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Effects of early rearing history on selected endocrine and immune functions in juvenile Pacific salmonids Salonius, Kira 1991

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EFFECTS OF EARLY REARING HISTORY ON SELECTED ENDOCRINE AND IMMUNE FUNCTIONS IN JUVENILE PACIFIC SALMONIDS by KIRA SALONIUS B.Sc, The University of New Brunswick, 1988 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Animal Science) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 1991 (c) Ki r a Salonius, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. (Signature) K i r a Salonius Department of Animal Science The University of British Columbia Vancouver, Canada Date October 10, 1991 DE-6 (2/88) ABSTRACT The e f f e c t s of d i f f e r e n t early rearing conditions on plasma C o r t i s o l concentration, immune function, hematological p r o f i l e , and disease resistance were examined i n populations of hatchery-reared and wild Chinook salmon (Oncorhynchus tshawytscha) and hatchery-reared, wild, and colonized coho salmon (0. kisutch). These features were examined during s m o l t i f i c a t i o n and a f t e r an acute s t r e s s . The e f f e c t of i n i t i a t i n g feeding with a wild-type di e t as compared to a commercially prepared d i e t was also examined i n chinook salmon f r y as one aspect of rearing history that i s d i f f e r e n t between f i s h reared i n the hatchery and those i n the wild. During s m o l t i f i c a t i o n and following periods of acute stress, wild chinook salmon and wild and colonized coho salmon had s i g n i f i c a n t l y higher concentrations of plasma C o r t i s o l . Hatchery-reared juveniles showed less s e n s i t i v i t y to stress and lower concentrations of plasma C o r t i s o l during the s m o l t i f i c a t i o n period and a f t e r an acute stress. Antibody producing c e l l (APC) number and disease resistance to Vibrio anguillarum were not s i g n i f i c a n t l y d i f f e r e n t between the hatchery and wild chinook salmon. These features were also s i m i l a r among the hatchery, wild and colonized coho salmon smolts, despite s i g n i f i c a n t l y higher leve l s of c i r c u l a t i n g C o r t i s o l i n the wild and colonized smolts. White blood c e l l to red blood c e l l (wbc/rbc) r a t i o s were s l i g h t l y higher i n wild f i s h than i n t h e i r hatchery-reared counterparts i n chinook and coho salmon juveniles. S i g n i f i c a n t elevations i n plasma C o r t i s o l concentration a f t e r an acute stress was s t i l l evident retained i n the wild and colonized coho salmon juveniles even a f t e r holding them for 6 months i n an a r t i f i c i a l rearing environment. Disease resistance i n the wild f i s h s i g n i f i c a n t l y decreased over that time. Following the 6-month rearing period, the i n i t i a l numbers of APC i n the wild f i s h were s i g n i f i c a n t l y higher than those i n t h e i r hatchery counterparts. This difference precludes the a b i l i t y to conclude that a cause-effect r e l a t i o n s h i p exists between a high C o r t i s o l response and decreased s p e c i f i c immune function. The use of a l i v e d i e t to i n i t i a t e feeding i n chinook salmon f r y compares favorably to feeding a commercial d i e t i n that the a c t i v i t y of lysozyme and wbc/rbc r a t i o s were higher i n th i s group. S p e c i f i c immune function was correlated with body weight while non-specific immune defense was not. There appear to be physiological differences between hatchery-reared, wild and colonized coho and chinook salmon. Rearing history may be a determinant i n the s u r v i v a l of hatchery-reared salmonids released into the natural environment. i v TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS X GENERAL INTRODUCTION 1 CHAPTER 1 C o r t i s o l and immune function i n chinook and salmon smolts 4 Introduction 5 Materials and Methods 11 Results 19 Discussion 34 CHAPTER 2 C o r t i s o l and s p e c i f i c immune responses to stress i n juvenile chinook and coho salmon 39 Introduction 4 0 Materials and Methods 42 Results 48 Discussion 60 CHAPTER 3 Diet and immune function at f i r s t feeding i n chinook salmon f r y 64 Introduction 65 Materials and Methods 70 Results 74 Discussion 80 GENERAL CONCLUSIONS AND RECOMMEDATIONS 84 REFERENCES /APPENDICES v i LIST OF TABLES Table 1.1. Size of wild and colonized coho smolts and t h e i r hatchery counterparts p r i o r to release 13 Table 1.2. Size of wild chinook salmon and of t h e i r hatchery counterparts p r i o r to release 13 Table 1.3. Size of wild and colonized coho salmon and t h e i r hatchery counterparts p r i o r to release 14 Table 2.1. Size of coho salmon juveniles a f t e r one month of rearing i n a common environment 45 Table 2.2. Size of chinook salmon juveniles a f t e r one month of rearing i n a common environment 45 Table 2.3. Size of coho salmon a f t e r 6 months rearing i n a common environment 45 LIST OF FIGURES Figure 1.1. Mean plasma C o r t i s o l concentrations i n May, 1990 of migrating wild chinook smolts and of t h e i r hatchery counterparts p r i o r to release 21 Figure 1.2. Mean plasma C o r t i s o l concentrations i n May, 1990 of migrating wild and colonized coho smolts and of t h e i r hatchery counterparts p r i o r to release 22 Figure 1.3. Mean plasma C o r t i s o l concentrations over the month of May, 1991 of migrating wild coho smolts and of t h e i r hatchery counterparts p r i o r to release 23 Figure 1.4. Mean hematocrit i n May, 1990 of migrating wild and colonized coho smolts and of t h e i r hatchery counterparts p r i o r to release 24 Figure 1.5. Mean hematocrit over the month of May, 1991 of migrating wild coho smolts and of t h e i r hatchery counterparts p r i o r to release 25 Figure 1.6. Mean hematocrit i n May, 1990 of migrating wild and chinook smolts and of t h e i r hatchery counterparts p r i o r to release 26 Figure 1.7. Mean lymphocyte, t o t a l leukocyte and thrombocyte r a t i o s i n May, 1990 of migrating wild chinook smolts and t h e i r hatchery counterparts p r i o r to release 27 Figure 1.8. Mean lymphocyte, t o t a l leukocyte and thrombocyte r a t i o s i n May, 1990 of migrating wild and colonized coho smolts and of t h e i r hatchery counterparts p r i o r to release 2 8 Figure 1.9. Mean lymphocyte, t o t a l leukocyte and thrombocyte r a t i o s i n May, 1991 of migrating wild coho smolts and of t h e i r hatchery counterparts p r i o r to release 2 9 Figure 1.10. Mean number of antibody producing c e l l s leukocytes i n May, 1991 of migrating wild smolts and t h e i r hatchery counterparts p r i o r to release 30 Figure 1.11. Mean number of receptor s i t e s for C o r t i s o l i n May, 1991 on leukocyte whole c e l l preparations of 7-10 anterior kidneys of migrating wild coho smolts and of t h e i r hatchery counterparts p r i o r to release 31 Figure 1.12. Total cumulative mortality (%) and time to death (d) for chinook salmon juveniles i n response to a challenge by bath immersion with Vibrio anguillarum 32 v i i i Figure 1.13. Total cumulative mortality (%) and time to death (d) for coho salmon juveniles i n response to a challenge by bath immersion with Vibrio anguillarum 33 Figure 2.1. Means + s.e. of plasma C o r t i s o l concentration of hatchery, wild and colonized coho salmon juveniles i n response to a 30-60 sec handling stress 49 Figure 2.2. Means + s.e. of hematocrit values of hatchery, wild and colonized coho salmon juveniles i n response to a 30-60 sec handling stress 50 Figure 2.3. Means + s.e. of the plasma C o r t i s o l concentration (ng/ml) of hatchery and wild chinook salmon juveniles i n response to a 30-60 sec handling stress 51 Figure 2.4. Means + s.e. of lymphocyte (top) and leukocyte (bottom) number per 10 3red blood c e l l s of hatchery and wild chinook salmon juveniles i n response to a 30-60 sec handling stress 52 Figure 2.5. Means + s.e. of hematocrit values of hatchery and wild chinook salmon juveniles i n response to a 30-60 sec handling stress 53 Figure 2.6. Means + s.e. of the plasma C o r t i s o l concentration of hatchery, wild and colonized chinook salmon juveniles i n response to a 30-60 sec handling stress a f t e r 6 months i n a common rearing environment 54 Figure 2.7. Means + s.e. of hematocrit values of hatchery, wild and colonized coho salmon juveniles i n response to a 30-60 sec handling stress a f t e r 6 months rearing i n a common environment 56 Figure 2.8. Means + s.e. of leukocyte number per 10^ red blood c e l l s of hatchery, wild and colonized coho salmon juveniles i n response to a 30-60 sec handling stress a f t e r 6 months rearing i n a common environment 57 Figure 2. 9. Means + s.e. of antibody producing c e l l (APC) number per 10°leukocytes i n the anterior kidney of hatchery, wild and colonized coho salmon juveniles i n response to a 3 0-60 sec handling stress a f t e r 6 months rearing i n a common environment 58 Figure 2.10. Cumulative mortality (%) and mean time to death (d) of hatchery, wild and colonized coho salmon juveniles i n response to a bath immersion challenge with V. anguillarum a f t e r 6 months rearing i n a common environment.... 59 i x Figure 3.1. Means + 1 s.e. of the number of antibody producing c e l l s (APC)/million viable leukocytes i n the head kidney of chinook salmon f r y fed eithe r l i v e or frozen k r i l l or a commercially prepared d i e t 75 Figure 3.2 Means + 1 s.e. of lysozyme a c t i v i t y (U) and concentration (jug/ml) i n the plasma of chinook salmon f r y fed e i t h e r l i v e or frozen k r i l l , or a commercially prepared d i e t 76 Figure 3.3 Hematological p r o f i l e of chinook salmon f r y fed eit h e r l i v e or frozen k r i l l or a commercially prepared d i e t (OMP) 77 Figure 3.4. a; Means + 1 s.e. of hematocrit and b; condition factor (CF.) of chinook salmon f r y fed either l i v e or frozen krill(£. pacifica), or a commercially prepared d i e t 7 8 X ACKNOWLEDGEMENTS I sincerely thank my supervisor, Dr. George Iwama for his guidance and consideration during the completion of t h i s degree and the writing of t h i s t h e s i s . I thank my committee members, e s p e c i a l l y Dr. Bruce Barton, for t h e i r time and e f f o r t s i n reviewing t h i s t h e s i s . The cooperation of Mr. Dave Huert, Mr. Eldon Stone, and Mr. Grant Ladoucer and other s t a f f at the DFO hatcheries during the f i e l d work that was a major part of t h i s study was very much appreciated. I thank Dr. J e f f Marliave and Mr. Don McKinlay for t h e i r collaboration i n the study that i s the t h i r d chapter of t h i s t h e s i s . I am deeply indebted to the graduate students and research assistants i n our laboratory; I benefited immeasurably from these associations. I thank my family for t h e i r support, e s p e c i a l l y my father, Peter Salonius for providing an admirable role model, and Stephen Hunter for providing a balance i n attitude that was as important. 1 General Introduction Intensive aquaculture i s used as a t o o l i n f i s h e r i e s management. The release of large numbers of intensively cultured juveniles to supplement wild f i s h stocks i s a common practice i n North America. Over the l a s t decade, s u r v i v a l rates (smolt to adult) of coho (Oncorhynchus kisutch) and chinook (O. tshawytscha) hatchery-reared salmon smolts have declined (Mclntyre, 1985). Survival of wild coho salmon i s reported to be 1.5 to 2 times higher than i n t h e i r hatchery counterparts (Felton et a l . , 1990). A s i m i l a r comparison involving chinook salmon on the Lewis River, Washington, (a t r i b u t a r y of the Columbia River) reported that wild f i s h had average su r v i v a l rates 2.6 times higher those of hatchery f i s h (Felton et a l . , 1990). These higher s u r v i v a l rates imply that differences between hatchery-reared and wild f i s h may s i g n i f i c a n t l y a f f e c t the su r v i v a l of hatchery f i s h i n the wild. Differences between wild and hatchery f i s h have been observed with respect to t h e i r morphology (Hjort and Schreck, 1982), behavior (Smith, 1987), and physiology (see Iwama et a l . , 1990, Shrimpton et a l . , 1990, Woodward and Strange, 1987). There may be a physiological advantage to rearing f i s h i n the wild environment. Reisenbichler (1988) found that stocks of salmon transferred into environments other than those i n which they were l o c a l l y adapted performed poorly i n comparison to indigenous populations and that wild f i s h from d i f f e r e n t r i v e r systems were more s i m i l a r to each other than a wild stock and i t s hatchery 2 counterpart. A f i s h that i s reared i n the wild may be phy s i o l o g i c a l l y adapted to survive those conditions, and, thus, p r e f e r e n t i a l l y suited to t h i s environment over others, including that of the hatchery. A series of experiments were conducted to t e s t the hypothesis that rearing conditions i n hatcheries could a f f e c t the surv i v a l of hatchery salmon smolts at the time of seawater entry. The e f f e c t s of rearing history on C o r t i s o l release, immune function and on disease resistance were examined i n hatchery-reared and wild chinook salmon and i n hatchery-reared, wild and colonized coho salmon. The f i r s t set of experiments, described i n Chapter 1, was a series of f i e l d experiments designed to describe the physiological state of salmonid smolts reared under d i f f e r e n t conditions just before entry into seawater. Endogenous C o r t i s o l concentration, immune function, and disease resistance were examined. The second set of experiments, described i n Chapter 2, examined the e f f e c t of stress-induced increase i n plasma C o r t i s o l concentration on immune function and disease resistance i n f i s h with d i f f e r e n t rearing h i s t o r i e s . The consistency of these responses following the transfer into and subsequent rearing of wild, colonized, and hatchery f i s h i n common environmental conditions was also examined. The t h i r d set of experiments, described i n Chapter 3, was designed to examine food type ( l i v e or prepared) as one aspect of intensive culture d i f f e r e n t from natural rearing that may influence survivorship. The possible e f f e c t s of feeding a l i v e d i e t as opposed to a 3 commercially prepared d i e t to chinook salmon f r y were studied. By undertaking these experiments, the hypothesis that physiological differences between salmon reared i n the natural environment and t h e i r hatchery-reared counterparts may be influence the sur v i v a l of hatchery releases i n the wild was tested. Several objectives were intended; these were: 1) to determine the e f f e c t of early rearing history on endogenous C o r t i s o l concentration, immune function and disease resistance i n salmonid juveniles at the time of seawater entry; 2) to examine the C o r t i s o l stress response and i t s e f f e c t on s p e c i f i c immune function and disease resistance i n these f i s h ; and 3) to examine the e f f e c t of d i f f e r e n t d i e t types on s p e c i f i c and non-s p e c i f i c immune defense i n salmon f r y . 