Hypoxic condition is a naturally occurring environmental stressor in aquatic ecosystems. However, due to modern anthropocentric activities, hypoxia has been increasing in prevalence and severity. Rainbow trout, a keystone species in many North American lakes, is hypoxia intolerant. As a result, this species is of particular concern when studying the effects of hypoxia on an organism’s physiological functioning. Chronic starvation was used as a tool to induce metabolic depression to determine the effect that depressed metabolic rate had on hypoxia tolerance. Juvenile rainbow trout were deprived of food for five weeks at 15oC. Each week, routine metabolic rate (RMR) and critical oxygen tension (Pcrit) were measured. Concomitantly, resting and post-hypoxia fish (8 h at ~50% air saturation) were sampled to measure metabolites in blood, liver and muscle, as well as enzyme activities in select tissues. Food deprivation resulted in a decrease in routine metabolic rate (RMR) and shift towards an increased reliance on aerobic metabolism. Pcrit decreased significantly following four weeks of food deprivation respectively, indicating that metabolic depression induced by food deprivation may confer an increased tolerance to low environmental oxygen concentration ([O2]). However, marginal metabolic scope (MMS), another indicator of hypoxia tolerance, did not change in response to metabolic depression. Furthermore, subjecting trout to O2 limitation resulted in mobilization of carbohydrates from the liver subsequently leading to hyperglycemia. This was likely a survival technique ensuring that if severe hypoxia ensues, anaerobic substrates are ready for transport to the necessary tissues. / Thesis (Master, Biology) -- Queen's University, 2011-10-12 23:21:04.517

No description available.

The three-spot wrasse (Halichoeres trimaculatus) is distributed in and around the coral reefs and shallow rocky areas in the tropical and subtropical Indo-Pacific regions. This species displays a distinct diurnal behavior, burrowing under the sand at dusk and emerging out of the sand at dawn, which appears to be synchronized to the photoperiod. In this thesis, the hypothesis tested was that this unique life-style subjected the animal to daily hypoxia exposure while under the sand at night. The measurements of oxygen concentration in the sand around the fish at night confirmed a complete lack of oxygen. The study had three specific objectives: i) obtain a tissue-specific temporal profile of the hypoxia-related molecular and biochemical responses in wrasse over a 24 h diurnal cycle, ii) determine the responses that were unique to sand dwelling and iii) determine if the responses seen at night in the sand are similar to an anoxic response in this species. Wrasse were maintained in a flow-through seawater aquaria (29 ±1°C), with sand at the bottom for the fish to hide, and kept under natural photoperiod. The fish were sampled at 10:00, 14:00, 18:00, 21:00, 24:00, 3:00, and 6:00 clock time and plasma and tissue (brain, liver, gill, heart and muscle) were collected to determine the molecular and biochemical responses over a 24 h period. Fish were also sampled from aquaria without sand at night to determine the responses that were specific to hiding in the sand, while fish exposed to nitrogen gas bubbling for 6 and 12 h served as the anoxic group. A partial cDNA sequence of the hypoxia-inducible factor (HIF)-1α and neuroglobin (two genes that are hypoxia-responsive) were cloned and sequenced from the liver and brain, respectively, and their expression was determined using real-time quantitative PCR. HIF-1α mRNA abundance was higher in the brain compared to the liver and the gills, while a clear pattern of diurnal change in tissue HIF-1α and brain neuroglobin gene expressions was not observed at night relative to the fish during the day. However, wrasse brain showed a significant reduction in glycogen content at night under the sand and this corresponded with a higher hexokinase activity and increased glucose level suggesting enhanced glycolytic capacity. The plasma glucose and lactate levels were significantly lower at night, while in sand, relative to the day. The lower plasma glucose at night corresponded with a significant drop in liver gluconeogenic capacity (reduction in phosphoenolpyruvate carboxykinase, a key gluconeogenic enzyme, activity), while the lower lactate levels support a lack of activity along with the absence of glycogen breakdown in the muscle. Overall, there was a reduction in the metabolic capacity in the gills, heart, liver and muscle, but not the brain, supporting a tissue-specific metabolic reorganization as an adaptive strategy to cope with sand-dwelling in the wrasse. The molecular and biochemical responses seen in the wrasse at night in the sand was dissimilar to that seen in fish exposed to anoxia, leading to the conclusion that this species is not experiencing a complete lack of oxygen while under the sand. Also, the lack of muscle movement associated with sand dwelling at night limits anaerobic glycolysis for energy production, thereby eliminating lactate accumulation that was evident in fish exposed to anoxia. Taken together, wrasse showed a tissue-specific difference in metabolic capacity at night while hiding under the sand. While the mechanism involved in this tissue-specific energy repartitioning at night is unclear, one hypothesis involves selective increase in blood flow to the brain, while limiting peripheral circulation, as a means to maintain oxygen and glucose delivery to this critical tissue while the fish is hiding under the sand. The higher metabolic capacity of the brain, but not other tissues, at night under the sand suggests that maintaining the brain function is essential for the diurnal life-style in this animal.

