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Climate change alters multiple abiotic environmental factors in aquatic environments but relatively little is known about their interacting impacts, particularly in developing organisms where these exposures have the potential to cause long-lasting effects. To explore these issues, we exposed developing killifish embryos (Fundulus heteroclitus) to 26 °C or 20 °C and 20 ppt or 3 ppt salinity in a fully-factorial design. After hatching, fish were transferred to common conditions of 20 °C and 20 ppt to assess the potential for persistent developmental plasticity. Warm temperature increased hatching success and decreased hatch time, whereas low salinity negatively affected hatching success, but this was only significant in fish developed at 20 °C. Temperature, salinity, or their interaction affected mRNA levels of genes typically associated with thermal and hypoxia tolerance (hif1a, hsp90b, hsp90a, hsc70, and hsp70.2) across multiple developmental timepoints. These differences were persistent into the juvenile stage, where the fish that developed at 26 °C had higher expression of hif1a, hsp90b, hsp90a, and hsp70.2 than fish developed at 20 °C, and this was particularly evident for the group developed at both high temperature and salinity. There were also long-lasting effects of developmental treatments on body size after four months of rearing under common conditions. Fish developed at low salinity or temperature were larger than fish developed at high temperature or salinity, but there was no interaction between the two factors. These data highlight the complex nature of the developmental effects of interacting stressors which has important implications for predicting the resilience of fishes in the context of climate change., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Thermal performance curves (TPCs) provide a framework for understanding the effects of temperature on ectotherm performance and fitness. TPCs are often used to test hypotheses regarding local adaptation to temperature or to develop predictions for how organisms will respond to climate warming. However, for aquatic organisms such as fishes, most TPCs have been estimated for adult life stages, and little is known about the shape of TPCs or the potential for thermal adaptation at sensitive embryonic life stages. To examine how latitudinal gradients shape TPCs at early life stages in fishes, we used two populations of Fundulus heteroclitus that have been shown to exhibit latitudinal variation along the thermal cline as adults. We exposed embryos from both northern and southern populations and their reciprocal crosses to eight different temperatures (15°C, 18°C, 21°C, 24°C, 27°C, 30°C, 33°C, and 36°C) until hatch and examined the effects of developmental temperature on embryonic and larval traits (shape of TPCs, heart rate, and body size). We found that the pure southern embryos had a right-shifted TPC (higher thermal optimum (T opt ) for developmental rate, survival, and embryonic growth rate) whereas pure northern embryos had a vertically shifted TPC (higher maximum performance (P max ) for developmental rate). Differences across larval traits and cross-type were also found, such that northern crosses hatched faster and hatched at a smaller size compared to the pure southern population. Overall, these observed differences in embryonic and larval traits are consistent with patterns of both local adaptation and countergradient variation., (© 2024 The Author(s). Journal of Experimental Zoology Part A: Ecological and Integrative Physiology published by Wiley Periodicals LLC.)

Common killifish Fundulus heteroclitus were acclimated to ecologically relevant temperatures (5, 15 and 33°C) and their maximum heart rate (f Hmax ) was measured at each acclimation temperature during an acute warming protocol. Acclimation to 33°C increased peak f Hmax by up to 32% and allowed the heart to beat rhythmically at a temperature 10°C higher when compared with acclimation to 5°C. Independent of acclimation temperature, peak f Hmax occurred about 3°C cooler than the temperature that first produced cardiac arrhythmias. Thus, when compared with previously published values for the critical thermal maximum of F. heteroclitus, the temperature for peak f Hmax was cooler and the temperature that first produced cardiac arrhythmias was similar to these critical thermal maxima. The considerable thermal plasticity of f Hmax demonstrated in the present study is entirely consistent with eurythermal ecology of killifish, as shown previously for another eurythermal fish Gillichthys mirabilis., (© 2019 The Fisheries Society of the British Isles.)

