Journal of Experimental Zoology Part A: Ecological and Integrative Physiology

Anoxia is a major stress for most vertebrates and frequently accompanies harsh winter conditions, particularly in species that spend much of the season frozen solid. North American freeze-tolerant wood frogs (Rana sylvatica) can survive several months without oxygen and endure whole-body freezing for up to eight months of the year, with [~]70% of total body water frozen as extracellular ice, yet revive when temperatures rise in spring. Survival depends on multiple adaptations, including tolerance of prolonged oxygen deprivation while frozen, when breathing and circulation are halted. A key strategy involves hepatic glycogen mobilization, producing large amounts of glucose that are distributed to tissues where it functions both as a cryoprotectant and as a substrate for anaerobic ATP production. The present study examines the role of histone lysine methylation and demethylation in regulating liver proteins under anoxic conditions. Relative protein expression of seven histone methyltransferases (ASH2L-S, ASH2L-L, RBBP5, SETD8, SMYD2, ESET, SETD1), six lysine demethylases (KDM1A, KDM3B, KDM4A, KDM4B, KDM5A, KDM5C), and eight histone marks (H3K4me1, H3K4me2, H3K9me3, H3K27me3, H3K36me3, H3K79me3, H4K20me1, H4K20me3) were evaluated in wood frog liver under control, 4-hour, and 24-hour anoxia exposures. The data indicate that histone lysine methylation and demethylation contribute significantly to transcriptional regulation under anoxia. Specifically, H3K4, H3K36, and H3K79 methylation were associated with transcriptional activation, whereas H3K9, H3K27, and H4K20 methylation correlated with transcriptional repression. These findings highlight the dynamic role of epigenetic regulation in supporting hypometabolism and stress adaptation in freeze-tolerant wood frogs.

The shift to terrestrial environments in ancestrally aquatic animals is often associated with key physiological and physical changes, including shifts in respiratory physiology and in some cases, even the evolution of completely novel respiratory structures. Examining how respiration operates across a gradient of submersion states in ancestrally aquatic terrestrial animals may shed light on how complex biological traits shift under different selective regimes. In this work, we begin exploring respiration in terrestrially-adapted land crabs that still use their gills to respire while underwater. We tested the relationship between aquatic respiratory rates, body size, and sex in red devil vampire crabs (Geosesarma hagen) at two ecologically-relevant temperatures. We found small females respire more than small males at 28{degrees}C, while large females respire more than large males at 21{degrees}C. Additionally, body size is a significant factor affecting respiratory rates of both sexes at 21{degrees}C and warmer temperatures significantly increase respiration in small crabs of both sexes. Interactions between these factors also led to emerging trends that can be explained by both physiological rules, such as reproductive investment and surface-to-volume ratios and heat transfer. We also report a temperature coefficient (Q10) of 1.52 for this species, showing an expected 52% change in respiratory and metabolic rate for every 10{degrees}C increase. This work also demonstrates the importance of understanding how and to what extent biological variables like sex and body size interact with abiotic environmental factors when measuring physiological traits in ectothermic invertebrate animals.

