Journal of Experimental Zoology Part A: Ecological and Integrative Physiology

Telomeres are key biomarkers of cellular ageing, yet their dynamics remain poorly studied in tropical and short-lived bat species. Here, we present the first investigation of telomere length across age in Molossus molossus, a tropical bat historically categorised as the shortest-lived bat on record. Through a multi-year mark-recapture study in Gamboa, Panama, we sampled 492 individuals (n = 317 females, 175 males) and documented a female M. molossus surviving to at least 13 years of age, more than doubling the previously reported maximum lifespan of 5.6 years. Across the population, relative telomere length (rTL) showed no overall significant decline with age. No evidence was found for sex-specific rates of telomere attrition. Rather these results suggest that males and females follow parallel age-related telomere trajectories, with any sex differences primarily reflecting differences in mean telomere length rather than ageing dynamics. Overall, the findings here challenge previous assumptions about the lifespan and ageing biology of M. molossus. They demonstrate that telomere maintenance is not limited to temperate bats, show that sex differences in telomere biology are subtle and species-specific, and reinforce the value of long-term field studies for understanding ageing processes in the wild.

Avian haemosporidia are blood parasites that can have dramatic fitness consequences on their hosts, including largescale population declines when introduced to naive hosts. Yet the physiological effects that accompany haemosporidian infection and underlie these fitness decrements are poorly characterized in most wild birds. Because haemosporidia destroy host red blood cells and consume host hemoglobin, they are predicted to have detrimental impacts on avian blood-oxygen transport and, as a result, reduce aerobic performance. However, the documented effects of infection on avian hematological traits vary across species and no effects have been demonstrated on avian aerobic performance to date. Here we quantified the physiological effects of haemosporidian infections on wild Pink-sided Juncos (Junco hyemalis mearnsi) breeding in northwestern Wyoming, USA. We assayed hematological traits (hemoglobin concentration and hematocrit) and aerobic performance (resting and summit metabolic rates, thermogenic endurance, and aerobic scope), then screened individuals for haemosporidian infection post-hoc (n = 106 adult juncos). We found that infection status did not correlate with any of the physiological indices that we measured, suggesting there is little cost of haemosporidian infection on either junco aerobic performance or energy budgets. Our results highlight the need for more studies of haemosporidia infections in a broader range of species and in a wider array of environmental contexts.

O_LIBody condition is an important life history challenge that directly impacts individual fitness and is particularly important for hibernating animals, whose maintenance of adequate body fat and mass is essential for survival. C_LIO_LIIt is well documented that symbiotic microorganisms play a vital role in animal physiology and behaviour. Recent work demonstrates that gut microbes are associated with fat accumulation and obesity; Firmicutes is consistently associated with obesity while Bacteroidetes is associated with leanness both in humans and other animals. C_LIO_LIThe focus of most microbiome studies has been on human health or involved lab reared animals used as a model system. However, these microbes likely are important for individual fitness in wild populations and provide potential mechanistic insights into the adaptability and survival of wildlife. C_LIO_LIHere we test whether symbiotic microorganisms within the phyla of Firmicutes and Bacteroidetes are associated with summer mass gain in an exceptionally well-studied wild population of yellow-bellied marmots (Marmota flaviventer) by quantifying microbial abundance over five years of fecal samples (2015 - 2019) collected during their summer active season. C_LIO_LIResults show that marmots with higher mass gain rates have a greater abundance of Firmicutes. In contrast, higher abundance of Bacteroidetes was associated with lower mass gain rates, but only for marmots living in harsher environments. Similar patterns were found at the family level where Ruminococcaceae, a member of Firmicutes, was associated with higher mass gain rates, and Muribaculaceae, a member of Bacteroidetes, was associated with lower mass gain rates, and similarly in harsher environments. C_LIO_LIAlthough correlative, these results highlight the importance of symbiotic gut microbiota to mass gain in the wild, a trait associated with survival and fitness in many taxonomic groups. C_LI

