Temperature can affect development and survival in oviparous squamates based on the conditions experienced within the nest. Because nest temperatures may vary unpredictably, females are expected to possess mechanisms that enhance embryo development and, consequently, reproductive success. During pregnancy, females can buffer embryos from unfavorable conditions by selecting body temperatures that, simultaneously, optimize embryonic development and their own physiological needs. When these temperatures do not coincide, an intergenerational thermal conflict may arise. In this context, females from thermally homogeneous habitats, where preferred temperatures may be less accessible, may face higher thermoregulatory costs. Here, we investigated how the occupation of thermally different habitats affected thermal preference during pregnancy, and how thermal preference affected reproductive investment in the lizard Tropidurus aff. torquatus. Specifically, we measured thermal preference of non-pregnant and pregnant females before and after egg laying, and we recorded clutch mass for each female. We hypothesized that females from a thermally homogeneous and warmer habitat (restinga site) would prefer higher temperatures than those from a thermally heterogeneous and cooler habitat (urban site). We also hypothesized that females that preferred higher temperatures would produce lighter clutches. While thermal preference did not differ between sites, pregnant females from the restinga preferred lower temperatures than non-pregnant ones. Females with higher preferred temperatures tended to produce lighter clutches in the restinga, and females from the restinga produced lighter clutches compared to urban females. Together, our results suggest that the elevated temperatures found in the restinga can impair embryo development and reduce reproductive investment.

Abstract Fever is a widespread and adaptive defence response that enhances immune performance through an increase in body temperature above normal values. In ectotherms, fever is expressed behaviourally through the selection of warmer microhabitats following infection, yet its magnitude and determinants vary widely across species and environments. Here, we performed a phylogenetically informed meta-analysis of behavioural fever in amphibians and reptiles to test whether its expression was shaped by climatic thermal variability, pathogen identity, and taxonomy. Specifically, we tested the hypotheses that (i) species from thermally variable environments would exhibit stronger behavioural fever than species from thermally stable environments, consistent with the climate variability hypothesis, and that (ii) reptiles would exhibit stronger fever responses than amphibians due to lower hydrothermal constraints. Across 47 studies encompassing 103 effect sizes, we found that behavioural fever is widespread but highly context-dependent. We found that evidence for behavioural fever was strongest in species from more thermally variable habitats, regardless of body size and phylogeny, suggesting that access to thermally heterogeneous landscapes and enhanced behavioural plasticity amplify the capacity to sustain febrile responses. Contrary to our hypothesis, amphibians exhibited stronger fever responses than reptiles, possibly reflecting differences in baseline thermoregulatory demands and environmental opportunity, or as a consequence of methodological artefacts. The expression of behavioural fever also varied with pathogen identity, with bacterial infections eliciting larger body temperature increases than fungal or viral challenges, although pathogen representation was uneven across studies. Together, our results support the idea that the capacity to express behavioural fever depends on access to thermally heterogeneous landscapes, and may vary according to pathogen biology. Ultimately, our study emphasises that temperature is not a background condition for host-pathogen interactions, but an active and environmentally contingent component of ectotherm immune defence in amphibians and reptiles.

The global amphibian decline caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd) threatens biodiversity. Some species, such as the bullfrog (Aquarana catesbeiana), can persist with infection without overt disease, yet the processes underlying this response remain unclear. Because bullfrogs are farmed worldwide under high-density conditions, understanding how production environments influence infection dynamics and immune response is essential. We assessed Bd dynamics in farmed and wild bullfrogs, focusing on immunity and environmental zoospore release. Individuals were exposed to two Bd lineages, and we compared infection loads, immune profiles, zoospore shedding, infection progression, and survival. Farmed bullfrogs consistently exhibited higher Bd loads and released more zoospores than wild individuals. Mortality did not differ between groups, suggesting the capacity to sustain high infection loads without immediate survival costs. These findings indicate that farming environments may increase pathogen amplification and environmental zoospore availability. The observed combination of high infection loads, elevated zoospore shedding, and sustained host survival suggests increased transmission risk under farming conditions. Given that bullfrog production systems may operate with variable biosafety standards, strengthened surveillance and containment practices could help reduce pathogen dissemination risks. This study provides insights into host–pathogen interactions in intensively farmed amphibians, with implications for conservation and disease management.

