Intraspecific variation in thermal performance curves for early development in Fundulus heteroclitus.

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 (Topt) for developmental rate, survival, and embryonic growth rate) whereas pure northern embryos had a vertically shifted TPC (higher maximum performance (Pmax) 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.

Genomic and Epigenomic Influences on Resilience Across Scales: lessons from the responses of fishes to environmental stressors.

Understanding the factors that influence the resilience of biological systems to environmental change is an increasingly critical concern in the face of increasing human impacts on ecosystems and the organisms that inhabit them. However, most considerations of biological resilience have focused at the community and ecosystem levels, whereas here we discuss how including consideration of processes occurring at lower levels of biological organization may provide insights into factors that influence resilience at higher levels of biological organization. Specifically, we explore how processes at the genomic and epigenomic levels may cascade up to influence resilience at higher levels and ask how the concepts of 'resistance', or the capacity of a system to minimize change in response to a disturbance, and 'recovery', or the ability of a system to return to its original state following a disturbance and avoid tipping points and resulting regime shifts map to these lower levels of biological organization. Overall, we argue that substantial changes at these lower levels may be required to support resilience at higher levels, using selected examples of genomic and epigenomic responses of fish to climate-change relevant stressors such as high temperature and hypoxia at the levels of the genome, epigenome and organism.

Through the looking glass: attempting to predict future opportunities and challenges in experimental biology.

To celebrate its centenary year, Journal of Experimental Biology (JEB) commissioned a collection of articles examining the past, present and future of experimental biology. This Commentary closes the collection by considering the important research opportunities and challenges that await us in the future. We expect that researchers will harness the power of technological advances, such as '-omics' and gene editing, to probe resistance and resilience to environmental change as well as other organismal responses. The capacity to handle large data sets will allow high-resolution data to be collected for individual animals and to understand population, species and community responses. The availability of large data sets will also place greater emphasis on approaches such as modeling and simulations. Finally, the increasing sophistication of biologgers will allow more comprehensive data to be collected for individual animals in the wild. Collectively, these approaches will provide an unprecedented understanding of 'how animals work' as well as keys to safeguarding animals at a time when anthropogenic activities are degrading the natural environment.

Heatwave resilience of juvenile white sturgeon is associated with epigenetic and transcriptional alterations.

Heatwaves are increasing in frequency and severity, posing a significant threat to organisms globally. In aquatic environments heatwaves are often associated with low environmental oxygen, which is a deadly combination for fish. However, surprisingly little is known about the capacity of fishes to withstand these interacting stressors. This issue is particularly critical for species of extreme conservation concern such as sturgeon. We assessed the tolerance of juvenile white sturgeon from an endangered population to heatwave exposure and investigated how this exposure affects tolerance to additional acute stressors. We measured whole-animal thermal and hypoxic performance and underlying epigenetic and transcriptional mechanisms. Sturgeon exposed to a simulated heatwave had increased thermal tolerance and exhibited complete compensation for the effects of acute hypoxia. These changes were associated with an increase in mRNA levels involved in thermal and hypoxic stress (hsp90a, hsp90b, hsp70 and hif1a) following these stressors. Global DNA methylation was sensitive to heatwave exposure and rapidly responded to acute thermal and hypoxia stress over the course of an hour. These data demonstrate that juvenile white sturgeon exhibit substantial resilience to heatwaves, associated with improved cross-tolerance to additional acute stressors and involving rapid responses in both epigenetic and transcriptional mechanisms.

Identification of upper thermal thresholds during development in the endangered Nechako white sturgeon with management implications for a regulated river.