The three-spot wrasse (Halichoeres trimaculatus) is distributed in and around the coral reefs and shallow rocky areas in the tropical and subtropical Indo-Pacific regions. This species displays a distinct diurnal behavior, burrowing under the sand at dusk and emerging out of the sand at dawn, which appears to be synchronized to the photoperiod. In this thesis, the hypothesis tested was that this unique life-style subjected the animal to daily hypoxia exposure while under the sand at night. The measurements of oxygen concentration in the sand around the fish at night confirmed a complete lack of oxygen. The study had three specific objectives: i) obtain a tissue-specific temporal profile of the hypoxia-related molecular and biochemical responses in wrasse over a 24 h diurnal cycle, ii) determine the responses that were unique to sand dwelling and iii) determine if the responses seen at night in the sand are similar to an anoxic response in this species. Wrasse were maintained in a flow-through seawater aquaria (29 ±1°C), with sand at the bottom for the fish to hide, and kept under natural photoperiod. The fish were sampled at 10:00, 14:00, 18:00, 21:00, 24:00, 3:00, and 6:00 clock time and plasma and tissue (brain, liver, gill, heart and muscle) were collected to determine the molecular and biochemical responses over a 24 h period. Fish were also sampled from aquaria without sand at night to determine the responses that were specific to hiding in the sand, while fish exposed to nitrogen gas bubbling for 6 and 12 h served as the anoxic group. A partial cDNA sequence of the hypoxia-inducible factor (HIF)-1α and neuroglobin (two genes that are hypoxia-responsive) were cloned and sequenced from the liver and brain, respectively, and their expression was determined using real-time quantitative PCR. HIF-1α mRNA abundance was higher in the brain compared to the liver and the gills, while a clear pattern of diurnal change in tissue HIF-1α and brain neuroglobin gene expressions was not observed at night relative to the fish during the day. However, wrasse brain showed a significant reduction in glycogen content at night under the sand and this corresponded with a higher hexokinase activity and increased glucose level suggesting enhanced glycolytic capacity. The plasma glucose and lactate levels were significantly lower at night, while in sand, relative to the day. The lower plasma glucose at night corresponded with a significant drop in liver gluconeogenic capacity (reduction in phosphoenolpyruvate carboxykinase, a key gluconeogenic enzyme, activity), while the lower lactate levels support a lack of activity along with the absence of glycogen breakdown in the muscle. Overall, there was a reduction in the metabolic capacity in the gills, heart, liver and muscle, but not the brain, supporting a tissue-specific metabolic reorganization as an adaptive strategy to cope with sand-dwelling in the wrasse. The molecular and biochemical responses seen in the wrasse at night in the sand was dissimilar to that seen in fish exposed to anoxia, leading to the conclusion that this species is not experiencing a complete lack of oxygen while under the sand. Also, the lack of muscle movement associated with sand dwelling at night limits anaerobic glycolysis for energy production, thereby eliminating lactate accumulation that was evident in fish exposed to anoxia. Taken together, wrasse showed a tissue-specific difference in metabolic capacity at night while hiding under the sand. While the mechanism involved in this tissue-specific energy repartitioning at night is unclear, one hypothesis involves selective increase in blood flow to the brain, while limiting peripheral circulation, as a means to maintain oxygen and glucose delivery to this critical tissue while the fish is hiding under the sand. The higher metabolic capacity of the brain, but not other tissues, at night under the sand suggests that maintaining the brain function is essential for the diurnal life-style in this animal.

