Jeffrey Richards
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Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.
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Ocean warming and eutrophication-driven hypoxia are two key threats marine organisms face with climate change and human activities. The impacts of warming and hypoxia on aerobic metabolism and respiratory capacity are thought to be a key mechanism underlying the susceptibility of marine organisms to these stressors, but how evolution and physiological tradeoffs affect these traits remains an area of intense investigation. Using intertidal sculpin fishes (Actinopterygii: Cottidae) as a model system, I studied how adaptation to the temperature- and oxygen-variable intertidal has shaped the temperature sensitivity of hypoxic respiratory capacity, a trait thought to shape biogeographic ranges in marine organisms broadly. Using the critical oxygen tension for standard metabolic rate (Pcrit) as a measure of hypoxic respiratory capacity, I found that tidepool-specializing species maintain greater capacity for oxygen uptake (i.e., lowest Pcrit) in warm, hypoxic water compared to subtidal congeneric species. Respiratory capacity and osmoregulation are linked in water breathing organisms through the osmorespiratory compromise, so I assessed whether improved respiratory capacity came with an osmoregulatory performance penalty in the tidepool-specialist Oligocottus maculosus. Based on diffusive water flux, an index of water permeability and a key component of osmoregulation, I found that O. maculosus suppressed water permeability in hypoxia but this suppression was lost during combined exposure to hypoxia and high temperature. These results suggest that the impacts of single vs. multistressor conditions may impact metabolic scope for ecologically-relevant activities in complex, difficult to predict ways. Lastly, based on the resurgent but controversial gill oxygen limitation hypothesis (GOLH), I analyzed whether gill surface area underlies the relationship between body size and respiratory capacity in O. maculosus. I did not find support for GOLH in O. maculosus, but hypermetric scaling of ventricle mass may contribute to the hypermetric scaling of maximum oxygen uptake rate in this species. Together my results suggest increased hypoxic respiratory capacity may be a potential evolutionary response to combined warming and hypoxia. An important avenue for further investigation is the impact of the interactions between respiratory capacity and linked processes such as osmoregulation found here on key ecologically-relevant performances such as growth and reproduction.
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Metabolic rate suppression during hibernation requires an orchestrated global reduction in metabolism while still matching O₂ supply and demand. Acidosis has been suggested as a mechanism for suppressing metabolic rate in hibernation, although the mechanism behind this process has not been thoroughly investigated. Findings from my thesis indicate that hemoglobin of both the facultative hibernator (Syrian hamster) and the obligate hibernator (13-lined ground squirrel) exhibit a reduced temperature sensitivity and increased Bohr effect relative to the non-hibernating rat, ultimately enhancing O₂ offloading at the tissues. I also found that in the obligate hibernator, due to metabolic state-dependent changes in intracellular buffering constituents, the ionisation ratio of intracellular imidazole (αim) did not vary between euthermia and hibernation. In the facultative hibernator, αim remained constant regardless of hibernation state. These findings contradict previous speculation that acidosis may suppress metabolism in quiescent tissues such as the brain and skeletal muscle during hibernation. Furthermore, mitochondrial respiration increased at low pH despite the reduced activity of electron transport system (ETS) enzymes at low pH. This suggests that, despite ETS enzyme activity being reduced at low pH, acidosis may reverse the inhibitory mechanisms that suppress mitochondrial respiration during steady-state hibernation, leading to the observed increase in mitochondrial respiration at low pH. Lastly, acidosis decreased cellular ATPase activity in the hibernating species but not in the rat. In the obligate hibernator, this inhibitory effect of acidosis on cellular ATPase activity was present regardless of hibernation state and assay temperature (37 – 10 °C). In the facultative hibernator, the effect of acidosis on ATPase activity was only present during euthermia and at warm temperatures. Taken together, these findings indicate that acidosis does not occur in quiescent tissues of hibernators or lower mitochondrial O₂ consumption. Instead, it may serve a role in matching O₂ supply to the high O₂ demand during arousal by increasing O₂ offloading by hemoglobin. Collectively, these data provide novel insight into the role of acidosis during hibernation that were previously uncharacterised.