Temperatures in aquatic ecosystems have been affected by anthropogenic activities such as agricultural and industrial water use, and climate change caused by greenhouse gas emissions. Changes in water temperature directly affect the cellular and organismal physiology of fishes because most fishes are ectotherms. These effects can take different forms, such as increased cellular stress and reactive oxygen species (ROS) production. However, the type and magnitude of response depend on the duration and the frequency of the exposure to elevated water temperature. In this study, we investigated the long-term effects of exposure to daily thermal fluctuations occurring during early-life stages of zebrafish, Danio rerio, on gene expression and CTmax in later developmental stages. To do so, wild-type zebrafish were exposed daily to a + 5{degrees}C fluctuation in temperature from ambient (28 {degrees}C) to 33 {degrees}C over the first 30 days post fertilization (dpf), before being held until 90 dpf at ambient temperature. The fish that experienced daily thermal fluctuation were compared to a control group that was kept at 28 {degrees}C throughout the experiment and sampled at the same timepoints. Samples were collected at 18 (larval), 30, 60 (juvenile), and 90 (adult) dpf to study the expression of heat shock proteins and oxidative stress genes. The thermotolerance of fish was tested using CTmax trials at 60 and 90 dpf. Daily thermal fluctuation over the first 30 dpf led to a significant increase in the expression of hsp47, gstp1a, sod1, and sod2 genes at 60 dpf, and hsp47, hsp90aa1, hsp90ab1, cat, glulb, gstp1a, sod1, and sod2 genes at 90 dpf. The only significant increase detected during the larval stage was glulb at 18 dpf. Fish that experienced thermal fluctuation also had a higher CTmax at 60 dpf, but this increased thermotolerance significantly decreased from 60 to 90 dpf, where it was not different between treatments. Overall, our study demonstrates that early-life thermal stress increased cellular stress responses and thermotolerance in zebrafish into later ontological stages.

Birds maintain unusually high circulating glucose levels compared with other vertebrates without developing the diabetic complications observed in mammals, yet the mechanisms underlying this resistance remain unclear. We investigated the effects of chronic glucose and methylglyoxal supplementation on physiological condition in zebra finches (Taeniopygia guttata), with particular emphasis on sex- and season-dependent variation in plasma biochemistry, haematology and immune traits. Ninety zebra finches (45 males, 45 females) were randomly assigned to control, glucose-supplemented (50 g/L), or methylglyoxal (8.33 g/L) drinking treatments. Over one year, we analysed plasma proteins, metabolites (glucose, uric acid, bile acids), tissue damage markers (AST, CK), electrolytes, and immune parameters (leukocyte profiles). Both supplementations increased plasma glucose concentrations, with methylglyoxal producing the strongest effect. More importantly, both treatments disrupted seasonal plasma protein dynamics, preventing the increase in total proteins and globulins normally observed in females during the reproductive period, which suggests alterations in reproductive-related protein metabolism. Glucose supplementation elevated the heterophil-to-lymphocyte (H/L) ratio in May and August, consistent with elevated physiological stress. In contrast, methylglyoxal supplementation reduced the H/L ratio in November and unexpectedly lowered plasma AST and CK concentrations in May, suggesting context-dependent protective effects on tissue integrity despite its well-established pro-oxidative properties, potentially through hormetic mechanisms. Supplementation also modified the calcium/phosphate balance, further supporting treatment effects on seasonal (reproductive) physiology. Overall, our findings demonstrate that glucose and methylglyoxal reshape physiological regulation in zebra finches in a strongly sex- and season-dependent manner rather than simply inducing generalized metabolic damage. These results provide new insights into avian resistance to glucose-associated physiological challenges and highlight the importance of considering both biological context and standardized haematological reference values when investigating glucose metabolism in birds.

Absorption of CO2 by global oceans is decreasing pH resulting in ocean acidification (OA). Impacts on shellfish have been documented in ecologically and commercially important species. We examined the influence of diet and OA between two populations of red abalone (Haliotis rufescens) a species of aquaculture importance and declining wild populations. Populations experience different exposure histories: strong upwelling (Van Damme, California [VD]) historically exposed to low-pH conditions and weak-intermittent upwelling (Santa Barbara, California [SB]). Abalone were cultured under control-pH or OA-conditions and fed crustose coralline algae (CCA) or diatoms used in aquaculture. We tested treatment effects of population, settlement diet, and OA-exposure on survival as influenced by larval-energy stores. Survival in both populations was enhanced by CCA when cultured under both treatment conditions; however, by later stages, this effect remained only for SB. SB had reduced post-settlement survival when cultured under OA-conditions, whereas post-settlement survival of VD was not. Diet affected the relationship between larval-energy and post-settlement survival; a positive relationship when fed diatoms and a negative relationship with CCA. The relationship between larval energy and post-settlement survival was stronger in VD. CCA enhanced juvenile growth in SB cultured abalone at both three-months and one-year post-settlement. Settlement diets can reduce the impacts of OA on early-life stages of abalone, but population differences driven by underlying energetics affect the consistency of this outcome. These findings illuminate the impacts from OA, suggesting populations may be at risk, and inform strategies for developing and sustaining shellfish aquaculture in the face of changing ocean conditions.