The Pacific hagfish (Eptatretus stoutii) is a representative of the most basal extant craniates, and is a marine osmoconformer with an extremely low metabolic rate (MO 2  = 475 µmol O 2 /kg/h at 12 °C). We investigated potential physiological trade-offs associated with compensatory changes in gill ventilation and perfusion when 12 °C-acclimated hagfish were acutely exposed to 7 °C or 17 °C, as reflected in diffusive unidirectional water flux ([Formula: see text], measured with tritiated water: 3 H 2 O), net ammonia flux (J amm ), and plasma ion and acid-base status. [Formula: see text] was high (~ 1.4 L/kg/h at 12 °C) in comparison to marine teleosts and elasmobranchs. MO 2 increased linearly with temperature (R 2  = 0.991), and was more sensitive (Q 10  = 3.22) in the 12-7 °C range than either J amm (1.86) or [Formula: see text] (1.35), but the pattern reversed from 12 to 17 °C (Q 10 s: MO 2  = 2.77, J amm  = 2.88, [Formula: see text] = 4.01). Heart rate, ventilatory index (a proxy for total ventilation), and coughing frequency also increased but with different patterns. At 17 °C, plasma [Ca 2+ ] and [Mg 2+ ] decreased, although osmolality increased, associated with elevations in plasma [Na + ] and [Cl - ]. Blood pH and PCO 2 were unaffected by acute temperature changes while [HCO 3 - ] increased. Hyperoxia (PO 2 , and had diverse effects on plasma ion and acid-base status. Our results suggest a clear osmorespiratory compromise occurring for the diffusive water fluxes as a result of acute temperature changes in this osmoconformer.amm , and had diverse effects on plasma ion and acid-base status. Our results suggest a clear osmorespiratory compromise occurring for the diffusive water fluxes as a result of acute temperature changes in this osmoconformer.

Life history strategies, physiological traits, and behavior are thought to covary along a "pace of life" axis, with organisms at the fast end of this continuum having higher fecundity, shorter lifespan, and more rapid development, growth, and metabolic rates. Countergradient variation represents a special case of pace of life variation, in which high-latitude organisms occupy the fast end of the continuum relative to low-latitude conspecifics when compared at a common temperature. Here, we use Atlantic killifish (Fundulus heteroclitus) to explore the role of mitochondrial properties as a mechanism underlying countergradient variation, and thus variation in the pace of life. This species is found along the Atlantic coast of North America, through a steep latitudinal thermal gradient. The northern subspecies has faster development, more rapid growth, higher routine metabolic rate, and higher activity than the southern subspecies when compared at a common temperature. The northern subspecies also has greater mitochondrial respiratory capacity in the liver, although these differences are not evident in other tissues. The increased respiratory capacity of liver mitochondria in northern fish is associated with increases in the activity of multiple electron transport complexes, which largely reflects an increase in the amount of inner mitochondrial membrane per mitochondrion in the northern fish. There are also differences in the lipid composition of liver mitochondrial membranes, including differences in cardiolipin species, which could also influence respiratory capacity. These data suggest that variation in mitochondrial properties could, at least in part, underlie variation in the pace of life in Atlantic killifish.

Colonization of freshwater habitats from marine environments exposes organisms to novel combinations of temperature and salinity, but little is known about physiological responses to the interactive effects of these stressors. Here, we examined the effects of temperature (14 versus 4 °C) and salinity (11 versus 0.3 ppt) on gill gene expression in marine, anadromous, and freshwater populations of threespine stickleback (Gasterosteus aculeatus). Expression of the epithelial calcium channel was not affected by temperature or salinity, but had significantly higher expression in the freshwater ecotype. The combination of low temperature and low salinity had non-additive effects on the expression of the Na + /H + exchanger. Fish exposed to the combination of low temperature and low salinity had expression levels similar to fish exposed to either factor in isolation. Expression of Na + ,K + -ATPase α-subunit was greater in fish exposed to low temperature and low salinity than in fish exposed to the factors separately, and this effect was the most pronounced in the marine ecotype. We also examined the interactive effects of salinity and temperature on gill morphology in the marine ecotype, and observed non-additive effects. Low temperature increased the size of the interlamellar cell mass in fish held at 11 ppt, but not at 0.3 ppt, and the effect of low salinity was in the opposite direction in fish at high and low temperatures. These data demonstrate interactive effects of temperature and salinity and highlight that overwintering in cold freshwater was likely a physiological challenge for marine stickleback as they colonized freshwater following the last glaciation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Phenotypic plasticity occurs at a variety of timescales, but little is known about the degree to which plastic responses at different timescales are associated with similar underlying molecular processes, which is critical for assessing the effects of plasticity on evolutionary trajectories. To address this issue, we identified differential gene expression in response to developmental temperature in the muscle transcriptome of adult threespine stickleback (Gasterosteus aculeatus) exposed to 12, 18 and 24°C until hatch and then held at 18°C for 9 months and compared these results to differential gene expression in response to adult thermal acclimation in stickleback developed at 18°C and then acclimated to 5 and 25°C as adults. Adult thermal acclimation affected the expression of 7,940 and 7,015 genes in response to cold and warm acclimation, respectively, and 4,851 of these genes responded in both treatments. In contrast, the expression of only 33 and 29 genes was affected by cold and warm development, respectively. The majority of the genes affected by developmental temperature were also affected by adult acclimation temperature. Many genes that were differentially expressed as a result of adult acclimation were associated with previously identified temperature-dependent effects on DNA methylation patterns, suggesting a role of epigenetic mechanisms in regulating gene expression plasticity during acclimation. Taken together, these results demonstrate similarities between the persistent effects of developmental plasticity on gene expression and the effects of adult thermal acclimation, emphasizing the potential for mechanistic links between plasticity acting at these different life stages., (© 2018 John Wiley & Sons Ltd.)