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The purpose of my thesis research was to explore the physiological and behavioural correlates and trade-offs associated with faster growth, and their consequences for phenotypic and ecological differentiation along productivity gradients in salmonids. I examined variation in behavioural, digestive and bioenergetic strategies between different ecotypes and species of wild salmonids that differ in ecological requirements. Juveniles of large-bodied piscivore vs. small-bodied insectivore rainbow trout differed sharply in bioenergetics and behaviour. Relative to insectivore fry, piscivores presented a pattern of faster growth, higher food intake, higher basal metabolism, higher growth efficiency and larger digestive organs, but also more proactive behaviours and greater digestive efficiency (e.g., lower digestive metabolism). Faster piscivore growth was traded-off against lower aerobic scope and presumably against lower survival when facing predators. Piscivore and insectivore integrated phenotypes largely differentiated along the slow-fast continuum of the Pace-Of-Life Syndrome framework (Réale et al., 2010). This result was consistent with specialization of the two ecotypes to rearing habitats that differ in productivity – insectivores occur in tributaries with low prey availability relative to the piscivore rearing environment. The coherence between fast growth and high habitat productivity also emerged as a key driver of the post-emergence dispersal of piscivore fry along a productivity gradient in the Lardeau River. Juvenile steelhead trout and coho salmon also differed in growth and bioenergetics. Relative to coho salmon, faster-growing steelhead trout had higher food consumption and digestive metabolism but lower growth efficiency, which differentiated the two species along an energy-maximizing (steelhead) vs. efficiency-maximizing (coho) continuum (Rosenfeld et al., 2020). This pattern was largely consistent with their specialization to adjacent habitats (pools for coho, riffles for steelhead) along increasing prey flux gradient in coastal streams where the species co-occur. Steelhead trout presented higher aerobic scope than coho salmon, which compensated for their elevated digestive metabolism and ultimately resulted in the convergence of aerobic budgets between the two species. Overall, I demonstrate: i) the existence of multiple sets of physiological trade-offs associated with growth differentiation in salmonids from individuals to populations and species; and ii) the consequences of integrated phenotypic differentiation for ecological specialization along natural productivity gradients.
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Variation in environmental oxygen (O₂) poses a significant physiological challenge to animals, not only because of the impact on aerobic metabolism, but also because it can lead to generation of potentially harmful reactive oxygen species (ROS). In this thesis, I aimed to investigate the interplay between two aspects of O2 use at the mitochondria, aerobic respiration and ROS metabolism, using species of intertidal sculpins (Cottidae, Actinopterygii) which are distributed along the marine intertidal zone, exposed to varying O2 conditions and vary in their tolerance to low O₂ (hypoxia). I first hypothesized that there would be a relationship between whole animal hypoxia tolerance and mitochondrial and cytochrome c oxidase (COX) O₂-binding affinity, whereby hypoxia tolerant sculpins would have higher mitochondrial and COX O₂-binding affinity than less hypoxia tolerant sculpins. This hypothesis was supported with functional analysis. In silico modelling of the COX catalytic core revealed that the variation in O₂ binding was related to interspecific differences in the interaction between COX3 and membrane phospholipid, cardiolipin, which could impact O₂ diffusion to its binding site. I then investigated whether intact mitochondria from hypoxia tolerant sculpins were able to use O₂ more efficiently such that phosphorylation efficiency was improved and ROS generation was reduced compared to mitochondria from less hypoxia tolerant sculpins. Although there were relationships between hypoxia tolerance and complex I and II dependencies, there were no interspecies differences in phosphorylation or mitochondrial coupling that would indicate differences in aerobic metabolism. Moreover, mitochondria from hypoxia tolerant sculpins generated more ROS under resting conditions and were more perturbed by in vitro redox and anoxia-recovery challenges. Finally, I confirmed consistent responses of mitochondria to in vivo responses with a whole animal study comparing ROS metabolism (redox status, mitochondrial H₂O₂, oxidative damage and scavenging capacity) between two sculpin species with different hypoxia tolerance to hypoxia, hyperoxia, with normoxia-recovery exposures. Taken together, my thesis demonstrates that hypoxia tolerance is associated with improved O₂ binding at the mitochondria and COX. Further, hypoxia tolerance in sculpins is associated with higher ROS generation compared to less tolerant species, suggesting a potentially important role of ROS in mediating hypoxia tolerance.