Birds exhibit longer lifespans than similarly sized mammals, despite having higher mass-adjusted blood glucose levels. This makes them a valuable model for the comparative study of the metabolic and physiological aspects of aging. Circulating glucose contributes to multiple pathological processes, primarily through glycation reactions and the formation of advanced glycation end-products (AGEs), as well as by promoting oxidative stress. These mechanisms are interconnected by feedback loops and play a key role in the development of age-related pathologies. To explore the causal role of glycaemia in avian ageing, we conducted a one-year dietary supplementation experiment in captive zebra finches. Birds received either glucose- or methylglyoxal-enriched water. Previously, we observed that chronic glucose supplementation in zebra finches increased mortality, an effect that did not appear to be mediated by the associated increase in plasma protein glycation or AGE levels. Therefore, the mechanisms underlying increased mortality in the glucose group remained unclear. In the present study, we investigated how glucose and methylglyoxal supplementation affect blood oxidative status and red blood cell telomere dynamics and apoptosis. We found that methylglyoxal supplementation decreased the non-enzymatic antioxidant capacity (OXY) of plasma and increased DNA damage, while glucose supplementation had no significant effect on oxidative stress, although circulating glucose levels influenced oxidative status in a sex-dependent manner. Males exhibited a positive correlation between glucose levels and organic hydroperoxides and protein carbonyls. Additionally, we report, for the first time in birds, a seasonal variation in telomere length, which was more pronounced in glucose-supplemented individuals, yet seemed independent of oxidative status. Apoptosis probability increased with both treatments, particularly with the methylglyoxal supplementation. These results highlight that glucose and methylglyoxal trigger different glucotoxicity-related pathways, with distinct effects on bird health and aging. However, the relationship between glucose supplementation and mortality remains still unclear and warrants further investigation.

Offspring phenotypes are often influenced by maternal age at reproduction. However, the persistence of such effects across multiple generations remains poorly understood, especially in rare cases where offspring of older mothers exhibit enhanced performance. Using the predatory mite Amblyseius herbicolus, we experimentally tested whether maternal age at reproduction influences offspring life history across two generations under contrasting dietary conditions. We found that grandmaternal (F0) and maternal (F1) age affected offspring (F2) developmental time and body size, but not hatching success, survival, or oviposition. The effect of maternal age on body size depended on grandmaternal age, suggesting that grandmaternal age mediates how mothers adjust offspring provisioning. Diet strongly influenced offspring traits but showed limited interaction with age effects. To our knowledge, this is the first study to demonstrate the persistence of an inverse Lansing effect across generations, whereby enhanced immature development is maintained without detectable trade-offs in reproduction or adult survival. These results identify maternal age as a key driver of transgenerational phenotypic variation, with important implications for population dynamics and responses to environmental change at the population level.