U.S. quail species are vulnerable to population declines as a result climate change, habitat loss, and habitat fragmentation; all of which can induce physiological stress. Additionally, population restoration techniques (PRTs), like translocations, also induce stress. Traditional stress assessments include capturing and handling birds to extract blood, methods which are inherently stressful and can compound stress analyses. However, the stress hormone corticosterone is metabolized from the blood and excreted in feces as fecal corticosterone metabolites (FCMs). FCMs have been used as a non-invasive measurement of stress in a variety of species, but must be validated for each species. The objective of this study was to biologically validate the use of FCMs as a non-invasive measurement of stress-hormone levels in California valley quail (Callipepla californica). Reference and treatment quail were acclimated for 3 wks in an outdoor aviary. Subsequently, treatment quail were subjected to a simulated, 48-h translocation, a common and stressful PRT. Fecal samples were collected every 4 h and processed using an enzyme immunoassay. Mean FCM concentrations of treatment quail (41.50 {+/-}16.13 ng/g) were higher than reference FCM concentrations (24.07 {+/-}10.4 ng/g). These results biologically validate the use of FCMs as a non-invasive method to assess stress hormone levels in California valley quail, demonstrate diurnal variation in quail stress levels, and confirms that quail translocations are a stressful PRT. Ultimately, this research validates a new non-invasive tool for stress measurement to advance quail research, management, and conservation. Lay summaryThis study biologically validates the use of fecal corticosterone metabolites as a non-invasive method for detecting stress in quail, demonstrates diurnal variation in quail stress levels, confirms that translocations elevate stress which likely impacts success, and establishes a new non-invasive tool for stress measurement in quail research, management, and conservation.

Amphibians that reproduce in early spring at northern latitudes may encounter environmental ice while migrating to their breeding sites. Due to the nucleation properties of ice, contact with environmental ice may induce rapid freezing of body tissues, which can cause irreversible damage to cells and lead to death. Although some species of salamanders are known to move over ice during early spring migration, freeze-intolerant species are expected to avoid physical contact with ice crystals to minimise the risk of freezing. Here, we documented the thermal biology of the freeze-intolerant blue-spotted salamander (Ambystoma laterale Hallowell, 1856) migrating at sub-zero temperatures in Algonquin Provincial Park, Ontario, Canada. During our surveys, we found sheltered, inactive, and migrating individuals; some in direct contact with ice. Our field measurements of skin temperature using high resolution thermal imaging suggest that A. laterale can sustain activity in a supercooled state (i.e., chilled below the freezing point of body fluids but not frozen). By migrating in a supercooled state, these salamanders may overcome the risk of freezing while simultaneously prolonging their breeding season and potentially avoiding predators.

Many different physiological changes have been observed in wild waterfowl during the flightless stage of wing moult, including a loss of body mass. Previously we established that captive barnacle geese (Branta leucopsis) underwent this characteristic decrease in body mass during their wing moult, even though they had unlimited and unrestricted access to food. In the present study we aimed to determine if this body mass loss during moult comprised mainly a reduction in fat stores, and to ascertain if the captive geese undergo pre-migratory and pre-winter fattening over a similar temporal scale to their wild conspecifics. The non-destructive technique of deuterium oxide isotope dilution was employed to provide repeated measurements of estimated fat deposition from a captive flock of fourteen barnacle geese. Birds were injected with deuterium oxide at 7 distinct intervals for one annual cycle. During the flightless period of the moult, body fat decreased by approximately 40% from the pre-moult value. During late-September and early October, body fat reached its highest point in the annual cycle, both as an absolute value and as a percentage of total body mass. We propose that while the energetic cost of wing moult is not the ultimate cause of fat loss in moulting barnacle geese, the approximate 212 g of fat catabolised during moult would provide sufficient energy to cover the cost of the replacement of the flight feathers, estimated to be 6384 kJ, over an approximate 42 day period. We conclude that the previously recorded increase in metabolism during moult in the geese, led to the use of endogenous fat reserves because the birds reduced rather than increased their food intake rates owing to the increased risk of predation when flightless. We also conclude that captive barnacle geese do undergo pre-winter and pre-migratory fattening, providing further evidence of the innate nature of these fat deposition cycles.Competing Interest StatementThe authors have declared no competing interest.View Full Text

