{"paper_id":"4cc391af-a17f-40ed-b877-44e5d7682b70","body_text":"Seasonal plasticity in the thermal sensitivity of metabolism but not water loss in a fossorial ectotherm | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Seasonal plasticity in the thermal sensitivity of metabolism but not water loss in a fossorial ectotherm Danilo Giacometti, Glenn J. Tattersall This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5478984/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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 acclimatisation (spring vs. autumn) affected the thermal sensitivity of standard metabolic rates (SMR), rates of evaporative water loss (EWL), and skin resistance to water loss ( R s ) in the spotted salamander ( Ambystoma maculatum ). We hypothesised that temperature would have both short- and long-term effects over traits (i.e., acute exposure to test temperatures and seasonal acclimatisation, respectively). After accounting for body mass and sex, we found that short-term changes in temperature led to an increase in SMR, EWL, and R s . Additionally, SMR and R s differed between seasons, but EWL did not. Sustaining low SMR and high R s in the spring may allow salamanders to allocate energy toward overwintering emergence and breeding while simultaneously maximising water conservation. By contrast, maintaining high SMR and low R s in the autumn may allow salamanders to forage aboveground on rainy nights to replenish energy reserves in preparation for the winter. 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 hydroregulatory requirements. Animal Physiology Acclimatisation amphibian energy budgets skin resistance water budgets Figures Figure 1 Figure 2 Introduction Shifts in temperature affect animals at all levels of organisation, from cellular processes to whole-organism performance (Tattersall et al. 2012 ). This is particularly true in ectotherms, which have limited capacity for metabolic heat production and depend on environmental temperatures to regulate physiological function (Bicego et al. 2007 ). Ectotherms have evolved different mechanisms to maintain performance in the face of varying temperatures. Behavioural responses can buffer the impact of thermal variability over physiology in the short term (e.g., hours), as demonstrated in toads that burrow in response to warming temperatures to minimise evaporative water loss (Hoffman and Katz 1989 ). By contrast, reversible physiological plasticity (e.g., acclimatisation) can aid in dealing with long-term shifts in temperature (Little and Seebacher 2016 ), especially in species with limited capacity for behavioural thermoregulation (Lowe et al. 2010 ). For example, several amphibians regulate energy expenditure to compensate for seasonal changes in temperature (Fitzpatrick 1973 ; Feder 1978 ; Kiss et al. 2009 ). While evidence suggests that ectotherms from highly variable climates should have greater plasticity potential than those from stable climates, there is still a dearth of knowledge about the mechanisms that underpin seasonal physiological adjustments, especially from an energetic perspective (Gatten Jr et al. 1992 ; Navas 1996 ; Sun et al. 2022 ). The maintenance of energy metabolism is one of the greatest challenges faced by ectotherms in thermally variable habitats, since changes in temperature can impact whether ATP production meets demand (Seebacher et al. 2010 ). In ectotherms, standard metabolic rates (SMR) represent the baseline costs for body maintenance under varying conditions (Gatten Jr et al. 1992 ). Typically, SMR rises with temperature as a consequence of thermodynamic effects on the biochemical reactions involved in energy turnover (Andrade 2016 ). The thermal sensitivity of SMR can be assessed through the temperature coefficient (Q 10 ), which describes changes in rate processes measured at contrasting temperatures across short time scales (Gatten Jr et al. 1992 ). Although the thermal sensitivity of SMR is ubiquitous in ectotherms (Carter et al. 2023 ), Q 10 values may vary intra- and interspecifically or according to the degree of thermal variability experienced (Gatten Jr et al. 1992 ; Kreiman et al. 2019 ). Indeed, the Q 10 of SMR has been recurrently used to explain differences in the ability to physiologically compensate for both short- and long-term shifts in temperature (reviewed in Havird et al. 2020 ). In the context of seasonality, assessing changes in the thermal sensitivity of SMR can clarify how ectotherms allocate energy toward different activities between seasons (e.g., reproduction or growth). For example, different species of salamanders (Amphibia: Caudata) exhibit metabolic compensation (Prosser 1969 ) in SMR between the activity and dormancy season, highlighting that compensatory energetic responses are key to survival (Fitzpatrick 1973 ; Fitzpatrick and Brown 1975 ; Feder 1978 ). Temperature can also have profound effects over evaporative water loss (EWL), particularly in amphibians (Lertzman-Lepofsky et al. 2020 ). Indeed, amphibians have a relatively thin skin that is supported by a unique vasculature that facilitates oxygen uptake and carbon dioxide excretion (Burggren and Moalli 1984 ). However, the ability to exchange gases through the skin comes at the expense of high EWL, which increases as a function of temperature (Spotila 1972 ). Thermal effects over EWL are mediated by the vapour pressure deficit (VPD), which measures the drying power of air, and is defined as the difference between the saturated water vapour pressure (WVP sat ) and the actual water vapour pressure (WVP act ) (Anderson 1936 ; Haynes 2016 ), since the skin interface where evaporation occurs will be a saturating conditions. Given that WVP sat increases with temperature, there should be greater evaporative demand at warmer than cooler temperatures (Riddell et al. 2019 ). Thus, amphibians exposed to a combination of warm temperatures and high VPD should pay higher hydroregulatory costs (Riddell et al. 2024 ). As a primary defence mechanism, some amphibians may enhance skin resistance to water loss ( R s ) in response to concomitant increases in temperature and VPD (Tracy et al. 2008 ; e.g., Riddell and Sears 2015 ; Senzano and Andrade 2018 ). However, the extent to which seasonality elicits plastic responses in the maintenance of water balance in amphibians is still unclear. For instance, research in salamanders indicated that both EWL and R s may respond to thermal acclimation (Riddell and Sears 2015 ; Riddell et al. 2018 ), whereas work in anurans suggested that neither trait varies between seasons (Davis et al. 2018 ; Maenaka et al. 2023 ). Ultimately, assessments of the effect of seasonality over EWL and R s are fundamental to improve our understanding of how amphibians cope with thermal and hydroregulatory challenges (Maenaka et al. 2023 ). Here, we evaluated how seasonal acclimatisation impacted the thermal sensitivity of energy and water balance in the spotted salamander ( Ambystoma maculatum ). Despite being fossorial and assumed to live in thermally stable habitats, seasonality influences many attributes in A . maculatum , including thermoregulation (Giacometti and Tattersall 2024 ), reproduction (Sexton et al. 1990 ), body condition (Moldowan et al. 2022 ), and activity patterns (Vasconcelos and Calhoun 2004 ). Aboveground activity in this species can be observed primarily in the spring and autumn (Vasconcelos and Calhoun 2004 ; Moldowan et al. 2022 ). In the spring, salamanders emerge from overwintering burrows and migrate toward bodies of water to breed at relatively low environmental temperatures (Moldowan et al. 2022 ). Individuals do not feed during breeding migration and the aquatic breeding period (Smallwood 1928 ) even after spending up to seven months underground (Moldowan et al. 2022 ). By contrast, aboveground activity in the autumn occurs at relatively higher environmental temperatures, and is likely associated with individuals foraging to replenish energy stores before the winter (Faccio 2003 ; Moldowan et al. 2022 ). Thus, while spring and autumn both fall within the activity period of A . maculatum , salamanders should have different energetic and hydroregulatory requirements between seasons. Although fossoriality did not impinge on amphibian metabolism (Giacometti and Tattersall 2023 ), evidence suggests that fossorial ectotherms have a blunted thermal sensitivity of SMR (Bars-Closel et al. 2018 ). Furthermore, while some anurans may minimise EWL by forming cocoons (Shoemaker 1988 ), modulation of R s is the primary physiological response to EWL in amphibians (Wygoda 1988 ; Prates and Navas 2009 ; Riddell and Sears 2015 ), at least in the short term. Whether these patterns are applicable in a seasonal context remains unexplored. To test the effect of seasonality over energy and water balance, we acclimatised A . maculatum to conditions that matched spring and autumn in nature (Moldowan et al. 2022 ). We then measured SMR, EWL, and R s at 2, 5, 10, 15, and 20°C, since these temperatures are biologically relevant across seasons to our study system (Giacometti and Tattersall 2024 ). Given that animals respond to simultaneous shifts in temperature and humidity, and warmer temperatures typically represent drier conditions in nature (Gates 1980 ; Riddell et al. 2019 ; Weaver et al. 2023 ), we designed our experiment such that an increase in temperature was followed by an increase in VPD. We hypothesised that temperature would have both short- and long-term effects over traits (i.e., acute exposure to test temperatures and seasonal acclimatisation, respectively) (Havird et al. 2020 ). For short-term effects, we predicted that SMR, EWL, and R s would increase with test temperature (Whitford and Hutchison 1963 ; Riddell and Sears 2015 ; Senzano and Andrade 2018 ). For long-term effects, we predicted that autumn-acclimatised salamanders would have higher SMR, EWL, and R s than spring-acclimatised salamanders across all test temperatures, following a pattern of inverse compensation (Packard 1972 ; Havird et al. 2020 ). We further predicted that the thermal sensitivity of SMR would differ between seasons, with overall lower Q 10 values in the spring than in the autumn, and lower sensitivity at cooler than warmer test temperatures (Fitzpatrick and Brown 1975 ). Lastly, we used our empirical EWL measurements to determine how long it would take for A . maculatum to reach their desiccation limit at each test temperature (Hall 1922 ). By examining the combined effects of temperature and humidity over physiology, we aim to elucidate the mechanisms behind the maintenance of energy and water balance between seasons (Chang and Hou 2005 ; Maenaka et al. 2023 ). Material and methods Salamander collection and husbandry On 15 May 2022, we collected 50 post-breeding, adult A . maculatum (26 females + 24 males) using a drift fence installed around Bat Lake, Algonquin Provincial Park, Ontario, Canada (45.5857°N, 78.5185°W). When we collected a salamander, we recorded its snout-vent length (SVL) with a tape measure, its body mass (M b ) with a Pesola™ spring scale, and its sex based on cloacal morphology (Petranka 1998 ). We assigned each individual a unique identification (ID) that was associated with a photograph of the dorsum of the salamander to create an individual-level recognition system based on spot patterns. To transfer the salamanders from Bat Lake to Brock University, we allocated them into plastic containers with ventilated fitted lids (34 cm x 19.6 cm x 12 cm). We kept 10 salamanders per container, totalling five containers used in transportation; each container had Sphagnum moss, pine needles and water. We placed the containers inside a transport box kept at ~ 4°C to avoid overheating and dehydration during transportation. In the lab, we housed salamanders in pairs within ventilated tanks that had coconut husk fibre, Sphagnum moss, and PVC pipe refuges; tanks were misted daily to maintain high humidity locally. We kept these tanks in a facility with controlled temperature, relative humidity, and photoperiod. We adjusted temperature and photoperiod seasonally to mimic their collection site soil conditions (Moldowan et al. 2022 ), while keeping relative humidity constantly at 70%. In the spring, we housed salamanders at 7°C and a 10h:14h light:dark cycle. During the summer, we kept them at 14°C and a 14h:12h light:dark cycle. In the autumn, we kept salamanders at 12°C, and a 12h:12h light:dark cycle. To transition between housing conditions, we increased (spring to summer) or decreased (summer to autumn) temperature by 1°C/day. We allowed the salamanders at least four weeks under the prevailing acclimatisation condition before starting our experiments. During their period in the lab, we fed salamanders twice a week with mealworms dusted in calcium and multivitamin powder, while water was available ad libitum . We weighed salamanders to the nearest 0.01 g every week with an analytical scale (Mettler Toledo, model PB602-S) to monitor changes in M b as an indicator of animal well-being; all animals maintained M b during the course of our experiments. Respirometry system We measured SMR and EWL with a flow-through open respirometry system. We used tubing attached to an electronic air pump to draw outside air into the lab, which was passed through a column of CO 2 absorbent (Amsorb®, Amsorb Plus), humidified, and directed to a dewpoint generator (Sable Systems International, model DG-4) set constantly to 1.5°C. The air excurrent from the dewpoint generator had a water vapour pressure of 0.681 kPa and a water vapour density (WVD) of 5.4 µg/mL, and was split into two channels with each running through a mass flow controller (Sable Systems International, model MFC-2) at a flow rate of 200 mL/min. Channel one was directed into a 500 mL glass respirometer containing a salamander and kept inside a temperature-controlled incubator (Thermo Fisher Scientific Inc., model 146E). We sealed the respirometer with a rubber stopper that had two syringe barrels used as ports for incurrent and excurrent air, respectively. Excurrent air left the respirometer toward an open manifold connected to a subsample line controlled by a solenoid switch. The subsampler directed air at a flow rate of 100 