Thesis (Ph. D.)--Miami University, Dept. of Zoology, 2009. / Title from second page of PDF document. Includes bibliographical references (p. 41-45).

This thesis is organized in “sandwich” format, as recommended by my supervisory committee. It consists of three main chapters. Chapter one is a general introduction and outlines the background information leading to the objectives and hypotheses of my thesis research. Chapter two is a manuscript prepared for submission to a peer-reviewed scientific journal. Chapter three is an overview of the major findings of this thesis, their implications in fish physiology and ecotoxicology, including suggestions of future directions of research. Appendix A contains data from an additional series of experiments that were conducted during my thesis but are not included as a full data chapter. It will be prepared for publication after my defence. / Hypoxia often occurs in aquatic ecosystems that receive effluent from municipal wastewater treatment plants (WWTP). WWTP effluent contains contaminants that could disrupt the complex physiological pathways fish use to cope with hypoxia (e.g., pharmaceuticals, polychlorinated biphenyls, and polycyclic aromatic hydrocarbons), but the effects of WWTP effluent on the physiological responses of fish to chronic hypoxia is poorly understood. We exposed mummichog killifish (Fundulus heteroclitus) to hypoxia (5 and 2 kPa O2) and/or WWTP effluent for 21 days in a full factorial design. We then measured hypoxia tolerance, whole-animal metabolism, gill morphology, haematology, and tissue metabolites. In clean water, killifish responded to chronic hypoxia with improvements in hypoxia tolerance – increases in time to loss of equilibrium at 0.5 kPa (tLOE) and decreases in critical O2 tension (Pcrit) – in association with increased gill surface area as a result of regression of the interlamellar cell mass (ILCM). Concurrent exposure to wastewater attenuated the increases in tLOE and gill remodeling in chronic hypoxia, and nearly depleted brain glycogen stores. Therefore, exposure to WWTP effluent can disrupt the physiological mechanisms fish use to cope with chronic hypoxia and impair hypoxia tolerance. My research suggests that the combination of stressors near WWTPs can have interactive effects on the physiology and health of fish. / Thesis / Master of Science (MSc) / Low oxygen conditions, known as ‘hypoxia’, frequently occur in aquatic ecosystems that receive municipal wastewater treatment plant (WWTP) effluent. WWTP effluent is a continuous and complex source of pollution, including contaminants that can disrupt fish physiology, affecting their ability to cope with stressors, like hypoxia. The effects of WWTP effluent on the responses of fish to chronic hypoxia are poorly understood. To address this research gap, I examined the effects of hypoxia and WWTP effluent on chronically exposed mummichog killifish. I provide evidence that combined exposure to hypoxia and wastewater affected hypoxia tolerance, gill structure, and depleted energy stores in the brain. My thesis demonstrates that WWTP effluent can disrupt mechanisms that fish use to cope with chronic hypoxia and impair hypoxia tolerance. These findings contribute to the existing body of work that documents the interactive effects of combined stressors in effluent-dominated ecosystems on the physiology and health of fish.

