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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, Kasetsart University, Thailand, 1964 M.Sc, Auburn University, Auburn, Alabama, 1972 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY We accept this thesis as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA in the Department of Zoology A p r i l , 1980. © Kamonporn Pawaputanon, 1980 In presenting th is thesis in par t ia l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the Library shal l make i t f ree ly avai lab le for reference and study. I further agree that permission for extensive copying of th i s thesis for scholar ly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publ icat ion of th is thesis for f inanc ia l gain shal l not be allowed without my written permission. Department of ZOT>\Q The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date E-6 B P 75-51 1 E ABSTRACT Sockeye salmon p a r a s i t i z e d with Salmincola calif'ovniensis"were compared experimentally with unparasitized f i s h of the same age to deter-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 stresses and (3) hematological e f f e c t s of t h i s p a r a s i t e on i t s f i s h host. I t was found that average i n f e c t i o n l e v e l s of 31.25 para-s i t e s per f i s h can reduce the weight of the f i s h host by almost 34 % within a period of 112 DPI. The rate of increase i n length of the i n f e c t e d f i s h group was slower than that of the non-infected f i s h group though 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 developed during the experimental period. The p a r a s i t i z e d f i s h were found to develop anemia, expressed by the reduction i n red c e l l counts, hemoglobin concentrations and hematocrit values. This anemic condition i s a t t r i b u t e d to hemodilution of the blood, r e s u l t i n g from damage to the g i l l and skin e p i t h e l i a , and, i n turn, leading to an osmotic imbalance between the water and the i n t e r n a l f l u i d s . In addition, the progressive reduction of the red c e l l s i n the c i r c u l a t i n g blood may be a r e s u l t of the absorption of p a r a s i t e metabolic secretions through the g i l l s or the b u l l a . Such absorption seems l i k e l y because of the observed v a r i a t i o n s of the c e l l s i n the l e u c o c y t i c system and the s i g n i f i c a n t increase i n lymphocytes, neutrophils and "granulocyte c e l l s " i n r e l a t i o n to i n f e c t i o n time. Furthermore, the blood of the i n f e c t e d f i s h c l o t t e d f a s t e r than that of the non-infected f i s h . During the course of i n f e c t i o n a marked increase was a l s o observed i n the number of thrombocytes. Parasitized f i s h were less able to cope with environmental stresses. A water temperature of 21°C was found to be the median l e t h a l temperature o infected f i s h . The swimming a b i l i t y of infected f i s h was also reduced. The parasitized f i s h reached 50% fatigue when they swam in water of a v e l o c i t y of 65 cm/sec for only 250 nin. The chance of surviv a l for the infected f i s h i n this high water v e l o c i t y i s only 6.6% over the period of 600 min. The a b i l i t y of the infected f i s h to transfer from fresh water to s a l t water was also affected. Mortality of the infected f i s h increased during this t r a n s i t i o n and these f i s h , as indicated by the s a l i n i t y prefer-ence test, also avoided high s a l i n i t y , suggesting that they may not have been ready to migrate. The c r i t i c a l period of i n f e c t i o n where marked differences were found i n a l l the parameters was that period when the parasites reached maximum size and a second i n f e c t i o n took place with 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 Fish 6 2. Parasite Source • • • • 6 3. Infection Techniques 6 3.1. Parasite Numbers • 7 3.2. Parasite-Days 10 SECTION I IMPACT ON. GROWTH AND WEIGHT 14 1. Introduction • 15 2. Materials and Methods 17 2.1. Experimental Design 17 2.2. Experimental Fish 17 2.3. Growth Study 18 2.4. Data Analysis 18 3. Results 19 4. Discussion 22 SECTION II IMPACT ON HOST'S TEMPERATURE TOLERANCE 29 1 . Introduction 30 2.. Materials and Methods 31 2.1. Experimental Fish 31 2.2. Experimental Procedure 31 3. Results 32 4. Discussion 40 SECTION III IMPACT ON HOST'S SWIMMING ABILITY 44 1 . Introduction ^5 2. Materials and Methods 46 iv 2.1. Experimental- F i s h 46 2.2. Experimental Procedures 47 2.2.1. C r i t i c a l Velocity 47 2.2.2. Fixed Velocity 48 3. Results 49 4. Discussion 58 SECTION IV IMPACT ON HOST'S SALINITY TOLERANCE 63 1. Introduction 64 '2. Materials and Methods 66 2.1. Experimental Fish 66 2.2. Experimental Procedures 66 2.2.1. S a l i n i t y Tolerance Test 67 2.2.2. S a l i n i t y Preference Test 67 3. Results 68 3.1. S a l i n i t y Tolerance Test 68 3.2. S a l i n i t y Preference Test 69 4. Discussion 74 SECTION V IMPACT ON BLOOD 78 1 . Introduction 79 2. Materials and Methods 81 2.1. Experimental Design 81 2.2. Experimental Fish 81 2.3. Blood Sampling Procedure 81 2.4. Staining Technique 82 2.5. Hematological Determinations 85 2.5.1. Hemoglobin Concentration 85 2.5.2. Hematocrit Value 86 2.5.3. Erythrocyte Osmotic F r a g i l i t y Test 86 2.5.4. Clo t t i n g Time 88 2.5.5. Total Blood C e l l Counts 88 2.5.6. Red Blood C e l l Count 89 2.5.7. Red C e l l Corpuscular Value 90 2.5.8. D i f f e r e n t i a l C e l l Count 90 2.5.9. S t a t i s t i c a l Analysis of Data 91 3. Results 91 3.1. Hemoglobin Concentration 91 3.2. Hematocrit Value 95 3.4. Clo t t i n g Time 95 3.5. Total Blood C e l l Count 105 3.6. Red Blood C e l l Count 105 3.7. Red Blood Corpuscular Values 111 3.8. White Blood C e l l Counts 111 3.9. D i f f e r e n t i a t i o n of Blood Cell s 111 3.9.1. D i f f e r e n t i a l C e l l Descriptions 111 3.9.1.1. Erythrocytic Series 111 3.9.1.2. Leucocytic Series 120 3.9.1.3. Thrombocytic Series 122 3.9.2. D i f f e r e n t i a l C e l l Counts 122 3.9.2.1. Erythrocytic Series 122 v 3.9.2.2. Leucocytic Series 123 3.9.2.3. Thrombocytic Series 144 4. Discussion 153 GENERAL DISCUSSION 161 REFERENCES 1 7 0 vi LIST OF TABLES Table Page I Lengths, weights and c o e f f i c i e n t of condition factors 25 II Variation of dissolved 0 2 and pH levels 33 III Percent mortality and cumulative mortality 39 IV Relation of swimming speed to the percent fatigued 50 V Mean fatigue time, percent f i s h fatigued and size of the f i s h host 55 VI Variation in the number of parasites 59 VII Percent cumulative mortality 72 VIII S a l i n i t y preference test 73 . IX Hemoglobin concentration g/dl 94 X Hematocrit value (%) 98 XI Percent NaCl and percent hemolysis in osmotic f r a g i l i t y test 101 XII Percent blood clotted 104 XIII Total blood c e l l , red blood c e l l and white blood c e l l counts 106 XIV Calculated MCHC, MCV and MCH 112 XV Ranges, means and S.D. of leucocytic c e l l s 119 XVI Percentage and number of-immature red c e l l s 138 XVII Mean and S.D. of leucocytic c e l l counts 141 v i i LIST OF FIGURES Figure Page 1 The relationships of parasite numbers, parasite-days and time 9 2 L i f e cycle of Salminoota californiensis 12 3 Length and weight in r e l a t i o n to time post i n f e c t i o n 21 4 Condition factor (K) during 112 DPI 24 5 pH in experimental aquaria 36 6 Dissolved 0 2 in.experimental aquaria 36 7 Percent mortality and percent cumulative mortality of 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 Relationships between fatigue times and % f i s h fatigued .... 54 10 Relationships between fatigue time and parasite days 56 11 Percent cumulative mortality in r e l a t i o n to s a l i n i t y 71 12 Blood Sampling Technique 84 13 Relationships between hemoglobin and DPI 93 14 Relationships between hematocrit value and DPI 97 15 Erythrocyte osmotic f r a g i l i t y of experimental f i s h 100 16 Blood Clotting Time , 102 17 Relationships 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 corpuscular volume (MCV) 114 v i i i Figure Page 20 Mean corpuscular hemoglobin (MCH) 114 21 Mean corpuscular hemoglobin concentration (MCHC) 115 22 Regression c o e f f i c i e n t s of white blood 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 leucocytic c e l l counts 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 neutrophil 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 " .... 150 28 Regression c o e f f i c i e n t s of thrombocyte counts 152 29 Host-parasite-environment relationships 167 ix LIST OF PLATES Plate Page I Immature and mature red c e l l s 125 II Mature red c e l l s and lymphocytes 127 III Small lymphocyte, large lymphocyte, monocyte, macrophage and neutrophils 129 IV Neutrophils 131 V "Granulocyte c e l l s " 133 VI Immature and mature thrombocytes 135 VII Mature thrombocytes 137 x ACKNOWLEDGEMENTS I would l i k e to express my sincere gratitude to my supervisor, Dr. J.R. Adams, who guided the research, patiently edited the manuscript and provided i n s p i r a t i o n , encouragement and moral support. Without his enthusiasm this study would not have been possible. I am also indebted to Drs. A.B. Acton, Z. Kabata, L. Margolis and D.J. Randall who served on my thesis committee. The thesis greatly bene-f i t e d from their c r i t i c i s m s . In p a r t i c u l a r , I am grateful for the encouragement and moral support of Drs. Z. Kabata and L. Margolis. I would also l i k e to acknowledge the many people at the P a c i f i c B i o l o g i c a l Station and P a c i f i c Environmental Institute who supplied the f i s h and f a c i l i t i e s for the research. I appreciated their invaluable assistance and I am extremely grateful for their friendship. I have been very fortunate to have continued assistance from my colleagues, Mr. L.R. Russell and Mr. G.S. Norman. Appreciation i s also extended to Mr. J.J. Kendall for his help in preparing this manuscript. Financial support was provided by the Canadian International Development Agency. 1 In natural water, sockeye salmon are found to carry parasites of many kinds: Protozoa, Myxosporida, Monogenea, Trematoda, Cestoidea, Acanthocephala, Hirudinoidea, Acarina and Copepoda (Margolis and Arthur, 1979). Among the p a r a s i t i c copepods, of which there are more than 1,000 species known from fishes throughout the world (Kabata, 1970), at least eight species of those copepods have been found on sockeye salmon. ( Margo-l i s and Arthur, 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 para s i t i z e d by only one species of t h i s genus, 5. californiensis (Dana, 1852). Kabata and Cousens (1977), who studied the host-parasite relationship between sockeye salmon and t h i s species of copepod, demon-strated the injurious effects r e s u l t i n g from the presence and a c t i v i t y of t h i s parasite. They noted that the severity of i t s effects depends upon the i n f e c t i o n s i t e . In juvenile sockeye salmon, the majority of the parasites are found i n the branchial c a v i t y , mainly on the branchial rim with some on the inner wall of the operculum. A few are attached to the g i l l filaments and almost a l l of these are immature. The g i l l s are found to have the strongest tissue response to t h i s parasite. The p r o l i f e r a t i o n of the g i l l epithelium leads to p a r t i a l or complete fusion of adjacent filaments. E p i t h e l i a l c e l l s become thic k e r , rendering them non-functional for gas and ion exchange. The effect 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 tissues 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 , losing i t s lamellar structure. Eventually, the structure of the bone i s destroyed. Normally 2 the effects of t h i s parasite are sublethal. Nevertheless, i t may be expected that t h i s parasite i s responsible for more than simple mechanical damage. Kabata (1970) reviewed the consequences of p a r a s i t i c Crustacea on fishes finding that they have ce r t a i n generalized effects on the host. These include: effects on weight and fat content, growth, metabolism, al 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 parasite can also lead to secondary i n f e c t i o n and dele-terious effects on the fis h e r y . j The l i t e r a t u r e contains several references to the loss 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 parasites 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) also found that the fat content of the l i v e r of haddock, severely p a r a s i t i z e d with Lernaeoceva, dropped by approximately 50%. Mann (1953) concluded from his observations that the parasite 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 , toxic secretions from the parasite can exert a great deal of ef f e c t on the f i s h host. Among them, metabolic disturbances are always found. Kollatsch (1959) has shown that a single Argulus larva i s capable of k i l l i n g Hyphessobryoon flammeus within three to four days. Considering the effect of parasites on blood character-i s t i c s , a blood-feeding parasite 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 parasites can also produce d e f i n i t e changes i n the blood of t h e i r hosts, for example, f i s h suffering from a severe i n f e c t i o n with Caligus have exhibited signs of anemia (Goregyad, 1955). Furthermore, Levnaea cypvlnacea. which attaches to the flank 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). Persistent loss 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 to aff e c t the repro-ductive capacity of the f i s h host (Kabata, 1958). Behavioral abnormalities were also observed i n many cases as i n fishes infected with AvguVus (Kollatsch, 1959). The introduction of secondary i n f e c t i o n as an a f t e r -math to the presence of crustacean parasites has been widely accepted and i s often referred 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 parasites induce negative repercussions i n the f i s h -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 single pond i n Ohio within two weeks (Tidd, 1933). Savage (1935) also reported that severe i n f e c t i o n of speckled trout (Salvelinus frontinalis) with Salmincola edwardsi resulted i n t h e i r death i n hatcheries i n Canada and the United States. After careful consideration 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 effects of the parasites on their hosts. The main d i f f i c u l t y l i e s i n our lack of knowledge regarding the normal condition of f i s h . Also, most investigators determined the effects of the parasite 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 studies. In many instances an investigation of the effects of a single parasite species on the host was undertaken without f i r s t perform-ing an examination to determine i f other species were also present on the host. These oversights possibly led to the contradictory 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 loss of weight i n infected f i s h ; however, Sherman and Wise (1961) found no such effect on the weight of Gadus morhua. Contradictory findings are very common i n hematological studies; Layman 4 (1957) mentions an increase in the number of neutrophils i n the blood of the tench {Tinea tinea) and the dace (Leuciscus leueisaus) infected with Ergasi-lus sieboldi, but Reichenback-Klinke et a l . (1968) did not f i n d any d i f f e r -ence between the blood c h a r a c t e r i s t i c s of parasite free Coregonus lavaretus and C. fera and the blood of f i s h carrying large numbers of Ergasilus. In l i g h t of previous research, does Salmineola, which attaches i t s e l f to an area of v i t a l importance have any generalized negative effects on i t s host? A study was, therefore, designed to c l a r i f y some aspects of the Salmin-aola-sockeye salmon host-parasite relationship by comparing (1) growth, (2) a b i l i t y to tolerate changes in temperature and s a l i n i t y , (3) swimming per-formance and (4) hematological c h a r a c t e r i s t i c s of infected versus non-infected f i s h under controlled conditions. These parameters were selected for the following reasons: growth is one of the factors frequently used to determine the severity of a para-s i t e ' s e f f e c t on the host (Schaperclaus, 1954) and hematological character-i s t i c s have been used as excellent indicators of physiological responses in many f i e l d s of study (Cope, 1961; Slicher, 1961; B a l l and Slicher, 1962 and Cardwell and Smith, 1971). Furthermore, sockeye salmon during their migra-tion to the sea must face major environmental changes. This parasite may reduce the f i s h e s ' a b i l i t y to withstand the changes they must face in nature. 5. GENERAL MATERIALS AND METHODS 6 In this section, materials and methods common to a l l parts of the study are described. Materials and methods s p e c i f i c to individual exper-iments w i l l be outlined i n the appropriate sections. 1. Experimental Fish Young sockeye salmon, Oncovhynchus nerka (Walbaum), were obtained from the federal government hatchery at Rosewall Creek, Vancouver Island, where they had been reared from f r y . The f i s h were transferred to the P a c i f i c Biological. Station in Nanaimo, B r i t i s h Columbia, kept in an aerated holding tank at ambient temperature and fed once a day to s a t i a t i o n with Oregon Moist P e l l e t s (3/32"). 2. Parasite Source In 1972 sockeye salmon smolts infected with the copepod Salmincola oaliforniensis (Dana, 1852) were brought to the P a c i f i c B i o l o g i c a l Station from Cultus Lake, B r i t i s h Columbia for the purpose of l i f e cycle 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 Station i n Nanaimo, were used as the source of i n f e c t i o n for the present studies. 3. Infection Techniques Egg sacs (with eggs at the pigment stage) were col l e c t e d from adult female Salmincola for the purpose of in f e c t i n g the stock of pa r a s i t i z e d f i s h . These eggs were placed in a dish of fresh water and refrigerated in a shaker bath at 12°C for two to four days or u n t i l the copepod larva hatched. The copepodids were released into the tank of the f i s h selected as the infected f i s h groups. 7 The experimental f i s h , divided into three groups according to the s p e c i f i c tests i n which they were to be used, were infected 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 to be used f o r growth and hematological studies, 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 for use i n temperature tolerance and swimming a b i l i t y t e s t s , was exposed to the larvae seven weeks p r i o r to the commencement of the tests i n temperature tolerance. Fish remaining from t h i s experiment were used i n the swimming a b i l i t y test conducted approximately f i v e months af t e r i n i t i a l exposure to the parasites. 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 infected three months p r i o r to that experiment. 3.1 Parasite Numbers . The p a r a s i t i z e d f i s h which were used for growth and hematological studies were also used i n evaluating the l e v e l of i n f e c t i o n . After the blood was sampled and the length and weight of the f i s h were recorded, the number of parasites i n the buccal cavity and on the skin was recorded for each f i s h . The mean numbers of parasites obtained at each sampling period 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 parasites during the experiment. I t was found that during the 3-41 Day Post Infection (DPI) period the number of copepods was 22.6 ± 3.2 per f i s h . At 41 DPI the majority of copepods were found with egg sacs but a few f i s h were found to be infected with copepods of the chalimus stages, in 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 parasite count f o r each f i s h increased very sharply with the occurrence of a new generation of parasites (indicated by the arrow with open c i r c l e i n Figure 1). At the chalimus stage, the parasite attaches to the g i l l filament by f r o n t a l filaments which do not provide a f i r m attachment. Figure 1 The relationships of parasite numbers, parasite-days and time Solid 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 in number of parasites. J indicates f i n d i n g of parasites of chalimus stage I indicates that copepods found on f i s h up to t h i s point * were adult indicates start of second i n f e c t i o n indicates a high count attributable to new generation of i n f e c t i o n P A R A S I T E D A Y S 6 10 This fact in combination with the further fact that male parasites die after mating can explain the noted tendency for the number of parasites per f i s h to decline. From 42 DPI to the end of the experiment (112 DPI), the mean number of the parasites 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 proportional not only to the number of parasites present but also to the length of time they have i n -fected the f i s h (Kabata and Cousens, 1977). Since the size of the parasite increases u n t i l i t reaches a f u l l y mature stage, i t i s more accurate to measure the degree of i n f e c t i o n by using parasite number in combination with "parasite-days" rather than alone. This l a t t e r measure (parasite-days) indicates the days that copepods of a given age are found on the f i s h host (Figure 2) and i s interpolated from the parasite number by means of the formula i l l u s t r a t e d below: Total parasite-days/fish = (P .xD ,)+(P _xD .)