Within host-microbiome-pathogen systems, the host chemical microenvironment is often overlooked despite its inherent role in host physiology. We used a multifaceted experimental approach encompassing culture-dependent and -independent methods, genomic and metagenomic analyses, and deep neural network modeling to assess the impact of host skin chemistry and the bacterial microbiome on the growth of Ophidiomyces ophidiicola (ophidiomycosis, snake fungal disease). Results suggest that host skin lipid chemistry (e.g., oleic acid, squalene) and bacteria isolated from wild snake skins (e.g., Chryseobacterium sp. and Stenotrophomonas maltophilia) suppress O. ophidiicola growth. Notably, the O. ophidiicola genome contains biosynthetic gene clusters (BGCs) that encode metabolites that may suppress host lipid production, facilitating fungal pathogenicity. A contrastive deep neural network produced a near-perfect alignment of snake skin lipid and microbiome profiles for both individual snakes and disease states. BGCs from bacterial genomes isolated from snake skin overlap with metagenome profiles from wild snakes and are associated with disease state. We highlight antifungal activity found in the diverse lipid milieu of snake skin and bacterial-fungal interactions that structure the skin microbiome. Our results illustrate a strong relationship among a fungal pathogen, the microbiome, and host skin lipid chemistry that may underpin disease susceptibility. Deep learning models accurately align multimodal data including snake skin lipid chemistry and microbiome-pathogen interactions in the snake fungal disease system.

Balancing survival and reproduction presents a fundamental evolutionary challenge, especially in extreme and unpredictable environments. Thermoregulatory behavior in particular imposes a costly trade-off, as time spent maintaining optimal body temperature precludes other essential activities and forces individuals to balance competing selective pressures. By combining field observations, behavioral assays, and gene expression profiling of red-sided garter snakes ( Thamnophis sirtalis parietalis ), we describe how an ectothermic vertebrate navigates this trade-off in an extreme thermal environment through a combination of finely tuned mechanisms of behavioral and physiological plasticity. Snakes demonstrated remarkably consistent critical thermal limits, with minimal individual variation, suggesting a hard physiological constraint. Importantly, behavioral temperature thresholds for voluntary withdrawal and courtship cessation occurred within 5 °C of lethal, demonstrating that males routinely operate perilously close to lethal body temperatures in order to maximize reproductive opportunities. Context-dependent plasticity allows males to prioritize mating opportunities over thermoregulation until survival becomes an immediate threat. Molecular analyses of multiple tissues revealed rapid, tissue-specific transcriptomic responses activated within one hour of thermal challenge, well before critical limits were reached. Heat shock proteins were universally upregulated across all tissues under both heat and cold stress, suggesting anticipatory protective mechanisms that support risky behavioral decisions rather than simply responding to thermal damage. Tissue-specific patterns of gene expression reflected functional priorities with liver showing extensive metabolic flexibility, heart maintaining conservative stability, while brain and testis appeared to balance critical functions with stress responses. These findings demonstrate precise thermal reaction norms that integrate behavioral and molecular plasticity to maximize reproductive effort without compromising survival. The narrow margin between behavioral thresholds and lethal limits suggests this system may already be approaching evolutionary constraints. While phenotypic plasticity currently buffers populations against extreme thermal variability, it may paradoxically limit long-term adaptive potential as increasing environmental temperatures further narrow safety margins. Precise thermal decision-making, rapid physiological responses, and context-dependent behavioral switching suggest an evolved solution to the problem of balancing competing selective pressures.