mL/min to a water vapour analyser (Sable Systems International, RH-300), and then to a respirometry system (Sable Systems International, FoxBox) that measured O 2 and CO 2 air content. We programmed the solenoid switch to be triggered for 5 min every 60 min, thereby diverting air from channel one to channel two, and providing baseline measurements of incurrent air. We discarded the initial 90 s after the switch was triggered to account for dead-space gas transit time (Sakich and Tattersall 2021 ). We connected our respirometry system to a computer with a data acquisition software (BIOPAC Systems Inc, Acqknowledge Data Acquisition and Analysis Software) that provided real-time, continuous measurements of O 2 (%), CO 2 (%), WVD (µg/mL), chamber temperature (°C), flow rate (mL/min), and baseline voltage (V). Thermal sensitivity of metabolism and water loss To assess seasonal changes in the thermal sensitivity of SMR and EWL, we conducted measurements at 2°C, 5°C, 10°C, 15°C, and 20°C in the spring and autumn (N = 10 individuals/temperature). In both seasons, we randomised the order of experiments and tested the same individuals at the same temperature. We allowed each individual a total of 4 h inside the respirometer and tested two individuals per day between 08h00–18h00, which represents the resting period for the study species. Prior to measurements, individuals were fasted for a week to ensure a postabsorptive state (Secor and Boehm 2006 ). We weighed each individual before and after an experiment to determine the percentage of M b loss during the experiment. We ran our experiments in total darkness, and continuously monitored the salamanders through a high-speed infrared webcam (AOS Technologies AG, model Promon U1000) that was mounted inside the incubator along with an infrared illuminator (wavelength = 850 nm; TVPSii, model TP-IRBP15). Because we kept the dew point constantly at 1.5°C, our experimental design allowed us to assess the combined effect of different temperatures and VPD over metabolism and water loss (Table 1 ). We determined the WVP sat , WVP act , and VPD at a given temperature (T) and percent relative humidity (RH) for our experimental conditions following (Haynes 2016 ): Table 1 Test temperature, relative humidity, saturated water vapour pressure, and vapour pressure deficit for each experimental condition. We achieved a combination of varying temperatures and humidity conditions by maintaining the dew point constantly at 1.5°C in our experiments. Temperature (°C) Relative humidity (%) Saturated water vapour pressure (kPa) Vapour pressure deficit (kPa) 2 96.48 0.706 0.025 5 78.04 0.873 0.192 10 55.44 1.228 0.547 15 39.92 1.706 1.025 20 29.12 2.339 1.658 $$\\:{WVP}_{sat}=6.11\\times\\:{10}^{\\left(\\frac{7.5\\times\\:T}{237.3+T}\\right)}$$ 1 , $$\\:{WVP}_{act}={WVP}_{sat}\\times\\:\\left(\\frac{RH}{100}\\right)$$ 2 , $$\\:VPD={WVP}_{sat}-{WVP}_{act}$$ 3 , We considered rates of carbon dioxide production (V̇CO 2 ; mL/h) as a proxy for SMR, and rates of water vapour production (V̇H 2 O; mg/h) as a proxy for EWL. We visually inspected the resulting graphs of O 2 , CO 2 , and WVD variation over time, and considered periods of 50.14 ± 5.28 min (mean ± standard deviation) of constancy in our calculations using equations described in (Lighton 2008 ): $$\\:{\\dot{V}{CO}_{2}=FR}_{i}\\left\\{\\left[\\frac{\\left\\{{[F}_{e}{CO}_{2}(1-{{F}_{i}O}_{2}-\\right.{{F}_{i}CO}_{2}-{{F}_{i}H}_{2}O)}{({1-F}_{e}{O}_{2}-{F}_{e}{CO}_{2}-{F}_{e}{H}_{2}O)}\\right]-{F}_{i}{CO}_{2}\\right\\}$$ 4 , $$\\:\\dot{V}{H}_{2}O={FR}_{i}\\{{WVD}_{e}-{WVD}_{i}\\}$$ 5 , where FR i is the incurrent flow rate, F e CO 2 is the fractional CO 2 concentration of the excurrent air, F i O 2 is the fractional O 2 concentration of the incurrent air, F i H 2 O is the fractional water vapour concentration of the incurrent air, F e O 2 is the fractional O 2 concentration of the excurrent air, F e H 2 O is the water vapour concentration of the excurrent air, F i CO 2 is the fractional CO 2 concentration of the incurrent air, WVD e is the water vapour density of the excurrent air, and WVD i is the water vapour density of the incurrent air. Due to low differences between F i O 2 and F e O 2 , we could not estimate V̇O 2 with confidence, especially at low temperatures, and therefore only report on V̇CO 2 . We calculated the Q 10 of mass-specific V̇CO 2 (mL/g/h) between 5–15°C and 10–20°C with the van’t Hoff equation: $$\\:{Q}_{10}={\\left({R}_{2}/{R}_{1}\\right)}^{\\frac{10}{{T}_{2}-{T}_{1}}}$$ 6 , where R 1 and R 2 are physiological rates measured at temperatures T 1 and T 2 , respectively. Days until desiccation Ambystoma maculatum can withstand a loss of 47% loss of M b via evaporation before losing function (Hall 1922 ). Based on our individual-specific data on M b and EWL, we calculated how long it would take for our individuals to reach their limit of desiccation across test temperatures. We determined time until desiccation (hours; H d ) using the equation: $$\\:{H}_{d}=\\frac{\\left(0.47\\right)\\times\\:{M}_{b}}{EWL}$$ 7 , Since EWL was expressed in g/h, we then divided H d by 24 to obtain an estimate of days until desiccation ( D d ): $$\\:{D}_{d=\\:\\raisebox{1ex}{${H}_{d}$}\\!\\left/\\:\\!\\raisebox{-1ex}{$24$}\\right.}$$ 8 , Resistance to water loss Total resistance ( R t ) to water loss is the combined outcome of skin ( R s ) and boundary layer resistances ( R b ) (Feder and Pinder 1988 ). Thus, to quantify the relative role of the skin in regulating water loss, one must determine R b . To this end, we used the framework proposed by (Spotila and Berman 1976 ), which consists of building agar replicas of one’s study system to empirically determine R b (i.e., from a free evaporating surface). We obtained custom built 3D-printed positive models of A . maculatum from Brock University’s Makerspace. These models matched the average SVL of males and females used in our study. We used a silicone-free mold maker (SilliNOT!®) to pour negative molds of the 3D-printed models, and then we used the molds to cast 5% agar replicas (Senzano et al. 2022 ) (Figure S1). Once the agar replicas were hardened, we measured their V̇H 2 O with the same method used with live salamanders. Based on our experimental conditions, salamanders experienced cool and humid conditions at 2°C that resulted in a low evaporative demand (Table 1 ). As such, we only calculated R t and R s at 5, 10, 15, and 20°C (N = 10 agar models per test temperature). To calculate R t (s/cm), we first estimated surface area ( S ; cm 2 ) based on (Whitford and Hutchison 1967 ): $$\\:S={{M}_{b}}^{\\left(0.694\\right)}$$ 9 , Then, we applied the equation postulated by (Spotila and Berman 1976 ): $$\\:{R}_{t}=VDD/{\\dot{VH}}_{2}{O}_{area}$$ 10 , where VDD is the vapour density deficit (i.e., the difference between WVD at the evaporating surface and the WVD of air temperature), and V̇H 2 O area is the area-specific V̇H 2 O (mg/cm 2 /h) of a salamander or an agar model. Given that agar replicas lack R s , their R t is made up solely by R b . Consequently, the R s of each individual salamander can be determined by: $$\\:{R}_{s}={R}_{t}-{R}_{b}$$ 11 , Statistical analyses We performed all analyses using R (version 4.4.1) in RStudio (version 2024.06.14) (R Core Team 2024 ) with a significance level of 0.05. To assess normality and homoscedasticity, we visually inspected Q-Q and P-P plots through the “fitdist” function from the fitdistrplus package (Delignette-Muller and Dutang 2015 ). To test our hypotheses, we fit linear mixed-effects models (LMMs) with the “lmer” function from the lme4 package (Bates et al. 2015 ). We considered log-transformed V̇CO 2 , log-transformed V̇H 2 O, D d , and R s as response variables in our models. We fit one model per response variable, while considering test temperature (continuous), season (categorical), sex (categorical), and log-transformed M b (continuous) as fixed terms in all models. We controlled for M b in our analyses to account for body size effects over physiological traits (Glazier 2009 ) except for the test with D d , since information on M b is needed to define D d . We included salamander ID as a random term in all models to account for multiple observations per individual. We evaluated model fit with the “check_model” function from the performance package (Lüdecke et al. 2021 ), and visualised fixed model effects through the “allEffects” function from the effects package (Fox et al. 2016 ). For each model, we computed marginal fixed effects with the “ggeffect” function from the ggeffects package (Lüdecke 2018 ). Finally, we created figures using the ggplot2 (Wickham 2016 ), ggbeeswarm (Clarke et al. 2023 ), and ggpubr (Kassambara and Kassambara 2020 ) packages. Results We found that V̇CO 2 increased with test temperature and was overall higher in the autumn than in the spring (Fig. 1 a) (Table 2 ). In both seasons, larger individuals had higher V̇CO 2 , although this effect was not mediated by sex (Table S1). The thermal sensitivity of mass-specific V̇CO 2 differed between seasons for the two temperature ranges considered (Spring: Q 10(5–15°C) = 1.08, Q 10(10–20°C) = 2.34; Autumn: Q 10(5–15°C) = 1.54, Q 10(10–20°C) = 1.33). Table 2 Summary of body size and physiological variables of Ambystoma maculatum studied in the spring and autumn. Values are presented as mean ± standard deviation. N = 10 individuals per test temperature in both seasons. 2°C 5°C 10°C 15°C 20°C Trait Autumn Spring Autumn Spring Autumn Spring Autumn Spring Autumn Spring M b (g) 12.60 ± 5.07 12.30 ± 3.43 12.70 ± 3.44 12.20 ± 3.40 15.10 ± 5.30 14.20 ± 4.71 11.10 ± 2.58 11.20 ± 2.38 11.90 ± 3.12 11.20 ± 3.32 M b loss (%) 0.76 ± 0.44 0.51 ± 0.24 2.11 ± 2.16 1.04 ± 1.11 1.79 ± 1.56 0.95 ± 0.35 2.19 ± 0.50 1.62 ± 0.26 2.76 ± 0.57 2.74 ± 0.79 S (cm 2 ) 96.20 ± 37.50 94.30 ± 25.40 94.30 ± 25.50 93.60 ± 25.30 115.09 ± 39.00 108.00 ± 34.80 85.60 ± 19.20 86.70 ± 17.70 91.50 ± 23.10 86.30 ± 24.70 V̇CO 2 (mL/h) 0.44 ± 0.25 0.34 ± 0.14 0.45 ± 0.21 0.37 ± 0.19 0.92 ± 0.53 0.33 ± 0.11 0.65 ± 0.41 0.41 ± 0.10 0.99 ± 0.52 0.62 ± 0.16 V̇H 2 O (mg/h) 1.05 ± 0.30 0.96 ± 1.02 6.78 ± 0.37 6.62 ± 1.54 18.70 ± 0.40 17.90 ± 2.30 32.30 ± 2.09 30.50 ± 3.46 49.20 ± 5.22 51.30 ± 4.86 R t (s/cm) — — 5.78 ± 1.77 6.26 ± 3.32 5.31 ± 1.54 8.00 ± 2.64 6.16 ± 2.13 9.49 ± 2.21 8.05 ± 4.19 7.50 ± 1.86 R s (s/cm) — — 3.35 ± 1.58 3.84 ± 2.97 2.73 ± 1.21 5.42 ± 2.57 4.24 ± 2.14 7.57 ± 2.15 6.91 ± 3.90 6.36 ± 2.23 M b = body mass, S = surface area, V̇CO 2 = rates of carbon dioxide production, V̇H 2 O = rates of cutaneous water loss, R t = total resistance to water loss, R s = skin resistance to water loss. Test temperature was the only factor affecting V̇H 2 O, with salamanders losing more water through evaporation with increasing test temperature (Fig. 1 b) (Table 2 ). Neither season, sex, nor M b explained the variation in V̇H 2 O (Table S2). Time until desiccation decreased as a function of temperature regardless of season and sex (Table S3) (mean ± standard deviation; 2°C = 266.73 ± 117.51 days, 5°C = 11.47 ± 14.66 days, 10°C = 8.76 ± 5.91 days, 15°C = 4.31 ± 1.05 days, 20°C = 2.96 ± 0.68 days). Although M b loss increased with test temperature, salamanders were never close to reaching their desiccation limit in our experiments (Table 2 ). Both test temperature and season affected R s , but with no contribution of sex or M b (Table S4). Specifically, R s increased in response to temperature and was overall higher in the spring than in the autumn (Fig. 2 ). Discussion In this study, we addressed the impact of seasonal acclimatisation on the thermal sensitivity of energy and water budgets in a fossorial salamander. After accounting for body size and sex, we found partial support for the hypothesis that temperature would have both short- and long-term effects over metabolism and water loss. Both SMR and R s increased with test temperature and differed between the spring and autumn, while EWL increased with test temperature but did not differ between seasons. We also demonstrated that the length of time necessary to reach the desiccation limit decreased as a function of temperature in A . maculatum . Together, our results demonstrate that thermal effects over physiology may occur through active plasticity and in response to acute changes in temperature. Such effects, however, may be trait-dependent. Furthermore, our study highlights that fossorial species may have different energetic and hydroregulatory requirements between seasons despite the prevailing assumption that fossorial ectotherms are buffered from thermal effects. Thermal and seasonal effects over metabolism In ectotherms, thermally-induced shifts in metabolism are the outcome of passive thermodynamic effects on the biochemical reactions that underpin energy turnover (Andrade 2016 ). The relationship between SMR and temperature has been assessed in several species of ectotherms (see Carter et al. 2023 ). As expected, we found a positive relationship between SMR and temperature (Gatten Jr et al. 1992 ), supporting the idea that an increase in temperature leads to an increase in the baseline costs for body maintenance. In our study, M b explained most of the variation in SMR along with temperature. Larger individuals had higher SMR, in concert with Kleiber’s law and the theory of metabolic scaling (Schmidt-Nielsen 1970 ; White et al. 2019 ). Our SMR values are similar to those previously reported for A . maculatum and other caudates (Whitford and Hutchison 1963 ; Gatten Jr et al. 1992 ), although we did not find differences in energy expenditure between sexes (Finkler et al. 2003 ). We found that SMR differed between seasons, with lower energy expenditure in the spring than in the autumn across all test temperatures. SMR followed a pattern of inverse compensation (Hazel and Prosser 1970 ), which is often attributed to the combined effect of passive and active plasticity over metabolism. In this scenario, SMR would increase with temperature through passive effects and this response would be amplified by active plasticity (Havird et al. 2020 ). Lowered SMR implies reduced energetic demand and expenditure (Giacometti et al. 2022 ), and sustaining low costs for body maintenance in the spring can be particularly beneficial for A . maculatum . Indeed, A . maculatum breed in the spring after overwintering underground for months (Madison 1997 ). Since A . maculatum do not feed while breeding (Smallwood 1928 ), salamanders are presumed to rely on energy stores obtained in the previous activity season to sustain performance. Thus, maintaining relatively low SMR in the spring could allow salamanders to allocate energy toward overwintering emergence, breeding migration, and reproduction without depleting energy reserves. This proposition is bolstered by our finding that spring-acclimatised salamanders had a lower Q 10 for SMR between 5°C and 15°C than between 10°C and 20°C. The former range of temperatures is more common at the onset of the breeding season (Moldowan et al. 2022 ), indicating that compensatory responses may have important fitness consequences (Packard 1972 ; Havird et al. 2020 ). Higher SMR in the autumn can be explained by overall warmer conditions and the fact that individuals forage to replenish energy stores in preparation for the winter (Faccio 2003 ; Moldowan et al. 2022 ). Thus, our data suggest that functional differences between seasons (i.e., breeding in the spring, provisioning in the autumn) are mediated by seasonal changes in energetic requirements. Importantly, we observed overall low Q 10 values for A . maculatum in both spring and autumn. In amphibians, low metabolic sensitivity is typically ascribed to tropical instead of temperate-zone species (Kreiman et al. 2019 ). However, blunted thermal sensitivity has been suggested as one of the physiological imprints of fossoriality in ectotherms (Bars-Closel et al. 2018 ) due to the thermal stability of underground environments. From an integrative perspective, the low thermal sensitivity of metabolism in A . maculatum corroborates the thermal generalist nature suggested for this species. Truly, A . maculatum are active across a broad thermal range (including sub-zero temperatures) (Giacometti and Tattersall 2024 ), which is possibly facilitated by their low energetic requirements and low thermal sensitivity across temperatures. While low metabolic sensitivity to temperature has been reported in other taxa (Fitzpatrick 1973 ; Gatten Jr et al. 1992 ; Bars-Closel et al. 2018 ), we still have a limited understanding of the suite of physiological and behavioural traits that allow fossorial ectotherms to sustain performance across a broad thermal range. Thermal effects over water loss and the role of the skin in limiting evaporation between seasons Our data supported the well-known pattern of EWL increasing with temperature in amphibians (Boutilier et al. 1992 ; Shoemaker et al. 1992 ). This effect is mediated by an increase in WVP sat at the skin interface with the air, which leads to higher VPD and greater evaporative demand at warmer than cooler temperatures (Riddell et al. 2019 ). Although body size and shape may contribute to EWL, stout-bodied salamanders like A . maculatum typically have lower EWL than slender and cylindrical ones (Spight 1968 ), which could explain why M b did not affect EWL in our study. Additionally, we used our EWL measurements to determine how long it would take for A . maculatum to reach their desiccation limit. Increasing temperatures resulted in a shorter time until desiccation in both spring and autumn, with salamanders expected to desiccate in less than three days at 20°C and a VPD of 1.658 kPa. By contrast, A . maculatum can persist within their desiccation tolerance for over 260 days at 2°C and a VPD of 0.025 kPa; these conditions are inferred to be found in overwintering burrows (Moldowan et al. 2022 ). Although A . maculatum may lose up to 14% of M b through evaporation when active under humid conditions (Giacometti and Tattersall 2024 ), terrestrial amphibians are generally more tolerant of dehydration than aquatic ones (Shoemaker et al., 1992 ; Thorson, 1955 ; Young et al., 2005 ). In turn, this could explain why neither time to desiccation or EWL differed between the spring and autumn in our study. Constancy of EWL between season suggests that salamanders should rely on modulation of R s and behaviour to mitigate possible detrimental effects of warming over the maintenance of water balance (Davies et al. 2015 ). Our analyses showed that A . maculatum increased R s in response to temperature, with overall higher R s in the spring than in the autumn. The mechanisms that underlie R s enhancement at warmer temperatures are still unclear, but could involve suppression of blood flow to capillaries underneath the epidermis or the thickening of chromatophore layers in the skin (Toledo and Jared 1993 ; Lillywhite 2006 ). Although salamanders do not secrete waterproofing mucus or build cocoons to limit EWL like some anurans (Withers 1998 ; Kröner et al. 2024 ), we provide evidence that A . maculatum may actively modulate R s in response to short- and long-term changes in temperature. The finding that salamanders maintained overall higher R s in the spring than in the autumn can be attributed to exposure to cooler acclimatisation conditions in the spring. Alternatively, the maintenance of low SMR in spring could lead to a coordinated lowering of blood flow to the skin which may contribute to R s enhancement. Salamanders never assumed a water-conserving posture (Semlitsch 1983 ) during our trials, which lends further support to the proposition that Ambystoma species have a high tolerance to dehydration induced by EWL (Hall 1922 ; Thorson 1955 ). Nonetheless, further work is still necessary to clarify the behavioural mechanisms through which salamanders maintain water balance, especially in fossorial species. Conclusions Temperature impacts the physiology and behaviour of ectotherms through both acute and prolonged effects (Tattersall et al. 2012 ). In A . maculatum , short-term changes in temperature led to an increase in SMR, EWL, and R s . Moreover, SMR and R s differed between seasons, but EWL did not. By maintaining relatively low SMR and high R s in the spring, A . maculatum may be able to allocate energy toward overwintering emergence and breeding while minimising water conservation. By contrast, relatively high SMR and low R s in the autumn could be associated with aboveground foraging on rainy nights (i.e., low evaporative demand) to replenish energy stores before the winter (Faccio 2003 ; Moldowan et al. 2022 ). Despite the prevailing assumption that fossorial ectotherms are buffered from thermal effects (Giacometti and Tattersall 2023 ), our study demonstrates that energetic and hydroregulatory requirements respond to both short- and long-term changes in temperature in the fossorial A . maculatum . Ultimately, our study contributes to the goal of elucidating the thermal dependency of physiology in fossorial ectotherms (Bars-Closel et al. 2018 ). Declarations Funding: DG was funded by a Doris White Memorial Bursary provided by Brock University. GJT was funded by a Natural Sciences and Engineering Research Council of Canada Discovery Grant (RGPIN-2020-05089). Ethics approval : All procedures were performed with permission from the Ministry of Northern Development, Mines, Natural Resources and Forestry (#1100575), Ontario Parks, and Brock University’s Animal Care Committee (Animal Use Protocol #22-03-04). Authors’ contribution: DG and GJT conceived the ideas and designed methodology; DG collected the data; DG analysed the data with input from GJT; DG and GJT led the writing of the manuscript. Both authors contributed critically to the drafts and gave final approval for publication. Conflicts of interest : None declared. Acknowledgements We thank Ontario Parks, the Ministry of Northern Development, Mines, Natural Resources and Forestry, the Algonquin Wildlife Research Station, Njal Rollinson, Kevin Kemmish, and Patrick Moldowan for facilitating access to our study animals. We thank Shawn Bukovac, Kristin Bray, Sarah Kehoe, and Natasha Hearn for assistance with animal care. We are grateful to Zak Mason from Brock University’s Makerspace and Alex Popescu for building the 3D printed models used in our study. Availability of data and code : Our data and code can be accessed from Brock University Dataverse: https://doi.org/10.5683/SP3/MQPFHT . References Anderson DB (1936) Relative humidity or vapor pressure deficit. Ecology 17:277–282. https://doi.org/10.2307/1931468 Andrade DV (2016) Temperature effects on the metabolism of amphibians and reptiles. In: de Andrade DV, Bevier CR, de Carvalho JE (eds) Amphibian and Reptile Adaptations to the Environment; Interplay between Physiology and Behavior. CRC, Boca Raton, pp 129–254 Bars-Closel M, Camacho A, Kohlsdorf T (2018) Shifts in space and time: ecological transitions affect the evolution of resting metabolic rates in microteiid lizards. J Exp Biol jeb 175661. https://doi.org/10.1242/jeb.175661 Bates D, Mächler M, Bolker B, Walker S (2015) Fitting Linear Mixed-Effects Models Using lme4. 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Physiol Biochem Zool 78:847–856. https://doi.org/10.1086/432152 Additional Declarations The authors declare no competing interests. Supplementary Files Supplementarymaterial.docx Supplementary material Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-5478984\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":379710678,\"identity\":\"2a748d18-5cce-46f3-b3e0-03df9b53be8a\",\"order_by\":0,\"name\":\"Danilo Giacometti\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAklEQVRIiWNgGAWjYDCCA2AEZX+osAFSjI0HcKlG1cLGzHBwxpk0kJYGgloYYFqYeVsOowpiA3wH2C8e+FFjE80v33/wMG/Debu17YeBtgBFcGmRPMBTcLDnWFruzDagw+buuJ287UwiUAtQpAGHFoMDPAkHeBsO5244xsxw4O2Z28lmB4BaGIEi+LQc/AtUsB+khbftXLLZ+YeEtLAfOAy2BRRivG0H7MxuELBF8jAPw2EZoMtnHEs2AAZycoLZDaAtCXj8wne8/fHHNzU2uf3NBx9/+FBhZ292Pv3hgw9AEVxaGJh5DFD4iWCVCbiUgwH7AxSuPV7Fo2AUjIJRMCIBAAeLcRTVdk9RAAAAAElFTkSuQmCC\",\"orcid\":\"https://orcid.org/0000-0002-3882-000X\",\"institution\":\"Brock University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Danilo\",\"middleName\":\"\",\"lastName\":\"Giacometti\",\"suffix\":\"\"},{\"id\":379710788,\"identity\":\"9e251df0-00ea-415f-8f4b-a79cce29966f\",\"order_by\":1,\"name\":\"Glenn J. 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In both panels, large dots represent mean marginal effects at a given temperature and bars indicate the corresponding 95% confidence intervals. Small dots show the mean predicted value for each individual salamander at a given temperature. Seasons are colour-coded, with autumn shown in yellow and spring shown in green.\\u003cbr\\u003e\\n\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure1MRandEWLasafunctionoftemperature.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5478984/v1/87443815f5d61c8a1df6b190.jpg\"},{\"id\":73444277,\"identity\":\"46fbd500-7d3e-409c-81ec-f5eddd23dc3d\",\"added_by\":\"auto\",\"created_at\":\"2025-01-10 04:35:36\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":539453,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThermal impacts on skin resistance (\\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e) to water loss in \\u003cem\\u003eAmbystoma maculatum\\u003c/em\\u003e measured in the autumn and spring. \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es \\u003c/sub\\u003eincreased with temperature and was overall higher in the spring than in the autumn. Large dots denote mean marginal effects at a given temperature and bars show the corresponding 95% confidence intervals. Small dots represent the mean predicted value for each individual salamander at a given temperature. Seasons are colour-coded (autumn = yellow, spring = green).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure2Skinresistanceasafunctionoftemperaturebetweenseasons.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5478984/v1/9c792368e61788636f581067.jpg\"},{\"id\":73444769,\"identity\":\"5aa8a3f9-073b-4035-88d2-8df839a6d564\",\"added_by\":\"auto\",\"created_at\":\"2025-01-10 04:43:38\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2346603,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5478984/v1/f112979a-fa1a-40e3-8d7f-8eeaf6556253.pdf\"},{\"id\":73444275,\"identity\":\"8ac11f6f-99c3-40ca-a5f8-f8ce4845587a\",\"added_by\":\"auto\",\"created_at\":\"2025-01-10 04:35:36\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1120227,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSupplementary material\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Supplementarymaterial.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5478984/v1/66425b7ec3270d36678dd3dc.docx\"}],\"financialInterests\":\"The authors declare no competing interests.