+ (P xD ) s1 s1 s2 s2 sn sn where P_ = number of parasites at a given stage D_ = number of days that a copepod at a given stage has been p a r a s i t i z i n g the f i s h (Figure 2) The parasite-days were plotted against time and a regression l i n e was drawn (y = 12.4 + .36 x). A high positive 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 , representing parasite numbers ( s o l i d l i n e ) , which w i l l be c a l l e d " l e v e l of i n f e c t i o n " and parasite-days (dotted line) "degree of i n f e c t i o n , " in Figure 1 leads to the observa-tion that the l a t t e r indicates the r e l a t i o n between the severity of infec t i o n and time (DPI) more accurately than does the former. The degree 11 Figure 2 L i f e cycle of Salnrincola oaliforniensvs This diagram i s adapted from Kabata and Cousens (1973). Time in brackets denotes duration of stages.- Since males die after mating only adult females are normally found on the f i s h . ADULT ( 5 d a y s ) t CHALIMUS IV (3 days) CHALIMUS III (2 days ) O" ADULT WITH EGG SACS ( 32- 45 days ) t CHALIMUS I ( 1 days) t COPE POD CHALIMUS II • ( ,1 days ) ADULT ( 14 days ) I CHALIMUSIV ( 4days ) CHALIMUSIII ( 2 days ) EGG AT PIGMENT STAGE of i n f e c t i o n was found to be constant at the rate of 7.5 parasite-days/day throughout the experiment. SECTION I IMPACT ON GROWTH AND WEIGHT 15 1. Introduction Many studies have repeatedly demonstrated that p a r a s i t i c i n f e c t i o n of f i s h may lead to growth retardation, weight loss, and a reduction in con-d i t i o n factor. P i t t and Grumdan (1957) found that yellow perch p a r a s i t i z e d with Liguta intestinatvs are markedly smaller than non-parasitized f i s h . Huggins (1959) attributed a stunted condition in a sample of black crappie (Pomox-is nigvomaoulatus) to Postodiplostomum minimum i n f e c t i o n . Lopukhina (1961) found that the length of rainbow trout parasitized with Triaenophovus avassus i s s i g n i f i c a n t l y less than that of non-parasitized f i s h . Uninfected Gasteosteous aculeatus were found to grow more rapidly than those parasitized with Schistocephalus solidus (Walkey and Meakins, 1970). Impairment of growth in juvenile sockeye salmon i s c l e a r l y the consequence of parasitism with Eubothrium salvelini (Boyce, 1979). • The studies of many researchers continue to stimulate interest in the effect of crustacean parasites on growth, weight and condition factors. Reduction in weight was observed by Scott (1909) and Kabata (1958) in whitings and other gadoids infected with Lernaeocera. Mann (1953) also found that Mevlangus parasitized with the same species were f i v e to ten percent under weight. Hotta (1962) observed the retardation.of growth by Caligus macavovi in Cololabis saura and Reichenbach-Klinke et a l . (1968) stated that Ergasilus sieboldi lowers the condition factor of Coregonus. The evidence points increasingly to the conclusion that several parasites exert an adverse e f f e c t on the growth, weight and condition factor of their f i s h host. However, there are contradictions which appear 16 in the l i t e r a t u r e i n d i c a t i n g that some parasites do not have any discernable effects.on t h e i r f i s h hosts. For example, Sproston and Hartley (1941) observed no differences between the condition factors of Gadus mevlangus infected with Lernaeooeva branahialis and those non-infected. K l e i n et a l . (1969) did not f i n d any s i g n i f i c a n t e ffects among rainbow trout infected by Crepidostomwn farionis. Lewis and Nickum (1964) also found no effect of Tosthodiptostomum minimum on the b l u e g i l l . Russell (1977) found only a s l i g h t decrease i n the growth rate of trout corresponding to an increase i n in f e c t i o n with Truttaedaonitis truttae. Surprisingly, Rao et a l . , (1972) even found that CatZa catla f i n g e r l i n g s , infected with black grub, seemed to gain weight. Interpretations from these studies are complicated by the fact that i n some cases, researchers f a i l e d to measure ei t h e r the l e v e l of in f e c t i o n or the relationships between the extent of i n j u r i e s and d i f f e r e n t levels of i n f e c t i o n . In some studies, the data were obtained from the f i e l d i n uncontrolled circumstances or the samples were too small. In comparing the effects of d i f f e r e n t parasites 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 into consideration. The effects of the parasite on the f i s h vary i n response to the number and/or size of parasites with which p a r t i c u l a r hosts are infected (Scott, 1929; Gross, 1935 and M i l l e r , 1945). Light infections may not produce any detectable effects on the host; high infections may create stress on the host and give an exagger ated condition. Furthermore, the c o n t r a - i n d i c a t i o n s may be due to differences among Iparasite species, f i s h species, f i s h age an environmental conditions. The attempt to evaluate the effects of Salmincota on growth and weight of juvenile 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 infected and non-infected f i s h of the same age and size were maintained under controlled experimental conditions. 17 2. Materials and Methods 2.1 Experimental Design Two experiments were conducted to investigate the effects of the para-s i t e on growth and blood c h a r a c t e r i s t i c s . The f i r s t experiment measured the effect between 14 and 112 days post i n f e c t i o n (DPI). Fish were sampled for growth and hematological determinations at approximately three day intervals for the f i r s t month, 14 day intervals for the second month and also at the end of the experiment (112 DPI). Twelve f i s h were sampled on each sampling day. After the i n i t i a t i o n of this experiment, a question arose about the possible occurrence of effects before 14 DPI. The second experiment was therefore assigned for the period of three to t h i r t y eight DPI, sample being taken every three days. 2.2 Experimental Fi s h Fourteen days before the f i r s t experiment was started, f i s h from the holding tank were transferred into two experimental tanks. They were size selected in order to keep this parameter constant. One hundred and f i f t y f i s h were used in each experimental tank. One tank was assigned for the control and the other for the infected group. Fish assigned to the i n -fected group were exposed to large numbers of copepod larvae (approximately 30,000 larvae) by the method described in the section of this paper e n t i -t l e d General Materials and Methods. Water supplied to these tanks was maintained at 9° ± 2°C by mixing water at ambient temperature with heated water. The tanks were equipped for water r e c i r c u l a t i o n and aeration. The second experiment was conducted one month after the i n i t i a t i o n of the f i r s t experiment. It was commenced on the t h i r d day after exposure of the f i s h to the copepod larvae. The average number of parasites 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 with Oregon Moist P e l l e t s with 3% body weight per day (recommended by Brett et a l . , 1969) for 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. l! I' 2.3 Growth Stujiy After the f i s h were sampled at random for hematological studies (as i described i n Section V), the length of each was measured to the nearest millimeter and t h e i r weights, to the nearest gram. Specific growth rate (G), c o e f f i c i e n t of condition (K) and absolute growth rate were computed to determine whether a relationship existed between the growth of the f i s h and the presence of the parasite using the formula: a) Specific growth rate (G) = log YT - log Yt X 100 T - t Where YT = f i n a l size at time T Yt = i n i t i a l size at time t b) Coefficient of condition (K) = W X 100 L 3 Where W and L = weight (g) and length (cm) c) Absolute growth rate = __ X 100 dt Where dW = weight difference 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 Analysis The mean lengths and weights of the control and infected f i s h groups were compared using the t - t e s t . S t a t i s t i c a l l y s i g n i f i c a n t differences were assessed at a confidence l e v e l of P = 0.05. Regression c o e f f i c i e n t s were 19 compared using analysis of variance. .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 control and infected 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 differences were found. For convenience of analysis and discussion, the data from the two experiment were pooled and represinted as a single experiment covering 3-112 DPI. 3. Results When the mean wet weights and lengths of infected and non-infected f i s h groups were compared, a s i g n i f i c a n t difference i n weight was found although there was no s i g n i f i c a n t difference i n length. The regression l i n e s were drawn to show the regression 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 infected and control f i s h samples during the period of 17-32 DPI were higher than those sampled during the period of 41-56 DPI which can be attributed not to growth, but to the fact that larger f i s h were sampled. The difference in weight becomes marked after 71 DPI (Figure 3, top graph). At the end of the experiment, the mean weight of the infected 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 control f i s h group, 28.34 ± 2.74 ( Table I) The percentage gain i n weight was computed (Haskell, 1959), the r e s u l t s showing that the non-infected f i s h group gained 25%, while the infected f i s h group l o s t 23.21%. I t was found that the parasite Salminoola reduced the potential weight of the f i s h host, juvenile sockeye salmon, by almost 34.86% at the end of the experiment (112 DPI), when they were infected at a l e v e l of 31.25 parasites per f i s h and the degree of i n f e c t i o n increased at the rate 20 Figure 3 Length and weight in r e l a t i o n to time post infection Comparison of length and weight between f i s h p arasitized with the p a r a s i t i c copepod, Salnrincola, and non-parasitized f i s h during the period of 112 DPI. Open units and broken lines indicate non-parasitized f i s h ; closed units and s o l i d lines indicate parasitized f i s h . 21 22 of 7.5 parasite-days/day over the period of 112 DPI. . Due to a slow growth rate and a short experimental period, s p e c i f i c growth rates (G) were found i n v a l i d as a measure to compare the growth of the infected and non-infected f i s h groups. Instead the condition factor or c o e f f i c i e n t of condition (K) was used. Mean Ks for both infected and non-infected f i s h were compared 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 = 0.05). The regression 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). The negative c o r r e l a t i o n of K (r = -.46) for the infected f i s h groups i l l u s t r a t e s a basic trend toward decreasing K with increasing DPI. Ks of the non-infected f i s h group were found quite constant (Figure 4, Table I) since both weight and length increased i n the same proportion 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 indicate that the i n f e c t i o n of juvenile sockeye salmon with i n i t i a l numbers of 22.6 ± 3.2 Salminaola 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 period of 112 DPI. The r e s u l t s i n Figure 3 and Table I indicate that both infected and control f i s h continue to grow although there appeared to be a s l i g h t decrease i n the rate of growth i n length among infected f i s h when compared with the control group. However, th i s difference 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. After 56 days, a negative relationship between weight and time was observed. This suggests that the reduction i n the weight of juvenile sockeye salmon depends not only on the parasite number but also on the number of days the parasites are found on the f i s h host. The degree of i n f e c t i o n (measured from the regression l i n e i n Figure 1) increased 23 Figure 4 Condition factor (K) during 112 DPI Regression l i n e s and regression c o e f f i c i e n t s of condition factor (K) of infected and non-infected f i s h groups. Open squares and broken li n e s indicate non-infected f i s h ; closed squares and s o l i d l i n e s , infected f i s h . C O N D I T I O N FACTOR ( K ) TABLE I Lengths, weights and c o e f f i c i e n t s of condition for infected and non-infected f i s h groups during the period of 112 DPI. Mean lengths and weights for each sampling period based on 12 f i s h . Time Means length Means weight Coefficient of d a y s mm g condition (K) Infected Control Infected Control Infected Control 3 145.8 + 6.18 145.7 + 9.44 24.04 + 4.35 22.59 + 4.47 .73 .73 6 146.4 + 5.59 146.4 + 9.47 22.80 + 3.94 23.90 + 4.02 .73 .76 10 147.8 + 3.19 145.8 + 4.43 19.69 + 2.68 23.10 + 3.39 .61 .74 13 152.5 + 6.55 145.1 + 3.58 23.88 + 3.22 25.47 ± 2.20 .67 .83 17 147.6 + 4.83 145.5 + 3.46 21.62 + 2.00 21.98 + 1.75 .67 .72 23 145.5 + 9.10 148.1 + 3.82 23.68 + 3.98 27.11 + 2.65 .77 .83 28 143.3 + 5.11 146.9 + 4.31 25.03 + 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 + 8.63 154.3 + 4.33 28.01 + 5.20 26.98 + 2.30 .78 .73 41 143.6 + 1.43 149.2 + 6.90 22.10 + 3.74 26.00 + 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 + 4.06 156.0 + 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 26 constantly throughout this experiment at 7.5 parasite-days/day. Each 7.5 parasite-days increase corresponded with a loss i n weight of almost .45 %. Corresponding to the time at which an e a s i l y observable difference between the weight of experimental and control groups of f i s h was noted (about 56 DPI) there was a dramatic increase in the number of parasites (Figure 1) in each f i s h . This was attributable to the hatching of copepods from the i n i t i a l i n f e c t i o n and th e i r attachment. This increase may not be proportional to the noted reduction of the respiratory area caused by the adult copepods. Since they attached themselves d i r e c t l y to the g i l l filaments, they may have interfered with normal respiratory function. The a b i l i t y of the f i s h to convert food into growth i s generally indicated by the "condition f a c t o r , " a measure of the relationship between length and weight. A comparison of the infected and control f i s h groups in this experiment indicates a decrease in the condition factor of the former. In the present case, however, the decrease in the c o n d i t i o n factor may be attributed not to a decreased a b i l i t y to convert food into growth but to a decrease in the amount of food consumed by the infected f i s h . A greater amount of unconsumed food was found at the bottom of the tank of the i n -fected f i s h than at the bottom of the tank of the non-infected group. Optimum growth of the f i s h depends upon their taking enough food to provide for basal metabolism, to replace broken down tissues and to supply normal tissue growth as well as energy required in various other a c t i v i t i e s . The presence and a c i t i v t y of the parasites may thus interfere with their normal growth. Food intake, however, i s not the only factor of importance a f f e c t -ing the growth and weight of the f i s h . 0 2 uptake must also be mentioned. In the present instance, this is a p a r t i c u l a r l y relevant factor since Salmincola causes direct damage to the g i l l s . Kabata and Cousens (19.77) i n -dicate that this parasite i s found in the g i l l cavity of juvenile sockeye salmon where at an early stage i t attaches i t s e l f to the g i l l filament, moving as an adult to a s i t e usually on the branchial rim or the inner side of the operculum where i t becomes more firml y a f f i x e d . The presence and a c t i v i t y of this parasite and the pressure i t exerts on the g i l l filaments leads to atrophy and eventual disappearance of the d i s t a l portions of these filaments. The adjacent parts of the missing filaments are affected by tissue reactions, becoming thick, and rendering them nonfunctional for r e s p i r a t i o n . These researchers further indicate that the extent of g i l l damage caused by SatnrincoZa depends on t h e i r number and s i z e . In the present experiment juvenile sockeye salmon were i n i t i a l l y infected with 22.6 Salminaola which increased to 31.25 per f i s h during the second part of the i n f e c t i o n . With infections of t h i s extent i t i s possible that at least 20% of the g i l l surface area was destroyed (Kabata and Cousens, 1977). Losses of t h i s magnitude can also be expected to affect the amount of 0 2 uptake. Since normal levels of 0_ must be maintained .to meet the re-quirement of basal metabolism, any interference with those levels w i l l upset the balance. Moreover, a reduction in the g i l l surface area r e s u l t s in a greater requirement for an increase i n the basal metabolic rate ( E l l i o t and Russert, 1949). It i s v i r t u a l l y certain that these f i s h did not obtain s u f f i c i e n t 0 2. The age of the f i s h host i s a further factor influencing the severity of the effects of p a r a s i t i z a t i o n in f i s h . P i t t and Grumdan (1957) noted that the rate of growth of infected f i s h was slowed to such an extent during the period of p a r a s i t i z a t i o n that the f i s h become stunted. Since the parasites continue to exert an effect throughout the l i f e of 28 their hosts subsequent to i n f e c t i o n , f i s h with the longest history of i n -fection showed the greatest retardation of growth. This observation of P i t t and Grumdan was confirmed by the present experiment. However, the dramatic increase in the degree of i n f e c t i o n in the present study points to the age of the parasite rather than to the age of the f i s h as the primary influence on the retardation of f i s h growth. The results of this experiment c l e a r l y indicate that Salmincola californiensis has a more pronounced effect on the weight and condition factor than on the length of sockey salmon, for the period of 112 DPI. This finding confirms some previous studies with p a r a s i t i c C r u s t a c e a such as those of Schaperclaus (1954) on tench, Tinea tinea parasitized with Erga-silus and of Reichenbach-Klinke et a l . (1968) on Covegonus lavaretus and C. feva infected with Ergasilus sieboldi. SECTION II IMPACT ON HOST'S TEMPERATURE TOLERANCE 30 1. Introduction Among those environmental factors which cause stress i n f i s h rapid or extensive temperature change i s one of the most severe i n i t s ef f e c t on f i s h physiology. Increasing water temperature above the optimum leads to the production of such adverse effects as r e s t r i c t e d swimming performance (Brett, 1964), interference with normal feeding habits ( P h i l l i p s et a l . , 1960), increased standard metabolism (Brett, 1964), and l i p i d composition (Lewis,1962). Kinne and Kinne (1962) also 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 raised. None of the changes which affect water q u a l i t y and can affect fresh-water f i s h e r i e s adversely i s generally believed to be more common and more s i g n i f i c a n t than reduction of dissolved 0 2. Doudoroff and Shumway (1967) claim that salmonid f i s h are among the most susceptible to reduction of 0_. Patterns of v a r i a t i o n i n the resistance of f i s h to 0 2 d e f i c i e n c i e s corre-lated with water temperature are also highly v a r i a b l e . Brett (1956) found that salmonids have a maximum upper l e t h a l temperature barely exceeding 25°C. He also 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 rate of a l l metabolic processes of f i s h so that t h e i r need for 0 2 can vary greatly with a change i n the temperature of t h e i r medium. Normally, most f i s h have the a b i l i t y to compensate for a reduction of dissolved 0 2 by making various physiological adjustments. Randall (1970) found increases i n breathing rate and ampli-tude and also i n the ventilation-perfusion 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. E l l i s (1937) also reported that respiratory compensation occurred in goldfish, yellow perch and other species of f i s h at an oxygen concentration only a l i t t l e below 5 mg/1 when the. temperature was between 20 and 25°C. These compensatory mechanisms require energy expenditures and the longer the f i s h i s in the s t r e s s f u l condition, the more energy i s required. This experiment attempted to f i n d out whether parasitism with Sal-mincola affects the a b i l i t y of juvenile sockeye salmon to withstand increa-ses in water temperature. The f i s h were subjected to d i f f e r e n t water temper-atures and their a b i l i t y to cope with each p a r t i c u l a r water temperature was evaluated in terms of mortality. 