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Animals rely on O₂ to balance cellular ATP supply and demand. In O₂-limited hypoxic environments, survival depends on the maintenance of this balance and is accomplished through some combination of aerobic metabolism, anaerobic metabolism and metabolic rate depression (MRD). My thesis studied how fishes combine these three metabolic strategies as a total hypoxic metabolic response (HMR) to survive hypoxic environments that vary in O₂ level (PwO₂) and duration. Calorimetry is required to accurately measure the metabolic rates (MR) of hypoxia (or anoxia)-exposed fishes that are partially reliant on anaerobic glycolysis and/or MRD. Thus, I started by building a novel calorespirometer that simultaneously measures indices of aerobic metabolism, anaerobic metabolism and MRD, and used it for the remainder of my thesis projects. Using goldfish, I found that time influences how PwO₂ affects HMR. Under acute and continually decreasing PwO₂ conditions, goldfish maintained routine O₂ uptake rates (ṀO₂) to ~3.0 kPa PwO₂ (i.e., Pcrit), but sustained routine MR to 0.5 kPa by up-regulating anaerobic glycolysis. Under constant hypoxia (1 or 4 h) at a variety of PwO₂s, however, goldfish maintained routine ṀO₂ to ~0.7 kPa and consequently reduced their reliance on anaerobic glycolysis. I confirmed this rapidly enhanced O₂ uptake ability in subsequent experiments by using different rates of hypoxia induction (RHI) to vary the amount of time goldfish spent at hypoxic PwO₂s. Gradual RHIs yielded greater lamellar surface areas, haemoglobin-O₂ binding affinities, and subsequently, lower Pcrits than rapid RHIs. However, goldfish only induced MRD below 0.7 kPa. To test the idea that MRD is reserved for extreme hypoxia, I compared two threespine stickleback populations from two isolated lakes: one that experiences deep, long-term hypoxia due to winterfreeze (Alta Lake), and the other that does not (Trout Lake). The two populations did not differ in Pcrit or capacities for anaerobic metabolism, but Alta Lake sticklebacks, which were 2-fold more hypoxia-tolerant than Trout Lake sticklebacks, employed hypoxia-induced MRD while Trout Lake sticklebacks did not. My results reveal that the HMR varies with an animal’s biology and the abiotic aspects of its natural hypoxic environment in a way that may optimize hypoxic survival.
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Variation in environmental conditions can sometimes impose severe limitations on organismal survival and reproductive success and organisms that live in variable environments have evolved complex traits to cope with or buffer against the environmental conditions. In my dissertation I examined how adaptive evolution and phenotypic plasticity play a role in the ability of nearshore species of sculpin to tolerate low levels of O₂ or hypoxia. I showed that constitutively expressed biochemical traits in the brain correlated with hypoxia tolerance among the different species independent of phylogenetic relationships, suggesting that these traits may have evolved via natural selection in response to hypoxia. A similar correlation was not seen in either the liver or the white muscle. Next, I showed that transcriptionally mediated phenotypic plasticity is likely associated with the difference in hypoxia tolerance between two species of sculpin. The hypoxia-tolerant tidepool sculpin (Oligocottus maculosus) did not alter gene transcription during the ecologically relevant time-frames of hypoxia exposure (up to 8 hours), in contrast to the hypoxia-intolerant silverspotted sculpin (Blepsias cirrhosus). This suggests that the tolerant species may not rely on phenotypic plasticity during a typical environmental hypoxia exposure and instead may rely on constitutively expressed or fixed traits for survival. Only if hypoxia persists do the hypoxia tolerant tidepool sculpins alter gene transcription, for which a large set of genes showed transcriptional patterns that were divergent to the hypoxia intolerant silverspotted sculpin. Lastly, I examined if similar transcriptional responses occur among three species of sculpin all with the same measured hypoxia tolerance. While a high proportion (65%) of clones showed similar transcription patterns among the species, a majority of genes associated with metabolism and protein production showed differences in both short and long exposures to hypoxia. As metabolism and protein production both play a major role in hypoxic survival, transcriptional differences in genes belonging to these biological processes suggests that the species likely use different mechanism to achieve similar overall hypoxia tolerance phenotype. Combined, this work demonstrates how phenotypic plasticity and adaptive evolution play a role in the variation of hypoxia tolerance among species of sculpin living in the nearshore environment.