O_LIAdaptive phenotypic plasticity can help organisms cope with global warming by increasing their heat tolerance, yet most research has focused on whole-organism traits. Much less is known about thermal plasticity at the gamete level despite increasing evidence that reproduction, and particularly sperm function, is especially sensitive to heat. C_LIO_LIWe tested for plasticity in sperm heat tolerance in two ways: via male thermal acclimation (pre-ejaculate stage) and via sperm cell heat hardening (post-ejaculate stage) in brown anole lizards (Anolis sagrei). We also measured plasticity in several other sperm and ejaculate traits in response to male thermal acclimation. C_LIO_LITo test for effects of male thermal acclimation, adult males were exposed to one of two ecologically realistic, fluctuating temperature regimes that either mimic cool spring conditions or projected future summer conditions for eight weeks. Throughout this period, we repeatedly measured sperm heat tolerance (LT50), baseline motility, and sperm count. We also measured sperm morphology at the end of thermal acclimation. We predicted that males in the warmer treatment would produce more heat tolerant sperm. C_LIO_LITo test for post-ejaculate heat hardening, we compared the heat tolerance of ejaculated sperm that either did or did not experience a high but non-lethal temperature prior to heat tolerance measurement. C_LIO_LIWe found no plasticity in sperm heat tolerance due to either male thermal acclimation or sperm cell heat shock. Long-term thermal acclimation of males did not increase sperm heat tolerance, nor change motility, sperm count, or sperm morphology. We also found no evidence for post-ejaculate heat hardening, as exposing sperm cells to mild sublethal heat shock did not enhance sperm heat tolerance. C_LIO_LIOur results indicate that gametic traits have limited capacity for plastic adjustment to thermal stress, which is broadly consistent with the low levels of thermal plasticity found for whole-organism thermal tolerance across ectotherms. This highlights the vulnerability of reproductive traits to rising temperatures and the importance of evolutionary and behavioral responses to buffer organisms from climate change. C_LI

Healthspan, the period of life where organisms are without frailty and/or disease, is a major focus of biogerontological research. To understand late-life decline and increased mortality risk, short-lived organisms such as nematode worms are commonly used. Pristionchus nematodes are established models for evolutionary developmental genetics research and show promise as systems for comparative and experimental study of ageing. To support this, we developed phenotypic ageing profiles for the evo-devo model Pristionchus pacificus and its little-studied congener Pristionchus fissidentatus. We find that various life history traits differ between P. pacificus and P. fissidentatus (lifespan, brood size, and reproductive period), demonstrating their utility for studying divergent ageing trajectories. Further, several traits are consistently impacted by age, including intestinal barrier function, body size, and locomotory ability. Additionally, in P. pacificus, rupture avoidance, cuticle integrity, and feeding rate decline with age, indicating dysregulation across many tissue types. Several age-linked patterns resemble those documented for Caenorhabditis elegans despite considerable evolutionary distance, suggesting conserved senescent processes across the Rhabditida family of nematodes. This work highlights similarities and differences in the impact of ageing in two Pristionchus nematodes and supports their development as models for evolutionary genetic study of senescence.

Crown-of-thorns sea star (CoTS), Acanthaster cf. solaris, outbreaks are a major cause of hard coral cover decline across the west Pacific, threatening coral reefs. Coral taxa vary in susceptibility to CoTS predation from preferred (Acropora spp.) to non-preferred (Porites spp.), yet the mechanisms underlying these differences are poorly understood. We investigated coral defenses during an ongoing CoTS outbreak in Mo'orea, French Polynesia by examining gene expression (including putative toxin genes) in healthy and actively predated colonies of a preferred (Acropora hyacinthus) and a non-preferred (Porites sp.) coral prey species. During predation, A. hyacinthus exhibited molecular signatures of cellular stress responses involving oxidative stress signalling, inflammation, and tissue proteolysis. In contrast, Porites sp. showed enrichment of genes involved in mitochondrial metabolic adjustment and aerobic metabolism, suggesting metabolic compensation to maintain cellular function. Furthermore, A. hyacinthus demonstrated a reactive defense behaviour by differentially expressing toxins (e.g., kunitz-type neurotoxins) while Porites sp. employed constitutive expression of all putative toxins regardless of active predation, suggesting a proactive defense strategy. Together, these findings suggest that preferred and non-preferred coral prey exhibit fundamentally different molecular and defensive strategies during CoTS predation, shedding light on the evolutionary arms race between corals and their predators.

