In this thesis, we examined the effects of the exposures to anthropogenic pollutants on the fish, primarily juvenile chinook salmon, immune system using newly and recently developed immune assays. In addition, we developed a new assay for measuring immunocompetence of fish. In the first chapter, the Alamar Blue assay was developed to quantify the proliferation of chinook salmon (Oncorhynchus tshawytscha) leukocytes. Isolated splenic and pronephric leukocytes were stimulated with different concentration of mitogens (LPS, PWM, and ConA) for various incubation times. Optimum cell culture conditions (cell density, mitogen concentration, and incubation time) for the Alamar Blue assay were evaluated by comparison with flow cytometric analysis. The Alamar Blue dye was non-toxic for leukocytes, and the assay proved to be able to quantify the mitogenic responses using LPS, but PWM and ConA. In the second chapter, we determined the effects and mechanisms by which p,p'- DDE exposure might affect the immune system of chinook salmon (Oncorhynchus tshawytscha). Isolated salmon splenic and pronephric leucocytes were incubated with different concentrations of p,p'-DDE, and cell viability, induction of apoptosis, and mitogenic responses were measured by flow cytometry and Alamar Blue assay. p,p'- DDE significantly reduced cell viability and proliferation and increased apoptosis. The effect of p,p'-DDE on pronephric leukocytes was more severe than on splenic leukocytes, likely because pronephric leucocytes had a higher proportion of granulocytes, cells that appear more sensitive to p,p'-DDE. The effect of p,p'-DDE on leucocytes appeared to vary between developmental stages or season. The mitogenic response of leukocytes of chinook salmon exposed to p,p'-DDE in vivo exhibited a biphasic dose-response relationship. Only leukocytes isolated from salmon treated with 59 ppm p,p'-DDE had a significantly lower percentage of Ig+ blasting cells than controls. Our results support the theory that exposure to chemical contaminants could lead to an increase in disease susceptibility and mortality of fish due to immune suppression. In the third chapter, we evaluated the direct effects of in vitro exposures to tributyltin (TBT), widely used biocide, on the cell mediated immune system of chinook salmon (Oncorhynchus tshawytscha). Splenic and pronephric leukocytes isolated from juvenile chinook salmon were exposed for 6, 24, or 96 hr to a concentration range of 0.03 0.1 mg TBT 1����� in cell cultures. Effects of TBT on cell viability, induction of apoptosis, and mitogenic responses were measured by flow cytometry. Splenic and pronephric leukocytes in the presence of TBT experienced a concentration-dependent decrease in the viability in cell cultures following the induction of apoptosis. In addition, pronephric lymphocytes exhibited a greater sensitivity to TBT exposure than pronephric granulocytes. The functional ability of splenic B-cells to undergo blastogenesis upon LPS stimulation was also significantly inhibited in the presence of 0.05, 0.07, or 0.10 mg 1����� of TBT in the cell cultures. Flow cytometric assay with the fluorescent conjugated monoclonal antibody against salmon surface immunoglobulin was employed for the conclusive identification of B-cell in the chinook salmon leukocytes. Our findings suggest that adverse effects of TBT on the function or development of fish immune systems could lead to an increase in disease susceptibility and its subsequent ecological implications. / Graduation date: 2004

Graduation date: 1992

Habitats for juvenile chinook salmon (Oncorhynchus tshawytscha) were observed at a high flow during the spring of 1989 and at a low flow during the spring of 1991 in the lower American River of California. Parameters of microhabitats in eight macrohabitats were measured. Mean column water velocity and total water depth variables were used to statistically compare juvenile chinook salmon microhabitat distributions between and among macrohabitat type/reach and flow combinations. Using mean column water velocity and total water depth as independent variables, each macrohabitat type/reach appeared to be unique. Mean column water velocity seemed to be a better descriptor of juvenile chinook salmon habitat selection than was total water depth. Riffles appeared to be the preferred macrohabitats for large non-schooling chinook salmon at both flow levels. Root wads, woody debris, and submerged terrestrial vegetation were utilized extensively and provided a significant cover for schooling chinook salmon during the high flow period. Habitat utilization by chinook salmon appears to be influenced by nonhydraulic variables. Application of models in current instream flow studies need to include factors such as water temperature and aquatic insect production. / Graduation date: 1993