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Cardiac failure occurs in most vertebrates including humans following even short hypoxia exposure due to an inability to match cardiac energy demand to the limited energy supply. In contrast, hypoxia-tolerant ectothermic vertebrates show the remarkable ability to maintain cardiac energy balance and stable cardiac function during prolonged exposure to severe hypoxia (cardiac hypoxia tolerance, CHT). I investigated how CHT is achieved and its relationship to whole-animal hypoxia tolerance using measurements at multiple physiological levels in two study models: 1) tilapia, a hypoxia-tolerant teleost, and 2) a two-species comparison of elasmobranchs with different hypoxia tolerance. I tested the hypothesis that CHT depends upon the depression of cardiac power output (PO) (i.e., cardiac energy demand) to a level lower than the cardiac maximum glycolytic potential (MGP). All species showed a hypoxic PO depression via bradycardia and my work generally supports this hypothesis. However, in tilapia, hypoxic PO depression is not necessarily required to maintain cardiac energy balance, contrary to previous suggestions, because of an exceptionally high MGP. Thus, in certain species, PO depression may primarily benefit CHT by minimizing fuel use and waste production. I also tested the hypothesis that greater hypoxia tolerance is associated with enhanced hypoxic O₂ supply and consequently enhanced cardiovascular function (i.e., less PO depression and improved cardiac energy balance). My work on elasmobranchs supported this hypothesis and also suggested a role for strategic cardiac O₂ supply via O₂ sparing resulting from metabolic rate depression (MRD) in non-essential tissues. Finally, my work on elasmobranchs showed that critical oxygen tension (Pcrit) predicts hypoxic blood O₂ transport, supporting the use of Pcrit as an indicator of hypoxia tolerance. Next, I tested the hypothesis that hypoxic PO depression is associated with the depression of whole-animal O₂ consumption rate below Pcrit. I found that this occurred in all species, suggesting that modulation of peripheral demand for blood flow (e.g., via MRD) may influence CHT. Finally, my work on in vivo and in situ cardiac responses in tilapia provided little evidence for the hypothesis that hypoxic modulation of aerobic energy production pathways, including provision of aerobic fuels (specifically, fatty acids), contributes to CHT.
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Master's Student Supervision
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
Atlantic salmon (Salmo salar) undergo a drastic change of environment during their lifecycle as they go from being salt-limited in freshwater to being water-limited in seawater. Fish prepare for this challenge by increasing various methods of water uptake and ion secretion. Currently the aquaculture industry prepares salmon for seawater entry by simulating the shift from a winter photoperiod to a summer photoperiod before transferring them into marine net pens at ~150g. Fish are then grown to the market size of ~4kg over the following 12-18 months. The marine net-pen environment is unpredictable and prone to high mortality events due to toxic algal blooms and periodic hypoxia, among other challenges. There is a strong interest on the part of the aquaculture industry in reducing the time salmon spend in net-pens by rearing them for longer in freshwater recirculating aquaculture systems to a much larger size prior to attempting to induce smoltification using photoperiod manipulations for seawater transfer. For this project, I aimed to use the industry standard light manipulation to generate fish that were ready for seawater entry at ~200g, ~500g, and ~1kg. Each size class had photoperiod-manipulated (PT) fish and control (C) fish to determine whether the light manipulation used in industry is effective in preparing salmon for seawater entry at larger sizes. Fish in seawater must drink water to offset passive water loss, so I measured drinking rates and plasma osmolytes to ask whether the amount that they drank was sufficient. As the intestine is a largely overlooked osmoregulatory organ, I investigated 3 different regions (proximal, mid, and distal intestines) of the intestine and measured Na⁺/K⁺-ATPase (NKA) activity to inform on the importance of photoperiod manipulation in growing larger fish for seawater transfer. Drinking rate did not differ between the PT and C fish, but did increase upon seawater transfer and was accompanied by adaptive changes in plasma osmolytes. NKA activity overall did not differ between treatments, but an increase in hindgut NKA activity was measured upon seawater transfer. The results of this thesis support the transfer of Atlantic salmon into the marine environment at larger sizes.
