o n t r o l f i s h group  00  99  F i g u r e 15  E r y t h r o c y t e osmotic  fragility  of experimental  fish  Osmotic f r a g i l i t y curve at 9°C of i n f e c t e d and c o n t r o l f i s h groups i n comparison w i t h the human blood (Davidson and Nelson, 1974). Arrow i n d i c a t e s % NaCl where 100% hemolysis o c c u r r e d .  101 TABLE XI  Percent NaCl used i n e r y t h r o c y t e osmotic f r a g i l i t y t e s t and % hemolysis of blood of sockeye salmon i n f e c t e d with Satmincola. The experimental f i s h were kept i n the water at 9°C.  % NaCl  % hemolysis  .30  97-100  .40  50-90  .45  5-45  .50  •  0-5  .55  0  i n Table X I I . The percent of blood c l o t t e d i n the h e p a r i n i z e d m i c r o c a p i l l a r y  tubes  i n c r e a s e d at a f a s t e r r a t e than that of the c o n t r o l f i s h group and reached 100% blood c l o t t e d w i t h i n 17 min (Figure 16). w i t h i n the f i r s t 100%  increase  appeared  11 min and t h e r e a f t e r , there was a gradual d e c l i n e u n t i l  of the blood c l o t t e d .  appeared  A sharp  Among the c o n t r o l f i s h  100% blood  clotting  a t 25 min and the r a t e of i n c r e a s e i n % b l o o d c l o t t i n g was q u i t e  d i f f e r e n t from that of i n f e c t e d f i s h . very slow r a t e d u r i n g the f i r s t that time  The % b l o o d c l o t t e d  increased at a  11 min, a sharp i n c r e a s e appearing  after  (Table X I I ) .  The r e s u l t s obtained from t h i s experiment time f o r the c o n t r o l f i s h  show that blood  i s longer than f o r the i n f e c t e d f i s h .  clotting When  measured from the h e p a r i n i z e d m i c r o c a p i l l a r y tubes, i t i s 8 min l o n g e r . It decrease  i s p o s s i b l e to conclude  that Satmincola  i n blood c l o t t i n g time f o r j u v e n i l e  h e a v i l y i n f e c t e d with t h i s  parasite.  can cause a s i g n i f i c a n t  sockeye  salmon, when they are  102  Figure  16  Blood C l o t t i n g Time Comparison between the b l o o d c l o t t i n g time of the i n f e c t e d and the c o n t r o l f i s h groups. The arrow i n d i c a t e s time when 100% of b l o o d i n m i c r o c a p i l l a r y tubes c l o t t e d , which denoted blood c l o t t i n g time f o r t h i s experiment. The arrow w i t h dotted l i n e i n d i c a t e s blood c l o t t i n g time of the c o n t r o l f i s h group. Number on each p o i n t i n d i c a t e s sample s i z e .  % BLOOD  ecu  CLOTTED  TABLE XII  Percent blood  Testing  time  c l o t t e d obtained from the blood of i n f e c t e d and c o n t r o l f i s h groups  Sample s i z e  Control % Blood  clotted  Sample s i z e  Infected % Blood  clotted  3  20  8.3  20  37.5  5  20  7.5  20  52.6  7  20  7.5  19  60.0  9  18  10.4  18  64.2  11  20  12.1  18  85.0  13  19  13.6  18  94.1  15  20  31.2  18  98.5  17  19  40.0  17  100.0  19  18  61.3  15  100.0  21  17  65.9  15  100.0  23  18  87.2  25  17  95.4  27  15  100.0  29  12  100.0  105 3.5  T o t a l Blood C e l l  Count  T o t a l b l o o d c e l l counts, r e c e i v e d from the C o u l t e r Counter are shown in. Table X I I I .  They were used only as a. b a s e l i n e to c a l c u l a t e the number of  red  and white blood c e l l s  blood c e l l s  further detail 3.6  in this  Red Blood C e l l  and t h i s need not be d i s c u s s e d i n  section.  Count  The c a l c u l a t e d r e d b l o o d c e l l counts are shown i n Table X I I I . numbers of c e l l s  from both i n f e c t e d and c o n t r o l f i s h groups were p l o t t e d  a g a i n s t time post i n f e c t i o n .  During the p e r i o d of 3-45 DPI, mean b l o o d c e l l  counts i n the i n f e c t e d f i s h appeared fish  (Figure  Mean  slightly  h i g h e r than i n the c o n t r o l  17). When a t - t e s t was used to compare the means of these two  groups, .no s t a t i s t i c a l l y  s i g n i f i c a n t d i f f e r e n c e was found.  The mean number of r e d b l o o d c e l l s  o b t a i n e d from the i n f e c t e d  fish  group shows a sharp decrease a f t e r 41 DPI (Figure 17). The r a t e of decrease appears to be g r e a t e s t d u r i n g the p e r i o d of 41-84 DPI and shows a tendency to d e c l i n e .  T h e r e a f t e r , the mean c e l l count of the i n f e c t e d f i s h  dropped from 1.21 x 10  s  cells/mm  3  to 9.02 x 10  s  cells/mm  3  (Table X I I I ) ,  while i n the c o n t r o l f i s h group, i t dropped only to 1.14 x 10 This i n d i c a t e s a highly s i g n i f i c a n t  group  6  cells/mm . 3  lowering i n the number of r e d c e l l s i n  the  i n f e c t e d f i s h group compared w i t h that i n the c o n t r o l f i s h group  ure  18). A r e d u c t i o n i n the r e d blood c e l l numbers i n the c o n t r o l  (Fig-  fish  group a l s o appeared but the decrease was very s m a l l when compared w i t h that i n the b l o o d of the i n f e c t e d f i s h group  (Figure 17).  The r e s u l t s obtained from t h i s experiment  i n d i c a t e that  Satmincola  can cause a p r o g r e s s i v e r e d u c t i o n of r e d c e l l numbers i n the c i r c u l a t i n g blood.  At the end of 112 DPI the number of r e d c e l l s  of the i n f e c t e d  group  was about 24% lower than a t the b e g i n n i n g of the experiment, whereas i n the c o n t r o l f i s h group the decrease over the same p e r i o d was only 4.7%.  TABLE X I I I  Counts of t o t a l  DPI  blood c e l l s , r e d blood c e l l s , and white blood c e l l s of i n f e c t e d and c o n t r o l f i s h groups'  T o t a l blood e e l s i n millions/mm  obtained from the blood  WBC cells/mm  RBC i n m i l l ions/mm  2  1  3  cells  3  3  S.D.  Infected  S.D.  Control  S.D.  1 .205  .185  24,162  4631  22,112  3911  .231  1.238  .174  20,784  3976  26,378  4286  1.208  .099  1.215  . 186  29,392  8321  24,413  2703  1 .229  1 .220  .224  1.200  .132  31,781  2611  29,455  4215  1.249  1 .239  1.224  . 181  1 .216  .205  25,529  4210  23,729  3810  23  1.247  1 .233  1.220  .115  1.203  . 155  27,473  3517  30,007  2756  28  1 .278  1.236  1.237  .271  1.212  . 148  26,411  6103  24,143  5632  32  1.264  1 .226  1.237  .119  1.200  .138  27,279  4218  26,168  7211  38  1 .258  1.227  1.235  .297  1.205  .065  23,345  2075  22,990  8439  41  1.233  1 .248  1.203  .063  1.120  . 158  30,123  4614  28,118  2175  56  1.162  1.187  1.135  .241  1. 160  .234  27,707  2903  27,342  4123  70  1.072  1 .189  1 .041  . 165  1.162  .235  31,811  2587  29,115  6287  84  1.064  1 .174  1.032  .231  1.150  . 198  32,175  3642  24,178  4319  112  0.953  1 . 175  0.922  .297  1. 149  .244  31,234  4341  26,139  3682  Control  3  1.235  1.225  1.212  . 132  6  1 .265  1.264  1.245  10  1 .237  1 .239  13  1.251  17  and  2  Infected  S.D.  Infected  Control  were c a l c u l a t e d . o  ON  107  Figure  17  R e l a t i o n s h i p s between red blood c e l l counts and DPI Mean numbers of r e d blood c e l l s obtained from i n f e c t e d and c o n t r o l f i s h groups. The s o l i d and dotted l i n e s are l i n e s of best f i t to i n d i c a t e the v a r i a t i o n of the red c e l l counts during the p e r i o d of 112 DPI. V e r t i c a l l i n e represents S.D. Number on each p o i n t r e p r e s e n t s the sample size.'  801  109  F i g u r e 18  Regression  coefficients  of red blood c e l l  counts  Comparison between r e g r e s s i o n c o e f f i c i e n t of the red blood c e l l s of the i n f e c t e d and n o n - i n f e c t e d f i s h groups.  RED  BLOOD CELL  COUNT  ( LOG  10)  111 3.7  Red Blood  Corpuscular  Values  C a l c u l a t e d MCV, MCH and MCHC a r e shown i n Tahle XIV.  