Impacts of sequential salinity and heat stress are recovery time-specific in freshwater crustacean, Daphnia pulicaria.

Stressors can interact to affect animal fitness, but we have limited knowledge about how temporal variation in stressors may impact their combined effect. This limits our ability to predict the outcomes of pollutants and future dynamic environmental changes. Elevated salinity in freshwater ecosystems has been observed worldwide. Meanwhile, heatwaves have become more frequent and intensified as an outcome of climate change. These two stressors can jointly affect organisms; however, their interaction has rarely been explored in the context of freshwater ecosystems. We conducted lab experiments using Daphnia pulicaria, a key species in lakes, to investigate how elevated salinity and heatwave conditions collectively affect freshwater organisms. We also monitored the impacts of various recovery times between the two stressors. Daphnia physiological conditions (metabolic rate, Na+-K+-ATPase (NKA) activity, and lipid peroxidation level) and life history traits (survival, fecundity, and growth) in response to salt stress as well as mortality in heat treatment were examined. We found that Daphnia responded to elevated salinity by upregulating NKA activity and increasing metabolic rate, causing a high lipid peroxidation level. Survival, fecundity, and growth were all negatively affected by this stressor. These impacts on physiological conditions and life history traits persisted for a few days after the end of the exposure. Heat treatments caused mortality in Daphnia, which increased with rising temperature. Results also showed that individuals that experienced salt exposure were more susceptible to subsequent heat stress, but this effect decreased with increasing recovery time between stressors. Findings from this work suggest that the legacy effects from a previous stressor can reduce individual resistance to a subsequent stressor, adding great difficulties to the prediction of outcomes of multiple stressors. Our work also demonstrates that cross-tolerance/susceptibility and the associated mechanisms remain unclear, necessitating further investigation.

WITHDRAWN: Utilizing comparative models in biomedical research.

The Publisher regrets that this article is an accidental duplication of an article that has already been published in Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, Volume 255, 2021, 110593, https://doi.org/10.1016/j.cbpb.2021.110593. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.

Behavioral and physiological evidence that increasing group size ameliorates the impacts of social disturbance.

Intra-group social stability is important for the long-term productivity and health of social organisms. We evaluated the effect of group size on group stability in the face of repeated social perturbations using a cooperatively breeding fish, Neolamprologus pulcher In a laboratory study, we compared both the social and physiological responses of individuals from small versus large groups to the repeated removal and replacement of the most dominant group member (the breeder male), either with a new male (treatment condition) or with the same male (control condition). Individuals living in large groups were overall more resistant to instability but were seemingly slower to recover from perturbation. Members of small groups were more vulnerable to instability but recovered faster. Breeder females in smaller groups also showed greater physiological preparedness for instability following social perturbations. In sum, we discover both behavioral and physiological evidence that living in larger groups helps to dampen the impacts of social instability in this system.

What do warming waters mean for fish physiology and fisheries?

Environmental signals act primarily on physiological systems, which then influence higher-level functions such as movement patterns and population dynamics. Increases in average temperature and temperature variability associated with global climate change are likely to have strong effects on fish physiology and thereby on populations and fisheries. Here we review the principal mechanisms that transduce temperature signals and the physiological responses to those signals in fish. Temperature has a direct, thermodynamic effect on biochemical reaction rates. Nonetheless, plastic responses to longer-term thermal signals mean that fishes can modulate their acute thermal responses to compensate at least partially for thermodynamic effects. Energetics are particularly relevant for growth and movement, and therefore for fisheries, and temperature can have pronounced effects on energy metabolism. All energy (ATP) production is ultimately linked to mitochondria, and temperature has pronounced effects on mitochondrial efficiency and maximal capacities. Mitochondria are dependent on oxygen as the ultimate electron acceptor so that cardiovascular function and oxygen delivery link environmental inputs with energy metabolism. Growth efficiency, that is the conversion of food into tissue, changes with temperature, and there are indications that warmer water leads to decreased conversion efficiencies. Moreover, movement and migration of fish relies on muscle function, which is partially dependent on ATP production but also on intracellular calcium cycling within the myocyte. Neuroendocrine processes link environmental signals to regulated responses at the level of different tissues, including muscle. These physiological processes within individuals can scale up to population responses to climate change. A mechanistic understanding of thermal responses is essential to predict the vulnerability of species and populations to climate change.

