Current and time-lagged effects of climate on innate immunity in two sympatric snake species.

Changing environments result in alterations at all levels of biological organization, from genetics to physiology to demography. The increasing frequency of droughts worldwide is associated with higher temperatures and reduced precipitation that can impact population persistence via effects on individual immune function and survival.We examined the effects of annual climate variation on immunity in two sympatric species of garter snakes from four populations in California over a seven-year period that included the record-breaking drought.We examined three indices of innate immunity: bactericidal competence (BC), natural antibodies (NABs), and complement-mediated lysis (CL).Precipitation was the only climatic variable explaining variation in immune function: spring precipitation of the current year was positively correlated to Thamnophis sirtalis BC and NABs, whereas spring precipitation of the previous year was positively correlated to T. elegans BC and NABs. This suggests that T. elegans experiences a physiological time-lag in response to reduced precipitation, which may reflect lack of capital for investment in immunity in the year following a dry year.In general, our findings demonstrate compelling evidence that climate can influence wild populations through effects on physiological processes, suggesting that physiological indices such as these may offer valuable opportunities for monitoring the effects of climate.

Embryonic heart rate correlates with maternal temperature and developmental stage in viviparous snakes.

Interactions between the environment and maternal and embryonic physiology can have critical ramifications for early-life phenotypes and survival in a range of species. A major component of the environment-maternal-embryonic nexus is the regulation of embryonic heart rate, which can have important ramifications for developmental phenology, but remains relatively unexplored in viviparous reptiles. The goal of this study was to test for a relationship between embryonic heart rate and maternal body temperature in two species of viviparous garter snakes. The embryonic heart rates of Thamnophis elegans and T. sirtalis were assessed using a field-portable ultrasound. For both T. elegans and T. sirtalis, embryonic heart rate was strongly correlated to maternal temperature. Interestingly, there was also a strong correlation between embryonic and maternal heart rate that was most likely mediated by a common response to maternal body temperature, in spite of the effects of handling during ultrasound on maternal heart rate. Furthermore, embryos at earlier developmental stages had lower heart rates. To our knowledge, this study is the first to explore embryonic heart rate in viviparous reptiles, providing a foundation for future work using ultrasonography to test ecological and evolutionary hypotheses related to developmental dynamics in free-ranging viviparous species.

The untapped potential of reptile biodiversity for understanding how and why animals age.

1. The field of comparative aging biology has greatly expanded in the past 20 years. Longitudinal studies of populations of reptiles with a range of maximum lifespans have accumulated and been analyzed for evidence of mortality senescence and reproductive decline. While not as well represented in studies of amniote senescence, reptiles have been the subjects of many recent demographic and mechanistic studies of the biology of aging. 2. We review recent literature on reptile demographic senescence, mechanisms of senescence, and identify unanswered questions. Given the ecophysiological and demographic diversity of reptiles, what is the expected range of reptile senescence rates? Are known mechanisms of aging in reptiles consistent with canonical hallmarks of aging in model systems? What are the knowledge gaps in our understanding of reptile aging? 3. We find ample evidence of increasing mortality with advancing age in many reptiles. Testudines stand out as slower aging than other orders, but data on crocodilians and tuatara are sparse. Sex-specific analyses are generally not available. Studies of female reproduction suggest that reptiles are less likely to have reproductive decline with advancing age than mammals. 4. Reptiles share many physiological and molecular pathways of aging with mammals, birds, and laboratory model organisms. Adaptations related to stress physiology coupled with reptilian ectothermy suggest novel comparisons and contrasts that can be made with canonical aging phenotypes in mammals. These include stem cell and regeneration biology, homeostatic mechanisms, IIS/TOR signaling, and DNA repair. 5. To overcome challenges to the study of reptile aging, we recommend extending and expanding long-term monitoring of reptile populations, developing reptile cell lines to aid cellular biology, conducting more comparative studies of reptile morphology and physiology sampled along relevant life-history axes, and sequencing more reptile genomes for comparative genomics. Given the diversity of reptile life histories and adaptations, achieving these directives will likely greatly benefit all aging biology.

The utility of reptile blood transcriptomes in molecular ecology.