From a conservation perspective, it is important to identify when sub-lethal temperatures begin to adversely impact an organism. However, it is unclear whether, during acute exposures, these cellular thresholds occur at similar temperatures to other physiological or behavioural changes. To test this, we estimated temperature preference (15.1 {+/-} 1.1 {degrees}C) using a shuttle box, thermal optima for aerobic scope (10-15 {degrees}C) using respirometry, agitation temperature (22.0 {+/-} 1.4 {degrees}C) as the point where a fish exhibits a behavioural avoidance response and the CTmax (28.2 {+/-} 0.4 {degrees}C) as the upper thermal limit for 1 yr old Brook Trout (Salvelinus fontinalis) acclimated to 10 {degrees}C. We then acutely exposed a different subset of fish to these temperatures and sampled tissues when they reached the target temperature or after 60 min of recovery at 10 {degrees}C. We used qPCR to estimate mRNA transcript levels of genes associated with heat shock proteins, oxidative stress, apoptosis, and inducible transcription factors. A major shift in the transcriptome response occurred near the agitation temperature, which may identify a link between the cellular stress response and the behavioural avoidance response.

Pacific Oysters (Magallana/Crassostrea gigas) are marine bivalves that are widely cultivated but increasingly experience summer mortality due to interacting stressors. Two major concerns are (1) the rising severity and frequency of marine heat waves and (2) disease outbreaks (e.g., OsHV-1). To better understand how multiple stressors influence oyster resilience and whether stress priming can improve survival, we tested the effects of parental immune challenge on offspring performance. We exposed broodstock to a Poly(I:C) immune challenge, reared their offspring to the seed stage, and assessed survival, growth, and metabolic responses under thermal stress in the lab. Offspring of immune-challenged parents showed higher growth rates during development. Under elevated temperatures, these offspring had higher survival than controls at 40{degrees}C, but lower survival at 42{degrees}C, suggesting thermal limits to priming benefits. Metabolic assays further revealed that at moderately elevated temperature (36{degrees}C), primed offspring had higher metabolic activity, whereas at higher temperature (40{degrees}C), they exhibited lower metabolic activity than controls. This pattern indicates that parental immune challenge may influence offspring metabolic flexibility, potentially enhancing thermal tolerance through an increased capacity for metabolic depression at extreme temperatures. Together, our results highlight cross-generational links between immune priming and thermal tolerance.

1. Increases in ultraviolet radiation (UVR) correlate spatially and temporally with global amphibian population declines and interact with other stressors such as disease and temperature. Declines have largely occurred in high-altitude areas associated with greater UVR and cooler temperatures. 2. UVR is a powerful mutagenic harming organisms largely by damaging DNA. When acutely exposed to UVR at cool temperatures, amphibian larvae have increased levels of DNA damage. Amphibians may be able to compensate for the depressive effects of temperature on DNA damage through thermal acclimatisation, but it is unknown whether they or other ectotherms have this capacity. 3. We reared striped marsh frog larvae (Limnodynastes peronii) in warm (25{degrees}C) and cool (15{degrees}C) temperatures under either a low or moderate daily dose of UVR (10 and 40 {micro}W cm-2 UV-B for 1 h at midday, respectively) for 18-20 days and then measured immediate DNA damage resulting from an acute high UVR dose (80 {micro}W cm-2 UV-B for 1.5 h) at a range of test temperatures (10, 15, 20, 25, and 30{degrees}C). 4. Larvae acclimated to 15{degrees}C and exposed to UVR at 15{degrees}C completely compensated UVR-induced DNA damage compared with 25{degrees}C acclimated larvae exposed to UVR at 25{degrees}C. Additionally, warm-acclimated larvae had higher CPDs than cold-acclimated larvae across test temperatures, which indicated a cost of living in warmer temperatures. In contrast, larvae reared under chronic elevated UVR levels showed no evidence of UVR acclimation resulting in lower DNA damage following an acute high UVR exposure. 5. Our finding that thermal acclimation in L. peronii larvae compensated UVR-induced DNA damage at low temperatures suggested that aquatic ectotherms living in cool temperatures may be more resilient to high UVR than previously realised. 6. We suggested individuals or species with less capacity for thermal acclimation of DNA repair mechanisms may be more at risk if exposed to changing thermal and UVR exposure regimes but cautioned that thermal acclimation of DNA repair mechanisms may not always be beneficial.