4 Chapter 1 Effects of early rearing h i s t o r y on the C o r t i s o l response and s p e c i f i c immune function i n chinook and coho salmon smolts. 5 Introduction Rearing history i s believed to play a major part i n determining ocean s u r v i v a l of stocked hatchery salmon. Fagerlund et a l . (1983) found that return rates of adult coho salmon were inversely proportional to t h e i r hatchery rearing density. Furthermore, Hosmer (1979) found that A t l a n t i c salmon (Salmo salar) reared i n water with a reduced flow rate had s i g n i f i c a n t l y reduced rates of returning adults. The f i s h reared i n Salmonid Enhancement Program (SEP) hatcheries are assumed to be g e n e t i c a l l y s i m i l a r to t h e i r wild counterparts because of the p o l i c y of SEP hatcheries to randomly se l e c t brood stock from returning adults. Any physiological differences observed are thought to be due to the divergence of physical conditions i n the a r t i f i c i a l rearing environment as compared to the natural environment. For example, differences i n conditions such as the temperature of the rearing water, dissolved oxygen concentrations, or rearing densities, or the presence of suspended s o l i d s may be important (see Appendix A for examples of conditions i n SEP hatcheries). Pickering (1989) suggested that conditions, such as those l i s t e d above, and those r e s u l t i n g from human perturbations r a r e l y occur i n a natural environment, and can cause a condition of chronic stress within a hatchery population. In the wild, variable conditions are the r u l e . Warren (1985) suggested that smolt qu a l i t y must be defined as conformance to requirements. In that regard, the character of f i s h desired then depends on the 6 objective of the a r t i f i c i a l propagation program. If one wishes to produce smolts that have high rates of s u r v i v a l i n the natural environment, they should resemble those wild f i s h that presumably are more successful at doing so. In physiological comparisons and performance t e s t s , the e f f e c t of p r i o r rearing on c e r t a i n parameters w i l l be affected by the present environment i n which the f i s h are held (Schreck, 1981). Thus, the study of the physiology of wild f i s h w i l l be affected by holding them i n an a r t i f i c i a l environment. In the following f i e l d experiments comparing naturally-reared and hatchery-reared juvenile salmon, the d i f f e r e n t environmental rearing conditions may be considered as the treatments. Performance t e s t s , such as a disease challenge, may be used to perturb the equilibrium state i n order to discern possible differences i n physiological states. In the spring of 1990, physiological differences i n c o r t i c o s t e r o i d concentrations and immune function were examined i n smolting populations of chinook and coho salmon reared i n d i f f e r e n t rearing environments. Physiological differences were once again examined i n 1991 i n the same coho salmon population. The three rearing h i s t o r i e s examined were wild rearing, colonized rearing, and hatchery rearing. Wild smolts were f i s h that developed from eggs spawned naturally i n the r i v e r . Hatchery smolts were f i s h a r t i f i c i a l l y propagated from eggs of returning adults i n the r i v e r and maintained for the entire rearing period i n a hatchery before smolt migration. Colonized (or outplanted) f i s h were of hatchery o r i g i n but were transported and released i n 7 the upper watershed of the r i v e r as f r y . At the time of smolt migration, colonized smolts w i l l have spent the larger proportion of t h e i r early rearing history i n the natural environment. Colonization occurred i n an area of the upper watershed where the outplanted f i s h were phy s i c a l l y separated from the wild f i s h to minimize competition between these groups. The management practice of colonization was undertaken within the coho salmon population, affording the opportunity to examine f i s h with three d i f f e r e n t rearing h i s t o r i e s i n 1990. In 1991 however, comparisons were made only between wild and hatchery coho smolts as very few colonized f i s h were obtained during the month of sampling. No colonization was undertaken by hatchery management i n the chinook salmon population under study. Physiological differences between wild and hatchery salmon may be the cause of d i f f e r i n g r e l a t i v e success rates of hatchery-reared and naturally-reared salmon to survive i n the wild. One aspect that a f f e c t s s u r v i v a l rates of f i s h i s disease. The a b i l i t y to generate an immune response d i r e c t l y a f f e c t s the health of the f i s h , and hence i s c l o s e l y related to t h e i r a b i l i t y to r e s i s t disease. Fish have been shown to be more susceptible to disease during periods of adverse environmental conditions and at the time of s m o l t i f i c a t i o n (Wedemeyer et a l . , 1984). Certain hormonal changes, such as those that occur during the physiological process of s m o l t i f i c a t i o n , have been shown to a f f e c t the immune system, and cause changes to haematopoietic tissues and c e l l s ( E l l i s , 1981). During the period leading up to seawater entry, there i s 8 an increase i n the concentration of endogenous co r t i c o s t e r o i d s i n the plasma of juvenile salmonids (Barton et a l . 1985; Young, 1989), a f f e c t i n g physiological changes such as increased Na + and C l ~ secretion, water permeability and ATPase ac t i v a t i o n on the g i l l (for review see Folmar and Dickoff, 1980). The primary c o r t i c o s t e r o i d of t e l e o s t f i s h i s C o r t i s o l (Barton and Iwama, 1991; Patino et a l . , 1987). During the period of s m o l t i f i c a t i o n , i n t e r r e n a l c e l l s producing C o r t i s o l i n the anterior kidney undergo hypertrophy and increase the secretion of t h i s hormone (Young, 1986). Increased secretion of C o r t i s o l i s also associated with the response to stress (Patino et a l , 1987). This elevation i n plasma C o r t i s o l has been shown to r e s u l t i n various types of immunosuppressive e f f e c t s i n tel e o s t s (McLeay, 1975; Ellsaesser and Clem, 1986; Tripp et a l . , 1987, Maule et a l . , 1987) . I t i s generally considered that endocrine receptors are involved i n the integration of the endocrine and immune systems (Coffey and Djeu, 1986). The eff e c t s of cor t i c o s t e r o i d s on the c e l l s of the immune system and the target tissues involved i n the physiological changes of s m o l t i f i c a t i o n i n salmon may be mediated s i m i l a r l y by s p e c i f i c c o r t i c o s t e r o i d receptors of these c e l l s (Maule, 1989). During s m o l t i f i c a t i o n , changes i n the a f f i n i t y and number of these receptors on leukocytes i n the anterior kidney have been observed (Maule, 1989). The net e f f e c t of exposure to C o r t i s o l was an increase i n the number of these receptors on leukocytes and may be responsible for the induction of immunosuppression at t h i s physiological state i n development 9 (Maule, 1989). Differences i n changes affected by C o r t i s o l may depend not only on the magnitude of the measurable c i r c u l a t i n g C o r t i s o l response, but on the s e n s i t i v i t y of the target tissues to C o r t i s o l (Pickering et a l , 1989). There i s evidence that the regulation of C o r t i s o l secretion i s mediated by negative feedback of C o r t i s o l on the hypothalmic-p i t u i t a r y - i n t e r r e n a l axis (Fryer and Peter, 1977). Continued C o r t i s o l secretion from the interrenal c e l l s w i l l i n h i b i t further i n t e r r e n a l response (Donaldson et a l . , 1981). Exposure to chronic stress has been shown to cause a decrease i n immune function despite acclimation of the f i s h to the stressor. Fish that were chro n i c a l l y stressed had le v e l s of c i r c u l a t i n g C o r t i s o l that were lower than those of the unstressed f i s h , while suppression of immunological defense systems continued (Pickering and Pottinger, 1989) . The f i r s t objective i n t h i s chapter was to esta b l i s h the magnitude of plasma C o r t i s o l changes as i t relates to s m o l t i f i c a t i o n i n naturally-reared and hatchery-reared juvenile salmonids. The second objective was to assess immune function and disease resistance at the time of smoltif i c a t i o n , and to determine the re l a t i o n s h i p between endogenous C o r t i s o l concentration and immune function at that time. The hypothesis tested was that enhanced in t e r r e n a l a c t i v i t y over the period of early rearing i n the hatchery environment ef f e c t s changes to interrenal tissue and t h e i r function, r e s u l t i n g i n a down regulation of these systems. This process may reduce the capacity of hatchery salmon i n a vari e t y of ways 10 to respond to challenges they must overcome i n order to survive, and, thus, be a p o t e n t i a l l y major determinant of s u r v i v a l at the time of seawater entry. 11 Materials and Methods Fish On May 16, 1990, juvenile wild chinook salmon smolts were netted from the Big Qualicum River during t h e i r nocturnal migration as they passed through open gates i n a f i s h counting fence. Colonized f i s h were distinguished from wild f i s h on the basis of s i z e . The following day, t h e i r hatchery counterparts were randomly sampled from the concrete raceways of the Big Qualicum River f i s h hatchery, Qualicum Beach, B r i t i s h Columbia (Department of Fisheries and Oceans, Canada). On May 26, 1990 and on May 10, 19, and 25, 1991, juvenile wild and colonized, coho salmon smolts and t h e i r hatchery counterparts were sampled as described above, from the Quinsam River and Quinsam River f i s h hatchery, near Campbell River, B r i t i s h Columbia. In 1990, approximately 1000 wild and hatchery chinook salmon and 1000 wild, colonized and hatchery coho salmon were brought to the Aquaculture Unit of the Department of Animal Science at the University of B r i t i s h Columbia. Wild, colonized and hatchery f i s h were held separately i n 1000-L oval tanks continuously supplied with dechlorinated Vancouver tap water (7-8°C, pH 6.1, t o t a l hardness 4.2 mg/L, [0 2] 10 ppm). Fish were fed a maintenance d i e t of EWOS grower p e l l e t s (EWOS Canada, Surrey^ B.C.) at 1 to 2 % body weight d a i l y . Wild f i s h were i n i t i a l y fed on freeze dried k r i l l (Argent Laboratories, 12 Richmond, B.C.) and gradually introduced to p e l l e t s over a period of two weeks. In July, 1990 these f i s h were subjected to disease challenge experiments. Experimental procedures and sampling Fish were ra p i d l y netted from t h e i r respective environments (naturally-reared f i s h from the r i v e r , hatchery-reared f i s h from t h e i r rearing channels) and transferred to a bucket containing a l e t h a l dose (200 mg/1) of buffered t r i c a i n e methanesulfonate (MS222). This dose has been shown to i n h i b i t a further r i s e i n plasma C o r t i s o l concentration i n salmon a f t e r anesthetization (Barton et a l . , 1986). After the f i s h were anesthetized, the caudal peduncle was severed and the blood was c o l l e c t e d i n heparinized c a p i l l a r y tubes. The plasma was separated by centrifugation within 15 min of c o l l e c t i o n and stored at -20°C for determination of plasma C o r t i s o l concentration. Fork length (Ln) and weight (W) were measured and used to calculate condition factor(K) using the following formula K=[W(g)/Ln3(cm) X 100] (Pickering and Duston, 1983). The basic body morphology of f i s h used i s presented i n Tables 1, 2 and 3. 13 Table 1.1. 1990. Mean ± S.E. of weight, length and condition factor of migrating wild and colonized coho salmon smolts and of t h e i r hatchery counterparts p r i o r to release, n=32. Group Weight Length condition factor (g) (cm) Hatchery 24.1 ± 1.0a 13.7 ± 0.2a 0.93 ± 0.01a Wild 8.7 + 0.5b 9 . 4 ± 0 . 2 b 0.95 + 0.02a Colonized 17.5 + 0.8C 12.5+0.2° 0.91 + 0.02a note: Values with superscripted l e t t e r s i n common are not s i g n i f i c a n t l y d i f f e r e n t between groups for each variable (p>0.05). Table 1.2. 1990. Mean + S.E. of weight, length and condition factor of migrating wild chinook salmon smolts and of t h e i r hatchery counterparts p r i o r to release, n=32. Group Weight Length condition factor (g) (cm) Hatchery 6.1 + 0.2a 8.3 + 0.1a 1.08 ± 0.01a Wild 3.2 ± 0.1b 6.8 ± 0.1b 1.01 ± 0.06a note: Values with superscripted l e t t e r s i n common are not s i g n i f i c a n t l y d i f f e r e n t between groups for each variable (p>0.05). 14 Table 1.3. 