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Surviving hypoxia exposure requires the reorganization of physiological processes to reduce overall metabolic rate and ATP consumption. Na⁺/K⁺ATPase (NKA), an enzyme found in all animal cell membranes is crucial for the maintenance of cellular homeostasis and is a major consumer of ATP. Some hypoxia-tolerant species are reported to actively downregulate gill NKA during hypoxia. To date, however, no studies have attempted to determine if there is a relationship between specifies-specific hypoxia tolerance and the regulation of NKA during hypoxia. Focusing on the role of NKA in hypoxia survival, the goals of my thesis were to: first, investigate the interspecific relationship between hypoxia tolerance and the hypoxia-induced changes in NKA activity among six species of marine sculpin (family Cottidae); and second, to investigate whether AMP-activated protein kinase (AMPK) plays a role in regulating NKA in tidepool sculpins (Oligocottus maculosus). I measured NKA activity in six sculpin species exposed to hypoxia over 72 hours. Out of all species examined, only the most hypoxia tolerant species (tidepool sculpin) showed the predicted downregulation and only in gill tissue. In the brain, contrary to my prediction to see a decrease in NKA activity, I observed an increase in two species and no directional pattern overall. I found no evidence for a relationship between hypoxia tolerance (as measured by Pcrit) and NKA downregulation. I further investigated the mechanism of NKA regulation in tidepool sculpin gills. I hypothesized that in tidepool sculpins, AMPK (an important protein for cellular energy sensing and regulation) regulates changes in NKA activity. I predicted that exposing tidepool sculpins to AICAR, a pharmacological activator of AMPK would result in a decrease in NKA activity compared to control, but my results did not reveal any evidence in support of this hypothesis. Overall, I showed that there are interspecific differences in how NKA activity is affected by hypoxia exposure in these species, and that NKA downregulation as a hypoxic response exists in the gill of tidepool sculpin but not across the board in marine sculpins. My work also provides new insights into the mechanism of NKA regulation in tidepool sculpins.
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The Arctic is currently undergoing a period of transformation brought on primarily by climate change. Climate-induced changes in sea ice have led to changes in primary productivity, prey abundance and distribution, and habitat availability. There are concerns that the Arctic could be ice free by 2040, meaning that there is urgency in determining the impacts that climate change will have on the organisms that inhabit the Arctic. Climate change, however, is not the only pressure that marine organisms are facing. Contaminants, such as polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and mercury (Hg) have been identified as contaminants of concern to wildlife in the Arctic. Little is known about the interaction between contaminants and climate change, but concerns have arisen that the two stressors may exacerbate each other. The goal of this thesis is to add to our current knowledge of the impacts of contaminants and climate change on the health of western Arctic beluga whales and to explore the potential interaction between the two stressors. I examined the mRNA expression of 12 genes involved in detoxification of xenobiotics, nutritional stress and metabolism as a proxy for beluga whale health. The relationship between mRNA expression and contaminant burdens, stable isotopes and fatty acid signatures, sea ice levels and body condition metrics were examined in liver and blubber samples taken between 2008 to 2017. A principal component analysis on gene expression resulted in factor 1 explaining 78% percent of the variance for blubber and 90% of the variance for liver. Factor 1 was found to be significantly related to ẟ¹³C in blubber. Analysis of fatty acid profiles using PCA revealed inter-annual clustering of the year with the highest sea ice extent as well as the year with the lowest sea ice extent. Analysis of individual genes revealed that sea ice extent, fatty acids, length, ẟ¹³C and Hg may contribute most to the overall variation in gene expression. Results suggest that physiology of Beaufort Sea beluga whales are affected by a combination of climate-induced changes in foraging patterns and environmental contaminants.