Despite a likely role in setting stress tolerance limits, food ration has received limited attention as an ecological factor affecting the cellular stress response (CSR). To study the interactive effects of food and temperature on the proteomic heat shock response, we acclimated intertidal mussels (Mytilus californianus) to four combinations of nearshore (low) and aquaculture (high) phytoplankton levels, combined with low (20 {degrees}C) and high (30 {degrees}C) aerial temperatures during daytime low tides. Mussels were then exposed to an acute (6 h), aerial heat stress (33 {degrees}C) and allowed to recover for 1 h and 25 h in pre-exposure conditions. Proteomic changes in the gill before and after heat shock were measured using label-free liquid-chromatography-mass spectrometry. Compared to other acclimation treatments, low-food-low-temperature (LTLF) mussels modified more splicing factors, heterogeneous nuclear ribonucleoproteins, ribosomal proteins, and translation initiation and elongation factors, suggesting systemic changes to RNA processing, selection, and translation. LTLF mussels also increased chaperones of the actin- and microtubule-associated cytoskeleton and proteins that mature along the endoplasmic reticulum to Golgi secretory pathway. Simultaneously, actin stress fiber formation at focal adhesions and the extracellular matrix, along with anchoring of microtubule-associated cilia, indicate a possible system-wide mechanical breakdown of the cytoskeleton. Signaling proteins causing cytoskeletal changes varied mainly in LTLF mussels and suggest a food-dependent role for prostaglandin synthesis. Overall, the acclimation-dependent proteomic changes show how thermal conditioning and food ration together will shape the CSR of mussels during heat waves. SUMMARY STATEMENTVariation in mRNA processing, translation, chaperoning proteins and stabilization of the actin- and microtubule-associated cytoskeleton establish food ration as a major environmental variable of the cellular stress response in mussel gill.

To better understand how animals cope with increasingly variable and challenging environments, there is a need to study how prolonged exposure to elevated glucocorticoid hormones (i.e. one mediator of the stress response) affects their physiology. While glucocorticoid elevation is known to increase oxidative stress and accelerate cellular ageing, there is evidence that king penguins (Aptenodytes patagonicus) can prevent oxidative stress during acute stress exposure, suggesting that species may differ in their sensitivity to glucocorticoids downstream negative effects. As king penguins thrive in a seemingly harsh environment, we hypothesized that they may be able to limit the deleterious effects usually associated with chronic glucocorticoid elevation, either through resistance (i.e. prevention of downstream negative effect) or resilience (i.e. rapid recovery following transient negative effect). To test this hypothesis, we experimentally elevated corticosterone levels in incubating king penguins and quantified treatment effects on a suite of physiological traits at multiple time points across incubation and early chick-rearing, up to ca. 2 months after implantation. Corticosterone-treated individuals showed a prolonged increase in corticosterone and decrease in body condition, confirming our treatment likely mimicked sustained stress exposure. Heterophil-to-lymphocyte ratio was only increased transiently, and there was no clear evidence that treatment influenced oxidative stress or telomere length maintenance. Plasma energy metabolites were mainly affected early after implantation, with rapid recovery over time. Overall, our results suggest that adult king penguins show at least moderate resistance and resilience to chronic corticosterone elevation, especially in preventing cellular integrity loss, though at-sea physiological effects remain to be determined.

Physiological adaptation across environmental gradients can contribute to ecological speciation by limiting performance outside locally optimal habitats. Intertidal systems provide strong natural thermal gradients, yet the extent to which thermal physiology contributes to divergence across shore height remains poorly resolved. We investigated cardiac thermal performance in two ecotypes of the marine snail Littorina saxatilis occupying different shore heights along the Galician coast (NW Spain): a wave-adapted ecotype on the lower and mid- shore and a crab-resistant ecotype on the mid- and upper shore. Using infrared photoplethysmography, we quantified heart rate responses in both a reciprocal field transplant experiment and laboratory thermal ramping trials. In the field, the Wave ecotype exhibited significantly higher heart rates than Crab ecotype snails under native mid-shore conditions and after 1 day of exposure to upper-shore conditions. However, after 4 days of exposure to the upper shore, Wave ecotype snails showed a marked reduction in cardiac activity, whereas Crab ecotype populations maintained stable heart rates across transplant locations and durations. In laboratory ramping experiments, Crab ecotypes displayed lower baseline cardiac activity and greater thermal insensitivity across the rising phase of the thermal response curve, while the Wave ecotype exhibited higher cardiac performance and an earlier decline in heartrate at high temperatures. Together, these results demonstrate pronounced ecotype divergence in cardiac thermal physiology and suggest that chronic exposure to upper-shore conditions compromises cardiac performance in the Wave ecotype. Such physiological differences likely contribute to vertical zonation and the evolution of barriers to gene flow between these ecotypes.