Thyroid hormone influences muscle mechanics in carp (Cyprinus carpio) independently from SERCA activity.

Thyroid hormone is a key regulator of metabolism, and in zebrafish, hypothyroidism decreases sustained and burst swimming performance. These effects are accompanied by decreases in both metabolic scope and the activity of sarco-endoplasmic reticulum ATPase (SERCA) in zebrafish. Our aim was to determine whether thyroid hormone affects skeletal muscle contractile function directly and whether these effects are mediated by influencing SERCA activity. We show that hypothyroidism reduces sustained locomotor performance but not sprint performance in carp (Cyprinus carpio). We accept our hypothesis that hypothyroidism reduces force production in isolated skeletal muscle, when compared with the thyroid hormone T2, but we reject the hypothesis that this effect is mediated by influencing SERCA activity. Blocking SERCA activity with thapsigargin reduced muscle fatigue resistance, but hypothyroidism had no effect on fatigue. Hence, thyroid hormone plays a role in determining isolated skeletal muscle mechanics, but its effects are more likely to be mediated by mechanisms other than affecting SERCA activity.

Skeletal muscle contractile function predicts activity and behaviour in zebrafish.

Locomotion facilitates behaviour and its underlying physiological mechanisms may therefore impact behavioural phenotypes. Metabolism is often thought to modulate locomotion and behaviour, but empirical support for this suggestion is equivocal. Muscle contractile function is directly associated with locomotion. Here, we test the hypotheses that muscle mechanics determine locomotor performance and activity in zebrafish (Danio rerio) and thereby also affect risk-taking behaviour. We show that there is a mechanistic link between muscle performance and behaviour by manipulating muscle contractile properties, which caused proportional changes in critical sustained swimming performance and, in an open arena, voluntary swimming speed, the proportion of time fish were active, and the latency to move. We modelled the relationships between muscle contractile properties, swimming performance, activity and behaviour with a partial least-squares path model. The latent variable 'muscle', formed by isolated muscle force production, stress, fatigue resistance and activation and relaxation rates, had a significant positive effect on swimming performance ('swim' reflected in sustained and sprint speeds). Together, muscle and swim had a significant positive effect on activity, and explained 71.8% of variation in the distance moved, time active and maximum voluntary speed in an open field. Activity had a significant positive effect on boldness, explaining 76.0% of variation in latencies to move and to approach a novel object. Muscle contractile function determines voluntary movement and we suggest that exploration and dispersal are functions of physiological and mechanical optimisation. Boldness therefore may be partly explained by the greater likelihood of faster fish to move further and encounter novel objects and conspecifics more quickly as a result.

Early effects of ageing on the mechanical performance of isolated locomotory (EDL) and respiratory (diaphragm) skeletal muscle using the work-loop technique.

Previous isolated muscle studies examining the effects of ageing on contractility have used isometric protocols, which have been shown to have poor relevance to dynamic muscle performance in vivo. The present study uniquely uses the work-loop technique for a more realistic estimation of in vivo muscle function to examine changes in mammalian skeletal muscle mechanical properties with age. Measurements of maximal isometric stress, activation and relaxation time, maximal power output, and sustained power output during repetitive activation and recovery are compared in locomotory extensor digitorum longus (EDL) and core diaphragm muscle isolated from 3-, 10-, 30-, and 50-wk-old female mice to examine the early onset of ageing. A progressive age-related reduction in maximal isometric stress that was of greater magnitude than the decrease in maximal power output occurred in both muscles. Maximal force and power developed earlier in diaphragm than EDL muscle but demonstrated a greater age-related decline. The present study indicates that ability to sustain skeletal muscle power output through repetitive contraction is age- and muscle-dependent, which may help rationalize previously reported equivocal results from examination of the effect of age on muscular endurance. The age-related decline in EDL muscle performance is prevalent without a significant reduction in muscle mass, and biochemical analysis of key marker enzymes suggests that although there is some evidence of a more oxidative fiber type, this is not the primary contributor to the early age-related reduction in muscle contractility.