Climate change-induced warming effects are already evident in river ecosystems, and projected increases in temperature will continue to amplify stress on fish communities. In addition, many rivers globally are impacted by dams, which have many negative effects on fishes by altering flow, blocking fish passage, and changing sediment composition. However, in some systems, dams present an opportunity to manage river temperature through regulated releases of cooler water. For example, there is a government mandate for Kenney dam operators in the Nechako river, British Columbia, Canada, to maintain river temperature <20°C in July and August to protect migrating sockeye salmon (Oncorhynchus nerka). However, there is another endangered fish species inhabiting the same river, Nechako white sturgeon (Acipenser transmontanus), and it is unclear if these current temperature regulations, or timing of the regulations, are suitable for spawning and developing sturgeon. In this study, we aimed to identify upper thermal thresholds in white sturgeon embryos and larvae to investigate if exposure to current river temperatures are playing a role in recruitment failure. We incubated embryos and yolk-sac larvae in three environmentally relevant temperatures (14, 18 and 21°C) throughout development to identify thermal thresholds across different levels of biological organization. Our results demonstrate upper thermal thresholds at 21°C across physiological measurements in embryo and yolk-sac larvae white sturgeon. Before hatch, both embryo survival and metabolic rate were reduced at 21°C. After hatch, sublethal consequences continued at 21°C because larval sturgeon had decreased thermal plasticity and a dampened transcriptional response during development. In recent years, the Nechako river has reached 21°C by the end of June, and at this temperature, a decrease in sturgeon performance is evident in most of the traits measured. As such, the thermal thresholds identified here suggest current temperature regulations may not be suitable for developing white sturgeon and future recruitment.

Hypoxia and High Temperature as Interacting Stressors: Will Plasticity Promote Resilience of Fishes in a Changing World?

AbstractDetermining the resilience of a species or population to climate change stressors is an important but difficult task because resilience can be affected both by genetically based variation and by various types of phenotypic plasticity. In addition, most of what is known about organismal responses is for single stressors in isolation, but environmental change involves multiple environmental factors acting in combination. Here, our goal is to summarize what is known about phenotypic plasticity in fishes in response to high temperature and low oxygen (hypoxia) in combination across multiple timescales, to ask how much resilience plasticity may provide in the face of climate change. There are relatively few studies investigating plasticity in response to these environmental stressors in combination; but the available data suggest that although fish have some capacity to adjust their phenotype and compensate for the negative effects of acute exposure to high temperature and hypoxia through acclimation or developmental plasticity, compensation is generally only partial. There is very little known about intergenerational and transgenerational effects, although studies on each stressor in isolation suggest that both positive and negative impacts may occur. Overall, the capacity for phenotypic plasticity in response to these two stressors is highly variable among species and extremely dependent on the specific context of the experiment, including the extent and timing of stressor exposure. This variability in the nature and extent of plasticity suggests that existing phenotypic plasticity is unlikely to adequately buffer fishes against the combined stressors of high temperature and hypoxia as our climate warms.

Integrative Approaches to Understanding Organismal Responses to Aquatic Deoxygenation.

AbstractOxygen bioavailability is declining in aquatic systems worldwide as a result of climate change and other anthropogenic stressors. For aquatic organisms, the consequences are poorly known but are likely to reflect both direct effects of declining oxygen bioavailability and interactions between oxygen and other stressors, including two-warming and acidification-that have received substantial attention in recent decades and that typically accompany oxygen changes. Drawing on the collected papers in this symposium volume ("An Oxygen Perspective on Climate Change"), we outline the causes and consequences of declining oxygen bioavailability. First, we discuss the scope of natural and predicted anthropogenic changes in aquatic oxygen levels. Although modern organisms are the result of long evolutionary histories during which they were exposed to natural oxygen regimes, anthropogenic change is now exposing them to more extreme conditions and novel combinations of low oxygen with other stressors. Second, we identify behavioral and physiological mechanisms that underlie the interactive effects of oxygen with other stressors, and we assess the range of potential organismal responses to oxygen limitation that occur across levels of biological organization and over multiple timescales. We argue that metabolism and energetics provide a powerful and unifying framework for understanding organism-oxygen interactions. Third, we conclude by outlining a set of approaches for maximizing the effectiveness of future work, including focusing on long-term experiments using biologically realistic variation in experimental factors and taking truly cross-disciplinary and integrative approaches to understanding and predicting future effects.