2. Materials and Methods 2.1 Experimental F i s h Fish used f o r t h i s experiment came from the stock of f i s h mentioned in the General Materials and Methods. Infection techniques were described in the same section. 2.2 Experimental Procedure One hundred experimental f i s h and 100 control f i s h were transferred into two separate tanks containing water of the same temperature. The water temperature of the two tanks was gradually adjusted by the addition of heated water at a rate of 1°C for each two day-interval, u n t i l i t reached 9°C. The f i s h were acclimated to these conditions for one week, and fed to s a t i a t i o n with Oregon Moist P e l l e t s . However, they were starved for three days prior to and throughout the course of the experiment in order to reduce the amount of waste products, the experimental aquaria having aeration 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 their average weight and length and also the le v e l and degree of i n f e c t i o n . Ten control and ten infected f i s h were transferred into each experimental aquarium. Four aquaria were used, each containing 20 gallons of temperature controlled water. The water temperature i n each aquarium was established at 9°C. The 0 2 content and pH of the water were measured i n each aquarium before the f i s h were introduced and throughout the experiment (Table I I ) . After the f i s h were transferred into the aquaria, they were l e f t to acclimate to t h e i r new environment for three days. During these three days, waste products from the bottom of the aquaria were siphoned out using a small p l a s t i c tube and water was replaced. After three days acclimation at 9°C, the f i s h were subjected to gradual increases in water temperatures u n t i l i t reached 23°C. During the course of the experiment, the dead f i s h (also those f a i l i n g to respond to the touch of a glass rod) were removed. The number of dead f i s h and l e v e l of i n f e c t i o n were recorded each day. 3. Results The average length and weight of ten randomly sampled f i s h were 15.1 ± 0.92 cm and 27.8 ± 4.6 g for the infected f i s h groups and 15.2 ± 0.89 cm and 31.4 ± 5.3 g for the control. According to the data, control f i s h were s l i g h t l y heavier than those in the infected group but there were 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 differences in length and weight between these two groups of f i s h . 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 f i s h (mainly adults with egg sacs) and degree of i n f e c t i o n was- 589.3 ± 28.1 parasite-days. Reduction of the g i l l surface area was not measured but erosion of the filaments was observed. Even with three days starvation prior to the experiment, the f i s h released some waste products into the aquaria, causing an increase i n the TABLE II Variation of dissolved 0 2 and pH levels during the experiment (1 month period) as the temperature increased from 9° to 23°C. Acclimated temperatures °C 1 1 12: 1.3 17 21 23* 23 23 0 2 ppm 12.25 11.40 10.5 10.9 8.5 8.15 7.45 7.43 pH 7.0 8.1 8.15 7.5 6.0 - - 5.9 * 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 replaced, the pH declined-and reached the o r i g i n a l l e v e l within 12 days. The pH gradually declined after the 12th day due to an increase i n dissolved G02 (the by-product of f i s h r e s p i r a t i o n and possibly f i s h excretion). However, at 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 for optimum f i s h health by E.P.A. (1973), although t h i s should not cause any concern since both infected and control f i s h groups were kept under the same conditions. The t o t a l amount of dissolved 0 2 i n the experimental aquaria de-cl i n e d as the water temperature increased (Figure 6) though i t remained higher than the amount of dissolved 0 2 i n normal fresh water (Whipple,1914) as a r e s u l t of the aeration of the experimenta aquaria. Measurement made on the 21st day of the experiment showed that the l e v e l of dissolved 0 2 i n the experimental aquaria had f a l l e n below the l e v e l of dissolved 0 2 i n normal fresh water at the same temperature. By the end of the experiment i t had dropped to 7.2 ppm. No mortality 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 infected f i s h showed signs of stress; they struggled and before los i n g t h e i r balance, swam with a great deal of a c t i v i t y . I n i t i a l mortality of infected 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 stress appeared i n both infected and non-infected f i s h groups and the mortality 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 in experimental aquaria Variation of pH during a 1 month period in the experimental aquaria a = l e v e l of pH recommended for optimum health of f i s h (E.P.A., 1973) Figure 6 Dissolved 0 2 in experimental aquaria Variation of the amount of dissolved 0 2 in the experimental aquaria with temperature increased from 9 to 23°C. The closed squares represent concentration of 0 2 measured in the experimental aquarium at the time indicated. Open squares represent concentration of dissolved 0 2 in fresh-water (derived from Whipple, 1914). 36 1 1 1 3 15 17 19~ 21 ~_3 23 TEMPERATURE (°C) 2 6 10 14 18 22 26 30 TIME IN DAYS Qj t t V I l IN EXPERIMENTAL AQUARIUM O—• SOIUIIIITT Of O , IN FRESH WATER I J. — i 1 1 I I I 11 13 15 17 19 21 23 23 TEMPERATURE (°C) 1 i — 1 1 1 1 1 i 2 6 10 14 18 22 26 30 TIME IN DAYS 37 Figure 7 Percent mortality and percent cumulative mortality of experimental  f i s h Variation of the mortality of infected and non-infected f i s h corresponding to temperature increases'from 9 to 23°C. The arrow denotes that from the indicated point, the water remained at 23°C u n t i l the end of the experiment. 8C TABLE III Percent mortality and cumulative mortality of the control and infected f i s h during the course of changing temperature from 9 to 23°C Control Fish Infected Fish Day Temperature °C Mortality Percent Cumulative 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 mortality reached 50% among infected 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 mortality rate occurred i n both f i s h groups (Figure 7, top graph). At the end of the experiment, the cumulative mortality among non-infected f i s h was 32.5% while i n the infected f i s h group, i t went up to 85%. A mortality rate of 62.5% was found among f i s h infected with an average of 27 Salmincola or 589 parasite-days 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 mortality found i n the non-infected group at the same temperature and time. The % mortality of both groups of f i s h was found to be highest during t h i s period. After the 24th day of the experiment, mortality tended to decline for both groups. This occurrence can be attributed to acclimation to the constant temperature of 23°C assigned for t h i s experiment. Only 22.5% of the non-infected group died at the temperature found to be the median l e t h a l temperature for 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% mortality was observed among the non-infected group. 4. Discussion The above r e s u l t s indicate that Salminoota reduces the a b i l i t y of juvenile sockeye salmon to withstand increases i n temperature. In healthy sockeye salmon, mortality occurs i f the water temperature exceeds 25°C but the mortality rate depends mainly on the acclimation time. The zone of tolerance can be extended i f the acclimation time for a p a r t i c u l a r temper-acture i s s u f f i c i e n t l y extended (Brett, 1956). However, the exact cause of death of f i s h exposed to high water temperatures i s not known with cer t a i n t y . Allanson and Noble (1964) agreed 41 with several previous researchers that osmotic stress may be the primary factor. I t has been shown by many workers that the addition of Na +, Mg + and Ca ions increased the resistance of f i s h to high temperature. Also Strawn and Dunn (1967) found that a sample of s a l t marsh Garrbusia affinis was more res i s t a n t to heat death than was a sample from fresh water. Increasing osmotic stress as a r e s u l t of increasing water temper-ature 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 highly dependent upon the ionic conditions of the body f l u i d . When the water temperature increases, the fish increase t h e i r metabolic a c t i v i t y and t h i s increase in turn demands an increase i n 0 2 consumption. Beamish (1964) demonstrated that a l l species of f i s h show a continual increase i n 0 2 consumption with increasing temper-ature . Temperature affects both the amount of 0 2 that can be dissolved in water and the amount f i s h require for metabolism. Higher water temper-atures increase 0 2 consumption but decrease the amount of 0 2 the water can dissolve. Generally the rate of 0 2 consumption tends to increase more than two-fold with a 10°C temperature r i s e . Brett (1964) has shown that the standard rate of 0 2 consumption of sockeye salmon r i s e s by a factor of almost f i v e when the temperature increases from 5 to 14°C and also that i t increases f a i r l y r apidly above 15°C to 220 mg/kg/hr at 25°C. The manner i n which temperature influences the survival of f i s h can now be c l e a r l y understood. The mortality of non-infected f i s h observed in the present experiment increased l i n e a r l y with 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) also suggest, a similar relationship between mortality rate and temperature. Among the infected f i s h group a linear increase in mortality asso-ciated with increasing temperature was also observed. Furthermore, the rate of increase in mortality was greater for t h i s group than that of the non-infected f i s h group. The mortality l e v e l among the infected f i s h group was found to be almost twice as high than that among the non-infected group at a water temperature of 23°C. Even more s i g n i f i c a n t , the mortality occurred e a r l i e r among this group on the 15th day of the experiment when the water temperature was only 17°C. At t h i s time the amount of dissolved 0 2 in the experimental tank was 11 ppm, about 2.3 ppm higher than the l e v e l of 0 2 that has been shown to l i m i t active metabolism. This r e s u l t c l e a r l y indicates that damage to the g i l l respiratory surface l i m i t s the amount of 0 2 uptake to a l e v e l below that required for basal metabolism. The osmotic stress i s not the only stress the f i s h experiences as a re s u l t of increasing water temperature. The very interference with the respiratory mechanism which the parasite causes to the f i s h , discussed e a r l i e r in section I, demands a higher l e v e l of 0 2 consumption than normal i f basal metabolism i s to be maintained. Compensatory mechanisms must be ca l l e d into play to increase the amount of 0 2 delivery to the tissues. Normally f i s h can cope with temperature and 0 2 changes which are not too severe by means of their respiratory compensatory mechanisms without there being any noticeable effect on mortality levels ( E l l i s , 1937; Privolnev, 1954; and Holeton and-Randall, 1967). Unfortunately, this parasite causes direct damage to the g i l l (as described in d e t a i l i n the previous section) and also interferes with the opercular movement and prevents complete closure of the operculum'. The 43 p o s s i b i l i t y of the infected f i s h delivering 0 2 adequately for the requirements of the tissue i s thereby greatly reduced; tissue anoxia and death are the usual results. SECTION III IMPACT ON HOST'S SWIMMING ABILITY 45 1. Introduction Swimming performance, a s i g n i f i c a n t parameter in evaluating f i s h s u r v i v a l , has been widely studied in r e l a t i o n to a number of important variables including f i s h size, body form (Bainbridge, 1958) and physical con-d i t i o n (Bainbridge, 1962; Thomas, 1964) and such environmental conditions as temperature, 0 2 , C02 and solution t o x i c i t y (Fry and Haet, 1948; Davis et al.., 1963; Brett, 1964, 1967). Many descriptions of the various components which characterize swimming movements in salmonids also exist (Bainbridge, 1960; Brett, 1958, 1964, 1967; Fry and Cox, 1970; Brett and Glass, 1973 and Jones et a l . , 1974). Owing to i t s importance and to the large volume of readily acces-s i b l e information of f i s h swimming a b i l i t y , some researchers have begun to incorporate swimming tests in their studies of host parasite relationships. Fox (1965) and Butler and Millemann (1971) used c r i t i c a l and fixed v e l o c i t y swimming tests to show that trout and salmon infected with large numbers of trematode metacercariae attained lower maximum sustained swimming speeds and fatigued faster than control f i s h . Lester (1971) also suggested that Schistocephalus aculeatus. affects the swimming performance of Gasterosteus. Smith and Margolis (1970) studied the effects of Eubothrium salvelini on the swimming a b i l i t y of sockeye salmon by measuring their performance in terms of distance t r a v e l l e d and found that sockeye smolt with parasites weighing up to 50% of the wet weight of the fish, swam only 2/3 as far as non-infected smolts of similar size. This r e s u l t was l a t e r confirmed by 46 Boyce (1979), who found that the infected 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 did the control f i s h . However, K l e i n et a l . (1969) could not f i n d any effects of an i n t e s t i n a l f l u k e , Crepidostomum farionis, on the stamina of rainbow trout. The eff e c t on the swimming a b i l i t y which Russell (1977) observed on the same f i s h infected with 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 lakes, sockeye salmon must sometimes swim against high water v e l o c i t i e s . Fish carrying a s i g n i f i c a n t number of parasites, p a r t i c u l a r l y parasites which exert a direct effect on the respiratory system, w i l l suffer from a decreased a b i l i t y to produce these variations i n swimming speed required during migration. The present experiment was conducted to determine whether or not the p a r a s i t i c copepod Salminaola i s responsible for 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. Materials and Methods 2.1 Experimental Fish The infected 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 infected for about f i v e months and carried large numbers of S. californiensis. An average of 67 of the parasites at various stages i n t h e i r l i f e cycles 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 parasites and kidney disease. Both control and infected f i s h were transferred from the P a c i f i c B i o l o g i c a l Station i n Nanaimo to the University of B r i t i s h Columbia and kept in separate tanks equipped with running water and 12-h photoperiods. The water i n these holding tanks was 9 ± 2°C. The f i s h were fed once a day to 47 s a t i a t i o n with Oregon Moist P e l l e t s . 2.2 Experimental Procedures The swimming performance tests were performed using a open c i r c u i t stamina chamber consisting of a 12.7 cm ID plexiglass tube, 86 cm i n length (Jones et a l . , 1974). A series of three 0.3 cm mesh grids at the upstream end of the chamber introduced microturbulence in the plexiglass tube, r e -s u l t i n g in 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 flow rate of about 100 cm/sec. At the downstream end, an e l e c t r i f i e d grid (5 vo l t ) stimulated the f i s h to swim. Fresh water was added to the system continuously and oxygen levels i n the water were maintained at saturation by the addition of a i r through airstones i n the chamber. During the experiment, water supplied fo the chamber was 8 ± 3°C. I t was c i r c u l a t e d by a variable speed pump which was also used to increase the v e l o c i t y of the water i n the chamber. Two types of swimming performance test were performed. These i n v o l -ved c r i t i c a l v e l o c i t y and fix e d v e l o c i t y tests respectively. 2.2.1 C r i t i c a l Velocity Due to the l i m i t e d number of infected f i s h a v a i l a b l e , c r i t i c a l v e l o c i t y tests were conducted only with twenty control f i s h with the intention of establishing a base l i n e f o r the cal c u l a t i o n of a fixed v e l o c i t y used i n testing the effect 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 control f i s h were introduced, f i v e at a time,into the stamina chamber, with water v e l o c i t y adjusted to 10 cm/sec. The f i s h were allowed to adapt to th i s new environment for about one hour p r i o r to testing to minimize the eff 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. at 60 minute 48 intervals as recoimuended by Brett (1967) u n t i l the f i s h became fatigued or exhausted and f e l l back against the e l e c t r i c g r i d . As they became fatigued, the f i s h were removed and t h e i r fatigue times were recorded. Total 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 following Jones et a l . (1974). V - c r i t . = V + (V, - V ) x tF P f P f l where = penultimate water v e l o c i t y (cm/sec) = f i n a l water v e l o c i t y (cm/sec) tF = time to fatigue at V_ (sec) TI = time between v e l o c i t y increments (sec). 2.2.2 Fixed Velocity This test was used to determine the eff e c t of SaZmincota on the swimming a b i l i t y of the infected f i s h i n comparison to that of the control f i s h group. The test was conducted at 90% of the calculated c r i t i c a l v e l o c i t y determined for the twenty control f i s h . The duration of the test was 600 min (Brett, 1967). Water was supplied to the chamber at 8 + 3 C. Control f i s h used i n the experiment were selected by s i z e such that they were s i m i l a r to infected f i s h , thereby minimizing the eff e c t of any si z e difference on swimming a b i l i t y (Bainbridge, 1958). Thirty control and infected f i s h were assigned for t h i s t e s t . Three f i s h from each group were tested at the same time. 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 NH3 l e v e l s . The introductory process was the same as i n the c r i t i c a l v e l o c i t y test 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 testing v e l o c i t y reached 90% of the calculated c r i t i c a l v e l o c i t y . The test v e l o c i t y was maintained for 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 to pre-vent disturbance to other f i s h . The fatigue time for each f i s h was recorded separately. For those f i s h which swam throughout the 600 min testing time, 600 min was assigned as "fatigue time." Length and weight of each f i s h were measured and the number of parasites was counted for each of the infected f i s h . 