Colonisation can expose organisms to novel combinations of abiotic and biotic factors and drive adaptive divergence. Yet, studies investigating the interactive effects of multiple abiotic factors on the evolution of physiological traits remain rare. Here we examine the effects of low salinity, low temperature, and their interaction on the growth of three North American populations of threespine stickleback (Gasterosteus aculeatus). In north-temperate freshwater habitats, stickleback populations experience a combination of low salinity and low winter temperatures that are not experienced by the ancestral marine and anadromous populations. Here we show that both salinity and temperature, and their interaction, have stronger negative effects on marine and anadromous populations than a freshwater population. Freshwater stickleback showed only a ~20% reduction in specific growth rate when exposed to 4 °C, while marine and anadromous stickleback showed sharp declines (82% and 74% respectively) under these conditions. The modest decreases in growth in freshwater stickleback in fresh water in the cold strongly suggest that this population has the capacity for physiological compensation to offset the negative thermodynamic effects of low temperature on growth. These results are suggestive of adaptive evolution in response to the interactive effects of low salinity and low temperature during freshwater colonisation.

Environmental temperature can greatly influence the homeostasis of ectotherms through its effects on biochemical reactions and whole-animal physiology. Elasmobranchs tend to be N limited and are osmoconformers, retaining ammonia and urea-N at the gills and using the latter as a key osmolyte to maintain high blood osmolality. However, the effects of temperature on these key processes remain largely unknown. We evaluated the effects of acute exposure to different temperatures (7°, 12°, 15°, 18°, 22°C) on oxygen consumption, ammonia, urea-N, and diffusive water fluxes at the gills of Squalus acanthias suckleyi. We hypothesized that as metabolic demand for oxygen increased with temperature, the fluxes of ammonia, urea-N, and 3 H 2 O at the gills would increase in parallel with those of oxygen. Oxygen consumption (overall [Formula: see text] from 7.5° to 22°C) and water fluxes (overall [Formula: see text]) responded to increases in temperature in a similar, almost linear, manner. Ammonia-N efflux rates varied the most, increasing almost 15-fold from 7.5° to 22°C ([Formula: see text]). Urea-N efflux was tightly conserved over the 7.5°-15°C range ([Formula: see text]) but increased greatly at higher temperatures, yielding an overall [Formula: see text]. These differences likely reflect differences in the transport pathways for the four moieties. They also suggest the failure of urea-N- and ammonia-N-conserving mechanisms at the gill above 15°C. Hyperoxia did not alleviate the effects of high temperature. Indeed, urea-N and ammonia-N effluxes were dramatically increased when animals were exposed to high temperatures in the presence of hyperoxia, suggesting that high partial pressure of oxygen may have caused oxidative damage to gill epithelial membranes.

Because of its profound effects on the rates of biological processes such as aerobic metabolism, environmental temperature plays an important role in shaping the distribution and abundance of species. As temperature increases, the rate of metabolism increases and then rapidly declines at higher temperatures - a response that can be described using a thermal performance curve (TPC). Although the shape of the TPC for aerobic metabolism is often attributed to the competing effects of thermodynamics, which can be described using the Arrhenius equation, and the effects of temperature on protein stability, this account represents an over-simplification of the factors acting even at the level of single proteins. In addition, it cannot adequately account for the effects of temperature on complex multistep processes, such as aerobic metabolism, that rely on mechanisms acting across multiple levels of biological organization. The purpose of this review is to explore our current understanding of the factors that shape the TPC for aerobic metabolism in response to acute changes in temperature, and to highlight areas where this understanding is weak or insufficient. Developing a more strongly grounded mechanistic model to account for the shape of the TPC for aerobic metabolism is crucial because these TPCs are the foundation of several recent attempts to predict the responses of species to climate change, including the metabolic theory of ecology and the hypothesis of oxygen and capacity-limited thermal tolerance., (© 2015. Published by The Company of Biologists Ltd.)

