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<p>Salmonids, such as rainbow trout, form social hierarchies, consisting of dominant and subordinate individuals, when in groups in environments with limiting resources, such as space or food. There were two main objectives for this thesis: to investigate the physiological consequences of being in a social hierarchy as well as to investigate whether grouped social status or individual behaviour most accurately recorded physiological data for a hierarchy and secondly, to investigate the behavioural and physiological changes of groups of rainbow trout exposed to ammonia concentrations which are above chronic protected guidelines.</p> <p>To create social hierarchies, groups of four fish were fed by a new method using a darkened feeding container, twice daily (morning and evening) for eight days. Each morning feeding was videotaped in order to record aggressive behaviour. Each aggressive act was scored, allowing for fish to be assigned a social status. For ammonia exposures, groups of fish were exposed to either 700, 1200 and 1500 µM total ammonia (or 2.97, 5.10, 6.37 µM NH3, respectively) 24 hours before first feeding and these concentrations were maintained throughout the experiment. On day 5 and day 10, physiological parameters were taken in fish fasted for 24-h in control and 700 µM total ammonia exposed hierarchies.</p> <p>Social hierarchies were created in all ammonia-free and 700 µM total ammonia groups, with no hierarchies formed in 1200 and 1500 µM total ammonia groups. In M.Sc. Thesis – Josias (Si) Grobler McMaster University – Department of Biology iv ammonia-free hierarchies, one fish would become dominant, while the three subordinate individuals would each assume a stable social rank and display similar physiology which was different from the dominant fish.</p> <p>Fish from the 700 µM total ammonia group showed reductions in various physiological parameters during period 1, however, these fish displayed similar values as what was reported in ammonia-free hierarchies during period 2. This suggests biochemical or physiological changes occurring in these fish in order to acclimate to the high ammonia environment</p> / Master of Science (MSc)

<p> The development of Na+ and Ca++ transport, ammonia excretion, and respiration in larval rainbow trout were studied over the period of yolk sac absorption and shortly after the onset of exogenous feeding. The gills and the yolk sac epithelium of larvae were examined for any indication of the presence of mitochondria-rich cells and/ or Na-K-ATPase activity. </p> <p> Whole body Na+ content of the larvae increased throughout yolk sac absorption and after the onset of exogenous feeding. During this period there was an increase in Na+ influx, efflux, transporter capacity Umax> and transporter affinity (Km). Jmax increased 4.6 times during yolk sac absorption, and increased another 2.8 times 248 degree·days after the onset of exogenous feeding. Km decreased over the first 168 degree·days and stayed constant for the remainder of the experimental period. The gills were the primary site of Na+ efflux. Na+ accumulation from the water began the first day post hatch. </p> <p> Ca++ transport capacity increased throughout yolk sac absorption, but then decreased after the onset of exogenous feeding. Jmaxca++ exhibited a 15.5 fold increase over 257 degree·days, which then decreased to 30% of that value after exogenous feeding commenced. There was a 162 fold increase in Ca++ Km over yolk sac absorption, which then decreased 15.6 times after the onset of exogenous feeding. Whole body Ca++ began to increase approximately 200 degree·days post-hatch and exhibited a large increase after the onset of exogenous feeding. It is possible that during yolk sac absorption the larvae does not begin to accumulate Ca++ from the external environment until its internal reserves are depleted, and with the onset of feeding the larvae then obtains its Ca++ from its diet. </p> <p> Ammonia excretion in larval trout increased 3.6 times over yolk sac absorption, and increased another 2.8 times after exogenous feeding commenced. There was no evidence of a link between Na+ uptake and NJ-:4+ excretion in larval fish as NJ-:4+ excretion was not stimulated in response to increasing external Na+ concentrations. Measurements of the ammonium concentration at different surfaces of the larvae revealed a larger ammonium concentration next to the gills than next to the yolk sac and skin. This may indicate that the gills are the primary site for ammonia excretion in the larval fish. </p> <P> Initially, branchial and cutaneous surfaces contributes to respiration, but as the gills grow and develop and the yolk sac decreases in size and the skin thickens, the contribution of the gills to this function increases. </p> <p> The gills of first day hatchlings are comprised of gill arches and filaments. Lamellae do not begin to develop until a few days after hatching. The gill filaments contain mitochondria-rich cells and have Na-K-ATPase activity as determined through binding of the fluorescent dye anthroylouabain. The yolk sac epithelium contained cells with concentrated regions of mitochondria, but significant Na-K-ATPase activity was not detected when compared to branchial staining. </p> / Thesis / Master of Science (MSc)