\",\"formattedTitle\":\"\\u003cp\\u003e\\u003cstrong\\u003eSeasonal plasticity in the thermal sensitivity of metabolism but not water loss in a fossorial ectotherm\\u003c/strong\\u003e\\u003c/p\\u003e\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eShifts in temperature affect animals at all levels of organisation, from cellular processes to whole-organism performance (Tattersall et al. \\u003cspan citationid=\\\"CR71\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e). This is particularly true in ectotherms, which have limited capacity for metabolic heat production and depend on environmental temperatures to regulate physiological function (Bicego et al. \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e). Ectotherms have evolved different mechanisms to maintain performance in the face of varying temperatures. Behavioural responses can buffer the impact of thermal variability over physiology in the short term (e.g., hours), as demonstrated in toads that burrow in response to warming temperatures to minimise evaporative water loss (Hoffman and Katz \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e1989\\u003c/span\\u003e). By contrast, reversible physiological plasticity (e.g., acclimatisation) can aid in dealing with long-term shifts in temperature (Little and Seebacher \\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e), especially in species with limited capacity for behavioural thermoregulation (Lowe et al. \\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e2010\\u003c/span\\u003e). For example, several amphibians regulate energy expenditure to compensate for seasonal changes in temperature (Fitzpatrick \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e1973\\u003c/span\\u003e; Feder \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e1978\\u003c/span\\u003e; Kiss et al. \\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e). While evidence suggests that ectotherms from highly variable climates should have greater plasticity potential than those from stable climates, there is still a dearth of knowledge about the mechanisms that underpin seasonal physiological adjustments, especially from an energetic perspective (Gatten Jr et al. \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e1992\\u003c/span\\u003e; Navas \\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e1996\\u003c/span\\u003e; Sun et al. \\u003cspan citationid=\\\"CR70\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eThe maintenance of energy metabolism is one of the greatest challenges faced by ectotherms in thermally variable habitats, since changes in temperature can impact whether ATP production meets demand (Seebacher et al. \\u003cspan citationid=\\\"CR59\\\" class=\\\"CitationRef\\\"\\u003e2010\\u003c/span\\u003e). In ectotherms, standard metabolic rates (SMR) represent the baseline costs for body maintenance under varying conditions (Gatten Jr et al. \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e1992\\u003c/span\\u003e). Typically, SMR rises with temperature as a consequence of thermodynamic effects on the biochemical reactions involved in energy turnover (Andrade \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e). The thermal sensitivity of SMR can be assessed through the temperature coefficient (Q\\u003csub\\u003e10\\u003c/sub\\u003e), which describes changes in rate processes measured at contrasting temperatures across short time scales (Gatten Jr et al. \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e1992\\u003c/span\\u003e). Although the thermal sensitivity of SMR is ubiquitous in ectotherms (Carter et al. \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e), Q\\u003csub\\u003e10\\u003c/sub\\u003e values may vary intra- and interspecifically or according to the degree of thermal variability experienced (Gatten Jr et al. \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e1992\\u003c/span\\u003e; Kreiman et al. \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). Indeed, the Q\\u003csub\\u003e10\\u003c/sub\\u003e of SMR has been recurrently used to explain differences in the ability to physiologically compensate for both short- and long-term shifts in temperature (reviewed in Havird et al. \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). In the context of seasonality, assessing changes in the thermal sensitivity of SMR can clarify how ectotherms allocate energy toward different activities between seasons (e.g., reproduction or growth). For example, different species of salamanders (Amphibia: Caudata) exhibit metabolic compensation (Prosser \\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e1969\\u003c/span\\u003e) in SMR between the activity and dormancy season, highlighting that compensatory energetic responses are key to survival (Fitzpatrick \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e1973\\u003c/span\\u003e; Fitzpatrick and Brown \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e1975\\u003c/span\\u003e; Feder \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e1978\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eTemperature can also have profound effects over evaporative water loss (EWL), particularly in amphibians (Lertzman-Lepofsky et al. \\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). Indeed, amphibians have a relatively thin skin that is supported by a unique vasculature that facilitates oxygen uptake and carbon dioxide excretion (Burggren and Moalli \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e1984\\u003c/span\\u003e). However, the ability to exchange gases through the skin comes at the expense of high EWL, which increases as a function of temperature (Spotila \\u003cspan citationid=\\\"CR68\\\" class=\\\"CitationRef\\\"\\u003e1972\\u003c/span\\u003e). Thermal effects over EWL are mediated by the vapour pressure deficit (VPD), which measures the drying power of air, and is defined as the difference between the saturated water vapour pressure (WVP\\u003csub\\u003esat\\u003c/sub\\u003e) and the actual water vapour pressure (WVP\\u003csub\\u003eact\\u003c/sub\\u003e) (Anderson \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1936\\u003c/span\\u003e; Haynes \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e), since the skin interface where evaporation occurs will be a saturating conditions. Given that WVP\\u003csub\\u003esat\\u003c/sub\\u003e increases with temperature, there should be greater evaporative demand at warmer than cooler temperatures (Riddell et al. \\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). Thus, amphibians exposed to a combination of warm temperatures and high VPD should pay higher hydroregulatory costs (Riddell et al. \\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). As a primary defence mechanism, some amphibians may enhance skin resistance to water loss (\\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e) in response to concomitant increases in temperature and VPD (Tracy et al. \\u003cspan citationid=\\\"CR74\\\" class=\\\"CitationRef\\\"\\u003e2008\\u003c/span\\u003e; e.g., Riddell and Sears \\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Senzano and Andrade \\u003cspan citationid=\\\"CR61\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). However, the extent to which seasonality elicits plastic responses in the maintenance of water balance in amphibians is still unclear. For instance, research in salamanders indicated that both EWL and \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e may respond to thermal acclimation (Riddell and Sears \\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Riddell et al. \\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e), whereas work in anurans suggested that neither trait varies between seasons (Davis et al. \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e; Maenaka et al. \\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). Ultimately, assessments of the effect of seasonality over EWL and \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e are fundamental to improve our understanding of how amphibians cope with thermal and hydroregulatory challenges (Maenaka et al. \\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eHere, we evaluated how seasonal acclimatisation impacted the thermal sensitivity of energy and water balance in the spotted salamander (\\u003cem\\u003eAmbystoma maculatum\\u003c/em\\u003e). Despite being fossorial and assumed to live in thermally stable habitats, seasonality influences many attributes in \\u003cem\\u003eA\\u003c/em\\u003e. \\u003cem\\u003emaculatum\\u003c/em\\u003e, including thermoregulation (Giacometti and Tattersall \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e), reproduction (Sexton et al. \\u003cspan citationid=\\\"CR63\\\" class=\\\"CitationRef\\\"\\u003e1990\\u003c/span\\u003e), body condition (Moldowan et al. \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e), and activity patterns (Vasconcelos and Calhoun \\u003cspan citationid=\\\"CR75\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e). Aboveground activity in this species can be observed primarily in the spring and autumn (Vasconcelos and Calhoun \\u003cspan citationid=\\\"CR75\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e; Moldowan et al. \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). In the spring, salamanders emerge from overwintering burrows and migrate toward bodies of water to breed at relatively low environmental temperatures (Moldowan et al. \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). Individuals do not feed during breeding migration and the aquatic breeding period (Smallwood \\u003cspan citationid=\\\"CR66\\\" class=\\\"CitationRef\\\"\\u003e1928\\u003c/span\\u003e) even after spending up to seven months underground (Moldowan et al. \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). By contrast, aboveground activity in the autumn occurs at relatively higher environmental temperatures, and is likely associated with individuals foraging to replenish energy stores before the winter (Faccio \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e; Moldowan et al. \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). Thus, while spring and autumn both fall within the activity period of \\u003cem\\u003eA\\u003c/em\\u003e. \\u003cem\\u003emaculatum\\u003c/em\\u003e, salamanders should have different energetic and hydroregulatory requirements between seasons. Although fossoriality did not impinge on amphibian metabolism (Giacometti and Tattersall \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e), evidence suggests that fossorial ectotherms have a blunted thermal sensitivity of SMR (Bars-Closel et al. \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). Furthermore, while some anurans may minimise EWL by forming cocoons (Shoemaker \\u003cspan citationid=\\\"CR64\\\" class=\\\"CitationRef\\\"\\u003e1988\\u003c/span\\u003e), modulation of \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e is the primary physiological response to EWL in amphibians (Wygoda \\u003cspan citationid=\\\"CR82\\\" class=\\\"CitationRef\\\"\\u003e1988\\u003c/span\\u003e; Prates and Navas \\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e; Riddell and Sears \\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e), at least in the short term. Whether these patterns are applicable in a seasonal context remains unexplored.\\u003c/p\\u003e \\u003cp\\u003eTo test the effect of seasonality over energy and water balance, we acclimatised \\u003cem\\u003eA\\u003c/em\\u003e. \\u003cem\\u003emaculatum\\u003c/em\\u003e to conditions that matched spring and autumn in nature (Moldowan et al. \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). We then measured SMR, EWL, and \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e at 2, 5, 10, 15, and 20\\u0026deg;C, since these temperatures are biologically relevant across seasons to our study system (Giacometti and Tattersall \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). Given that animals respond to simultaneous shifts in temperature and humidity, and warmer temperatures typically represent drier conditions in nature (Gates \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e1980\\u003c/span\\u003e; Riddell et al. \\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e; Weaver et al. \\u003cspan citationid=\\\"CR76\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e), we designed our experiment such that an increase in temperature was followed by an increase in VPD. We hypothesised that temperature would have both short- and long-term effects over traits (i.e., acute exposure to test temperatures and seasonal acclimatisation, respectively) (Havird et al. \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). For short-term effects, we predicted that SMR, EWL, and \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e would increase with test temperature (Whitford and Hutchison \\u003cspan citationid=\\\"CR78\\\" class=\\\"CitationRef\\\"\\u003e1963\\u003c/span\\u003e; Riddell and Sears \\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Senzano and Andrade \\u003cspan citationid=\\\"CR61\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). For long-term effects, we predicted that autumn-acclimatised salamanders would have higher SMR, EWL, and \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e than spring-acclimatised salamanders across all test temperatures, following a pattern of inverse compensation (Packard \\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e1972\\u003c/span\\u003e; Havird et al. \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). We further predicted that the thermal sensitivity of SMR would differ between seasons, with overall lower Q\\u003csub\\u003e10\\u003c/sub\\u003e values in the spring than in the autumn, and lower sensitivity at cooler than warmer test temperatures (Fitzpatrick and Brown \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e1975\\u003c/span\\u003e). Lastly, we used our empirical EWL measurements to determine how long it would take for \\u003cem\\u003eA\\u003c/em\\u003e. \\u003cem\\u003emaculatum\\u003c/em\\u003e to reach their desiccation limit at each test temperature (Hall \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e1922\\u003c/span\\u003e). By examining the combined effects of temperature and humidity over physiology, we aim to elucidate the mechanisms behind the maintenance of energy and water balance between seasons (Chang and Hou \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2005\\u003c/span\\u003e; Maenaka et al. \\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e).\\u003c/p\\u003e\"},{\"header\":\"Material and methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eSalamander collection and husbandry\\u003c/h2\\u003e \\u003cp\\u003eOn 15 May 2022, we collected 50 post-breeding, adult \\u003cem\\u003eA\\u003c/em\\u003e. \\u003cem\\u003emaculatum\\u003c/em\\u003e (26 females\\u0026thinsp;+\\u0026thinsp;24 males) using a drift fence installed around Bat Lake, Algonquin Provincial Park, Ontario, Canada (45.5857\\u0026deg;N, 78.5185\\u0026deg;W). When we collected a salamander, we recorded its snout-vent length (SVL) with a tape measure, its body mass (M\\u003csub\\u003eb\\u003c/sub\\u003e) with a Pesola\\u0026trade; spring scale, and its sex based on cloacal morphology (Petranka \\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e1998\\u003c/span\\u003e). We assigned each individual a unique identification (ID) that was associated with a photograph of the dorsum of the salamander to create an individual-level recognition system based on spot patterns. To transfer the salamanders from Bat Lake to Brock University, we allocated them into plastic containers with ventilated fitted lids (34 cm x 19.6 cm x 12 cm). We kept 10 salamanders per container, totalling five containers used in transportation; each container had \\u003cem\\u003eSphagnum\\u003c/em\\u003e moss, pine needles and water. We placed the containers inside a transport box kept at ~\\u0026thinsp;4\\u0026deg;C to avoid overheating and dehydration during transportation.