Patricia Schulte
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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.
Anthropogenic climate change is causing rising average temperatures and increased thermal variability across aquatic environments. These impacts may be particularly concerning for early life-stages of fishes as their thermal windows are thought to be narrower than those of adults. However, relatively little is known about how these early-life stages will respond to predicted temperature changes with global warming. Therefore, I investigated the effects of different temperature regimes on early life-stages and how they respond to these environments both acutely and by utilizing developmental plasticity in two subspecies of Fundulus heteroclitus, a topminnow that inhabits intertidal saltmarshes along the Atlantic coast of North America. I generated thermal performance curves (TPC) for development in embryos of two F. heteroclitus subspecies reared at a series of constant temperatures and found evidence consistent with both local adaptation and countergradient variation between the subspecies. I also showed that F. heteroclitus reared at different temperatures had altered hypoxia tolerance and hif1α mRNA transcript abundance, but I observed no change in thermal tolerance. This finding demonstrates that developmental cross-tolerance can occur in F. heteroclitus. However, these differences did not persist at the age of 1 year, highlighting reversible plasticity. I then examined how fluctuating thermal regimes during development affected embryonic and larval phenotypes. I demonstrated development under fluctuating temperatures can alter performance in ways that cannot always be predicted based on performance generated at constant temperatures. Furthermore, I showed the fish reared under fluctuating temperatures had altered growth, thermal tolerance, and hypoxia tolerance, which were associated with long-lasting transcriptomic effects that persisted even in a common environment. However, high thermal variability during development had lasting negative consequences on phenotypes as the result of deleterious plasticity. Taken together, my research demonstrates that F. heteroclitus utilize developmental plasticity as a mechanism to cope with changing temperatures during early development. However, there are limitations to this plasticity which are highlighted in the reversible and deleterious plasticity I detected.
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How organisms respond to environmental change is a fundamental question in biology, critical to our understanding of how animals will cope with climate change. Determining the climate change resilience of a species or population is a challenging task, as resilience can be influenced by phenotypic plasticity and genetic variation. Specifically in aquatic habitats, organisms encounter rapid environmental changes including variations in temperature, oxygen availability, and salinity which occur across different spatial and temporal scales. My thesis aimed to explore how plasticity and adaptation influence resilience to environmental change across these scales in three different fish species.The three species I studied were Atlantic killifish (Fundulus heteroclitus), white sturgeon (Acipenser transmontanus), and rainbow trout (Oncorhynchus mykiss). I measured whole-animal physiology and underlying molecular mechanisms to assess responses to climate change stressors across developmental stages and populations. In killifish and white sturgeon, I found that exposure to climate change stressors during development had lasting phenotypic effects and affected the plasticity of many traits. These studies underlined the importance of considering developmental stage when evaluating stressor effects, as individuals must persist despite environmental changes during sensitive life stages to survive. Also, in white sturgeon and killifish I demonstrated the impacts of simultaneous stressors on whole-animal performance and the underlying genetic mechanisms highlighting the detrimental impact of compounding stressors. To investigate the role genetic variation plays on the ability of fishes to cope with environmental change I assessed how adaptation to different environments can affect the stress response or survival in novel environments. I found that local adaptation can influence the magnitude and plasticity of the stress-axis in killifish, highlighting the potential for adaptive tradeoffs between the stress response, body condition, and survival in the wild. In rainbow trout, I found that adaptation to hatchery environments impacts offspring phenotypes and decreases survival in the wild, highlighting the interplay between adaptation and coping with novel stressors. My thesis contributes to our understanding of the basic biological question, “how do fishes cope with environmental change?”, and also provides evidence for concrete actions and conservation-management recommendations during climate change.