Reptiles and other nonmammalian vertebrates have transcriptionally active nucleated red blood cells. If blood transcriptomes can provide quantitative data to address questions relevant to molecular ecology, this could circumvent the need to euthanize animals to assay tissues. This would allow longitudinal sampling of animals' responses to treatments, as well as sampling of protected taxa. We developed and annotated blood transcriptomes from six reptile species and found on average 25,000 proteins are being transcribed in the blood, and there is a CORE group of 9,282 orthogroups that are found in at least four of six species. In comparison to liver transcriptomes from the same taxa, approximately two-thirds of the orthogroups were found in both blood and liver; and a similar percentage of ecologically relevant gene groups (insulin and insulin-like signalling, electron transport chain, oxidative stress, glucocorticoid receptors) were found transcribed in both blood and liver. As a resource, we provide a user-friendly database of gene ids identified in each blood transcriptome. Although on average 37% of reads mapped to haemoglobin, importantly, the majority of nonhaemoglobin transcripts had sufficient depth (e.g., 97% at ≥10 reads) to be included in differential gene expression analysis. Thus, we demonstrate that RNAseq blood transcriptomes from a very small blood sample (<10 µl) is a minimally invasive option in nonmammalian vertebrates for quantifying expression of a large number of ecologically relevant genes that would allow longitudinal sampling and sampling of protected populations.

Convergence in reduced body size, head size, and blood glucose in three island reptiles.

Many oceanic islands harbor diverse species that differ markedly from their mainland relatives with respect to morphology, behavior, and physiology. A particularly common morphological change exhibited by a wide range of species on islands worldwide involves either a reduction in body size, termed island dwarfism, or an increase in body size, termed island gigantism. While numerous instances of dwarfism and gigantism have been well documented, documentation of other morphological changes on islands remains limited. Furthermore, we lack a basic understanding of the physiological mechanisms that underlie these changes, and whether they are convergent. A major hypothesis for the repeated evolution of dwarfism posits selection for smaller, more efficient body sizes in the context of low resource availability. Under this hypothesis, we would expect the physiological mechanisms known to be downregulated in model organisms exhibiting small body sizes due to dietary restriction or artificial selection would also be downregulated in wild species exhibiting dwarfism on islands. We measured body size, relative head size, and circulating blood glucose in three species of reptiles-two snakes and one lizard-in the California Channel Islands relative to mainland populations. Collating data from 6 years of study, we found that relative to mainland population the island populations had smaller body size (i.e., island dwarfism), smaller head sizes relative to body size, and lower levels of blood glucose, although with some variation by sex and year. These findings suggest that the island populations of these three species have independently evolved convergent physiological changes (lower glucose set point) corresponding to convergent changes in morphology that are consistent with a scenario of reduced resource availability and/or changes in prey size on the islands. This provides a powerful system to further investigate ecological, physiological, and genetic variables to elucidate the mechanisms underlying convergent changes in life history on islands.

Use of field-portable ultrasonography reveals differences in developmental phenology and maternal egg provisioning in two sympatric viviparous snakes.

A thorough understanding of the life cycles underlying the demography of wild species is limited by the difficulty of observing hidden life-history traits, such as embryonic development. Major aspects of embryonic development, such as the rate and timing of development, and maternal-fetal interactions can be critical features of early-life fitness and may impact population trends via effects on individual survival. While information on development in wild snakes and lizards is particularly limited, the repeated evolution of viviparity and diversity of reproductive mode in this clade make it a valuable subject of study. We used field-portable ultrasonography to investigate embryonic development in two sympatric garter snake species, Thamnophis sirtalis and Thamnophis elegans in the Sierra Nevada mountains of California. This approach allowed us to examine previously hidden reproductive traits including the timing and annual variation in development and differences in parental investment in young. Both species are viviparous, occupy similar ecological niches, and experience the same annual environmental conditions. We found that T. sirtalis embryos were more developmentally advanced than T. elegans embryos during June of three consecutive years. We also found that eggs increased in volume more substantially across developmental stages in T. elegans than in T. sirtalis, indicating differences in maternal provisioning of embryos via placental transfer of water. These findings shed light on interspecific differences in parental investment and timing of development within the same environmental context and demonstrate the value of field ultrasonography for pursuing questions relating to the evolution of reproductive modes, and the ecology of development.

Integrating behaviour into the pace-of-life continuum: Divergent levels of activity and information gathering in fast- and slow-living snakes.