\\u003c/p\\u003e \\u003cp\\u003eIn the lab, we housed salamanders in pairs within ventilated tanks that had coconut husk fibre, \\u003cem\\u003eSphagnum\\u003c/em\\u003e moss, and PVC pipe refuges; tanks were misted daily to maintain high humidity locally. We kept these tanks in a facility with controlled temperature, relative humidity, and photoperiod. We adjusted temperature and photoperiod seasonally to mimic their collection site soil conditions (Moldowan et al. \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e), while keeping relative humidity constantly at 70%. In the spring, we housed salamanders at 7\\u0026deg;C and a 10h:14h light:dark cycle. During the summer, we kept them at 14\\u0026deg;C and a 14h:12h light:dark cycle. In the autumn, we kept salamanders at 12\\u0026deg;C, and a 12h:12h light:dark cycle. To transition between housing conditions, we increased (spring to summer) or decreased (summer to autumn) temperature by 1\\u0026deg;C/day. We allowed the salamanders at least four weeks under the prevailing acclimatisation condition before starting our experiments. During their period in the lab, we fed salamanders twice a week with mealworms dusted in calcium and multivitamin powder, while water was available \\u003cem\\u003ead libitum\\u003c/em\\u003e. We weighed salamanders to the nearest 0.01 g every week with an analytical scale (Mettler Toledo, model PB602-S) to monitor changes in M\\u003csub\\u003eb\\u003c/sub\\u003e as an indicator of animal well-being; all animals maintained M\\u003csub\\u003eb\\u003c/sub\\u003e during the course of our experiments.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eRespirometry system\\u003c/h3\\u003e\\n\\u003cp\\u003eWe measured SMR and EWL with a flow-through open respirometry system. We used tubing attached to an electronic air pump to draw outside air into the lab, which was passed through a column of CO\\u003csub\\u003e2\\u003c/sub\\u003e absorbent (Amsorb\\u0026reg;, Amsorb Plus), humidified, and directed to a dewpoint generator (Sable Systems International, model DG-4) set constantly to 1.5\\u0026deg;C. The air excurrent from the dewpoint generator had a water vapour pressure of 0.681 kPa and a water vapour density (WVD) of 5.4 \\u0026micro;g/mL, and was split into two channels with each running through a mass flow controller (Sable Systems International, model MFC-2) at a flow rate of 200 mL/min. Channel one was directed into a 500 mL glass respirometer containing a salamander and kept inside a temperature-controlled incubator (Thermo Fisher Scientific Inc., model 146E). We sealed the respirometer with a rubber stopper that had two syringe barrels used as ports for incurrent and excurrent air, respectively. Excurrent air left the respirometer toward an open manifold connected to a subsample line controlled by a solenoid switch. The subsampler directed air at a flow rate of 100 mL/min to a water vapour analyser (Sable Systems International, RH-300), and then to a respirometry system (Sable Systems International, FoxBox) that measured O\\u003csub\\u003e2\\u003c/sub\\u003e and CO\\u003csub\\u003e2\\u003c/sub\\u003e air content. We programmed the solenoid switch to be triggered for 5 min every 60 min, thereby diverting air from channel one to channel two, and providing baseline measurements of incurrent air. We discarded the initial 90 s after the switch was triggered to account for dead-space gas transit time (Sakich and Tattersall \\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). We connected our respirometry system to a computer with a data acquisition software (BIOPAC Systems Inc, Acqknowledge Data Acquisition and Analysis Software) that provided real-time, continuous measurements of O\\u003csub\\u003e2\\u003c/sub\\u003e (%), CO\\u003csub\\u003e2\\u003c/sub\\u003e (%), WVD (\\u0026micro;g/mL), chamber temperature (\\u0026deg;C), flow rate (mL/min), and baseline voltage (V).\\u003c/p\\u003e\\n\\u003ch3\\u003eThermal sensitivity of metabolism and water loss\\u003c/h3\\u003e\\n\\u003cp\\u003eTo assess seasonal changes in the thermal sensitivity of SMR and EWL, we conducted measurements at 2\\u0026deg;C, 5\\u0026deg;C, 10\\u0026deg;C, 15\\u0026deg;C, and 20\\u0026deg;C in the spring and autumn (N\\u0026thinsp;=\\u0026thinsp;10 individuals/temperature). In both seasons, we randomised the order of experiments and tested the same individuals at the same temperature. We allowed each individual a total of 4 h inside the respirometer and tested two individuals per day between 08h00\\u0026ndash;18h00, which represents the resting period for the study species. Prior to measurements, individuals were fasted for a week to ensure a postabsorptive state (Secor and Boehm \\u003cspan citationid=\\\"CR58\\\" class=\\\"CitationRef\\\"\\u003e2006\\u003c/span\\u003e). We weighed each individual before and after an experiment to determine the percentage of M\\u003csub\\u003eb\\u003c/sub\\u003e loss during the experiment. We ran our experiments in total darkness, and continuously monitored the salamanders through a high-speed infrared webcam (AOS Technologies AG, model Promon U1000) that was mounted inside the incubator along with an infrared illuminator (wavelength\\u0026thinsp;=\\u0026thinsp;850 nm; TVPSii, model TP-IRBP15). Because we kept the dew point constantly at 1.5\\u0026deg;C, our experimental design allowed us to assess the combined effect of different temperatures and VPD over metabolism and water loss (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). We determined the WVP\\u003csub\\u003esat\\u003c/sub\\u003e, WVP\\u003csub\\u003eact\\u003c/sub\\u003e, and VPD at a given temperature (T) and percent relative humidity (RH) for our experimental conditions following (Haynes \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e):\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eTest temperature, relative humidity, saturated water vapour pressure, and vapour pressure deficit for each experimental condition. We achieved a combination of varying temperatures and humidity conditions by maintaining the dew point constantly at 1.5\\u0026deg;C in our experiments.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"4\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTemperature (\\u0026deg;C)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eRelative humidity (%)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eSaturated water vapour pressure (kPa)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eVapour pressure deficit (kPa)\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e96.48\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.706\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.025\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e5\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e78.04\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.873\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.192\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e10\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e55.44\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e1.228\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.547\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e15\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e39.92\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e1.706\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1.025\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e20\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e29.12\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e2.339\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1.658\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003cdiv id=\\\"Equ1\\\" class=\\\"Equation\\\"\\u003e \\u003cdiv format=\\\"TEX\\\" class=\\\"mathdisplay\\\" id=\\\"FileID_Equ1\\\" name=\\\"EquationSource\\\"\\u003e\\n$$\\\\:{WVP}_{sat}=6.11\\\\times\\\\:{10}^{\\\\left(\\\\frac{7.5\\\\times\\\\:T}{237.3+T}\\\\right)}$$\\u003c/div\\u003e \\u003cdiv class=\\\"EquationNumber\\\"\\u003e1\\u003c/div\\u003e\\u003c/div\\u003e,\\u003cdiv id=\\\"Equ2\\\" class=\\\"Equation\\\"\\u003e\\u003cdiv format=\\\"TEX\\\" class=\\\"mathdisplay\\\" id=\\\"FileID_Equ2\\\" name=\\\"EquationSource\\\"\\u003e\\n$$\\\\:{WVP}_{act}={WVP}_{sat}\\\\times\\\\:\\\\left(\\\\frac{RH}{100}\\\\right)$$\\u003c/div\\u003e\\u003cdiv class=\\\"EquationNumber\\\"\\u003e2\\u003c/div\\u003e\\u003c/div\\u003e,\\u003cdiv id=\\\"Equ3\\\" class=\\\"Equation\\\"\\u003e\\u003cdiv format=\\\"TEX\\\" class=\\\"mathdisplay\\\" id=\\\"FileID_Equ3\\\" name=\\\"EquationSource\\\"\\u003e\\n$$\\\\:VPD={WVP}_{sat}-{WVP}_{act}$$\\u003c/div\\u003e\\u003cdiv class=\\\"EquationNumber\\\"\\u003e3\\u003c/div\\u003e\\u003c/div\\u003e,\\u003c/p\\u003e \\u003cp\\u003eWe considered rates of carbon dioxide production (V̇CO\\u003csub\\u003e2\\u003c/sub\\u003e; mL/h) as a proxy for SMR, and rates of water vapour production (V̇H\\u003csub\\u003e2\\u003c/sub\\u003eO; mg/h) as a proxy for EWL. We visually inspected the resulting graphs of O\\u003csub\\u003e2\\u003c/sub\\u003e, CO\\u003csub\\u003e2\\u003c/sub\\u003e, and WVD variation over time, and considered periods of 50.14\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;5.28 min (mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard deviation) of constancy in our calculations using equations described in (Lighton \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e2008\\u003c/span\\u003e):\\u003cdiv id=\\\"Equ4\\\" class=\\\"Equation\\\"\\u003e\\u003cdiv format=\\\"TEX\\\" class=\\\"mathdisplay\\\" id=\\\"FileID_Equ4\\\" name=\\\"EquationSource\\\"\\u003e\\n$$\\\\:{\\\\dot{V}{CO}_{2}=FR}_{i}\\\\left\\\\{\\\\left[\\\\frac{\\\\left\\\\{{[F}_{e}{CO}_{2}(1-{{F}_{i}O}_{2}-\\\\right.{{F}_{i}CO}_{2}-{{F}_{i}H}_{2}O)}{({1-F}_{e}{O}_{2}-{F}_{e}{CO}_{2}-{F}_{e}{H}_{2}O)}\\\\right]-{F}_{i}{CO}_{2}\\\\right\\\\}$$\\u003c/div\\u003e\\u003cdiv class=\\\"EquationNumber\\\"\\u003e4\\u003c/div\\u003e\\u003c/div\\u003e,\\u003cdiv id=\\\"Equ5\\\" class=\\\"Equation\\\"\\u003e\\u003cdiv format=\\\"TEX\\\" class=\\\"mathdisplay\\\" id=\\\"FileID_Equ5\\\" name=\\\"EquationSource\\\"\\u003e\\n$$\\\\:\\\\dot{V}{H}_{2}O={FR}_{i}\\\\{{WVD}_{e}-{WVD}_{i}\\\\}$$\\u003c/div\\u003e\\u003cdiv class=\\\"EquationNumber\\\"\\u003e5\\u003c/div\\u003e\\u003c/div\\u003e,\\u003c/p\\u003e \\u003cp\\u003ewhere FR\\u003csub\\u003ei\\u003c/sub\\u003e is the incurrent flow rate, F\\u003csub\\u003ee\\u003c/sub\\u003eCO\\u003csub\\u003e2\\u003c/sub\\u003e is the fractional CO\\u003csub\\u003e2\\u003c/sub\\u003e concentration of the excurrent air, F\\u003csub\\u003ei\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e is the fractional O\\u003csub\\u003e2\\u003c/sub\\u003e concentration of the incurrent air, F\\u003csub\\u003ei\\u003c/sub\\u003eH\\u003csub\\u003e2\\u003c/sub\\u003eO is the fractional water vapour concentration of the incurrent air, F\\u003csub\\u003ee\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e is the fractional O\\u003csub\\u003e2\\u003c/sub\\u003e concentration of the excurrent air, F\\u003csub\\u003ee\\u003c/sub\\u003eH\\u003csub\\u003e2\\u003c/sub\\u003eO is the water vapour concentration of the excurrent air, F\\u003csub\\u003ei\\u003c/sub\\u003eCO\\u003csub\\u003e2\\u003c/sub\\u003e is the fractional CO\\u003csub\\u003e2\\u003c/sub\\u003e concentration of the incurrent air, WVD\\u003csub\\u003ee\\u003c/sub\\u003e is the water vapour density of the excurrent air, and WVD\\u003csub\\u003ei\\u003c/sub\\u003e is the water vapour density of the incurrent air. Due to low differences between F\\u003csub\\u003ei\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e and F\\u003csub\\u003ee\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e, we could not estimate V̇O\\u003csub\\u003e2\\u003c/sub\\u003e with confidence, especially at low temperatures, and therefore only report on V̇CO\\u003csub\\u003e2\\u003c/sub\\u003e.\\u003c/p\\u003e \\u003cp\\u003eWe calculated the Q\\u003csub\\u003e10\\u003c/sub\\u003e of mass-specific V̇CO\\u003csub\\u003e2\\u003c/sub\\u003e (mL/g/h) between 5\\u0026ndash;15\\u0026deg;C and 10\\u0026ndash;20\\u0026deg;C with the van\\u0026rsquo;t Hoff equation:\\u003cdiv id=\\\"Equ6\\\" class=\\\"Equation\\\"\\u003e\\u003cdiv format=\\\"TEX\\\" class=\\\"mathdisplay\\\" id=\\\"FileID_Equ6\\\" name=\\\"EquationSource\\\"\\u003e\\n$$\\\\:{Q}_{10}={\\\\left({R}_{2}/{R}_{1}\\\\right)}^{\\\\frac{10}{{T}_{2}-{T}_{1}}}$$\\u003c/div\\u003e\\u003cdiv class=\\\"EquationNumber\\\"\\u003e6\\u003c/div\\u003e\\u003c/div\\u003e,\\u003c/p\\u003e \\u003cp\\u003ewhere \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003e1\\u003c/sub\\u003e and \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003e2\\u003c/sub\\u003e are physiological rates measured at temperatures \\u003cem\\u003eT\\u003c/em\\u003e\\u003csub\\u003e1\\u003c/sub\\u003e and \\u003cem\\u003eT\\u003c/em\\u003e\\u003csub\\u003e2\\u003c/sub\\u003e, respectively.\\u003c/p\\u003e\\n\\u003ch3\\u003eDays until desiccation\\u003c/h3\\u003e\\n\\u003cp\\u003e \\u003cem\\u003eAmbystoma maculatum\\u003c/em\\u003e can withstand a loss of 47% loss of M\\u003csub\\u003eb\\u003c/sub\\u003e via evaporation before losing function (Hall \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e1922\\u003c/span\\u003e). Based on our individual-specific data on M\\u003csub\\u003eb\\u003c/sub\\u003e and EWL, we calculated how long it would take for our individuals to reach their limit of desiccation across test temperatures. We determined time until desiccation (hours; \\u003cem\\u003eH\\u003c/em\\u003e\\u003csub\\u003ed\\u003c/sub\\u003e) using the equation:\\u003cdiv id=\\\"Equ7\\\" class=\\\"Equation\\\"\\u003e\\u003cdiv format=\\\"TEX\\\" class=\\\"mathdisplay\\\" id=\\\"FileID_Equ7\\\" name=\\\"EquationSource\\\"\\u003e\\n$$\\\\:{H}_{d}=\\\\frac{\\\\left(0.47\\\\right)\\\\times\\\\:{M}_{b}}{EWL}$$\\u003c/div\\u003e\\u003cdiv class=\\\"EquationNumber\\\"\\u003e7\\u003c/div\\u003e\\u003c/div\\u003e,\\u003c/p\\u003e \\u003cp\\u003eSince EWL was expressed in g/h, we then divided \\u003cem\\u003eH\\u003c/em\\u003e\\u003csub\\u003ed\\u003c/sub\\u003e by 24 to obtain an estimate of days until desiccation (\\u003cem\\u003eD\\u003c/em\\u003e\\u003csub\\u003ed\\u003c/sub\\u003e):\\u003cdiv id=\\\"Equ8\\\" class=\\\"Equation\\\"\\u003e\\u003cdiv format=\\\"TEX\\\" class=\\\"mathdisplay\\\" id=\\\"FileID_Equ8\\\" name=\\\"EquationSource\\\"\\u003e\\n$$\\\\:{D}_{d=\\\\:\\\\raisebox{1ex}{${H}_{d}$}\\\\!