Regression  l i n e s were drawn from the c a l c u l a t e d MCV (Figure 19), MCH (Figure 20) and MCHC (Figure 21). MCV  No s t a t i s t i c a l l y  s i g n i f i c a n t d i f f e r e n c e s were found-in the  and MCH of the blood of i n f e c t e d and c o n t r o l f i s h groups.  Only the MCHC  value of the i n f e c t e d f i s h blood was found to be s l i g h t l y lower (P = .1) than that of the blood of the c o n t r o l f i s h 3.8  White Blood The  cytes  Cell  group.  Counts  c a l c u l a t e d t o t a l number of white blood c e l l s ,  (WBC-T) i s shown i n Table X I I I .  i n c l u d i n g thrombo-  A s i g n i f i c a n t l y higher number of  WBC-T was found i n the i n f e c t e d f i s h group than i n the c o n t r o l group 22).  An i n c r e a s e i n WBC-T must have come from the s i g n i f i c a n t  (Figure  increase i n  lymphocyte, n e u t r o p h i l (Table XV) or thrombocyte counts which w i l l be discussed i n a l a t e r section. 3.9  D i f f e r e n t i a t i o n of Blood The  Cells  c e l l s were i d e n t i f i e d  i n order to analyze  numbers between i n f e c t e d and n o n - i n f e c t e d 3.9.1  differences i n c e l l  fish.  D i f f e r e n t i a l Cell Descriptions  3.9.1.1  Erythrocytic Series Immature E r y t h r o c y t e  ( P l a t e I, F i g u r e a-d). diameter  The c e l l  shape v a r i e s from round to o v a l  A small round immature red c e l l  ( P l a t e I, F i g u r e a ) .  Oval c e l l s vary  i s 6.5 ± 1.72 urn i n  i n s i z e from 10.39 ± 2.4 urn  i n length and 8.52 ± 2.2 um i n width to the same s i z e as mature e r y t h r o c y t e s , that i s 12.9 ± 3.2 um i n l e n g t h and 7.1 ± 3.4 i n width F i g u r e b-d).  With other types of s t a i n b a s o p h i l i c cytoplasm  seen only i n the e a r l y stage of e r y t h r o c y t e s the c e l l s  (Plate I, can be c l e a r l y  ( P l a t e I , F i g u r e a and b) but  i n P l a t e I, F i g u r e b and c cannot be e a s i l y d i f f e r e n t i a t e d  from  TABLE XIV  C a l c u l a t e d MCHC, MCV and MCH of the blood of i n f e c t e d and c o n t r o l  Control  Infected DPI  MCHC  %  MCH Pg  f i s h groups  MCV u 3  MCHC  MCH  %  P8  MCV u 3  3  19.84  72.64  366.3  19.24  73.69  382.5  6  17.23  71 .44  364.4  19.01  69.62  365.1  10  19.77  72.19  365.0  18.91  72.86  384.0  13  19.19  63.13  362.3  18.67  72.50  389.1  17  18.11  66.63  631.9  18.46  70.97  385.6  23  18.67  68.36  365.6  18.63  73.56  394.8  28  19.49  65.94  338.9  18.19  71.45  392.7  32  18.00  64.75  323.7  18.39  70.01  380.5  38  19.15  66.20  331.0  18.31  72.80  397.4  41  18.10  70. 13  384.8  19.02  72.13  379.0  56  18.47  70.61  364.5  19.12  75.68  395.6  70  19.39  76.95  396.8  18.20  70.03  384.2  84  18.90  75.05  371.5  18.50  75.60  396.8  112  18.20  74.00  341.1  18.95  76.24  360.2  113  Figure  19  Mean c o r p u s c u l a r volume (MCV) Comparison between MCV of the blood of i n f e c t e d and n o n - i n f e c t e d f i s h groups.  F i g u r e 20  Mean c o r p u s c u l a r hemoglobin  (MCH)  Comparison between MCH of the blood of i n f e c t e d and n o n - i n f e c t e d f i s h groups.  fish  •  •  D---Q  Q  D  0  * ~""' "o •  40  20  71_.l_t_0001_X_  J=  1 3  -  a  *  '  60  infected control  ""  " y « 49.9+.0O9X  120  80  TIME IN DAYS  •  •  D---Q  0  •a--  20  40  60  TIME IN DAYS  80  infected control  y o  3 8 2 . 3 2 +.0004 X  Y=  3 6 8 . 2 9 * .0001 X  100  —I 120  115  F i g u r e 21  Mean c o r p u s c u l a r hemoglobin c o n c e n t r a t i o n  (MCHC)  Comparison of the MCHC of the blood of i n f e c t e d and n o n - i n f e c t e d f i s h groups.  • • o—a 21  infected control  -t  20 H _N  y  19  u X u  -E  = 18.05 + . 0 0 2 X  H y =  i9._ - .005  x  18-  17H  I  20  40  I  80  60 T I M E IN  DAYS  100  120  117  F i g u r e 22  . R e g r e s s i o n c o e f f i c i e n t s of white blood c e l l  counts  Comparison of r e g r e s s i o n c o e f f i c i e n t s of white blood c e l l counts of the blood of i n f e c t e d and n o n - i n f e c t e d f i s h groups.  WBC  COUNT  LOG  10  TABLE XV  Ranges, means and S.D. of l e u c o c y t i c  C e l l Types  cells  obtained from i n f e c t e d and c o n t r o l f i s h groups at 112 DPI  Ranges (%)  Control  Infected Mean ± S.D.  80.35 - 95.42  89,.88 + 7.41  88.75 + 9.01  74.26 - 94.38  84..32 + 5.74  83.92 + 6.82  3.04 -  6.09  4..56 + 1.97  4.03 + 0.9  Neutrophils  7.01 -  9.20  8.. 10 + 1.02  8.39 + 0.98  Monocytes  0.58 -  1.30  + 0.27 1 .07 ,  1.01 + 0. 19  1.21 -  5.40  3,.28 + 0.95  3.35 + 0.62  0.02 -  0.07  0,.04 + 0.008  0.03 + 0.007  Total  lymphocytes  small  lymphocytes  large  lymphocytes  "Granulocyte Macrophages  cells"  120 the mature r e d c e l l . m a t u r i t y of the c e l l .  B a s o p h i l i c cytoplasm can be used to i n d i c a t e the The l e s s b a s o p h i l i c , the more mature the c e l l i s .  Mature E r y t h r o c y t e  T h i s i s an o v a l c e l l .  ± 2.5 um i n l e n g t h and 7.0 ± 1.28 um i n width f) .  The c e l l  s i z e i s 13.0  ( P l a t e I, F i g u r e a,  b, e and  The nucleus s t a i n s dark, p u r p l e with HDQ s t a i n and dark blue w i t h Wright's  stain.  The cytoplasm s t a i n s orange-brown with HDQ and yellow-green w i t h  Wright's  stain.  Immature e r y t h r o c y t e s c o n t a i n many more m i t o c h o n d r i a  I I , F i g u r e b) than do mature c e l l s 3-9.1.2  (Plate  (Plate I I , Figure a ) .  Leucocytic Series Two types of c e l l s are found i n the blood o f j u v e n i l e sockeye  agranulocytes and g r a n u l o c y t e s .  salmon:  The m a j o r i t y of the c e l l s are a g r a n u l o c y t i c  cells. Agranulocytes Small lymphocyte  These are s m a l l , round or almost round  ure c - f ) with a s i z e range from 8.2 ± 3.15 to 12.6 ± 4.0 um. occupies v i r t u a l l y p h i l i c cytoplasm  the whole c e l l ,  (Plate I I , F i g The nucleus  l e a v i n g only a very narrow r i m of baso-  ( P l a t e I I , F i g u r e d ) . The chromatin meshwork  appears  l o o s e r than that of mature r e d c e l l s but was s i m i l a r to that of .immature red  cells  (Plate I I , Figure d).  Small lymphocytes  f r a y e d margins ( P l a t e I I , F i g u r e e ) , or pseudopodia Large  lymphocyte  Figure b ) . range from  Normally  eye salmon.  (Plate I I I ,  they are l a r g e r than mature r e d c e l l s with a s i z e The nucleus, which i s o f t e n found w i t h a deep  i n d e n t a t i o n , occupies almost  Monocyte  (Plate I I , Figure f ) .  These c e l l s are round or almost round  14.5 ± 1 . 7 2 um.  cytoplasm.  are o f t e n found with  The chromatin  the e n t i r e c e l l  l e a v i n g only a small r i m of  i s a c o a r s e l y meshed s t r a n d .  These are very few i n number i n the b l o o d of j u v e n i l e They have a s i z e range  sock-  from 8.7 - 10.1 um (Plate I I I , F i g u r e c ) .  121 The nucleus occupies h a l f or s l i g h t l y more than h a l f of the c e l l volume and i s o f t e n notched o r h o r s e s h o e -  loose s t r a n d s .  shaped.  Nuclear chromatin appears  The cytoplasm s t a i n e d l i g h t  as f a i r l y  gray-blue w i t h HDQ s t a i n .  mally i t appears with the formation of the t h i r d lobe of the nucleus Macrophage  In a l l the s l i d e s examined i n t h i s experiment  c e l l s of t h i s type were observed c e l l s were observed  seven  i n the i n f e c t e d f i s h group a t each sampling p e r i o d . A l l  measured but these c e l l s appeared also irregular  (arrow).  i n the c o n t r o l group and only one or two  of them were of very i r r e g u l a r shape ( P l a t e I I I , F i g u r e d ) .  was  only  Nor-  Size was not  l a r g e r than mature red c e l l s .  i n shape and s e v e r a l vacuoles were found  The nucleus  i n the cytoplasm.  Granulocytes Neutrophil  S i m i l a r to or l a r g e r than mature r e d c e l l s .  