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Ammonia, which is excreted across the gills, is the major nitrogenous waste of fish. It is also a toxicant. My thesis focuses on how and why ammonia influences breathing in fish, using the phylogenetically ancient Pacific hagfish (an agnathan) and the rainbow trout (a teleost) as models. I first characterized the unique breathing mechanism of hagfish, demonstrating a two-phase unidirectional system with a fast suction velar pump for inhalation through the nostril and a much slower force pump for exhalation through gill pouches. High environmental ammonia (HEA) causes an initial hypoventilation, sometimes apnea, and a later sustained hyperventilation. The hypoventilation is independent from responses to O₂ and CO₂ and is mediated by external receptors. The hyperventilation is mediated by increased blood ammonia, detected internally, similar to previous findings on teleosts. In trout, I confirmed an assumption in the literature that ventilation would not affect ammonia excretion. However, I then used chronic internal ammonia loading to upregulate the ammonia transport system (rhesus glycoproteins) in the gills. This removed diffusion limitation so that ammonia excretion became sensitive to ventilation. Hyperventilating trout, therefore, excrete more ammonia. After developing a new less invasive system for the direct measurement of ventilation, I used it to show that HEA hyperventilation is not immediate, but develops gradually, mediated by internal receptors. Indeed, specific application of HEA to the external surface of the gills causes a transient acute hypoventilation, again as in hagfish. Direct application of ammonia to the hindbrain causes hyperventilation by the stimulation of central chemoreceptors, while peripheral chemoreceptors in the gills (neuroepithelial cells) sense increased plasma ammonia. In humans, ammonia buildup in the brain similarly stimulates breathing. I conclude that a role for ammonia in ventilatory control is probably ubiquitous in vertebrates, including the oldest extant representatives (hagfish). Initial hypoventilation is protective against the uptake of a toxicant, while sustained hyperventilation is beneficial at times of internal ammonia loading such post-exercise recovery, and after feeding. This hyperventilation will facilitate not only greater ammonia excretion, but also the greater O₂ uptake needed to recover from exercise and to metabolically process food.
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The fish gill is a multipurpose organ that plays a central role in gas exchange, ion regulation, acid-base balance and nitrogenous waste excretion. Effective gas transfer requires a large surface area and thin water-to-blood diffusion distance, but such structures also promote diffusive ion and water movements between blood and water that challenge the maintenance of hydromineral balance. Therefore, a functional conflict exists between gas exchange and ionic and osmotic regulation at the gill.The overarching goal of my thesis was to examine the trade-offs associated with the optimization of these different functions (i.e. the osmorespiratory compromise) in species with diverse osmoregulatory strategies, when exposed to environmental stressors such as hypoxia, changes in temperature and salinity. To address this I have used three species of fish that are phylogenetically, ecologically and physiologically diverse, the Atlantic killifish (teleost), the Pacific hagfish (myxine) and the Pacific spiny dogfish (elasmobranch).My results show that salinity influences the capacity to regulate oxygen consumption at low oxygen and hypoxia tolerance in the killifish. Acclimation to fresh water resulted in a lowering of the lamellar respiratory surface area and a higher percentage of the gill lamellae covered by an interlamellar cell mass. These responses could be adaptations to aid survival in hypo-osmotic waters as freshwater-acclimated fish showed a greater ability to downregulate transcellular gill permeability to both ions and water when exposed to hypoxia in comparison to their seawater-acclimated counterparts. However, at salinities ranging from fresh water to 100% sea water, plasma ion concentration and osmolality were unaffected by hypoxia. I also found that there is a strong interaction between gill permeability to gases and to ions and water in hagfish, an osmoconforming marine species in which the osmorespiratory compromise had never been investigated. An increase in gill permeability to urea, ammonia, and water was also seen in the dogfish exposed to elevated temperature, indicating a disruption in the nitrogen conservation mechanisms at the gill. In summary, this thesis has expanded the range of species in which the osmorespiratory compromise has been investigated, and has provided new insights into the mechanisms involved.