Canada is the fourth largest producer of farmed salmon in the world, with Atlantic salmon being the major species cultivated. Paradoxically British Columbia (BC), which borders the Pacific Ocean, is the major producer province where Atlantic salmon was introduced in the mid-80’s. Escaped salmon may constitute a threat to natural populations of Pacific salmon as they compete for the same resources such as food and spawning territory. A potential solution to the aquaculture industry would be to further develop the aquaculture of native species in the region. The work presented here used semi-natural spawning channels to evaluate the effects of breeding strategies and early-rearing environments on the immune performance of Chinook salmon. Breeding strategy was tested analyzing artificial hatchery practices versus semi-natural propagation in spawning channels. Early-rearing environmental assessment contrasted indoor plastic hatchery tanks with outdoor gravelled-bottom spawning channels. A disease challenge involving over 1400 fish showed interaction effects between breeding strategy and rearing environment. Fish artificially mated presented a disease susceptibility influenced by the rearing environment. The contrary occurred in the offspring of self-breeding brood stock in the spawning channels, as no differences were observed in their susceptibility to the disease regardless of rearing environment. Monitoring of anti-Vibrio anguillarum antibodies during the disease challenge and a follow up of the survivors in sea net pens further confirmed the interaction between breeding strategy and rearing environment. Gene expression in pre- and post-infected artificially propagated fish showed differential gene expression when analyzed with a 695-gene cDNA microarray for Chinook salmon. Genotyping of major histocompatibility (MH) class II β1 alleles showed a tendency of a higher heterozygosity in survivors as expected, as well as a general tendency of a higher heterozygosity in semi-naturally propagated fish. The latter is likely a direct consequence of MH-linked mate choice, which was recently described in Chinook salmon (Neff et al., 2008). To further characterize the mating system of Chinook salmon in the spawning channels, brood stock were genotyped at 12 microsatellite loci. Females and males were found to mate randomly with regards to genetic pairwise relatedness, but they tended to mate with fish of similar condition as revealed by their pairwise differences in Fulton’s condition factor. This work demonstrated that genotype-by-environment interactions can modify the disease resistance of Chinook salmon. More importantly, these effects were seen after just one round of semi-natural spawning of domesticated hatchery fish, suggesting that further studies on spawning channels may highlight other hidden benefits. Therefore, breeding strategy and early-rearing environment should be considered when propagating cultured stocks. The use of more natural propagation methods such as spawning channels could improve the immune performance of Chinook salmon and help to expand the aquaculture of this native species in BC.

Marine and freshwater environments support numerous species of teleost fish with a wide and diverse range of reproductive strategies. Despite the considerable interest in fish reproduction, our knowledge regarding ejaculate traits and factors affecting them is limited. Using computer-assisted sperm analysis (CASA) I measured ejaculate traits (sperm swimming speed, motility, path trajectory, longevity and concentration) from sexually mature chinook salmon (Oncorhynchus tshawytscha) activated in freshwater and ovarian fluid. I also looked at these ejaculate traits in relation to measures of male quality (body condition) and investment into reproduction (relative testes mass). Furthermore, I determined the chemical composition of seminal and ovarian fluid and looked at the effect these fluids have on sperm behaviour. A considerable amount of intraspecific variation existed in all ejaculate traits measured, and investment into reproduction (relative testes mass) was dependent on male body condition, as males in better condition had relatively larger testes. However, these males did not have superior quality ejaculates or ejaculates with a higher density of spermatozoa; hence the potential reproductive advantage of having larger relative testes in this species remains unknown and requires further investigation. In addition, a positive relation between sperm longevity and sperm swimming speed was observed defying the expected trade-off between ejaculate traits according to theory. There was also a weak negative trend in our data between body condition and sperm swimming speed, linearity, and longevity. All sperm traits measured were greatly enhanced when activated in a solution containing ovarian fluid (a viscous fluid which is excreted with the egg batch during spawning) from female chinook salmon. Interestingly, sperm swimming speed activated in fresh water only accounted for < 12% of the observed variation in mean sperm swimming speed in ovarian fluid. This result suggests the sperm traits measured in fresh water are not relevant to those same traits measured in ovarian fluid, so caution should be applied when comparing the potential for individual males to fertilize ova when sperm traits are activated in water, especially in studies of sperm competition in an externally fertilising species. Sperm competition between males is known to strongly influence sperm and ejaculate traits, but less is known about female sperm choice after copulation via a process called cryptic female choice (CFC). In CFC, females may have the ability to favour the sperm of one male over another and bias fertilisation accordingly. To test whether ovarian fluid could act as a mechanism of CFC in an externally fertilising fish species, I measured sperm traits from each male activated in the ovarian fluids from different females. I found that mean sperm swimming speed, longevity, and path trajectory differed significantly among males, but most importantly, the pattern of within-male variation in these traits also varied significantly among males in response to different females’ ovarian fluids. This result suggests that ovarian fluid may be a potential mechanism of CFC whereby females differentially enhance the swimming speed of sperm from different males. In addition, I found that sperm longevity was negatively correlated with variation in [Ca²⁺] and [Mg²⁺] concentration in the ovarian fluid, while percent motility increased with increasing concentration of [Mg²⁺]. These observations provide a possible chemical basis for cryptic female choice whereby female ovarian fluid differentially influences the behaviour of sperm from different males and thus their fertilisation success. This finding is particularly exciting, as we may have uncovered a potential mechanism of CFC in an externally fertilising species, which is poorly understood. In addition, results from this study suggest new directions for genetic studies to provide direct evidence for CFC. For example, does sperm selection via ovarian fluid promote favoured genetic combinations that enhance male reproductive success?