In fishes, performance failure at high temperature is thought to be due to a limitation on oxygen delivery (the theory of oxygen and capacity limited thermal tolerance, OCLTT), which suggests that thermal tolerance and hypoxia tolerance might be functionally associated. Here we examined variation in temperature and hypoxia tolerance among 41 families of Atlantic salmon (Salmo salar), which allowed us to evaluate the association between these two traits. Both temperature and hypoxia tolerance varied significantly among families and there was a significant positive correlation between critical maximum temperature (CTmax) and hypoxia tolerance, supporting the OCLTT concept. At the organ and cellular levels, we also discovered support for the OCLTT concept as relative ventricle mass (RVM) and cardiac myoglobin (Mb) levels both correlated positively with CTmax (R(2)=0.21, P<0.001 and R(2)=0.17, P=0.003, respectively). A large RVM has previously been shown to be associated with high cardiac output, which might facilitate tissue oxygen supply during elevated oxygen demand at high temperatures, while Mb facilitates the oxygen transfer from the blood to tissues, especially during hypoxia. The data presented here demonstrate for the first time that RVM and Mb are correlated with increased upper temperature tolerance in fish. High phenotypic variation between families and greater similarity among full- and half-siblings suggests that there is substantial standing genetic variation in thermal and hypoxia tolerance, which could respond to selection either in aquaculture or in response to anthropogenic stressors such as global climate change.

Thermal performance curves (TPCs) describe the effects of temperature on biological rate processes. Here, we use examples from our work on common killifish (Fundulus heteroclitus) to illustrate some important conceptual issues relating to TPCs in the context of using these curves to predict the responses of organisms to climate change. Phenotypic plasticity has the capacity to alter the shape and position of the TPCs for acute exposures, but these changes can be obscured when rate processes are measured only following chronic exposures. For example, the acute TPC for mitochondrial respiration in killifish is exponential in shape, but this shape changes with acclimation. If respiration rate is measured only at the acclimation temperature, the TPC is linear, concealing the underlying mechanistic complexity at an acute time scale. These issues are particularly problematic when attempting to use TPCs to predict the responses of organisms to temperature change in natural environments. Many TPCs are generated using laboratory exposures to constant temperatures, but temperature fluctuates in the natural environment, and the mechanisms influencing performance at acute and chronic time scales, and the responses of the performance traits at these time scales may be quite different. Unfortunately, our current understanding of the mechanisms underlying the responses of organisms to temperature change is incomplete, particularly with respect to integrating from processes occurring at the level of single proteins up to whole-organism functions across different time scales, which is a challenge for the development of strongly grounded mechanistic models of responses to global climate change., (© The Author 2011. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved.)

Behavioral thermoregulation can allow ectotherms to buffer the effects of changes in environmental temperature, and thus an organism's preferred temperature is thought to be under strong selection. However, this contention has seldom been tested. We used common killifish Fundulus heteroclitus from high-latitude (northern) and low-latitude (southern) populations to investigate intraspecific variation in thermal preference and its relationship to habitat temperature. We quantified the preferred temperatures of northern and southern killifish populations acclimated to three temperatures (5 degrees , 15 degrees , and 25 degrees C) to evaluate two alternative hypotheses for the evolution of differences in thermal preference among latitudinally separated populations: local thermal adaptation, which predicts that organisms from high latitudes should prefer lower temperatures than individuals from lower latitudes, versus countergradient variation, which predicts that high-latitude organisms should prefer higher temperatures to compensate for shorter growing seasons. All killifish selected their final thermal preferendum within 4 h. Southern killifish and killifish acclimated to warmer temperatures had greater variability in selected temperature. This increase in variability was the result of an increase in interindividual variation in preferred temperature rather than a reduction in the precision of temperature selection in these groups. Northern killifish preferred significantly higher temperatures than southern fish (30.6 degrees vs. 29.0 degrees C, respectively, when calculated on the basis of the temperature selected consistently for at least 30 min; 28.4 degrees vs. 26.5 degrees C, respectively, when calculated on the basis of the mean temperature occupied), regardless of acclimation temperature. These data are not consistent with local adaptation in thermal preference but instead can be better explained by countergradient variation in thermal preference in killifish.