Juvenile rainbow trout were chronically exposed to acidified softwater, alone and in combination with a slight temperature increase in order to understand the possible effects of global warming and environmental acidification in freshwater fish. The second goal was to determine the role of diet in the response to acid stress and elevated temperatures. In the first two exposures, a simulated global warming scenario (+2°C) was applied for 90 days in winter (8-12 °C), in the presence and absence of sublethal acidity (pH 5.2). In the first trial, fish were fed to satiation twice daily, while in the second trial, fish were fed only 1% of their wet body weight every four days (~0.25% daily). A slight increase in temperature caused a marked increase in oxygen consumption, nitrogenous waste excretion and growth, although there did not appear to be any specific pH effects. During the Satiation Exposure, fish exposed to low pH especially at slightly elevated temperatures had increased appetites compared to non-acid exposed fish. This increased appetite suggested that NaCl losses brought about by low pH exposure, stimulated appetite in some way, thereby alleviating any ionoregulatory disturbances. During the Limited Ration Exposure, ionoregulatory disturbances occurred during low pH exposure, with more dramatic effects in fish at slightly elevated temperatures. Trout maintained on a limited diet had a higher mortality rate, lower plasma and whole body Na⁺ and Cl⁻ concentrations, and elevated cortisol levels compared with fish fed to satiation. Thus, it became clear that fish could use food to compensate for the stresses of increased temperature and low pH. The third exposure was conducted to determine whether food simply provided the necessary fuel to meet the increased cost of living in a low pH environment, or whether food directly provided the dietary salts necessary to replace branchial ion loss. Diets were formulated at two levels of energy (regular: 16.31MJ/kg or low: 9.77MJ/kg) and two levels of NaCl (regular: 263 mmols/kg or low: 43 mmols/kg) using a factorial design (2x2=4 treatments). In addition, a fifth group of fish were not fed during the exposure. All five groups of fish were challenged with pH 4.0 to induce a rapid ionoregulatory disturbance and then held at pH 5.2 for the next 28 days. During this month, fish were fed 0.6% of their body weight of one of the four diets. Fish fed the low salt diets incurred typical long-term ionoregulatory disturbances with decreased whole body Na+, K+ and Cl concentrations. These effects were not seen in fish fed regular salt diets, regardless of energy content, showing that it is the salt content of the food rather than the energy content which is critical in protecting against the deleterious effects of low environmental pH. Interestingly, fish fed the regular energy/low salt diet had high cortisol levels and increased mortality while fish fed the regular salt diets, low energy/low salt diets and starved fish did not have a high rate of mortality. These results may have been due to differences in metabolic rate and therefore oxygen consumption (M₀₂). Starved fish had the lowest M₀₂. Fish fed the regular energy diet had increased post-prandial M₀₂ due to the specific dynamic action evoked in fish by ingestion of protein-rich food. An increase in oxygen consumption may have caused an increase in branchial ion loss, thereby exacerbating the ionoregulatory deficit associated with chronic acid exposure. This is detrimental when dietary salts are unavailable to replace branchial losses. Overall, the salt content of food may play an important role in ameliorating the deleterious effects of chronic low pH, while the energy content of food may complicate the response. / Thesis / Master of Science (MS)

Thesis / Master of Science (MS)

This thesis examined the effects of exhaustive exercise and post-exercise recovery on metabolism and respiratory gas exchange in juvenile rainbow trout, in relation to the classical oxygen (O₂) debt hypothesis (Hill & Lupton, 1923). The initial study was a detailed quantitative analysis of the metabolic cost of post-exercise recovery in terms of O₂ and ATP equivalents. A 5 min bout of exhaustive exercise resulted in about a 2.0-2.5 fold increase in O₂ consumption (MO₂), a 6-8 fold increase in whole-body lactate (LAC) levels and a near depletion in whole-body glycogen (GLY), adenosine triphosphate (ATP) and creatine phosphate (CP) stores. Recovery of MO₂, LAC and GLY was usually complete by 6 h, though GLY did not always return to resting levels. Recovery of resting whole-body ATP required 1.0-1.5 h, whereas restoration of CP required only 5 min. Quantitative budgets of the cost of recovery were prepared, based on two opposite assumptions. The first scenario (A) assumed that all the GLY resynthesized came from LAC, and that the remaining 25% of the total LAC cleared was oxidized. This scenario accounted for 48% of the excess post-exercise O₂ consumption (EPOC) and 94% of the ATP budget. The alternative scenario (B), attributed 100% of the EPOC to LAC oxidation (64% of the total LAC cleared), while the remaining LAC (36%) was resynthesized into GLY. As scenario B accounted for only 35% of the ATP budget, scenario (A) appeared more probable. Subsequent experiments attempted to experimentally dissociate LAC disappearance from EPOC, via repetitive exercise bouts and prior GLY depletion. A 2nd bout of exhaustive exercise, given 6 h after the 1st, significantly reduced EPOC by 40%, whereas metabolite status (LAC disappearance and GLY, ATP, and CP restoration) remained essentially unchanged. In contrast, prior GLY depletion by 5 days starvation significantly lowered the LAC burden while EPOC remained unchanged. Neither the two scenarios of the theoretical analysis could adequately explain the relationships between EPOC and the various metabolites. It is likely that some blend of the two approaches may be more realistic. Nevertheless, these approaches all point to the conclusion that the EPOC is not directly related to the metabolism of LAC after exercise in the rainbow trout. Thus, in contrast to the classical O₂ debt hypothesis, LAC disappearance does not determine the magnitude EPOC after exhaustive exercise in rainbow trout. / Thesis / Master of Science (MS)