Parasites can have profound effects on their hosts, and those effects can be altered by changing environmental conditions. The dinoflagellate Hematodinium sp. is a common and deadly parasite of the crab Chionoecetes bairdi, a species vulnerable to rising ocean temperatures. To examine the impact of parasitism under various temperature conditions, infected crabs (n = 9) were held under three temperature regimes (4{degrees}C, 7.5{degrees}C, and 10{degrees}C) for 17 days. RNAseq was performed on samples from three timepoints, and the relationships of temperature and time to gene expression were examined. Transcriptomes for C. bairdi and Alveolata symbiotes were created, and genes linked to immune function were identified within both host and parasite. Within the host, 1721 contigs were differentially expressed in response to a temperature increase, with 86% of these increased in expression. In total, 3013 contigs linked to temperature response were identified. Additionally, numerous changes in biological processes were observed in Hematodinium over the course of the experiment, including development and microtubule-based processes and ribosomal assembly. Through understanding the impact of changes in temperature on gene expression within both Hematodinium and infected C. bairdi, we provide a more complete picture of the response of these species to rising ocean temperatures.

Telomere length (TL) is increasingly used in ecology as a biomarker of individual quality and environmental stress, yet research on non-model species with complex life histories remains limited. Because TL varies among tissues and across ages in a species-specific manner, identifying non-lethal tissues that reliably reflect whole-organism telomere dynamics is essential for longitudinal telomere studies in the field. This study aimed to evaluate tissue-specific TL in Japanese eel (Anguilla japonica), an endangered catadromous fish. We first mapped the chromosomal distribution of telomeric sequences using fluorescent in situ hybridization (FISH), the first application of this method in this species. We then tested whether muscle and caudal fin, which can be sampled easily and non-lethally, can serve as suitable proxy tissues for TL measurements in wild individuals. Relative telomere length (RTL) was quantified by qPCR in blood, brain, caudal fin, gonads, heart, liver, and muscle. FISH analysis confirmed telomeric repeats at all chromosomal ends, with only weak interstitial signals on three chromosomal pairs unlikely to affect qPCR-based estimates. A generalized additive mixed model and Wilcoxons signed-rank tests revealed significant inter-tissue differences: RTL was shortest in the brain and muscle and longest in liver, blood and caudal fin. Muscle and caudal fin RTL were significantly correlated with RTL in many other tissues, supporting their use as proxy tissues for longitudinal TL monitoring, including responses to environmental variation. Both total length and age were tested as explanatory variables for RTL, and the model including total length showed a better fit than the age-based model. Non-linear relationships between RTL and total length observed in several tissues suggest physiological shifts associated with growth and sexual differentiation. Overall, these findings advance understanding of telomere dynamics in eels and establish muscle and caudal fin as suitable tissues for repeated, non-lethal TL assessment in ecological and conservation contexts.