Abstract Long-term winter dormancy in ectotherms (brumation) defines the annual cycle of many temperate-zone reptiles, yet the transcriptional regulation that supports survival across months of cold and aphagy remains poorly understood. We generated time-resolved transcriptomic profiles of liver and testis from male red-sided garter snakes ( Thamnophis sirtalis parietalis ) at five timepoints spanning the eight-month brumation cycle: pre-brumation, early, mid-, and late brumation, and post-arousal under continued aphagy. Time-course negative-binomial regression (maSigPro) followed by gene-set enrichment analysis identified 3,715 transcripts in liver and 5,828 in testis with significant temporal expression structure organized into five overarching temporal patterns: sustained downregulation, downregulation with post-arousal recovery, sustained upregulation, brumation-specific upregulation and cyclic modulation. Liver showed coordinated upregulation of fatty acid mobilization enzymes (ATGL, FOXO1, PPARα, CPT1A) and gluconeogenic regulators (CREBBP, PCK1) coincident with sustained low temperatures. Additionally, low temperature transcriptional activity was suggestive of a shift toward hepatic lipid mobilization and alanine-supported gluconeogenesis. Testis showed sustained suppression of meiosis, reproduction, and DNA-metabolism gene sets that did not fully recover at arousal consistent with this species’ dissociated reproductive pattern. Both tissues showed coordinated upregulation of stress-response pathways involving heat-shock proteins, HIF1α and a glutathione-based antioxidant defense. Interestingly, three vitellogenin transcripts and 17β-hydroxysteroid dehydrogenases associated with estradiol-favoring steroid metabolism were upregulated in male liver during late brumation, which is not expected during natural physiology in adult males. Together these data support a framework in which temperature- and starvation-associated transcriptional programs contribute to survival of one of the longest, coldest brumations documented in a squamate. Summary statement A time-resolved transcriptomic analysis of liver and testis spanning eight months of winter brumation in Thamnophis sirtalis parietalis reveals gene expression patterns consistent with a temperature-associated shift toward hepatic lipid mobilization, sustained reproductive suppression, and vitellogenin response in males.

Within host-microbiome-pathogen systems, the host chemical microenvironment is often overlooked despite its inherent role in host physiology. We used a multifaceted experimental approach encompassing culture-dependent and independent methods, metagenomic and genomic data, and deep neural network modeling to assess the impact of host skin lipid chemistry and the bacterial microbiome on the growth of Ophidiomyces ophidiicola (ophidiomycosis, snake fungal disease). Results suggest that host skin lipid chemistry (e.g., oleic acid, squalene) and bacteria isolated from wild snake skins (e.g., Chryseobacterium sp. and Stenotrophomonas maltophilia ) suppress O. ophidiicola growth. Notably, the O. ophidiicola genome contains biosynthetic gene clusters (BGCs) that encode metabolites that may suppress host lipid production, facilitating fungal pathogenicity. The contrastive deep neural network produced a near-perfect alignment of snake skin lipid and microbiome profiles for both individual snakes and disease states. BGCs from bacterial genomes isolated from snake skin overlap with metagenome profiles from wild snakes and correlate with disease state. We highlight antifungal activity found in the diverse lipid milieu of snake skin and bacterial-fungal interactions (BFIs) that structure the skin microbiome. Our results illustrate a strong relationship among a fungal pathogen, the microbiome, and host skin lipid chemistry. ### Competing Interest Statement The authors have declared no competing interest. U.S. National Science Foundation, https://ror.org/021nxhr62, EF-2125065, CAREER 2236580, REU 1757493, DEB 1933925 Tennessee Wildlife Resources Agency, State Wildlife Grant #58209 Middle Tennessee State University, https://ror.org/02n1hzn07 Fulbright Visiting Scholar Program University of Florida, https://ror.org/02y3ad647 US Department of Agriculture, 58‐6066‐4‐047, 6066‐21310‐005‐052‐S

As global temperatures continue to rise, understanding the impacts of warming environments has become increasingly important. Temperature is especially relevant for ectothermic organisms which depend upon consistent and predictable annual temperature cycles for reproduction and development. However, additional research is required in this area to elucidate the potential impacts of climate change on future generations. To understand how projected increases in environmental temperatures may impact reproductive outcomes within natural populations of ectothermic vertebrates, we manipulated minimum ambient temperatures during gestation in Red-sided garter snakes (Thamnophis sirtalis parietalis). Wild snakes were collected in the Interlake region of Manitoba, Canada during their spring mating season and allowed to mate in controlled conditions. For the duration of gestation, mated females were placed into one of two ambient thermal conditions: temperatures emulating those found in the species' natural habitat or temperatures with a consistent 5 °C increase to match end-of-century climate change projections. We recorded observations for each litter and all neonates resulting from controlled mating trials. We observed no difference in litter sizes or birth rates between thermal conditions. However, we observed a significant reduction in gestation length and significant increase to neonate body mass and body condition associated with increased ambient temperatures. These results suggest that increased minimum temperatures during gestation may confer reproductive benefits for the northern populations of this species even under the most extreme current modeled warming predictions. We discuss the broader implications of this effect, including possible negative ecological outcomes.