O coração é um órgão notável por sua flexibilidade estrutural e metabólica em resposta a variações de demanda. Na dormência sazonal, a interrupção da alimentação, associada à inatividade física e à acentuada redução da frequência cardíaca, ocasiona uma inibição da demanda sobre a função do órgão e, provavelmente, uma reorganização estrutural e metabólica do tecido cardíaco. Estes aspectos foram investigados ao longo do ciclo anual de atividades em lagartos teiú Tupinambis merianae, com o objetivo de examinar as alterações de capacidade funcional cardíaca dadas por ajustes da massa, estrutura e composição do tecido, por regulação do fluxo de substratos energéticos em vias de produção de energia e por mudanças da composição de ácidos graxos dos fosfolipídios das membranas. Grupos de animais jovens foram mortos em diferentes fases do primeiro ciclo anual e após 20 dias de jejum na fase ativa e o ventrículo cardíaco foi removido e pesado. Um fragmento da parede ventricular foi retirado, transferido para fixador e utilizado posteriormente para a confecção de cortes histológicos de 10 μm de espessura que foram analisados utilizando-se método estereológico. O restante do tecido ventricular foi congelado em N2 líquido e conservado em freezer -80 ºC. Os teores de água, proteína total e solúvel e lipídio total foram medidos por meio de ensaios padrão; as atividades máximas de enzimas foram medidas por espectrofotometria em condições saturantes de substratos e cofatores; e o perfil de ácidos graxos dos lipídios neutros e polares foi determinado por cromatografia gasosa. No início do outono, a massa ventricular relativa é 0,16% e aumenta 31% até o final desta fase, quando o miocárdio esponjoso possui aspecto denso e poucos espaços lacunares que ocupam cerca de 8% da área total do corte. Este arranjo é mantido na dormência, quando a massa ventricular relativa aumenta 29% em relação ao final do outono, e no início do despertar, quando a massa ventricular relativa diminui para valores semelhantes aos do final do outono. Após a retomada da alimentação, a massa ventricular relativa volta a exibir uma porcentagem comparável a da dormência, juntamente com um pequeno aumento da área de lacunas no miocárdio esponjoso. Na primavera, a massa ventricular relativa é de 0,24% e o miocárdio esponjoso possui aspecto extremamente reticulado, com 29% da área total do corte ocupada por espaços lacunares. Animais ativos submetidos a jejum apresentam redução de 19% da massa ventricular relativa em relação a animais alimentados. A densidade numérica de cardiomiócitos na camada esponjosa é 37% menor na dormência em relação à atividade de primavera, resultando em um volume calculado de um cardiomiócito nesta fase 52% maior em relação à atividade de primavera. A análise do teor de água, proteínas totais e solúveis não indica variação ao longo do ciclo anual, com exceção de uma tendência ao aumento do teor de água na dormência e de uma tendência à redução do teor de proteínas solúveis após o despertar e ingestão de água e no grupo de animais ativos submetidos ao jejum. Na atividade de outono e dormência de inverno a concentração de proteínas miofibrilares é reduzida em relação à atividade de primavera e aumenta no início do despertar após a ingestão de água. A concentração de lipídios totais é menor na dormência e despertar em relação à atividade de outono e no grupo de animais submetidos a jejum em relação a animais alimentados. As enzimas glicolíticas PK e LDH não variam ao longo do ciclo anual, enquanto a CS, indicadora da capacidade aeróbia, exibe forte tendência ao aumento na dormência, e a HOAD, enzima da β-oxidação lipídica, encontra-se inibida na dormência e no despertar em relação ao outono. Em contraste, com exceção da LDH que também não varia, a PK e a CS diminuem, enquanto que a HOAD é mantida constante após jejum na fase ativa. As variações do perfil de ácidos graxos da fração lipídica neutra sugerem que ácidos graxos insaturados são preferencialmente mobilizados das reservas do miocárdio durante a dormência e início do despertar, enquanto que no jejum durante a fase ativa as diferentes classes de ácidos graxos são equitativamente mobilizadas. A composição de ácidos graxos da fração lipídica polar exibe uma notável constância ao longo do ciclo anual, sugerindo que os ajustes à dormência sazonal não afetam de modo abrangente os fosfolipídios do tecido cardíaco e, portanto, não sugerem um papel preponderante de mudanças da composição lipídica das membranas na regulação metabólica sazonal nos teiús. Além disso, o contraste em relação às alterações observadas em mamíferos hibernantes