Juvenile rainbow trout were chronically exposed to cadmium in hard water, soft water, and in calcium-supplemented soft water in order to understand the effects of long term cadmium exposure in freshwater fish. A particular goal was to characterize changes in gill cadmium burden and the cadmium-binding properties of the gills during chronic sublethal exposures, so as to examine the applicability of the acute gill surface metal binding model or Biotic Ligand Model to trout chronically exposed to cadmium. Trout were exposed for 30 days to sublethal concentrations of cadmium in: a) moderately hard, Hamilton tap water (Ca = 1000 μM), b) synthetic soft water (Ca = 130 μM), or c) calcium-supplemented soft water (Ca = 260, 470, 770, and 1200 μM Ca). For both the hard and soft water cadmium exposures, no effects were observed on growth, swimming performance, and whole body ions. Growth and whole body and plasma Ca²⁺ concentrations were similar for all treatments in the calcium-supplemented soft water experiment; however, swimming performance was significantly reduced for the 470 μM Ca + Cd exposed fish. Acclimation to cadmium occurred in the hard water and lower concentrations of calcium-supplemented exposures but not in the soft water exposure. Cadmium accumulation was greatest in kidneys and gills and was directly related to cadmium exposure concentration. Tissue metal burdens were reduced with increased water calcium concentrations. Affinity of the gill for cadmium and the number of binding sites for cadmium decreased at higher water calcium concentrations. Affinity of the gill for Cd decreased with chronic cadmium exposure but binding site numbers increased with chronic Cd exposure. The acute gill binding model or Biotic Ligand Model, originally developed in soft water, was successfully applied to fish in both hard and soft water; however, complications arose when extending the model to fish chronically exposed to cadmium at various water calcium concentrations. / Thesis / Master of Science (MS)

Thesis / Master of Science (MS)

The expression of the estradiol-responsive genes in the liver of rainbow trout, Salmo gairdnerii, has been studied as a system which may permit quantitative measure of adverse sublethal effects of various xenobiotics. Upon administration of 17 β-estradiol to male or immature female rainbow trout, the expression of two genes are markedly enhanced. One of these genes codes for the egg yolk precursor protein, vitellogenin, whereas the other codes for another, as yet unidentified, protein. Complementary DNA (cDNA) genes of these two proteins have been employed as probes to characterize the dose-response relationship as well as the time course induction of these two classes of transcripts in liver both in vivo and in vitro. The maximal net transcription of pRTC 2 occurs at 20 ug β -estradiol per 100 gram fish body weight whereas those encoded for by pRTC 5 display maximal transcription at 5 μg β-estradiol per 100 gram fish body weight. With doses exceeding the dose required for maximal induction of transcripts, both classes of transcripts reach their maximal levels between 4 and 8 hours after primary and secondary induction. Transcripts homologous to pRTC 5 return to control levels by 16 hours and 2 days after primary and secondary induction respectively. Those homologous to pRTC 2 however, were not observed to recover to normal levels in the primary induction even 16 days after stimulation with estradiol. However, upon secondary induction pRTC 2 transcripts returned to control levels 8 days post-inoculation with estradiol. The effect of various xenobiotics on the induction of these two estradiol-responsive genes was investigated. Pretreatment with β-naphthoflavone resulted in neither the induction or repression of both classes of transcripts. Administration of Kepone prior to a subsequent stimulation with estradiol, resulted in a decrease in the accumulation of pRTC 2 transcripts only. In contrast, an isomer of DDT, p,p'-DDT, was found to enhance the expression of pRTC 5 transcripts in fish subsequently administered β-estradiol. Oncogenes have been found to be activated in transformed cells or cells treated with various xenobiotics. In this study, I have shown that the cellular oncogene, pRTC-myc 1-81, is expressed in the liver at an elevated level in response to estradiol treatment. The two classes of polyadenylated transcripts were detected (2.5 kb and 5.6 kb). The three estradiol-responsive genes mentioned above (pRTC 2, pRTC 5, and pRTC-myc 1-81) were also shown to be transcribed in an established rainbow trout hepatoma cell line. Transcriptional activities of all three genes, as well as the translational activity of vitellogenin, were diminished upon growth in serum stripped of endogenous hormones. Further characterization of this rainbow trout hepatoma cell line is warranted so that it may be employed in a sensitive and quantitative bioassay for assessing the pathobiochemical effects of environmental xenobiotics on fish reproduction. Such an index may be beneficial for establishing adequate guidelines for water contaminants that potentially could affect aquatic and human reproductive success. / Thesis / Master of Science (MS)