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The freshwater turtle, Trachemys scripta is considered one of the most anoxia-tolerant vertebrates because of its ability to survive months at cold temperatures in the complete absence of oxygen. When deprived oxygen, mitochondria from anoxia intolerant organisms become one of the largest cellular energy consumers because of the reverse functioning of the F₁F₀-ATPase (complex V), which hydrolyzes ATP to pump protons out of the mitochondrial matrix, quickly depleting cellular ATP and leading to cellular death. T. scripta has previously shown to inhibit complex V in response to anoxia exposure, but the regulatory mechanism is still unknown. The goal of this thesis was to explore the mitochondrial response to anoxia in T. scripta. My first objective was to deduce the mechanism responsible for the severe downregulation of Complex V. In heart, brain, and liver tissue from anoxic exposed turtles, complex V activity was significantly reduced to more than 80% compared with normoxic controls. Employing a proteomics approach, I determined that three subunits of complex V (ATP5A1, ATP5F1, and MT-ATP5J), all associated with the peripheral stalk, decreased in protein expression in response to anoxia. Increasing assay buffer pH, in an attempt to strip Inhibitory Factor-1 (IF₁) from complex V did not increase enzyme activity of normoxic or anoxic exposed samples, but decreasing pH
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Aquaculture of salmon worldwide is a 15.3 billion dollar industry and the majority of fish are produced in net-pen systems in coastal waters. Recently producers have begun investigating the feasibility of moving salmon production onto land and into recirculating aquaculture systems (RAS). The major downsides to RAS are the startup and operational costs; however the ability to optimize many environmental variables to enhance growth and feed conversion, something impossible to do in net-pen systems, may help defray these otherwise prohibitive costs. Salinity may be the most important of these variables due to the metabolic cost of osmoregulation, which has been estimated to account for 5-50% of routine metabolic rate. Decreased osmoregulatory costs could result in a greater allocation of energy toward growth, thus shortening production times and improving feed conversion efficiency. To establish an optimal salinity for growth in salmon, seven replicate, 15,000 liter RAS were constructed at the University of British Columbia’s InSEAS research facility. I conducted a preliminary study to validate that each system was able to control water quality parameters and yield similar levels of growth and feed conversion in coho salmon (Oncorhynchus kisutch). I then conducted salinity trials with Atlantic (Salmo salar) and coho salmon. Fish were grown in five salinities ranging from freshwater to seawater (0, 5, 10, 20, 30 ppt) for approximately five months. Growth rates and feed conversion ratios (FCR) were measured throughout the trial. The fastest growth rate and lowest FCR in coho salmon was at 10 ppt, which is approximately isosmotic to the blood. Growth rate of coho at intermediate salinities was almost double that at 0 or 30 ppt through the first growth period. This trend was not seen during the second coho growth period, possibly due to a size-dependent or density effect. Unexpectedly, salinity had no effect on growth rate and FCR in Atlantic salmon, although growth rates were consistent with those seen in industry. This research will help further move salmon production out of the oceans and onto land, alleviating some of the environmental costs associated with salmon grown in the oceans.
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British Columba (BC) has a well-established lake-stocking program that relies on hatchery-reared rainbow trout (Oncorhynchus mykiss) from multiple wild and domesticated strains. These strains are stocked into BC lakes as diploids and triploids and, in general, high mortality rates are common after lake stocking due to environmental conditions. Of particular concern is that some lakes in BC are approaching a pH of 9.5, and it is not known if strain and ploidy affects the physiological responses of trout to high pH exposure. The goal of this thesis is to understand the effects of pH exposure, in both soft and hard water, on wild and domestic strains of trout as diploids and triploids. In soft water, high pH exposure resulted in more than 40% loss of equilibrium in the wild strains of trout while the Fraser Valley domesticated strain had fewer than 10% of individuals lose equilibrium overall. There were no clear differences between ploidies in loss of equilibrium. High pH exposure caused significant increases in plasma and tissue ammonia, with no differences between strains or ploidies in ammonia accumulation. In the brain, glutamine increased in response to high pH exposure and glutamate decreased suggesting a protective mechanism of glutamine production in high pH. Plasma lactate accumulated in all groups, suggesting an increase in anaerobic metabolism as a result of high pH exposure. There were no physiological differences between high pH exposure in hard and soft water among the strains tested. However, triploid rainbow trout suffered a greater loss of equilibrium than diploid trout, occurring in conjunction with a significant elevation of brain ammonia in triploid rainbow trout when compared to diploid trout in high pH water. Overall, the results of this thesis demonstrate an effect of strain on high pH tolerance in trout, but the differences in tolerance appear to not be explained by differences in ion regulation and ammonia balance.