Canada is the fourth largest producer of farmed salmon in the world, with Atlantic salmon being the major species cultivated. Paradoxically British Columbia (BC), which borders the Pacific Ocean, is the major producer province where Atlantic salmon was introduced in the mid-80’s. Escaped salmon may constitute a threat to natural populations of Pacific salmon as they compete for the same resources such as food and spawning territory. A potential solution to the aquaculture industry would be to further develop the aquaculture of native species in the region. The work presented here used semi-natural spawning channels to evaluate the effects of breeding strategies and early-rearing environments on the immune performance of Chinook salmon. Breeding strategy was tested analyzing artificial hatchery practices versus semi-natural propagation in spawning channels. Early-rearing environmental assessment contrasted indoor plastic hatchery tanks with outdoor gravelled-bottom spawning channels. A disease challenge involving over 1400 fish showed interaction effects between breeding strategy and rearing environment. Fish artificially mated presented a disease susceptibility influenced by the rearing environment. The contrary occurred in the offspring of self-breeding brood stock in the spawning channels, as no differences were observed in their susceptibility to the disease regardless of rearing environment. Monitoring of anti-Vibrio anguillarum antibodies during the disease challenge and a follow up of the survivors in sea net pens further confirmed the interaction between breeding strategy and rearing environment. Gene expression in pre- and post-infected artificially propagated fish showed differential gene expression when analyzed with a 695-gene cDNA microarray for Chinook salmon. Genotyping of major histocompatibility (MH) class II β1 alleles showed a tendency of a higher heterozygosity in survivors as expected, as well as a general tendency of a higher heterozygosity in semi-naturally propagated fish. The latter is likely a direct consequence of MH-linked mate choice, which was recently described in Chinook salmon (Neff et al., 2008). To further characterize the mating system of Chinook salmon in the spawning channels, brood stock were genotyped at 12 microsatellite loci. Females and males were found to mate randomly with regards to genetic pairwise relatedness, but they tended to mate with fish of similar condition as revealed by their pairwise differences in Fulton’s condition factor. This work demonstrated that genotype-by-environment interactions can modify the disease resistance of Chinook salmon. More importantly, these effects were seen after just one round of semi-natural spawning of domesticated hatchery fish, suggesting that further studies on spawning channels may highlight other hidden benefits. Therefore, breeding strategy and early-rearing environment should be considered when propagating cultured stocks. The use of more natural propagation methods such as spawning channels could improve the immune performance of Chinook salmon and help to expand the aquaculture of this native species in BC.