1991. Mean ± S.E. of weight, length and condition factor of migrating wild and colonized coho salmon smolts and of t h e i r hatchery counterparts p r i o r to release n=24, except for the colonized group (n=6). Group Weight Length condition factor (g) (cm) (W/Ln3X100) Hatchery May 10 May 19 May 25 21.9 ± 1.4a 23.3 + 1.6a 26.0 + 1.7a 13.1 ± 0.2a 13.3 + 0.2a 13.8 + 0.3a 0.95 0.98 0.96 +1 +1 + 0.01a 0.01a 0.01a Wild May 10 May 19 May 25 14.1 ± 0.6b 10.6 + 0.6b 9.3 + 0.6b 11.4 + 0.2b 10.3 + 0.2b 9.8 + 0.2b 0.94 0.97 0.98 + + + 0.01a 0.02a 0.01a Colonized* May 10 20.8 + 1.7a 13.0 + 3.0a 0.94 + 0.02a (n=6) note: Values with superscripted l e t t e r s i n common are not s i g n i f i c a n t l y d i f f e r e n t between groups for each variable (p>0.05). * In 1991, i n s u f f i c e n t numbers of colonized f i s h were netted from the r i v e r to provide adequate samples for further experimentation. 15 Plasma C o r t i s o l determination Plasma C o r t i s o l was determined by radioimmunoassay [ 1 2 5 I ] (Baxter Healthcare Corporation, Cambridge Massachusetts. C l i n i c a l Assay No. 529) based on competitive binding p r i n c i p l e s (Foster and Dunn, 1974). Hematology Hematocrit values were determined as percent packed c e l l volume a f t e r centrifugation for 5 minutes at 11,500 revolutions per minute (RPM); Model MB microhematocrit centrifuge, International Equipment Co., Needham Heights, Ma., U.S.A.). The r a t i o of t o t a l leukocyte, lymphocyte, and thrombocyte to 1000 erythrocytes was manually determined from Wright-Giemsa stained blood smears by counting erythrocytes (average 637 per s l i d e , range 475 to 802), lymphocytes and other leukocytes i n 9 random f i e l d s per s l i d e ( i . e . per f i s h ) using Yasutake and Wales (1983) as a reference. Assay of s p e c i f i c immune response in vitro To assess the a b i l i t y of lymphocytes to generate s p e c i f i c antibodies, we established c e l l cultures of leukocytes as described by Tripp et a l . (1987) and Maule et a l . (1989). Duplicate 50 / i l samples of anterior kidney tiss u e homogenate containing 2 X 107 leukocytes/ml were incubated for 7 d i n 96 16 well m i c r o t i t r e plates with 50 fil tissue culture media (TCM) containing the antigen trinitrophenalated (TNP)-lipopolysaccharide (LPS) or TCM only (negative c o n t r o l s ) . See Appendix B f o r recipes and a detailed protocol for t h i s assay, and Appendix C for the conjugation technique of TNP to LPS. When immunized against TNP-LPS, the lymphocytes secreting anti-TNP antibody can be detected by the Cunningham plaque assay (Cunningham and Szenberg, 1968). At the time of the plaque assay , a 50 fj.1 aliquot of the c e l l culture suspension i n each well was mixed with 10 jul coho salmon serum complement and 10 jzl of TNP-tagged sheep red blood c e l l s (SRBC) and put i n sealed Cunningham chambers at 17 °C for 2h. The antigen-antibody complexes activate the complement cascade and lyse the SRBC membranes, causing the appearance of holes i n the lawn of TNP-tagged SRBC, each hole corresponding to an antibody producing c e l l (APC). The number of viable leukocytes at the time of the assay was determined using a hemacytometer and trypan blue dye exclusion. Results were expressed as number of APC per m i l l i o n viable leukocytes, (see Appendix B for d e t a i l s ) . C o r t i c o s t e r o i d receptor assay This assay i s based on competitive binding of a saturable, high a f f i n i t y , low capacity binding of r a d i o - l a b e l l e d [ 3H] triamcinolone acetonide (TA), a synthetic C o r t i s o l , and cold TA. The whole leukocyte binding study was c a r r i e d out according to the procedure of Maule and Schreck (1990). Tissues were 17 harvested from the anterior kidney as previously described (Tripp et a l . , 1987). Tissue homogenates were obtained from up to 10 coho salmon smolts per sample i n order to obtain enough c e l l s to carry out the assay. The f i n a l concentration of leukocytes i n a suspension of TCM (Appendix B) was 3.0-4.6 X 10 7 leukocytes/ml. To determine the number of high a f f i n i t y C o r t i s o l receptors, samples were incubated i n 3H TA or 100-fold excess cold TA and r a d i o - l a b e l l e d TA. Total binding was determined by combining 0.1 ml of the leukocyte suspension, and 0.05 ml TCM, and 0.05 ml 3H TA dissolved i n TCM and incubating for 2 h. Non-specific binding was determined by incubating 0.01 ml of the leukocyte suspension i n 0.05 ml excess cold TA for 1 h and then with 3H TA for an additional 2 h. S p e c i f i c binding was determined by subtracting non-specific binding from the t o t a l binding. The f i n a l incubation volume was 0.2 ml and contained 4-10 X 10 6 c e l l s and 0.5-2.0 nM 3H-TA. Samples were counted on a LKB rack beta s c i n t i l l a t i o n counter, model 1214. A computer software program (McPherson, 1985} was used to analyze the binding data to obtain the maximum number of s i t e s (W m a x) per leukocyte based on Scatchard plo t analyses (Scatchard, 1949). Disease challenge t e s t Duplicate treatment groups of f i s h were exposed to Vibrio anguillarum ( Strain RI, T.P.T. Evelyn, P a c i f i c B i o l o g i c a l Stn., Nanaimo, B.C.) by the bath immersion method (Gould et a l . , 1979) at concentrations of 1 x 10 7 colony forming units (cfu)/ml for 18 the chinook challenge (mean water temperature, 8 C) and 5 x 10° cfu/ml for the coho challenge (mean water temperature, 8°C). Control groups were treated s i m i l a r l y , but without the addition of V. anguillarum to the immersion bath. At the time of the challenge, the c e l l mass of V. anguillarum i n culture was estimated by i t s o p t i c a l density at 540 nm. A quantitative estimation of the concentration of bacteria i n solution was determined by plate counts (Ramey, 1985). M o r t a l i t i e s were removed d a i l y and deaths due to the pathogen were confirmed by i s o l a t i o n of V. anguillarum from the kidney on tirypticase soy agar (TSA) plates (4% TSA (Difco), 0.5% NaCl). Data were expressed as percent t o t a l mortality and mean time to death. Data analysis Data were subjected to analysis of variance and where s i g n i f i c a n t differences were found, the Tukey t e s t (Steel and Torr i e , 1990) was used to i d e n t i f y s i g n i f i c a n t l y d i f f e r e n t treatment means. C o r t i s o l receptor values were compared with a Student's t t e s t (Duncan et a l . , 1983). These analyses were performed with the SYSTAT s t a t i s t i c a l program (Wilkinson, 1988). As the mortality i n the duplicate treatments of the disease challenges was not s i g n i f i c a n t l y d i f f e r e n t within groups, the re s u l t s were pooled and then analyzed using contingency tables (chi-square analysis) to t e s t for independence of the means (Duncan et a l . , 1983). S t a t i s t i c a l s i g n i f i c a n c e was taken at the 5 % l e v e l i n a l l t e s t s . 19 Results In 1990, migrating wild chinook salmon smolts had s i g n i f i c a n t l y higher plasma C o r t i s o l concentrations than t h e i r hatchery counterparts (Fig. 1.1). Migrating wild and colonized coho smolts had higher plasma C o r t i s o l concentrations than t h e i r hatchery counterparts but these differences were not s i g n i f i c a n t (Fig. 1.2). In 1991, the elevation i n concentration of plasma C o r t i s o l i n the hatchery coho smolts was transient, and by May 19 and 25, concentrations were s i g n i f i c a n t l y lower than that of the wild smolts (Fig. 1.3). Hematocrit values were s i g n i f i c a n t l y lower i n hatchery-reared coho salmon smolts than the naturally-reared 1990 coho salmon smolts (Fig. 1.4). In 1991, the hatchery reared coho smolts had s i g n i f i c a n t l y lower hematocrit values by May 25, the l a s t sampling period (Fig. 1.5) when C o r t i s o l concentrations were also lowered (Fig. 1.3). Hematocrit values were not s i g n i f i c a n t l y d i f f e r e n t between 1990 wild and hatchery chinook smolts (Fig. 1.6) . Ratios of lymphocyte and t o t a l leukocytes were higher i n the wild chinook smolts than i n t h e i r hatchery counterparts but these differences were not s i g n i f i c a n t (Fig. 1.7). This trend of higher lymphocyte and t o t a l leukocytes r a t i o s i n wild f i s h was consistent i n the 1990 coho smolts, but the colonized smolts had the lowest r a t i o s of a l l coho groups (Fig. 1.8). In 1991, the trend of higher lymphocyte and t o t a l leukocyte r a t i o s i n wild f i s h was again consistent with those of the 1990 coho and chinook 20 smolts (Fig. 1.9). In 1990, hatchery coho smolts had s i g n i f i c a n t l y higher thrombocyte r a t i o s than i n the wild or colonized smolts (Fig. 1.8). This feature was not observed i n the following year, or i n the chinook smolts. In 1991, tissues were taken to obtain a measure of s p e c i f i c immune response and C o r t i s o l receptor s i t e number of anterior kidney leukocytes. Antibody producing c e l l (APC) number was not s i g n i f i c a n t l y d i f f e r e n t between wild and hatchery coho smolts, although numbers were consistently higher i n the anterior kidney of the wild smolts (Fig. 1.10). The number of receptor s i t e s ( N m a x ) for C o r t i s o l on leukocytes i n wild smolts was almost two times that found i n the hatchery smolts (Fig. 1.11). Salmon stocks brought into a common environment at U.B.C. for one month showed no difference i n t o t a l mortality i n response to the disease challenge with V. anguillarum between the wild and hatchery chinook smolts (Fig. 1.12) or the wild, colonized or hatchery coho smolts (Fig. 1.13). This challenge was not used in 1991 because no d i s c e r n i b l e differences i n s u r v i v a l were seen between hatchery-reared and naturally-reared smolts of either stock, and l a t e r challenges with the coho juveniles indicated that disease resistance i n the wild and colonized f i s h were adversely affected by the rearing environment (Figure 2.7). 21 140.0 j ^ 130.0-120.0-\ 110.0-^ 100.0-^ 90.0-80.0-70.0-60.0-50.0-40.0-30.0-20.0-10.0-0.0--o cn o o o £ CO _o Q. Hatchery Wild Figure 1.1. Mean plasma C o r t i s o l concentrations i n May, 1990 of migrating wild chinook smolts and of t h e i r hatchery counterparts p r i o r to release, ± 1 s.e. Values with d i f f e r e n t superscripts are s i g n i f i c a n t l y d i f f e r e n t , n=32. 22 a E 70.0 j 65.0-60.0 -j-55.0--cn 50.0---S 45.0 o o u o E _D C L 40.0 35.0 + 30.0 25.0-20.0-15.0-10.0 5.0 T 0.0 Hatchery Wild Colonized Figure 1.2. Mean plasma C o r t i s o l concentrations i n May, 1990 of migrating wild and colonized coho smolts and of t h e i r hatchery counterparts p r i o r to release, ± 1 s.e. Values with d i f f e r e n t superscripts are s i g n i f i c a n t l y d i f f e r e n t , n=32. 23 cn c o CO o o co Q_ 140.0 j 130.0--120.0-1 10.0-100.0-90.0-80.0-70.0-60.0-50.0-40.0-30.0-20.0-10.0-0.0 :-K\N hatchery I—I wild May 10 May 19 May 25 Figure 1.3. Mean plasma C o r t i s o l concentrations over the month of May, 1991 of migrating wild coho smolts and of t h e i r hatchery counterparts p r i o r to release, + 1 s.e. Values with d i f f e r e n t superscripts are s i g n i f i c a n t l y d i f f e r e n t , n=10. 24 Figure 1.4. Mean hematocrit (% packed c e l l volume) i n May, 1990 of migrating wild and colonized coho smolts and of t h e i r hatchery counterparts p r i o r to release, + 1 s.e. Values with d i f f e r e n t superscripts are s i g n i f i c a n t l y d i f f e r e n t , n=32. 25 Figure 1.5. Mean hematocrit (% packed c e l l volume) over the month of May, 1991 of migrating wild coho smolts and of t h e i r hatchery counterparts p r i o r to release, + 1 s.e. Values with d i f f e r e n t superscripts are s i g n i f i c a n t l y d i f f e r e n t , n=24. 26 Figure 1.6. Mean hematocrit (% packed c e l l volume) i n May, 1990 of migrating wild and chinook smolts and of t h e i r hatchery counterparts p r i o r to release, + 1 s.e. Values with d i f f e r e n t superscripts are s i g n i f i c a n t l y d i f f e r e n t , n=3 2. i 27 Figure 1.7. Mean lymphocyte, t o t a l leukocyte and thrombocyte r a t i o s (no. c e l l s per 103 erythrocytes) i n May, 1990 of migrating wild chinook smolts and of t h e i r hatchery counterparts p r i o r to release, + 1 s.e. Values with d i f f e r e n t superscripts are s i g n i f i c a n t l y d i f f e r e n t , n=12. 28 ~o o -a o _o .a •o CO o o o CO E a c co o CO 40 35-30-25 20-15 10-0 a A . H W C H W C ZD lymphocyte ratio ZD leukocyte ratio v/>* thrombocyte ratio H W C Figure 1.8. Mean lymphocyte, t o t a l leukocyte and thrombocyte r a t i o s (no. c e l l s per 103 erythrocytes) i n May, 1990 of migrating wild and colonized coho smolts and of t h e i r hatchery counterparts p r i o r to release, ± 1 s.e. Values with d i f f e r e n t superscripts are s i g n i f i c a n t l y d i f f e r e n t , n=12. 29 Figure 1.9. Mean lymphocyte, t o t a l leukocyte and thrombocyte r a t i o s (no. c e l l s per 103 erythrocytes) i n May, 1991 of migrating wild coho smolts and of t h e i r hatchery counterparts p r i o r to release, + 1 s.e. Values with d i f f e r e n t superscripts are s i g n i f i c a n t l y d i f f e r e n t , n=12. 30 CO <D -+-> o o c o o CL < 120.0 j 1 l O . O -IOO.O-QO.O--80.0-70.0-60.0-50.0-40.0-30.0-20.ol-IO.0-o.o 1 ESS hatchery I 1 wild May 19 May 25 Figure 1.10. Mean number of antibody producing c e l l s (APC)/ 10° leukocytes in Hay, 1991 of migrating wild smolts and of t h e i r hatchery counterparts p r i o r to release, ± 1 s.e. Values with d i f f e r e n t superscripts are s i g n i f i c a n t l y d i f f e r e n t , n=12. 31 CD O CO CD -i~> 'co o Q. <U O O CO 3.0E4T 2.5E4 2.0E4 1.5E4 1.0E4 o 5000.0 0.0 Hatchery Wild Figure 1.11. Mean number of receptor s i t e s f o r C o r t i s o l i n May, 1991 on leukocyte whole c e l l preparations of 7-10 an t e r i o r kidneys of migrating wild coho smolts (n=2) and of t h e i r hatchery counterparts (n=3) p r i o r to release, + 1 s.e. Values with d i f f e r e n t superscripts are s i g n i f i c a n t l y d i f f e r e n t . 