round c e l l s with a s i z e range and f ; P l a t e IV, F i g u r e a - f ) .  10.1-13.4 urn i n diameter  have 2 to 5 l o b e s , r a r e l y seen.  (Plate I I I , F i g u r e e  N e u t r o p h i l s of j u v e n i l e sockeye  to have two types of n u c l e i : segmented and banded.  These a r e  salmon appear  Segmented n u c l e i always  5 lobes were more common, n u c l e i w i t h 2  lobes were  Sometimes, c e l l s with n u c l e i with more than 5 lobes were seen  ( P l a t e I I I , F i g u r e f ; P l a t e IV, F i g u r e a ) .  Lobes were connected  to each  other by a v e r y f i n e f i l a m e n t of n u c l e a r m a t e r i a l s (see arrow i n P l a t e I I I , Figure e ) .  In n e u t r o p h i l s w i t h banded n u c l e i , the nucleus was always  to be t w i s t e d or bent  ( P l a t e IV, F i g u r e b, d, e and f ) . The nucleus  found stains  v i o l e t and has chromatin c o n s i s t i n g of i r r e g u l a r patches of l i g h t and dark staining.  The cytoplasm and the granules s t a i n very l i g h t blue w i t h HDQ  stain. "Granulocyte c e l l s "  Some c e l l s resembled  d e s c r i b e d by others (Lehmann and Stu'renberg, 1977), however, I was unable i d e n t i f i c a t i o n was d i f f i c u l t .  " e o s i n o p h i l s " and " b a s o p h i l s "  1975; G o l o v i n a , 1976; and E l l i s ,  to s t a i n granules i n these c e l l s and thus These u n i d e n t i f i a b l e c e l l s were put i n t o a  122 category c a l l e d 3.9.1.3  "granulocyte c e l l s . "  Thrombocytic S e r i e s Thrombocytes were found a t s e v e r a l stages of m a t u r i t y i n the c i r c u -  l a t i n g blood of sockeye mediate  salmon, ranging from immature  ( s p h e r i c a l ) to i n t e r -  ( s l i g h t l y oval) to f u l l y mature ( e l o n g a t e ) .  Immature Form  These are very small round c e l l s  with a s i z e range 6.4-7.3 um i n diameter.  ( P l a t e VI, F i g u r e a)  These c e l l s and s m a l l lymphocytes  are m o r p h o l o g i c a l l y s i m i l a r but they can be d i f f e r e n t i a t e d w i t h HDQ. circulating  b l o o d of j u v e n i l e sockeye  be s m a l l e r than small lymphocytes. the whole  salmon, these c e l l s are o f t e n found to  The very dense purple nucleus  occupies  cell.  Intermediate Form form  In the  These c e l l s are normally l a r g e r than the immature  ( P l a t e VI, F i g u r e b, c and d) w i t h a s i z e range 7.2-9.4 um i n diameter.  The cytoplasm appeared  colorless.  This c e l l  i s o f t e n found w i t h  pseudopodia  ( P l a t e VI, F i g u r e d ) . Mature Form to elongated. like  In the e a r l y stage of m a t u r i t y the c e l l s appear o v a l or r o d -  ( P l a t e VI, F i g u r e  indented VII,  These c e l l s vary i n s i z e and shape from o v a l t o rod l i k e  e and f ) .  (Plate VI, Figure f ) .  F i g u r e a a r e o f t e n found  cytoplasm s t a i n s very l i g h t  N u c l e i of these c e l l s o f t e n appear  C e l l s i n P l a t e VI, F i g u r e e and f and P l a t e  i n the blood o f j u v e n i l e sockeye  salmon.  blue-gray w i t h evidence of very f i n e  The  granules.  Mature thrombocytes o f t e n have long cytoplasmic p r o j e c t i o n s at one pole ( P l a t e V I I , F i g u r e a ) . They sometimes appear i n groups of 2, 3, or 4 c e l l s (Plate VII, F i g u r e c ) . 3.9.2 3.9.2.1  Differential  Cell  Counts  Erythrocytic Series Immature Red C e l l  of both  Percentage  and number of immature r e d c e l l s  i n f e c t e d and c o n t r o l f i s h groups are shown i n Table XVI.  Mean  123 numbers of c e l l s of these two groups of f i s h were compared u s i n g a t - t e s t and were found to be s l i g h t l y d i f f e r e n t Mature Red C e l l  (P = 0.1).  No comparison between the mature r e d c e l l count of  the b l o o d of the i n f e c t e d and c o n t r o l f i s h groups were made but only the t o t a l red c e l l counts were compared. 3.9.2.2  Leucocytic  Series  The t o t a l l e u c o c y t e counts (Thrombocyte not i n c l u d e d ) of the blood of both i n f e c t e d and c o n t r o l f i s h groups  (Table XVII) were p l o t t e d  time (DPI) and the r e g r e s s i o n c o e f f i c i e n t s were compared.  A  significantly  h i g h e r number of l e u c o c y t i c c e l l s appeared i n the blood of i n f e c t e d than i n that of the c o n t r o l f i s h  against  fish  (Figure 24).  A c c o r d i n g to the d i f f e r e n c e i n f u n c t i o n of each type of l e u c o c y t i c cell,  the number of c e l l s f o r each group was  counted s e p a r a t e l y as shown  i n Table XVII. Agranulocytes Lymphocyte XV).  Small lymphocytes were dominant  among lymphocytes  (Table  These comprised 89.88 ± 7.4 of the t o t a l l e u c o c y t i c c e l l s w h i l e l a r g e  lymphocytes made up only 4.56  ± 1.97.  Both small and l a r g e  lymphocytes  were analyzed together i n the t o t a l lymphocyte counts (Table XVII and ure 25).  Fig-  T o t a l lymphocyte counts were found to be s i g n i f i c a n t l y h i g h e r i n  the blood of the i n f e c t e d f i s h group than i n that of the c o n t r o l group.  The  i n c r e a s e i n t h i s count seems to come from the small lymphocytes r a t h e r than the l a r g e ones.  D i f f e r e n c e i n the number of c e l l s was  clearly  shown at  70-112 DPI. Monocyte and Macrophage  A very small number of monocytes and macro-  phages were found i n the c i r c u l a t i n g b l o o d of the j u v e n i l e sockeye  salmon.  At some sampling p e r i o d s n e i t h e r monocyte nor macrophage were observed  124  C e l l s shown i n P l a t e s I - V I I , a r e from the blood of n o n - i n f e c t e d j u v e n i l e sockeye salmon, s t a i n e d with d i f f e r e n t types•of s t a i n . Those which are not s p e c i f i e d , are s t a i n e d with HDQ s t a i n . C e l l s are photographed under o i l immersion with a L e i t z microscope and Wild Photo Automat or L e i t z camera coupled with l i g h t meter.  Plate I  (a) Immature e r y t h r o c y t e ( e a r l y s t a g e ) . Note the c e l l s i z e i s s m a l l e r than the mature e r y t h r o c y t e . (b) Immature e r y t h r o c y t e ( l a t e r s t a g e ) , c e l l almost the same s i z e as the mature e r y t h r o c y t e and becomes more o v a l . (c) Immature erythrocyte. Note the c e l l with l e s s b a s o p h i l i c cytoplasm than the c e l l i n d, which i n d i c a t e s more m a t u r i t y . (d) Immature e r y t h r o c y t e (intermediate s t a g e ) . Note the chromatin p a t t e r n i s more c o a r s e l y mesh. L i g h t area can be seen. (e) Mature e r y t h r o c y t e w i t h small r o d shaped mitochondria. (f) Mature and immature e r y t h r o c y t e s w i t h Wright's s t a i n . Note mitochondria c o u l d not be seen.  125  PLATE I  d  126  Plate II  (a) Mature e r y t h r o c y t e showing mitochondria (arrow). Note s m a l l e r number of mitochondria i n comparison with immature erythrocyte. (b) Immature e r y t h r o c y t e ( i n t e r m e d i a t e stage) with numerous mytochondria (arrow). (c) Small lymphocyte with t y p i c a l l i g h t area (arrow). (d) Small lymphocyte. Arrow i n d i c a t e s a very narrow r i m of b a s o p h i l i c cytoplasm, (e) Small lymphocyte with f r a y e d margin, f r e q u e n t l y encountered i n the c i r c u l a t i n g b l o o d . ( f ) Small lymphocyte with the formation of pseudopodia (arrow) i n f r e q u e n t l y encountered i n the b l o o d .  