Accurately measuring body temperature is critical for understanding how ectotherms interact with their thermal environment. In amphibians, traditional methods like cloacal thermometry require restraint, potentially altering body temperature through stress and heat transfer from human hands. Here, we used infrared thermography to examine how short-term gentle handling (2 min) impacted skin temperatures (Tskin) in two species of mole salamanders (Ambystoma laterale and A. maculatum) by combining field and lab experiments. In the field, we randomly assigned salamanders to a handled or control group, whereas in the lab salamanders were assigned to a heating plate set to hand temperature or a control group. We also scored salamander activity (active vs. inactive) in both settings. In the field, handling increased Tskin in both species, with the head warming up more than the dorsum. Moreover, A. laterale experienced an overall greater magnitude of thermal change (ΔTskin) than A. maculatum due to body size differences. In the lab, contact with the heating plate increased Tskin, with head Tskin being consistently warmer than dorsal Tskin. We also found that ΔTskin was greater for the handled than the heating plate group (ΔTskin = 4.86 ± 1.21 °C vs. 1.72 ± 0.70 °C, respectively), suggesting that handling may amplify heat gain. Warmer salamanders were more prone to be active regardless of whether they were handled or in contact with a heating plate. Our results demonstrate that short-term handling can cause rapid warming that exceeds rates used in heat stress studies, with implications for data reliability and amphibian welfare in thermal biology assays.

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 sub-zero temperatures, possibly in a supercooled state (i.e., chilled below the freezing point of body fluids but not frozen). By migrating in such conditions, these salamanders may overcome the risk of freezing while simultaneously prolonging their breeding season and potentially avoiding predators.

Ectotherms from highly seasonal habitats should have enhanced potential for physiological plasticity to cope with climatic variability. However, whether this pattern is applicable to fossorial ectotherms, who are potentially buffered from thermal variability, is still unclear. Here, we evaluated how seasonal acclimation (spring vs. autumn) in the lab affected the thermal sensitivity of standard metabolic rates (SMR) and rates of evaporative water loss (EWL) in the spotted salamander (Ambystoma maculatum). We hypothesised that temperature would have both acute and prolonged effects over traits (i.e., exposure to test temperatures and seasonal acclimation, respectively). After accounting for body mass and sex, we found that acute changes in temperature led to an increase in SMR and EWL. Additionally, SMR differed between seasons, but EWL did not. Salamanders had lower SMR in the spring, suggesting that energy may be allocated toward overwintering emergence and breeding. By contrast, maintaining higher SMR in the autumn may allow salamanders to forage aboveground on rainy nights to replenish energy reserves in preparation for the winter. The seasonal constancy of EWL suggests that salamanders should rely on behavioural rather than physiological modulations to mitigate possible detrimental effects of warming over the maintenance of hydric state. Despite the common assumption that fossorial ectotherms are buffered from thermal effects, our study shows that functional differences between seasons (i.e., breeding in the spring and provisioning in the autumn) are accompanied by seasonal changes in energetic and hydric requirements.

Garter Snakes (Thamnophis) are the most widespread reptiles in North America, although evidence suggests that thermal preference has not diverged much among populations or species. To shed light on how thermal decisions influence local habitat use by the Eastern Garter Snake (Thamnophis sirtalis sirtalis (Linnaeus, 1758)), we measured the thermal profiles of three habitats differing in canopy cover: open peat, mixed shrub, and closed forest. We installed biophysical models to record operative temperatures at a fine scale and assess habitat thermal quality. We also used coverboards to survey habitat usage. While the open canopy offered the highest thermal quality, we recorded the greatest number of snakes in the mixed shrub, which had a lower thermal quality. Since environmental temperatures regularly exceeded the upper thermal limit of T. s. sirtalis in the open canopy, snakes might favour the use of habitats that minimize the odds of overheating. Therefore, open habitats potentially restrict snakes’ activity window and may not be thermally attractive. Our data show that T. s. sirtalis use habitats that vary in thermal quality, but warmer habitats are not necessarily better. Rather, snakes preferentially seek areas that offer a mix of open and closed canopies to suit their thermoregulatory needs.