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 plotted against per cent fatigued (Figure P). F i f t y per cent of the f i s h fatigued at the v e l o c i t y of 70 cm/sec. The approximate 5 and.95% fatigue levels occurred at 40 and 87 cm/sec respectively. The v e l o c i t y used to test the difference between the swimming a b i l i -ty of the infected and control 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 control f i s h , i . e . 65 cm/sec. I t i s apparent from Figure 9 that no f i s h fatigued during the f i r s t 45 min at the fi x e d v e l o c i t y (65 cm/sec). Thereafter, infected f i s h started to fatigue e a r l i e r than control f i s h . They became 50% fatigued when forced to swim at 65 cm/sec for about 250 min. At t h i s time only 7% of the control f i s h were fatigued. The control f i s h did not reach the 50% fatigue l e v e l u n t i l they had swum longer than 500 min. At the end of the 600 min test i n g time, 14 control f i s h , compared with only two infected f i s h , were s t i l l swimming (Table V). These r e s u l t s indicate that the infected f i s h have less a b i l i t y to swim at high v e l o c i t i e s than do the control f i s h . The l e v e l of i n f e c t i o n was also found to affect the swimming a b i l i t y of f i s h . Figure 10 indicates a negative c o r r e l a t i o n (-.73) between time to TABLE IV Relation of swimming speed to the per cent fatigued for 20 sockeye salmon of mean size 14.47+ 3.8 cm and 26.62 ± 5.15 g tested at 8 ± 3°C for 400 min. V e l o c i t i e s were raised by 10 cm/sec every 60 min. Testing v e l o c i t y (cm/sec) 40 50 60 70 80 90 Testing time (min) 60-120 120-180 180-240 240-300 300-360 360-400 Fatigue times (min) 115 170 195 290 315 360 225 295 322 400 235 295 340 298 340 300 346 300 350 355 Mean c r i t i c a l v e l o c i t y 49.1 58.3 66.3 79.35 86.32 95.0 Grand mean c r i t i c a l v e l o c i t y 72.39 (n=20) I t'r O f ' 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 f i s h C r i t i c a l v e l o c i t y of juvenile sockeye salmon obtained fron 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 for 400 min i n t e r v a l . S W I M M I N G VELOCITY ( L / s e c ) S W I M M I N G V E L O C I T Y ( c m / s e c ) ho 53 Figure 9 Relationships between fatigue times and % f i s h fatigued-Comparison of the fatigue times of infected and control f i s h groups at a fixed v e l o c i t y (65 cm/sec) at 8 i 2°C with 600 min testing time. The numbers in brackets indicate the numbers of f i s h s t i l l swimming at the end of testing time. Arrow indicates the time when 50% of f i s h were found fatigued. TABLE V Mean fatigue times, % f i s h fatigued and size of the infected and control f i s h tested at the fixed ' v e l o c i t y of 65.0 cm/sec at 8 i 2°C for 600 min testing time. Infected Control Lgue time % Fatigue Size Fatigue time % ',• Fatigue Size L(cm) W(g) L (cm) 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 6002 52.8 14.17 27.8 531.0 82.5 13.4 24.8 571.0 85.8 14.2 24.3 600 1 92.4 15.4 31.29 l 2 infected f i s h s t i l l swimming at the end of testing t ime. 2 14 control f i s h s t i l l swimming at the end of testing time. Ln U i 56 Figure 10 Relationships between fatigue time and parasite-days Regression l i n e indicates the relationships between fatigue time and parasite-days during the 600 min testing time at fixed v e l o c i t y . V e r t i c a l l i n e = standard deviation. Points on the top of the graph indicate the fatigue times of f i s h with average infections of about 400 parasite-days. 58 fatigue and parasite-days. Fatigue time gradually declined when the parasite-days increased from 640 to about 1170 (Table VI). It is interesting to note that no f i s h fatigued during the period of about 300 to 500 min and that f i s h fatigued between 500 to 600 min testing time were found to have average number of parasite-days of 407 ± 28. This indicated that there is a negative c o r r e l a t i o n between parasite-days and fatigue time (Figure 10). It can be concluded that when juvenile sockeye salmon, parasitized with Salmincola for 640-1230 parasite-days, are forced to swim at high v e l o c i t y of 65 cm/sec, the chance of survival for the infected f i s h i s only 6.6%. 4. Discussion The adverse effects exerted on the swimming a b i l i t y of juvenile sockeye salmon by Salnrineola observed i n the present experiment demonstrated that p a rasitized f i s h have less a b i l i t y to tolerate active swimming than do non-infected sockeye. The results also confirm that this a b i l i t y is related to the le v e l of i n f e c t i o n . Increased levels and degrees of i n f e c t i o n increase the amount of respiratory area destroyed. This, in turn, l i m i t s the amount of 0 2 which can be taken up by the f i s h . The increased metabolic a c t i v i t y associated with active swimming demands an increase in 0 2 uptake. Moreover, the requirements for 0 2 increase very rapidly with increases in swimming speed (Brett, 1964). However, the reduction in the g i l l respiratory area obviously causes a reduction i n the amount of 0 2 that can be absorbed. Whenever the amount of 0 2 required by the tissue exceeds the 0 2 uptake, f i s h fatigue and eventually death r e s u l t s . Demands for increased 0 2 up-take among parasitized f i s h not only come from increasing a c t i v i t y (active swimming) but also from the stress exerted on the f i s h host by the TABLE VI Variation in the number of parasites, calculated as parasite days, found in the fatigued f i s h . Mean fatigue time Mean parasite days Grand mean mm 45.0 1139.0 62.5 1130.5 86.0 900.0 116.25 1074.5 138.0 1170.0 169.0 960.0 224.0 750.0 240.5 982.5 267.5 922.5 279.0 640.0 495.0 410.0 531.0 365.5 571.0 413.0 600.0 440.5 976 ± 112 407 ± 28 parasite. Lester (1971) has found a difference between 0 2 consumption during the active swimming of sticklebacks infected with Sohistoaephalus solidus and non-infected ones. Various stresses are associated with the presence and a c t i v i t y of th i s parasite. F i r s t l y , most important among these i s the dir e c t damage caused to the g i l l surface area. Secondly, r e l a t i v e to the s i t e of in f e c -t i o n , there w i l l be an interference with both the respiratory and locomo-tory mechanisms. The locomotory mechanism can be observed much more c l e a r l y i n sockeye f r y than in the juvenile 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 . Interference with the movement of the f i n or damage to the f i n w i l l upset the locomotory balance of the f i s h . Thirdly, 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 skin. Lester 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 sticklebacks caused by Gyrodaotylus may disrupt normal functions of the epithelium such as i t s providing a b a r r i e r for ionic exchange and may eventually lead to the death of the f i s h . If t h i s i s the case Salmincola should have a si m i l a r e f f e c t on i t s host. In cases where there i s e p i t h e l i a l damage esse n t i a l ions such + ++ + as Na , Ca and Mg tend to escape from the e p i t h e l i a l c e l l s leading to a reduction i n the t o t a l concentration of e l e c t r o l y t e s (Lockwood, 1964) and consequently lead to the development of osmotic stress. Under stress conditions the adrenal cortex of f i s h i s activated (Fagerlund, 1967; Hoar, 1975) r e s u l t i n g i n the release of Glucocorticoids which break down protein into glucose. The more body protein that breaks down, the weaker the f i s h becomes. 61 In cases of heavy i n f e c t i o n (such as in this experiment), the f i s h w i l l be subjected to the various stresses mentioned e a r l i e r . In addition the reduction in the g i l l surface area, combined with i n e f f e c t i v e function-ing of the compensatory mechanism to maintain 0 2 uptake, d r a s t i c a l l y reduces the a b i l i t y of the f i s h to cope with active swimming. Fatigue among these f i s h occurs e a r l i e r than among those without parasites. Among f i s h with l i g h t i n f e c t i o n s , i f the area of the g i l l surface is not too great and the f i s h can make use of their compensatory mechanisms, normal 0 2 uptake may not be upset and fatigue w i l l not occur. When active swimming i s required the amount of 0 2 needed by the f i s h increases and the f i s h have to increase the effectiveness of th e i r 0 2 uptake mechanisms, which in turn, leads to an increase in energy expenditure. Furthermore, other stresses may occur during long periods of swimming in water of high veloc-i t y . Physiological responses to these stresses may activate the adrenal cortex, lead to protein breakdown and consequently, to the f i s h becoming too week to cope with further active metabolic a c t i v i t y . Then fatigue r e s u l t s . Because of the v a r i a t i o n in the degree of infe c t i o n (Table VII) i t was not possible to predict the exact c r i t i c a l fatigue time for juvenile sockeye salmon swimming in highspeed water (65 cm/sec). However, a positive 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 fatigue time was obtained. Some of the l i t e r a t u r e shows that parasites other than g i l l para-sites exert a marked e f f e c t on the swimming a b i l i t y of f i s h : Schtstoeeph&lus solidus plerocercoid on stickleback (Lester, 1971); Eubothvium saZvelini in sockeye salmon (Smith, 1973; Boyce, 19 79); Nanophyetus salminoola in salmonids (S. gaivdnevi and 0. kisutoh) (Butler and Millemann, 1971) and Bubophorus aonfusus in rainbow trout (Fox, 1965). The damage to the f i s h 62 host caused by these parasites i s not well understood but Wood and Yasutake (1956) found evidence of marked obstruction and mechanical injury 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 tissue and g a l l bladder as the r e s u l t of N. salmincola i n f e c t i o n . This may indicate that the damage i s related to the migration of the cercariae through the tissues or to i t s obstruction of the i n t e r n a l organs. Salmincola, a g i l l parasite, can be used as a good example of how parasites exert adverse effects on the swimming a b i l i t y of t h e i r f i s h hosts. These effects have never before been studied i n r e l a t i o n to g i l l parasites. The res u l t s of t h i s experiment should therefore, be useful as a guideline for future studies of the impact of g i l l parasites 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 salmon move from a fresh water to a s a l t water environ-ment, a profound change takes place in their physiological condition. The transformation process i s very complex. Hoar (1976) refers to smolt transformation as a seasonal phenomenon. For quite some time i t has been understood that the seasonal progression of photoperiods 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 factor c o n t r o l l i n g the onset of parr-smolt transformation. Beside the photoperiod, Zaugg and McLain (1976) have demonstrated the important influence of temperature on the duration of the smolt phase in yearling coho salmon. In order to tolerate environmental changes, a spectacular change must take place p r i o r to commencing seaward migration. Parry (1966) who studied the osmotic adaptation of the f i s h , referred to the p o s s i b i l i t y of a mechanism for osmoregulation developing during the juvenile l i f e of the salmon. The same author also mentioned that the a b i l i t y to tolerate envi-ronmental change i s a faculty which may develop during the entire period of juvenile l i f e though i t may change suddenly p r i o r to the seaward migration. In a sea water environment, where s a l t concentrations are much greater than those within the f i s h ' s body, the natural tendency i s for water to flow from the f i s h to i t s environment by osmosis. To maintain water content within the f i s h , water must be imbibed and excess s a l t must 65 be eliminated. The excretion and absorption of chloride v i a the g i l l s i s a matter of some int e r e s t . Copeland (1948) presented evidence of a c e l l type on the g i l l filament of Fundulus responsible for chloride transfer. Further studies by the same author i n 1950 suggested that t h i s c e l l i s l a b i l e and sensitive to blood-salt l e v e l s . According to the observation of Kessel and Beams (1962) these c e l l s exist embedded in the s t r a t i f i e d e p i t h e l i a l layer. Datta Munchi (1964) found these c e l l s at the base of and among the secondary g i l l lamellae. Electron microscopic studies by Threadgold and Houston (1964) implicated a special e p i t h e l i a l c e l l r i c h i n mitochondria as being responsi-ble for the secretory function. These c e l l s increased i n numbers at the parr-smolt transformation but became reduced during the post-smolt stage. This c e l l has been referred to many times as the "chloride secretory c e l l . " P r i o r to migration, the necessary transformation or reorganization of body function 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 juvenile sockeye salmon, which quickly grow to smolt size and can adapt to sea water as underyearlings (Kennedy et a l . , 1976). 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 relationships can be traced back i n the l i t e r a t u r e for 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 laboratory, 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 Salminoola causes any reduction i n the t o l e r -ance to s a l i n i t y changes. To my knowledge, no studies of the effect of parasites on f i s h s a l i n i t y tolerance e x i s t at present. 66 2. Materials and Methods 2.1. Experimental Fish The yea r l i n g sockeye salmon used in t h i s experiment came from Rose-wal l Hatchery. They were transferred to the P a c i f i c B i o l o g i c a l Station and kept i n running water at ambient temperature. Before the copepod larva were introduced, 10 f i s h were randomly selected and examined, and found to be free of parasites and b a c t e r i a l kidney disease. Then the f i s h were divided into two groups with approximately 200 f i s h i n each, and were put into two sepa-rate tanks. A large number of copepod larva (hatched i n the laboratory) were introduced to the f i s h chosen to be "infected f i s h group". Both i n f e c -ted and control 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 started. 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 prob-lems 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 exper-imental f i s h , both control and infected, were held i n tanks equipped with a r t i f i c i a l l i g h t (diffused incandescent lamps at the top of the tanks) to control photoperiods. There were four sequential photoperiods (12-h, 14-h, 16-h and 17-h) each"lasting two weeks. After eight weeks, the f i s h were kept on a long daylight photoperiod (17-h) u n t i l the experiment was started at.the beginning of June. The water i n the holding tanks was controlled at 12 ± 3°C by mixing ambient and heated water with 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 with Oregon Moist P e l l e t s . Two types of experiments were conducted to test whether the p a r a s i t i c copepod has any additional effects on juvenile sockeye salmon while they are migrating: the tests were a s a l i n i t y tolerance test and a s a l i n i t y preference te s t . 67 2.2.1. S a l i n i t y Tolerance Test This test was performed to see whether Salmineola affects the a b i l i t y 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 July i n eight small c y l i n d r i c a l tanks. The fresh and s a l t water supplies for the experimental tanks came from separate pipes and were mixed i n a connecting pipe before being passed into the temperature c o n t r o l l e r . Each pipe had i t s own valve for adjusting t I . the amount of water required for each experimental period. The tanks were supplied with water at a constant temperature of 12°C. A l l of the experimental tanks were set outdoors. At the beginning of the experiment the tanks were supplied with fresh running water at a constant flow rate of 60 gal/h from the top of the tank. The overflow water was drained out through the overflow pipe, each tank therefore obtain-ing new water at a l l times. Ten infected f i s h and ten control f i s h were introduced together into each of the experimental tanks and l e f t to adjust to the new environmental conditions for one week. By the gradual addition 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 f i s h were maintained i n f u l l strength sea-water for 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 after 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 period. S a l i n i t y tolerance was measured by recording the time of death, the mortality then being expressed i n terms of % cumulative mortality. 2.2.2. S a l i n i t y Preference Test This test was conducted using a method modified a f t e r Houston (1957) in an aquarium (18x32x18 in.) which was divided into two compartments of 68 equal volume by a central p a r t i t i o n 14 i n . high. A water "bridge" 2.5 i n . high was created over the central p a r t i t i o n enabling the f i s h to move be-tween the compartments. The aquarium was set in a larger oval tank in which running water of constant temperature (10°C) was kept at 1/2 the height of the aquarium to insure that the water temperature in the aquarium was kept constant throughout the experiment. The sides of the aquarium were covered with black p l a s t i c to prevent disturbances and the top was covered with a glass cover to allow observation 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 almost to the top of the par-t i t i o n with fresh water and the second compartment with sea water for the experimental, and fresh'water for the control group. Four r e p l i c a t i o n s were made for the experimental and two for the control experiment. For each experiment, f i v e infected and f i v e non-infected f i s h were put into the fresh water compartment and l e f t over night. The next day water was slowly added to the bottom of the fresh water compartment through a glass tube u n t i l a water bridge 2.5 i n . high was established over the central p a r t i t i o n . For the control experiment, both o r i g i n a l and alternate compartments were f i l l e d with fresh water. Illumination was at the center of the room about 20 feet from the testing aquarium; i t had no noticeable effect on the behavior of the f i s h . The number of f i s h in each compartment was determined for six periods at 30, 60, 90, 120, 180 and 240 min. The percentage of f i s h , either infected or control, in the seawater compartment was taken as a measure of the preference of the f i s h for seawater. 3. Results 3.1. S a l i n i t y Tolerance Test Observations on the behavior of the f i s h after each period of i n -creasing s a l i n i t y in the experimental tank indicated that both infected and 69 non-infected f i s h swam in a normal fashion, with no struggling or fatigue, when the s a l i n i t y was between 0-15%o. Only 1.5% mortality occurred i n the infected f i s h group at t h i s time. After the s a l i n i t y was adjusted to 20%., some of the infected f i s h appeared to lose t h e i r equilibrium; active swimming and struggling appeared occasionally. At the end of the f i v e day period with 20% owater s a l i n i t y , 22.5% mortality was found among the infected f i s h ( F i g -ure 11, Table VII) as compared to only 1.5 among the non-infected group. The mortality i n the infected group increased r a p i d l y to 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 point, the mortality among the control 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 infected f i s h survived, as compared with a su r v i v a l rate of almost 90% among the non-infected f i s h . The r e s u l t s of t h i s experiment show that non-parasitized juvenile sockeye salmon have a high a b i l i t y to tolerate s a l i n i t y changes. P a r a s i t i z a -t i o n with an average of 24 Salminoola 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 control experiment. I n i t i a l l y , both infected and non-infected f i s h had a marked tendency to remain i n the o r i g i n a l compartments. 