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Epigenetic mechanisms such as DNA methylation have been proposed as an important source of variation that can influence phenotypic plasticity and adaptive evolutionary processes, yet little is known about the role of DNA methylation in an ecological or evolutionary context in vertebrates. In this thesis I examine the effects of the environment and sex on DNA methylation and gene expression patterns in the threespine stickleback fish (Gasterosteus aculeatus), an ecological and evolutionary model system that has been used to study mechanisms involved in the evolution of adaptive phenotypes in novel environments. The dynamic regulation of DNA methylation and gene expression patterns during early developmental periods plays an important role in cell differentiation and establishing adult phenotypes. Here I demonstrate that adult DNA methylation and gene expression patterns are modified in response to the temperature and salinity experienced during development. Similarly, maternal stress can have long-term effects on neurodevelopment and the behavior of offspring that can influence offspring performance and population evolutionary trajectories. I demonstrate that the effects of maternal stress on the brain transcriptome differ between adult male and female stickleback offspring. These sex-specific effects of maternal stress suggest that male and female offspring may respond differently to maternal stress exposure, which could have important implications when assessing the long-term ecological and evolutionary impacts of stress across generations. DNA methylation has also been proposed to play a key role in regulating sexually dimorphic phenotypes and in the evolution of sex determination mechanisms. I compare genome-wide DNA methylation patterns between male and female stickleback and identify apparent differential methylation on the stickleback sex chromosome that correspond to the regions of genetic divergence between the X and Y chromosome. These data provide evidence of a potential role of DNA methylation in the evolution of sex chromosomes in vertebrates. Taken together, these data demonstrate that there is a complex relationship between genetic, epigenetic, and transcriptomic processes that are dynamically regulated during development and in response to environmental cues, and that epigenetic processes may be involved in regulating evolutionary processes.
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Ambient temperature is a pervasive environmental stressor for ectotherms, with effects from individual atoms to the population level. Indeed, the effects of temperature on organismal performance are suggested to constrain species’ geographic distributions. Aerobic metabolism is proposed to underlie the thermal limits of organisms, with thermal constraints occurring at the level of the mitochondrion due to its position at the terminus of the O₂ transport cascade and as the primary site of cellular ATP production. Despite this theoretical link, there is limited understanding of the relationship between mitochondrial function and thermal tolerance, particularly for interacting responses among multiple biological timescales.I used two subspecies of Atlantic killifish (Fundulus heteroclitus) to characterize mitochondrial responses to acute thermal shifts following thermal acclimation to 5, 15, and 33 °C, and putative local adaptation. Northern killifish exhibited higher liver mitochondrial respiratory capacity and lower mitochondrial O2 binding affinity when compared to the southern subspecies. Subspecies variation in mitochondrial function was associated with differences in electron transport system (ETS) complex IV capacity. Decreasing acclimation temperature increased liver mitochondrial respiratory capacity and decreased mitochondrial O₂ binding affinity in both subspecies. Thermal acclimation effects on liver mitochondrial respiratory capacity were associated with ETS complex I. In contrast, heart and brain mitochondrial respiratory capacity decreased following acclimation to both high and low thermal extremes and did not differ between subspecies. Thermal acclimation effects on liver mitochondrial performance were not associated with increased reactive oxygen species production or a loss of mitochondrial proton motive force at high assay temperatures. Liver mitochondrial membrane composition varied in response to thermal acclimation and differed between subspecies, with thermal acclimation effects being largely consistent between subspecies. Mitochondrial lipid remodeling was primarily associated with changes in specific phospholipid species, suggesting a role for targeted membrane remodeling as a mechanism underlying variation in mitochondrial function.My data provide evidence for variation in mitochondrial function as a mechanism that differentiates aerobic and thermal performance between F. heteroclitus subspecies and that is involved in thermal acclimation responses. These mitochondrial responses likely underlie the aerobic performance limits of ectotherms and influence species’ fitness and geographic distributions.