Osmorespiratory compromise in an elasmobranch: oxygen consumption, ventilation and nitrogen metabolism during recovery from exhaustive exercise in dogfish sharks (Squalus suckleyi).

The functional trade-off between respiratory gas exchange versus osmolyte and water balance that occurs at the thin, highly vascularized gills of fishes has been termed the osmorespiratory compromise. Increases in gas exchange capacity for meeting elevated oxygen demands can end up favoring the passive movement of osmolytes and water, potentially causing a disturbance in osmotic balance. This phenomenon has been studied only sparsely in marine elasmobranchs. Our goal was to evaluate the effects of exhaustive exercise (as a modulator of oxygen demand) on oxygen consumption (MO2), branchial losses of nitrogenous products (ammonia and urea-N), diffusive water exchange rates, and gill ventilation (frequency and amplitude), in the Pacific spiny dogfish (Squalus suckleyi). To that end, MO2, osmolyte fluxes, diffusive water exchange rate, and ventilation dynamics were first measured under resting control conditions, then sharks were exercised until exhaustion (20 min), and the same parameters were monitored for the subsequent 4 h of recovery. While MO2 nearly doubled immediately after exercise and remained elevated for 2 h, ventilation dynamics did not change, suggesting that fish were increasing oxygen extraction efficiency at the gills. Diffusive water flux rates (measured over 0-2 h of recovery) were not affected. Ammonia losses were elevated by 7.6-fold immediately after exercise and remained elevated for 3 h into recovery, while urea-N losses were elevated only 1.75-fold and returned to control levels after 1 h. These results are consistent with previous investigations using different challenges (hypoxia, high temperature) and point to a tighter regulation of urea-N conservation mechanisms at the gills, likely due to the use of urea as a prized osmolyte in elasmobranchs. Environmental hyperoxia offered no relief from the osmorespiratory compromise, as there were no effects on any of the parameters measured during recovery from exhaustive exercise.

Gene expression and latitudinal variation in the stress response in Fundulus heteroclitus.

Atlantic killifish, Fundulus heteroclitus, are intertidal marsh fish found along the east coast of North America. Associated with the thermal gradient along this coast, northern and southern killifish populations are known to differ in morphology, behavior, and physiology, including in their cortisol stress response. Our goal was to explore population differences in the stress response and identify underlying molecular mechanisms. We measured responses to both acute and repeated stress in plasma cortisol, stress axis mRNA expression, and body condition in northern and southern killifish. Following an acute stressor, the southern population had higher cortisol levels than the northern population but there was no difference between populations following repeated stress. In the brain, both corticotropin releasing factor and its binding protein had higher expression in the southern than the northern population, but the northern population showed more changes in mRNA levels following a stressor. In the head kidney, Melanocortin 2 Receptor and steroidogenic acute regulatory protein mRNA levels were higher in the southern population suggesting a larger capacity for cortisol synthesis than in the northern fish. Lastly, the glucocorticoid receptor GR1 mRNA levels were greater in the liver of southern fish, suggesting a greater capacity to respond to cortisol, and GR2 had differential expression in the head kidney, suggesting an interpopulation difference in stress axis negative feedback loops. Southern, but not northern, fish were able to maintain body condition following stress, suggesting that these differences in the stress response may be important for adaptation across latitudes.