32 100-r 90-v—' 80--f ' 70-~o •+-> i _ 60-o E 50-CD 40-> _o 30-Z3 E 20-o 10-0* • •A .0 hatchery wild 4 5 6 7 8 9 1011 1 2 1 3 1 4 1 5 1 6 Time (days) Figure 1.12. Tot a l cumulative mortality (%) and time to death (d) f o r chinook salmon juveniles i n response to a challenge by bath immersion with 1 X 10 7 cfu/ml of V i b r i o anguillarum. Data represent means of pooled duplicate treatments, n=64. Values are not s i g n i f i c a n t l y d i f f e r e n t . 33 >> i d ~5 •+-> l . o E CD > _o ZJ E Z5 o 60-55- A 50- • 45- H 40-35-30-25-20-15-10-5-01 •A hatchery d wild H colonized A-A- 1 = 1 8 9 10 1112 13 14 15 16 (days) Figure 1.13. Total cumulative mortality (%) and time to death (d) f o r coho salmon juveniles i n response to a challenge by bath immersion with 5 X 10 6 cfu/ml of v i b r i o anguillarum. Data represent means of pooled duplicate treatments, n=64. Values are not s i g n i f i c a n t l y d i f f e r e n t . 34 Discussion The s i g n i f i c a n t differences i n plasma C o r t i s o l concentrations between both wild and colonized smolts and the hatchery smolts are too large to assume that t h i s change i s p r e c i p i t a t e d by the difference i n sampling time (wild and colonized f i s h migrated across the f i s h counting fence at night, necessitating the sampling at t h i s time and hatchery f i s h were sampled the following day to keep the sampling time of these f i s h as close as that of the wild f i s h ) . I t i s more l i k e l y that higher plasma C o r t i s o l concentrations are a t t r i b u t a b l e to physiological differences between the wild and colonized smolts and t h e i r hatchery-reared counterparts. Several possible explainations for differences seen i n the l e v e l s of plasma C o r t i s o l between wild and colonized smolts and t h e i r hatchery-reared counterparts can be ruled out. F i r s t , differences cannot be attributed to differences i n body weight of the f i s h . Other workers have found no c o r r e l a t i o n between i n d i v i d u a l f i s h weight, interrenal response, and plasma C o r t i s o l concentrations i n salmonid smolts (Barton et a l , 1985; Young, 1986; Mazur, 1991). Second, differences cannot be attributed to diurnal variations i n plasma C o r t i s o l concentrations. Thorpe et a l . (1986), found d i e l and seasonal changes i n plasma C o r t i s o l concentrations i n coho salmon. At c e r t a i n times of the year, they found nocturnal r e s t i n g plasma C o r t i s o l concentrations to be 2X higher than those measured during the day, but others have found l i t t l e diurnal 35 difference i n resting C o r t i s o l concentrations (Barton et a l . , 1986). Also, the seasonal elevation i n plasma C o r t i s o l during smoltif i c a t i o n has been shown to be 10-fold higher than those found at other times i n the year (Young, 1989), therefor t h i s would be more i n f l u e n t i a l i n causing plasma C o r t i s o l elevations. Third, differences cannot be attributed to higher plasma clearance rates of C o r t i s o l i n hatchery f i s h . Shrimpton (pers. comm., Dept. Zoology, U.B.C.) found no difference i n the metabolic clearance rates of C o r t i s o l i n the same wild and hatchery f i s h used i n t h i s study to explain the observed divergence of plasma C o r t i s o l concentrations between the wild and hatchery f i s h . The difference i n plasma C o r t i s o l concentration may be due to a decreased secretion of C o r t i s o l from interrenal c e l l s of hatchery-reared f i s h . If hatchery practices induce stress, sustained stimulus of the interrenal c e l l s i n early rearing associated with t h i s chronic stress may r e s u l t i n desensitization of the interrenal t i s s u e , or tissu e atrophy, mediated by the negative feedback i n h i b i t i o n of C o r t i s o l on these tissues ( E l l i s , 1981). This desensitization may also be affected by a reduced secretion of adrenocorticotrophic hormone from the p i t u i t a r y or C o r t i s o l releasing factor from the hypothalamus (Barton et a l . , 1987). The series of samples taken i n the spring of 1991 from the coho salmon smolts indicated that l e v e l s of C o r t i s o l i n the hatchery f i s h were highest on the f i r s t sampling date and were s i g n i f i c a n t l y lower than those of the wild and colonized f i s h on the l a s t two sampling periods. Others have found that when the 36 migration i n c l i n a t i o n or natural state of readiness for seawater entry i s ignored and f i s h are held back, they w i l l lose there migratory response and revert to a parr-type physiology (Folmar and Dickoff, 1981; Schreck, 1981). Although C o r t i s o l i s only one of many predictive indicators of the physiological state of s m o l t i f i c a t i o n , reversion may have occurred i n the hatchery-reared coho smolts. Wild smolts also had higher numbers of c o r t i c o s t e r o i d receptors on anterior kidney leukocytes than did the hatchery smolts. The level s of C o r t i s o l receptors were higher.than those previously reported for other salmonids (Maule and Schreck, 1990) and t h i s may have been because of the higher degree of non-s p e c i f i c binding i n crude whole c e l l preparations. Higher non-s p e c i f i c binding could have elevated the absolute number of receptors present (A. Maule, pers. comm.), but would not change the r e l a t i v e differences i n receptor numbers found between hatchery and wild f i s h . In coho salmon, the physiological changes associated with s m o l t i f i c a t i o n are thought to be mediated by increased C o r t i s o l receptor s i t e number on the target tissues (Maule, 1989). I t i s in t e r e s t i n g that both receptor s i t e number and C o r t i s o l concentrations were higher i n the wild coho smolts. There was no s i g n i f i c a n t difference found i n the number of APC i n the anterior kidney between wild and hatchery coho smolts. There appeared to be no rel a t i o n s h i p between increased C o r t i s o l concentration associated with the smolting response i n the wild f i s h and decreased APC numbers i n t h i s experiment. Levels of c i r c u l a t i n g t o t a l leukocytes were also not s i g n i f i c a n t l y 37 d i f f e r e n t between the wild f i s h and the hatchery f i s h and, i n fa c t , were s l i g h t l y higher i n the wild f i s h than those i n the hatchery f i s h , despite the higher concentration of c i r c u l a t i n g C o r t i s o l i n the wild f i s h . Maule and Schreck (1990), could not a t t r i b u t e the r e d i s t r i b u t i o n and number of leukocytes i n lymphoid tissues s o l e l y to the action of C o r t i s o l and indicated that other factors may be involved i n mediating immunological changes i n response to C o r t i s o l at s m o l t i f i c a t i o n and following acute stress. The mechanism involved i n causing immunosuppression may be d i f f e r e n t during the period of s m o l t i f i c a t i o n , or, a l t e r n a t i v e l y , wild f i s h may i n i t i a l l y have had higher numbers of these c e l l s i n c i r c u l a t i o n . C a s t i l l a s and Smith (1977) also found higher i n i t i a l l e v e l s of lymphocytes i n populations of wild trout than those found i n hatchery trout. In order to perform disease challenges, f i s h were brought into and held i n a common environment, necessitating the introduction of wild f i s h to a r t i f i c i a l conditions. Disease challenges showed no difference i n mortality between the wild and the hatchery f i s h . Wild f i s h may have been compromised i n an a r t i f i c i a l rearing environment and as such were predisposed to disease s i m i l a r to the hatchery f i s h . The problems of studying immune function during periods of s m o l t i f i c a t i o n are confounded by possible hormonal changes that accompany that process. I t appears that hormonal e f f e c t s could occur with d i f f e r i n g magnitude between wild and hatchery f i s h . There i s l i t t l e known about the i n t e r a c t i v e e f f e c t s of C o r t i s o l and other hormones that change during s m o l t i f i c a t i o n . I t i s 38 unclear i f increases i n plasma C o r t i s o l associated with the smolting process caused immunosuppression such as that which occurs i n response to stress. I t i s clear, however, that there are lower l e v e l s of c i r c u l a t i n g C o r t i s o l concentrations and C o r t i s o l receptor s i t e numbers i n hatchery-reared f i s h as compared to f i s h reared i n the wild. This may indicate a lower s e n s i t i v i t y of tissues to environmental cues r e s u l t i n g i n a lowered response to s t i m u l i i n hatchery-reared smolts. 39 Chapter 2: Ef f e c t of rearing history on the C o r t i s o l and s p e c i f i c immune responses to stress i n juvenile chinook and coho salmon. 40 Introduction Salmonid f i s h are sens i t i v e to handling and respond with a stress-associated secretion of C o r t i s o l ( Donaldson, 1981; Schreck, 1981; Barton and Iwama, 1991), the primary c o r t i c o s t e r o i d associated with stress i n teleosts (Patino et a l . , 1987). Elevation of blood C o r t i s o l has been shown to increase the s u s c e p t i b i l i t y of some salmonids to infectious diseases (Wedemeyer et a l . , 1984; Pickering and Pottinger, 1989) and there i s evidence to suggest that t h i s e f f e c t may be mediated by suppression of the immune system by C o r t i s o l (Maule et a l . , 1987; Tripp et a l . , 1987). I t has been shown that physiological l e v e l s of C o r t i s o l can cause a decrease i n the l e v e l s of c i r c u l a t i n g lymphocytes (McLeay, 1975) and that the a b i l i t y to generate antibody i s reduced as early as 4 h a f t e r stress (Maule et a l . , 1989). Others have compred the stress response of wild and hatchery f i s h (Woodward and Strange, 1987; Iwama et a l . , 1991) and found marked differences. The objective of t h i s experiment was to examine the e f f e c t of early rearing history ( i e . hatchery, wild, and colonized) on the magnitude of the stress response, as determined by changes i n plasma C o r t i s o l concentration, and the secondary e f f e c t s of C o r t i s o l on physiological features important to disease resistance i n salmon. A series of three experiments examined f i s h with d i f f e r e n t rearing h i s t o r i e s . Experiment 1 examined the magnitude of the stress response, determined by increase i n plasma C o r t i s o l 41 concentration of hatchery, wild and colonized juvenile coho salmon a f t e r 1 month i n a common environment. Experiment 2 was si m i l a r to Experiment 1 but examined changes i n hematological features i n hatchery and wild chinook salmon juveniles following the stress. Experiment 3 examined the magnitude of the C o r t i s o l response and i t s e f f e c t on hematological parameters and s p e c i f i c immunological function of hatchery, wild, and colonized coho salmon juveniles a f t e r 6 months of rearing i n a common environment. 42 Materials and Methods Fish In June, 1990, wild and colonized coho salmon f r y , trapped from the Quinsam r i v e r , and t h e i r hatchery counterparts were transported from the Quinsam River Fish Hatchery, B r i t i s h Columbia and kept i n 1000-L oval tanks continuously supplied with dechlorinated Vancouver tap water (pH 6.1, Total hardness 4.2 mg/L, [0 2] 10 ppm) at ambient temperatures ranging from 12°C i n August to 4°C i n December. Fish were fed a maintenance d i e t of EWOS grower p e l l e t s (EWOS Canada Ltd., Surrey, B r i t i s h Columbia) at 1 to 2 % body weight d a i l y . Wild f i s h were started on freeze dried k r i l l (Argent Laboratories, Richmond, B r i t i s h Columbia) and gradually introduced to p e l l e t s over a period of 2 weeks. In July, 1990 these f i s h were used i n Experiment 1. Also i n June, 1990 juvenile wild chinook salmon, trapped from the Big Qualicum r i v e r , and t h e i r hatchery counterparts were transported from the Big Qualicum River Fish Hatchery and held s i m i l a r l y to f i s h i n Experiment 1. After 1 month i n t h i s common environment, these f i s h were used i n Experiment 2. For Experiment 3, hatchery, wild, and colonized coho salmon juveniles were transferred to 200-L oval tanks with the same water source except that the temperature was maintained at a constant 9°C. Fish were allowed to acclimate to the new conditions for 2 weeks. The experiments were conducted i n February, 1991, af t e r 6 months i n a common environment. 43 Experimental procedures and sampling In each experiment the f i s h were acutely stressed by rap i d l y netting and holding them out of the water for 30-60 seconds and returned to t h e i r tanks. This approach has been demonstrated to evoke a large r i s e i n plasma C o r t i s o l within a r e l a t i v e l y short time i n salmonids and other fishes (see Barton and Iwama, 1991). Fish were sampled at 8 and 30 h following the handling stress i n Experiment 1. In Experiment 2, a t h i r d sampling period was added at 4 h following handling, i n case elevations i n plasma C o r t i s o l were being missed by sampling at 8 h post-stress. In Experiment 3, the primary objective of the experiment was to examine the e f f e c t of the r i s e i n plasma C o r t i s o l concentration on the immune function and to see i f the trend i n response was s i m i l a r i n the wild, hatchery, and colonized f i s h a f t e r 6 months. For that reason only a 4 h sample was taken. At the time of sampling, f i s h were rapidly netted and transferred to a bucket containing a l e t h a l dose (200 mg/1) of buffered t r i c a i n e methanesulfonate (MS222). This dose has been shown to i n h i b i t a further r i s e i n plasma C o r t i s o l concentration i n salmon a f t e r anesthetization (Barton et a l . , 1986). Control f i s h were sampled s i m i l a r l y from t h e i r home tanks. After the f i s h were anesthetized, the caudal peduncle was severed and the blood was c o l l e c t e d i n heparinized c a p i l l a r y tubes. The plasma was separated by centrifugation and stored at -20°C for determination of C o r t i s o l concentration. Fork length (Ln) and weight (W) were recorded and were used to determine the condition factor (K) of 44 the f i s h by t h i s equation (K=W/Ln3 X 100). The si z e of f i s h used i n each experiment i s presented i n Tables 2.1, 2.2 and 2.3. 