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High pH is physiologically stressful for Rainbow Trout, causing poor survival when fish are stocked into high pH lakes. To assess the relative contributions of genetic variation and phenotypic plasticity in high pH tolerance in Rainbow Trout, I examined high pH (pH 9.5) tolerance in three Rainbow Trout strains (Blackwater River, Eagle Lake and Fraser Valley Domestics) under four different rearing conditions: 1) near-neutral hatchery conditions (pH 7.2) from fertilization; 2) pH 8.5 from fertilization; 3) pH 8.8 from fertilization; and 4) near-neutral hatchery conditions from fertilization followed by acclimation to pH 8.8 for one month prior to testing (at fry and yearling). In general, I found that either rearing or acclimating fish to elevated pH improved high pH tolerance. Variation among strains was observed only at the fry life stage. I performed a genome wide association study to identify genetic variation that may be associated with differences in pH tolerance among strains. The results suggest that pH tolerance is likely controlled polygenically. To assess mechanisms underlying phenotypic plasticity in high pH tolerance, gill gene expression of fish reared under control conditions and those acclimated to pH 8.8 were compared using RNA-Seq. There were 140 genes that were significantly differentially expressed in response to high pH, but the most dramatic results were the strong interaction effects between pH and strain suggesting that each strain compensates for high pH conditions in different ways. Finally, the variation among strains and rearing treatments observed within the laboratory was tested in natural lakes. In general, short-term net pen trials were consistent with laboratory results showing higher pH tolerance in fish reared at or acclimated to elevated pH levels. Long-term survival trials indicate that the large differences in survival in natural lakes between strains mask subtler effects of prior exposure to high pH and require further investigation. My data suggest that it is the remarkable plasticity of Rainbow Trout rather than a specific strain or genotype which has the greatest effect on high pH tolerance, and that modifications of hatchery practices could be used to improve survival of stocked Rainbow Trout in high pH lakes.
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Colonization of new environments exposes organisms to novel combinations of abiotic factors that have the potential to negatively affect fitness. Organisms may be able to cope with these changes in abiotic factors using existing phenotypic plasticity, or the novel environment may drive adaptive divergence, but the role of phenotypic plasticity in assisting or hindering the process of local adaptation remains unclear. This dissertation contributes to addressing this topic by examining the interactive effects of multiple abiotic factors on phenotypic plasticity and the evolution of physiological traits, which is an area that has received relatively little study. Specifically, I explored the roles of salinity and temperature in driving divergence during freshwater colonization using marine, anadromous, and derived freshwater populations of the threespine stickleback, Gasterosteus aculeatus. In north-temperate freshwater habitats, stickleback experience a combination of low salinity and low winter temperatures that is not experienced by the ancestral marine and anadromous forms which overwinter at sea. Overall, the results of this work are consistent with adaptive evolution in response to the interactive effects of low salinity and low temperature during freshwater colonization. My results showed that both salinity and temperature, and the interaction between them, had stronger negative effects on the growth of marine and anadromous populations compared to the freshwater population. Using a whole-transcriptome approach, I also detected differentiation in gene expression patterns between populations, particularly in processes important for changes in gill structure and permeability. Based on these data I hypothesize that freshwater stickleback have less permeable gills in fresh water, which may result in less energy use for osmoregulation, providing a physiological mode by which freshwater stickleback save energy, resulting in superior growth in cold fresh water. Both marine and freshwater stickleback showed interactive effects of low temperature and salinity on gill morphology, and marine stickleback exhibited substantial increases in the expression of Na⁺,K⁺-ATPase in cold fresh water, whereas more modest responses were observed in the freshwater ecotype, which may indicate increased energetic costs of osmoregulation in the marine population and potentially contribute to the growth deficits exhibited by these fish in cold fresh water.