The ability to control hydration state is essential for terrestrial species, especially amphibians, which are highly susceptible to dehydration. Here, we examined how temperature (17°C versus 22°C) influenced behavioural hydroregulation in spotted salamanders (Ambystoma maculatum) using a laboratory humidity gradient. Salamanders defended a constant vapour pressure deficit (VPD) between temperatures by targeting higher relative humidity at 22°C than at 17°C, possibly to compensate for increased evaporative demand at warmer temperatures. Individuals selecting higher VPDs experienced greater evaporative water loss (EWL), with larger salamanders losing more water than smaller ones after accounting for temperature. Together, these results highlight a trade-off among body size, humidity preference and desiccation tolerance. Salamanders also rehydrated faster at 22°C than 17°C, highlighting temperature-dependent water uptake rates. Our finding that salamanders regulated a constant driving force of evaporation between temperatures suggests they are able to detect rates of EWL. Local evaporative cooling of the skin is a plausible mechanism: if moist-skinned ectotherms show local evaporative cooling on exposed surfaces but not on those in contact with the substrate, then the temperature gradient between dorsal and ventral skin could act as an effective cue to detect the drive for EWL. Ultimately, our study underscores the complexity of amphibian hydroregulation and emphasises the role of behaviour in maintaining hydration state.

Animals rely on physiological and behavioral processes to maintain thermal balance. Some animals, however, bear structures that help dissipate excess heat when body temperatures rise. Although widespread in animals, animal weapons—exaggerated morphological structures with multiple characteristics that can make them good at dissipating heat—have rarely been studied in the context of thermoregulation. Here, we investigated whether the horns of the Rhinoceros Beetle ( Megasoma gyas ) acted as a thermal window. We heated live and dead beetles to 30°C and allowed them to cool to 20°C while measuring surface temperature changes in four body regions: the cephalic and thoracic horns, the scutellum, and the abdomen. If horns actively dissipated heat, they would show the lowest cooling rate among body regions. Contrary to this expectation, we found that the cephalic horn had the highest cooling rate, followed by the abdomen, thoracic horn, and scutellum, respectively. This suggests that the horns are not used for active heat dissipation in M. gyas . The low cooling rate of the scutellum can be explained by the presence of large flight muscles in the thorax, which play a role in heat generation, but could also aid in heat dissipation by pumping hemolymph across tagmata or through the low-insulated cuticle to prevent thoracic overheating. We also demonstrate that beetles show regional heterothermy even in the absence of exercise or stress. As such, we propose that regional heterothermy may result from both active (control of hemolymph flow) and passive (heat dissipation through poorly insulated structures) processes within individuals. ### Competing Interest Statement The authors have declared no competing interest.

Assessments of the interplay between physiology and whole-organism performance are fundamental to understand how individuals function in different ecological contexts. Here, we investigated the relationship between locomotor performance, androgen levels, and metabolic capacity of muscle tissues in the lizard Tropidurus catalanensis. We hypothesized that faster individuals would exhibit higher circulating androgen concentrations and greater metabolic capacity in skeletal and cardiac muscles, regardless of body size. We measured morphological variables, maximum sprint speed (v), plasma testosterone concentration, and the maximum activity of the enzymes lactate dehydrogenase (LDH) and citrate synthase (CS) in the gastrocnemius, iliofibularis, and cardiac muscles of adult males. We found that intraspecific variations in v were not explained by body size, plasma testosterone concentration, nor by the activity of LDH or CS in skeletal muscles. The absence of an effect of testosterone on locomotion suggests that androgen concentrations may change in response to other factors, such as environmental stressors or reproductive state. Our results indicated that the fastest lizards also had the highest CS activity in the heart. This relationship suggests that cardiac oxidative capacity plays an important role in clearing metabolites in the postexercise recovery phase. We also found a positive relationship between CS and LDH in all tissues, suggesting a functional complementarity between glycolytic and aerobic pathways that should be relevant in situations that require rapid alternation between bursts of speed and endurance, such as predator evasion or thermoregulation. Ultimately, our results highlight the importance of integrating performance and physiological traits to understand interactions between animals and their environment.