Winter poses harsh physiological challenges for insects living in temperate ponds due to the combination of low temperatures and reduced oxygen levels (hypoxia). Despite these stressors, water boatmen (Hesperocorixa sp.), backswimmers (Notonecta sp.) and diving beetles (Laccophilus maculosus) remain active year-round in eastern Nova Scotia, including in ice- covered ponds. However, the underlying mechanisms that allow pond insects to survive overwintering have not been widely studied. We hypothesized that cold and hypoxia tolerance would improve from September to March in these insects, with changes in whole-animal and biochemical correlates of tolerance to both stressors. We collected insects from the field and stocked them in outdoor freshwater mesocosms. Every two months, between September 2023 and April 2024, we characterized whole animal responses and biochemical changes. Whole insect cold tolerance assays indicated a trend of improved ability to sustain voluntary muscle control (CTmin) at lower temperatures in winter-collected insects and higher internal fluid freezing temperatures (SCP). There were no changes in whole animal correlates of hypoxia tolerance over time (surface respiration frequency, submersion and surface time). Potential cryoprotectants like proline, myo-inositol and trehalose increased in concentration in multiple insect taxa during winter but were relatively low compared to terrestrial insect studies. As lactate dehydrogenase activity did not change, there is little evidence that the insects experienced functional hypoxia sufficient to induce anaerobic metabolism. Overall, this research has established a baseline cold and hypoxia tolerance dataset in understudied pond insects. Summary StatementPond insects remain active during low temperature and low oxygen conditions associated with ice cover in winter, supported by physiological and biochemical changes.

Long-term winter dormancy in ectotherms (brumation) defines the annual cycle of many temperate-zone reptiles, yet the transcriptional regulation that supports survival across months of cold and aphagy remains poorly understood. We generated time-resolved transcriptomic profiles of liver and testis from male red-sided garter snakes (Thamnophis sirtalis parietalis) at five timepoints spanning the eight-month brumation cycle: pre-brumation, early, mid-, and late brumation, and post-arousal under continued aphagy. Time-course negative-binomial regression (maSigPro) followed by gene-set enrichment analysis identified 3,715 transcripts in liver and 5,828 in testis with significant temporal expression structure organized into five overarching temporal patterns: sustained downregulation, downregulation with post-arousal recovery, sustained upregulation, brumation-specific upregulation and cyclic modulation. Liver showed coordinated upregulation of fatty acid mobilization enzymes (ATGL, FOXO1, PPAR, CPT1A) and gluconeogenic regulators (CREBBP, PCK1) coincident with sustained low temperatures. Additionally, low temperature transcriptional activity was suggestive of a shift toward hepatic lipid mobilization and alanine-supported gluconeogenesis. Testis showed sustained suppression of meiosis, reproduction, and DNA-metabolism gene sets that did not fully recover at arousal consistent with this species dissociated reproductive pattern. Both tissues showed coordinated upregulation of stress-response pathways involving heat-shock proteins, HIF1 and a glutathione-based antioxidant defense. Interestingly, three vitellogenin transcripts and 17{beta}-hydroxysteroid dehydrogenases associated with estradiol-favoring steroid metabolism were upregulated in male liver during late brumation, which is not expected during natural physiology in adult males. Together these data support a framework in which temperature- and starvation-associated transcriptional programs contribute to survival of one of the longest, coldest brumations documented in a squamate. Summary statementA time-resolved transcriptomic analysis of liver and testis spanning eight months of winter brumation in Thamnophis sirtalis parietalis reveals gene expression patterns consistent with a temperature-associated shift toward hepatic lipid mobilization, sustained reproductive suppression, and vitellogenin response in males.