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Environmental temperatures impact the performance of ectothermic organisms, such that changes in performance with temperature often influence range limits. Aerobic scope (the difference between maximum and standard aerobic metabolic rates) has been proposed as a physiological mechanism that may underlie the effects of temperature on performance. However, the effects of phenotypic plasticity and genetic divergence on aerobic scope curves, and the physiological mechanisms that constrain changes in the shapes of these curves remain poorly understood. Here, I assess the responses of aerobic scope to temperature in two subspecies of the eurythermal Atlantic killifish (Fundulus heteroclitus) through measurements of routine and maximum oxygen consumption. I demonstrate that killifish maintain aerobic scope over wide ranges of temperatures even during acute thermal exposures (5-33°C), but that thermal acclimation increases aerobic scope within optimal temperature ranges and at extreme temperatures (Chapter 2). Differences in aerobic scope as a result of thermal acclimation and intraspecific divergence in killifish are primarily associated with differences in routine oxygen consumption. Northern killifish have higher routine oxygen consumption than southern killifish, whereas cold-acclimated killifish have lower routine oxygen consumption than acutely cold-exposed warm-acclimated killifish. I also demonstrate that intraspecific variation in routine oxygen consumption is not associated with differences in acute thermal tolerance and hypoxia tolerance in admixed killifish (Chapter 3), and that decreases in routine oxygen consumption as a result of cold acclimation are paralleled by lower expression levels of genes involved in oxidative phosphorylation in muscle tissue (Chapter 4). Interestingly, despite these changes in oxidative phosphorylation gene expression, positive regulators of mitochondrial biogenesis are induced in cold-acclimated killifish (Chapters 4 & 5), suggesting that changes in mitochondrial volume density may improve oxygen delivery to mitochondria rather than compensating the effects of low temperature on cellular respiration. Taken together, my data indicate that cold acclimation may result in inverse compensation of metabolism in killifish, whereas intraspecific divergence results in countergradient variation in metabolism (i.e., thermal compensation). These opposite patterns may reflect latitudinal differences in selection associated with overwinter survival, and may contribute to the differences between the killifish subspecies that maintain intraspecific range boundaries.
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Hybrid zones act as natural experiments that can provide insights into the factors governing species formation and maintenance. In order to investigate these factors, I examined a hybrid zone between two subspecies of Atlantic killifish, Fundulus heteroclitus. Previous research has shown that these subspecies differ both genetically and phenotypically, but very little work had examined the hybrid zone between them. I used a suite of genetic markers to describe the genetic pattern within this hybrid zone and laboratory breeding experiments to investigate the forces responsible for its maintenance. Based on hybrid indices calculated using microsatellite and SNP (single nucleotide polymorphism) loci, a trimodal hybrid zone located in Beaverdam Creek (in the Metedeconk river system in New Jersey) separates the two subspecies of killifish, suggesting that while some F1 hybrids are produced, backcross types are rare. This pattern persisted across several sampling sites and across two years, suggesting that this pattern was not a sampling artifact. By investigating the geographical patterns of genetic variation in 30 SNPs along the Atlantic coast, I found that clines in mitochondrial DNA markers and in SNPs in several nuclear genes with mitochondrially-associated functions were coincident, concordant and exceptionally steep compared to those of other loci. I used tension zone analyses to conclude that these clines are likely being maintained either by selection or by assortative mating. The observed cytonuclear disequilibria also suggested a role for cytonuclear epistasis in maintaining this hybrid zone. Within Beaverdam Creek, there was no genetic differentiation between samples taken at locations differing in temperature and salinity, suggesting that habitat specialization on these abiotic variables is not involved in the maintenance of the hybrid zone. However, my results from a "choice" breeding experiment among individuals originating from the extremes of the species' distributions suggested a possible role for positive assortative mating. Taken together, my research provides evidence that differentiation in mitochondrial properties resulting in selection or assortative mating could be involved in the maintenance of distinct subspecies of F. heteroclitus, and points to a potential general role for divergence in energy metabolism as a mechanism in promoting or maintaining species differences.