45 Table 2.1. Experiment 1: Mean + s.e. of weight, length, and condition factor of coho salmon juveniles reared one month i n a common environment, n=36. weight length condition factor (g) (cm) Hatchery 22. .5 + 2. .0 a 14. .0 + 0. .4a 0. .85 + 0. .02a Wild 10. .4 + 1. . o b 10. .1 + 0. .3b 0. .91 + 0 . ,02a Colonized 18. .6 + 1. .4a 12. .5 + 0. ,3 C 0. .95 + 0. ,03a note: Values with superscripts that are d i f f e r e n t indicates s i g n i f i c a n t differences between groups, p<0.05. Table 2.2. Experiment 2: Mean ± s.e. of weight, length, and condition factor of chinook salmon juveniles reared one month i n a common environment, n=30 • weight length condition factor (g) (cm) Hatchery 6.9 ± 0.3 a 8.8 + 0.1 a 1.0 ± 0.01 a Wild 4.8 ± 0.2 b 7.8 ± 0.1 b 1.0 ± 0.02a note: Values with d i f f e r e n t superscripts indicates s i g n i f i c a n t difference i n that parameter between groups, p<0.05. Table 2.3. Experiment 3: Mean + s.e. of weight, length, and condition factor of juveniles reared 6 months i n a common environment, n=3 6. weight length condition factor (g) (cm) Hatchery 82.4 + 9.0a 19.8 + 0.7 a 1.01 + 0.03 a Wild 37.1 + 2.9 b 15.2 + 0.3 b 1.04 + 0.02a Colonized 55.3 + 7.7 b 17.2 + 0.8 b 1.02 + 0.02a note: Values with superscript that are d i f f e r e n t indicates s i g n i f i c a n t differences between groups, p<0.05. 46 Plasma C o r t i s o l determination Plasma C o r t i s o l was determined by radioimmunoassay as previously described i n Chapter 1: Materials and Methods. Hematology Hematocrit values and d i f f e r e n t i a l white blood c e l l counts were determined according to the methods described i n Chapter 1: Materials and Methods. Assay of s p e c i f i c immune response in vitro In Experiment 3, the e f f e c t of the stress-related r i s e i n plasma C o r t i s o l on the a b i l i t y of lymphocytes to generate s p e c i f i c antibody was assessed. We established c e l l cultures of leukocytes as previously described (Tripp et a l . , 1989, Maule et a l . , 1989). Duplicate anterior kidney tissue samples were incubated for each f i s h i n tiss u e culture media and treated as described i n Chapter 1: Materials and Methods. Disease challenge te s t In Experiment 3, duplicate treatment groups of f i s h not subjected to the handling stress were exposed to Vibrio anguillarum at a concentration of 1.3 x 106 cfu/ml as described 47 i n Chapter 1: Materials and Methods. Data analysis A l l data were subjected to analysis of variance and where s i g n i f i c a n t differences were found the Tukey t e s t (Steel and Torr i e , 1980) was used to i d e n t i f y s i g n i f i c a n t differences between treatment means. These analyses were performed with the SYSTAT s t a t i s t i c a l program (Wilkinson, 1988). As the mortality i n the duplicate treatments of the disease challenge was not s i g n i f i c a n t l y d i f f e r e n t within groups, the re s u l t s were pooled and analyzed using contingency tables (chi-square analysis) to te s t for independence of the means (Duncan et a l . , 1983). S t a t i s t i c a l s i g n i f i c a n c e was taken at the 5 % l e v e l i n a l l t e s t s . 48 Results In experiment 1, the plasma C o r t i s o l concentration of wild and colonized coho salmon was s i g n i f i c a n t l y elevated over control l e v e l s at 8 h. There was no s i g n i f i c a n t elevation i n plasma C o r t i s o l i n the hatchery f i s h over control l e v e l s at 8 h. In a l l groups, except the wild group, the 30 hour post-stress C o r t i s o l concentration was not d i f f e r e n t from i t s control (Fig. 2.1). No s i g n i f i c a n t changes were apparent i n hematocrit values at 8 h following stress (Fig. 2.2). In experiment 2, the plasma C o r t i s o l concentrations of wild chinook salmon were s i g n i f i c a n t l y elevated at both 4 and 8 h aft e r the acute stress. There was no s i g n i f i c a n t elevation i n plasma C o r t i s o l concentration i n the hatchery f i s h at any sampling time a f t e r the acute stress (Fig. 2.3). The hematological p r o f i l e of both the wild and hatchery chinook salmon showed a transient increase i n lymphocyte and leukocyte r a t i o s at the 4 h and 8 h post-stress sampling times. At 30 hours post-stress the lymphocyte and leukocyte r a t i o s were below those of the control, but t h i s decrease 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 (Fig. 2.4). Hematocrit values of the control f i s h were not s i g n i f i c a n t l y d i f f e r e n t from the post-stress hematocrit values at any sampling time (Fig. 2.5). In experiment 3, wild and colonized coho salmon juveniles had s i g n i f i c a n t l y elevated plasma C o r t i s o l concentrations at 4 and 8 h following acute stress while the hatchery f i s h had no 49 Figure 2.1. Means + s.e. of plasma C o r t i s o l concentration (ng/ml) of hatchery, wild and colonized coho salmon juveniles i n response to a 30-60 sec handling s t r e s s . Values with d i f f e r e n t superscripts are s i g n i f i c a n t l y d i f f e r e n t from t h e i r controls, n=32. 50 65 -r A A hatchery colonized wild 35 Oh control 8 h 30 h Figure 2.2. Means + s.e. of hematocrit values (% packed c e l l volume) of hatchery, wild and colonized coho salmon juveniles i n response to a 30-60 sec handling s t r e s s . Values with d i f f e r e n t superscripts are s i g n i f i c a n t l y d i f f e r e n t from t h e i r controls, n=32. 51 Figure 2.3. Means + s.e. of the plasma C o r t i s o l concentration (ng/ml) of hatchery and wild chinook salmon juveniles i n response to a 30-60 sec handling s t r e s s . Values with d i f f e r e n t superscripts are s i g n i f i c a n t l y d i f f e r e n t from t h e i r controls, n=32. 52 Figure 2.4. Means + s.e. of lymphocyte (top) and leukocyte (bottom) number per 10 3 red blood c e l l s of hatchery and wi l d chinook salmon juveniles i n response to a 30-60 sec handling s t r e s s . Values with d i f f e r e n t superscripts are s i g n i f i c a n t l y d i f f e r e n t from t h e i r c o n t r o l s , n=12. 53 Figure 2.5. Means ± s.e. of hematocrit values (% packed c e l l volume) of hatchery and wild chinook salmon juveniles i n response to a 30-60 sec handling s t r e s s . Values with d i f f e r e n t superscripts are s i g n i f i c a n t l y d i f f e r e n t from t h e i r c o n t r o l s , n=32. 54 c o co o o o E CO Q. 300 j 280-260-240-220-200-180-160-140-120-100-80-60-40-201 oJ-control 4 h after stress Hatchery Wild Colonized Figure 2.6. Means + s.e. of the plasma Cortisol concentration (ng/ml) of hatchery, wild and colonized chinook salmon juveniles i n response to a 30-60 sec handling stress a f t e r 6 months i n a common rearing environment. Values with d i f f e r e n t superscripts are significantly d i f f e r e n t from t h e i r controls, n=12. 55 s i g n i f i c a n t increase i n plasma C o r t i s o l at these times (Fig. 2.6) . No s i g n i f i c a n t changes were evident i n most of the hematological c h a r a c t e r i s t i c s , including hematocrit values (Fig. 2.7) , although lymphocyte and leukocyte r a t i o s were decreased from the control values 4 hours following acute stress (Fig. 2.8) . There was a s i g n i f i c a n t decrease i n APC number i n the anterior kidney of the wild f i s h . This decrease was seen i n the hatchery and colonized groups but 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 (Fig. 2.9). I n i t i a l l e v e l s of APC were highest i n the wild than the other two groups (Fig. 2.9). In response to a disease challenge of unstressed (0 h) f i s h , t o t a l cumulative mortality was s i g n i f i c a n t l y higher i n the wild f i s h than i n the hatchery-reared f i s h . Mortality i n the colonized group was intermediate to that i n the hatchery and wild groups, and not s i g n i f i c a n t l y d i f f e r e n t from either (Fig. 2.10). These r e s u l t s are d i f f e r e n t than those of 5 months e a r l i e r when there was no s i g n i f i c a n t difference i n mortality between the hatchery, wild, and colonized f i s h (Fig. 1.13). Figure 2.7. Means + s.e. of hematocrit values (% packed c e l l volume) of hatchery, wild and colonized coho salmon juveniles i n response to a 30-60 sec handling stress a f t e r 6 months rearing i n a common environment. Values with d i f f e r e n t superscripts are s i g n i f i c a n t l y d i f f e r e n t from t h e i r controls, n=12. 57 32 T Hatchery Wild Colonized Figure 2.8. Means ± s.e. of leukocyte number per 10 J red blood c e l l s of hatchery, wild and colonized coho salmon juveniles i n response to a 30-60 sec handling stress a f t e r 6 months rearing i n a common environment. Values with d i f f e r e n t superscripts are s i g n i f i c a n t l y d i f f e r e n t from t h e i r controls, n=12. Figure 2. 9. Means ± s.e. of antibody producing c e l l (APC) number per 10 6 leukocytes i n the anterior kidney of hatchery, wild and colonized coho salmon juveniles i n response to a 30-6 sec handling stress a f t e r 6 months rearing i n a common environment. Values with d i f f e r e n t superscripts are s i g n i f i c a n t l y d i f f e r e n t from t h e i r controls, n=12. 59 CD > _0 Z3 E D ( J wild hatchery colonized A^A-A-A-A Time (days) Figure 2.10. Cumulative mortality (%) and mean time to death (d) of hatchery, wild and colonized coho salmon juveniles i n response to a bath immersion challenge with V. anguillarum at a concentration of 1.3 X 10 6 cfu/ml a f t e r 6 months rearing i n a common environment, Values with d i f f e r e n t superscripts are s i g n i f i c a n t l y d i f f e r e n t from t h e i r controls, n=42. 60 Discussion Certain r e s u l t s were common to a l l these experiments. At none of the sampling times did the chinook or coho hatchery juveniles respond to the handling stress with s i g n i f i c a n t elevation i n plasma C o r t i s o l concentration, a disturbance that consistently produced a s i g n i f i c a n t increase i n naturally-reared (wild chinook and coho salmon, and colonized coho salmon) juveniles. In coho salmon juveniles reared i n a common environment for s i x months, the stress response of the f i s h reared i n the natural environment (wild and colonized f i s h ) was si m i l a r , i n d i c a t i n g that longer rearing i n a common environment did not a f f e c t the magnitude of the response to an acute stress. Repetitive stress i n salmonids can r e s u l t i n a negative feedback of C o r t i s o l on the hypothalmic-pituitary-interrenal axis and i n h i b i t further i n terrenal response (Donaldson et a l . , 1981, Barton et a l . , 1987). Enhanced interrenal a c t i v i t y from stress i n early freshwater rearing may have prolonged secondary e f f e c t s on the function of both interrenal and lymphoid tissues of hatchery-reared salmonids. I speculate that t h i s mechanism may be the cause of the lack of measured response of hatchery f i s h to stress, or that the duration of the elevation of plasma C o r t i s o l so b r i e f i n nature that i t was missed by the sampling protocol. Immunosuppression may occur despite the fac t that plasma C o r t i s o l remains low i n response to an acute stress, such as the one used i n t h i s study. Hatchery juveniles are subjected to d a i l y disturbances i n early rearing that I have shown evoke a 61 s i g n i f i c a n t stress response i n wild f i s h . The cumulative stresses to salmonids i n early fresh water rearing may be responsible for a low C o r t i s o l stress response i n hatchery f i s h that i s lower than and not t y p i c a l of t h e i r wild or colonized ( i n the case of the coho salmon) counterparts. However, assuming that these f i s h were of s i m i l a r genetic stock, the p o s s i b i l i t y cannot be discounted that there was sel e c t i o n within the wild f i s h and within colonized hatchery releases for a high C o r t i s o l stress response. Adaptation to stress may have induced a low C o r t i s o l response i n the hatchery-reared f i s h . There may be some se l e c t i v e advantage of a high C o r t i s o l stress response i n terms of s u r v i v a l i n the natural environment. Studies have recently been published where the C o r t i s o l stress response has been measured i n order to obtain an i n d i r e c t measure of disease resistance. Fevoldeh et a l . (1991) hypothesized that, due to the immunosuppressive e f f e c t s of C o r t i s o l , high C o r t i s o l response groups would have lower s u r v i v a l rates. However, they actually found that c e r t a i n immune responses were higher i n the high C o r t i s o l stress response groups. Maule et a l . (1989) found both immunosuppressive and immunostimulatory e f f e c t s of stress i n chinook salmon. The tendency to equate a high stress response with a lowered immune capacity and resistance to disease may be erroneous. Indeed, i n t h i s study, re s t i n g numbers of APC were highest i n the wild f i s h , a high C o r t i s o l response group. Although the re l a t i o n s h i p between an elevation i n plasma C o r t i s o l and resultant immunosuppression (Tripp et a l . , 1987) holds i n t h i s study, the character of f i s h with the potential of high 62 i n t e r r e n a l a c t i v i t y may also be to have p o t e n t i a l l y elevated function of lymphoid c e l l s and t i s s u e s . In the second experiment, the t r a n s i t o r y increase i n t o t a l leukocyte and lymphocyte r a t i o s i n the wild and hatchery chinook salmon may have been caused by increased plasma catecholamine concentrations at 4 h and 8 h following acute stress. Increases - i n the concentration of plasma epinephrine have been shown to cause splenic contraction and release of red blood c e l l s and i t may also increase the number of c i r c u l a t i n g white blood c e l l s i n the blood (Perry and Kincaid, 1989). In the coho salmon stress experiment a f t e r 6 months of rearing i n the a r t i f i c i a l environment, wild f i s h had s i g n i f i c a n t l y higher mortality than the hatchery f i s h . Results of t h i s challenge are comparable to a s i m i l a r challenge on these same f i s h 5 months e a r l i e r that showed no difference i n t o t a l mortality between the groups. The response to acute stress was unchanged over the 6 month period and i t appears that chronic stress may have reduced the survivorship of wild f i s h to the disease challenge. Mortality i n the colonized group was less than i n the wild group, but the difference 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 . There was a change i n r e l a t i v e mortality between hatchery and wild f i s h to the Vibrio anguillarum challenge. In other studies, mortality due to V. anguillarum has been shown to be stress-mediated and a r e l i a b l e indicator of adverse conditions (Wedemeyer and McLeay, 1981). This may have been a f f e c t i n g the mortality of the naturally-reared f i s h i n the a r t i f i c i a l environment. 