127  128  Plate III  (a) Small lymphocyte showing c o r a s e l y meshed chromatin strands with a l t e r n a t i n g l i g h t and dark areas under Wright's stain. (b) Large lymphocyte with deep i n d e n t a t i o n of nucleus (arrow). Note c e l l i s normally l a r g e r than the mature e r y t h r o c y t e . (c) Monocyte, note lobed nucleus and formation of the t h i r d lobe (arrow). (d) Macrophage with some small vacuoles (arrow). I n f r e q u e n t l y encountered i n the b l o o d . (e) N e u t r o p h i l , one of the t y p i c a l appearances (5 lobed n u c l e u s ) . Arrow i n d i c a t e s the f i n e n u c l e a r m a t e r i a l connecting one lobe to another. (f) N e u t r o p h i l , nucleus with more than 5 l o b e s . I n f r e q u e n t l y found i n the b l o o d .  130  P l a t e IV  (a) N e u t r o p h i l , showing nucleus with more than 5 l o b e s . (b) N e u t r o p h i l , showing n u c l e a r f i l a m e n t s , (c) Neutrop h i l , nucleus with 2 l o b e s , i n f r e q u e n t l y encountered i n the b l o o d . (d) N e u t r o p h i l with bent nucleus. (e) N e u t r o p h i l , note the c e l l s i z e i s almost twice the s i z e of mature e r y t h r o c y t e . T h i s p i c t u r e shows f i n e granules r e g u l a r l y appear i n the cytoplasm, normally found i n a l l the c e l l types of n e u t r o p h i l s . (f) N e u t r o p h i l w i t h t w i s t e d n u c l e u s .  131 PLATE I V  132  Plate V  (a) to ( f ) are termed " g r a n u l o c y t e c e l l s " but t h e i r p r e c i s e nature could not be determined.. (a) and (c) more o f t e n found than any other c e l l s but (f) i s very r a r e l y found.  133  134  P l a t e VI  (a) Immature thrombocyte (very e a r l y s t a g e ) , cytoplasm i s not normally seen. (b) Immature thrombocyte ( e a r l y s t a g e ) , note very pale cytoplasm (arrow). (c) Immature thrombocyte, note cytoplasm may appear i r r e g u l a r (arrow). (d) Immature thrombocyte ( i n t e r m e d i a t e s t a g e ) , note pseudopodium formation a t one of the c e l l (arrow). (e) and ( f ) are mature thrombocytes.  136  P l a t e VII  (a) and (b) are mature thrombocytes at a c t i v e stage. (c) Mature thrombocytes, f r e q u e n t l y found i n a group of 4-5.  PLATE V I I  TABLE XVI  Percentage and number of immature red c e l l s  observed i n the blood of i n f e c t e d and c o n t r o l f i s h  Infected DPI  Z  groups.  Control cells/mm  3  %  cells/mm  3  .76  9211  .75  9037  6  .35  4537  .53  6561  10  1.18  14254  1.00  19440  13  .32  3904  .83  9960  17  .36  4406  .57  6931  23  .37  4514  .37  4451  28  .39  4824  .67  8120  32  .43  5319  .43  5160  38  .22  2717  .20  2410  41  .44  5293  .60  6720  56  .45  5107  .38  4408  70  .78  8119  .56  6507  84  1.27  13106  .40  4600  112  .98  9035  .64  7353  3  Co CO  139  F i g u r e 23  Regression c o e f f i c i e n t s of immature red c e l l  counts  Comparison between r e g r e s s i o n c o e f f i c i e n t s of immature red c e l l counts of the blood of i n f e c t e d and noni n f e c t e d f i s h groups.  1.2  TABLE XVI I Heans t Small UHl 1  Lymphocyte  S.D.  of d i f f e r e n t  L a r g e l.y mphocy t e Coitt ro 1  Inl ec ted  Control  Infected  19 . 7 l 3.5  22.912 .8  1.031 .05  1.2111.5  leucocyte  (nuut s ( c e l l s  N eul rnji.hi 1 (.'on t ro 1  l n f e c ted  in thousands/mi) ) f o r i n f e c t e d and c o n t r o l 1  Monocy ttInfected  1 .481 .23  1 .621 . 19  .241 .01  Control  "Cranulocyt e c e l l " Infected  Comrol  fish  groups.  Macrophage Infected  .281.02  1.481 .33  1 .541 .34  .0541.006  Total  Control  Infected  Control  .0481 .007  24 1 4 .6  22 1 3.9 26 1 4.2  6  17 .212 . 7  21 .413 .4  1 .071.23  1.161..23  1 . 39t.17  1 .541 . 15  .211 .07  .261.04  1.531 .45  1 .361 .33  .0521.003  -  20 1 3 .9  10  2 1.914 . 2  20 .013 .5  1.041 .38  1.431. 19  2 .041 . 19  1 .4 11 . 13  .261 .04  .241.06  1.421 .21  1 .381 .30  .0461.002  .0561 .004  29 1 8 .3  24 1  11  25 . a n .5  24 .412 .9  1.351 .09  1.291. 16  2 .251 .08  1 . 781.24  -  .281.06  1.651 .42  1 .451 .36  .0531.006  -  31 t  2 .6  29 1 4.2  25 1 4 .2  23 1 3.8  17  22 .411 .6  2 1 .514. . 1  1.021 . 17  1.161. 19  1 .861 .05  1 .511. .21  .261 .03  .221.04  1.601 . 18  1 .461 .28  .0611.001  2 1  2 1 .912 . 3  25 .315. .2  1 .111. 16  1.251. 09  1 . 791.32  1 . B i t . 18  -  .211.07  1.481 .09  1 .401 .39  .0581.004  -  28  21..412. .9  28 .411. .4  1.091 .52  1.201. 42  1 .811 . 18  1 .621. . 14  .271 .07  .261.08  1.451 .26  1 .591 .24  .0531.006  .0571 .003  26 1 6  12  2 J. I l l ..7  21..511. 3  1.131.. 14  18  20. 214. 3  18. 413. 9  0.961. 17  '. 1  24 .B l S . 1  23. 712. 6  56  23. 611. 4  23. 012. 4  ;o  27. 212. 6  24 .412. 3  84  27. 116. 2  1 12  26. 813. 0  1.161. 1 7  .27  27 1 3 .5  30 1 2.7  |  24 1 5.6  1 . 761. 12  1 .561. 23  .261 .06  .251.02  1.491 .42  1 .521 . 17  .0521.003  .0511 .009  27 1 4 .2  1 .621 . 17  1 . 321. 39  .241 .06  .231.01  1.401 .31  1 .551 .39  .0421.004  -  26 1 7.2  0 . 9 8 1 . 32  23 1 2 .0  22 1 8.4  1.221..38  1.241. 31  2 .081 .28  1 .681. 15  .311 .04  .291.03  1.531 .42  1 .321 .32  .0511.002  .0621..007  30 1 4 .6  28 1 2.3  1.051. 42  1.251. 35  1..971 . 19  1.621. 16  .291 .01  .271.07  1.481 . 16  1 .651 .42  .0521.003  -  27 1 2 .9  1 . 3 0 1 . 27  2..321..23  1 . 721.18  .301 .03  .281.04  1.701 . 18  1.451 .43  .0581.003  -  27 1 4.1  1.29*. 07  31 1 2 .5  29 1 6.2  21. 212. 7  1. 121.42  1.161. 39  2. 341. .26  1 .441. 32  .311 .07  .251.02  1.731,.27  1 . 4 31.4 1  .04 11.004  .0631..001  32 1 3..6  24 1 4.3  21. 911. 6  1. 111.42  1.191. 40  2. 0 7 l . 20  1 . 5 3 1 . 37  -  .261.05  1. 741..40  1 .351. .39  .0511.002  .0611. 006  31 1 4..3  26 1 3.6  142  F i g u r e 24  Regression  c o e f f i c i e n t s of l e u c o c y t i c  cell  counts  Comparison between r e g r e s s i o n c o e f f i c i e n t s of l e u c o c y t i c c e l l counts ( c e l l s i n thousands) i n the blood of i n f e c t e d and n o n - i n f e c t e d f i s h groups.  LEUCOCYTIC cells in  o  CELL  COUNTS  thousand  144 (Table X V I I ) .  T h e r e f o r e , no comparisons were attempted.  Granulocytes Neutrophil  They were found to be the commonest g r a n u l o c y t e i n the  c i r c u l a t i n g blood of j u v e n i l e sockey  (Table XV and X V I I ) .  When the number  of c e l l s were compared between the blood of the i n f e c t e d and c o n t r o l  fish  groups, n e u t r o p h i l counts from the blood of the i n f e c t e d f i s h group were s i g n i f i c a n t l y h i g h e r than those i n the c o n t r o l f i s h group c e l l s were found to have i n c r e a s e d from 1.4 ± .2 x 10 .24 x 10  cells/mm .  3  3  "Granulocyte C e l l s "  A l l of the c e l l s  were counted and analyzed together under  of  cells/mm  3  The  to 2.20  ±  N e u t r o p h i l s i n c r e a s e d by almost 35% i n the i n f e c t e d  f i s h w h i l e those of the c o n t r o l f i s h decreased by  (Figure 27).  3  (Figure 26).  5.6%.  shown i n P l a t e V, F i g u r e a - f  the t i t l e  of " g r a n u l o c y t e c e l l s "  V a r i a t i o n i n the c e l l counts were very h i g h d u r i n g the p e r i o d  3 to about 60 DPI.  Then the mean c e l l  counts from the b l o o d of the i n -  f e c t e d f i s h group appeared h i g h e r than those from the c o n t r o l f i s h A s i g n i f i c a n t d i f f e r e n c e was  group.  o b t a i n e d when the r e g r e s s i o n c o e f f i c i e n t s were  compared. 3.9.2.3  Thrombocytic  Series  The mean number of thrombocytes blood was  fish  found to be h i g h e r than i n the n o n - i n f e c t e d f i s h b l o o d a t the be-  g i n n i n g of the experiment. lines  (of a l l forms) i n the i n f e c t e d  The r e g r e s s i o n c o e f f i c i e n t s of both r e g r e s s i o n  (Figure 28) were compared and found to be s i g n i f i c a n t l y  (P = 0.05). cells/mm . 3  cells/mm . 3  The mean v a l u e of the thrombocyte At the end of the experiment, i t was  pseudopodia  count at 3 DPI was found to be 23.3 x  T h i s means that the number of thrombocytes  times as a r e s u l t of the p a r a s i t i c  infection.  