Growth genes are implicated in the evolutionary divergence of sympatric piscivorous and insectivorous rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Identifying ecologically significant phenotypic traits and the genomic mechanisms that underly them are crucial steps in understanding traits associated with population divergence. We used genome-wide data to identify genomic regions associated with key traits that distinguish two ecomorphs of rainbow trout (Oncorhynchus mykiss)-insectivores and piscivores-that coexist for the non-breeding portion of the year in Kootenay Lake, southeastern British Columbia. "Gerrards" are large-bodied, rapidly growing piscivores with high metabolic rates that spawn north of Kootenay Lake in the Lardeau River, in contrast to the insectivorous populations that are on average smaller in body size, with lower growth and metabolic rates, mainly forage on aquatic insects, and spawn in tributaries immediately surrounding Kootenay Lake. We used pool-seq data representing ~ 60% of the genome and 80 fish per population to assess the level of genomic divergence between ecomorphs and to identify and interrogate loci that may play functional or selective roles in their divergence. RESULTS: Genomic divergence was high between sympatric insectivores and piscivores ([Formula: see text] = 0.188), and in fact higher than between insectivorous populations from Kootenay Lake and the Blackwater River ([Formula: see text] = 0.159) that are > 500 km apart. A window-based [Formula: see text] analysis did not reveal "islands" of genomic differentiation; however, the window with highest [Formula: see text] estimate did include a gene associated with insulin secretion. Although we explored the use of the "Local score" approach to identify genomic outlier regions, this method was ultimately not used because simulations revealed a high false discovery rate (~ 20%). Gene ontology (GO) analysis identified several growth processes as enriched in genes occurring in the ~ 200 most divergent genomic windows, indicating many loci of small effect involved in growth and growth-related metabolic processes are associated with the divergence of these ecomorphs. CONCLUSION: Our results reveal a high degree of genomic differentiation between piscivorous and insectivorous populations and indicate that the large body piscivorous phenotype is likely not due to one or a few loci of large effect. Rather, the piscivore phenotype may be controlled by several loci of small effect, thus highlighting the power of whole-genome resequencing in identifying genomic regions underlying population-level phenotypic divergences.

Acute measures of upper thermal and hypoxia tolerance are not reliable predictors of mortality following environmental challenges in rainbow trout (Oncorhynchus mykiss).

Anthropogenic climate change threatens freshwater biodiversity and poses a challenge for fisheries management, as fish will increasingly be exposed to episodes of high temperature and low oxygen (hypoxia). Here, we examine the extent of variation in tolerance of acute exposure to these stressors within and among five strains of rainbow trout (Oncorhynchus mykiss) currently being used or under consideration for use in stocking programmes in British Columbia, Canada. We used incipient lethal oxygen saturation (ILOS) as an index of acute hypoxia tolerance, critical thermal maximum (CTmax) as an index of acute upper thermal tolerance and mortality following these two acute exposure trials to assess the relative resilience of individuals and strains to climate change-relevant stressors. We measured tolerance across two brood years and two life stages (fry and yearling), using a highly replicated design with hundreds of individuals per strain and life stage. There was substantial within-strain variation in CTmax and ILOS, but differences among strains, although statistically significant, were small. In contrast, there were large differences in post-trial mortality among strains, ranging from less than 2% mortality in the most resilient strain to 55% mortality in the least resilient. There was a statistically significant, but weak, correlation between CTmax and ILOS at both life stages for some strains, with thermally tolerant individuals tending to be hypoxia tolerant. These data indicate that alternative metrics of tolerance may result in different conclusions regarding resilience to climate change stressors, which has important implications for stocking and management decisions for fish conservation in a changing climate.

Intraspecific variation in tolerance of warming in fishes.