\\\\left/\\\\:\\\\!\\\\raisebox{-1ex}{$24$}\\\\right.}$$\\u003c/div\\u003e\\u003cdiv class=\\\"EquationNumber\\\"\\u003e8\\u003c/div\\u003e\\u003c/div\\u003e,\\u003c/p\\u003e\\n\\u003ch3\\u003eResistance to water loss\\u003c/h3\\u003e\\n\\u003cp\\u003eTotal resistance (\\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003et\\u003c/sub\\u003e) to water loss is the combined outcome of skin (\\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e) and boundary layer resistances (\\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003eb\\u003c/sub\\u003e) (Feder and Pinder \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e1988\\u003c/span\\u003e). Thus, to quantify the relative role of the skin in regulating water loss, one must determine \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003eb\\u003c/sub\\u003e. To this end, we used the framework proposed by (Spotila and Berman \\u003cspan citationid=\\\"CR69\\\" class=\\\"CitationRef\\\"\\u003e1976\\u003c/span\\u003e), which consists of building agar replicas of one\\u0026rsquo;s study system to empirically determine \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003eb\\u003c/sub\\u003e (i.e., from a free evaporating surface). We obtained custom built 3D-printed positive models of \\u003cem\\u003eA\\u003c/em\\u003e. \\u003cem\\u003emaculatum\\u003c/em\\u003e from Brock University\\u0026rsquo;s Makerspace. These models matched the average SVL of males and females used in our study. We used a silicone-free mold maker (SilliNOT!\\u0026reg;) to pour negative molds of the 3D-printed models, and then we used the molds to cast 5% agar replicas (Senzano et al. \\u003cspan citationid=\\\"CR62\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e) (Figure S1). Once the agar replicas were hardened, we measured their V̇H\\u003csub\\u003e2\\u003c/sub\\u003eO with the same method used with live salamanders. Based on our experimental conditions, salamanders experienced cool and humid conditions at 2\\u0026deg;C that resulted in a low evaporative demand (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). As such, we only calculated \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003et\\u003c/sub\\u003e and \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e at 5, 10, 15, and 20\\u0026deg;C (N\\u0026thinsp;=\\u0026thinsp;10 agar models per test temperature).\\u003c/p\\u003e \\u003cp\\u003eTo calculate \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003et\\u003c/sub\\u003e (s/cm), we first estimated surface area (\\u003cem\\u003eS\\u003c/em\\u003e; cm\\u003csup\\u003e2\\u003c/sup\\u003e) based on (Whitford and Hutchison \\u003cspan citationid=\\\"CR79\\\" class=\\\"CitationRef\\\"\\u003e1967\\u003c/span\\u003e):\\u003cdiv id=\\\"Equ9\\\" class=\\\"Equation\\\"\\u003e\\u003cdiv format=\\\"TEX\\\" class=\\\"mathdisplay\\\" id=\\\"FileID_Equ9\\\" name=\\\"EquationSource\\\"\\u003e\\n$$\\\\:S={{M}_{b}}^{\\\\left(0.694\\\\right)}$$\\u003c/div\\u003e\\u003cdiv class=\\\"EquationNumber\\\"\\u003e9\\u003c/div\\u003e\\u003c/div\\u003e,\\u003c/p\\u003e \\u003cp\\u003eThen, we applied the equation postulated by (Spotila and Berman \\u003cspan citationid=\\\"CR69\\\" class=\\\"CitationRef\\\"\\u003e1976\\u003c/span\\u003e):\\u003cdiv id=\\\"Equ10\\\" class=\\\"Equation\\\"\\u003e\\u003cdiv format=\\\"TEX\\\" class=\\\"mathdisplay\\\" id=\\\"FileID_Equ10\\\" name=\\\"EquationSource\\\"\\u003e\\n$$\\\\:{R}_{t}=VDD/{\\\\dot{VH}}_{2}{O}_{area}$$\\u003c/div\\u003e\\u003cdiv class=\\\"EquationNumber\\\"\\u003e10\\u003c/div\\u003e\\u003c/div\\u003e,\\u003c/p\\u003e \\u003cp\\u003ewhere VDD is the vapour density deficit (i.e., the difference between WVD at the evaporating surface and the WVD of air temperature), and V̇H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003earea\\u003c/sub\\u003e is the area-specific V̇H\\u003csub\\u003e2\\u003c/sub\\u003eO (mg/cm\\u003csup\\u003e2\\u003c/sup\\u003e/h) of a salamander or an agar model. Given that agar replicas lack \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e, their \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003et\\u003c/sub\\u003e is made up solely by \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003eb\\u003c/sub\\u003e. Consequently, the \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e of each individual salamander can be determined by:\\u003cdiv id=\\\"Equ11\\\" class=\\\"Equation\\\"\\u003e\\u003cdiv format=\\\"TEX\\\" class=\\\"mathdisplay\\\" id=\\\"FileID_Equ11\\\" name=\\\"EquationSource\\\"\\u003e\\n$$\\\\:{R}_{s}={R}_{t}-{R}_{b}$$\\u003c/div\\u003e\\u003cdiv class=\\\"EquationNumber\\\"\\u003e11\\u003c/div\\u003e\\u003c/div\\u003e,\\u003c/p\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistical analyses\\u003c/h2\\u003e \\u003cp\\u003eWe performed all analyses using R (version 4.4.1) in RStudio (version 2024.06.14) (R Core Team \\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e) with a significance level of 0.05. To assess normality and homoscedasticity, we visually inspected Q-Q and P-P plots through the \\u0026ldquo;fitdist\\u0026rdquo; function from the fitdistrplus package (Delignette-Muller and Dutang \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e). To test our hypotheses, we fit linear mixed-effects models (LMMs) with the \\u0026ldquo;lmer\\u0026rdquo; function from the lme4 package (Bates et al. \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e). We considered log-transformed V̇CO\\u003csub\\u003e2\\u003c/sub\\u003e, log-transformed V̇H\\u003csub\\u003e2\\u003c/sub\\u003eO, \\u003cem\\u003eD\\u003c/em\\u003e\\u003csub\\u003ed\\u003c/sub\\u003e, and \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e as response variables in our models. We fit one model per response variable, while considering test temperature (continuous), season (categorical), sex (categorical), and log-transformed M\\u003csub\\u003eb\\u003c/sub\\u003e (continuous) as fixed terms in all models. We controlled for M\\u003csub\\u003eb\\u003c/sub\\u003e in our analyses to account for body size effects over physiological traits (Glazier \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e) except for the test with \\u003cem\\u003eD\\u003c/em\\u003e\\u003csub\\u003ed\\u003c/sub\\u003e, since information on M\\u003csub\\u003eb\\u003c/sub\\u003e is needed to define \\u003cem\\u003eD\\u003c/em\\u003e\\u003csub\\u003ed\\u003c/sub\\u003e. We included salamander ID as a random term in all models to account for multiple observations per individual. We evaluated model fit with the \\u0026ldquo;check_model\\u0026rdquo; function from the performance package (L\\u0026uuml;decke et al. \\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e), and visualised fixed model effects through the \\u0026ldquo;allEffects\\u0026rdquo; function from the effects package (Fox et al. \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e). For each model, we computed marginal fixed effects with the \\u0026ldquo;ggeffect\\u0026rdquo; function from the ggeffects package (L\\u0026uuml;decke \\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). Finally, we created figures using the ggplot2 (Wickham \\u003cspan citationid=\\\"CR80\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e), ggbeeswarm (Clarke et al. \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e), and ggpubr (Kassambara and Kassambara \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e) packages.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003eWe found that V̇CO\\u003csub\\u003e2\\u003c/sub\\u003e increased with test temperature and was overall higher in the autumn than in the spring (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ea) (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). In both seasons, larger individuals had higher V̇CO\\u003csub\\u003e2\\u003c/sub\\u003e, although this effect was not mediated by sex (Table S1). The thermal sensitivity of mass-specific V̇CO\\u003csub\\u003e2\\u003c/sub\\u003e differed between seasons for the two temperature ranges considered (Spring: Q\\u003csub\\u003e10(5\\u0026ndash;15\\u0026deg;C)\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;1.08, Q\\u003csub\\u003e10(10\\u0026ndash;20\\u0026deg;C)\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;2.34; Autumn: Q\\u003csub\\u003e10(5\\u0026ndash;15\\u0026deg;C)\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;1.54, Q\\u003csub\\u003e10(10\\u0026ndash;20\\u0026deg;C)\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;1.33).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eSummary of body size and physiological variables of \\u003cem\\u003eAmbystoma maculatum\\u003c/em\\u003e studied in the spring and autumn. Values are presented as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard deviation. \\u003cem\\u003eN\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;10 individuals per test temperature in both seasons.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"11\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" class=\\\"colspec\\\" colname=\\\"c7\\\" colnum=\\\"7\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" class=\\\"colspec\\\" colname=\\\"c8\\\" colnum=\\\"8\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" class=\\\"colspec\\\" colname=\\\"c9\\\" colnum=\\\"9\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" class=\\\"colspec\\\" colname=\\\"c10\\\" colnum=\\\"10\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" class=\\\"colspec\\\" colname=\\\"c11\\\" colnum=\\\"11\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c3\\\" namest=\\\"c2\\\"\\u003e \\u003cp\\u003e2\\u0026deg;C\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c5\\\" namest=\\\"c4\\\"\\u003e \\u003cp\\u003e5\\u0026deg;C\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c7\\\" namest=\\\"c6\\\"\\u003e \\u003cp\\u003e10\\u0026deg;C\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c9\\\" namest=\\\"c8\\\"\\u003e \\u003cp\\u003e15\\u0026deg;C\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c11\\\" namest=\\\"c10\\\"\\u003e \\u003cp\\u003e20\\u0026deg;C\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTrait\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eAutumn\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eSpring\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eAutumn\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eSpring\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eAutumn\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eSpring\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003eAutumn\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c9\\\"\\u003e \\u003cp\\u003eSpring\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c10\\\"\\u003e \\u003cp\\u003eAutumn\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c11\\\"\\u003e \\u003cp\\u003eSpring\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eM\\u003csub\\u003eb\\u003c/sub\\u003e (g)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e12.60\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;5.07\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e12.30\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.43\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e12.70\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.44\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e12.20\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.40\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e15.10\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;5.30\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e14.20\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.71\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e11.10\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.58\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c9\\\"\\u003e \\u003cp\\u003e11.20\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.38\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c10\\\"\\u003e \\u003cp\\u003e11.90\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.12\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c11\\\"\\u003e \\u003cp\\u003e11.20\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.32\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eM\\u003csub\\u003eb\\u003c/sub\\u003e loss (%)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0.76\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.44\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.51\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.24\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e2.11\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.16\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e1.04\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.11\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1.79\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.56\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0.95\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.35\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e2.19\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.50\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c9\\\"\\u003e \\u003cp\\u003e1.62\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.26\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c10\\\"\\u003e \\u003cp\\u003e2.76\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c11\\\"\\u003e \\u003cp\\u003e2.74\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.79\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eS\\u003c/em\\u003e (cm\\u003csup\\u003e2\\u003c/sup\\u003e)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e96.20\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;37.50\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e94.30\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;25.40\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e94.30\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;25.50\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e93.60\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;25.30\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e115.09\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;39.00\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e108.00\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;34.80\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e85.60\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;19.20\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c9\\\"\\u003e \\u003cp\\u003e86.70\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;17.70\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c10\\\"\\u003e \\u003cp\\u003e91.50\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;23.10\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c11\\\"\\u003e \\u003cp\\u003e86.30\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;24.70\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eV̇CO\\u003csub\\u003e2\\u003c/sub\\u003e (mL/h)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0.44\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.25\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.34\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.14\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.45\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.21\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0.37\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.19\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0.92\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.53\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0.33\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.11\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e0.65\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.41\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c9\\\"\\u003e \\u003cp\\u003e0.41\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.10\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c10\\\"\\u003e \\u003cp\\u003e0.99\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.52\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c11\\\"\\u003e \\u003cp\\u003e0.62\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.16\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eV̇H\\u003csub\\u003e2\\u003c/sub\\u003eO (mg/h)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1.05\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.30\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.96\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.02\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e6.78\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.37\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e6.62\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.54\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e18.70\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.40\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e17.90\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.30\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e32.30\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.09\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c9\\\"\\u003e \\u003cp\\u003e30.50\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.46\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c10\\\"\\u003e \\u003cp\\u003e49.20\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;5.22\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c11\\\"\\u003e \\u003cp\\u003e51.30\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.86\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003et\\u003c/sub\\u003e (s/cm)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026mdash;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e\\u0026mdash;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e5.78\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.77\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e6.26\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.32\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e5.31\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.54\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e8.00\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.64\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e6.16\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.13\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c9\\\"\\u003e \\u003cp\\u003e9.49\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.21\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c10\\\"\\u003e \\u003cp\\u003e8.05\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.19\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c11\\\"\\u003e \\u003cp\\u003e7.50\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.86\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e (s/cm)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u0026mdash;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e\\u0026mdash;\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e3.35\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.58\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e3.84\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.97\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e2.73\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.21\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e5.42\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.57\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e4.24\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.14\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c9\\\"\\u003e \\u003cp\\u003e7.57\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.15\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c10\\\"\\u003e \\u003cp\\u003e6.91\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.90\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c11\\\"\\u003e \\u003cp\\u003e6.36\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.23\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003ctfoot\\u003e \\u003ctr\\u003e\\u003ctd colspan=\\\"11\\\"\\u003eM\\u003csub\\u003eb\\u003c/sub\\u003e = body mass, \\u003cem\\u003eS\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;surface area, V̇CO\\u003csub\\u003e2\\u003c/sub\\u003e = rates of carbon dioxide production, V̇H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u0026thinsp;=\\u0026thinsp;rates of cutaneous water loss, \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003et\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;total resistance to water loss, \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;skin resistance to water loss.\\u003c/td\\u003e\\u003c/tr\\u003e \\u003c/tfoot\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eTest temperature was the only factor affecting V̇H\\u003csub\\u003e2\\u003c/sub\\u003eO, with salamanders losing more water through evaporation with increasing test temperature (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eb) (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). Neither season, sex, nor M\\u003csub\\u003eb\\u003c/sub\\u003e explained the variation in V̇H\\u003csub\\u003e2\\u003c/sub\\u003eO (Table S2). Time until desiccation decreased as a function of temperature regardless of season and sex (Table S3) (mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard deviation; 2\\u0026deg;C\\u0026thinsp;=\\u0026thinsp;266.73\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;117.51 days, 5\\u0026deg;C\\u0026thinsp;=\\u0026thinsp;11.47\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;14.66 days, 10\\u0026deg;C\\u0026thinsp;=\\u0026thinsp;8.76\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;5.91 days, 15\\u0026deg;C\\u0026thinsp;=\\u0026thinsp;4.31\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.05 days, 20\\u0026deg;C\\u0026thinsp;=\\u0026thinsp;2.96\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.68 days). Although M\\u003csub\\u003eb\\u003c/sub\\u003e loss increased with test temperature, salamanders were never close to reaching their desiccation limit in our experiments (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). Both test temperature and season affected \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e, but with no contribution of sex or M\\u003csub\\u003eb\\u003c/sub\\u003e (Table S4). Specifically, \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e increased in response to temperature and was overall higher in the spring than in the autumn (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eIn this study, we addressed the impact of seasonal acclimatisation on the thermal sensitivity of energy and water budgets in a fossorial salamander. After accounting for body size and sex, we found partial support for the hypothesis that temperature would have both short- and long-term effects over metabolism and water loss. Both SMR and \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e increased with test temperature and differed between the spring and autumn, while EWL increased with test temperature but did not differ between seasons. We also demonstrated that the length of time necessary to reach the desiccation limit decreased as a function of temperature in \\u003cem\\u003eA\\u003c/em\\u003e. \\u003cem\\u003emaculatum\\u003c/em\\u003e. Together, our results demonstrate that thermal effects over physiology may occur through active plasticity and in response to acute changes in temperature. Such effects, however, may be trait-dependent. Furthermore, our study highlights that fossorial species may have different energetic and hydroregulatory requirements between seasons despite the prevailing assumption that fossorial ectotherms are buffered from thermal effects.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eThermal and seasonal effects over metabolism\\u003c/h2\\u003e \\u003cp\\u003eIn ectotherms, thermally-induced shifts in metabolism are the outcome of passive thermodynamic effects on the biochemical reactions that underpin energy turnover (Andrade \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e). The relationship between SMR and temperature has been assessed in several species of ectotherms (see Carter et al. \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). As expected, we found a positive relationship between SMR and temperature (Gatten Jr et al. \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e1992\\u003c/span\\u003e), supporting the idea that an increase in temperature leads to an increase in the baseline costs for body maintenance. In our study, M\\u003csub\\u003eb\\u003c/sub\\u003e explained most of the variation in SMR along with temperature. Larger individuals had higher SMR, in concert with Kleiber\\u0026rsquo;s law and the theory of metabolic scaling (Schmidt-Nielsen \\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e1970\\u003c/span\\u003e; White et al. \\u003cspan citationid=\\\"CR77\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). Our SMR values are similar to those previously reported for \\u003cem\\u003eA\\u003c/em\\u003e. \\u003cem\\u003emaculatum\\u003c/em\\u003e and other caudates (Whitford and Hutchison \\u003cspan citationid=\\\"CR78\\\" class=\\\"CitationRef\\\"\\u003e1963\\u003c/span\\u003e; Gatten Jr et al. \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e1992\\u003c/span\\u003e), although we did not find differences in energy expenditure between sexes (Finkler et al. \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eWe found that SMR differed between seasons, with lower energy expenditure in the spring than in the autumn across all test temperatures. SMR followed a pattern of inverse compensation (Hazel and Prosser \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e1970\\u003c/span\\u003e), which is often attributed to the combined effect of passive and active plasticity over metabolism. In this scenario, SMR would increase with temperature through passive effects and this response would be amplified by active plasticity (Havird et al. \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). Lowered SMR implies reduced energetic demand and expenditure (Giacometti et al. \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e), and sustaining low costs for body maintenance in the spring can be particularly beneficial for \\u003cem\\u003eA\\u003c/em\\u003e. \\u003cem\\u003emaculatum\\u003c/em\\u003e. Indeed, \\u003cem\\u003eA\\u003c/em\\u003e. \\u003cem\\u003emaculatum\\u003c/em\\u003e breed in the spring after overwintering underground for months (Madison \\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e1997\\u003c/span\\u003e). Since \\u003cem\\u003eA\\u003c/em\\u003e. \\u003cem\\u003emaculatum\\u003c/em\\u003e do not feed while breeding (Smallwood \\u003cspan citationid=\\\"CR66\\\" class=\\\"CitationRef\\\"\\u003e1928\\u003c/span\\u003e), salamanders are presumed to rely on energy stores obtained in the previous activity season to sustain performance. Thus, maintaining relatively low SMR in the spring could allow salamanders to allocate energy toward overwintering emergence, breeding migration, and reproduction without depleting energy reserves. This proposition is bolstered by our finding that spring-acclimatised salamanders had a lower Q\\u003csub\\u003e10\\u003c/sub\\u003e for SMR between 5\\u0026deg;C and 15\\u0026deg;C than between 10\\u0026deg;C and 20\\u0026deg;C. The former range of temperatures is more common at the onset of the breeding season (Moldowan et al. \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e), indicating that compensatory responses may have important fitness consequences (Packard \\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e1972\\u003c/span\\u003e; Havird et al. \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eHigher SMR in the autumn can be explained by overall warmer conditions and the fact that individuals forage to replenish energy stores in preparation for the winter (Faccio \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e; Moldowan et al. \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). Thus, our data suggest that functional differences between seasons (i.e., breeding in the spring, provisioning in the autumn) are mediated by seasonal changes in energetic requirements. Importantly, we observed overall low Q\\u003csub\\u003e10\\u003c/sub\\u003e values for \\u003cem\\u003eA\\u003c/em\\u003e. \\u003cem\\u003emaculatum\\u003c/em\\u003e in both spring and autumn. In amphibians, low metabolic sensitivity is typically ascribed to tropical instead of temperate-zone species (Kreiman et al. \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). However, blunted thermal sensitivity has been suggested as one of the physiological imprints of fossoriality in ectotherms (Bars-Closel et al. \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e) due to the thermal stability of underground environments. From an integrative perspective, the low thermal sensitivity of metabolism in \\u003cem\\u003eA\\u003c/em\\u003e. \\u003cem\\u003emaculatum\\u003c/em\\u003e corroborates the thermal generalist nature suggested for this species. Truly, \\u003cem\\u003eA\\u003c/em\\u003e. \\u003cem\\u003emaculatum\\u003c/em\\u003e are active across a broad thermal range (including sub-zero temperatures) (Giacometti and Tattersall \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e), which is possibly facilitated by their low energetic requirements and low thermal sensitivity across temperatures. While low metabolic sensitivity to temperature has been reported in other taxa (Fitzpatrick \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e1973\\u003c/span\\u003e; Gatten Jr et al. \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e1992\\u003c/span\\u003e; Bars-Closel et al. \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e), we still have a limited understanding of the suite of physiological and behavioural traits that allow fossorial ectotherms to sustain performance across a broad thermal range.