The evolution of endothermy is explained by thyroid hormone-mediated responses to cold in early vertebrates.

The evolution of endothermy is one of the most intriguing and consistently debated topics in vertebrate biology, but the proximate mechanisms that mediated its evolution are unknown. Here, we suggest that the function of thyroid hormone in regulating physiological processes in response to cold is key to understanding the evolution of endothermy. We argue that the capacity of early chordates to produce thyroid hormone internally was the first step in this evolutionary process. Selection could then act on the capacity of thyroid hormone to regulate metabolism, muscle force production and cardiac performance to maintain their function against the negative thermodynamic effects of decreasing temperature. Thyroid-mediated cold acclimation would have been the principal selective advantage. The actions of thyroid hormone during cold acclimation in zebrafish are very similar to its role during endothermic thermogenesis. The thyroid-mediated increases in metabolism and locomotor performance in ectotherms eventually resulted in sufficient heat production to affect body temperature. From this point onwards, increased body temperature per se could be of selective advantage and reinforce thyroid-induced increases in physiological rates. Selection for increased body temperature would promote those mechanisms that maximise heat production, such as increased Na(+)/K(+)-ATPase activity, futile cycling by SERCA, and mitochondrial uncoupling, all of which are regulated by thyroid hormone. The specific end point of this broader evolutionary process would be endothermic thermoregulation. However, considering the evolution of endothermy in isolation is misleading because the selective advantages that drove the evolutionary process were independent from endothermy. In other words, without the selective advantages of thyroid-mediated cold acclimation in fish, there would be no endotherms.

Thyroid hormone regulates cardiac performance during cold acclimation in zebrafish (Danio rerio).

Limitations to oxygen transport reduce aerobic scope and thereby activity at thermal extremes. Oxygen transport in fish is facilitated to a large extent by cardiac function so that climate variability may reduce fitness by constraining the performance of the heart. In zebrafish (Danio rerio), thyroid hormone (TH) regulates skeletal muscle function and metabolism in response to thermal acclimation. Here, we aimed to determine whether TH also regulates cardiac function during acclimation. We used propylthiouracil and iopanoic acid to induce hypothyroidism in zebrafish over a 3 week acclimation period to either 18 or 28°C. We found that cold-acclimated fish had higher maximum heart rates and sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) activity than warm-acclimated fish. Hypothyroid treatment significantly decreased these responses in the cold-acclimated fish, but it did not affect the warm-acclimated fish. TH did not influence SERCA gene transcription, nor did it increase metabolic rate, of isolated whole hearts. To verify that physiological changes following hypothyroid treatment were in fact due to the action of TH, we supplemented hypothyroid fish with 3,5-diiodothryronine (T2) or 3,5,3'-triiodothyronine (T3). Supplementation of hypothyroid fish with T2 or T3 restored heart rate and SERCA activity to control levels. We also show that, in zebrafish, changes in cardiac output in response to warming are primarily mediated by heart rate, rather than by stroke volume. Thus, changes in heart rate are important for the overall aerobic capacity of the fish. In addition to its local effects on heart phenotype, we show that TH increases sympathetic tone on the heart at rest and during maximum exercise. Our findings reveal a new pathway through which fish can mitigate the limiting effects of temperature variability on oxygen transport to maintain aerobic scope and promote thermal tolerance.

Thyroid hormone actions are temperature-specific and regulate thermal acclimation in zebrafish (Danio rerio).