The corticotropin-releasing factor (CRF) system is a major neural regulator of stress responses in vertebrates. However, stress-related roles for the CRF system in other tissues--and whether these roles vary between stressor types--remain unclear. To address this gap, we first characterized the CRF system in the gills and intestine of rainbow trout (Oncorhynchus mykiss) and then evaluated how it is transcriptionally regulated following either an immune (vaccination) or osmotic (seawater transfer) stressor. Additionally, since the CRF system is involved in food intake regulation, we also evaluated whether feeding state affects the intestinal CRF system. Vaccination against Vibrio anguillarum reduced CRF system activity in the intestine--as indicated by elevations in CRF binding protein transcripts paired with reductions in ligand (crfa2) and receptor (crfr1b) transcripts--but did not affect the gill CRF system. In contrast, seawater transfer caused the abundance of most CRF system transcripts to increase in the middle (but not posterior) portion of the intestine, while transcript levels of CRF binding proteins and receptors in the gills declined. Finally, levels of CRF system transcripts in the intestine varied with feeding state in a region-specific manner. In the middle intestine, transcript levels of most components declined with fasting and increased when feeding was resumed, whereas the opposite pattern occurred in the posterior intestine. Overall, our results implicate the peripheral CRF system as a stressor- and epithelial tissue-specific modulator of immune and osmoregulatory functions in teleosts.

Most organisms have evolved mechanisms to reduce the negative effects of toxins in their diet. Some animals that are toxin specialists, such as dendrobatid poison frogs, have amino acid substitutions in proteins targeted by the toxins that prevent or limit the toxins ability to bind and exert its bioactive effects. The Phantasmal poison frog, Epipedobates tricolor, has amino acid substitutions in its neuronal nicotinic acetylcholine receptors that were previously shown to provide resistance to the highly potent neurotoxin epibatidine in vitro. However, it is unclear whether E. tricolor resists the physiological effects of epibatidine in vivo. To investigate this, we examined the effects of epibatidine exposure on the locomotion and behavior of E. tricolor. We performed whole-animal performance assays and behavioral evaluations at multiple time points following the administration of high yet biologically relevant levels of epibatidine. These assays were followed by alkaloid quantification to track chemical concentrations in the skin. Epipedobates tricolor exhibited similar locomotor performance and behavior at both epibatidine doses compared to controls and regardless of the quantity of alkaloid accumulated into the skin. However, we observed an impact of low-percentage ethanol solutions on behavior when compared to water controls, as well as general impacts of handling stress (regardless of the administered solution type), which should be considered in future experimental designs. Overall, we demonstrate that E. tricolor likely avoids a physiological fitness trade-off between toxin ingestion and the defensive benefits of epibatidine sequestration. Our study suggests that the ability to ingest toxins involves multi-faceted resistance mechanisms. Highlights- Whole-animal performance assay shows no impact of epibatidine on frog behavior - Epipedobates tricolor is 20-5000 times more resistant to epibatidine than mice - Assays demonstrate that frogs are sensitive to 6% ethanol and handling stress