different 8.3 x 10  10  3  i n c r e a s e d about  four  The i n t e r m e d i a t e c e l l s w i t h  ( P l a t e VI, F i g u r e d) seemed to appear more f r e q u e n t l y i n the  blood of the i n f e c t e d  fish.  3  145  F i g u r e 25  Regression c o e f f i c i e n t s of lymphocyte counts Comparison of r e g r e s s i o n c o e f f i c i e n t s of lymphocyte counts of the blood of i n f e c t e d and c o n t r o l f i s h groups.  146  (oi o o D S i N n c o ajjooHdWjn  147  F i g u r e 26  Regression c o e f f i c i e n t s of n e u t r o p h i l counts Comparison between r e g r e s s i o n c o e f f i c i e n t s n e u t r o p h i l counts i n the blood of i n f e c t e d n o n - i n f e c t e d f i s h groups.  of and  TIME  IN  DAYS  -p00  149  F i g u r e 27  Regression c o e f f i c i e n t s of counts of " g r a n u l o c y t e  cells"  Comparison between r e g r e s s i o n c o e f f i c i e n t s of "granulocyte c e l l s " of the blood of i n f e c t e d and n o n - i n f e c t e d f i s h groups.  151  F i g u r e 28  R e g r e s s i o n c o e f f i c i e n t s of thrombocyte  counts  Comparison between r e g r e s s i o n c o e f f i c i e n t s of thrombocyte counts of the blood of i n f e c t e d and n o n - i n f e c t e d f i s h groups.  THROMBOCYTE  o  COUNT  L O G 10  153 4.  Discussion It  i s g e n e r a l l y agreed that the a c t i v i t y of b l o o d - f e e d i n g p a r a s i t e s  induces anemia, and t h i s c o n d i t i o n has a l s o been observed  i n a few cases of  p a r a s i t i z a t i o n with p a r a s i t e s which do not r e l y on b l o o d f o r n u t r i t i o n . However the r e s u l t s of t h i s experiment c r u s t a c e a n Satmincola  induces s i g n i f i c a n t a l t e r a t i o n i n the b l o o d c h a r a c t e r -  i s t i c s of j u v e n i l e sockey of  salmon.  That i n f e c t i o n leads to the development  anemia was i n d i c a t e d i n t h i s experiment  cells,  by the r e d u c t i o n i n r e d b l o o d  i n hemoglobin c o n c e n t r a t i o n and a l s o i n hematocrit v a l u e . There are at l e a s t  cause anemia. or  s t r o n g l y i n d i c a t e that the p a r a s i t i c  around  Firstly,  three p o s s i b l e ways i n which t h i s p a r a s i t e can  i t may r e s u l t from d i r e c t damage to c e l l s at the s i t e  the s i t e of i n f e c t i o n .  The s e v e r i t y of the e f f e c t s of t h i s p a r a -  s i t e depend upon the s i t e of i n f e c t i o n and a c t i v i t y o f the p a r a s i t e . major s i t e o f i n f e c t i o n w i t h t h i s p a r a s i t e i n j u v e n i l e sockeye the g i l l  cavity.  The  salmon i s i n  In f r y , the p a r a s i t e s a r e normally found on the g i l l  fila-  ments which they e v e n t u a l l y leave f o r s i t e s of more permanent attachment adults. organ  The l e s i o n s on the g i l l  as  f i l a m e n t which are caused by the attachment  ( f r o n t a l f i l a m e n t ) of the p a r a s i t e and the e p i t h e l i a l damage caused by  the f e e d i n g a c t i v i t y of the p a r a s i t e may l e a d to hemorrhaging as observed in  tench i n f e c t e d w i t h Evgasilus  sieboldi  hemorrhaging, however, was not observed i t may r e s u l t from the h e m o d i l u t i o n . tween g i l l  (Einszporn-Orecka,  i n the present experiment.  s u r f a c e area and body mass must e x i s t  t h i s balance and lead to h e m o d i l u t i o n .  v a t i o n of Hines and S p i r a  i f the f i s h  i s to maintain will  T h i s i s confirmed by the obser-  (1973) on carp that Ichthyophthirius  and i n c r e a s e s i n serum K .  Secondly,  Any damage t o the g i l l s  r e g u l a t o r y d i s t u r b a n c e s which l e d to s i g n i f i c a n t decreases Mg  Severe  I t i s known that a balanced r a t i o be-  proper osmotic r e g u l a t i o n of i n t e r n a l f l u i d s . upset  1970).  caused osmoi n serum Na  and  In cases of severe h e m o d i l u t i o n , such as  154 r e s u l t i n g from kidney d i s e a s e , Iwama (1977) found that MCV  values of the  blood of the i n f e c t e d f i s h are h i g h e r than those of h e a l t h y f i s h . i n the present experiment,  no s t a t i s t i c a l l y  However,  s i g n i f i c a n t d i f f e r e n c e was  found  i n the MCV.  T h i s r e s u l t should i n d i c a t e that h e m o d i l u t i o n d i d not  But from my  p o i n t of view, t h i s seems u n l i k e l y , s i n c e the p r o g r e s s i v e r e -  d u c t i o n of red blood c e l l s per u n i t volume observed ( F i g u r e s 17 and  occur.  i n the i n f e c t e d  fish  18) i n d i c a t e s the p o s s i b i l i t y of an i n c r e a s e i n blood volume  along with a r e d u c t i o n of the red c e l l numbers.  In the f i r s t case, an  i n c r e a s e i n red blood c e l l volume r e s u l t i n g from the h y p o t o n i c i t y of the surrounding plasma occurs and, However, i f a decrease  t h e r e f o r e , an i n c r e a s e i n MCV  i s obtained.  i n the number of red c e l l a l s o occurs at the same  time, there w i l l be a r e d u c t i o n i n the r a t e of i n c r e a s e of the hematocrit value. any  In that case, no d i f f e r e n c e  i n MCV  s i g n i f i c a n t d i f f e r e n c e i n the MCH  i n f e c t e d f i s h groups found. variations  (Davidsohn  f i s h shows a s l i g h t l y  Neither  of the blood of i n f e c t e d and  Consequently,  and Nelson,  w i l l be observed.  1974).  the MCV  and MCH  show s i m i l a r  lower v a l u e , not s i g n i f i c a n t l y d i f f e r e n t  to the p r o g r e s s i v e d e s t r u c t i o n of the red c e l l s . r e s u l t from the d e s t r u c t i o n of the red c e l l s  e f f e c t s on the blood c e l l s they may r e d u c t i o n i n the number of c e l l s may  reach the hemopoietic  .05),  attributed  T h i r d l y , anemia may  themselves.  also  There were some the p a r a s i t e through  the b u l l a and a l s o the metabolic e x c r e t i o n from the p a r a s i t e may i n t o the c i r c u l a t i n g b l o o d .  infected  (P =  I t can a l s o be  i n d i c a t i o n s of m e t a b o l i t e exchange between the f i s h and  absorbed  non-  MCHC of the blood of the  than the blood of the n o n - i n f e c t e d f i s h groups.  was  I f these m e t a b o l i t e s have  have been pathogenic  d i r e c t l y destroy them, l e a d i n g to a  i n the c i r c u l a t i n g b l o o d .  P o s s i b l y , they  t i s s u e , i n t e r f e r i n g with the normal f u n c t i o n of  the t i s s u e , l e a d i n g to a r e d u c t i o n of the c e l l s  i n the c i r c u l a t i n g b l o o d .  T h i s i s supported by the study by Romestand and T r i l l e s  (1977) of  fish  155 p a r a s i t i z e d w i t h a cymothoid l a t i n g e r y t h r o c y t e s and  isopod.  suggested  They found a decrease  that t h i s was  i n the c i r c u -  a good i n d i c a t i o n of  inter-  ference with the normal p r o d u c t i o n of b l o o d . I t is. concluded  that Satmincola  t i o n s i n j u v e n i l e sockeye  californiensis  causes anemic c o n d i -  salmon, i n d i c a t e d by a r e d u c t i o n i n red c e l l s ,  hemoglobin c o n c e n t r a t i o n and hematocrit v a l u e s .  T h i s c o n d i t i o n may  be  a t t r i b u t e d to h e m o d i l u t i o n r e s u l t i n g from damage to the g i l l s and the  skin  caused by t h i s p a r a s i t e and p o s s i b l y a l s o by metabolic e x c r e t i o n s of the p a r a s i t e , d i r e c t damage to the blood c e l l s or i n t e r f e r e n c e w i t h the f u n c t i o n of the hemopoietic The  tissue.  