Sexual selection is often invoked to explain the evolution of extravagant morphologies, such as antlers and horns. While the focus is typically on the process of exaggeration of these traits, the functional impact of exaggeration remains a topic of debate. One aspect that has been largely overlooked is how exaggerated structures might impact thermal biology. For example, as a hollow (i.e., non-vascularized or non-perfused) structure increases in size, its surface area and volume change, potentially impacting its ability to obtain and dissipate heat passively. However, if the exaggerated structure is vascularized, or in the case of arthropods, has hemolymph perfusion, then it may be actively used as a thermal radiator to avoid overheating in instances of thermal stress. Based on these and additional examples, we propose that morphological exaggeration may influence how arthropods manage heat exchange with the environment. Ultimately, individuals that bear exaggerated structures may develop ecological innovations that, due to selection or as a corollary effect, maximize effectiveness of thermoregulation. Our essay is divided into four sections. First, we delve on how exaggerated structures, particularly animal weapons, may impact how organisms exchange heat with the environment, and the implications for whole-organism thermoregulation. Second, we use beetles and fiddler crabs to provide experimental evidence of how structural exaggeration may influence thermal biology. Third, we examine macroecological data from arthropods to explore how the size of sexually selected morphologies varies with changes in environmental temperature. Finally, we synthesize these pieces of evidence to identify significant ecological implications and gaps in knowledge. Through this essay, we aim to ignite discussion on how morphological changes driven by sexual selection can lead to innovations not only in the functional role of morphologies but also in the thermal biology of individuals.

Mounting evidence suggests that temperature seasonality plays a pivotal role in shaping the thermal biology of ectotherms. However, we still have a limited understanding of how amphibians maintain thermal balance in the face of varying temperatures, especially in fossorial species. Due to thermal buffering underground, theory predicts relaxed selection pressure over thermoregulation in fossorial ectotherms. As a result, fossorial ectotherms typically show low thermoregulatory precision and low evidence of thermotactic behaviours when tested in laboratory thermal gradients. In this study, we evaluated how temperature selection ( T sel) and behavioural thermoregulation differed between seasons in the fossorial Spotted Salamander ( Ambystoma maculatum ). By comparing thermoregulatory parameters between the activity and overwintering seasons, we provide evidence that A . maculatum engages in active behavioural thermoregulation despite its fossorial habit. In both seasons, we found T sel to be consistently offset higher than prevailing thermal conditions. Thermoregulation differed between seasons, with salamanders having higher T sel and showing greater evidence of thermophilic behaviours in the active season compared to the overwintering season. Our study highlights that the combination of behavioural and thermal biology measurements is a necessary step to better understand the mechanisms that underlie body temperature control in amphibians. Ultimately, our study provides a broader understanding of thermoregulation in amphibians, particularly in the context of behavioural responses to seasonality in fossorial species.Summary statement By comparing thermoregulatory parameters between seasons, we demonstrate that the Spotted Salamander engages in active behavioural thermoregulation despite being fossorial.### Competing Interest StatementThe authors have declared no competing interest.

To decide whether to remain underground or to emerge from overwintering, fossorial ectotherms simultaneously process environmental, gravitational and circannual migratory cues. Here, we provide an experimental framework to study the behaviour of fossorial ectotherms during soil temperature inversion - a phenomenon that marks the transition between winter and spring - based on three non-mutually exclusive hypotheses (thermoregulation, negative geotaxis and migration restlessness). Using a vertical thermal gradient, we evaluated how temperature selection (Tsel), activity and vertical position selection differed under simulated soil temperature inversion (contrasting the active versus overwintering thermal gradients) in the spotted salamander (Ambystoma maculatum). Salamanders had different Tsel and activity levels between gradients, but selected similar heights regardless of thermal gradient orientation. Negative geotaxis may explain responses to changes in vertical thermal gradient orientation, with migratory restlessness contributing to differences in activity levels. Ultimately, our work should benefit those who aim to better understand the biology of fossorial ectotherms.