sugere que, nestes, os ajustes seriam mais relacionados com a adaptação às baixas temperaturas corpóreas típicas da hibernação. A análise de regressão indica uma variação do conteúdo dos ácidos graxos C18:1n-9, C22:5n-6 e C22:6n-3 em função da massa corpórea dos jovens teiús e as mudanças do padrão alométrico sugerem uma relação entre o conteúdo destes ácidos graxos e as diferenças de taxa metabólica em animais de diferentes massas corpóreas, observadas em determinadas fases do ciclo anual de atividades e após o jejum durante a fase ativa. / The structural and metabolic flexibility of cardiac response to a variable physiological demand is notable. During seasonal dormancy, interruption of feeding together with inactivity and reduced heart beating, cause a large decrease of demand which probably brings about structural and metabolic heart tissue reorganization. These aspects were studied during the annual cycle in young tegu lizards Tupinambis merianae to investigate the hypothesis of seasonal changes of the heart capacities given by adjustments of tissue mass, structure and composition, by regulation of flux of substrates in the pathways of energy production, and by changes in the composition of fatty acids of tissue membranes. Groups of animals were killed in selected phases during the first year cycle of young tegus and after a 20 days fasting period during spring activity. Heart ventricle was removed and weighed and a tissue sample was collected and transfered to fixative solution, being used to obtain tissue slices of 10μm width for histological analysis with stereological tools. The remaining tissue was cut and split into aliquots, frozen in liquid N2 and stored at -80ºC. Later, the aliquots were used to assess the content of water, total and soluble proteins, and total lipids, by standard assays, the maximum activity of enzymes by spectrophotometry, and neutral and polar fatty acids profiles by gas chromatography. In early fall, the relative mass ventricle is 0.16%, and 31% increased in late fall, when the spongy myocardium appears dense and with few lacunar spaces which area corresponds to 8% of slice total área. During dormancy, the ventricle mass increases further 29%, decreasing to values of late fall during early arousal. After food intake, mass ventricle is again increased together with a small increase of the lacunar spaces, which appear highly expanded later in spring (29% of the total area), when tissue mass is 0,24% increased in relation to early fall. Unlike dormancy, fasting during spring caused a decrease of 19% of the ventricle mass. The cardiomyocytes density in the spongy layer is 37% decreased during dormancy while estimated cell volume is 52% increased, in relation to spring activity. There was no seasonal changes in the content of water and proteins in the groups analysed, except to a tendency to increase in the water content during dormancy, and to decrease in the soluble proteins in early arousal and in fasted animals. Myofibrillar protein is lower during fall and dormancy in relation to spring, increasing soon in early arousal after water intake. Total lipids decrease in the tissue during dormancy in relation to late fall by similar proportion than after fasting during spring. The glycolytic enzymes PK e LDH are unchanged during the year cycle, whereas the mitochondrial CS shows a tendency to increase, and HOAD, a β-oxidation enzyme, is decreased during dormancy and early arousal, in relation to fall. Unlike, PK and CS are decreased, while HOAD is unchanged after a period of fasting during spring. Fatty acids (FA) profiles of neutral lipids suggest that unsaturated FA are preferentially mobilized during dormancy and arousal, whereas all FA would be equally used during spring fasting. FA of polar lipids are remarkably constant during the year, suggesting that membrane FA in the heart tissue are not generally affected by season, and thus, results do not support a predominant role played by compositional changes of membranes in metabolic depression in the tegu. In addition, the otherwise distinct findings with hibernating mammals suggest that changes of FA composition in these animals would be an adaptation to the low body temperature of torpor, rather than mechanism of metabolic inhibition. Regression analysis indicate significant relationships of C18:1n-9, C22:5n-6, and C22:6n-3 contents as a function of body mass in young tegus, and changes in the allometric patterns are consistent with a putative relationship between these FA levels and the scaling of mass specific metabolic rates of young tegus during the year cycle.