Populations of marine species that respond differently to ocean acidification offer natural reservoirs of biodiversity that can be leveraged for conservation efforts and to sustain marine food systems. The molecular and physiological traits associated with tolerance to acidification must first be identified. This study leveraged oysters from three phenotypically distinct populations of the Olympia oyster, Ostrea lurida, but that were bred and reared in common conditions for four years. We assessed their growth, reproductive development, and transcriptional response to acidification within and across generations. Responses reveal energetic trade-offs that reflect unique physiotypes previously observed among populations. The population with the slowest growth but high survival rates, oysters from Dabob Bay, mounted the largest transcriptional response to acidification without effects to growth and reproduction. A moderate response was observed in the population with fastest growth rate but lowest fecundity (Fidalgo Bay). Oyster Bay, the population with highest fecundity but lowest survival rates, did not respond at the transcript level. Oyster Bay was also the only population for which acidification negatively affected growth and reproductive development. While exposure to acidification did not affect gene expression in the next generations larval stage, it did result in larger larvae in the Oyster Bay population, which could partially alleviate negative effects of acidification in the wild for that population. Given the distinct transcriptional response of the Dabob Bay population to acidification and its high survival rates in previous studies, we then identified genes that were uniquely expressed in Dabob Bay oysters compared to the other populations. Genes involved in antibacterial and antiviral processes, metabolism, growth, and reproduction were uniquely expressed in Dabob Bay, and many similar functions were identified in both adults and larvae, which provides insight into the mechanisms behind a stress-tolerant oyster population. The population-specific physiotypes and responses to acidification illustrate the diversity of physiological strategies in O. lurida that balance the energetic demands of growth, reproduction, cellular maintenance, and offspring viability. Taken together this study reveals that there are distinct physiotypes among marine invertebrate populations on small geographic scales with implications for species resilience to acidification and other environmental stressors.

Maternal thyroid hormones (THs) are known to be crucial in embryonic development in humans, but their influence on other, especially wild, animals remains poorly understood. So far, the studies that experimentally investigated the consequences of maternal THs focused on short-term effects, while early organisational effects with long-term consequences, as shown for other prenatal hormones, could also be expected. In this study, we aimed at investigating both the short- and long-term effects of prenatal THs in a bird species, the Japanese quail Coturnix japonica. We experimentally elevated yolk TH content (the prohormone T4, and its active metabolite T3, as well as a combination of both hormones). We analysed hatching success, embryonic development, offspring growth and oxidative stress as well as their potential organisational effects on reproduction, moult, and oxidative stress in adulthood. We found that eggs injected with both hormones had a higher hatching success compared with control eggs, suggesting conversion of T4 into T3 by the embryo. We detected no other clear short-term or long-term effects of yolk THs. These results suggest that yolk thyroid hormones are important in the embryonic stage of precocial birds, but other short- and long-term consequences remain unclear. Research on maternal thyroid hormones will greatly benefit from studies investigating how embryos use and respond to this maternal signalling. Long-term studies on prenatal THs in other taxa in the wild are needed for a better understanding of this hormone-mediated maternal pathway.

1.The aim of this study was to investigate the effect of ocean acidification (OA) and warming (OW) as well as the transgenerational effect of OA on larval and juvenile growth and metabolism of a large economically important fish species with a long generation time. Therefore we incubated European sea bass from Brittany (France) for two generations (>5 years in total) under current and predicted OA conditions (PCO2: 650 and 1700 {micro}atm). In the F1 generation both OA condition were crossed with OW (temperature: 15-18{degrees}C and 20-23{degrees}C). We found that OA alone did not affect larval or juvenile growth and OW increased developmental time and growth rates, but OAW decreased larval size at metamorphosis. Larval routine metabolic rate (RMR) and juvenile standard metabolic rate (SMR) were significantly lower in cold compared to warm conditioned fish and also lower in F0 compared to F1 fish. We did not find any effect of OA on RMR or SMR. Juvenile PO2crit was not affected by OA, OW or OAW in both generations. We discuss the potential underlying mechanisms resulting in beneficial effects of OW on F1 larval growth and RMR and in resilience of F0 and F1 larvae and juveniles to OA, but on the other hand resulting in vulnerability of F1, but not F0 larvae to OAW.. With regard to the ecological perspective, we conclude that recruitment of larvae and early juveniles to nursery areas might decrease under OAW conditions but individuals reaching juvenile phase might benefit from increased performance at higher temperatures. Summary statementWe found that OA did not affect developmental time, growth, RMR and SMR, while OW increased these traits. OAW decreased larval size at metamorphosis. We discuss underlying mechanisms and the ecological perspective resulting from these results and conclude that recruitment to nursery areas might decrease under OAW conditions but individuals reaching juvenile phase might benefit from increased performance at higher temperatures in Atlantic waters.