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 in both groups of f i s h (Table V I I I ) . However, i n the experimental group, both infected and non-infected f i s h were r e l a t i v e l y inactive during the preliminary resting period before the water l e v e l i n the fresh water compartment was increased; a few infected f i s h were swimming slowly close to the water surface. When the water bridge was 70 Figure 11 Percent cumulative mortality i n r e l a t i o n to s a l i n i t y Per cent cumulative mortality of infected and control 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% o) for 15 days. S A L I N I T Y % o 5 10 15 2 0 2 5 - 2 8 - 3 0 . 1 1 • 1 1 1 TABLE VII Percent cumulative mortality of infected and non-infected f i s h during the s a l i n i t y tolerance test S a l i n i t y % 5 10 15 20 25 28-30 28-30 28-30 28-30 Time Mean length cm days Non-infected Infected 5 -10 -15 - 14.2 20 13.7 13.6 25 - 13.4 30 14.1 14.7 35 13.2 13.5 40 14.9 14.0 45 13.3 14.0 % Cumulative M o r t a l i t y 1 Non-infected Infected Mean parasite per f i s h 1.5 1.5 4.5 6.0 9.0 10.5 1.5 22.5 43.5 61 .5 65.5 88.5 90.0 23.2 22.9 25.6 24.2 20.7 29.2 23.4 1 T o t a l number of test f i s h = 80 *LT 50 occurred between the s a l i n i t y of 25-28%„ TABLE VIII S a l i n i t y preference test for infected and non-infected juvenile sockeye salmon at 12°C :ing period S a l i n i t y l Percentage of f i s h in the Percentage of f i s h in the % salt water compartments alternate fresh 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 = fresh water compartment SC = s a l t water compartment 1 = s a l i n i t y is measured at the center of each compartment 2 = control experiment CO 74 established, the infected f i s h crossed the central p a r t i t i o n to the sea water before the control f i s h during the i n i t i a l observation. Both infected and non-infected f i s h swam back and forth across the central p a r t i t i o n . Occa-s i o n a l l y , darting, perhaps r e s u l t i n g from the interference by the parasites, interrupted the normal behavior of the f i s h ; t his lasted for a few minutes before the f i s h returned to the normal resting state. The number of f i s h , either infected or non-infected, in the a l t e r -nate s a l t water compartment and the intensity of the in f e c t i o n for the infected f i s h were recorded at the end of each testing period. The results are shown in Table VIII. Similar calculations were made for the control experiment. The percentage of non-infected f i s h in the s a l t water compart-ment gradually increased. At the end of the experiment (240 min testing time) about 75% of the non-infected f i s h appeared in this compartment. The infected f i s h seemed to avoid high s a l i n i t y water. Only 25% of the infected f i s h remained successfully in the alternate s a l t water compartment. 4. Discussion Because of their well developed a b i l i t y to osmoregulate salmonid f i s h can move from fresh to s a l t water with great e f f i c i e n c y . Cpnte et a l . (1966) demonstrated this a b i l i t y in coho salmon by immersing them in sea water. The f i s h in that experiment successfully adapted to sharp increases in osmotic concentration from 143 to about 400 mOsmol followed by a return to near fresh water l e v e l s . This suggests that mortality among the non-infected juvenile sockeye salmon (Figure 11) in the present experiment does not res u l t from osmotic stress. It may, however, be attributed to the absence of food, a condition established in the present experiment. The drastic increase in the mortality of the"infected f i s h probably indicates that their e f f i c i e n c y to trave l from fresh to s a l t water i s 75 disrupted. This can be attributed to a reduction in their a b i l i t y to osmo-regulate as w i l l be discussed below. It has been reported that the development of the a b i l i t y of anadromous f i s h to osmoregulate. in sea water depends upon a p r i o r physiological meta-morphosis referred to as the parr-smolt transformation (Black, 1951; Houston, 1961; Houston and Threadgold, 1963). Apart from environmental changes, regulation of osmotic and ionic concentrations i n the blood appears to be one of the most important factors i n this transformation. 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 structure. Any damage to the g i l l would probably disrupt that a b i l -i t y leading either to a decrease in the number of these c e l l e s or to the hinderance of the i r e f f i c i e n c y i n performing their secretory function. Fur-thermore such damage may prevent the elevation of g i l l Na, K stimulated ATPase a c t i v i t y which has been related to seaward migration (Baggerman, 1960; Otto and Mclnery, 1970; Zaugg and McLain, 1972). Severe injury to the e p i t h e l i a l c e l l s , g i l l s and skin, would upset the normal e p i t h e l i a l c e l l functions, such as acting as an ion bar r i e r and may eventually lead to a decrease in the concentration of e l e c t r o l y t e s , most importantly of sodium, which play a key role in the normal neuro-transmission of stress signals from the brain (Baker, 1966) . If this is the case, the f i s h would be unable to endure the stresses. Fish size appears to rel a t e to the process of s m o l t i f i c a t i o n (Elson, 1957). Wagner et a l . (1969) made a very interesting observation in the i r study of osmotic and ionic regulation in chinook salmon. The salmon, which were growing more rapidly than normal, possessed regulatory systems which were either more functional with respect to a given s a l t gradient or capable of being i n i t i a t e d more quickly in response to changes in environmental 76 s a l i n i t y . Hoar (1976) agreed with the conclusions i n these e a r l i e r papers that larger f i s h are more re 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 size 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 , hindering t h e i r a b i l i t y to tolerate s a l i n i t y changes during seaward migration. 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 test the infected 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 concentration difference between the blood and the water. Again energy expenditure i s demanded to 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 ther from the parasites 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 fact 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 2 demand, which the infected f i s h are normally less able to meet. The threshold number of parasites 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 mortality rate among the infected f i s h during a s a l i n i t y tolerance t e s t . U n t i l better r e s u l t s are obtained, t h i s number can presumably be used as the number of parasites 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 transfer from fresh water to s a l t water. An incomplete parr-smolt transformation process i n the infected f i s h i s confirmed by the avoidance a c t i v i t y observed among those infected f i s h during the s a l i n i t y preference t e s t . This raises the int e r e s t i n g question 77 of what factor or factors r e a l l y have the greatest impact on this readiness. This question would have to be considered separately and quantified in a future investigation. SECTION V IMPACT ON BLOOD 79 1. Introduction Among important indicators of the effects of the environment on f i s h health, hematological studies have received p a r t i c u l a r attention, f i s h hema-tology i s becoming an increasingly useful tool for the fishery b i o l o g i s t and research ichthyologist. To est a b l i s h the basic f i t n e s s of f i s h for exper-imental purposes, Cope (1961) suggested several observations including some hematological measurements, should be made. Si m i l a r l y , Johnson (1968) suggested that with the standardization of techniques, procedures, and normal values for varying environmental conditions and laboratory conditions, f i s h hematology may be used for a variety of studies including that of popu-lations in ex i s t i n g and changing environmental conditions. Slicher (1961) and B a l l and Slicher (1962) used blood as an excellent indicator of physiological responses in endocrinological studies. Katz (1950) has used blood counts to evaluate the diets of f i s h on the grounds that the number of erythrocytes responds quickly to some dietary d e f i c i e n c i e s . Bouck and B a l l (1966) found hematology a useful tool for monitoring stress levels in f i s h exposed to aquatic p o l l u t i o n . Because hematological tech-niques substantially help with d i f f e r e n t i a l diagnosis, e s p e c i a l l y in cases with similar c l i n i c a l pictures, numerous f i s h b i o l o g i s t s have employed hematological procedures to assess the condition of the f i s h . The determination of blood parameters involves several techniques. Estimates of the number of blood 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 various disadvantages in this approach however, modern electronic methods have taken over in human hematology and the same procedure is now generally used 80 in f i s h hematology. Along with c e l l counts, hemoglobin determination is often used to analyze blood conditions and i t is.the simplest method for detecting anemia. A l l the methods used for hemoglobin determination in human blood have been t r i e d with the f i s h blood. At present, cyanmethemoglobin is considered to be the method of choice (Larsen and Snieszko, 1961; Larsen, 1964; and Muleahy, 1970). Microhematocrit values are nearly always employed as a hematological index since only a small amount of blood i s needed for the determination. In addition to red blood c e l l c h a r a c t e r i s t i c s , white blood c e l l s have been used with accuracy in monitoring stress response (Swift and Lloyd, 1974; Soivio and Oikari, 1976 and McLeay and Gordon, 1'977), in assessing the health of f i s h (Blaxhall, 1972 and Hickey, 1976), and in assessing disease r e s i s t -ance of f i s h (Watson et a l . , 1956 and Corbel, 1975), Belova (1966) suggested that the white blood c e l l count is a more r e l i a b l e indicator of unfavorable environmental conditions and stresses than other hematological parameters. The role of white blood c e l l types i n the hematological defense mechanism of f i s h and the effects of environmental stresses on the outbreak of disease are becoming increasingly more clear (Snieszko, 1974; Corbel, 1975; E l l i s et a l . , 1976). However, evaluation of the nature and extent of these i n f l u -ence are hampered by contradictions i n the terminology encountered i n the l i t e r a t u r e . These contradictions can be attributed mainly to the staining techniques used. However, advances i n immunology, i n electron microscopy and i n staining techniques have suggested that these contradictions may soon be resolved. Terminology regarding the white blood c e l l s has become less confusing. Formerly, i t had been generally agreed that lymphocytes and thrombocytes were very similar morphologically. Ferguson. (1976), however, has demonstrated recently that lymphocytes and thrombocytes are not only d i s s i m i l a r 81 morphologically, but are also not related developmentally. Some contra-dictions in terminology and consequently even a question about the existence of some of the c e l l s remained. McKnight (1966) claimed that no eosinophils or monocytes were found in the f i s h he examined but Lester and Daniels (1976) demonstrated the occurrence of these c e l l s , using.electron microscopy. On considering the many reports on the occurrence of white blood c e l l s and the terminology used to describe them, i t seems that most workers assisted by the descriptions available in the l i t e r a t u r e have independantly developed th e i r own terminology. There i s no general agreement upon s p e c i f i c d e f i n i t i o n s for most of the c e l l s . However, because of the noted s e n s i t i v i t y and r a p i d i t y of response of the white blood c e l l s to any environmental changes, they have received much attention from many researchers. Some of them have used f i s h blood to evaluate the nature of the effects of parasites and diseases on f i s h (Watson et a l . , 1956; Weinreb, 1958; Enomato, 1969; Mawdesley-Thomas, 1969; Carbery, 1970 and Einszporn-Orecka, 1970). The present experiments attempted to study the effects of the crustacean parasite Salnrinaola, on the blood of sockeye salmon. 2. Materials and Methods 2.1. Experimental Design This experiment has the same experimental design as described i n Section I. 2.2. Experimental F i s h The f i s h used for hematological determinations were the same group as was used for the growth study described in d e t a i l in Section I. 2.3. Blood Sampling Procedure Fish were removed at random one at a time from the experimental tanks with a small net and placed in a 1:10,000 solution of the anesthetic MS-222 (Smith and B e l l , 1967) for f i v e minutes. After removal from the anesthetic, 82 they were blotted dry with a paper towel. The blood was then removed by the following syringe method. A clean, dry, 1 ml. syringe (needle size 1/2" no. 26 gauge) i s inserted through the musculature in the area between the dorsal and adipose f i n s and just above the l a t e r a l l i n e . In this position, the needle w i l l i n i t i a l l y contact the vertebrae. The needle i s then c a r e f u l l y lowered u n t i l i t touches the dorsal aorta which l i e s under the vertebral column (Figure 12). Blood i s drawn by c a p i l l a r y action into the syringe. This technique ensures that the blood is shielded from contamination. Various blood anticoagulants have been used by other researchers: oxalate c r y s t a l (McCay, 1929), ammonium oxalate (Schlicher, 1927), sodium oxalate ( F i e l d et a l . , 1943), sodium c i t r a t e (Catton, 1951), EDTA (Mulcahy, 1970), and heparin (Yokayama, 1947; Hesser, 1960; Summerflet, 1967). Heparin i s highly recommended f o r f i s h blood. Heparinized c a p i l l a r y tubes were therefore used in a l l blood sampling to delay blood c l o t t i n g . The f i r s t drop of blood from the syringe was discarded. One drop was then smeared on each of three slides for the d i f f e r e n t i a l c e l l count. The blood l e f t i n the syringe was transferred into micropipettes for t o t a l blood c e l l count, into Sahli pipettes for hemoglobin determination and into microcapillary tubes for hematocrit determination. 2.4. Staining Technique The smeared sl i d e s were l e f t overnight (air dried) and then stained with a Modified Wright-Giemsa stain (Halico D i f f Quik Stain) as described below. The slides were f i r s t dipped 'into a f i x a t i v e solution (Diff Quik no. 68451 A) for 5 one-second dips. Any excessive f i x a t i v e solution on the slides was allowed to drain before immersion into solution I (Diff Quik no. 68451 B). This procedure was repeated with solution II (Diff Quik no. 83 Figure 12 Blood Sampling Technique This figure shows the needle inserted through the muscle tissue and penetrating the dorsal aorta. When the needle i s inserted into the blood vessel, the blood w i l l be drawn through c a p i l l a r y action. 85 68451 C). The slides were dipped 5 times into each one of these two solu-tions in a fashion similar to the dipping in the f i x a t i v e solution. Imme-diately after dipping into solution I I , the slides were rinsed in d i s t i l l e d water (pH 6.8) for 1 min. and allowed to dry. Increasing the number of dippings into Solutions I and I I , as recommended by the manufacturer for better staining of eosinophils and baso-p h i l s of human blood, was t r i e d with some smeared s l i d e s . However, no improvement in d i f f e r e n t i a t i o n was noted. The following stains were also t r i e d in order to i d e n t i f y the white blood c e l l s Wright's stain Giemsa stain Pappenheim stain Peroxidase tes t s . Buffy coat was also smeared on glass s l i d e s which were stained in Halico D i f f Quik Stain and also in the other stains mentioned above. 2.5. Hematological Determinations 2.5.1. Hemoglobin Concentration Owing to i t s extensive use in f i s h hematology (Larsen and Snieszko, 1961; Larsen, 1964; Mulcahy, 1970) the Cyanmethemoglobin method was used for hemoglobin (Hb) determination in this experiment. The solution used for Hb determination was Drabkin's diluent solution Sodium bicarbonate (NaHC03) 1.0 g. Potassium cyanide (KCN) ' 0.05 g. Potassium ferricyanide (K 3Fe(CN)g) 0.2 g. D i s t i l l e d water to make 1000 ml. Ferricyanide converts hemoglobin iron from the ferrous to the f e r r i c state to form methemoglobin, which combines with potassium cyanide to produce the stable cyanmethemoglobin. The absorption values of the hemoglobin from 86 this solution were measured with a photoelectric colorimeter using an absorption band in the region of 540 nm. The Hb concentration (g/dl 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 established 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 at 540 nm. The absorbance of each standard was then plotted against i t s concentration. The hemoglobin concentration of a blood sample was measured by d i -l u t i n g 0.02 ml. of blood from a Sahli pipette into 10 ml. cuvettes containing 5 ml. of cyanmethemoglobin reagent, mixing the solution well and allowing i t to stand at room temperature for 5 minutes to permit the formation of cyanme-themoglobin. A subsample was then transferred to a smaller cuvette, in which the absorbance was measured at 540 nm. and compared to the standard curve. This gave the hemoglobin concentration in g/dl. 2.5.2 Hematocrit Value Heparinized microcapillary tubes (77 mm. in length 1.1-1.2 mm. ID, 0.2 ± 0.02 mm. wall) approximately 3/4 f i l l e d with blood, were spun in a microcentrifuge at 3000 rpm. Five tubes were used for each sampled f i s h . The hematocrit values were then computed using the following formula: Hematocrit value (%) = ^  x 100 L2 where L_ i s the height of packed red c e l l s i n mm. and L 2 i s the height of the t o t a l blood specimen. The grey-white layer (buffy layer) above the packed red c e l l s was included in L_. The mean of the 5 samples was c a l -culated . 2.5.3. Erythrocyte Osmotic F r a g i l i t y Test The osmotic f r a g i l i t y test provides an indication of the f r a g i l i t y of the red c e l l s . Suspended in a hypotonic solution of sodium chloride red 87 c e l l s take up water, swell, become spheroid and, after reaching a c r i t i c a l volume, eventually burst. Fish used for this experiment were not the same as those described in section V. They came from the same stock but they were infected with the copepodid larva 2 months p r i o r to the experiment. Only 100 f i s h were assigned to each of the experimental tanks (control and infected f i s h were kept in separate tanks at the same temperature of 9°C). The average f i s h size and weight at the end of 2 months were 17.83 ± 1.97 cm, and 59.5 ± 4.3 g for the control and 16.46 ± 2.01 cm and 55.47 ± 5.0 g for the i n -fected group. The average number of parasites per f i s h was 23.9 ± 4.6 and 547 ± 48 parasite-days. Blood was sampled using the technique described e a r l i e r , although the syringe and needle size had to be changed to 3 1/2 ml. and 25 gauge respectively according to f i s h size and the amount of blood required for the test. For the quantitative method of measuring osmotic f r a g i l i t y , sodium chloride solutions 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 chloride at pH 7.4. Five ml. of each solution was transferred into 13 tubes and 0.05 ml. of blood was added to each of the tubes. The tubes were immediately mixed and centrifuged at 3000 rpm. for 5 minutes. The degree of hemolysis was recorded by measuring the absorption values of the mixed d i l u t i o n , (further di l u t e d 1 to 5) using a spectro-photometer adjusted 7 to a wave length, of 540 nm. The % hemolysis was obtained by comparing the experimental tube with a standard tube of d i s t i l l e d water in which hemolysis was 100%. Blood from 20 infected f i s h was compared with the blood of 20 control f i s h . 