Phenotypic plasticity is an important aspect of an organism's response to environmental change that often requires the modulation of gene expression. These changes in gene expression can be quantitative as a result of increases or decreases in the amounts of specific transcripts, or qualitative as a result of the expression of alternative transcripts from the same gene (e.g., via alternative splicing of pre-mRNAs). Although the role of quantitative changes in gene expression in phenotypic plasticity is well known, relatively few studies have examined the role of qualitative changes. Here, we use skeletal muscle RNA-seq data from Atlantic killifish (Fundulus heteroclitus), threespine stickleback (Gasterosteus aculeatus) and zebrafish (Danio rerio) to investigate the extent of qualitative changes in gene expression in response to cold. Fewer genes demonstrated alternative splicing than differential expression as a result of cold acclimation; however, differences in splicing were detected for between 426 and 866 genes depending on species, indicating that large numbers of qualitative changes in gene expression are associated with cold acclimation. Many of these alternatively spliced genes were also differentially expressed, and there was functional enrichment for involvement in muscle contraction among the genes demonstrating qualitative changes in response to cold acclimation. Additionally, there was a common group of 29 genes with coldacclimation- mediated changes in splicing in all three species, suggesting that there may be a set of genes with expression patterns that respond qualitatively to prolonged cold temperatures across fishes. [ABSTRACT FROM AUTHOR]

Thermal effects on mitochondrial efficiency and ATP production can influence whole-animal thermal tolerance and performance. Thus, organisms may have the capacity to alter mitochondrial processes through acclimation or adaptation to mitigate these effects. One possible mechanism is through the action of uncoupling proteins (UCPs), which can decrease the proton-motive force independent of the production of ATP. To test this hypothesis, we examined the mRNA expression patterns of UCP isoforms and characterized the effects of thermal acclimation and putative local thermal adaptation on mitochondrial capacity, proton leak and P/O ratios in two subspecies of Atlantic killifish (Fundulus heteroclitus). Ucp1 was the dominant isoform in liver and was more highly expressed in northern killifish. We found that cold acclimation increased mitochondrial capacity (state III and maximum substrate oxidation capacity), state II membrane potential, proton leak and P/O ratios in northern, but not southern, killifish liver mitochondria. Palmitate-induced mitochondrial uncoupling was detected in northern, but not southern, killifish liver mitochondria, consistent with the differences in Ucp mRNA expression between the subspecies. Taken together, our data suggest that mitochondrial function is more plastic in response to thermal acclimation in northern killifish than in southern killifish and that UCP1 may play a role in regulating the proton-motive force in northern, but not southern, killifish in response to thermal acclimation. These data demonstrate the potential for adaptive variation in mitochondrial plasticity in response to cold. [ABSTRACT FROM AUTHOR]

The resilience of organisms to climate change through adaptive evolution is dependent on the extent of genetically based variation in key phenotypic traits and the nature of genetic associations between them. For aquatic animals, upper thermal tolerance and hypoxia tolerance are likely to be a important determinants of sensitivity to climate change. To determine the genetic basis of these traits and to detect associations between them, we compared naturally occurring populations of two subspecies of Atlantic killifish, Fundulus heteroclitus, that differ in both thermal and hypoxia tolerance. Multilocus association mapping demonstrated that 47 and 35 single nucleotide polymorphisms (SNPs) explained 43.4% and 51.9% of variation in thermal and hypoxia tolerance, respectively, suggesting that genetic mechanisms underlie a substantial proportion of variation in each trait. However, no explanatory SNPs were shared between traits, and upper thermal tolerance varied approximately linearly with latitude, whereas hypoxia tolerance exhibited a steep phenotypic break across the contact zone between the subspecies. These results suggest that upper thermal tolerance and hypoxia tolerance are neither phenotypically correlated nor genetically associated, and thus that rates of adaptive change in these traits can be independently fine‐tuned by natural selection. This modularity of important traits can underpin the evolvability of organisms to complex future environmental change. For aquatic organisms, variation in the ability to tolerate high temperatures and low oxygen levels is likely to influence resilience to climate change. Here, we use populations of killifish to show that naturally occurring variations in upper thermal tolerance and hypoxia tolerance have genetic bases, and that these two traits are not functionally or genetically correlated. This genetic variation provides the material needed for the action of natural selection, but the lack of trait association could limit rapid adaptation to combined stressors. However, this lack of association potentially provides flexibility to fine‐tune adaptive responses to specific local conditions. [ABSTRACT FROM AUTHOR]