63 In these experiments, naturally-reared salmon juveniles were shown to have d i f f e r e n t physiological responses to acute stress than t h e i r hatchery counterparts. I t appears that the high C o r t i s o l response has been retained i n the naturally reared f i s h while t h i s response was l o s t i n the f i s h reared i n the hatchery. In view of the p o s s i b i l i t y that the natural environment seems to sel e c t for t h i s type of response, the strategy of enhancement by colonization appears to be a viable and economical way to supplement wild populations and increase s u r v i v a l rates i n hatchery releases from the time of seaward migration onward. 64 Chapter 3 : The e f f e c t of d i e t on immune function at f i r s t feeding i n developing chinook salmon. 65 Introduction: The d i e t of intensively cultured f i s h plays a v i t a l part i n determining t h e i r health and well being. Present commercial d i e t formulations of salmonid feeds are generally considered to be n u t r i t i o n a l l y sound. Indeed, the standard d i e t used to i n i t i a t e feeding i n SEP hatcheries (Plotnikoff et a l . , 1985) and the commercial d i e t examined i n t h i s study have been shown to contain adequate l e v e l s of vitamins and minerals (Felton et a l . , 1990), as well as adequate l e v e l s of less l a b i l e components of f i s h d i e t s . Given t h i s , however, the feeding of commercial diets has been shown to cause decreased immune function i n salmonids (Blazer and Wolke, 1984). One absolute difference between the feeding of a l i v e d i e t and a formulated d i e t i s the degree of ra n c i d i t y present i n commercially prepared di e t s , an aspect of the d i e t that i s absent from a l i v e d i e t . Fish feeds and f i s h tissues contain r e l a t i v e l y high concentrations of highly unsaturated f a t t y acids. These are important components of c e l l membranes and are vulnerable to l i p i d peroxidation and resultant tissue damage (Tacon, 1985; L a l l , 1990). The autoxidation of l i p i d s i s one of the most deleterious changes that may occur to f i s h d i e t s . Oxidized l i p i d may have adverse e f f e c t s on f i s h health due to the destruction of vitamin A and E and other e s s e n t i a l nutrients (Hung et a l . , 1980). A review of various adverse e f f e c t s of n u t r i t i o n a l t o x i c i t i e s and d e f i c i e n c i e s on the immune function i n f i s h was recently written by Landolt (1989). 66 A second type of study that helps to c l a r i f y the e f f e c t of oxidized dietary l i p i d s on the immune system are those that examine the moderate depletion of vitamin E, vitamin C and selenium from the d i e t . The importance of these factors as antioxidants has been well studied. Vitamin E terminates the free r a d i c a l reaction because i t i s an e f f i c i e n t scavenger of free r a d i c a l s . Selenium i s a necessary component of glutathione (GSH) peroxidase, which i s able to convert f a t t y acid hydroperoxides to innocuous hydroxy f a t t y acids ( B e l l and Cowey, 1985). One the most important functions of vitamin C i s that i t quenches oxygen r a d i c a l s a r i s i n g from c e l l u l a r r e s p i r a t i o n (Hardie et a l . , 1991). Studies have shown that feeding diets d e f i c i e n t i n selenium have resulted i n an impairment of the antigenic response i n rodents (Purnham et a l . , 1983). Diets d e f i c i e n t i n both vitamin E and selenium i n A t l a n t i c salmon during the f i r s t four weeks of feeding resulted i n twice the mortality of the controls with adequate leve l s of these components (Poston et a l . , 1976). Diets with moderate depletion of vitamin C, vitamin E and selenium did not cause gross or h i s t o l o g i c pathology i n A t l a n t i c salmon or rainbow trout (O. mykiss), yet had more subtle e f f e c t s on disease resistance and immune function (Blazer and Wolke, 1984; Hardie et a l . , 1991). E a r l i e r studies with coho salmon, where there was in c l u s i o n of rancid l i p i d into the d i e t without causing gross or h i s t o l o g i c change, found no e f f e c t of the r a n c i d i t y factor on the immune response (Forster et a l . , 1988). The majority of studies on the e f f e c t s of r a n c i d i t y on f i s h 67 health, with the noted exception of Poston et a l . (1976), involved experimenting with f i s h that had reached immunological maturity, known to occur soon a f t e r the onset of f i r s t feeding i n rainbow trout (Tatner, 1986) and masu salmon (O. masou) (Fuda et a l . , 1991). The study described herein attempted to examine the e f f e c t of a commercial d i e t compared to a l i v e or fresh frozen d i e t of k r i l l on the immune response at t h i s c r i t i c a l period i n the development of lymphoid organs and ontogeny of immunological responsiveness. The period following swim-up i n salmonid fishes i s one characterized by a rapid rate of growth (Brett, 1979). During the normal course of metabolism, the autoxidation of the highly unsaturated f a t t y acids i n formulated commercial feeds gives r i s e to free r a d i c a l s that are toxic or p o t e n t i a l l y t o x i c , highly reactive metabolites of oxygen ( B e l l and Cowey, 1985). In f i s h , damage from these metabolites i s ameliorated by a mu l t i l e v e l defense system including GSH, previously mentioned, and superoxide dismutase (SOD). The glutathione peroxidase systems that protect f i s h tissues from damage by hydroperoxides are one to two orders of magnitude lower i n salmonid l i v e r and kidney tissues than i n mammals and are nearly absent i n the gut (Tappel et a l . , 1982). Autoxidized dietary l i p i d s would be necessarily absent i n the d i e t of wild salmon as they consume l i v e prey (Marliave, 1989). During the period of accelerated growth and increased food consumption following swim-up i n hatchery-reared salmonids, production of free r a d i c a l s from oxidized foodstuffs may override 68 the capacity of the natural defense systems to cause damage to developing lymphoid ti s s u e s . There i s some evidence that feeding chum salmon (O. keta) a l i v e d i e t from swim-up has lowered mortality i n response to a b a c t e r i a l challenge, despite the fa c t that the swim-up f r y fed the l i v e d i e t were on a lower rat i o n l e v e l (Marliave, 1989). Because of the dependence of young salmonids on the non-s p e c i f i c immune system (NSIS) during the period before the immunological maturation of non-specific immune defenses (Grace et a l . , 1980), lysozyme a c t i v i t y was examined i n the chinook salmon f r y i n each of the d i e t treatments as well the s p e c i f i c immune function of the anterior kidney leukocytes. Lysozyme i s an enzymatic component of the NSIS, located i n the lysosomes of neutrophils and macrophages, and i s secreted into the blood by these c e l l s (Mock and Peters, 1990). Lysozyme i s active i n breaking down the peptidoglycan layer of b a c t e r i a l c e l l walls (Murray and Fletcher, 1976). I t i s also thought to be e f f e c t i v e against gram-negative organisms through the s y n e r g i s t i c action with complement and other enzymes (Hjelmeland et a l . , 1983). Lysozyme a c t i v i t y has been found to be higher i n leukocyte-rich tissues at points where the r i s k of b a c t e r i a l i n f e c t i o n i s high, such as i n mucus and the g i l l s (Lindsay, 1986). The haemolytic plaque assay determines the number of leukocytes i n the lymphoid tiss u e of the kidney capable of an antibody response to a hapten-carrier stimulus and i s a standard technique for quantifying antibody producing c e l l numbers i n mammalian immunology (Roitt et a l . , 1985). I t has been 69 characterized for use i n f i s h as an assay with a narrow s t a t i s t i c a l margin and a high s e n s i t i v i t y (Anderson, 1990) and was used i n the present study as a t o o l to determine immunocompetence. The objective of t h i s study was to compare the e f f e c t on the immune response of juvenile chinook salmon of i n i t i a t i n g feeding with either a commercial d i e t or a wild-type d i e t of l i v e or fresh frozen k r i l l , without s p e c i f i c regard for r a t i o n l e v e l or standardization of n u t r i t i o n a l composition of these d i e t s . 70 Materials and Methods Fish and Diets Juvenile chinook salmon were hatched and reared to swim-up at the Capilano Hatchery, North Vancouver, at which time feeding was i n i t i a t e d on one of three treatments; Oregon Moist P e l l e t (OMP) commercial s t a r t e r , l i v e ocean euphausids (sel e c t i v e gear was used that fished almost exclusively for Euphausia pacifica), or commercially prepared, fresh frozen, bulk k r i l l (Murex brand (Euphausia pacifica), Aquafoods, Langley, B.C.)* The l i v e d i e t and frozen k r i l l treatments were i n i t i a t e d on day 46 post-hatch, 3 and 11 days, respectively, a f t e r the OMP treatment. After 11 days, the l i v e d i e t treatment was replaced with fresh frozen k r i l l . P r a c t i c a l considerations li m i t e d the feeding of l i v e d i e t for more than 11 days. Fish were held i n Capilano Hatchery troughs with 45,000-50,000 f i s h per treatment. Fry were held from hatch i n heated spring water maintained at a constant temperature of 10°C. During the course of feeding the l i v e d i e t , the euphausiids were introduced into the water where they were a c t i v e l y captured by the f r y . The frozen k r i l l were supplied to the f r y i n netted bags, hung every 0.6 m i n the trough. The three treatment groups were sampled on three consecutive days; groups were OMP-91 days post-hatch, l i v e diet-88 days post-hatch, and frozen ^ r i l l - 8 0 days post-hatch ( f i s h mean wt + S.E.: OMP di e t ; 3.06' £ 0.09 g, l i v e d i e t ; 2.03 ± 0.07 g, frozen k r i l l ; 1.56 + 0.05 g). 71 Experimental procedures and sampling Fish (n=30) from each treatment were rapidl y netted and transferred to a bucket containing a l e t h a l dose (200 mg/1) of buffered t r i c a i n e methanesulfonate (MS222). After the f i s h were anesthetized, the caudal peduncle was severed and the blood was co l l e c t e d i n heparinized c a p i l l a r y tubes. The plasma was separated by centrifugation for 5 minutes at 11,500 RPM (Model MB microhematocrit centrifuge, International Equipment Co., MA, U.S.A.) and stored at -20°C p r i o r to determination of lysozyme a c t i v i t y . Fork length and weight were measured and used to determine condition factor (K) (Pickering and Duston, 1983). Hematology Hematocrit and d i f f e r e n t i a l white blood c e l l counts were made as previously d i s c r i b e d i n Chapter 1: Materials and Methods. S p e c i f i c immune response To assess the a b i l i t y of lymphocytes to generate s p e c i f i c antibody, c e l l cultures of leukocytes were established as previously described i n Chapter 1, according to Tripp et a l . (1987) and Maule et a l . . (1989). In order to achieve a concentration of 2 X 10 7 leukocytes/ml, anterior kidney tissue homogenates of f i v e f i s h were pooled. Cultures were treated as in Chapter 1: Materials and Methods. 72 Plasma lysozyme determination Lysozyme a c t i v i t y was determined using the modified lysoplate assay of Ossermann and Lawlor (1966). This assay i s based on the l y s i s of the l y o p h i l i z e d , lysozyme-sensitive, Gram-pos i t i v e bacterium Micrococcus lysodeikticus (Sigma) i n agarose g e l . Agar plates were prepared by suspending 0.6 g/L M. lysodeikticus, 1.17 g/L NaCl, and 5 g/L agarose (Sigma) i n 1 L Phosphate Buffer (see Appendix B). The agarose was dissolved by heating and 25 ml was poured into P e t r i plates and allowed to s o l i d i f y . Ten-/xl samples of plasma or standard were put into wells (diameter about 3 mm) punched i n the agar. The diameter of the zone of cle a r i n g around the well was measured a f t e r incubation i n a moist chamber at 22-25°C for 17 h. The lysozyme a c t i v i t y of each plasma sample was calculated using logarithmic regression analysis, employing the formula Y=3.971ogX-2.87 (r 2=0.921), where Y= the diameter of the area cleared by the l y s i s (mm) , and X= the lysozyme a c t i v i t y (U) based on a external standard curve using hen egg white lysozyme (HEWL, Sigma). The a c t i v i t y of HEWL was determined using a turbimetric method (Grinde, 1989) at pH 6.0. The s p e c i f i c a c t i v i t y of HEWL varies with pH and must be determined at the pH of the assay. By t h i s method, one unit of lysozyme a c t i v i t y (U) i s defined as the amount of enzyme causing a decrease i n the absorbance of a suspension of M. lysodeikticus (0.2 mg/ml 0.06M phosphate buffer, pH 6.0) of 0.001/min when the reaction i s c a r r i e d out at 23°C and an absorbance of 530 nm. I t was found that 1 lysozyme unit (U)/ml 73 corresponded to 2.2 /xg of HEWL. Although the corresponding lysozyme concentrations (fig/ml) of the plasma samples were calculated and reported i n the r e s u l t s , there has not yet been an international standardization of these methods. Although the use of the commercially available HEWL i s universal as a standard i n assays for f i s h lysozyme, the pH and t o n i c i t y of the buffers used are variable. Thus the concentrations of lysozyme determined i n t h i s study are r e l a t i v e and may not be comparable to those of other investigators. Data analysis A l l data were subjected to analysis of variance and where s i g n i f i c a n t differences were found, Tukey's t e s t (Steel and Torrie, 1980) was used to i d e n t i f y s i g n i f i c a n t differences between treatment means. These analyses were performed with the SYSTAT s t a t i s t i c a l program (Wilkinson, 1988). S t a t i s t i c a l s i g n i f i c a n c e was taken at the 5 % l e v e l i n a l l t e s t s . 