Intraspecific variation in key traits such as tolerance of warming can have profound effects on ecological and evolutionary processes, notably responses to climate change. The empirical evidence for three primary elements of intraspecific variation in tolerance of warming in fishes is reviewed. The first is purely mechanistic that tolerance varies across life stages and as fishes become mature. The limited evidence indicates strongly that this is the case, possibly because of universal physiological principles. The second is intraspecific variation that is because of phenotypic plasticity, also a mechanistic phenomenon that buffers individuals' sensitivity to negative impacts of global warming in their lifetime, or to some extent through epigenetic effects over successive generations. Although the evidence for plasticity in tolerance to warming is extensive, more work is required to understand underlying mechanisms and to reveal whether there are general patterns. The third element is intraspecific variation based on heritable genetic differences in tolerance, which underlies local adaptation and may define long-term adaptability of a species in the face of ongoing global change. There is clear evidence of local adaptation and some evidence of heritability of tolerance to warming, but the knowledge base is limited with detailed information for only a few model or emblematic species. There is also strong evidence of structured variation in tolerance of warming within species, which may have ecological and evolutionary significance irrespective of whether it reflects plasticity or adaptation. Although the overwhelming consensus is that having broader intraspecific variation in tolerance should reduce species vulnerability to impacts of global warming, there are no sufficient data on fishes to provide insights into particular mechanisms by which this may occur.

Mitochondria and the thermal limits of ectotherms.

Temperature is a critical abiotic factor shaping the distribution and abundance of species, but the mechanisms that underpin organismal thermal limits remain poorly understood. One possible mechanism underlying these limits is the failure of mitochondrial processes, as mitochondria play a crucial role in animals as the primary site of ATP production. Conventional measures of mitochondrial performance suggest that these organelles can function at temperatures much higher than those that limit whole-organism function, suggesting that they are unlikely to set organismal thermal limits. However, this conclusion is challenged by recent data connecting sequence variation in mitochondrial genes to whole-organism thermal tolerance. Here, we review the current state of knowledge of mitochondrial responses to thermal extremes and ask whether they are consistent with a role for mitochondrial function in shaping whole-organism thermal limits. The available data are fragmentary, but it is possible to draw some conclusions. There is little evidence that failure of maximal mitochondrial oxidative capacity as assessed in vitro sets thermal limits, but there is some evidence to suggest that temperature effects on ATP synthetic capacity may be important. Several studies suggest that loss of mitochondrial coupling is associated with the thermal limits for organismal growth, although this needs to be rigorously tested. Most studies have utilized isolated mitochondrial preparations to assess the effects of temperature on these organelles, and there remain many untapped opportunities to address these questions using preparations that retain more of their biological context to better connect these subcellular processes with whole-organism thermal limits.

Ionoregulatory aspects of the hypoxia-induced osmorespiratory compromise in the euryhaline Atlantic killifish (Fundulus heteroclitus): the effects of salinity.

The osmorespiratory compromise is a physiological trade-off between the characteristics of the gill that promote respiratory gas exchange and those that limit passive flux of ions and water with the environment. In hypoxia, changes in gill blood flow patterns and functional surface area that increase gas transfer can promote an exacerbation in ion and water flux. Our goal was to determine whether the osmorespiratory compromise is flexible, depending on environmental salinity (fresh, isosmotic and sea water) and oxygen levels (hypoxia) in euryhaline killifish, Fundulus heteroclitus Plasma ion concentrations were minimally affected by hypoxia, indicating a maintenance of osmoregulatory homeostasis. In freshwater killifish, hypoxia exposure reduced branchial Na+/K+-ATPase and NEM-sensitive ATPase activities, as well as diffusive water flux rates. Unidirectional Na+ influx and Na+ efflux decreased during hypoxia in freshwater, but net Na+ flux remained unchanged. Net loss rates of Cl-, K+ and ammonia were also attenuated in hypoxia, suggesting both transcellular and paracellular reductions in permeability. These reductions appeared to be regulated phenomena as fluxes were restored immediately in normoxia. Na+ flux rates increased during hypoxia in 11 ppt, but decreased in 35 ppt, the latter suggesting a similar response to hypoxia to that in freshwater. In summary, freshwater and seawater killifish experience a reduction in gill permeability, as seen in other hypoxia-tolerant species. Fish acclimated to isosmotic salinity increased Na+ influx and efflux rates, as well as paracellular permeability in hypoxia, responses in accord with the predictions of the classic osmorespiratory compromise.