The hydration state of animals vying for reproductive success may have implications for the tempo and mode of sexual selection, which may be salient in populations that experience increasing environmental fluctuations in water availability. Using red-sided garter snakes as a model system, we tested the effect of water supplementation on courtship, mating behavior, and copulatory plug (CP) production during a drought year. Over 3 days of mating trials, water-supplemented males (WET males, n = 45) outperformed a control group that was not supplemented with water (DRY males, n = 45). Over 70% of WET males mated but just 33% of DRY males did so. As a group, WET males mated 79 times versus 28 times by DRY males. On the last day of mating trials, over 70% of WET males were still courting, with 19 of them mating, whereas less than 25% of DRY males were courting and only one mated. CP deposition accounted for 4-6% of the mass lost by mating males, but hydration did not affect CP mass or water content. These findings suggest that, in years of low water availability, the number of courting males and the intensity of their courtship declines, thereby affecting sexual selection and conflict, at least within that year. Water availability may have profound effects on the evolution of sexually selected traits. We show that hydration state predicts mating success, and persistence of courtship during a drought year. Our work suggests that strength of sexual selection on males fluctuates dramatically between years and is very likely to change with climate change.

In lizards and snakes, the kidneys produce seminal fluid in addition to having osmoregulatory functions. Therefore, in response to polyandry, kidney mass should be under selection regimes such as those influencing testes. Male red-sided garter snakes deposit a kidney-derived copulatory plug that functions in sperm competition. We first tested for intersexual differences in kidney mass and allometry in one population and found that males had kidneys twice as heavy as those of females, consistent with stronger selection on male kidney mass. Previous studies have shown that male size enhances mating success in one-on-one competition prevalent in small mating aggregations. We then examined the relationship between body size, kidney mass and testes mass in two populations with low (LD) and high (HD) mating aggregation densities. Males from the HD population had heavier testes and heavier kidneys compared with males from the LD population; HD males were also smaller in body size. Our results suggest that the strength of sexual selection, and possibly the balance between pre- and postcopulatory selection, varies in response to population size or density. However, more replication is needed to confirm the generality of these results within red-sided garter snakes and other squamate reptiles.

Telomeres, tandem repeats of TTAGGG at the ends of chromosomes, are highly dynamic structures that shorten in response to a variety of factors, including organismal stress and tissue-specific growth rates. Cell turnover rates are frequently linked to their functions, resource availability and telomere dynamics. Using male red-sided garter snakes, Thamnophis sirtalis parietalis, as a model, we investigated the relationship between telomere length in sperm cells, blood cells telomere length and a growth proxy (age-adjusted body length and mass). This relationship is interesting because snakes exhibit indeterminate growth and because these garter snakes have a dissociated reproductive cycle where spermatogenesis occurs months prior to the mating season. In this study, we determined sperm telomere length (STL) and male age using qPCR and skeletochronology, respectively. Sperm telomere length correlated positively with snout-vent length (SVL) and with age-adjusted SVL as a proxy for growth rate (residuals of size against age regression, hereafter growth), but not with age. Although an individual's STL is correlated with blood telomere length (BTL), sperm telomeres are 60% longer than blood telomeres. In previous work, we have shown that BTL is shorter in older males and unrelated to SVL or any growth rate proxies. We hypothesized that STL is related to growth and SVL because growth and sperm production both occur during summer when resources are most abundant and stress lowest. This study is the first to compare telomere dynamics between cell types in a snake and supports growing evidence that telomere dynamics may be highly tissue-specific and driven by the life-history strategy of an organism.

Reptiles signal to conspecifics using lipids in their skin, primarily to enable mate tracking and assessment. The isolation of these lipids has utility in research focused on evolutionary patterns and mechanisms of chemical communication, in addition to understanding the waterproofing role of lipids in the evolution of terrestrial life. In an applied approach, such skin-based cues have potential use for wildlife managers dealing with invasive species. The main steps for quantifying reptile skin lipids in the protocol presented here include extraction, total lipid determination, and fractionation via column chromatography, the latter process resulting in purified eluates of compounds which can then either be analyzed to assign compound identifications (e.g., gas chromatography-mass spectrometry [GC-MS]) and/or used directly in more refined bioassays. Skin lipids can be extracted from living skin, shed skin, or dead whole animals, using nonpolar organic solvents (e.g., hexane, benzene, toluene). Extraction solubilizes the lipids and, then, the solvent can be evaporated to yield a measurable lipid-only extract. Fractionation involves the separation of the total lipid extract into specific eluates via traditional column chromatography. The total lipid extract is first bound to a substrate-based column (e.g., alumina) and, then, individual eluates ("fractions") of solvent at specific volumes are passed sequentially through the column to elute sets of compounds from the lipid mixture based on common polarity. The fractions progress in polarity at a standardized sequence by increasing the relative amount of polar solvent (e.g., diethyl ether) in nonpolar solvent. In this manuscript, we describe several methods for extracting skin lipids of reptiles and, then, provide a standard protocol for isolating different sets of compounds based on polarity, using traditional column chromatography. Whole lipid extracts or specific fractions can, then, be used in bioassays to determine any biological activity elicited by the compounds therein.