An animal's life history, physiology, and behaviour can be shaped by selection in a manner that favours strong associations among these aspects of an integrated phenotype. Recent work combining animal personality and life-history theory proposes that animals with faster life-history strategies (i.e., fast growth, high annual reproductive rate, short lifespan) should exhibit higher general activity levels relative to those with slower life-history strategies, but empirical tests of within-species variation in these traits are lacking. In garter snakes from ecotypes which are known to differ in ecology, life-history strategy, and physiology, we tested for differences in tongue-flick rate as a measure of information gathering and movement patterns as a measure of general activity. Tongue flicks and movement were strongly positively correlated and both behaviours were repeatable across trials. Snakes from the fast-living ecotype were more active and showed evidence of habituation. The slow-living ecotype maintained low levels of activity throughout the trials. We propose that environmental factors, such as high predation, experienced by the fast-living ecotype select for both increased information-gathering and activity levels to facilitate efficient responses to repeated challenges. Thus, we offer evidence that behaviour is an important component of co-evolved suites of traits forming a general pace-of-life continuum in this system.

Geographic variation and within-individual correlations of physiological stress markers in a widespread reptile, the common garter snake (Thamnophis sirtalis).

Characterizing the baseline and stress-induced hormonal, metabolite, and immune profiles of wild animals is important to assess the impacts of variable environments, including human-induced landscape changes, on organismal health. Additionally, the extent to which these profiles are coordinated across physiological systems within individuals remains an important question in understanding how stressors can differentially affect aspects of an individual's physiology. Here, we present data from wild populations of the common garter snake (Thamnophis sirtalis) on both baseline and stress-induced biomarkers: plasma corticosterone (CORT) concentration, plasma glucose concentration, and whole blood heterophil:lymphocyte ratio. Using a standardized restraint protocol with individuals from populations in disparate portions of this species' range - the Sierra Nevada Mountains of California and the plains of Iowa - we collected blood plasma samples at nine time points over three days. Both CORT and glucose response curves differed between georegions, with Iowa snakes attaining higher glucose concentration and maintaining elevated CORT and glucose levels for a longer duration. Additionally, both the total amount and proportional increases of CORT and glucose were lower in larger and therefore older snakes, suggesting ontogenetic shifts in stress perception or response. Within-individual correlation among the three physiological indicators was significant at the time of capture, absent after 3h in captivity, and partially restored after 3days in captivity, demonstrating the effect of stress on the relationships among these physiological systems. Together, these results provide further evidence for the great physiological flexibility of ectothermic tetrapods in maintaining homeostasis across a range of factors.

Physiological indices of stress in wild and captive garter snakes: correlations, repeatability, and ecological variation.

Glucocorticoids and leukocyte ratios have become the most widespread variables employed to test hypotheses regarding physiological stress in wild and captive vertebrates. Little is known, however, regarding how these two indices of stress covary in response to stressors, their repeatability within individuals, and differences in response time upon capture. Furthermore, few studies compare stress indices between captive and wild populations, to assess potential alteration of stress physiology in captivity. To address these issues, we examined corticosterone (CORT) and heterophil to lymphocyte (H:L) ratios in two ecotypes of the garter snake Thamnophis elegans. We found that CORT and H:L ratios were not correlated within individuals, and both variables showed little or no repeatability over a period of months. CORT levels, but not H:L ratios, were higher for individuals sampled after 10min from the time of capture. However, both variables showed similar patterns of ecotypic variation, and both increased over time in gravid females maintained in captivity for four months. We suggest that CORT and H:L ratios are both useful, but disparate indices of stress in this species, and may show complex relationships to each other and to ecological and anthropogenic variables.

Rates of molecular evolution vary in vertebrates for insulin-like growth factor-1 (IGF-1), a pleiotropic locus that regulates life history traits.