\\u003c/p\\u003e \\u003cp\\u003e \\u003cem\\u003eThermal effects over water loss and the role of the skin in limiting evaporation between seasons\\u003c/em\\u003e \\u003c/p\\u003e \\u003cp\\u003eOur data supported the well-known pattern of EWL increasing with temperature in amphibians (Boutilier et al. \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e1992\\u003c/span\\u003e; Shoemaker et al. \\u003cspan citationid=\\\"CR65\\\" class=\\\"CitationRef\\\"\\u003e1992\\u003c/span\\u003e). This effect is mediated by an increase in WVP\\u003csub\\u003esat\\u003c/sub\\u003e at the skin interface with the air, which leads to higher VPD and greater evaporative demand at warmer than cooler temperatures (Riddell et al. \\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). Although body size and shape may contribute to EWL, stout-bodied salamanders like \\u003cem\\u003eA\\u003c/em\\u003e. \\u003cem\\u003emaculatum\\u003c/em\\u003e typically have lower EWL than slender and cylindrical ones (Spight \\u003cspan citationid=\\\"CR67\\\" class=\\\"CitationRef\\\"\\u003e1968\\u003c/span\\u003e), which could explain why M\\u003csub\\u003eb\\u003c/sub\\u003e did not affect EWL in our study. Additionally, we used our EWL measurements to determine how long it would take for \\u003cem\\u003eA\\u003c/em\\u003e. \\u003cem\\u003emaculatum\\u003c/em\\u003e to reach their desiccation limit. Increasing temperatures resulted in a shorter time until desiccation in both spring and autumn, with salamanders expected to desiccate in less than three days at 20\\u0026deg;C and a VPD of 1.658 kPa. By contrast, \\u003cem\\u003eA\\u003c/em\\u003e. \\u003cem\\u003emaculatum\\u003c/em\\u003e can persist within their desiccation tolerance for over 260 days at 2\\u0026deg;C and a VPD of 0.025 kPa; these conditions are inferred to be found in overwintering burrows (Moldowan et al. \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). Although \\u003cem\\u003eA\\u003c/em\\u003e. \\u003cem\\u003emaculatum\\u003c/em\\u003e may lose up to 14% of M\\u003csub\\u003eb\\u003c/sub\\u003e through evaporation when active under humid conditions (Giacometti and Tattersall \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e), terrestrial amphibians are generally more tolerant of dehydration than aquatic ones (Shoemaker et al., \\u003cspan citationid=\\\"CR65\\\" class=\\\"CitationRef\\\"\\u003e1992\\u003c/span\\u003e; Thorson, \\u003cspan citationid=\\\"CR72\\\" class=\\\"CitationRef\\\"\\u003e1955\\u003c/span\\u003e; Young et al., \\u003cspan citationid=\\\"CR83\\\" class=\\\"CitationRef\\\"\\u003e2005\\u003c/span\\u003e). In turn, this could explain why neither time to desiccation or EWL differed between the spring and autumn in our study.\\u003c/p\\u003e \\u003cp\\u003eConstancy of EWL between season suggests that salamanders should rely on modulation of \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e and behaviour to mitigate possible detrimental effects of warming over the maintenance of water balance (Davies et al. \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e). Our analyses showed that \\u003cem\\u003eA\\u003c/em\\u003e. \\u003cem\\u003emaculatum\\u003c/em\\u003e increased \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e in response to temperature, with overall higher \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e in the spring than in the autumn. The mechanisms that underlie \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e enhancement at warmer temperatures are still unclear, but could involve suppression of blood flow to capillaries underneath the epidermis or the thickening of chromatophore layers in the skin (Toledo and Jared \\u003cspan citationid=\\\"CR73\\\" class=\\\"CitationRef\\\"\\u003e1993\\u003c/span\\u003e; Lillywhite \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e2006\\u003c/span\\u003e). Although salamanders do not secrete waterproofing mucus or build cocoons to limit EWL like some anurans (Withers \\u003cspan citationid=\\\"CR81\\\" class=\\\"CitationRef\\\"\\u003e1998\\u003c/span\\u003e; Kr\\u0026ouml;ner et al. \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e), we provide evidence that \\u003cem\\u003eA\\u003c/em\\u003e. \\u003cem\\u003emaculatum\\u003c/em\\u003e may actively modulate \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e in response to short- and long-term changes in temperature. The finding that salamanders maintained overall higher \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e in the spring than in the autumn can be attributed to exposure to cooler acclimatisation conditions in the spring. Alternatively, the maintenance of low SMR in spring could lead to a coordinated lowering of blood flow to the skin which may contribute to \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e enhancement. Salamanders never assumed a water-conserving posture (Semlitsch \\u003cspan citationid=\\\"CR60\\\" class=\\\"CitationRef\\\"\\u003e1983\\u003c/span\\u003e) during our trials, which lends further support to the proposition that \\u003cem\\u003eAmbystoma\\u003c/em\\u003e species have a high tolerance to dehydration induced by EWL (Hall \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e1922\\u003c/span\\u003e; Thorson \\u003cspan citationid=\\\"CR72\\\" class=\\\"CitationRef\\\"\\u003e1955\\u003c/span\\u003e). Nonetheless, further work is still necessary to clarify the behavioural mechanisms through which salamanders maintain water balance, especially in fossorial species.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Conclusions\",\"content\":\"\\u003cp\\u003eTemperature impacts the physiology and behaviour of ectotherms through both acute and prolonged effects (Tattersall et al. \\u003cspan citationid=\\\"CR71\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e). In \\u003cem\\u003eA\\u003c/em\\u003e. \\u003cem\\u003emaculatum\\u003c/em\\u003e, short-term changes in temperature led to an increase in SMR, EWL, and \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e. Moreover, SMR and \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e differed between seasons, but EWL did not. By maintaining relatively low SMR and high \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e in the spring, \\u003cem\\u003eA\\u003c/em\\u003e. \\u003cem\\u003emaculatum\\u003c/em\\u003e may be able to allocate energy toward overwintering emergence and breeding while minimising water conservation. By contrast, relatively high SMR and low \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e in the autumn could be associated with aboveground foraging on rainy nights (i.e., low evaporative demand) to replenish energy stores before the winter (Faccio \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e; Moldowan et al. \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). Despite the prevailing assumption that fossorial ectotherms are buffered from thermal effects (Giacometti and Tattersall \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e), our study demonstrates that energetic and hydroregulatory requirements respond to both short- and long-term changes in temperature in the fossorial \\u003cem\\u003eA\\u003c/em\\u003e. \\u003cem\\u003emaculatum\\u003c/em\\u003e. Ultimately, our study contributes to the goal of elucidating the thermal dependency of physiology in fossorial ectotherms (Bars-Closel et al. \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e).\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003ch2\\u003eFunding:\\u003c/h2\\u003e \\u003cp\\u003eDG was funded by a Doris White Memorial Bursary provided by Brock University. GJT was funded by a Natural Sciences and Engineering Research Council of Canada Discovery Grant (RGPIN-2020-05089).\\u003c/p\\u003e \\u003cp\\u003e \\u003cstrong\\u003e \\u003cem\\u003eEthics approval\\u003c/em\\u003e:\\u003c/strong\\u003e \\u003cp\\u003e All procedures were performed with permission from the Ministry of Northern Development, Mines, Natural Resources and Forestry (#1100575), Ontario Parks, and Brock University\\u0026rsquo;s Animal Care Committee (Animal Use Protocol #22-03-04).\\u003c/p\\u003e \\u003c/p\\u003e\\u003ch2\\u003eAuthors\\u0026rsquo; contribution:\\u003c/h2\\u003e \\u003cp\\u003eDG and GJT conceived the ideas and designed methodology; DG collected the data; DG analysed the data with input from GJT; DG and GJT led the writing of the manuscript. Both authors contributed critically to the drafts and gave final approval for publication.\\u003c/p\\u003e \\u003cp\\u003e \\u003cstrong\\u003e \\u003cem\\u003eConflicts of interest\\u003c/em\\u003e:\\u003c/strong\\u003e \\u003cp\\u003eNone declared.\\u003c/p\\u003e \\u003c/p\\u003e\\u003ch2\\u003eAcknowledgements\\u003c/h2\\u003e \\u003cp\\u003eWe thank Ontario Parks, the Ministry of Northern Development, Mines, Natural Resources and Forestry, the Algonquin Wildlife Research Station, Njal Rollinson, Kevin Kemmish, and Patrick Moldowan for facilitating access to our study animals. We thank Shawn Bukovac, Kristin Bray, Sarah Kehoe, and Natasha Hearn for assistance with animal care. We are grateful to Zak Mason from Brock University\\u0026rsquo;s Makerspace and Alex Popescu for building the 3D printed models used in our study.\\u003c/p\\u003e\\u003ch2\\u003eAvailability of data\\u003c/h2\\u003e \\u003cp\\u003e \\u003cem\\u003eand code\\u003c/em\\u003e: Our data and code can be accessed from Brock University Dataverse: \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.5683/SP3/MQPFHT\\u003c/span\\u003e\\u003cspan address=\\\"10.5683/SP3/MQPFHT\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eAnderson DB (1936) Relative humidity or vapor pressure deficit. 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Physiol Biochem Zool 78:847\\u0026ndash;856. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1086/432152\\u003c/span\\u003e\\u003cspan address=\\\"10.1086/432152\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":true,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"Brock University\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":true,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Acclimatisation, amphibian, energy budgets, skin resistance, water budgets\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-5478984/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-5478984/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eEctotherms 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 acclimatisation (spring vs. autumn) affected the thermal sensitivity of standard metabolic rates (SMR), rates of evaporative water loss (EWL), and skin resistance to water loss (\\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e) in the spotted salamander (\\u003cem\\u003eAmbystoma maculatum\\u003c/em\\u003e). We hypothesised that temperature would have both short- and long-term effects over traits (i.e., acute exposure to test temperatures and seasonal acclimatisation, respectively). After accounting for body mass and sex, we found that short-term changes in temperature led to an increase in SMR, EWL, and \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e. Additionally, SMR and \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e differed between seasons, but EWL did not. Sustaining low SMR and high \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e in the spring may allow salamanders to allocate energy toward overwintering emergence and breeding while simultaneously maximising water conservation. By contrast, maintaining high SMR and low \\u003cem\\u003eR\\u003c/em\\u003e\\u003csub\\u003es\\u003c/sub\\u003e in the autumn may allow salamanders to forage aboveground on rainy nights to replenish energy reserves in preparation for the winter. 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 hydroregulatory requirements.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Seasonal plasticity in the thermal sensitivity of metabolism but not water loss in a fossorial ectotherm\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-01-10 04:35:31\",\"doi\":\"10.21203/rs.3.rs-5478984/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"de94b91a-3223-470b-81db-42f3a9b4fc51\",\"owner\":[],\"postedDate\":\"January 10th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[{\"id\":40435152,\"name\":\"Animal Physiology\"}],\"tags\":[],\"updatedAt\":\"2025-01-10T04:35:31+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-01-10 04:35:31\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-5478984\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-5478984\",\"identity\":\"rs-5478984\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}