Jeffrey Richards

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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