A feeding experiment was designed to examine how fish growth rates are affected by the size abundance of pelagic zooplankton. Juvenile chinook salmon, Oncorhynchus tshawytscha , were raised in 90 m³ mesh enclosures in Saanich Inlet, B.C. The enclosures permitted ample water and zooplankton circulation while retaining 5-6 gram juvenile salmon. The enclosed fish grew at an average rate of 1.8% wet body weight/day for a six week experimental period. Weekly growth rates ranged from 3.9%/day while food was abundant, to -0.5%/day when food was scarce. Several analytical methods were used to establish a relationship between fish gr.owth and the size and abundance of zooplankton in the enclosures. There is a strong relationship between the fish growth rates and the abundance of 1.4-4.5mm prey. Rates of successful search vary directly with the size and inherent contrast of a prey item. A minimum rate of successful search of 2.0m³/hour was estimated from a functional response curve for salmon feeding on 1.4-4.5mm zooplankton. This value is discussed in relation to a salmonids physical capabilities and results from previous field studies and tank experiments. Daily growth increments on the otoliths of the enclosed fish were examined with respect to daily variations in water temperature and zooplankton abundance. Extremes in food abundance appear to have a significant and consistent effect on the spacing of the growth rings. However, water temperatures would have to be kept constant in order to establish any closer relationship between food abundance and otolith growth rings. / Science, Faculty of / Zoology, Department of / Graduate

Incremental growth rings in X-rays of salmon vertebrae have been used since the 1980s to age Pacific salmon (Oncorhynchus spp.) remains from archaeological sites in the Pacific Northwest. These age estimates, paired with generalized life history patterns, have been used to determine salmon species, season of capture and in turn season of site occupation. This approach relies on a variety of assumptions, the most fundamental of which is that rings represent true years. Archaeologists using vertebral age determination techniques have failed to adequately test this assumption and present their methodologies. This thesis assesses the validity of using incremental growth structures in Chinook salmon (Oncorhynchus tshawytscha) vertebrae to determine the age at death of fish represented in archaeological sites. This project develops criteria and a protocol for the identification of true annuli and tests these identifications on a collection of modern Chinook salmon of known age. Finally, this protocol is applied to archaeological remains of Chinook from Cathlapotle (45CL1). Three collections of modern known age fish (N=121) were used to evaluate and test approaches to aging Chinook salmon with vertebrae. These collections contained juvenile and adult Chinook from throughout Washington and Oregon. I evaluated a variety of methods for viewing rings including magnified surface images, X-ray images and thin sections to determine which is the most accurate, reliable and efficient, also considering the extent of specimen destruction. Rings visible in X-rays were found to reflect the internal structure of vertebrae rather than annular growth. The number of these internal walls did not correspond to the known ages of fish and are therefore not true annuli. Criteria previously described by salmon fisheries biologist were used to isolate annuli (on the centrum surface) on the Hanford reach collection (N=46). In a test for accuracy 39 (85%) were aged correctly. In a test of reliability utilizing five additional readers all but 14 cases showed discrepancies among readers. Results of the test of reliability were not as successful as other researchers in fisheries biology but given the high accuracy rate the method remains valid. The final goal of this project was to determine the feasibility of applying the surface ring method of age determination to archaeological collections. Archaeological salmon vertebrae from Cathlapotle (45CL1) on the lower Columbia River, Washington state were utilized. One hundred salmon vertebrae were selected and classified to species according to Huber et al. (2011); 89 were identified as Chinook. Of these, 39 had sufficient preservation of the surface to view and interpret incremental rings. Three ages were identified 3, 4 and 5 year olds. This ageing protocol can be applied to archaeological Chinook salmon vertebrae to estimate age of ancient Chinook salmon. Additional work is needed on other salmon species to demonstrate the methods validity across all salmonid species. This analysis has great potential for modeling salmon paleo-life history by contributing data from salmon populations prior to the major impacts of the 19th and 20th century. This is particularly valuable for salmon conservation because information on size, the timing and duration of freshwater emigration as well as the age of spawning and death is critical to the management of hatchery and wild salmon populations.