Mitochondrial performance may play a role in setting whole-animal thermal tolerance limits and their plasticity, but the relative roles of adjustments in mitochondrial performance across different highly aerobic tissues remain poorly understood. We compared heart and brain mitochondrial responses to acute thermal challenges and to thermal acclimation using high-resolution respirometry in two locally adapted subspecies of Atlantic killifish (Fundulus heteroclitus). We predicted that 5°C acclimation would result in compensatory increases in mitochondrial performance, while 33°C acclimation would cause suppression of mitochondrial function to minimize the effects of high temperature on mitochondrial metabolism. In contrast, acclimation to both 33 and 5°C decreasedmitochondrial performance compared with fish acclimated to 15°C. These adjustments could represent an energetic cost-saving mechanism at temperature extremes. Acclimation responses were similar in both heart and brain; however, this effect was smaller in the heart, which might indicate its importance in maintaining whole-animal thermal performance. Alternatively, larger acclimation effects in the brain might indicate greater thermal sensitivity compared with the heart. We detected only modest differences between subspecies that were dependent on the tissue assayed. These data demonstrate extensive plasticity in mitochondrial performance following thermal acclimation in killifish, and indicate that the extent of these responses differs between tissues, highlighting the importance and complexity of mitochondrial regulation in thermal acclimation in eurytherms. [ABSTRACT FROM AUTHOR]

Adjustments of aerobic metabolic processes are critical components of organismal responses to environmental change that require tight coordination between the nuclear and mitochondrial genomes. Intraspecific differences in mitochondrial genotype can affect gene transcription in both genomes. Thus, variation in mitochondrial genotype may be associated with differences in the plasticity of gene expression when organisms are faced with changes in environmental conditions. Cold acclimation is known to result in metabolic responses involving increases in mitochondrial amount and capacity, suggesting that low temperatures may pose a particular challenge when coordinating the functions of the nuclear and mitochondrial genomes. In this study, we utilized RNA-seq to assess transcriptome-wide gene expression in the muscle of Atlantic killifish ( Fundulus heteroclitus) from a population that contains segregating variation in mitochondrial genotype. We examined gene expression plasticity in response to 5 °C acclimation and the effects of mitochondrial genotype on this plasticity. Cold acclimation resulted in changes in gene expression consistent with up-regulation of genes involved in many cellular functions, including spliceosomal and proteasomal processes, and with down-regulation of genes involved in extracellular matrix, muscle contraction and oxidative phosphorylation functions. There were few differences in gene expression between killifish with different mitochondrial genotypes: 14 genes demonstrated significant interactions between mitochondrial genotype and acclimation temperature and three genes demonstrated effects of mitochondrial genotype alone. These results indicate that variation in mitochondrial genotype has modest effects on gene expression; the majority of which are revealed as differences in plasticity as a result of environmental change. [ABSTRACT FROM AUTHOR]

Human activities are increasing both the frequency of hypoxic episodes and the mean temperature of aquatic ecosystems, but few studies have considered the possibility that acclimation to one of these stressors could improve the ability to cope with the other stressor. Here, we used Atlantic killifish, Fundulus heteroclitus, to test this hypothesis. Hypoxia tolerance was measured as time to loss of equilibrium in hypoxia (LOEhyp) at 0.4 kPa oxygen. Time to LOEhyp declined from 73.3±6.9 min at 15°C to 2.6±3.8 min at 23°C, and at 30°C no fish could withstand this level of hypoxia. Prior acclimation to warm temperatures significantly increased time to LOEhyp. Hypoxia tolerance of the southern subspecies of killifish, F heteroclitus heteroclitus, was greater than that of the northern subspecies, F heteroclitus macrolepidotus, measured both as critical oxygen tension (Phyp) and as time to LOEhyp. Warm acclimation offset the negative effects of temperature on time to LOEhyp to a similar extent in the two subspecies. Warm acclimation increased total lamellar surface area of the gill in both subspecies as a result of regression of an interlamellar cell mass (ILCM). However, differences in total lamellar surface area could not explain differences in time to LOEhyp between the subspecies, suggesting that the lower time to LOEhyp of northern fish is related to their higher routine metabolic rate. These data suggest that thermal plasticity in gill morphology can improve the capacity of this species to tolerate hypoxia, and shows how existing plasticity may help organisms to cope with the complex interacting stressors that they will encounter with increasing frequency as our climate changes. [ABSTRACT FROM AUTHOR]

Abiotic factors can act as barriers to colonization and drive local adaptation. During colonization, organisms may cope with changes in abiotic factors using existing phenotypic plasticity, but the role of phenotypic plasticity in assisting or hindering the process of local adaptation remains unclear. To address these questions, we explore the role of winter conditions in driving divergence during freshwater colonization and the effects of plasticity on local adaptation in ancestral marine and derived freshwater ecotypes of threespine stickleback ( Gasterosteus aculeatus). We found that freshwater-resident stickleback had greater tolerance of acute exposure to low temperatures than marine stickleback, but these differences were abolished after acclimation to simulated winter conditions (9L:15D photoperiod at 4 °C). Plasma chloride levels differed between the ecotypes, but showed a similar degree of plasticity between ecotypes. Gene expression of the epithelial calcium channel (ECaC) differed between ecotypes, with the freshwater ecotype demonstrating substantially greater expression than the marine ecotype, but there was no plasticity in this trait under these conditions in either ecotype. In contrast, growth (assessed as final mass) and the expression of an isoform of the electroneutral Na+/H+ exchanger (NHE3) exhibited substantial change with temperature in the marine ecotype that was not observed in the freshwater ecotype under the conditions tested here, which is consistent with evolution of these traits by a process such as genetic assimilation. These data demonstrate substantial divergence in many of these traits between freshwater and marine stickleback, but also illustrate the complexity of possible relationships between plasticity and local adaptation. [ABSTRACT FROM AUTHOR]