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Understanding how complex traits evolve is critical for understanding how animals meet environmental challenges. In my dissertation I studied the mechanisms by which prolonged swimming performance (Ucrit), a complex whole-animal performance trait, has evolved among ancestral anadromous-marine and derived non-migratory stream-resident ecotypes of threespine stickleback (Gasterosteus aculeatus). I showed that stream-resident populations from Bonsall and West Creeks have evolved a decreased Ucrit, but via different genetic mechanisms, and that three additional wild stream-resident populations also had low Ucrits. Collectively, these data are consistent with a role for natural selection in the evolution of a reduced capacity for prolonged swimming after freshwater colonization.I next determined which candidate morphological, physiological, and biochemical traits evolved in conjunction with these decreases in Ucrit capacity in Bonsall and West Creek stream- resident populations. I found that a number of traits predicted to influence Ucrit in fishes evolved as predicted in both stream-resident populations. To further assess the associations between these candidate traits and Ucrit, I compared the genetic architecture of Ucrit with the genetic architecture of candidate traits by comparing F1 hybrids to pure F1 crosses. I found that a number of candidate traits had a similar genetic architecture as Ucrit, but that many of these traits were population-specific. These data suggest that non-parallel genetic, morphological and physiological mechanisms may contribute to the evolution of similar performance capacities.To test the associations between candidate traits and Ucrit, I correlated traits with Ucrit in Bonsall Creek F2 hybrids. In F2 hybrids the complete linkage of all divergent traits in F1 crosses is partially broken apart. I found that only four candidate traits (ventricle mass, adductor mass, and adductor and abductor citrate synthase activities) significantly regressed against Ucrit in F2 hybrids, accounting for 17.9% of variation in Ucrit. These data suggest that, when dissociated from other traits, many candidate traits do not have a strong effect on Ucrit, additional unmeasured traits are likely to influence Ucrit, and that many traits are necessary to reach a high Ucrit. This dissertation provides a clear empirical example of the patterns of evolution in a complex trait and its underlying mechanisms.
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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.
Understanding how morphology is shaped by captive breeding and rearing of fish is becoming increasing important for policy-making surrounding the conservation of aquatic species, as hatchery supplementation becomes more and more commonplace and the potential for impacts on wild populations grows. Therefore, the objective of this thesis was to explore how genetics and environment shape the morphological phenotypes of domestic-wild hybrid salmonids. First, I used photos of F2 hybrid rainbow trout parr after a single growing season in a semi-natural environment to determine whether “domestic” or “wild” phenotypes predominate under these conditions. I found differences in overall body shape, particularly in the head and deepness of the body between the domestic and wild fish. Some phenotypes in the F2s were more similar to those of one or the other of the grandparents reared in hatchery conditions, and others were entirely different, likely due to the differences in food availability, presence of predation, water velocity, etc. in the semi-wild environment compared to a hatchery environment. Next, I used photos of F2 offspring of domestic and wild strains of coho salmon to do a similar analysis, which revealed an extremely fast-growing group of juveniles. I then used next-generation genomic sequencing (PoolSeq) to attempt to find SNPs correlated with this fast growth trait. I found three large regions of differentiation between the fast growing and slow growing F2 fish, with enrichment for processes such as musculoskeletal development, omega peptidase activity, and folate transmembrane transport. The results of my first chapter indicate that the environment has extremely large impacts on morphological phenotypes; however, as shown by my second chapter, genotype can create drastic differences in morphology when rearing conditions are identical. The results of this thesis illustrate the interacting effects of genetics and environment on morphological phenotypes in salmonids.
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Little is known about how variation in functional traits affects the performance and ecological success of a population. Here, I investigate the importance of intraspecific variation and plasticity in two traits related to coping with environmental change: the cortisol-mediated stress response and whole-organism upper thermal tolerance. Using the Atlantic killifish (Fundulus heteroclitus) as an experimental model, I quantified variation between populations, between individuals, and within individuals in these traits. By analyzing video footage of northern and southern killifish after exposure to handling stress, I found evidence of differences in boldness and exploratory behaviour between populations, but no evidence for population differences in the behavioural response to stress. Behavioural differences between populations were associated with variation in the expression of a glucocorticoid receptor, GR1, in the liver between the two populations. Next, I used a repeated measures experiment to assess the extent of inter-individual variation in critical thermal maximum (CTmax) and determine whether it is a repeatable trait across different acclimation temperatures. I found significant inter-individual variation, and in accordance with other studies of repeatability in CTmax, I found high repeatability at a moderate acclimation temperature; however, acclimation to temperatures these fish experience in mid-summer in their natural environment resulted in a significant decrease in repeatability. Moreover, I found that CTmax was not repeatable across acclimation temperatures, which suggests that measures of the heritability of upper thermal tolerance are only relevant to the temperature at which they are determined. There was substantial inter-individual and intra-individual variation in plasticity of thermal tolerance, which could provide opportunities for adaptation in this trait. Taken together, these studies demonstrate that there is substantial intraspecific variation and plasticity in traits involved in responses to the environment, which has implications for how this species may respond to climate change.
