Electrofishing is a sampling tool commonly used by fisheries researchers. While much is known about the effects of electroshock on fish physiology, consequences to the immune system and disease progression have not received attention. To understand the effects of electroshock on immune function we undertook a comparison of electroshock and handling stress in regards to selected immune functions and disease progression in juvenile spring chinook salmon (Oncorhynchus tshawytscha). The handling stress treatment was included to insure the responsiveness of the fish. Our objectives were to determine the effects of electroshock on immune function, determine the mechanism of any observed alteration, and to determine the effects of electroshock on disease progression. Skin mucous lysozyme concentrations were not affected by exposure to electroshock. Respiratory burst activity may be enhanced in leukocytes immediately after an in vitro electroshock. Any effect in vivo, however, appears to be brief given the lack of differences observed 3 h after exposure. The specific immune response, measured as the ability of anterior kidney leukocytes to generate antibody producing cells (APC), was suppressed 3 h after electroshock, but recovered within 24 h. This response was similar in timing and magnitude to that of fish subjected to an acute handling stress. The mechanism of suppression is hypothesized to be via elevation of plasma cortisol concentrations. The ability to generate APC may be suppressed 7 d after electroshock, but it is not evident what mechanism is responsible for this suppression. There was some evidence that the progression of a Renibacterium salmoninarum (RS) infection was altered after exposure to an electroshock. Exposure to electroshock did not have a clear affect on the severity of infection or the number of mortalities, but may have accelerated the time to death in infected fish that died. The limited duration of specific immune suppression and lack of effect on mortality in RS infected fish lead us to conclude that electrofishing under the conditions we tested is a safe procedure in regards to immunity and disease. / Graduation date: 2000

Environmental factors such as chemical contamination can have immunomodulatory effects on the immune response of fish and may be contributing to the decline in salmonid populations by augmenting disease susceptibility. Xenobiotics can interfere with the immune system at several levels of complexity, and different immune cells and processes have variable sensitivity to pollutants. For this reason, a suite of tests is required to evaluate immunomodulatory mechanisms. In this thesis, I formulated and calibrated an assay for the detection of humoral immunity for chinook salmon (Oncorhynchus tshawvtscha). Subsequently, I used this technique in conjunction with other immune and endocrine assays to detect effects of embryonic exposure to o,p'-DDE, a known environmental estrogen. The technique combines exposure of whole animals or leukocyte cultures to immunomodulatory agents/conditions with in vitro mitogenic activation of B-lymphocytes. The proportion of leukocytes undergoing blastogenesis following in vitro stimulation with lipopolysaccaride (LPS) was quantified by flow cytometric analysis of forward and side scatter properties. In addition, I used a fluorescein isothiocyanate labeled anti-rainbow trout surface immunoglobin monoclonal antibody (anti-RBT SIgM-FITC) to determine the ability of the lymphoblasts to express surface immunoglobin (SIgM) through flow cytometry. I used the assay to evaluate the effects of short-term exposures to o,p'-DDE during early life history stages on the long-term immune competence of fall chinook salmon. Immersion of chinook salmon eggs in 10 ppm o,p'-DDE for 1 h at fertilization followed by 2 h at hatch caused significant reductions in the ability of splenic leukocytes to undergo blastogenesis and express SIgM upon in vitro stimulation with LPS one year after treatment (ANOVA, P<0.05). The concentration of o,p'-DDE in fry treated with 10 ppm o,p'-DDE was 0.92 ��g g����� lipid one month post first feeding. The chemical persisted through development and, one year after exposure, levels in juvenile muscle tissue were 0.94 ��g g����� lipid. Mortality rate, time to hatch, fish size, sex ratios, gonadal development, plasma estradiol and 11-ketotestosterone concentrations were not affected by treatment with o,p'-DDE. In addition, neither plasma lysozyme concentration, nor mitogenic response of splenic leukocytes to concanavallin A or polyinosinic-polycytidylic acid were influenced by the treatment. A short period of exposure to an estrogenic chemical during early periods of development induced long term effects on humoral immune competence of chinook salmon. I discuss the possibility that the xenobiotic is exerting its activity through steroid-mediated pathways. / Graduation date: 2002

Graduation date: 1995

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. / Graduation date: 2001

Graduation date: 1992

Wild populations of fish are faced with a multitude of stressors, which may include human interaction, toxins, and disease. Benzo(a)pyrene (BaP), a known carcinogen and immunotoxin, has been reported in the stomach contents of juvenile chinook salmon, Oncorhynchus tshawytscha, in urban waterways. We investigated the impact of chronic dietary exposure of environmentally relevant levels of BaP on the immune system and cytochrome P4501A1 (CYP1A1) expression in juvenile chinook salmon. Two experiments were carried out in which juvenile fish were fed food treated with ethanol (control diet), low or high concentrations of BaP, or cortisol. In the first experiment we measured mitogen-stimulated proliferation of splenic leukocytes using flow cytometry and a colorimetric assay using Alamar Blue[superscript TM] Susceptibility to a microsporidian parasite, Loma salmonae, was evaluated in the second experiment by quantification of xenomas in the gills. Hepatic CYP1A1 and plasma cortisol were measured in both experiments. No significant trends were found in leukocyte mitogen activation or plasma cortisol between treatments or days. However, western blot analysis of CYP1A1 concentration in liver revealed interesting patterns of induction: in cortisol fed groups CYP1A1 was <20% of control on all days, groups fed low levels of BaP were 250% of control values on days 8 and 21 then dropped below control values on day 29, and groups fed high levels of BaP had less CYP1A1 than controls on all days. Similar patterns of CYP1A1 levels were found in the second experiment, and diseased control groups showed about a 55% decrease in CYP1A1 concentration when compared with non-diseased control groups. Susceptibility to L. salmonae was significantly higher in groups receiving cortisol. Whereas there was no effect of the high BaP dose, the low BaP dose appeared to increase disease susceptibility. This study supports concerns of stress and toxin induced immune dysfunction in wild populations of fish. / Graduation date: 2004

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