A rapid intrinsic heart rate resetting response with thermal acclimation in rainbow trout, Oncorhynchus mykiss.

We examined cardiac pacemaker rate resetting in rainbow trout following a reciprocal temperature transfer. In the original experiment, performed in winter, 4°C-acclimated fish transferred to 12°C reset intrinsic heart rate after just 1 h (from 56.8±1.2 to 50.8±1.5 beats min-1); 12°C-acclimated fish transferred to 4°C reset intrinsic heart rate after 8 h (from 33.4±0.7 to 37.7±1.2 beats min-1). However, in a replicate experiment, performed in the summer using a different brood year, intrinsic heart rate was not reset, even after 10 weeks at a new temperature. Using this serendipitous opportunity, we compared mRNA expression changes of a suite of proteins in sinoatrial node (SAN), atrial and ventricular tissues after both 1 h and longer than 3 weeks for both experimental acclimation groups to identify those changes only associated with pacemaker rate resetting. Of the changes in mRNA expression occurring after more than 3 weeks of warm acclimation and associated with pacemaker rate resetting, we observed downregulation of NKA α1c in the atrium and ventricle, and upregulation of HCN1 in the ventricle. However, in the SAN there were no mRNA expression changes unique to the fish with pacemaker rate resetting after either 1 h or 3 weeks of warm acclimation. Thus, despite identifying changes in mRNA expression of contractile cardiac tissues, there was an absence of changes in mRNA expression directly involved with the initial, rapid pacemaker rate resetting with warm acclimation. Importantly, pacemaker rate resetting with thermal acclimation does not always occur in rainbow trout.

Evolutionary Physiology and Genomics in the Highly Adaptable Killifish (Fundulus heteroclitus).

By investigating evolutionary adaptations that change physiological functions, we can enhance our understanding of how organisms work, the importance of physiological traits, and the genes that influence these traits. This approach of investigating the evolution of physiological adaptation has been used with the teleost fish Fundulus heteroclitus and has produced insights into (i) how protein polymorphisms enhance swimming and development; (ii) the role of equilibrium enzymes in modulating metabolic flux; (iii) how variation in DNA sequences and mRNA expression patterns mitigate changes in temperature, pollution, and salinity; and (iv) the importance of nuclear-mitochondrial genome interactions for energy metabolism. Fundulus heteroclitus provides so many examples of adaptive evolution because their local population sizes are large, they have significant standing genetic variation, and they experience large ranges of environmental conditions that enhance the likelihood that adaptive evolution will occur. Thus, F. heteroclitus research takes advantage of evolutionary changes associated with exposure to diverse environments, both across the North American Atlantic coast and within local habitats, to contrast neutral versus adaptive divergence. Based on evolutionary analyses contrasting neutral and adaptive evolution in F. heteroclitus populations, we conclude that adaptive evolution can occur readily and rapidly, at least in part because it depends on large amounts of standing genetic variation among many genes that can alter physiological traits. These observations of polygenic adaptation enhance our understanding of how evolution and physiological adaptation progresses, thus informing both biological and medical scientists about genotype-phenotype relationships. © 2020 American Physiological Society. Compr Physiol 10:637-671, 2020.

The effect of acute warming and thermal acclimation on maximum heart rate of the common killifish Fundulus heteroclitus.

Common killifish Fundulus heteroclitus were acclimated to ecologically relevant temperatures (5, 15 and 33°C) and their maximum heart rate (fHmax ) was measured at each acclimation temperature during an acute warming protocol. Acclimation to 33°C increased peak fHmax 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 fHmax 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 fHmax was cooler and the temperature that first produced cardiac arrhythmias was similar to these critical thermal maxima. The considerable thermal plasticity of fHmax demonstrated in the present study is entirely consistent with eurythermal ecology of killifish, as shown previously for another eurythermal fish Gillichthys mirabilis.

Mitochondria, sex and variation in routine metabolic rate.