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Global climate change threatens to reduce available habitat for cold-water fish such asrainbow trout (Oncorhynchus mykiss). Phenotypic plasticity might help individuals to cope, butmay not be sufficient and survival could require adaptation in situ. Here, I assessed levels ofphenotypic variation in thermal and hypoxia tolerance that could act as the substrate foradaptation. Furthermore, I also characterized the extent of thermal plasticity in these traits inmultiple strains of rainbow trout. To characterize phenotypic variation, I used a common gardenbreeding approach with 25 family crosses in each of several strains of British Columbia rainbowtrout, assessing critical thermal maximum (CTmax) and incipient lethal oxygen saturation (ILOS).Using California strains, I investigated the extent of thermal plasticity in CTmax, ILOS, and (incollaboration) standard and maximum metabolic rate, absolute aerobic scope (AAS), criticaloxygen tension (Pcrit), and measures of cardiac performance such as maximum heart rate (ƒHmax)and cardiac gene expression. I found little among but large within-strain variation in CTmax andILOS, whereas post-trial mortality clearly differentiated the strains. There was little correlationbetween upper thermal and hypoxia tolerance at the individual level. I observed significantplasticity in CTmax, with associated declines in AAS and ƒHmax and increases in the expressionof stress-related genes. However, plasticity in all these measures reached a limit at a high butecologically relevant temperature. Taken together, these findings suggest that thermal plasticitywill not be sufficient to allow rainbow trout to cope with climate change, but that troutpopulations possess substantial phenotypic variation in climate-change relevant traits that mayallow adaptation in situ. However, differences between strains were not evident for all traits, andmanagers will need to take a multifaceted approach when examining the effects of climatechange on natural and stocked strains of rainbow trout.
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Environmental temperature can greatly impact the functioning of ectothermic organisms through effects on mitochondria, which are crucial to aerobic metabolism. Changes in temperature have the potential to influence mitochondrial ATP production and production of reactive oxygen species (ROS), both of which are influenced by the activity of the mitochondrial electron transport system, which generates the proton gradient necessary for mitochondrial ATP production. Thus, I hypothesized that ectothermic organisms have a mechanism for modulating the proton gradient in the face of changes in environmental temperature to maintain ATP production, and that this mechanism may act through uncoupling proteins (UCPs) which can cause a decrease in the proton gradient independent of the production of ATP. Here, I investigate changes in UCPs and mitochondrial function following thermal acclimation in two populations of the eurythermal Atlantic killifish, Fundulus heteroclitus. I show that UCP mRNA expression is tissue-specific, changes with thermal acclimation, and differs between two populations of killifish. However, these changes vary depending on the isoform, tissue, and population (Chapter 2). I also demonstrate that changes in UCP function are not necessarily consistent with changes in mRNA expression in isolated liver and brain mitochondria, but that UCP function may differ in liver between the two populations (Chapter 3). Cold-acclimated northern killifish increase liver mitochondrial capacity and coupling as indicated by increases in state III, respiratory control and ADP/O ratios (Chapter 3). Interestingly, I also observed increases in proton conductance in isolated liver mitochondria from cold-acclimated northern killifish as indicated by increased O₂ consumption rate at a common membrane potential (Chapter 3). Mitochondrial properties in southern killifish did not differ with thermal acclimation.Taken together, my data suggest that UCPs may play a role in thermal acclimation, although there is not a clear connection between UCP mRNA expression and function. Furthermore, my data indicate that northern killifish may have a greater capacity to respond to low temperature acclimation than southern killifish, suggesting a potential role for adaptive variation in mitochondrial responses to temperature.