The desert-adapted frogs Cyclorana platycephala and Cyclorana maini survive long periods of inhospitably hot and dry conditions by retreating underground and aestivating. While aestivating they suspend food and water intake as well as physical activity, depress their metabolic rate by ~80 %, and form cocoons that protect them against desiccation. How these frogs function during this exceptional state is largely unknown. This work characterized a number of physiological parameters in three metabolic states spanning their natural metabolic range: during aestivation (depressed metabolism), at rest (normal metabolism), and where possible, during exercise (elevated metabolism). The primary objective was to identify by comparison, physiological adjustments in these parameters to metabolic depression, as well as the scope of these parameters in frogs capable of aestivation. The parameters measured for C. maini were (a) the glucose transport kinetics and (b) the fluid balance of an extensive number of their individual organs. For C. platycephala, the parameters measured were (a) the activity of the cardiovascular system as indicated by heart rate and blood pressure and (b) the roles of pulmonary and cutaneous respiratory systems in gas exchange

Maintaining thermal balance is essential for all homeotherms but can be especially challenging for pinnipeds which must regulate over a variety of ambient temperatures and habitats as part of their life history. Young pinnipeds, with their immature physiology and inexperience, have the additional expense of needing to allocate energy for growth while still dealing with a thermally stressful aquatic environment. With the immense environmental and physiological pressures acting on juvenile age-classes, declines in prey resources would be particularly detrimental to survival. The goal of the present study was to examine the metabolic and thermoregulatory capabilities of juvenile Steller sea lions to better understand how changing prey resources indirectly impact juvenile age classes. Data collected from captive Steller sea lions suggest that changes in body mass and body composition influence the thermoregulatory capabilities of smaller sea lions in stationary and flowing water. Serial thermal images taken of sea lions after emergence from the water show vasoconstriction of the flippers compared to the body trunk to help minimize heat loss. Despite this ability to vasoconstrict, sea lions in poor body condition displayed a reduced tolerance for colder water temperatures, suggesting that decreases in prey availability which affect insulation may limit survival in younger sea lions. If reductions in prey availability (i.e., nutritional stress) were impacting western Alaskan populations, a reduction in energetic expenditures would be expected in these animals to cope. Measures of resting metabolism in juvenile free-ranging Steller sea lions across Alaska showed no differences between eastern and western capture locations, suggesting no evidence of metabolic depression in declining western stocks of sea lions. Finally, thermal costs predicted by a thermal balance model were compared to actual costs measured in the present study. Model output reliably predicted thermoregulatory costs for juvenile Steller sea lions under certain environmental conditions. Basic physiological measurements combined with the predictive power of modeling will allow for greater exploration of the environmental constraints on juvenile Steller sea lions and identify directions of future study.

Maintaining thermal balance is essential for all homeotherms but can be especially challenging for pinnipeds which must regulate over a variety of ambient temperatures and habitats as part of their life history. Young pinnipeds, with their immature physiology and inexperience, have the additional expense of needing to allocate energy for growth while still dealing with a thermally stressful aquatic environment. With the immense environmental and physiological pressures acting on juvenile age-classes, declines in prey resources would be particularly detrimental to survival. The goal of the present study was to examine the metabolic and thermoregulatory capabilities of juvenile Steller sea lions to better understand how changing prey resources indirectly impact juvenile age classes. Data collected from captive Steller sea lions suggest that changes in body mass and body composition influence the thermoregulatory capabilities of smaller sea lions in stationary and flowing water. Serial thermal images taken of sea lions after emergence from the water show vasoconstriction of the flippers compared to the body trunk to help minimize heat loss. Despite this ability to vasoconstrict, sea lions in poor body condition displayed a reduced tolerance for colder water temperatures, suggesting that decreases in prey availability which affect insulation may limit survival in younger sea lions. If reductions in prey availability (i.e., nutritional stress) were impacting western Alaskan populations, a reduction in energetic expenditures would be expected in these animals to cope. Measures of resting metabolism in juvenile free-ranging Steller sea lions across Alaska showed no differences between eastern and western capture locations, suggesting no evidence of metabolic depression in declining western stocks of sea lions. Finally, thermal costs predicted by a thermal balance model were compared to actual costs measured in the present study. Model output reliably predicted thermoregulatory costs for juvenile Steller sea lions under certain environmental conditions. Basic physiological measurements combined with the predictive power of modeling will allow for greater exploration of the environmental constraints on juvenile Steller sea lions and identify directions of future study.