c r i t i c a l p e r i o d of i n f e c t i o n ,  i n d i c a t e d by a sharp decrease i n  red blood c e l l numbers (Figure 17), hemoglobin c o n c e n t r a t i o n (Figure 13) hematocrit values  ( F i g u r e 14), c o i n c i d e d with the p e r i o d  and  d u r i n g which the  copepod reached maximum growth and with the appearance of a secondary  gener-  a t i o n of p a r a s i t e s . Before d i s c u s s i n g the response z a t i o n of the f i s h by Satmincola,  of the white blood c e l l s  i t i s a p p r o p r i a t e to d i s c u s s and compare  the i n f o r m a t i o n on white .blood c e l l s which appears the r e s u l t s of t h i s  i n the l i t e r a t u r e  with  experiment.  Many c o n t r a d i c t o r y statements in l i t e r a t u r e .  to p a r a s i t i -  Nonetheless,  about white blood c e l l s have  appeared  as a r e s u l t of more recent developments, i n -  creased a t t e n t i o n has been given to the use of hematology as an  indicator  of the e f f e c t s of the environment on f i s h h e a l t h and,  consequently,  there  have been many advances i n h e m a t o l o g i c a l techniques.  Both terminology  and  hence d i s c u s s i o n s about white blood c e l l s have become l e s s c o n t r a d i c t o r y . I t i s now  r e c o g n i z e d that lymphocytes and  mrophology and f u l f i l l  quite different  c y t e s , i n both mature and  thrombocytes are d i s s i m i l a r i n  f u n c t i o n s (Ferguson,  1976).  immature forms, were d i f f e r e n t i a b l e  Thrombo-  i n the present  156 experiment, although the immature thrombocyte, which was o f t e n  encountered,  could not be d i f f e r e n t i a t e d from the small lymphocyte d e s c r i b e d i n the literature. While many workers denied  that monocytes e x i s t e d i n f i s h b l o o d  (Jakowska, 1956; McKnight, 1966; Wienreb and Wienreb, 1969; McLeay, 1970; Klontz,  1972; McCarthy et a l . , 1973) s e v e r a l others have r e p o r t e d them  (Lehmann and Stiirenberg, 1975; E l l i s , Budd, 1979).  1976; Ferguson, 1976 and L e s t e r and  Using the d e s c r i p t i o n s p r o v i d e d i n these p u b l i c a t i o n s , mono-  c y t e s were i d e n t i f i e d  i n the blood of j u v e n i l e sockeye salmon i n t h i s  experiment. In the present study, macrophages were r a r e l y found l a t i n g b l o o d of the experimental  fish.  i n the c i r c u -  A c c o r d i n g to the d e f i n i t i o n of  macrophage formulated by Van F u r t h e t a l . (1972),  this c e l l  i s mainly  l o c a l i z e d w i t h i n c o n n e c t i v e and other t i s s u e h a b i t a t s and i s not normally present  i n the c i r c u l a t i n g b l o o d .  this c e l l  However, many o t h e r workers have  found  i n the c i r c u l a t i n g blood of f i s h .  Amongst g r a n u l o c y t e s , n e u t r o p h i l s are the b e s t d e s c r i b e d and have been observed  i n most f i s h examined.  however, that n e u t r o p h i l s were absent t i o n seems most unusual.  Gardner and Y e v i c h  (1969) claimed  from c y p r i n o d o n t s .  T h i s c e l l was found  This  observa-  o f t e n i n the c i r c u l a t i n g  blood of salmon and the s t a i n i n g technique used i n t h i s experiment  clearly  demonstrated the c h a r a c t e r i s t i c s of n e u t r o p h i l s as d e s c r i b e d i n the l i t e rature. U n f o r t u n a t e l y , e o s i n o p h i l s and b a s o p h i l s a r e l e s s w e l l d e s c r i b e d , and  I was unable  to p o s i t i v e l y  i d e n t i f y these c e l l s  Studies by Watson et a l . (1956),  Ostroumova (1960), Lukina  and Haynes (1975) L e s t e r and Desser c o n t r a d i c t one another  i n sockeye salmon b l o o d .  (1975),  i n v a r i o u s ways.  (1965),  Davies  and L e s t e r and D a n i e l s  (1976)  Lehmann and Stiirenberg (1975)  157 c l e a r l y demonstrated Papenheim s t a i n .  the presence of these two c e l l s  T h i s s t a i n was  i n rainbow  trout using  a l s o used with the b l o o d of sockeye  salmon  i n the present experiments, but the c e l l s c o u l d not be d i f f e r e n t i a t e d . difficulty  i n d i f f e r e n t i a t i n g between these two  granules remain u n s t a i n e d . Duthie  (1939), Drury  types of c e l l  The  i s that the  L e s t e r and D a n i e l s (1976) however, agreed w i t h  (1951) and Catton  (1951) that e o s i n o p h i l s are common  i n the b l o o d and t i s s u e s of c e r t a i n t e l e o s t s but were unable to i d e n t i f y them w i t h c e r t a i n t y because to take up the s t a i n .  the granules a p p a r e n t l y vary i n t h e i r  ability  They noted that the e a r l i e r d e s c r i p t i o n s were proba-  b l y based on c e l l s found i n the t i s s u e . I t i s probable that these two of sockeye them was  types of c e l l s a l s o e x i s t  i n the b l o o d  salmon and that the f a i l u r e of the present experiment  due to t h e i r i n a b i l i t y t o take up the s t a i n used.  to d e t e c t  A number of c e l l s  which were q u i t e s i m i l a r m o r p h o l o g i c a l l y to those d e s c r i b e d i n the  literature  were observed however, and these have been c l a s s i f i e d as " g r a n u l o c y t e c e l l s " and await f u t h e r , more r e f i n e d  differentiation.  It has been known f o r q u i t e some time that the number of c i r c u l a t i n g white b l o o d c e l l s can be a f f e c t e d by environmental and p h y s i o l o g i c a l Reduction i n the number of c i r c u l a t i n g white b l o o d c e l l s appears  i n response  to an i n c r e a s e i n the l e v e l of c i r c u l a t i n g p i t u i t a r y ACTH (Mcleay, The  same author found that environmental s t r e s s on f i s h i n 0  c o n d i t i o n s l e d to a decrease i n lymphocyte  1973).  deficient  numbers and B l a x h a l l  an i n c r e a s e i n e o s i n o p h i l s r e l a t e d to environmental s t r e s s . r a p i d response to environmental changes,  2  factors.  (1972) noted  Because of such  white b l o o d c e l l s have come to be  used more o f t e n f o r e v a l u a t i n g the nature and depth of the e f f e c t s of p a r a s i t e s on f i s h .  As y e t , the mechanisms i n v o l v e d i n the v a r i a t i o n s i n each  type of c e l l have not been c l e a r l y e x p l a i n e d . is l i k e l y  to prove  significant  i n t h i s regard.  C e l l u l a r f u n c t i o n , however,  158 Some t h e o r i e s about the f u n c t i o n a l r o l e of lymphocytes  which may be  a p p l i c a b l e to our understanding of t h i s phenomenon have been e s t a b l i s h e d . The most widely accepted of these i s one proposed by Y o f f e y (1962), views the lymphocyte to immunological cells  as a m u l t i p o t e n t i a l hemopoietic stem c e l l which  stimuli.  responds  White (1963) a l s o commented that the e x e c u t i v e  i n the immunological  system are c i r c u l a t i n g lymphocytes.  u l a r immunological f u n c t i o n of the lymphocyte species of f i s h i n c l u d i n g salmon. lymphocytes  which  This p a r t i c -  has been noted i n v a r i o u s  T h e r e f o r e an i n c r e a s e i n the number of  i n the b l o o d of sockeye salmon i n f e c t e d w i t h Salminaola  must be  a t t r i b u t e d to the f u n c t i o n they p l a y i n the immunological response. would seem to be the case that when a lymphocyte i t r e c o g n i z e s , i t undergoes  c o n t a c t s an a n t i g e n which  r a p i d change, producing a l a r g e number of  daughter c e l l s which a l s o respond t o that a n t i g e n . the number o f lymphocytes  It  Hence, an i n c r e a s e i n  caused by the p a r a s i t e would i n d i c a t e the uptake  of m e t a b o l i t e s from the p a r a s i t e i n t o the c i r c u l a t i n g system.  