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Exposure to high environmental pH is physiologically stressful for fish. In British Columbia, this has led to low survivorship among Rainbow Trout stocked into alkaline lakes. Early studies have shown promising results for stocking the progeny of brood stock collected in high pH lakes into similar alkaline environments. Here I follow up by characterizing the high pH tolerance of fish with parents collected from an alkaline lake, Stump Lake. I also look at the effects of acclimation and rearing fish at pH 8.8 on subsequent pH 9.5 tolerance. I found that this population had a short time to loss of equilibrium, with only 10% of fish remaining after a 3 day exposure to pH 9.5. Acclimation resulted in significant improvements to tolerance and rearing resulted in almost none of the fish losing equilibrium over a 3 day exposure. A genome wide association study on non-acclimated and acclimated individuals did not show any significant genetic marker associations with high pH tolerance. However this analysis did identify some potential SNPs associated with genes involved in acid-base regulation, muscle function, neural signaling, and DNA transcription in the non-acclimated fish. The pH 8.8 acclimated fish only showed association with genes involved in neural signaling and DNA transcription. These data suggest that acclimation may remove limitations associated with some of these other processes. Overall the Stump Lake population does not appear to have genetic adaptations that improve tolerance to high pH exposure, but can improve tolerance through acclimation to moderately high pH.
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Episodes of hypoxia are becoming more common along the British Columbia (BC) coast especially in the late summer. When dissolved oxygen drops below optimum levels, fish survival, growth and reproduction are affected; moreover, hypoxia can be lethal to fish, resulting in economic losses to salmon farmers. As a first step towards addressing this challenge for BC salmon farmers, the objectives of this study were to characterize variation in hypoxia tolerance in Atlantic salmon (Salmo salar) under culture conditions and identify the genetic basis of this variation in the strains of salmon used by the aquaculture producer Marine Harvest Canada. Using time-to-loss-of-equilibrium (LOE) following exposure to acute hypoxia (2.1 mg/L) as an index of hypoxia tolerance, I show that there are significant differences in hypoxia tolerance within and between the strains of Atlantic salmon examined. For adults in seawater, time-to-LOE at 2.1 mg/L DO ranged from 4.6 min to 126.9 min, and the McConnell strain had better hypoxia tolerance than the Mowi strain. A similar pattern was observed for smolts in freshwater, with time-to-LOE ranging from 4.5 min before 2.1 mg/L DO was reached to 355.4 min at 2.1 mg/L DO. Genotyping-by-sequencing (GBS) was used to identify single-nucleotide polymorphism (SNP) markers in these strains for use in a genome-wide association study (GWAS). GWAS in adult fish in seawater revealed two SNPs associated with hypoxia tolerance using genome-wide FDR correction, and six SNPs associated with hypoxia tolerance using chromosome-wide FDR correction. In contrast, GWAS in smolts in freshwater identified one SNP using genome-wide FDR correction and one SNP using chromosome-wide FDR correction. There was no overlap in the SNPs identified as associated with hypoxia tolerance at these two life stages. In addition, I identified four significant SNPs associated with body mass in adults with chromosome-wide FDR correction and two SNPs associated with body mass with genome-wide FDR correction and fifty-eight SNPs associated with body mass with chromosome-wide FDR correction. These findings provide promise for follow-up work on SNP markers that could potentially be used for marker-assisted selection to improve hypoxia tolerance and growth in Atlantic salmon.
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The oxygen-limited thermal tolerance (OLTT) hypothesis suggests that an organism’s upper and lower thermal tolerance limits are linked to the point at which the organism cannot extract enough oxygen from its environment to meet its metabolic demands. Therefore, one conclusion that could be drawn from this hypothesis is that an organism’s metabolic rate (Mo₂) should be inversely correlated with the maximum environmental temperature it can tolerate. This is because organisms with a higher demand for oxygen will be the first to experience a mismatch between oxygen supply and demand as the amount of dissolved oxygen in water decreases with increasing temperature. The validity of this hypothesis was tested using the common killifish, Fundulus heteroclitus, from a contact zone between the two subspecies: the Northern Fundulus heteroclitus macrolepidotus and the Southern Fundulus heteroclitus heteroclitus. I found that there was a significant inverse correlation (p=0.0192) between the critical thermal maximum (CTMax) and routine metabolic rate (RMR) after controlling for the weight of fish, which supports the OLTT hypothesis. The predictive power of this relationship, however, was low (R²=0.16). In addition, there was a significant effect of collection location on CTMax (p
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