Temperature seasonality plays a pivotal role in shaping the thermal biology of ectotherms. However, we still have a limited understanding of how ectotherms maintain thermal balance in the face of varying temperatures, especially in fossorial species. Due to thermal buffering underground, thermal ecology theory predicts relaxed selection pressure over thermoregulation in fossorial ectotherms. As a result, fossorial ectotherms typically show low thermoregulatory precision and low evidence of thermotactic behaviours in laboratory thermal gradients. Here, we evaluated how temperature selection (Tsel) and associated behaviours differed between seasons in a fossorial amphibian, the spotted salamander (Ambystoma maculatum). By comparing thermoregulatory parameters between the active and overwintering seasons, we show that A. maculatum engages in active behavioural thermoregulation despite being fossorial. In both seasons, Tsel was consistently offset higher than acclimatization temperatures. Thermoregulation differed between seasons, with salamanders having higher Tsel and showing greater evidence of thermophilic behaviours in the active compared with the overwintering season. Additionally, our work lends support to experimental assumptions commonly made but seldom tested in thermal biology studies. Ultimately, our study demonstrates that the combination of careful behavioural and thermal biology measurements is a necessary step to better understand the mechanisms that underlie body temperature control in amphibians.

By living underground, fossorial animals may be challenged by limited gas exchange due to prolonged exposure to low oxygen levels (hypoxia) and carbon dioxide buildup (hypercarbia) in their burrows. The negative effects of hypoxia and hypercarbia generally relate to changes in breathing and energy metabolism. In mammals, reduced metabolic rates are a common adaptation to a fossorial lifestyle. It is unclear, however, whether fossoriality exerted a similar selection pressure over the metabolism of fossorial ectotherms. In this study, we tested whether fossorial amphibians showed reduced metabolic rates compared to non-fossorial and aquatic ones in a phylogenetic framework. We found that whole-organism resting metabolic rates varied according to body mass and temperature. However, our analyses did not support the hypothesis of lowered energy expenditure in fossorial species. We suggest that the intrinsically low energetic requirements of amphibians, coupled with their relatively small body sizes, and ability to breathe through both lungs and skin, potentially circumvented the energetic and respiratory challenges imposed by fossoriality. Our analyses further suggest that species from higher latitudes have higher metabolic rates than those from lower latitudes regardless of lifestyle. This finding supports a mechanism of negative compensation in metabolic responses, whereby species from cooler habitats would be able to sustain relatively high activity levels despite thermal constraints. This view of energetics in the context of fossoriality integrates central tenets of eco-physiological theory (metabolic scaling, metabolic variation along environmental gradients) and comparative physiology (control of bimodal breathing). Ultimately, our work contributes to a broader understanding of the metabolic correlates in vertebrate ectotherms.