Recent research conducted throughout the Northern California Current (NCC) on the ecology of Pacific salmon (Oncorhynchus spp.) indicates that variable ocean conditions affect the community composition of zooplankton in the nearshore environment which, in turn, can affect the quality of prey for fish, sea birds and mammals. Interannual variability in the quality and composition of the copepod community in the NCC during early marine residency of some Pacific salmon populations is related to survival to adulthood. However, copepods make up a small portion of the diet of coho and Chinook salmon, and the mechanistic linkages between ocean climate, zooplankton composition and salmon prey remain unclear. Parasite analysis provides a supplement to traditional diet analysis that can describe the foraging history of a host species. Coho salmon (O. kisutch) and Chinook salmon (O. tshawytscha) serve as hosts to an array of marine parasites acquired through consumption of infected intermediate hosts such as copepods, euphausiids, and planktivorous fishes. Causing little or no harm to their salmon host, the presence of trophically transmitted parasites provides information on the dietary history of their salmonid host beyond the 24 hours associated with traditional diet analysis. This study (1) examined differences in feeding behavior of coho and Chinook salmon during their early marine residency using both stomach and parasite community analyses and (2) tested the hypothesis that variability in ocean circulation patterns (measured through the Pacific Decadal Oscillation, sea surface temperature (SST) and Bakun's upwelling index) and copepod species composition are related to variability in the community structure of trophically transmitted marine parasites found in juvenile salmon. I compared the abundance and species composition of parasites recovered from juvenile Columbia River coho and upper Columbia River summer and fall Chinook salmon captured off the coast of Washington from 2002 to 2009. I also compared interannual variability in parasite assemblages to physical and biological indices of ocean conditions. Coho and Chinook salmon consumed similar prey taxa; however, the species richness and abundance of trophically transmitted parasites indicated that Chinook salmon consumed a greater diversity and abundance of infected prey. In addition, differences in the abundance of fish in the diet and Anisakis simplex, a parasitic nematode known to infect salmon through fish consumption, suggest that Chinook salmon consistently consumed more fish prey than coho. In contrast, coho appeared to consume more euphausiids as indicated by stomach content analysis and increased abundance of the euphausiid parasite, Rhadinorhynchus trachuri. Shifts in the parasite community composition of both coho and Chinook salmon were related to interannual variability in SST and the biomass of southern-origin copepods (r > 0.7, P < 0.05). The acanthocephalan R. trachuri and a tetraphyllid cestode were associated with "warm" SSTs and greater biomass of lipid-poor, subtropical copepods while the nematode A. simplex was more abundant in years of "cold" SST and a relatively low biomass of subtropical copepods. These results provide novel insight into differences in the diet of Columbia River coho and Chinook salmon and illustrate linkages between ocean climate, zooplankton community composition and salmon diet during early marine residency. / Graduation date: 2012

A series of experiments with Aeromonas salmonicida and infectious hematopoietic necrosis virus (IHNV) were carried out to determine dynamics of the spread of infection in chinook salmon (1.2-1.98g) and rainbow trout (1.2-3.1g). It was found in experiments with A. salmonicida that fish infected by bath immersion became infectious at 4 days postexposure (dpe), one day prior to dying from furunculosis. In cohabitation experiments with a single infected fish donor, an average of 75% disease specific mortality was obtained. There was suggestive evidence that there is a positive relationship between holding volumes and furunculosis prevalence in cohabitation experiments with single donor fish. Median day to infection was inversely correlated with density. The threshold density at density of 1.97 fish/L was approximately 30 times less than the density of 0.08 fish/L, 13.33 and 320 fish respectively. Reproductive ratio (R₀) and transmission coefficient (β) in the furunculosis epizootic were 3.23 and 0.021 (individuals*day)⁻¹ respectively. The mortality rate (α) of infected animals was 28.7% per day. The models constructed successfully mirrored the results of laboratory experiments. Data produced by simulation of the models were significantly associated with the data obtained from laboratory experiments for susceptible (S) class and also for infected class. In similar experiments carried out with IHNV, it was found that donor fish became infectious 3 dpe. The virus levels in donor fish and prevalence levels were also highly associated. Smaller volumes of that led to higher prevalence levels than observed in bigger volumes with 60 or 30 fish in each. A single donor fish was able to transfer virus to recipient fish. However, unlike the A. salmonicida experiment, transmission was insufficient to initiate a full-scale infectious hematopoietic (IHN) epizootic. Estimated parameters for dynamics of infection were approximately half of the values for A. salmonicida (R₀=2.57,β=0.008 (individuals*day)⁻¹ and α=0.15). The models constructed for IHNV spread were used to simulate the results of density experiment. However, it was not possible to test the association between susceptible and infected classes due to inadequate number of infected fish. / Graduation date: 2001