Sleep is a ubiquitous, yet highly variable, behavior across species. The duration and timing of sleep are influenced by ecological demands and dietary context. In the mosquito Aedes aegypti, a blood-feeding insect with specialized nutritional requirements, the relationship between feeding and sleep remains poorly understood. Here, we investigated how blood-derived dietary protein influences sleep regulation. Using postural analysis, videography, and arousal-threshold assays, we established that immobility bouts of greater than 10 minutes reliably define sleep in Ae. aegypti. Mosquitoes lacking the circadian clock gene cycle still maintained daily sleep rhythms but exhibited reduced sleep duration and heightened overall activity. Infrared activity monitoring revealed that blood-fed females showed a marked increase in sleep beginning immediately after feeding and persisting for several days, accompanied by reduced locomotor activity. Notably, this sleep elevation lasted well beyond the cessation of previously reported host-seeking phases, raising the possibility of distinct phases of opportunistic versus targeted host pursuit. To determine the dietary basis of this effect, we tested mosquitoes fed a bovine serum albumin (BSA)-based diet. BSA feeding alone was sufficient to mimic the sleep-promoting and activity-reducing effects of blood, suggesting dietary protein is a major nutritional regulator. Moreover, RNAi-mediated knockdown of the leucokinin receptor (Lkr), which has previously been associated with fluid homeostasis and feeding behavior, resulted in enhanced sleep and reduced activity, implicating mosquito LK signaling in the modulation of postprandial sleep. Together, these findings demonstrate that blood-derived proteins drive sustained increases in sleep and reductions in locomotor activity in Ae. aegypti. This work positions Ae. aegypti as a model for dissecting nutrient-specific regulation of sleep and highlights potential adaptive functions of protein-induced quiescence, such as energy conservation and predator avoidance. More broadly, it provides insight into how specialized diets shape the neural and behavioral architecture of sleep.

Climate-driven warming is challenging the physiological limits of aquatic ectotherms, with cardiac performance emerging as one of the key determinants of thermal tolerance. Cardiac function relies on mitochondrial ATP production, and mitochondrial dysfunction has been linked to cardiac failure at critical temperatures. However, mitochondria are plastic and may represent a target for interventions aimed at improving thermal tolerance in fish. Exercise training improves whole-animal performance in fish, including cardiac thermal performance, and improves mitochondrial function in other taxa. However, its effects on the thermal sensitivity of cardiac mitochondria remain unknown. This study investigated whether exercise-training alters cardiac mitochondrial performance at optimal and critical temperatures in rainbow trout Oncorhynchus mykiss. Farmed rainbow trout were subjected to a four-week exercise training regime, while control fish remained under standard rearing conditions. Cardiac mitochondrial respiration was assessed in permeabilised heart fibres at 16{degrees}C (optimal growth temperature) and 26{degrees}C (temperature associated with cardiac arrhythmia) and several biochemical and nuclear indicators were measured. No significant differences were detected between treatments for any measured variable. However, trained fish generally exhibited higher maximal respiratory capacities and respiratory control ratios, particularly at the elevated temperature, suggesting subtle improvements in mitochondrial function despite considerable inter-individual variation. Temperature influenced mitochondrial performance, increasing proton leak and reducing coupling efficiency. These findings demonstrate that cardiac mitochondrial function is thermally sensitive and represents a potential targeted for improving thermal resilience in aquaculture species.

Parental care can have pervasive effects on offsprings neurodevelopment. Parent-offspring interactions are often modulated by the neuropeptide oxytocin, which is responsible for the development of social bonds. The development of the oxytocinergic system is dependent on the quality of parental care during the post-natal phase. However, it is yet unknown how post-natal direct interactions can influence the development of the oxytocinergic pathway. Here we tested how an obligate parental care behaviour, tadpole transport in poison frogs, influences the development of the oxytocinergic pathway. To this end, we quantified whole brain expression of oxytocin receptor and oxytocin precursor throughout three developmental stages of A. femoralis tadpoles, before, during and after tadpole transport. Our results show an overall downregulation during tadpole transport, which indicates that during transport tadpoles enter a dormant state to slow down development until they are placed in water. Interestingly, the expression of oxytocin precursor did not vary between the three developmental stages. This might indicate that oxytocin is being recruited during transport, but does not lead to neurodevelopmental changes. In sum, here we present the first evidence of a dormant state during tadpole transport which might be an adaptive response to the terrestrial reproduction in poison frogs.