BACKGROUND: Thyroid hormone (TH) is best known for its role in development in animals, and for its control of metabolic heat production (thermogenesis) during cold acclimation in mammals. It is unknown whether the regulatory role of TH in thermogenesis is derived in mammals, or whether TH also mediates thermal responses in earlier vertebrates. Ectothermic vertebrates show complex responses to temperature variation, but the mechanisms mediating these are poorly understood. The molecular mechanisms underpinning TH action are very similar across vertebrates, suggesting that TH may also regulate thermal responses in ectotherms. We therefore aimed to determine whether TH regulates thermal acclimation in the zebrafish (Danio rerio). We induced hypothyroidism, followed by supplementation with 3,5-diiodothyronine (T2) or 3,5,3'-triiodothyronine (T3) in zebrafish exposed to different chronic temperatures. We measured whole-animal responses (swimming performance and metabolic rates), tissue-specific regulatory enzyme activities, gene expression, and free levels of T2 and T3. RESULTS: We found that both T3 and the lesser-known T2, regulate thermal acclimation in an ectotherm. To our knowledge, this is the first such study to show this. Hypothyroid treatment impaired performance measures in cold-acclimated but not warm-acclimated individuals, whereas supplementation with both TH metabolites restored performance. TH could either induce or repress responses, depending on the actual temperature and thermal history of the animal. CONCLUSIONS: The low sensitivity to TH at warm temperatures could mean that increasing temperatures (that is, global warming) will reduce the capacity of animals to regulate their physiologies to match demands. We suggest that the properties that underlie the role of TH in thermal acclimation (temperature sensitivity and metabolic control) may have predisposed this hormone for a regulatory role in the evolution of endothermy.

Thyroid hormone links environmental signals to DNA methylation.

Environmental conditions experienced within and across generations can impact individual phenotypes via so-called 'epigenetic' processes. Here we suggest that endocrine signalling acts as a 'sensor' linking environmental inputs to epigenetic modifications. We focus on thyroid hormone signalling and DNA methylation, but other mechanisms are likely to act in a similar manner. DNA methylation is one of the most important epigenetic mechanisms, which alters gene expression patterns by methylating cytosine bases via DNA methyltransferase enzymes. Thyroid hormone is mechanistically linked to DNA methylation, at least partly by regulating the activity of DNA methyltransferase 3a, which is the principal enzyme that mediates epigenetic responses to environmental change. Thyroid signalling is sensitive to natural and anthropogenic environmental impacts (e.g. light, temperature, endocrine-disrupting pollution), and here we propose that thyroid hormone acts as an environmental sensor to mediate epigenetic modifications. The nexus between thyroid hormone signalling and DNA methylation can integrate multiple environmental signals to modify phenotypes, and coordinate phenotypic plasticity at different time scales, such as within and across generations. These dynamics can have wide-ranging effects on health and fitness of animals, because they influence the time course of phenotypic adjustments and potentially the range of environmental stimuli that can elicit epigenetic responses. This article is part of the theme issue 'Endocrine responses to environmental variation: conceptual approaches and recent developments'.

Endocrine responses to environmental variation.

Hormones regulate most physiological functions and life history from embryonic development to reproduction. In addition to their roles in growth and development, hormones also mediate responses to the abiotic, social and nutritional environments. Hormone signalling is responsive to environmental changes to adjust phenotypes to prevailing conditions. Both hormone levels and receptor densities can change to provide a flexible system of regulation. Endocrine flexibility connects the environment to organismal function, and it is central to understanding environmental impacts and their effect on individuals and populations. Hormones may also act as a 'sensor' to link environmental signals to epigenetic processes and thereby effect phenotypic plasticity within and across generations. Many environmental parameters are now changing in unprecedented ways as a result of human activity. The knowledge base of organism-environmental interactions was established in environments that differ in many ways from current conditions as a result of ongoing human impacts. It is an urgent contemporary challenge to understand how evolved endocrine responses will modulate phenotypes in response to anthropogenic environmental impacts including climate change, light-at-night and chemical pollution. Endocrine responses play a central role in ecology, and their integration into conservation can lead to more effective outcomes. This article is part of the theme issue 'Endocrine responses to environmental variation: conceptual approaches and recent developments'.

Thyroid hormone regulates muscle function during cold acclimation in zebrafish (Danio rerio).