Furthermore,  Hines and S p i r a  (1973) observed a c q u i r e d immunity i n carp i n f e c t e d with  Ichthyophthir-Cus  .  G o l o v i n a (1976), however, found a decrease r a t h e r than an  i n c r e a s e i n the number of c i r c u l a t i n g lymphocytes t i o n of carp w i t h Daatylogyrus  as a r e s u l t of the i n f e c -  but she d i d not p r o v i d e a c l e a r e x p l a n a t i o n  f o r t h i s decrease. The i n c r e a s e i n n e u t r o p h i l s r e s u l t i n g from Salmincola.  infection  must be e x p l a i n e d i n terms of i n f i l t r a t i o n of the n e u t r o p h i l s to the i n j u r i ous t i s s u e .  A number of r e s e a r c h e r s have observed t h i s inflammatory  i n the course of s t u d i e s of b a c t e r i a l , protozoan and copepod even as a r e s u l t of t a g g i n g (Weinreb, and Jones,  1973; Hines and S p i r a ,  response  i n f e c t i o n s and  1959; Thorpe and Roberts, 1972; Joy  1973; L e s t e r and D a n i e l s ,  1976).  The  inflammatory response i n f i s h i s b a s i c a l l y s i m i l a r to that i n mammals a l though  i t i s l e s s i n t e n s e and slow both i n appearance  and i n r e s o l u t i o n  159 (Finn and N i e l s o n ,  1971).  In a d d i t i o n to t h e i r r o l e on the  inflammatory  response, n e u t r o p h i l s i n humans are p r i m a r i l y a s s o c i a t e d w i t h the phagocytosis  of microorganisms and  was  i n rainbow t r o u t by Watson et a l . (1963) and F i n n and  reported  (1971). likely  A highly significant  other f o r e i g n m a t e r i a l s .  initial  increase  This function Nielson  i n the number of n e u t r o p h i l s would  i n d i c a t e the presence of t h i s l a t t e r f u n c t i o n i n a d d i t i o n to  the  inflammatory response. In humans there i s a long r e c o g n i z e d and  parasitic  infection.  earlier, this c e l l mental f i s h cell out.  because of the d i f f i c u l t i e s  could not be d i f f e r e n t i a t e d i n the blood  i n t h i s experiment.  (under the category The  Unfortunately,  a s s o c i a t i o n between e o s i n o p h i l s  of the  explained  experi-  Hence e v a l u a t i o n of the v a r i a t i o n of  of "granulocyte  i n c r e a s e i n the "granulocyte  this  c e l l s " ) c o u l d not be v a l i d l y c a r r i e d  c e l l s " i n t h i s experiment may  be  due  not only to i n c r e a s e i n the number of e o s i n o p h i l s ; i t c o u l d a l s o have r e s u l t e d from i n c r e a s e s  in basophils  cannot go beyond t h i s p o i n t u n t i l d e s c r i p t i o n of the c e l l s  i n the  Some of those who discussed that the and  other  f u r t h e r study can provide  "granulocyte  claimed  types of c e l l s .  i n two  eosinophils exist  Acanthocephalus.  Golovina  response to Dactylogyms appeared i n the b l o o d The  was  a clear  i n f i s h blood Bullock  i n the number of e o s i n o p h i l s i n brook and  species of catostomid  Discussion  c e l l s " series.  the e f f e c t s of the p a r a s i t e on t h e i r number. increase  reduction  and  (1963) noted  rainbow t r o u t  a response to i n f e c t i o n  by  (1976) observed that the most c h a r a c t e r i s t i c  i n carp was  an  i n c r e a s e i n the e o s i n o p h i l s which  during n e c r o s i s of the g i l l t i s s u e .  other obvious r e s u l t of the presence of Salmincola  i n blood c l o t t i n g time i n the i n f e c t e d f i s h .  c o u l d be a t t r i b u t e d to the c i r c u l a t i n g blood  have  increase  was  a  T h i s phenomenon  i n the number of thrombocytes i n the  (Gardner and Y e v i c h ,  1969;  Wardle, 1971).  160 Observation of the r e d and white blood c e l l s indicated  i n this  experiment  that damage caused to the f i s h as a r e s u l t of p a r a s i t i z a t i o n i s  not c o n f i n e d to mechanical damage a l o n e . and changes  i n the white b l o o d  cells.  I t goes f u r t h e r  to cause anemia  GENERAL DISCUSSION  162  The  r e s u l t s of t h i s study show that the p a r a s i t e  californiensis  not only causes mechanical damage to i t s f i s h h o s t , but a l s o  that i t adversely  a f f e c t s the f i s h ' s h e a l t h .  t a t i n g , and even l e t h a l . increase  Salminoola  I t s e f f e c t s can be d e b i l i -  The presence of t h i s p a r a s i t e can cause a d r a s t i c  i n the r a t e of m o r t a l i t y among i n f e c t e d f i s h  i n f e c t i o n i s high  i f the degree of  enough or when i t a c t s s y n e r g i s t i c a l l y with other  environ-  mental s t r e s s e s . In order to provide a c l e a r p i c t u r e of the p a r a s i t e ' s e f f e c t on f i s h health  i n the presence of other environmental s t r e s s e s , t h e i r i n t e r a c t i o n s  are c l a r i f i e d discussed  i n the accompanying f l o w chart  (Figure 29). They are a l s o  i n d e t a i l below.  First  of a l l , i t should be kept i n mind that the pathogenic e f f e c t s  of the p a r a s i t e are not c o n f i n e d  to the g i l l s .  found on t h e . f i n s , the f i n - b a s e ,  the s k i n and the b r a n c h i a l  majority  are i n the f i n - b a s e s .  In f r y , the p a r a s i t e can be  In j u v e n i l e s , they have a s i m i l a r  u t i o n but are found mainly i n the b r a n c h i a l r e g i o n .  In l i g h t estimate of g i l l be about 20%.  pathology i n the  w i l l be emphasized.  of the study by Kabata and Cousens (1977), a reasonable surface  area d e s t r u c t i o n  With such extensive  i n the present experiment would  damage, i t i s obvious that gas exchange  as w e l l as other p h y s i o l o g i c a l f u n c t i o n s must be d i s t u r b e d . salmonid f i s h ,  distrib-  As j u v e n i l e sockeye  salmon were used i n the experiment, the p a r a s i t e - i n d u c e d r e g i o n of the g i l l s  r e g i o n but the  sockeye salmon are a c t i v e , t h e r e f o r e ,  i s much h i g h e r than i n more sedentary s p e c i e s .  L i k e any other  tissue 0  2  consumption  Under normal circumstances,  163 f i s h can i n c r e a s e t h e i r 0 mechanism.  2  consumption by making use o f a compensatory  When an i n c r e a s e d amount of 0  2  i s r e q u i r e d , f i s h can i n c r e a s e  the flow of water over the g i l l s by i n c r e a s i n g the r a t e and amplitude of o p e r c u l a r movement.  When Satmincola  i s present,  the o p e r c u l a r movement  appears to be i n t e r f e r e d w i t h due to the s i z e and the s i t e of i n f e c t i o n . For example, the attachment o f t h i s p a r a s i t e along the b r a n c h i a l r i m can prevent  the movement and/or complete c l o s u r e of the operculum.  This  condi-  t i o n would c e r t a i n l y a f f e c t the amount of water p a s s i n g through the g i l l s . A l s o i n severe thelial  i n f e c t i o n with extensive  c e l l s adjacent  gill  surface d e s t r u c t i o n , the e p i -  to the damage are e q u a l l y n o n - f u n c t i o n a l  i n gas  transfer. G i l l d e s t r u c t i o n diminishes chamber  (Hughes, 1964) and c e r t a i n l y , l i m i t s the amount of 0  a l s o upsets of g i l l  the amount o f water p a s s i n g through the  osmoregulation  area a v a i l a b l e .  s i n c e the s a l t balance Firstly,  leads to a l e a k of l a r g e molecular (Hunn, 1964).  +  and CI  It  or s k i n e p i t h e l i a  weight molecules e s p e c i a l l y p r o t e i n s  Secondly, i t may i n t e r f e r e with the p r o d u c t i o n and/or normal i n t e r f e r i n g with the t r a n s p o r t  ions a g a i n s t the c o n c e n t r a t i o n g r a d i e n t .  