Variation in the metabolic costs associated with organismal maintenance may play a key role in determining fitness, and thus these differences among individuals are likely to be subject to natural selection. Although the evolvability of maintenance metabolism depends on its underlying genetic architecture, relatively little is known about the nature of genetic variation that underlies this trait. To address this, we measured variation in routine metabolic rate (MO2routine ), an index of maintenance metabolism, within and among three populations of Atlantic killifish, Fundulus heteroclitus, including a population from a region of genetic admixture between two subspecies. Polygenic association tests among individuals from the admixed population identified 54 single nucleotide polymorphisms (SNPs) that were associated with MO2routine , and these SNPs accounted for 43% of interindividual variation in this trait. However, genetic associations with MO2routine involved different SNPs if females and males were analysed separately, and there was a sex-dependent effect of mitochondrial genotype on variation in routine metabolism. These results imply that there are sex-specific genetic mechanisms, and potential mitonuclear interactions, that underlie variation in MO2routine . Additionally, there was evidence for epistatic interactions between 17% of the possible pairs of trait-associated SNPs, suggesting that epistatic effects on MO2routine are common. These data demonstrate not only that phenotypic variation in this ecologically important trait has a polygenic basis with considerable epistasis among loci, but also that these underlying genetic mechanisms, and particularly the role of mitochondrial genotype, may be sex-specific.

Beyond the Powerhouse: Integrating Mitonuclear Evolution, Physiology, and Theory in Comparative Biology.

Eukaryotes are the outcome of an ancient symbiosis and as such, eukaryotic cells fundamentally possess two genomes. As a consequence, gene products encoded by both nuclear and mitochondrial genomes must interact in an intimate and precise fashion to enable aerobic respiration in eukaryotes. This genomic architecture of eukaryotes is proposed to necessitate perpetual coevolution between the nuclear and mitochondrial genomes to maintain coadaptation, but the presence of two genomes also creates the opportunity for intracellular conflict. In the collection of papers that constitute this symposium volume, scientists working in diverse organismal systems spanning vast biological scales address emerging topics in integrative, comparative biology in light of mitonuclear interactions.

The osmorespiratory compromise: physiological responses and tolerance to hypoxia are affected by salinity acclimation in the euryhaline Atlantic killifish (Fundulus heteroclitus).

The characteristics of the fish gill that maximize gas exchange are the same that promote diffusion of ions and water to and from the environment; therefore, physiological trade-offs are likely to occur. Here, we investigated how salinity acclimation affects whole-animal respiratory gas exchange during hypoxia using Fundulus heteroclitus, a fish that inhabits salt marshes where salinity and oxygen levels vary greatly. Salinity had marked effects on hypoxia tolerance, with fish acclimated to 11 and 35 ppt showing much longer time to loss of equilibrium (LOE) in hypoxia than 0 ppt-acclimated fish. Fish acclimated to 11 ppt (isosmotic salinity) exhibited the greatest capacity to regulate oxygen consumption rate (MO2 ) under hypoxia, as measured through the regulation index (RI) and Pcrit At 35 ppt, fish had a higher routine metabolic rate (RMR) but a lower RI than fish at 11 ppt, but there were no differences in gill morphology, ventilation or blood O2 transport properties between these groups. In contrast, 0 ppt-acclimated fish had the highest ventilation and lowest O2 extraction efficiency in normoxia and hypoxia, indicating a higher ventilatory workload in order to maintain similar levels of MO2 These differences were related to alterations in gill morphology, where 0 ppt-acclimated fish had the smallest lamellar surface area with the greatest epithelial cell coverage (i.e. thicker lamellae, longer diffusion distance) and a larger interlamellar cell mass, contrasting with 11 ppt-acclimated fish, which had overall the highest respiratory surface area. The alteration of an array of physiological parameters provides evidence for a compromise between salinity and hypoxia tolerance in killifish acclimated to freshwater.