Thyroid hormone (TH) is a universal regulator of growth, development and metabolism during cold exposure in mammals. In zebrafish (Danio rerio), TH regulates locomotor performance and metabolism during cold acclimation. The influence of TH on locomotor performance may be via its effect on metabolism or, as has been shown in mammals, by modulating muscle phenotypes. Our aim was to determine whether TH influences muscle phenotypes in zebrafish, and whether this could explain changes in swimming capacity in response to thermal acclimation. We used propylthiouracil and iopanoic acid to induce hypothyroidism in zebrafish over a 3-week acclimation period to either 18 or 28°C. To verify that physiological changes following hypothyroid treatment were in fact due to the action of TH, we supplemented hypothyroid fish with 3,5-diiodothryronine (T2) or 3,5,3'-triiodothyronine (T3). Cold-acclimated fish had significantly greater sustained swimming performance (Ucrit) but not burst speed. Greater Ucrit was accompanied by increased tail beat frequency, but there was no change in tail beat amplitude. Hypothyroidism significantly decreased Ucrit and burst performance, as well as tail beat frequency and SERCA activity in cold-acclimated fish. However, myofibrillar ATPase activity increased in cold-acclimated hypothyroid fish. Hypothyroid treatment also decreased mRNA concentrations of myosin heavy chain fast isoforms and SERCA 1 isoform in cold-acclimated fish. SERCA 1 mRNA increased in warm-acclimated hypothyroid fish, and SERCA 3 mRNA decreased in both cold- and warm-acclimated hypothyroid fish. Supplementation with either T2 or T3 restored Ucrit, burst speed, tail beat frequency, SERCA activity and myosin heavy chain and SERCA 1 and 3 mRNA levels of hypothyroid fish back to control levels. We show that in addition to regulating development and metabolism in vertebrates, TH also regulates muscle physiology in ways that affect locomotor performance in fish. We suggest that the role of TH in modulating SERCA1 expression during cold exposure may have predisposed it to regulate endothermic thermogenesis.

Low Glycolysis Is Neuroprotective during Anoxic Spreading Depolarization (SD) and Reoxygenation in Locusts.

Migratory locusts enter a reversible hypometabolic coma to survive environmental anoxia, wherein the cessation of CNS activity is driven by spreading depolarization (SD). While glycolysis is recognized as a crucial anaerobic energy source contributing to animal anoxia tolerance, its influence on the anoxic SD trajectory and recovery outcomes remains poorly understood. We investigated the effects of varying glycolytic capacity on adult female locust anoxic SD parameters, using glucose or the glycolytic inhibitors 2-deoxy-d-glucose (2DG) or monosodium iodoacetate (MIA). Surprisingly, 2DG treatment shared similarities with glucose yet had opposite effects compared with MIA. Specifically, although SD onset was not affected, both glucose and 2DG expedited the recovery of CNS electrical activity during reoxygenation, whereas MIA delayed it. Additionally, glucose and MIA, but not 2DG, increased tissue damage and neural cell death following anoxia-reoxygenation. Notably, glucose-induced injuries were associated with heightened CO2 output during the early phase of reoxygenation. Conversely, 2DG resulted in a bimodal response, initially dampening CO2 output and gradually increasing it throughout the recovery period. Given the discrepancies between effects of 2DG and MIA, the current results require cautious interpretations. Nonetheless, our findings present evidence that glycolysis is not a critical metabolic component in either anoxic SD onset or recovery and that heightened glycolysis during reoxygenation may exacerbate CNS injuries. Furthermore, we suggest that locust anoxic recovery is not solely dependent on energy availability, and the regulation of metabolic flux during early reoxygenation may constitute a strategy to mitigate damage.

Evolution of mitochondrial-encoded cytochrome oxidase subunits in endothermic fish: the importance of taxon-sampling in codon-based models.

While endothermy is ubiquitous in birds and mammals, it is not exclusive to these most recently arisen vertebrate classes. The ability to warm specific organs and/or tissues above ambient temperature (regional endothermy) has evolved at least three times in phylogentically discrete fish lineages: lamnid sharks (Lamnidae), tunas (Scombridae) and billfishes (Istiophoridae and Xiphidae). Given the links between endothermy and metabolic rate, we looked for evidence of convergent molecular evolution in mtDNA-encoded cytochrome c oxidase (COX) subunits in each of these discrete lineages. We found no evidence that the endothermic phenotype in fishes is driven or accompanied by molecular convergence. Though we found little evidence for positively-selected sites in any of the lineages in any subunit, the conclusions were sensitive to the choice of maximum-likelihood model. Several sites identified by Naïve Empirical Bayes (NEB) were not found when Bayes Empirical Bayes (BEB) was employed. As well, conclusions were profoundly influenced by taxon-sampling. Several of the putative sites of positive selection in COX II were no longer apparent as we augmented taxon sampling. The lack of convergent molecular evolution in these remarkable taxa, combined with the profound influence of model choice and taxon sampling provide a cautionary note on the use of rates of non-synonymous to synonymous mutations (dN/dS) to explore questions of the evolution of physiological function.