Life-history strategies vary dramatically between the sexes, which may drive divergence in sex-specific senescence and mortality rates. Telomeres are tandem nucleotide repeats that protect the ends of chromosomes from erosion during cell division. Telomeres have been implicated in senescence and mortality because they tend to shorten with stress, growth and age. We investigated age-specific telomere length in female and male red-sided garter snakes, Thamnophis sirtalis parietalis . We hypothesized that age-specific telomere length would differ between males and females given their divergent reproductive strategies. Male garter snakes emerge from hibernation with high levels of corticosterone, which facilitates energy mobilization to fuel mate-searching, courtship and mating behaviours during a two to four week aphagous breeding period at the den site. Conversely, females remain at the dens for only about 4 days and seem to invest more energy in growth and cellular maintenance, as they usually reproduce biennially. As male investment in reproduction involves a yearly bout of physiologically stressful activities, while females prioritize self-maintenance, we predicted male snakes would experience more age-specific telomere loss than females. We investigated this prediction using skeletochronology to determine the ages of individuals and qPCR to determine telomere length in a cross-sectional study. For both sexes, telomere length was positively related to body condition. Telomere length decreased with age in male garter snakes, but remained stable in female snakes. There was no correlation between telomere length and growth in either sex, suggesting that our results are a consequence of divergent selection on life histories of males and females. Different selection on the sexes may be the physiological consequence of the sexual dimorphism and mating system dynamics displayed by this species.

Male reproductive success is dependent on a correlated suite of traits related to a species' ecology and mating system dynamics. Closely related species differing in their mating systems and ecology, such as the garter snakes (Thamnophis), are ideal for studying the correlated evolution of sexually selected traits. Here, we compare the degree of sexual size dimorphism (SSD), copulatory behavior, copulatory plug size, and traits associated with sperm competition between two sympatric and closely related Thamnophis species, T. sirtalis and T. radix with divergent mating aggregation size and density. Our findings indicate that T. sirtalis has greater female-biased SSD, shorter copulations, and larger, more strongly adhering copulatory plugs than T. radix. Our finding that T. sirtalis have longer sperm and higher numbers of sperm per ejaculate is further evidence of more intense sperm competition in this species than in T. radix. However, this reduced number of sperm in the ejaculate means that T. radix males are likely capable of more matings per season than T. sirtalis. This result may reflect differences in feeding during the breeding season (obligate aphagy in T. sirtalis) and the potential for sperm loss in T. radix during prolonged copulations that are prevented in T. sirtlais by their substantial copulatory plugs. Our findings demonstrate that ecological and mating system dynamics have the capacity to strongly influence correlated selection of pre- and postcopulatory traits.

Reproductive effort is an important aspect of life history as reproductive success is arguably the most important component of fitness. Males tend to compete for access to females and, in the process, expend their energetic capital on mate searching, male-male competition and courtship rather than directly on offspring. Red-sided garter snakes, Thamnophis sirtalis parietalis, are an exceptional model for studying energetic costs of courtship and mating as they fast during the spring mating season, which segregates the cost of energy acquisition from the cost of courtship and mating. However, measuring an individual male's metabolic rate during courtship is complicated by the fact that male courtship behaviour in red-sided garter snakes is dependent on both the detection of a female sexual attractiveness pheromone and on facilitated courtship (i.e. vigorous courtship is only exhibited in the presence of other males). Thus, traditional techniques of placing a mask over the head of individuals would prevent male courtship behaviour, and single animals placed in a flow-through chamber would not yield ecologically realistic levels of courtship, which are only seen in the context of a mating aggregation in this species. Because of these difficulties, we placed groups of males in a flow-through metabolic chamber together with a single female whose respiratory gases were vented outside the chamber to yield a whole-group metabolic rate during competitive courtship. We also measured the standard metabolic rates (SMR) of the males individually for comparison with active metabolic rates. Conservative estimates of peak group metabolic rates during courtship are 10-20 times higher than resting group metabolic rate, which was 1.88 times higher than SMR. These measurements, coupled with the fact that these males are aphagous during the breeding, indicates that costs of courtship may be high for males and has implications for the male mating tactics in this system. (C) 2017 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.