Insulin-like growth factor-1 (IGF-1) is a member of the vertebrate insulin/insulin-like growth factor/relaxin gene family necessary for growth, reproduction, and survival at both the cellular and organismal level. Its sequence, protein structure, and function have been characterized in mammals, birds, and fish; however, a notable gap in our current knowledge of the function of IGF-1 and its molecular evolution is information in ectothermic reptiles. To address this disparity, we sequenced the coding region of IGF-1 in 11 reptile species-one crocodilian, three turtles, three lizards, and four snakes. Complete sequencing of the full mRNA transcript of a snake revealed the Ea-isoform, the predominant isoform of IGF-1 also reported in other vertebrate groups. A gene tree of the IGF-1 protein-coding region that incorporated sequences from diverse vertebrate groups showed similarity to the species phylogeny, with the exception of the placement of Testudines as sister group to Aves, due to their high nucleotide sequence similarity. In contrast, long-branch lengths indicate more rapid divergence in IGF-1 among lizards and snakes. Additionally, lepidosaurs (i.e., lizards and snakes) had higher rates of non-synonymous:synonymous substitutions (dN/dS) relative to archosaurs (i.e., birds and crocodilians) and turtles. Tests for positive selection on specific codons within branches and evaluation of the changes in the amino acid properties, suggested positive selection in lepidosaurs on the C domain of IGF-1, which is involved in binding affinity to the IGF-1 receptor. Predicted structural changes suggest that major alterations in protein structure and function may have occurred in reptiles. These data propose new insights into the molecular co-evolution of IGF-1 and its receptors, and ultimately the evolution of IGF-1's role in regulating life-history traits across vertebrates.

Corticosterone and pace of life in two life-history ecotypes of the garter snake Thamnophis elegans.

Glucocorticoids are main candidates for mediating life-history trade-offs by regulating the balance between current reproduction and survival. It has been proposed that slow-living organisms should show higher stress-induced glucocorticoid levels that favor self-maintenance rather than current reproduction when compared to fast-living organisms. We tested this hypothesis in replicate populations of two ecotypes of the garter snake (Thamnophis elegans) that exhibit slow and fast pace of life strategies. We subjected free-ranging snakes to a capture-restraint protocol and compared the stress-induced corticosterone levels between slow- and fast-living snakes. We also used a five-year dataset to assess whether baseline corticosterone levels followed the same pattern as stress-induced levels in relation to pace of life. In accordance with the hypothesis, slow-living snakes showed higher stress-induced corticosterone levels than fast-living snakes. Baseline corticosterone levels showed a similar pattern with ecotype, although differences depended on the year of study. Overall, however, levels of glucocorticoids are higher in slow-living than fast-living snakes, which should favor self-maintenance and survival at the expense of current reproduction. The results of the present study are the first to relate glucocorticoid levels and pace of life in a reptilian system and contribute to our understanding of the physiological mechanisms involved in life-history evolution.

Social and demographic effects of anthropogenic mortality: a test of the compensatory mortality hypothesis in the red wolf.

Whether anthropogenic mortality is additive or compensatory to natural mortality in animal populations has long been a question of theoretical and practical importance. Theoretically, under density-dependent conditions populations compensate for anthropogenic mortality through decreases in natural mortality and/or increases in productivity, but recent studies of large carnivores suggest that anthropogenic mortality can be fully additive to natural mortality and thereby constrain annual survival and population growth rate. Nevertheless, mechanisms underlying either compensatory or additive effects continue to be poorly understood. Using long-term data on a reintroduced population of the red wolf, we tested for evidence of additive vs. compensatory effects of anthropogenic mortality on annual survival and population growth rates, and the preservation and reproductive success of breeding pairs. We found that anthropogenic mortality had a strong additive effect on annual survival and population growth rate at low population density, though there was evidence for compensation in population growth at high density. When involving the death of a breeder, anthropogenic mortality was also additive to natural rates of breeding pair dissolution, resulting in a net decrease in the annual preservation of existing breeding pairs. However, though the disbanding of a pack following death of a breeder resulted in fewer recruits per litter relative to stable packs, there was no relationship between natural rates of pair dissolution and population growth rate at either high or low density. Thus we propose that short-term additive effects of anthropogenic mortality on population growth in the red wolf population at low density were primarily a result of direct mortality of adults rather than indirect socially-mediated effects resulting in reduced recruitment. Finally, we also demonstrate that per capita recruitment and the proportion of adults that became reproductive declined steeply with increasing population density, suggesting that there is potential for density-dependent compensation of anthropogenically-mediated population regulation.

Helper effects on pup lifetime fitness in the cooperatively breeding red wolf (Canis rufus).

