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 1−1 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 1−1 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.

Stress, including extreme or rapidly changing temperatures, are known to have deleterious effects on fish health and physiology. This thesis examines the combined effects of elevated acclimation temperature and acute handling stress on the number of antibody producing cells, plasma lysozyme concentrations, and the number of pronephric leukocytes in juvenile chinook salmon (Oncorhynchus tshawytscha). An additional goal of this thesis was to explore the effects of a temperature fluctuation, as a potential instigator of thermal shock, on innate immunity in wild fall chinook salmon of the Columbia River, specifically to determine if there are effects on plasma lysozyme concentrations and on the frequencies of lymphocytes, neutrophils, and thrombocytes in circulation. Finally, based on results found in an experiment involving elevated acclimation temperature, the relationship between the number of antibody producing cells and fish body weight was examined. Plasma lysozyme concentrations and the number of pronephric leukocytes were both affected by acclimation to 21°C compared to 13°C. While a positive relationship was found between temperature and lysozyme, an inverse relationship was found between temperature and the number of pronephric leukocytes. Plasma lysozyme concentrations, the number of pronephric leukocytes, and the number of antibody producing cells did not respond to the stressor, and the combination of elevated temperature and stress did not have an additive effect on any of the physiological or immunological variables studied. Differences between controls and temperature-treated fish were not detected among individual time points throughout a temperature fluctuation experiment, despite overall responses in plasma lysozyme concentrations and the frequencies of circulating lymphocytes. The frequencies of circulating neutrophils and thrombocytes did not respond to the thermal stressor. Finally, a significant positive relationship was detected between the number of antibody producing cells (assessed by a hemolytic plaque assay) and body weight among non-stressed fish acclimated to 21°C and 13°C. Regardless of acclimation temperature, these results emphasize the importance of the standardization of fish size for immunological experiments. Results from this thesis suggest that some components of innate immunity are affected by elevated acclimation temperatures and that the adaptive immune system is affected by acclimation temperature differently in small and large fish.