In natural environments two or more abiotic parameters often vary simultaneously, and interactions between covarying parameters frequently result in unpredictable, non-additive biological responses. To better understand the mechanisms and consequences of interactions between multiple stressors it is important to study their effects on both survival and performance. The splashpool copepod Tigriopus californicus tolerates extremely variable abiotic conditions and exhibits a non-additive, antagonistic interaction resulting in higher survival when simultaneously exposed to high salinity and acute heat stress. Here, we investigated T. californicus response in activity and oxygen consumption under simultaneous manipulation of salinity and temperature to identify if this interaction also arises in these sublethal measures of performance. Oxygen consumption and activity rates decreased with increasing assay salinity. Oxygen consumption also sharply increased in response to acute transfer to lower salinities, an effect that was absent upon transfer to higher salinities. Elevated temperature led to reduced rates of activity overall, resulting in no discernible impact of increased temperature on routine metabolic rates. This suggests that swimming activity has a non-negligible effect on copepods metabolic rates and must be accounted for in metabolic studies. Temperature also interacted with assay salinity to affect activity and with acclimation salinity to affect routine metabolic rates upon acute salinity transfer, implying that the sublethal impacts of these co-varying factors are also not predictable from experiments that study them in isolation. Summary StatementTemperature and salinity interact to affect metabolic rate in the copepod Tigriopus californicus, but the stressors individual effects and their interaction are complicated by concurrent changes in activity.

While immunity is frequently suppressed when birds engage in strenuous migratory flights, whether and how immunity changes during the rapid accumulation of energy stores in preparation for migration remains largely unknown. Here, we induced pre-migratory fattening through controlled changes of daylight in common quails Coturnix coturnix and regularly assessed changes in a marker of constitutive innate immunity (Leukocyte Coping Capacity or LCC) and measures of body composition (lean and fat mass). LCC responses were highest in the mid-fattening phase and lowest when fattening was completed. At mid-fattening, we also found that the birds that kept a higher proportion of lean mass (i.e. accumulated less fat) had the highest LCC peaks. Our results indicate that migratory birds undergo rapid immunological changes as they accumulate energy stores for migration and propose that this could be due to competing or trade-off processes between metabolic remodelling and innate immune system function. Summary statementImmunity is vital when migrating to new environments. It is costly and competivite to other physiological processes. Here we bring new evidence on this process in migratory birds.

The passive dissolution of anthropogenically produced CO2 into the ocean system is reducing ocean pH and changing a suite of chemical equilibria, with negative consequences for some marine organisms, in particular those that bear calcium carbonate shells. Although our monitoring of these chemical changes has improved, we have not developed effective tools to translate observations, which are typically of the pH and carbonate saturation state, into ecologically relevant predictions of biological risks. One potential solution is to develop bioindicators: biological variables with a clear relationship to environmental risk factors that can be used for assessment and management. Thecosomatous pteropods, a group of pelagic shelled marine gastropods, whose biological responses to CO2 have been suggested as potential bioindicators of OA owing to their sensitivity to acidification in both laboratory and the natural environment. Using five CO2 exposure experiments, occurring across 4 seasons and running for up to 15 days, we describe a consistent relationship between saturation state, shell transparency, and duration of exposure, as well as identify a suite of genes that could be used for biological monitoring. We clarify variations in thecosome responses due to seasonality, resolving prior uncertainties and demonstrating the range of their phenotypic plasticity. These biomarkers of acidification stress can be implemented into ecosystem models and monitoring programs in regions where pteropods are found, while the approach will serve as an example for other regions on how to bridge the gap between point-based chemical monitoring and biologically relevant assessments of ecosystem health. Summary StatementDespite seasonal variability, pteropods exposed to acidification over multiple seasons reveal consistent patterns in gene expression and shell condition that can be used as bioindicators of ocean acidification stress.