Plasticity of Performance Curves in Ectotherms: Individual Variation Modulates Population Responses to Environmental Change.

Many ectothermic animals can respond to changes in their environment by altering the sensitivities of physiological rates, given sufficient time to do so. In other words, thermal acclimation and developmental plasticity can shift thermal performance curves so that performance may be completely or partially buffered against the effects of environmental temperature changes. Plastic responses can thereby increase the resilience to temperature change. However, there may be pronounced differences between individuals in their capacity for plasticity, and these differences are not necessarily reflected in population means. In a bet-hedging strategy, only a subsection of the population may persist under environmental conditions that favour either plasticity or fixed phenotypes. Thus, experimental approaches that measure means across individuals can not necessarily predict population responses to temperature change. Here, we collated published data of 608 mosquitofish (Gambusia holbrooki) each acclimated twice, to a cool and a warm temperature in random order, to model how diversity in individual capacity for plasticity can affect populations under different temperature regimes. The persistence of both plastic and fixed phenotypes indicates that on average, neither phenotype is selectively more advantageous. Fish with low acclimation capacity had greater maximal swimming performance in warm conditions, but their performance decreased to a greater extent with decreasing temperature in variable environments. In contrast, the performance of fish with high acclimation capacity decreased to a lesser extent with a decrease in temperature. Hence, even though fish with low acclimation capacity had greater maximal performance, high acclimation capacity may be advantageous when ecologically relevant behaviour requires submaximal locomotor performance. Trade-offs, developmental effects and the advantages of plastic phenotypes together are likely to explain the observed population variation.

Physiological Performance Curves: When Are They Useful?

This review serves as an introduction to a special issue of Frontiers in Physiology, focused on the importance of physiological performance curves across phylogenetic and functional boundaries. Biologists have used performance curves to describe the effects of changing environmental conditions on animal physiology since the late 1800s (at least). Animal physiologists have studied performance curves extensively over the past decades, and there is a good foundation to understanding how the environment affects physiological functions of individuals. Our goal here was to build upon this research and address outstanding questions regarding the mutability and applicability of performance curves across taxonomic groups and levels of biological organization. Performance curves are not fixed at a taxonomic, population, or individual level - rather they are dynamic and can shift in response to evolutionary pressures (e.g., selection) and epigenetic programming (e.g., plasticity). The mechanisms underlying these shifts are being increasingly used to predict the efficacy with which plasticity and heritability of performance curves can render individuals and populations less vulnerable to climate change. Individual differences in physiological performance curves (and plasticity of performance curves) can also have cascading effects at higher levels of biological organization. For instance, individual physiology likely influences group behaviors in non-additive ways. There is a need therefore to extend the concept of performance curves to social interactions and sociality. Collectively, this special issue emphasizes the power of how within- and between-individual shifts in performance curves might scale up to the population-, species-, and community-level dynamics that inform conservation management strategies.

Utilizing comparative models in biomedical research.

This review serves as an introduction to a Special Issue of Comparative Biochemistry and Physiology, focused on using non-human models to study biomedical physiology. The concept of a model differs across disciplines. For example, several models are used primarily to gain an understanding of specific human pathologies and disease states, whereas other models may be focused on gaining insight into developmental or evolutionary mechanisms. It is often the case that animals initially used to gain knowledge of some unique biochemical or physiological process finds foothold in the biomedical community and becomes an established model. The choice of a particular model for biomedical research is an ongoing process and model validation must keep pace with existing and emerging technologies. While the importance of non-mammalian models, such as Caenorhabditis elegans, Drosophila melanogaster, Danio rerio and Xenopus laevis, is well known, we also seek to bring attention to emerging alternative models of both invertebrates and vertebrates, which are less established but of interest to the comparative biochemistry and physiology community.