Patricia Schulte

Professor

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Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - May 2019)
Environmental epigenomics in stickleback : plasticity of DNA methylation and gene expression patterns across time scales (2018)

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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Intraspecific variation and thermal acclimation effects on mitochondrial function in a eurythermal teleost (Fundulus heteroclitus) (2018)

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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Contributions of genetic variations and phenotypic plasticity to variation in high pH tolerance in Rainbow Trout (2017)

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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Phenotypic plasticity and divergence in physiological traits during freshwater colonization in threespine stickleback (Gasterosteus aculeatus) (2017)

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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Aerobic metabolism and temperature : intraspecific variation and thermal acclimation in Atlantic killifish (2016)

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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Genetic characterization of a hybrid zone in killifish (Fundulus heteroclitus) : evidence for assortative mating or selection against hybrids (2013)

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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The evolution of swimming capacity among migratory and non-migratory populations of the threespine stickleback (Gasterosteus aculeatus) (2012)

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 (2010 - 2018)
The role of mitochondrial uncoupling in temperature responses in Atlantic killifish, Fundulus heteroclitus (2018)

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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A genetic basis of adaptation to high pH in Rainbow Trout (2017)

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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An investigation into the genetic basis of variation in hypoxia tolerance in Atlantic salmon (2017)

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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Characterization of Fundulus heteroclitus embryonic cell lines and their applications to fish health (2014)

Common killifish, or mummichogs (Fundulus heteroclitus), are a species of estuarine teleost that are widely used in comparative physiology, toxicology and embryology. Their ability to withstand extreme environmental conditions, widespread distribution, and relatively sedentary nature, makes them ideal as sentinel species of estuarine health. However, the lack of cell lines derived from F. heteroclitus places limitations on the utility of this species in environmental research. In contrast, cell cultures derived from other model organisms have assisted and facilitated our understanding of the effects that environmental contaminants have on organisms in vitro. The development and use of novel F. heteroclitus cell lines for toxicological and parasitological applications is reported here. Continuous proliferating cells were derived from pre-hatch embryos of killifish and have been maintained for 3 years. Three stable cell lines were obtained from the head and body tissues of F. heteroclitus; these stable cell lines have been dubbed KilliFish Embryo 1, 3, and 5 (KFE-1, KFE-3, KFE-5). All three cell lines have been characterized for origin and functionality, as well as for applications in toxicology, studying effects of model chemical pollutants, and in parasitology to evaluate a cod-infecting microsporidia that has been an emerging pathogen of concern, for their ability to infect and grow in cell lines derived from sentinel species. KFE-1 has characteristics of neuroepithelial cells, whereas KFE-3 are possibly liver derived cells, and KFE-5 are distinctly myogenic, as this line has cells that appear to be striated muscle cells. Like intact F. heteroclitus, these cell lines can withstand a wide temperature range from 4°C to 37°C. Mechanisms of thermotolerance and ability to withstand salinity and hypoxia as well as chemical toxicity tolerance could be readily studied with these new cell lines.

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Determining the relative amounts of prey in Steller sea lion (Eumetopias jubatus) diet using real-time PCR (2010)

Determining diets of pinnipeds by visually identifying prey remains recovered in faecal samples is challenging because of differences in digestion and passage rates of hard parts. Analyzing the soft matrix of faecal material using DNA-based techniques is an alternative means to identify prey species consumed, but published techniques are largely non-quantitative, which limits their applicability. I developed and validated a real-time PCR technique using species-specific mitochondrial DNA primers to quantify the diets of Steller sea lions (Eumetopias jubatus). I first demonstrated that the proportions of prey tissue DNA in mixtures of DNA isolated from four prey species could be estimated within a margin of ~12% of the percent in the mix. These prey species included herring Clupea palasii, eulachon Thaleichthyes pacificus, squid Loligo opalescens and rosethorn rockfish Sebastes helvomaculatus. I then applied real-time PCR to DNA extracted from faecal samples obtained from Steller sea lions that had been fed 11 different combinations of herring, eulachon, squid and Pacific ocean perch rockfish (Sebastes alutus), ranging from 7-75% contributions to a meal mix (by wet weight). The difference between the average percentage estimated by real-time PCR and the percentage of prey consumed was generally less than 12% for all diets fed when percentages of prey consumed were corrected for differences in mtDNA density among the prey items. My findings indicate that real-time PCR can detect the quantity of prey consumed for a variety of complex diets and prey species, including cephalopods and fish.

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Oxygen-Limited Thermal Tolerance in Hybrid Killifish, Fundueus heteroclitus (2010)

No abstract available.

 

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