d i e n t which e x i s t s between blood and water, coupled  with  The osmotic  can l e a d t o a net uptake of water and a subsequent  gra-  the improper  f u n c t i o n i n g of the c h l o r i d e s e c r e t o r y c e l l s , and extensive  The  absorbed.  i s a f f e c t e d by the amount  damage to the g i l l  f u n c t i o n i n g of the c h l o r i d e s e c r e t o r y c e l l s , of N a  2  gill  damage,  hemodilution.  anemic c o n d i t i o n of the i n f e c t e d f i s h can be explained as a  r e s u l t of hemodilution.  However, there a r e other p o s s i b l e a e t i o l o g i c a l  agents, as no s t a t i s t i c a l l y Volume was observed  s i g n i f i c a n t d i f f e r e n c e i n Mean  (as d i s c u s s e d  i n s e c t i o n V).  Corpuscular  The anemic c o n d i t i o n of  the i n f e c t e d f i s h c o u l d be due to the r e d u c t i o n of red c e l l numbers i n the c i r c u l a t i n g blood.  T h i s may be a t t r i b u t e d to a t o x i c s e c r e t i o n r e l e a s e d  164 by the p a r a s i t e .  T h i s substance may  the b u l l a , as m e t a b o l i t e the b u l l a  (Kabata and  through the g i l l  get  i n t o the c i r c u l a t i n g blood  exchange has p r e v i o u s l y been observed to occur v i a  Cousens, 1977), or i t may  epithelia.  hemopoietic t i s s u e s and  be absorbed from the water  This " t o x i n " might a l t e r n a t e l y a f f e c t  i n t e r f e r e w i t h the normal p r o d u c t i o n  However, t h i s i s u n l i k e l y as immature red c e l l s were found to be  s l i g h t l y , but not  significantly  The widely accepted  increased.  i n the  into circulating  Salmincota of the f i s h .  uptake i s upset (Hughes, 1964).  i n d i c a t e s the p o s s i b i l i t y of  is attributed, f i r s t l y ,  t h i s normally  the growth  to a c o n s i d e r a b l e amount  homeostatis,  while  the amount  as the r e s u l t of the damage to the g i l l r e s p i r a t o r y Secondly, the i n f e c t e d f i s h i s u n l i k e l y to consume an more o f t e n observed l e f t at the bottom  of tanks c o n t a i n i n g i n f e c t e d f i s h than those these  metabolite  blood.  adequate amount of food as food was  Therefore,  (as  increase in  has a l s o been found to d e l e t e r i o u s l y a f f e c t  This r e s u l t  re-  cells.  of energy which must be expended to maintain  area  circu-  immunological f u n c t i o n of lymphocytes  t h e i r numbers i n i n f e c t e d f i s h ,  2  blood  However, i t  d i s c u s s e d e a r l i e r i n s e c t i o n V) coupled w i t h a s i g n i f i c a n t  of 0  of e r y t h r o c y t e s .  s t i m u l a t e s the hemopoietic t i s s u e to i n c r e a s e p r o d u c t i o n  s u l t i n g i n a r a i s e d l e v e l of immature red  absorption  the  i n the c i r c u l a t i n g  i s p o s s i b l e that the p r o g r e s s i v e r e d u c t i o n of the r e d c e l l s l a t i n g blood  through  containing non-infected  i n f e c t e d f i s h must make use of t h e i r energy r e s e r v e s  r e s u l t s i n the r e d u c t i o n of weight and  fish. and  growth of the i n f e c t e d  fish. Under an environmental change such as an i n c r e a s e i n water tempera t u r e , f i s h i n c r e a s e metabolic  a c t i v i t y and r e q u i r e more 0  2  ( B r e t t , 1964).  High water temperature, however, l i m i t s the amount of d i s s o l v e d 0 . 2  f o r e , i n the case of extensive  There-  r e d u c t i o n of the r e s p i r a t o r y s u r f a c e due  to  165 severe i n f e c t i o n by t h i s p a r a s i t e , i t i s l i k e l y 0  2  uptake may not be met.  t h a t the r e q u i r e d amount of  F i s h p a r a s i t i z e d w i t h approximately 31  were found to have a c r i t i c a l  l e t h a l temperature of only 21°C.  Swimming performance n o r m a l l y i n c r e a s e s w i t h r i s i n g ature  (Brett,  1958).  Salnrincola  water  temper-  In h i g h v e l o c i t y water, the swimming a b i l i t y of the  f i s h under a c o n t r o l temperature c o n d i t i o n was found t o be l e s s than i n the non-infected f i s h .  T h e r e f o r e , the a b i l i t y o f the i n f e c t e d f i s h to swim i n  water of h i g h temperature and h i g h v e l o c i t y must be much l e s s than that o f non-infected f i s h . the  However, the r e d u c t i o n of the g i l l  only f a c t o r l i m i t i n g the swimming a b i l i t y  s u r f a c e area i s not  of the f i s h .  The anemic  con-  d i t i o n caused by t h i s p a r a s i t e a l s o reduces the maximum swimming speed. Jones  (1971) demonstrated that i n an anemic  condition,  i n which the hemato-  c r i t v a l u e was reduced t o h a l f or o n e - t h i r d , a 40% r e d u c t i o n i n maximum swimming speed of t r o u t was observed. Osmoregulation r e s u l t i n g from the damage caused by t h i s p a r a s i t e may be one of the problems of f i s h d u r i n g the course of m i g r a t i o n .  The  r e s u l t s o b t a i n e d i n the present experiment a l s o i n d i c a t e that the i n f e c t e d f i s h have l e s s a b i l i t y infected.  to move from f r e s h water to s a l t water than do non-  In a d d i t i o n , i n f e c t e d f i s h a v o i d h i g h s a l i n i t y .  Physiological  t r a n s f o r m a t i o n of p a r a s i t i z e d f i s h f o r smolting was not measured i n t h i s experiment.  However, C l a r k e and Blackburn (1977) have proposed a seaward  c h a l l e n g e t e s t as a s e n s i t i v i t y index to measure the smolting of the f i s h . If  i n f u t u r e s t u d i e s , t h i s parameter i s c o n s i d e r e d f o r measuring the  r e a d i n e s s of the i n f e c t e d f i s h  to migrate, we w i l l be i n a b e t t e r  to answer the q u e s t i o n as to whether  position  or not t h i s p a r a s i t e prevents f i s h  from m i g r a t i n g . In  t h i s study f u n g a l i n f e c t i o n s were observed on .some of the  infected f i s h .  Secondary i n f e c t i o n s such as fungus or b a c t e r i a a r e  166 observed  i n most cases of e x t e r n a l p a r a s i t i c c o n d i t i o n s ; these may  l e a d to  an i n c r e a s e i n the s e v e r i t y of the e f f e c t of the p a r a s i t e on the f i s h h o s t . Finally, oatifovn-iensis  i t would be reasonable to conclude  a f f e c t s growth and b l o o d c h a r a c t e r i s t i c s  S e v e r i t y of the e f f e c t  infection.  Salmincola of the f i s h h o s t .  i s i n c r e a s e d w i t h r e l a t i o n t o : the l e v e l of  i n f e c t i o n , environmental secondary  that  s t r e s s e s , and p o s s i b l y with the occurence of  167  F i g u r e 29  Host-parasite-environment  relationships  Conclusion of the r e s u l t s of t h i s experiment. square i n d i c a t e s h o s t - p a r a s i t e r e l a t i o n s h i p s .  Doubled  Food consumpt. Temperature Parasitized fish  Osmotic stresses  Growth  Mechanical Fish b l o o d  k — — —  —  —  —I m i U b o l i t a  Salinity  damage  O.uptake Fish  Swimming ability  health  Mortality  Secondary infection  •(Migration  /*7  REFERENCES  170  A l l a n s o n , B.R. and R.G. Noble. 1964. (Peters) to h i g h temperature.  The t o l e r a n c e of Utopia mossambica Trans. Amer. F i s h . Soc. 93:323-332.  Baggerman, B. 1960. S a l i n i t y p r e f e r e n c e , t h y r o i d a c t i v i t y and the seaward m i g r a t i o n of f o u r s p e c i e s of P a c i f i c Salmon Onaorhynchus. J. Fish. Res. Bd. Can. 17:295-322. B a i n b r i d g e , R. 1958. The speed of swimming of f i s h as r e l a t e d t o s i z e and to the frequency and amplitude of the t a i l beat. J . Exp. B i o l . 35: 109-133. .  1960.  Speed and stamina i n three f i s h e s .  J . Exp. B i o l .  37:129-  153. .  1962.  T r a i n i n g speed and stamina i n t r o u t .  J . Exp. 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