74 Results S p e c i f i c immunological function of anterior kidney leukocytes was higher i n f i s h from the OMP treatment than i n those from either the l i v e or frozen k r i l l treatments (Fig. 3.1). Although the use of pooled anterior kidney samples as c e l l suspensions precluded the regression of APC number against i n d i v i d u a l f i s h weights. When the mean APC number was regressed against the average weights of the f i s h used across a l l d i e t treatments, there was a s i g n i f i c a n t c o r r e l a t i o n between APC number i n the anterior kidney and f i s h weight ( r 2 = 0.941). Non-specific immunological function as measured by lysozyme a c t i v i t y , was higher i n f i s h from the l i v e d i e t treatment than i n those from the frozen k r i l l treatment but not s i g n i f i c a n t l y higher than that seen i n the OMP treatment group (Fig. 3.2). There was no c o r r e l a t i o n between weight of the f i s h and lysozyme a c t i v i t y . There was no difference i n the l e v e l s of lysozyme a c t i v i t y found i n t h i s study and those reported for adult salmonids and where reported, the plasma lysozyme concentrations i n the l i t e r a t u r e were comparable to values obtained i n t h i s study (Lindsay, 1986; Mock and Peters, 1990). The hematological p r o f i l e s of the f i s h from the three d i e t treatments were not s i g n i f i c a n t l y d i f f e r e n t from each other, yet i n a l l of the wbc/rbc r a t i o s measured (lymphocyte, leukocyte, and thrombocyte) the l i v e and frozen k r i l l treated f i s h had higher r a t i o s of these c e l l s than the OMP fed f i s h with the highest values i n the l i v e k r i l l - f e d f i s h (Fig. 3.3). 75 to © o c o o < 180.0 160.0 140.0 120.0 100.0 80.0 60.0 40.0 20.0 0.0-a b JL OMP Live Krili Figure 3.1. Means ± 1 s.e. of the number of antibody producing c e l l s (APC)/million v i a b l e leukocytes i n the head kidney of chinook salmon f r y fed eithe r l i v e or frozen k r i l l (E.pacifica), or a commercially prepared d i e t (OMP). Values with superscipts that are d i f f e r e n t are 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. n=12? 20.0 18.0-16.0--14.0-12.0-10.0-8.0-6.0-4.0 2.0 + 0.0 ab OMP Live Krill -35.0 30.0 4-25.0 20.0 - 15.0 -10.0 -5.0 0.0 Figure 3.2 Means ± 1 s.e. of lysozyme a c t i v i t y (U) and concentration (/ig/ml) i n the plasma of chinook salmon f r y fed eit h e r l i v e or frozen k r i l l (E. pacifica), or a commercially prepared d i e t (OMP). Values with superscipts that are d i f f e r e n t are 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. n=12. 77 to 26 t P L F P L F P L F P = pellets, L = live, F = frozen Figure 3.3 Hematological p r o f i l e of chinook salmon f r y fed either l i v e or frozen k r i l l (E. pacifica), or a commercially prepared d i e t (OMP). Values with superscipts that are d i f f e r e n t are 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. n=6. 78 1a, > o CL O o o £ CD 1 b. o o E o cn O 45.0 40.0 35.0 30.0 25.0 20.0 15.0 10.0 5.0 0.0-1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 OMP Live Kril OMP Live Krill Figure 3 .4 . a; Means ± 1 s.e. of hematocrit (% packed c e l l volume) and fc>; condition factor (C.F.) of chinook salmon f r y fed ei t h e r l i v e or frozen k r i l l ( J B . pacifica) , or a commercially prepared d i e t (OMP). Values with l e t t e r s i n common are not 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. n=30. 79 The f i s h of the OMP treatment had a higher mean hematocrit than either the l i v e or frozen k r i l l - t r e a t e d f i s h (Fig. 3.4a). Hematocrit values from the two l a t t e r treatments were not s i g n i f i c a n t l y d i f f e r e n t i n t h i s respect. The mean weight of the f i s h i n the OMP treatment (3.06 + 0.09) was s i g n i f i c a n t l y higher than that of the l i v e fed (2.03 ± 0.07) or the frozen k r i l l - f e d (1.56 ± 0.05) f i s h . Fish of the l a t t e r two treatments were also s i g n i f i c a n t l y d i f f e r e n t i n mean weight, but not i n condition factor (Fig. 3.4b). The OMP treated f i s h had a s i g n i f i c a n t l y higher condition factor than both the l i v e fed d i e t f i s h or the frozen k r i l l fed f i s h (Fig. 3.4b). 80 Discussion As the f i s h i n each d i e t treatment were ponded on d i f f e r e n t days, the OMP fed f r y i n i t i a t e d feeding 11 days before the frozen k r i l l fed f r y and 4 days before the l i v e fed f r y . As a r e s u l t , the p hysiological and immunological state of development i n the OMP group appeared to be more advanced than that of the other groups. The e f f e c t of feeding a d i e t free of r a n c i d i t y on the s p e c i f i c immune system cannot be discerned from the l e v e l s of APC determined i n t h i s study. The s p e c i f i c immunological response appeared to be c l o s e l y correlated with weight at the early stages of development. As t h i s study shows, the presence of lymphocytes and lymphoid tiss u e may not herald immunological maturity i n terms of the s p e c i f i c immune response. Tatner (1986) suggested that a c r i t i c a l mass of immunocompetent c e l l s must be reached before a f i s h can mount a f u l l antibody response to a stimulus. Indeed, f r y have been shown to be refractory to vaccination with V. anguillarum i n each of two separate studies, where a d i f f e r e n t chronological age was determined as the time of the appearance of immunological response (Manning et a l . , 1982). Manning et a l . (1982) found i n both of t h e i r studies that the t r a n s i t i o n to a immunocompetent state coincided with the period of t r a n s i t i o n from a r e l a t i v e l y low growth rate to a higher growth rate, at the onset of f i r s t feeding. The number of APC i n the head kidney of the OMP fed f r y indicated they had reached an immunocompetent l e v e l at 91 days 81 post-hatch, yet the number of APC i n the l i v e fed f r y , although only 3 days behind, did not indicate immunocompetence. The high c o r r e l a t i o n of APC number with weight indicates that s p e c i f i c immune response i s dependent on the attainment of a c r i t i c a l s i z e and less on the s p e c i f i c age i n these f i s h . Other studies have also shown t h i s to be true (Tatner, 1986; Fuda et a l . , 1991). In l i g h t of the apparent dependence of the development of s p e c i f i c immunological maturity on f i s h weight, non-specific immune defenses may be r e l a t i v e l y more important during early ontogeny of the s p e c i f i c immune system. Indeed, for a period of time i n immunological development, salmonid f r y are e n t i r e l y dependent on nonspecific defense mechanisms for protection against invading pathogens (Grace et a l . , 1980; Fletcher, 1982; Manning et a l . , 1982). Yousif et a l . (1991) have shown that the nonspecific factor examined i n t h i s study, lysozyme, i s present i n salmonid eggs. The NSIS i n the chinook salmon f r y i n t h i s study was f u l l y active at 80-91 days post-hatch and, thus, independent of the f i s h weights. A s l i g h t advantage of feeding a l i v e d i e t on the l e v e l of lysozyme a c t i v i t y i n the chinook salmon f r y was apparent. Higher r a t i o s of white blood c e l l / r e d blood c e l l (WBC/RBC) i n the l i v e fed f r y also may indicate a s l i g h t advantage i n terms of disease resistance of using t h i s d i e t type to i n i t i a t e feeding i n salmonids. The differences i n mean weights of the f i s h i n each d i e t treatment resulted, i n part, from the staggered swim-up times, but i t i s also possible that ra t i o n was r e s t r i c t e d i n the l i v e k r i l l - f e d and frozen k r i l l - f e d f i s h . The delivery system for the 82 frozen k r i l l may have allowed only the more aggressive f r y to become satiated. Also, on two occasions, the trawler was unable to f i s h the l i v e k r i l l and, thus, the f i s h remained unfed for two of the eleven days on l i v e d i e t . The mean condition factors of the f i s h i n the l i v e and fresh frozen k r i l l treatment groups does not indicate that these f i s h were lim i t e d n u t r i t i o n a l l y , although ra t i o n l e v e l may have contributed to t h e i r lower weights r e l a t i v e to the OMP fed f r y . Control of the factors such as swim-up time and r a t i o n l e v e l would be better achieved i n a laboratory s i t u a t i o n and further studies should incorporate t h i s into the design. Although t h i s study f a i l e d to demonstrate that a l i v e d i e t i s b e n e f i c i a l i n terms of s p e c i f i c immune response, or that possible r a n c i d i t y was detrimental to immunological development i t does show that feeding of a l i v e d i e t to salmonids compares favorably t o feeding a prepared d i e t with respect to c i r c u l a t i n g leukocyte numbers and lysozyme a c t i v i t y i n plasma, even at a lower r a t i o n l e v e l . I t also supports the suggestion that the time of f i r s t feeding i n chinook salmon f r y i s an important period of immunological maturation, a period also characterized by rapid growth. If there i s a period during physiological maturation where even a small degree of r a n c i d i t y i n prepared di e t s would cause sublethal e f f e c t s such as immunosuppression, i t i s probably at t h i s time. The high growth rate (brought about by the onset of feeding) could cause an increase i n the amount of autoxidated l i p i d s i n the gut as a natural r e s u l t of metabolizing the unsaturated f a t t y acids i n prepared diets and overwhelm the 83 natural antioxidant systems, thereby allowing damage to occur the developing lymphoid ti s s u e s . 84 General Conclusions and Recommendations The findings of these experiments suggest that the plasma C o r t i s o l increase i n response to stress and environmental s t i m u l i are d i f f e r e n t between both wild and colonized salmonid f i s h and t h e i r hatchery counterparts. The mechanism behind the lower C o r t i s o l response of hatchery f i s h may be down regulation of the C o r t i s o l production by the interrenal tissue brought about by chronic stress i n the hatchery rearing environment. There i s an ind i c a t i o n that wild and colonized f i s h may have a better i n t r i n s i c a b i l i t y to mount an immune response. In wild and colonized f i s h however, the immune response may be affected by C o r t i s o l mediated immunosuppression during smoltification- and during chronic stress i n a common a r t i f i c i a l rearing environment. C o r t i s o l may a f f e c t immune function d i f f e r e n t l y at d i f f e r e n t physiological stages, such as s m o l t i f i c a t i o n . Wild and colonized juvenile f i s h have a d i f f e r e n t physiological character than t h e i r hatchery-reared counterparts with regard to C o r t i s o l response. This character appears to be determined by the nature of the early rearing environment and remains unchanged a f t e r an extended period i n a common environment. Disease resistance of wild and colonized f i s h decreases over the course of holding them i n an a r t i f i c i a l environmental rearing environment, possibly as a d i r e c t r e s u l t of C o r t i s o l mediated immunosuppression. In veiw of the negative e f f e c t s of cumulative stressors i n 85 intensive culture on developing interrenal and lymphoid ti s s u e , further work to f i n d ways of mitigating stress i n the hatchery environment may produce hatchery f i s h with a higher capacity to survive the rig o r s of the natural environment. Feeding a l i v e d i e t to f i s h was found to compare favorably with feeding a commercial d i e t , even at a lower r a t i o n l e v e l . This may be one modification that could be incorporated into hatchery rearing of salmonids to achieve a more "wild-type" f i s h . Other modifications of the hatchery environment might include using natural water temperature p r o f i l e s , providing exercise by varying flow rates, and by allowing the aggressive capture of l i v e prey. A portion of the d i e t fed to swim-up f r y could include marine euphausiids, which may prevent any possible deleterious e f f e c t s of r a n c i d i t y i n prepared feed on the development of the immune system. This approach would be an economically viable prospect to undertake for coastal B r i t i s h Columbia salmon enhancement f a c i l i t i e s that are close to a seawater source. P h y s i o l o g i c a l l y d i f f e r e n t wild f i s h are presumably the r e s u l t of sele c t i o n i n the natural environment and might be considered as a model by which to measure the qua l i t y of hatchery smolts destined fo r release. In intensive culture of salmonids reared to supplement natural populations, i t may be necessary for hatcheries to adapt new p o l i c i e s to improve the s u r v i v a l of t h e i r hatchery releases and produce f i s h that are more l i k e t h e i r wild counterparts. 