Ectotherms that maintain thermal balance in the face of varying climates should be able to colonise a wide range of habitats. In lizards, thermoregulation usually appears as a variety of behaviours that buffer external influences over physiology. Basking species rely on solar radiation to raise body temperatures and usually show high thermoregulatory precision. By contrast, species that do not bask are often constrained by climatic conditions in their habitats, thus having lower thermoregulatory precision. While much focus has been given to the effects of mean habitat temperatures, relatively less is known about how seasonality affects the thermal biology of lizards on a macroecological scale. Considering the current climate crisis, assessing how lizards cope with temporal variations in environmental temperature is essential to understand better how these organisms will fare under climate change. Activity body temperatures (Tb ) represent the internal temperature of an animal measured in nature during its active period (i.e. realised thermal niche), and preferred body temperatures (Tpref ) are those selected by an animal in a laboratory thermal gradient that lacks thermoregulatory costs (i.e. fundamental thermal niche). Both traits form the bulk of thermal ecology research and are often studied in the context of seasonality. In this study, we used a meta-analysis to test how environmental temperature seasonality influences the seasonal variation in the Tb and Tpref of lizards that differ in thermoregulatory strategy (basking versus non-basking). Based on 333 effect sizes from 137 species, we found that Tb varied over a greater magnitude than Tpref across seasons. Variations in Tb were not influenced by environmental temperature seasonality; however, body size and thermoregulatory strategy mediated Tb responses. Specifically, larger species were subjected to greater seasonal variations in Tb , and basking species endured greater seasonal variations in Tb compared to non-basking species. On the other hand, the seasonal variation in Tpref increased with environmental temperature seasonality regardless of body size. Thermoregulatory strategy also influenced Tpref , suggesting that behaviour has an important role in mediating Tpref responses to seasonal variations in the thermal landscape. After controlling for phylogenetic effects, we showed that Tb and Tpref varied significantly across lizard families. Taken together, our results support the notion that the relationship between thermal biology responses and climatic parameters can be taxon and trait dependent. Our results also showcase the importance of considering ecological and behavioural aspects in macroecological studies. We further highlight current systematic, geographical, and knowledge gaps in thermal ecology research. Our work should benefit those who aim to understand more fully how seasonality shapes thermal biology in lizards, ultimately contributing to the goal of elucidating the evolution of temperature-sensitive traits in ectotherms.

Thermal biology research compares field with laboratory data to elucidate the evolution of temperature-sensitive traits in ectotherms. The hidden challenge of many of these studies is discerning whether animals actively thermoregulate, since motivation is not typically assessed. By studying the behaviours involved in thermoregulation, we can better understand the mechanisms underlying body temperature control. Using an integrative approach, we assessed the thermoregulatory and thermotactic behaviours of two sympatric snake species with contrasting life histories: the generalist Eastern Garter Snake (Thamnophis sirtalis sirtalis ( Linnaeus, 1758 )) and the semi-fossorial Northern Red-bellied Snake (Storeria occipitomaculata occipitomaculata ( Storer, 1839 )). We expected that thermoregulatory behaviours would be optimized based on life history, in that T. s. sirtalis would show higher evidence for thermally oriented behaviours than S. o. occipitomaculata due to its active nature. Thamnophis sirtalis sirtalis actively thermoregulated, had higher thermal preferences (29.4 ± 2.5 vs. 25.3 ± 3.6 °C), and was more active than S. o. occipitomaculata, which showed relatively low evidence for thermotaxis. Our results build on the notion that evaluating movement patterns and rostral orientation towards a heat source can help ascertain whether animals make thermally motivated choices. Our data provide insight into the thermoregulatory strategies used by snakes with different life histories and maximize the information provided by behavioural thermoregulation experiments.

The Kalotermitidae Cryptotermes brevis (Walker) presents colonies that lack a true worker caste. They have totipotent worker-like individuals named pseudergates. Few studies have characterized the morphology of immature instars, including pseudergates. In order to identify these instars and characterize the pseudergates, we conducted a comparison between morphometric and morphological variations among immature individuals of C. brevis colonies. Juvenile hormone analog (JHA) was used in the first instar nymphs to induce regressive molts and compare morphological differences between nymphs and pseudergates. Results showed the existence of three larval instars and four nymphal instars. These immatures were morphologically characterized. Individuals classified as third instar larvae presented white body, 10 to 12 antennal articles, absent or small non-pigmented compound eyes, and absence of wing buds. Pseudergates presented pigmented abdomen and sclerotized cuticle, 10 to 12 antennal articles, and absent or small compound eyes, and few specimens had large pigmented compound eyes and absence of wing buds. First instar nymphs had pigmented abdomen and sclerotized cuticle, 10 to 12 antennal articles, both large non-pigmented and pigmented compound eyes, the presence of wing buds. Bioassays using JHA on first instar nymphs resulted in a large percentage of nymph-soldier intercastes. We concluded that abdomen pigmentation and sclerotized cuticle are good characters to differentiate pseudergates from larvae and the absence of wing buds is a good character to differentiate pseudergates from nymphs. Our findings not only contribute to the basic biological and morphological information of this species but also help to identify correctly pseudergates in further studies that involve applied bioassays.