Although the term environmental stress is used across multiple fields in biology, the inherent ambiguity associated with its definition has caused confusion when attempting to understand organismal responses to environmental change. Here I provide a brief summary of existing definitions of the term stress, and the related concepts of homeostasis and allostasis, and attempt to unify them to develop a general framework for understanding how organisms respond to environmental stressors. I suggest that viewing stressors as environmental changes that cause reductions in performance or fitness provides the broadest and most useful conception of the phenomenon of stress. I examine this framework in the context of animals that have evolved in highly variable environments, using the Atlantic killifish, Fundulus heteroclitus, as a case study. Consistent with the extreme environmental variation that they experience in their salt marsh habitats, killifish have substantial capacity for both short-term resistance and long-term plasticity in the face of changing temperature, salinity and oxygenation. There is inter-population variation in the sensitivity of killifish to environmental stressors, and in their ability to acclimate, suggesting that local adaptation can shape the stress response even in organisms that are broadly tolerant and highly plastic. Whole-organism differences between populations in stressor sensitivity and phenotypic plasticity are reflected at the biochemical and molecular levels in killifish, emphasizing the integrative nature of the response to environmental stressors. Examination of this empirical example highlights the utility of using an evolutionary perspective on stressors, stress and stress responses. [ABSTRACT FROM AUTHOR]

Loss of aerobic scope at high and low temperatures is a physiological mechanism proposed to limit the thermal performance and tolerance of organisms, a theory known as oxygen-and capacity-limited thermal tolerance (OCLTT). Eurythermal organisms maintain aerobic scope over wide ranges of temperatures, but it is unknown whether acclimation is necessary to maintain this breadth. The objective of this study was to examine changes in aerobic scope in Fundulus heteroclitus, a eurythermal fish, after acclimation and acute exposure to temperatures from 5° to 33°C. The range of temperatures over which aerobic scope was nonzero was similar in acclimated and acutely exposed fish, suggesting that acclimation has modest effects on the thermal breadth of aerobic scope. However, in acclimated fish, there was a clear optimum temperature range for aerobic scope between 25° and 30°C, whereas aerobic scope was relatively constant across the entire temperature range with acute temperature exposure. Therefore, the primary effect of acclimation was to increase aerobic scope between 25° and 30°C, which paradoxically resulted in a narrower temperature range of optimal performance in acclimated fish compared to acutely exposed fish. There was only weak evidence for correlations between the thermal optimum of aerobic scope and the thermal optimum of measures of performance (specific growth rate and gonadosomatic index), and indicators of anaerobic metabolism (lactate accumulation and lactate dehydrogenase activity) only increased at high temperatures. Together these data fit many, but not all, of the predictions made by OCLTT. [ABSTRACT FROM AUTHOR]

As global temperatures rise, there is a growing need to understand the physiological mechanisms that determine an organism's thermal niche. Here, we test the hypothesis that increases in mitochondrial capacity with cold acclimation and adaptation are associated with decreases in thermal tolerance using two subspecies of killifish (Fundulus heteroclitus) that differ in thermal niche. We assessed whole-organism metabolic rate, mitochondrial amount and mitochondrial function in killifish acclimated to several temperatures. Mitochondrial enzyme activities and mRNA levels were greater in fish from the northern subspecies, particularly in cold-acclimated fish, suggesting that the putatively cold-adapted northern subspecies has a greater capacity for increases in mitochondrial amount in response to cold acclimation. When tested at the fish's acclimation temperature, maximum ADP-stimulated (State III) rates of mitochondrial oxygen consumption in vitro were greater in cold-acclimated northern fish than in southern fish but did not differ between subspecies at higher acclimation temperatures. Whole-organism metabolic rate was greater in fish of the northern subspecies at all acclimation temperatures. Cold acclimation also changed the response of mitochondrial respiration to acute temperature challenge. Mitochondrial oxygen consumption was greater in cold-acclimated northern fish than in southern fish at low test temperatures, but the opposite was true at high test temperatures. These differences were reflected in whole-organism oxygen consumption. Our data indicate that the plasticity of mitochondrial function and amount differs between killifish subspecies, with the less high-temperature tolerant, and putatively cold adapted, northern subspecies having greater ability to increase mitochondrial capacity in the cold. However, there were few differences in mitochondrial properties between subspecies at warm acclimation temperatures, despite differences in both whole-organism oxygen consumption and thermal tolerance at these temperatures. [ABSTRACT FROM AUTHOR]