Once a year, penguins undergo a catastrophic moult replacing their entire plumage during a fasting period on land or on sea-ice during which time individuals can lose 45% of their body mass. In penguins, new feather synthesis precedes the loss of old feathers leading to an accumulation of two feathers layers (double coat) before the old plumage is shed. We hypothesize that the combination of the high metabolism required for new feathers synthesis and the potentially high thermal insulation linked to the double coat could lead to a thermal challenge requiring additional peripheral circulation to thermal windows to dissipate extra-heat. To test this hypothesis, we measured the surface temperature of different body regions of captive Gentoo penguins (Pygoscelis papua) throughout the moult under constant environmental conditions. The surface temperature of the main body trunk decreased during the initial stages of the moult, therefore suggesting a higher thermal insulation. On the opposite, the periorbital region, a potential proxy of core temperature in birds, increased during these same early moulting stages. The surface temperature of bill, flipper and foot (thermal windows) tended to initially increase during the moult period, highlighting the likely need for extra heat dissipation in moulting penguins. These results raise questions regarding the thermoregulatory capacities of wild penguins during the challenging period of moulting on land in the current context of global warming.

Orbicella faveolata, commonly known as the mountainous star coral, is a dominant reef-building species in the Caribbean, but populations have suffered sharp declines since the 1980s due to repeated bleaching and disease-driven mortality. Prior research has shown that inshore adult O. faveolata populations in the Florida Keys are able to maintain high coral cover and recover from bleaching faster than their offshore counterparts. However, whether this origin-specific variation in thermal resistance is heritable remains unclear. To address this knowledge gap, we produced purebred and hybrid larval crosses from O. faveolata gametes collected at two distinct reefs in the Upper Florida Keys, a nearshore site (Cheeca Rocks, CR) and an offshore site (Horseshoe Reef, HR), in two different years (2019, 2021). We then subjected these aposymbiotic larvae to severe (36 {degrees}C) and moderate (32 {degrees}C) heat challenges to quantify their thermal tolerance. Contrary to our expectation based on patterns of adult thermal tolerance, HR purebred larvae survived better and exhibited gene expression profiles that were less driven by stress response under elevated temperature compared to purebred CR and hybrid larvae. One potential explanation could be compromised reproductive output of CR adult colonies due to repeated summer bleaching events in 2018 and 2019, as gametes originating from CR in 2019 contained less storage lipids than those from HR. These findings provide an important counter-example to the current selective breeding paradigm, that more tolerant parents will yield more tolerant offspring, and highlight the importance of adopting a holistic approach when evaluating larval quality for conservation and restoration purposes.

The intensity and duration of heat waves, as well as average global temperatures, are expected to increase due to climate change. Heat waves can cause physiological stress and reduce fitness in animals. Species can reduce overheating risk through phenotypic plasticity, which allows them to raise their thermal tolerance limits over time. This mechanism could be important for ectotherms whose body temperatures are directly influenced by available environmental temperatures. Geckos are a large, diverse group of ectotherms that vary in their thermal habitats and times of daily activity, which could affect how they physiologically adjust to heat waves. Data on thermal physiology are scarce for reptiles, with only one study in geckos. Understanding thermal tolerance and plasticity, and their relationship, is essential for understanding how some species are able to adjust or adapt to changing temperatures. In this study, we estimated thermal tolerance and plasticity, and their interaction, in the crepuscular gecko, Eublepharis macularius, a species that is emerging as a model for reptile biology. After estimating basal thermal tolerance for 28 geckos, thermal tolerance was measured for each individual a second time at several timepoints (3, 6, or 24 h) to determine thermal tolerance plasticity. We found that thermal tolerance plasticity (1) does not depend on the basal thermal tolerance of the organism, (2) was highest after 6 hours from initial heat shock, and (3) was negatively influenced by individual body mass. Our findings contribute to the increasing body of work focused on understanding the influence of biological and environmental factors on thermal tolerance plasticity in organisms and provide phenotypic data to further investigate the molecular basis of thermal tolerance plasticity in organisms.