86 References Anonymous, 1981. Nutrient requirements of coldwater fishes (Nutrient requirements of domestic animals. No. 16). National Academy Press, Washington. Anderson, D.P. 1990. Immunological indicators: E f f e c t s of environmental stress on immune protection and disease outbreaks Am. Fish Soc. Symp. 8: 33-50. Barton, B.A. and G.K. Iwama. 1991. Physiological changes i n f i s h from stress i n aquaculture with emphasis on the response and e f f e c t of c o r t i c o s t e r o i d s . Ann. Rev. Fish Dis. 1: 3-26. Barton, B.A., C.B. Schreck and L.D. Barton. 1987. 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Org. 10: 45-49. 94 Appendix A: Known physical parameters of the hatchery and r i v e r rearing environments. Big Oualicum f i s h hatchery, Oualicum Beach,. B.C., 1990 Chinook salmon from the Big Qualicum f i s h hatchery had been reared at densities of 0.9 kg*m~3 at ponding i n January to 10.5 kg*m~3 just p r i o r to release. Fish were ponded i n concrete raceways. Water temperature was 5°C at ponding time and increased to 11°C just p r i o r to release. Fish were maintained during t h i s period on Oregon Moist P e l l e t (OMP) at the rate of 0.82 % to 2.93% of body weight per day as they increased i n s i z e . F i s h were not vaccinated and received no prophylactic treatments. No measurements were taken at the r i v e r or were available from hatchery s t a f f . Ouinsam f i s h hatchery, near Campbell River, B.C.,1990/91 Coho salmon from the Quinsam River f i s h hatchery were ponded i n earthen ponds and fed OMP at a average r a t i o n l e v e l of 1-2 % body weight per day. Parameters are available from March u n t i l f i s h were released i n June. Flow loading, or biomass per flow rate, was 0.13 kg/L per min at the beginning of t h i s period and 0.25 kg/L per min at time of release. Dissolved oxygen was an average of 9 ppm at the outflow i n March and decreased to 7-8 i n May. The water source was ground water and remained a constant 10°C throughout t h i s period. Non-filterable residue averaged 0.2 mg/L at the inflow and increased s t e a d i l y from 0.6 mg/L i n March to 2.0 mg/L i n May at the outflow. Hatchery f i s h were not 95 vaccinated and received no prophylactic treatment for 3 months pr i o r to release. Water i n the r i v e r was considered saturated at a l l times; [0 2] 11-12 ppm. Temperature increased s t e a d i l y from a March low of 3°C to a high of 15°C i n June. Conditions i n the hatchery i n 1991 were s i m i l a r to those i n 1990 except i n March, 1991, [0 2] i n the ponds was 8.35 ppm and declined further to 7.47 ppm at the end of May. 96 Appendix B Part 1: Protocol for the Hemolytic Plaque Assay Recipes for media, buffers, and solutions are given i n Part 2 of t h i s Appendix. A l l chemicals are from Sigma Chemical Co, St. Lewis, Mo, U.S.A., unless otherwise noted. a. Tissue extraction. Head kidney tissue was excised a s e p t i c a l l y and placed i n 12 X 75 mm s t e r i l e culture medium (TCM) i n a volume dependent on the size of the f i s h tissue (usually 0.5-2.0 ml for smolts). This was done while working i n a laminer flow hood ( or with a UV hood i n the f i e l d ) . A l l surfaces were wiped with 70 % EtOH including the skin surface of the f i s h . Tissues were excised on a bed of paper towel soaked with EtOH. With a flamed s c a l p e l , a ventral i n c i s i o n was made, extending from the pectoral f i n s to the p e l v i c f i n s . With a re-flamed s c a l p e l , a dorsal second i n c i s i o n was made behind the operculum extending to just below the backbone. The head was grasped and stripped away head with the attached organs to expose the triangular head kidney. The head kidney was removed from the back musculature with s t e r i l e flamed instruments. This was best done with tweezers i n smaller f i s h and pinching i t out or i n larger f i s h (> 30 g) by using a sc a l p e l and scraping the tissu e out to break the connective t i e s to the back musculature. Fish were kept on ice u n t i l sampled and a l l samples were kept i n sealed culture tubes on ice before processing. C e l l s were dissociated from tiss u e by aspirating the tissue 97 with a s t e r i l e 1 cc tuberculin syringe (Benton Dickinson and Co., Rutherford, New Jersey). After the connective tissue and other t i s s u e debris s e t t l e d out, the c e l l suspension was transferred to a new tube and, i f p r a c t i c a l , i e . i f the sample i s large enough that some c e l l loss i s acceptable, the tube i s centrifuged at 500g X 10 min (Beckman Model TJ6, Beckman Instruments, Palo Alto, Ca, U.S.A.) and the supernatent poured o f f and replaced with fresh TCM. Tubes were vortexed to resuspend c e l l s . b. Establishment of c e l l cultures. Approximate c e l l numbers were established by manual c e l l counts by the trypan blue exclusion method and a hemacytometer. A fi x e d volume eppendorf and s t e r i l e yellow t i p s were used to remove 10 /il from the culture tubes and were put to a well of a 96 well culture plate containing 90 /il trypan blue ( 0.4% i n PBS ) and 100 /Ltl PBS. Contents were mixed with a Pasteur pipet and added to a hemacytometer chamber. Live WBC's were counted and the concentration of c e l l s was calculated as follows; # cells/ml = 100 (# of c e l l s i n 16 squares) X 20 ( d i l u t i o n factor) X 10 6. A f i n a l concentration of 2 X 10 7 wbc/ml was desired. At t h i s point i f the concentration of c e l l s was too high a d i l u t i o n was made with TCM. 50 /il of the c e l l suspension was added to wells of a 96 well culture plate (Falcon) containing eit h e r 50 /il of TCM/ TNP-LPS (test wells) or TCM (background c o n t r o l s ) . Cultures were incubated i n a gas box (American S c i e n t i f i c Products) purged with blood gas (10% C0 2, 10% 0 2 , 80% N 2) for 7 days at 17°C. C e l l s 98 were fed on alternate days with feeding c o c k t a i l (10-20 /i l / w e l l ) . The gas box was purged with blood gas a f t e r each feeding. c. Haptenation of sheep red blood c e l l s (SRBC). SRBC's were obtained from an adult ewe (Sheep Unit, Department of Animal Science, U.B.C.). SRBC were stored i n a 1:1.2 r a t i o of blood to Alsever's buffer and were successfully maintained up to 1 month i n the r e f r i g e r a t o r . For haptenation, approximately 2.5 ml of t h i s mixture i s pipetted into a centrifuge tube and centrifuged for 5 min X 1200g X 4° C. The supernatent was discarded leaving 0.5 ml packed c e l l s . The packed c e l l s were then washed 3X with modified b a r b i t o l buffer (MBB), brought up to a 10 ml volume and centrifuged as before. After the f i n a l wash and spin, the TNP solution ( 200 fil aqueous TNP i n 3.5 ml cacodylate buffer) was added to the SRBC. The l i g h t s e n s i t i v e TNP must be kept foil-covered. The mixture was vortexed and mixed on a nutator (Clay-Adams) at room temperature for 20 minutes. Immediately a f t e r mixing, the tube was centrifuged as before and the supernatent discarded (should be orange i n colour) and c e l l s resuspended i n a g l y c y l c l y c i n e solution (3.7 mg g l y c y l g l y c i n e i n 5.83 ml MBB). The tube was centrifuged as before and the supernatent aspirated (this time i t should be yellow). The c e l l s were washed with MBB twice and resuspended i n MBB for storage i n the r e f r i g e r a t o r u n t i l use. Haptenated SRBC have been used with success for up to 4 days following haptenation. 99 d. Harvest assay l.SRBC's were washed twice with MBB as described above i n the haptenation protocol and brought up to 3.5 ml volume i n MBB af t e r the f i n a l wash and spin. 2. Dilute complement was prepared from frozen (-80°C) adult coho serum obtained i n November, 1989, from Capilano Hatchery, North Vancouver. Based on the r e s u l t s of s e r i a l d i l u t i o n s of the complement with MBB i t was found that a 1 i n 10 d i l u t i o n produced the desired l e v e l of a c t i v i t y i n a two hour time frame when combined with the hapenated SRBC. Complement was kept on ice throughout the assay. 3. C e l l cultures were prepared for the assay as follows; Tissue culture plates were removed from the gas box and centrifuged at 500g X 10 min at 4°C. The supernatent was discarded and fresh TCM was added back. Culture plate sealers (Dynatech Inc., C h a n t i l l y , Va., U.S.A.) were placed over the wells and the plate was vortexed to resuspend the c e l l s . The volume added back depended on the number of c e l l s i n the culture and the number of plaques one wanted to count. 4. Cunningam chambers were f i l l e d as follows; Working with 10-15 wells at a time, 50 / i l of one well containing the c e l l suspension, 10 / i l TNP-SRBC, and 10 / i l d i l u t e complement was added to a new tissue culture well. The contents of the well were 100 gently mixed with a Pasteur pipet and added to one side of a Cunningham chamber. After the second chamber was s i m i l a r l y f i l l e d , the edges were then sealed with melted p a r a f f i n by c a r e f u l l y dipping the sides of the chamber i n wax. Chambers were incubated at 17°C X 1.5-2 hours. 5. Plaque counts were made using dark f i e l d microscopy at 2.5X and questionable plaques checked for the presence of a lymphocyte i n the center at 10X. Results were expressed as number of plaques (APC)/10 6 viable WBC (the concentration of viable WBC must be determined at the time of the assay). There can be a 100 f o l d reduction i n viable c e l l numbers over the course of the seven day culture incubation. 101 Appendix B Part 2: Recipes Tissue Culture Medium Make up a s e p t i c a l l y i n a s t e r i l e 75 cm2 t i s s u e culture f l a s k (Corning): 172.0 ml RPMI 1640 Medium (without L-Glutamine) 20.0 ml Fetal Bovine Serum (Gibco Laboratories, Long Island, New York) 2.0 ml Non-Essential Amino Acids (Whittaker, Bioproducts, Walkersville, Ma) 2.0 ml Sodium Pyruvate (Whittaker) 2.0 ml L-Glutamine (Whittaker) 2.0 ml AUC Supplement (see below) 2.0 ml G Supplement (see below) 0.1 ml 2-Mercaptoethanol 0.1 M 0.2 ml Gentamicin Sulphate (Whittaker) Store ingredients i n the r e f r i g e r a t o r , keep L-Glutamine frozen i n s t e r i l e aliquots and only unthaw as much as needed. AUC Supplement Dissolve i n 100 ml Minimal Essential Medium (M.E.M, Gibco) 0.1 g Adenosine 0.1 g Uridine 0.1 g Cytosine F i l t e r s t e r i l i z e using a 0.45 u f i l t e r unit (Nalgene Brand, Nalge Co., Rochester, N.Y.) or equivalent and r e f r i g e r a t e i n 10-20 ml aliquots. G Supplement 0.1 g Guanosine i n 100 ml M.E.M.. Warm in water bath at 37°C to dissolve guanosine and then f i l t e r s t e r i l i z e . Feeding Cocktail 7.50 ml feeding " s t o c k t a i l " (see below) 3.75 ml Fetal Bovine Serum 0.50 ml AUC Supplement 0.50 ml G Supplement Thaw frozen feeding " s t o c k t a i l " and combine a s e p t i c a l l y with the other ingredients. Store r e f r i g e r a t e d . 102 Feeding " S t o c k t a i l " 70 ml RPMI 1640 10 ml Essential Amino Acids (Whittaker) 5 ml Non-essential Amino Acids 5 ml Dextrose solution (200 mg/ml manicure H 20) 5 ml L-Glutamine Combine the ingredients together and adjust the pH to 7.2 using 10 N NaOH. F i l t e r s t e r i l i z e and freeze i n 15 ml aliquots. Phosphate Buffered Saline (PBS) Dissolve the following i n 1 L nanopure H 20: 1.00 g KH 2P0 4 8.50 g NaCl 9.25 g Na 2HP0 4 adjust pH to 7.3-7.4. Phospahate Buffered Saline (pH 6.0. 0.06 M) Stock A: 8.28 g NaH 2P0 4*H 20 i n 1 L nanopure H 20 Stock B: 8.52 g Na 2HP0 4 i n 1 L nanopure H 20 Use 438.5 ml of Stock A and 61.5 ml Stock B make up to 1 L with nanopure H 20. Modified Barbitol Buffer (MBB) To make one 1 L of 5X stock; 1 v i a l of Barbitol Buffer, add 1 part MBB 5X stock to 4 parts PBS. pH should be 7.3-7.4. Alse v e r / s Solution (Anticoagulant) Dissolve the following i n 100 ml H 20; 2.05 g Glucose 0.80 g Tri-sodium c i t r a t e (anhyd.) 0.42 g Sodium chloride Adjust the pH to 6.1 with 10 % c i t r i c acid solution. S t e r i l i z e by f i l t r a t i o n . C o l l e c t blood into 1.2 volumes of Alsever's solution. 103 Cacodylate Buffer To prepare a 0.28 M solution of (CH 3) 2As(0)ONa'H 20 (sodium cacodylate); use 2.24 grams i n 50 ml of H20, adjust the pH to 6.9-7.1 with 0.2 M HC1. Appendix C: Conjugation of TNP to LPS. Solution A: 100 mg LPS (w) i n 5.0 ml cacodylate buffer (pH 6.9, 0.28M) A 5ml syringe was used to i n j e c t the cacodylate buffer into the bottle containing l y s o p h i l i z e d powder. Adjust pH to 11.5 with 5 N NaOH, fi n e tune with IN HC1 i f needed. Solution B: 60 mg of p i c r y l s u l f o n i c acid (TNBS) i n 5.0 ml cacodylate buffer and mix on a nutator (Clay-Adams) i n a f o i l wrapped container. Add solution B dropwise to solution A while shaking. Place on a nutator for 2 h. 15 cm of d i a l y s i s tubing (6-8 Kd cutoff) was soaked i n PBS. One end was clamped with d i a l y s i s tubing and the TNP-LPS mixture was poured i n the tube. The other end was then clamped and placed i n 1 L of PBS i n a f o i l wrapped erlenmeyer f l a s k with a s t i r bar. This was dialyzed for 4 hours against PBS. The PBS was then renewed and the solution was again dialyzed overnight. The PBS was replaced with RPMI and l e f t to dialyze f o r 24 hours. The contents of the d i a l y s i s tubing were 104 transferred into a f o i l wrapped centrifuge tube and pasteurized at 70°C for 40 min. i n a water bath. The TNP-LPS was transferred a s e p t i c a l l y into a f o i l wrapped serum bottle with a septum and clamped metal top and stored i n the r e f r i g e r a t o r . The concentration of t h i s stock bottle was 10 mg/ml. The working concentration was 1.0 /ig/ml i n TCM ( c e l l s were plated at 0.5 /xg/ml). 

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