88 2.5.4. C l o t t i n g Time This experiment was done following the osmotic f r a g i l i t y test using the remaining experimental f i s h from that test (these f i s h had been infected 3 months). The average number of parasites was 29 ± 4.5 per f i s h , while the average length and weight were 17.92 ± 1.73 cm and 58.91 ± 3.6 g for the con t r o l and 16.20 ± 2.17 cm and 53.24 ± 4.9 g for the infected group (n = 10). Pla i n c a p i l l a r y tubes were used i n i t i a l l y to determine c l o t t i n g time However, due to rapid blood c l o t t i n g , e s p e c i a l l y of blood from the infected f i s h , the results from the i n i t i a l c l o t t i n g time test were deemed unreliable The experiment was repeated using heparinized microcapillary tubes to prolong blood c l o t t i n g . After blood was removed from the f i s h by the technique described e a r l i e r , i t was immediately transferred into microcapil-lary tubes. Each' tube was f i l l e d to approximately 4/5 of i t s volume. Blood from one f i s h was s u f f i c i e n t to f i l l at least 25 tubes. At the same time, a stopwatch was started. A short segment of the c a p i l l a r y tubes was broken off every 2 minutes u n t i l a f i b r i n thread was seen to connect the fragments, thus denoting the end point of the experiment. Every 2 minutes a record was made of those with cl o t t e d blood and those where c l o t t i n g had not yet occurred. The c a p i l l a r y tubes of clotted blood were recorded. 100% blood clotted was denoted by the time when a l l tubes were cl o t t e d . 2.5.5. Total Blood C e l l Counts Immediately after sampling, blood was transferred into micropipettes using 5, 10 ul micropipettes for each sampled f i s h . Blood from each pipette was diluted in a bot t l e containing 100 ml. of 0.9% f i l t e r e d sodium chloride to make a 1:10,000 suspension. A Coulter Counter was used to count the number of c e l l s in a 1/2 ml. suspension of sampled blood. The aperture of the glass cylinder was 100 um. 89 A coincidence correction was made by r e f e r r i n g to a chart supplied by the manufacturer. The number of c e l l s per cubic millimeter of blood was then calculated. 2.5.6. Red Blood C e l l Count As mentioned in the blood sampling procedure, the second, t h i r d and fourth drops of blood were each smeared on one of three glass slides using the "two-slide method." This method involved a second s l i d e held at an angle of 35-40 degrees to the centre of the f i r s t s l i d e which was brought back against the blood u n t i l i t spread by c a p i l l a r y action along the i n t e r -face between the two s l i d e s . The "spread s l i d e " ( s t i l l maintained at an angle of 35-40 degrees) was then pushed forward at a moderate speed so that blood spread evenly along the other s l i d e . The completed smeared sl i d e s were a i r dried and then stained (using a procedure to be described in the next section). The stained smears were examined under o i l immersion at a magnifica-tion of 1500 x. Red blood c e l l s (both immature and mature) and white blood 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 for each s l i d e ( f i e l d s containing no greater than 100 c e l l s but no less than 20 c e l l s were counted). The t o t a l numbers of RBC and WBC from three slides were then calcu-lated. According to the t o t a l blood c e l l counts received from the Coulter Counter described e a r l i e r , the number of red cells/mm 3 can be calculated using the formula: J L x K RBC c e l l s / mm3 = W+R 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 mm3 from Coulter Counter. 9 0 2 . 5 . 7 . Red C e l l Corpuscular Value Three erythrocytic indices were calculated from the hematocrit, hemo-globin and RBC value using the following formula: i i r™mr\ Hematocrit (%) x 10 Mean corpuscular volume (MCVj = r — — • —-.———\ -,—r% v RBC count (millions/mm 3) v* i u i /UPD\ Hemoglobin g/dl x 10 Mean corpuscular hemoglobin (MCH; = -.. — • 7 — r \ * 6 RBC count (million s/mm3) Mean corpuscular hemoglobin = Hemoglobin g/dl x 1 0 0 concentration (MCHC) Hematocrit (%) 2 . 5 . 8 . D i f f e r e n t i a l C e l l Count A l l the c e l l types were c l a s s i f i e d as far as possible according to the stains used in this experiment and following the description Golovina ( 1 9 7 6 ) , Lester and Daniels ( 1 9 7 6 ) , Lehmann and Sturenberg ( 1 9 7 5 ) , E l l i s ( 1 9 7 7 ) , Einszporn-Orecka ( 1 9 7 3 ) , Watson et a l . ( 1 9 6 3 ) , Wienreb ( 1 9 5 8 ) and Katz ( 1 9 4 9 ) . In d i f f e r e n t i a l c e l l counts the c e l l s were d i f f e r e n t i a t e d from the smeared sl i d e s stained with Halico D i f f Quik which was found to be an appropriate stain for the blood of sockeye salmon because more c e l l s could be i d e n t i f i e d with this s t a i n . I d e n t i f i c a t i o n of c e l l s during counting was aided by applying the following c r i t e r i a : Immature red c e l l - round to oval c e l l with round to oval nucleus l i g h t blue-grey Mature red c e l l - an oval c e l l with oval nucleus, 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 rim of blue cytoplasm, frayed margin Large lymphocyte - similar to the above c e l l but larger, no frayed margin, may not have an indented nucleus 91 Neutrophil - large c e l l with lobed nucleus and granular cytoplasm Monocyte < - round c e l l with eccentric and deeply c l e f t nucleus and very loose chromatin Macrophage - as large as or larger than RBC, with a number of vacuoles Thrombocyte immature - very dense nucleus, pale cytoplasm, may or may not form pseudopodia mature - a c e l l with packed eccentric oval nucleus, cytoplasmic granules s t a i n very l i g h t l y 2.5.9. S t a t i s t i c a l Analysis of Data The means of each hematological parameter of the infected groups were compared with those of the control group using student's t - t e s t . S t a t i s t i c -a l l y s i g n i f i c a n t differences were measured at a confidence l e v e l of P = 0.05. Linear regression l i n e s were drawn and regression c o e f f i c i e n t s were compared using analysis of variance. The data from some of the hematological tests with non-homogeneous varianc were transformed, using log 10. 3. Results 3.1. Hemoglobin Concentration Mean hemoglobin concentration of infected f i s h was compared with concentration of the control group using a t - t e s t . I t appeared that the concentration of Hb was s i g n i f i c a n t l y lower (P = 0.05) i n the infected than i n the control group (Figure 13). The Hb concentration dropped from 8.81 ± 0.1 to 6.90 ± 0.17 g/dl (Table IX) i n infected f i s h during the experimental period of 112 days, while i n the control group i t remained constant within the range of 8.60-8.86 g/dl. during the same period of time (Figure 13). During the period of 3-41 DPI there were variations i n the Hb con centration i n both control and infected f i s h groups but there were no s i g n i f -icant differences. After 41 DPI, the Hb concentration was greatly reduced. 92 Figure 13 Relationships between hemoglobin and DPI Comparison of the hemoglobin concentrations of the blood of infected and control f i s h groups i n r e l a -tion to in f e c t i o n time. Each point represents mean values. V e r t i c a l l i n e represents S.D. Number on each point represents sample size. H E M O G L O B I N C O N C E N T R A T I O N g m / dl o C6 TABLE IX Hemoglobin concentration g/dl measured from the blood of infected and non-infected f i s h groups during the 3 - 112 DPI mean S.D. sample size me an S.D. sample size 10 13 17 Day Post Infection 23 28 32 38 41 56 70 84 112 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 .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 8.83 8.62 8.86 8.70 8.63 8.85 8.66 8.50 8.70 8.62 8.88 8.56 8.60 8.70 .15 .12 .15 .14 .18 12 12 12 23 24 .13 .17 .19 .15 .14 .18 .20 .15 .17 22 24 23 23 20 11 10 12 11 I C = Infected f i s h group = Control f i s h group 95 These results c l e a r l y indicate that Satmincola exerts an adverse effect on juvenile sockeye salmon by causing the reduction of Hb concentra-tion in the infected f i s h . During the period of 112 DPI, when the f i s h were parasitized with 31.25 Satmincola, Hb concentration i n the infected f i s h dropped by 20%. 3.2. Hematocrit Value No s i g n i f i c a n t difference between the blood of infected and control f i s h groups (Figure 14) was found in the mean hematocrit values during the period of 3-41 DPI. The reduction of the hematocrit value of the infected f i s h group appeared after 41 DPI, and gradually declined toward the end of the experiment. At 112 DPI i t had dropped to 34.51% while in the control group., i t remained at 46.21% (Table X). It i s obvious that Satmincola can cause a reduction in hematocrit value of infected juvenile sockeye salmon by 22.2% during a period of 112 days when the f i s h were infected with an average of 31.25 Satmincola per f i s h . 3.3. Erythrocyte Osmotic F r a g i l i t y There was no s i g n i f i c a n t difference between the erythrocyte osmotic f r a g i l i t y of the infected and the control groups (P=.05). Both the i n -fected and the control groups reached 100% hemolysis with the % NaCl at 0.30 (Table XI), which i s very similar to the osmotic f r a g i l i t y of normal human blood (Figure 15). 3.4. Clotting Time In preliminary tests in which p l a i n c a p i l l a r y tubes were used the blood of the control f i s h c lotted within 3-4 min (Figure 16). The heparinized microcapillary tubes can prolong blood c l o t t i n g from 3-4 min to almost 25 min in the blood of the control f i s h . The results are shown 96 Figure 14 Relationships between hematocrit value and DPI Variation in hematocrit values of the blood of infected and control f i s h groups during the experimental period of 3-112 DPI. Each point represents a mean value. V e r t i c a l l i n e represents S.D. Number on each point represents sample size. TABLE X Hematocrit value (%) of infected and control f i s h groups during 112 DPI Day Post Infection 3 6 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 I 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 sample ) 2 n n u 2 Q 2 2 ^ n 20 20 10 9 8 8 size 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 C 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 u n n ^ u 2 2 2 3 2 3 2 3 2 Q u ] Q u u s lze I = Infected f i s h group C = Control f i s h group 00 99 Figure 15 Erythrocyte osmotic f r a g i l i t y of experimental f i s h Osmotic f r a g i l i t y curve at 9°C of infected and control f i s h groups in comparison with the human blood (David-son and Nelson, 1974). Arrow indicates % NaCl where 100% hemolysis occurred. 101 TABLE XI Percent NaCl used in erythrocyte osmotic f r a g i l i t y test and % hemolysis of blood of sockeye salmon infected with Satmincola. The experimental f i s h were kept in the water at 9°C. % NaCl % hemolysis .30 97-100 .40 50-90 .45 5-45 .50 • 0-5 .55 0 in Table XII. The percent of blood c l o t t e d in the heparinized microcapillary tubes increased at a faster rate than that of the control f i s h group and reached 100% blood c l o t t e d within 17 min (Figure 16). A sharp increase appeared within the f i r s t 11 min and thereafter, there was a gradual decline u n t i l 100% of the blood c l o t t e d . Among the control f i s h 100% blood c l o t t i n g appeared at 25 min and the rate of increase i n % blood c l o t t i n g was quite di f f e r e n t from that of infected f i s h . The % blood c l o t t e d increased at a very slow rate during the f i r s t 11 min, a sharp increase appearing a f t e r that time (Table XII). The results obtained from this experiment show that blood c l o t t i n g time for the control f i s h is longer than for the infected f i s h . When measured from the heparinized microcapillary tubes, i t i s 8 min longer. It is possible to conclude that Satmincola can cause a s i g n i f i c a n t decrease i n blood c l o t t i n g time for juvenile sockeye salmon, when they are heavily infected with this parasite. 102 Figure 16 Blood C l o t t i n g Time Comparison between the blood c l o t t i n g time of the infected and the control f i s h groups. The arrow indicates time when 100% of blood in microcapillary tubes c l o t t e d , which denoted blood c l o t t i n g time for this experiment. The arrow with dotted l i n e indicates blood c l o t t i n g time of the control f i s h group. Number on each point indicates sample size. % BLOOD CLOTTED ecu TABLE XII Percent blood c l o t t e d obtained from the blood of infected and control f i s h groups Testing time Control Sample size % Blood clotted Infected Sample size % 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 Total Blood C e l l Count Total blood c e l l counts, received from the Coulter Counter are shown in. Table XIII. They were used only as a. baseline to calculate the number of red blood c e l l s and white blood c e l l s and this need not be discussed in further d e t a i l in this section. 3.6 Red Blood C e l l Count The calculated red blood c e l l counts are shown in Table XIII. Mean numbers of c e l l s from both infected and control f i s h groups were plotted against time post i n f e c t i o n . During the period of 3-45 DPI, mean blood c e l l counts in the infected f i s h appeared s l i g h t l y higher than i n the control f i s h (Figure 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 difference was found. The mean number of red blood c e l l s obtained from the infected f i s h group shows a sharp decrease a f t e r 41 DPI (Figure 17). The rate of decrease appears to be greatest during the period of 41-84 DPI and shows a tendency to decline. Thereafter, the mean c e l l count of the infected f i s h group dropped from 1.21 x 10 s cells/mm 3 to 9.02 x 10 s cells/mm 3 (Table XIII), while in the control f i s h group, i t dropped only to 1.14 x 106 cells/mm 3. This indicates a highly s i g n i f i c a n t lowering in the number of red c e l l s in the infected f i s h group compared with that in the control f i s h group (Fig-ure 18). A reduction in the red blood c e l l numbers in the control f i s h group also appeared but the decrease was very small when compared with that in the blood of the infected f i s h group (Figure 17). The results obtained from this experiment indicate that Satmincola can cause a progressive reduction of red c e l l numbers in the c i r c u l a t i n g blood. At the end of 112 DPI the number of red c e l l s of the infected group was about 24% lower than at the beginning of the experiment, whereas in the control f i s h group the decrease over the same period was only 4.7%. TABLE XIII Counts of t o t a l blood c e l l s , red blood c e l l s , and white blood c e l l s obtained from the blood of infected and control f i s h groups' DPI Total blood eels in millions/mm 3 c e l l s RBC1 in m i l l ions/mm3 WBC2 cells/mm 3 Infected Control Infected S.D. Control S.D. Infected S.D. Control S.D. 3 1.235 1.225 1.212 . 132 1 .205 .185 24,162 4631 22,112 3911 6 1 .265 1.264 1.245 .231 1.238 .174 20,784 3976 26,378 4286 10 1 .237 1 .239 1.208 .099 1.215 . 186 29,392 8321 24,413 2703 13 1.251 1 .229 1 .220 .224 1.200 .132 31,781 2611 29,455 4215 17 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 and 2 were calculated. o ON 107 Figure 17 Relationships between red blood c e l l counts and DPI Mean numbers of red blood c e l l s obtained from infected and control f i s h groups. The s o l i d and dotted lines are lines of best f i t to indicate the v a r i a t i o n of the red c e l l counts during the period of 112 DPI. V e r t i c a l l i n e represents S.D. Number on each point represents the sample size.' 801 109 Figure 18 Regression c o e f f i c i e n t s of red blood c e l l counts Comparison between regression c o e f f i c i e n t of the red blood c e l l s of the infected and non-infected f i s h groups. RED BLOOD CELL C O U N T ( L O G 10) 111 3.7 Red Blood Corpuscular Values Calculated MCV, MCH and MCHC are shown in Tahle XIV. Regression li n e s were drawn from the calculated MCV (Figure 19), MCH (Figure 20) and MCHC (Figure 21). 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 differences were found-in the MCV and MCH of the blood of infected and control f i s h groups. Only the MCHC value of the infected 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 control f i s h group. 3.8 White Blood C e l l Counts The calculated t o t a l number of white blood c e l l s , including thrombo-cytes (WBC-T) i s shown in Table XIII. A s i g n i f i c a n t l y higher number of WBC-T was found in the infected f i s h group than in the control group (Figure 22). An increase in WBC-T must have come from the s i g n i f i c a n t increase in lymphocyte, neutrophil (Table XV) or thrombocyte counts which w i l l be discussed in a la t e r section. 3.9 D i f f e r e n t i a t i o n of Blood Cel l s The c e l l s were i d e n t i f i e d in order to analyze differences in c e l l numbers between infected and non-infected f i s h . 3.9.1 D i f f e r e n t i a l C e l l Descriptions 3.9.1.1 Erythrocytic Series Immature Erythrocyte The c e l l shape varies from round to oval (Plate I, Figure a-d). A small round immature red c e l l i s 6.5 ± 1.72 urn in diameter (Plate I, Figure a). Oval c e l l s vary in size from 10.39 ± 2.4 urn in length and 8.52 ± 2.2 um in width to the same size as mature erythro-cytes, that i s 12.9 ± 3.2 um in length and 7.1 ± 3.4 in width (Plate I, Figure b-d). With other types of stain basophilic cytoplasm can be c l e a r l y seen only in the early stage of erythrocytes (Plate I, Figure a and b) but the c e l l s i n Plate I, Figure b and c cannot be ea s i l y d i f f e r e n t i a t e d from TABLE XIV Calculated MCHC, MCV and MCH of the blood of infected and control f i s h groups Infected Control DPI MCHC MCH MCV MCHC MCH MCV % Pg u 3 % P8 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 corpuscular volume (MCV) Comparison between MCV of the blood of infected and non-infected f i s h groups. Figure 20 Mean corpuscular hemoglobin (MCH) Comparison between MCH of the blood of infected f i s h and non-infected f i s h groups. • • i n f e c t e d D - - - Q c o n t r o l Q D 0 1 3 J= 71_.l_t_0001_X_ * ~""'a"o - * ' "" • " y « 4 9 . 9 + . 0 O 9 X 80 20 40 60 120 TIME IN DAYS • • i n f e c t e d D - - - Q c o n t r o l 0 y o 382 .32 +.0004 X •a--Y = 3 6 8 . 2 9 * .0001 X 20 40 60 80 100 —I 120 TIME IN DAYS 115 Figure 21 Mean corpuscular hemoglobin concentration (MCHC) Comparison of the MCHC of the blood of infected and non-infected f i s h groups. 21 -t 20 H • • infected o—a contro l _N 19 H u X u -E 18-y = 18.05 + .002 X y = i9._ - .005 x 1 7 H 100 I 20 40 I 60 80 120 TIME IN DAYS 117 Figure 22 . Regression c o e f f i c i e n t s of white blood c e l l counts Comparison of regression c o e f f i c i e n t s of white blood c e l l counts of the blood of infected and non-infected f i s h groups. W B C C O U N T L O G 10 TABLE XV Ranges, means and S.D. of leucocytic c e l l s obtained from infected and control f i s h groups at 112 DPI C e l l Types Ranges (%) Control Infected Mean ± S.D. Total lymphocytes 80.35 - 95.42 89, .88 + 7.41 88.75 + 9.01 small lymphocytes 74.26 - 94.38 84. .32 + 5.74 83.92 + 6.82 large lymphocytes 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 1 , .07 + 0.27 1.01 + 0. 19 "Granulocyte c e l l s " 1.21 - 5.40 3, .28 + 0.95 3.35 + 0.62 Macrophages 0.02 - 0.07 0, .04 + 0.008 0.03 + 0.007 120 the mature red c e l l . Basophilic cytoplasm can be used to indicate the maturity of the c e l l . The less basophilic, the more mature the c e l l i s . Mature Erythrocyte This i s an oval c e l l . The c e l l size i s 13.0 ± 2.5 um in length and 7.0 ± 1.28 um in width (Plate I, Figure a, b, e and f) . The nucleus stains dark, purple with HDQ stain and dark blue with Wright's st a i n . The cytoplasm stains orange-brown with HDQ and yellow-green with Wright's stain. Immature erythrocytes contain many more mitochondria (Plate II, Figure b) than do mature c e l l s (Plate I I , Figure a). 