Abstract: Common killifish, Fundulus heteroclitus, are found in marshes and estuaries along the Atlantic coast of North America from Newfoundland to Florida. Although these habitats are highly productive, they are also characterized by variation in a number of abiotic stressors, including temperature, salinity, oxygen, and anthropogenic toxicants, which vary substantially in both space and time. In order to survive in these habitats, killifish must be able to cope with these stressors, both individually and in combination. There is substantial evidence to suggest that populations of F. heteroclitus have undergone local adaptation to multiple abiotic stressors, including temperature, salinity, and toxicants, but most studies have examined the effects of single stressors in isolation. Here I review some of the studies on local adaptation in F. heteroclitus, focusing on the molecular basis of local adaptation to abiotic stressors, and the acute responses to these stressors both singly and in combination. This work demonstrates that there are substantial interactions between the responses to both natural and anthropogenic stressors at the cellular level. [Copyright &y& Elsevier]

Populations of common killifish, Fundulus heteroclitus, are distributed along the Atlantic coast of North America through a steep latitudinal thermal gradient. We examined intraspecific variation in whole-animal thermal tolerance and its relationship to the heat shock response in killifish from the northern and southern extremes of the species range. Critical thermal maxima were significantly higher in southern than in northern fish by ∼1.5°C at a wide range of acclimation temperatures (from 2–34°C), and critical thermal minima differed by ∼1.5°C at acclimation temperatures above 22°C, converging on the freezing point of brackish water at lower acclimation temperatures. To determine whether these differences in whole-organism thermal tolerance were reflected in differences in either the sequence or regulation of the heat shock protein genes (hsps) we obtained complete cDNA sequences for hsc70, hsp70-1 and hsp70-2, and partial sequences of hsp90α and hsp90β. There were no fixed differences in amino acid sequence between populations in either hsp70-1 or hsp70-2, and only a single conservative substitution between populations in hsc70. By contrast, there were significant differences between populations in the expression of many, but not all, of these genes. Both northern and southern killifish significantly increased hsp70-2 levels above control values (Ton) at a heat shock temperature of 33°C, but the magnitude of this induction was greater in northern fish, suggesting that northern fish may be more susceptible to thermal damage than are southern fish. In contrast, hsp70-1 mRNA levels increased gradually and to the same extent in response to heat shock in both populations. Hsc70 mRNA levels were significantly elevated by heat shock in southern fish, but not in northern fish. Similarly, the more thermotolerant southern killifish had a Ton for hsp90α of 30°C, 2°C lower than that of northern fish. This observation combined with the ability of southern killifish to upregulate hsc70 in response to heat shock suggests a possible role for these hsps in whole-organism differences in thermal tolerance. These data highlight the importance of considering the complexity of the heat shock response across multiple isoforms when attempting to make linkages to whole-organism traits such as thermal tolerance. [ABSTRACT FROM AUTHOR]

We used differential display PCR to identify hepatic genes responsive to handling stress and genes that differ in expression between populations of a fish, Fundulus heteroclitus, from different thermal environments. Despite substantial inter-individual variation, we cloned 20 putatively stress-regulated bands from Northern fish, 10 of which had high similarity to genes of known function. We selected five of these genes for further analysis based on their known roles in the stress response. Three of these genes (glucokinase, serine-threonine kinase 10 and cRAF) were confirmed as stress-responsive using real-time PCR. These genes increased in expression in response to a 7-day chronic stress protocol in fish from the Southern population of F. heteroclitus, but did not change significantly in fish from the Northern population. These three genes also differed in expression between populations in control fish, suggesting a link between the response to chronic stress and inter-population differences in gene expression in unstressed laboratory-acclimated fish. Two genes that did not respond to stress (glycogen synthase kinase and warm acclimation-related protein (WAP)) also differed between populations. Expression of WAP was eight-fold higher in Southern than in Northern fish, consistent with a previously suggested role for this gene in thermal acclimation or adaptation in fish. [Copyright &y& Elsevier]