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It has been long suggested that hypoxia tolerant species should have a great capacity to generate energy through anaerobic pathways to maintain energy balance when oxygen is limited; however, this assertion has not been rigorously tested. In the present study, I characterized hypoxia tolerance in 12 groups representing 10 species from the genera Danio and Devario (with three strains of D. rerio) and examined whether there is a phylogenetically independent relationship between variation in hypoxia tolerance and anaerobic capacity as judged by enzyme activity and anaerobic substrate concentrations present in various tissues. Hypoxia tolerance was assessed using two measures: time to loss of equilibrium (LOE) and the oxygen tension that yields 50% LOE in a group of fish over 8 hr (TLE₅₀). Time to LOE to low oxygen was very sensitive to changes in water PO₂, with no LOE seen over 8 hr in some species at 16 torr (2.1 kPa) and complete LOE within 30 min at 8 torr (1.1 kPa). At 12 torr (1.6 kPa) however, there was significant variation in time to LOE among all the species investigated. In three species (Danio rerio, Danio albolineatus and Danio choprai) time to LOE at 12 torr showed the same pattern of hypoxia tolerance as TLE₅₀. Despite the variation in hypoxia tolerance seen among the species under study, there was very little variation in the critical oxygen tension (Pcrit), which is the environmental PO₂ at which fish transition from an oxyregulating strategy to an oxyconforming strategy. Routine Ṁ₀₂ varied between the species, but the variation was primarily explained by body size and not hypoxia tolerance. Anaerobic energy capacity was estimated by measuring maximal enzyme activities of pyruvate kinase (PK), lactate dehydrogenase (LDH) and creatine phosphokinase (CPK), and concentrations of glycogen and glucose in muscle, liver and brain, plus creatine phosphate (CrP) and ATP in muscle. Through comparative analysis, I showed that the variation in hypoxia tolerance seen among species was related to some aspects of anaerobic energy metabolism, but not in a consistent fashion, indicating that other factors contribute to describing the variation in hypoxia tolerance.
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To determine what may contribute to the poorer survival of triploid (3n) trout in lake stocking programs relative to their diploid (2n) counterparts, we compared whole animal performance in response to environmental challenges in juvenile 2n and 3n fish from four wild strains and one domestic strain of rainbow trout. Spanning four years (2008, 2009, 2010, and 2011), wild fish were caught from nature and spawned in-hatchery along with hatchery-reared domestic trout. Offspring from all strains were raised to eight months as both 2n and 3n and exposed to low oxygen, swimming, and high temperature challenges. The only measure of performance to show a consistent difference between 2n and 3n individuals across all strains was time to loss of equilibrium (LOE) as a result of hypoxia exposure (~10% air saturation, 16 torr). Triploid trout always showed a shorter time to LOE (by 15-86% depending on the strain) relative to their 2n counterparts, with the exception of lake reared trout which showed no significant differences between 2n and 3n time to LOE. Additionally, there were no consistent effects of ploidy on critical oxygen tension, ṀO2, critical swimming speed (Ucrit), critical thermal maxima (CTMax), or muscle enzyme activities. We observed significant effects of strain on all performance measures except for CTMax. In general, the Fraser Valley domestic strain had higher Ucrit, higher ṀO2, and greater muscle enzyme activities than did Blackwater, Tzenzaicut, and Pennask wild conspecifics, suggesting that domestication affects a variety of traits in addition to growth rates.
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The ability to undergo metabolic rate suppression (MRS) markedly improves chances of survival during aquatic hypoxia. In this thesis, I specifically tested the hypothesis that AMP-activated protein kinase (AMPK) initiates MRS in hepatocytes from the common goldfish (Carassius auratus). My first goal was to investigate the responses of isolated hepatocytes to changes in O₂. Goldfish hepatocytes showed a gradual decrease in cellular oxygen consumption rate (MO₂) as O₂ was decreased from normoxia (~310 µM O₂) down to the apparent P₉₀ of 13 µM, below which there was a steep decline in MO₂. The apparent P₉₀ for hepatocyte respiration matched published measurements of venous [O₂], which suggests that hepatocyte MO₂ in vivo may be regulated by O₂. To address the relationship between AMPK and MRS, several drugs were used to manipulate AMPK activity. I was able to activate AMPK with 5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) under normoxic conditions, which caused a reduction in MO₂; this decrease was mediated through a decrease in protein synthesis rate via eukaryotic elongation factor 2 (eEF2) phosphorylation. Specifically, a maximal 7.5-fold activation of AMPK resulted in a 24% reduction in MO2, thus supporting the notion that AMPK activation initiates MRS. We then used compound C, a general protein kinase inhibitor, in an attempt to reverse the AICAR effects on AMPK activation, but compound C did not reverse the effects of AICAR. A recently discovered specific AMPK activator, A769662, was also used to manipulate AMPK activity. However, at all doses, A769662 failed to activate AMPK. Nevertheless, whenever I was able to activate AMPK via AICAR incubation, there was a consistent lowering of metabolic rate. Thus I have provided evidence to support the hypothesis that AMPK is important in the initiation of MRS in goldfish hepatocytes.
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