3-9.1.2 Leucocytic Series Two types of c e l l s are found i n the blood of juvenile sockeye salmon: agranulocytes and granulocytes. The majority of the c e l l s are agranulocytic c e l l s . Agranulocytes Small lymphocyte These are small, round or almost round (Plate I I , F i g -ure c-f) with a size range from 8.2 ± 3.15 to 12.6 ± 4.0 um. The nucleus occupies v i r t u a l l y the whole c e l l , leaving only a very narrow rim of baso-p h i l i c cytoplasm (Plate I I , Figure d). The chromatin meshwork appears looser than that of mature red c e l l s but was similar to that of .immature red c e l l s (Plate I I , Figure d). Small lymphocytes are often found with frayed margins (Plate I I , Figure e), or pseudopodia (Plate II, Figure f ) . Large lymphocyte These c e l l s are round or almost round (Plate I I I , Figure b). Normally they are larger than mature red c e l l s with a size range from 14.5 ±1.72 um. The nucleus, which is often found with a deep indentation, occupies almost the entire c e l l leaving only a small rim of cytoplasm. The chromatin i s a coarsely meshed strand. Monocyte These are very few in number in the blood of juvenile sock-eye salmon. They have a size range from 8.7 - 10.1 um (Plate III, Figure c ) . 121 The nucleus occupies half or s l i g h t l y more than half of the c e l l volume and is often notched orhorse -shoe shaped. Nuclear chromatin appears as f a i r l y loose strands. The cytoplasm stained l i g h t gray-blue with HDQ sta i n . Nor-mally i t appears with the formation of the t h i r d lobe of the nucleus (arrow). Macrophage In a l l the slides examined in th i s experiment only seven c e l l s of this type were observed in the control group and only one or two c e l l s were observed in the infected f i s h group at each sampling period. A l l of them were of very ir r e g u l a r shape (Plate I I I , Figure d). Size was not measured but these c e l l s appeared larger than mature red c e l l s . The nucleus was also i r r e g u l a r in shape and several vacuoles were found in the cytoplasm. Granulocytes Neutrophil Similar to or larger than mature red c e l l s . These are round c e l l s with a size range 10.1-13.4 urn in diameter (Plate I I I , Figure e and f; Plate IV, Figure a - f ) . Neutrophils of juvenile sockeye salmon appear to have two types of n u c l e i : segmented and banded. Segmented nu c l e i always have 2 to 5 lobes, 5 lobes were more common, nu c l e i with 2 lobes were rarely seen. Sometimes, c e l l s with n u c l e i with more than 5 lobes were seen (Plate I I I , Figure f; Plate IV, Figure a). Lobes were connected to each other by a very fine filament of nuclear materials (see arrow i n Plate III, Figure e). In neutrophils with banded n u c l e i , the nucleus was always found to be twisted or bent (Plate IV, Figure b, d, e and f ) . The nucleus stains v i o l e t and has chromatin consisting of irregular patches of l i g h t and dark staining. The cytoplasm and the granules stain very l i g h t blue with HDQ stain. "Granulocyte c e l l s " Some c e l l s resembled "eosinophils" and "basophils" described by others (Lehmann and Stu'renberg, 1975; Golovina, 1976; and E l l i s , 1977), however, I was unable to stain granules in these c e l l s and thus i d e n t i f i c a t i o n was d i f f i c u l t . These uni d e n t i f i a b l e c e l l s were put into a 122 category c a l l e d "granulocyte c e l l s . " 3.9.1.3 Thrombocytic Series Thrombocytes were found at several stages of maturity in the c i r c u -l a t i n g blood of sockeye salmon, ranging from immature (spherical) to i n t e r -mediate ( s l i g h t l y oval) to f u l l y mature (elongate). Immature Form These are very small round c e l l s (Plate VI, Figure a) with a size range 6.4-7.3 um in diameter. These c e l l s and small lymphocytes are morphologically 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 with HDQ. In the c i r c u l a t i n g blood of juvenile sockeye salmon, these c e l l s are often found to be smaller than small lymphocytes. The very dense purple nucleus occupies the whole c e l l . Intermediate Form These c e l l s are normally larger than the immature form (Plate VI, Figure b, c and d) with a size range 7.2-9.4 um in diameter. The cytoplasm appeared c o l o r l e s s . This c e l l i s often found with pseudopodia (Plate VI, Figure d). Mature Form These c e l l s vary in size and shape from oval to rod l i k e to elongated. In the early stage of maturity the c e l l s appear oval or rod-l i k e (Plate VI, Figure e and f ) . Nuclei of these c e l l s often appear indented (Plate VI, Figure f ) . C e l l s in Plate VI, Figure e and f and Plate VII, Figure a are often found in the blood of juvenile sockeye salmon. The cytoplasm stains very l i g h t blue-gray with evidence of very f i n e granules. Mature thrombocytes often have long cytoplasmic projections at one pole (Plate VII, Figure a). They sometimes appear i n groups of 2, 3, or 4 c e l l s (Plate VII, Figure c ) . 3.9.2 D i f f e r e n t i a l C e l l Counts 3.9.2.1 Erythrocytic Series Immature Red C e l l Percentage and number of immature red c e l l s of both infected and control f i s h groups are shown in Table XVI. Mean 123 numbers of c e l l s of these two groups of f i s h were compared using a t-test and were found to be s l i g h t l y d i f f e r e n t (P = 0.1). Mature Red C e l l No comparison between the mature red c e l l count of the blood of the infected and control 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 leucocyte counts (Thrombocyte not included) of the blood of both infected and control f i s h groups (Table XVII) were plotted against time (DPI) and the regression c o e f f i c i e n t s were compared. A s i g n i f i c a n t l y higher number of leucocytic c e l l s appeared in the blood of infected f i s h than in that of the control f i s h (Figure 24). According to the difference in function of each type of leucocytic c e l l , the number of c e l l s for each group was counted separately as shown in Table XVII. Agranulocytes Lymphocyte Small lymphocytes were dominant among lymphocytes (Table XV). These comprised 89.88 ± 7.4 of the t o t a l leucocytic c e l l s while large lymphocytes made up only 4.56 ± 1.97. Both small and large lymphocytes were analyzed together in the t o t a l lymphocyte counts (Table XVII and F i g -ure 25). Total lymphocyte counts were found to be s i g n i f i c a n t l y higher in the blood of the infected f i s h group than in that of the control group. The increase in this count seems to come from the small lymphocytes rather than the large ones. Difference in the number of c e l l s was c l e a r l y shown at 70-112 DPI. Monocyte and Macrophage A very small number of monocytes and macro-phages were found in the c i r c u l a t i n g blood of the juvenile sockeye salmon. At some sampling periods neither monocyte nor macrophage were observed 124 C e l l s shown in Plates I-VII, are from the blood of non-infected juvenile sockeye salmon, stained with d i f f e r e n t types•of s t a i n . Those which are not spe c i f i e d , are stained with HDQ st a i n . Cells are photo-graphed under o i l immersion with a L e i t z microscope and Wild Photo Auto-mat or L e i t z camera coupled with l i g h t meter. Plate I (a) Immature erythrocyte (early stage). Note the c e l l size i s smaller than the mature erythrocyte. (b) Immature ery-throcyte (later stage), c e l l almost the same size as the mature erythrocyte and becomes more oval. (c) Immature erythrocyte. Note the c e l l with less basophilic cytoplasm than the c e l l i n d, which indicates more maturity. (d) Immature erythrocyte (intermediate stage). Note the chromatin pattern i s more coarsely mesh. Light area can be seen. (e) Mature erythrocyte with small rod shaped mitochondria. (f) Mature and immature erythrocytes with Wright's stain. Note mitochondria could not be seen. 125 PLATE I d 126 Plate II (a) Mature erythrocyte showing mitochondria (arrow). Note smaller number of mitochondria in comparison with immature erythrocyte. (b) Immature erythrocyte (intermediate 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 indicates a very narrow rim of basophilic cytoplasm, (e) Small lymphocyte with frayed margin, frequently encountered in the c i r c u l a t i n g blood. (f) Small lympho-cyte with the formation of pseudopodia (arrow) infrequently encountered in the blood. 127 128 Plate III (a) Small lymphocyte showing corasely meshed chromatin strands with alternating l i g h t and dark areas under Wright's sta i n . (b) Large lymphocyte with deep indentation of nucleus (arrow). Note c e l l i s normally larger than the mature erythrocyte. (c) Monocyte, note lobed nucleus and formation of the t h i r d lobe (arrow). (d) Macrophage with some small vacuoles (arrow). Infrequently encountered in the blood. (e) Neutrophil, one of the t y p i c a l appearances (5 lobed nucleus). Arrow indicates the fine nuclear material connecting one lobe to another. (f) Neutrophil, nucleus with more than 5 lobes. Infrequently found in the blood. 130 Plate IV (a) Neutrophil, showing nucleus with more than 5 lobes. (b) Neutrophil, showing nuclear filaments, (c) Neutro-p h i l , nucleus with 2 lobes, infrequently encountered in the blood. (d) Neutrophil with bent nucleus. (e) Neutrophil, note the c e l l size i s almost twice the size of mature erythrocyte. This picture shows fine granules regularly appear in the cytoplasm, normally found i n a l l the c e l l types of neutrophils. (f) Neutrophil with twisted nucleus. 131 PLATE IV 132 Plate V (a) to (f) are termed "granulocyte c e l l s " but their precise nature could not be determined.. (a) and (c) more often found than any other c e l l s but (f) i s very rarely found. 133 134 Plate VI (a) Immature thrombocyte (very early stage), cytoplasm is not normally seen. (b) Immature thrombocyte (early stage), note very pale cytoplasm (arrow). (c) Immature thrombocyte, note cytoplasm may appear ir r e g u l a r (arrow). (d) Immature thrombocyte (intermediate stage), note pseudopodium formation at one of the c e l l (arrow). (e) and (f) are mature thrombocytes. 136 Plate VII (a) and (b) are mature thrombocytes at active stage. (c) Mature thrombocytes, frequently found in a group of 4-5. PLATE V I I TABLE XVI Percentage and number of immature red c e l l s observed in the blood of infected and control f i s h groups. Infected Control DPI Z cells/mm 3 % cells/mm 3 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 Co CO 139 Figure 23 Regression c o e f f i c i e n t s of immature red c e l l counts Comparison between regression c o e f f i c i e n t s of immature red c e l l counts of the blood of infected and non-infected f i s h groups. 1.2 TABLE XVI I Heans t S.D. of d i f f e r e n t leucocyte (nuut s ( c e l l s in thousands/mi) 1 ) f o r i n f e c t e d and c o n t r o l f i s h groups. UHl Small Lymphocyte Inl ec ted Control Large l.y Infected mphocy t e Coitt ro 1 N lnfec eul rnji ted .hi 1 (.'on t ro 1 Monocy tt-Infected Control "Cranulocyt Infected e c e l l " Comrol Macrophage Infected Control Total Infected Control 1 19 . 7l3 .5 22.912 .8 1.031 .05 1.2111.5 1 .481 .23 1 .621 . 19 .241 .01 .281.02 1.481 .33 1 .541 .34 .0541.006 .0481 .007 24 1 4 .6 22 1 3.9 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 26 1 4.2 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 .27 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 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 - 25 1 4 .2 23 1 3.8 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 - 27 1 3 .5 30 1 2.7 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 | 24 1 5.6 12 2 J. I l l . .7 21. .511. 3 1.131. . 14 1.161. 1 7 1 . 761 . 12 1 .561. 23 .261 .06 .251.02 1.491 .42 1 .521 . 17 .0521.003 .0511 .009 27 1 4 .2 26 1 7.2 18 20. 214. 3 18. 413. 9 0.961. 17 0 . 9 8 1 . 32 1 .621 . 17 1 . 321. 39 .241 .06 .231.01 1.401 .31 1 .551 .39 .0421.004 - 23 1 2 .0 22 1 8.4 '. 1 24 . BlS. 1 23. 712. 6 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 56 23. 611. 4 23. 012. 4 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 27 1 4.1 ;o 27. 212. 6 24 . 412. 3 1.29*. 07 1.301. 27 2. .321. .23 1 . 721. 18 .301 .03 .281.04 1.701 . 18 1 .451 .43 .0581.003 - 31 1 2 .5 29 1 6.2 84 27. 116. 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 1 12 26. 813. 0 21. 911. 6 1. 111. 42 1.191. 40 2. 07l . 20 1 .531. 37 - .261.05 1. 741. .40 1 .351. .39 .0511.002 .0611. 006 31 1 4. .3 26 1 3.6 142 Figure 24 Regression c o e f f i c i e n t s of leucocytic c e l l counts Comparison between regression c o e f f i c i e n t s of leuco-c y t i c c e l l counts ( c e l l s i n thousands) in the blood of infected and non-infected f i s h groups. LEUCOCYTIC CELL C O U N T S cells in thousand o 144 (Table XVII). Therefore, no comparisons were attempted. Granulocytes Neutrophil They were found to be the commonest granulocyte in the c i r c u l a t i n g blood of juvenile sockey (Table XV and XVII). When the number of c e l l s were compared between the blood of the infected and control f i s h groups, neutrophil counts from the blood of the infected f i s h group were s i g n i f i c a n t l y higher than those i n the control f i s h group (Figure 26). The c e l l s were found to have increased from 1.4 ± .2 x 103 cells/mm 3 to 2.20 ± .24 x 103 cells/mm 3. Neutrophils increased by almost 35% in the infected f i s h while those of the control f i s h decreased by 5.6%. "Granulocyte C e l l s " A l l of the c e l l s shown in Plate V, Figure a - f were counted and analyzed together under the t i t l e of "granulocyte c e l l s " (Figure 27). Variation in the c e l l counts were very high during the period of 3 to about 60 DPI. Then the mean c e l l counts from the blood of the i n -fected f i s h group appeared higher than those from the control f i s h group. A s i g n i f i c a n t difference was obtained when the regression 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 (of a l l forms) in the infected f i s h blood was found to be higher than in the non-infected f i s h blood at the be-ginning of the experiment. The regression c o e f f i c i e n t s of both regression lines (Figure 28) were compared 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 = 0.05). The mean value of the thrombocyte count at 3 DPI was 8.3 x 103 cells/mm 3. At the end of the experiment, i t was found to be 23.3 x 103 cells/mm 3. This means that the number of thrombocytes increased about four times as a result of the p a r a s i t i c i n f e c t i o n . The intermediate c e l l s with pseudopodia (Plate VI, Figure d) seemed to appear more frequently in the blood of the infected f i s h . 145 Figure 25 Regression c o e f f i c i e n t s of lymphocyte counts Comparison of regression c o e f f i c i e n t s of lymphocyte counts of the blood of infected and control f i s h groups. 146 (oi ooDSiNnco ajjooHdWjn 147 Figure 26 Regression c o e f f i c i e n t s of neutrophil counts Comparison between regression c o e f f i c i e n t s of neutrophil counts in the blood of infected and non-infected f i s h groups. T I M E IN DAYS -p-00 1 4 9 Figure 27 Regression c o e f f i c i e n t s of counts of "granulocyte c e l l s " Comparison between regression c o e f f i c i e n t s of "granulocyte c e l l s " of the blood of infected and non-infected f i s h groups. 151 Figure 28 Regression c o e f f i c i e n t s of thrombocyte counts Comparison between regression c o e f f i c i e n t s of thrombocyte counts of the blood of infected and non-infected f i s h groups. T H R O M B O C Y T E COUNT LOG 10 o 153 4. Discussion It is generally agreed that the a c t i v i t y of blood-feeding parasites induces anemia, and this condition has also been observed i n a few cases of p a r a s i t i z a t i o n with parasites which do not rely on blood for n u t r i t i o n . However the results of this experiment strongly indicate that the p a r a s i t i c crustacean 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 blood character-i s t i c s of juvenile sockey salmon. That i n f e c t i o n leads to the development of anemia was indicated in this experiment by the reduction in red blood c e l l s , in hemoglobin concentration and also in hematocrit value. There are at least three possible ways in which this parasite can cause anemia. F i r s t l y , i t may res u l t from direct damage to c e l l s at the s i t e or around the s i t e of i n f e c t i o n . The severity of the effects of this para-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 of the parasite. The major s i t e of i n f e c t i o n with t h i s parasite i n juvenile sockeye salmon i s in the g i l l cavity. In f r y , the parasites are normally found on the g i l l f i l a -ments which they eventually leave for sites of more permanent attachment as adults. The lesions on the g i l l filament which are caused by the attachment organ (fr o n t a l filament) of the parasite and the e p i t h e l i a l damage caused by the feeding a c t i v i t y of the parasite may lead to hemorrhaging as observed in tench infected with Evgasilus sieboldi (Einszporn-Orecka, 1970). Severe hemorrhaging, however, was not observed i n the present experiment. Secondly, i t may res u l t from the hemodilution. It i s known that a balanced r a t i o be-tween g i l l surface area and body mass must exist i f the f i s h i s to maintain proper osmotic regulation of internal f l u i d s . Any damage to the g i l l s w i l l upset this balance and lead to hemodilution. This i s confirmed by the obser-vation of Hines and Spira (1973) on carp that Ichthyophthirius caused osmo-regulatory disturbances which led to s i g n i f i c a n t decreases in serum Na and Mg and increases in serum K . In cases of severe hemodilution, such as 154 r e s u l t i n g from kidney disease, Iwama (1977) found that MCV values of the blood of the infected f i s h are higher than those of healthy f i s h . However, in the present experiment, 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 difference was found in the MCV. This r e s u l t should indicate that hemodilution did not occur. But from my point of view, this seems un l i k e l y , since the progressive re-duction of red blood c e l l s per unit volume observed in the infected f i s h (Figures 17 and 18) indicates the p o s s i b i l i t y of an increase in blood volume along with a reduction of the red c e l l numbers. In the f i r s t case, an increase in red blood c e l l volume r e s u l t i n g from the hypotonicity of the surrounding plasma occurs and, therefore, an increase in MCV i s obtained. However, i f a decrease in the number of red c e l l also occurs at the same time, there w i l l be a reduction in the rate of increase of the hematocrit value. In that case, no difference in MCV w i l l be observed. Neither was any s i g n i f i c a n t difference in the MCH of the blood of infected and non-infected f i s h groups found. Consequently, the MCV and MCH show similar variations (Davidsohn and Nelson, 1974). MCHC of the blood of the infected f i s h shows a s l i g h t l y lower value, not s i g n i f i c a n t l y d i f f e r e n t (P = .05), than the blood of the non-infected f i s h groups. It can also be attributed to the progressive destruction of the red c e l l s . Thirdly, anemia may also r e s u l t from the destruction of the red c e l l s themselves. There were some indications of metabolite exchange between the f i s h and the parasite through the b u l l a and also the metabolic excretion from the parasite may have been absorbed into the c i r c u l a t i n g blood. If these metabolites have pathogenic effects on the blood c e l l s they may d i r e c t l y destroy them, leading to a reduction in the number of c e l l s in the c i r c u l a t i n g blood. Possibly, they may reach the hemopoietic tissue, i n t e r f e r i n g with the normal function of the tissue, leading to a reduction of the c e l l s i n the c i r c u l a t i n g blood. This i s supported by the study by Romestand and T r i l l e s (1977) of f i s h 155 parasitized with a cymothoid isopod. They found a decrease in the c i r c u -l a t i n g erythrocytes and suggested that t h i s was a good indication of i n t e r -ference with the normal production of blood. It is. concluded that Satmincola californiensis causes anemic condi-tions in juvenile sockeye salmon, indicated by a reduction in red c e l l s , hemoglobin concentration and hematocrit values. This condition may be attributed to hemodilution r e s u l t i n g from damage to the g i l l s and the skin caused by this parasite and possibly also by metabolic excretions of the parasite, d i r e c t damage to the blood c e l l s or interference with the function of the hemopoietic tissue. The c r i t i c a l period of i n f e c t i o n , indicated by a sharp decrease in red blood c e l l numbers (Figure 17), hemoglobin concentration (Figure 13) and hematocrit values (Figure 14), coincided with the period during which the copepod reached maximum growth and with the appearance of a secondary gener-ation of parasites. Before discussing the response of the white blood c e l l s to p a r a s i t i -zation of the f i s h by Satmincola, i t i s appropriate to discuss and compare the information on white .blood c e l l s which appears in the l i t e r a t u r e with the results of this experiment. Many contradictory statements about white blood c e l l s have appeared in l i t e r a t u r e . Nonetheless, as a r e s u l t of more recent developments, i n -creased attention has been given to the use of hematology as an indicator of the effects of the environment on f i s h health and, consequently, there have been many advances in hematological techniques. Both terminology and hence discussions about white blood c e l l s have become less contradictory. It i s now recognized that lymphocytes and thrombocytes are d i s s i m i l a r in mrophology and f u l f i l l quite d i f f e r e n t functions (Ferguson, 1976). Thrombo-cytes, in both mature and immature forms, were d i f f e r e n t i a b l e i n the present 156 experiment, although the immature thrombocyte, which was often encountered, could not be d i f f e r e n t i a t e d from the small lymphocyte described i n the l i t e r a t u r e . While many workers denied that monocytes existed in f i s h blood (Jakowska, 1956; McKnight, 1966; Wienreb and Wienreb, 1969; McLeay, 1970; Klontz, 1972; McCarthy et a l . , 1973) several others have reported them (Lehmann and Stiirenberg, 1975; E l l i s , 1976; Ferguson, 1976 and Lester and Budd, 1979). Using the descriptions provided i n these publications, mono-cytes were i d e n t i f i e d in the blood of juvenile sockeye salmon in this experiment. In the present study, macrophages were rarely found in the c i r c u -l a t i n g blood of the experimental f i s h . According to the d e f i n i t i o n of macrophage formulated by Van Furth et a l . (1972), this c e l l is mainly l o c a l i z e d within connective and other tissue habitats and i s not normally present in the c i r c u l a t i n g blood. However, many other workers have found this c e l l in the c i r c u l a t i n g blood of f i s h . Amongst granulocytes, neutrophils are the best described and have been observed in most f i s h examined. Gardner and Yevich (1969) claimed however, that neutrophils were absent from cyprinodonts. This observa-tion seems most unusual. This c e l l was found often in the c i r c u l a t i n g blood of salmon and the staining technique used i n th i s experiment c l e a r l y demonstrated the ch a r a c t e r i s t i c s of neutrophils as described in the l i t e -rature. Unfortunately, eosinophils and basophils are less well described, and I was unable to p o s i t i v e l y identify these c e l l s in sockeye salmon blood. Studies by Watson et a l . (1956), Ostroumova (1960), Lukina (1965), Davies and Haynes (1975) Lester and Desser (1975), and Lester and Daniels (1976) contradict one another in various ways. Lehmann and Stiirenberg (1975) 157 c l e a r l y demonstrated the presence of these two c e l l s i n rainbow trout using Papenheim st a i n . This stain was also used with the blood of sockeye salmon i n the present experiments, but the c e l l s could not be d i f f e r e n t i a t e d . The d i f f i c u l t y i n d i f f e r e n t i a t i n g between these two types of c e l l i s that the granules remain unstained. Lester and Daniels (1976) however, agreed with Duthie (1939), Drury (1951) and Catton (1951) that eosinophils are common in the blood and tissues of certain teleosts but were unable to i d e n t i f y them with certainty because the granules apparently vary in their a b i l i t y to take up the s t a i n . They noted that the e a r l i e r descriptions were proba-bly based on c e l l s found in the ti s s u e . It i s probable that these two types of c e l l s also exist i n the blood of sockeye salmon and that the f a i l u r e of the present experiment to detect them was due to th e i r i n a b i l i t y to take up the st a i n used. A number of c e l l s which were quite similar morphologically to those described in the l i t e r a t u r e were observed however, and these have been c l a s s i f i e d as "granulocyte c e l l s " and await futher, more refined d i f f e r e n t i a t i o n . It has been known for quite some time that the number of c i r c u l a t i n g white blood c e l l s can be affected by environmental and physiological f a c t o r s . Reduction in the number of c i r c u l a t i n g white blood c e l l s appears in response to an increase in 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, 1973). The same author found that environmental stress on f i s h i n 0 2 d e f i c i e n t conditions led to a decrease in lymphocyte numbers and Blaxhall (1972) noted an increase in eosinophils related to environmental stress. Because of such rapid response to environmental changes, white blood c e l l s have come to be used more often for evaluating the nature and depth of the effects of para-sites on f i s h . As yet, the mechanisms involved in the variations i n each type of c e l l have not been c l e a r l y explained. C e l l u l a r function, however, is l i k e l y to prove s i g n i f i c a n t in this regard. 158 Some theories about the functional role of lymphocytes which may be applicable to our understanding of th i s phenomenon have been established. The most widely accepted of these i s one proposed by Yoffey (1962), which views the lymphocyte as a multipotential hemopoietic stem c e l l which responds to immunological s t i m u l i . White (1963) also commented that the executive c e l l s in the immunological system are c i r c u l a t i n g lymphocytes. This p a r t i c -ular immunological function of the lymphocyte has been noted in various species of f i s h including salmon. Therefore an increase i n the number of lymphocytes in the blood of sockeye salmon infected with Salminaola must be attributed to the function they play in the immunological response. It would seem to be the case that when a lymphocyte contacts an antigen which i t recognizes, i t undergoes rapid change, producing a large number of daughter c e l l s which also respond to that antigen. Hence, an increase i n the number of lymphocytes caused by the parasite would indicate the uptake of metabolites from the parasite into the c i r c u l a t i n g system. Furthermore, Hines and Spira (1973) observed acquired immunity in carp infected with Ichthyophthir-Cus . Golovina (1976), however, found a decrease rather than an increase in the number of c i r c u l a t i n g lymphocytes as a re s u l t of the i n f e c -tion of carp with Daatylogyrus but she did not provide a clear explanation for this decrease. The increase in neutrophils r e s u l t i n g from Salmincola. i n f e c t i o n must be explained in terms of i n f i l t r a t i o n of the neutrophils to the i n j u r i -ous tissue. A number of researchers have observed t h i s inflammatory response in the course of studies of b a c t e r i a l , protozoan and copepod infections and even as a r e s u l t of tagging (Weinreb, 1959; Thorpe and Roberts, 1972; Joy and Jones, 1973; Hines and Spira, 1973; Lester and Daniels, 1976). The inflammatory response i n f i s h i s b a s i c a l l y similar to that in mammals a l -though i t is less intense and slow both in appearance and i n resolution 159 (Finn and Nielson, 1971). In addition to their role on the inflammatory response, neutrophils in humans are primarily associated with the i n i t i a l phagocytosis of microorganisms and other foreign materials. This function was reported in rainbow trout by Watson et a l . (1963) and Finn and Nielson (1971). A highly s i g n i f i c a n t increase in the number of neutrophils would l i k e l y indicate the presence of this l a t t e r function in addition to the inflammatory response. In humans there i s a long recognized association between eosinophils and p a r a s i t i c i n f e c t i o n . Unfortunately, because of the d i f f i c u l t i e s explained e a r l i e r , this c e l l could not be d i f f e r e n t i a t e d in the blood of the experi-mental f i s h in this experiment. Hence evaluation of the v a r i a t i o n of this c e l l (under the category of "granulocyte c e l l s " ) could not be v a l i d l y c arried out. The increase in the "granulocyte c e l l s " i n this experiment may be due not only to increase in the number of eosinophils; i t could also have re -sulted from increases in basophils and other types of c e l l s . Discussion cannot go beyond this point u n t i l further study can provide a clear description of the c e l l s in the "granulocyte c e l l s " series. Some of those who claimed eosinophils exist in f i s h blood have discussed the effects of the parasite on t h e i r number. Bullock (1963) noted that the increase in the number of eosinophils in brook and rainbow trout and in two species of catostomid was a response to i n f e c t i o n by Acanthocephalus. Golovina (1976) observed that the most c h a r a c t e r i s t i c response to Dactylogyms in carp was an increase in the eosinophils which appeared in the blood during necrosis of the g i l l t i ssue. The other obvious r e s u l t of the presence of Salmincola was a reduction in blood c l o t t i n g time in the infected f i s h . This phenomenon could be attributed to the increase in the number of thrombocytes in the c i r c u l a t i n g blood (Gardner and Yevich, 1969; Wardle, 1971). 160 Observation of the red and white blood c e l l s in this experiment indicated 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 confined to mechanical damage alone. It goes further to cause anemia and changes i n the white blood c e l l s . GENERAL DISCUSSION 162 The results of this study show that the parasite Salminoola californiensis not only causes mechanical damage to i t s f i s h host, but also that i t adversely affects the f i s h ' s health. Its effects can be d e b i l i -tating, and even l e t h a l . The presence of t h i s parasite can cause a dr a s t i c increase in the rate of mortality among infected f i s h i f the degree of inf e c t i o n i s high enough or when i t acts s y n e r g i s t i c a l l y with other environ-mental stresses. In order to provide a clear picture of the parasite's e f f e c t on f i s h health i n the presence of other environmental stresses, t h e i r interactions are c l a r i f i e d i n the accompanying flow chart (Figure 29). They are also discussed in d e t a i l below. F i r s t of a l l , i t should be kept in mind that the pathogenic e f f e c t s of the parasite are not confined to the g i l l s . In f r y , the parasite can be found on the.fins, the fin-base, the skin and the branchial region but the majority are in the fin-bases. In juveniles, they have a sim i l a r d i s t r i b -ution but are found mainly in the branchial region. As juvenile sockeye salmon were used in the experiment, the parasite-induced pathology i n the region of the g i l l s w i l l be emphasized. In l i g h t of the study by Kabata and Cousens (1977), a reasonable estimate of g i l l surface area destruction in the present experiment would be about 20%. With such extensive damage, i t i s obvious that gas exchange as well as other physiological functions must be disturbed. Like any other salmonid f i s h , sockeye salmon are active, therefore, tissue 0 2 consumption is much higher than in more sedentary species. Under normal circumstances, 163 f i s h can increase their 0 2 consumption by making use of a compensatory mechanism. When an increased amount of 0 2 i s required, f i s h can increase the flow of water over the g i l l s by increasing the rate and amplitude of opercular movement. When Satmincola is present, the opercular movement appears to be interfered with due to the size and the s i t e of in f e c t i o n . For example, the attachment of this parasite along the branchial rim can prevent the movement and/or complete closure of the operculum. This condi-tion would c e r t a i n l y a f f e c t the amount of water passing through the g i l l s . Also in severe in f e c t i o n with extensive g i l l surface destruction, the e p i -t h e l i a l c e l l s adjacent to the damage are equally non-functional in gas transfer. G i l l destruction diminishes the amount of water passing through the chamber (Hughes, 1964) and c e r t a i n l y , l i m i t s the amount of 0 2 absorbed. It also upsets osmoregulation since the s a l t balance i s affected by the amount of g i l l area a v a i l a b l e . F i r s t l y , damage to the g i l l or skin e p i t h e l i a leads to a leak of large molecular weight molecules e s p e c i a l l y proteins (Hunn, 1964). Secondly, i t may inte r f e r e with the production and/or normal functioning of the chloride secretory c e l l s , i n t e r f e r i n g with the transport of Na + and CI ions against the concentration gradient. The osmotic gra-dient which exists between blood and water, coupled with the improper functioning of the chloride secretory c e l l s , and extensive g i l l damage, can lead to a net uptake of water and a subsequent hemodilution. The anemic condition of the infected f i s h can be explained as a res u l t of hemodilution. However, there are other possible 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 s i g n i f i c a n t difference in Mean Corpuscular Volume was observed (as discussed in section V). The anemic condition of the infected f i s h could be due to the reduction of red c e l l numbers in the c i r c u l a t i n g blood. This may be attributed to a toxic secretion released 164 by the parasite. This substance may get into the c i r c u l a t i n g blood through the b u l l a , as metabolite exchange has previously been observed to occur v i a the b u l l a (Kabata and Cousens, 1977), or i t may be absorbed from the water through the g i l l e p i t h e l i a . This "toxin" might alternately affect the hemopoietic tissues and interfere with the normal production of erythrocytes. However, this i s u n l i k e l y as immature red c e l l s in the c i r c u l a t i n g blood were found to be s l i g h t l y , but not s i g n i f i c a n t l y increased. However, i t is possible that the progressive reduction of the red c e l l s in the c i r c u -l a t i n g blood stimulates the hemopoietic tissue to increase production r e -s u l t i n g in a raised l e v e l of immature red c e l l s . The widely accepted immunological function of lymphocytes (as discussed e a r l i e r i n section V) coupled with a s i g n i f i c a n t increase in th e i r numbers in infected f i s h , indicates the p o s s i b i l i t y of metabolite absorption into c i r c u l a t i n g blood. Salmincota has also been found to deleteriously a f f e c t the growth of the f i s h . This r e s u l t i s attributed, f i r s t l y , to a considerable amount of energy which must be expended to maintain homeostatis, while the amount of 0 2 uptake i s upset as the r e s u l t of the damage to the g i l l respiratory area (Hughes, 1964). Secondly, the infected f i s h i s u n l i k e l y to consume an adequate amount of food as food was more often observed l e f t at the bottom of tanks containing infected f i s h than those containing non-infected f i s h . Therefore, these infected f i s h must make use of their energy reserves and this normally results in the reduction of weight and growth of the infected f i s h . Under an environmental change such as an increase in water temper-ature, f i s h increase metabolic a c t i v i t y and require more 0 2 (Brett, 1964). High water temperature, however, l i m i t s the amount of dissolved 0 2. There-fore, in the case of extensive reduction of the respiratory surface due to 165 severe i n f e c t i o n by this parasite, i t i s l i k e l y that the required amount of 0 2 uptake may not be met. Fish parasitized with approximately 31 Salnrincola 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 normally increases with r i s i n g water temper-ature (Brett, 1958). In high 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 control temperature condition was found to be less than i n the non-infected f i s h . Therefore, the a b i l i t y of the infected f i s h to swim in water of high temperature and high v e l o c i t y must be much less than that of non-infected f i s h . However, the reduction of the g i l l surface area i s not the only factor l i m i t i n g the swimming a b i l i t y of the f i s h . The anemic con-d i t i o n caused by this parasite also reduces the maximum swimming speed. Jones (1971) demonstrated that i n an anemic condition, in which the hemato-c r i t value was reduced to half or one-third, a 40% reduction in maximum swimming speed of trout was observed. Osmoregulation r e s u l t i n g from the damage caused by this parasite may be one of the problems of f i s h during the course of migration. The results obtained i n the present experiment also indicate that the infected f i s h have less a b i l i t y to move from fresh water to sa l t water than do non-infected. In addition, infected f i s h avoid high s a l i n i t y . Physiological transformation of par a s i t i z e d f i s h for smolting was not measured in th i s experiment. However, Clarke and Blackburn (1977) have proposed a seaward challenge test 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 in future studies, this parameter i s considered for measuring the readiness of the infected f i s h to migrate, we w i l l be in a better position to answer the question as to whether or not this parasite prevents f i s h from migrating. In this study fungal infections were observed on .some of the infected f i s h . Secondary infections such as fungus or bacteria are 166 observed in most cases of external p a r a s i t i c conditions; these may lead to an increase in the severity of the effect of the parasite on the f i s h host. F i n a l l y , i t would be reasonable to conclude that Salmincola oatifovn-iensis a f f e c t s growth and blood c h a r a c t e r i s t i c s of the f i s h host. Severity of the effect i s increased with r e l a t i o n to: the l e v e l of i n f e c t i o n , environmental stresses, and possibly with the occurence of secondary i n f e c t i o n . 167 Figure 29 Host-parasite-environment relationships Conclusion of the results of this experiment. Doubled square indicates host-parasite relationships. Food consumpt. G r o w t h P a r a s i t i z e d f ish Fish b l o o d k — — — — — —I miUbol i ta Osmot ic s t r e s s e s M e c h a n i c a l d a m a g e F ish h e a l t h O . u p t a k e M o r t a l i t y S e c o n d a r y i n f e c t i o n T e m p e r a t u r e Sa l in i ty S w i m m i n g a b i l i t y •(Migration /*7 REFERENCES 170 Allanson, B.R. and R.G. Noble. 1964. The tolerance of Utopia mossambica (Peters) to high temperature. Trans. Amer. F i s h . Soc. 93:323-332. Baggerman, B. 1960. S a l i n i t y preference, thyroid a c t i v i t y and the seaward migration of four species of P a c i f i c Salmon Onaorhynchus. J . F i s h . Res. Bd. 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