The evolutionary maintenance of cooperative breeding systems is thought to be a function of relative costs and benefits to breeders, helpers and juveniles. Beneficial effects of helpers on early-life survivorship and performance have been established in several species, but lifetime fitness benefits and/or costs of being helped remain unclear, particularly for long-lived species. We tested for effects of helpers on early- and late-life traits in a population of reintroduced red wolves (Canis rufus), while controlling for ecological variables such as home-range size and population density. We found that the presence of helpers in family groups was positively correlated with pup mass and survival at low population density, but negatively correlated with mass/size at high density, with no relation to survival. Interestingly, mass/size differences persisted into adulthood for both sexes. While the presence of helpers did not advance age at first reproduction for pups of either sex, females appeared to garner long-term fitness benefits from helpers through later age at last reproduction, longer reproductive lifespan and a greater number of lifetime reproductive events, which translated to higher lifetime reproductive success. In contrast, males with helpers exhibited diminished lifetime reproductive performance. Our findings suggest that while helper presence may have beneficial short-term effects in some ecological contexts, it may also incur long-term sex-dependent costs with critical ramifications for lifetime fitness.

Developmental plasticity of immune defence in two life-history ecotypes of the garter snake, Thamnophis elegans - a common-environment experiment.

1. Ecoimmunological theory predicts a link between life-history and immune-defence strategies such that fast-living organisms should rely more on constitutive innate defences compared to slow-living organisms. An untested assumption of this hypothesis is that the variation in immune defence associated with variation in life history has a genetic basis. 2. Replicate populations of two life-history ecotypes of the garter snake Thamnophis elegans provide an ideal system in which to test this assumption. Free-ranging snakes of the fast-living ecotype, which reside in lakeshore habitats, show higher levels of three measures of constitutive innate immunity than those of the slow-living ecotype, which inhabit meadows around the lake. Although this pattern is consistent with the ecoimmunological pace-of-life hypothesis, environmental differences between the lakeshore and meadow habitats could also explain the observed differences in immune defence. 3. We performed a common-environment experiment to distinguish between these alternatives. Snakes born and raised in common-environment conditions reflected the immune phenotype of their native habitats when sampled at 4 months of age (i.e. fast-living lakeshore snakes showed higher levels of natural antibodies, complement activity and bactericidal competence than slow-living meadow snakes), but no longer showed differences when 19 months old. 4. This suggests that the differences in innate immunity observed between the two ecotypes have an important - and likely age-specific - environmental influence, with these immune components showing developmental plasticity. A genetic effect in early life may also be present, but further research is needed to confirm this possibility and therefore provide a more definitive test of the ecoimmunological pace-of-life hypothesis in this system.

Evolutionary ecology of endocrine-mediated life-history variation in the garter snake Thamnophis elegans.

The endocrine system plays an integral role in the regulation of key life-history traits. Insulin-like growth factor-1 (IGF-1) is a hormone that promotes growth and reproduction, and it has been implicated in the reduction of lifespan. IGF-1 is also capable of responding plastically to environmental stimuli such as resource availability and temperature. Thus pleiotropic control of life-history traits by IGF-1 could provide a mechanism for the evolution of correlated life-history traits in a new or changing environment. An ideal system in which to investigate the role of IGF-1 in life-history evolution exists in two ecotypes of the garter snake Thamnophis elegans, which derive from a single recent ancestral source but have evolved genetically divergent life-history characteristics. Snakes from meadow populations near Eagle Lake, California (USA) exhibit slower growth rates, lower annual reproductive output, and longer median adult lifespans relative to populations along the lakeshore. We hypothesized that the IGF-1 system has differentiated between these ecotypes and can account for increased growth and reproduction and reduced survival in lakeshore vs. meadow snakes. We tested for a difference in plasma IGF-1 levels in free-ranging snakes from replicate populations of each ecotype over three years. IGF-1 levels were significantly associated with adult body size, reproductive output, and season in a manner that reflects established differences in prey ecology and age/size-specific reproduction between the ecotypes. These findings are discussed in the context of theoretical expectations for a tradeoff between reproduction and lifespan that is mediated by pleiotropic endocrine mechanisms.

An empirical test of evolutionary theories for reproductive senescence and reproductive effort in the garter snake Thamnophis elegans.

Evolutionary theory predicts that differential reproductive effort and rate of reproductive senescence will evolve under different rates of external mortality. We examine the evolutionary divergence of age-specific reproduction in two life-history ecotypes of the western terrestrial garter snake, Thamnophis elegans. We test for the signature of reproductive senescence (decreasing fecundity with age) and increasing reproductive effort with age (increasing reproductive productivity per gram female) in replicate populations of two life-history ecotypes: snakes that grow fast, mature young and have shorter lifespans, and snakes that grow slow, mature late and have long lives. The difference between life-history ecotypes is due to genetic divergence in growth rate. We find (i) reproductive success (live litter mass) increases with age in both ecotypes, but does so more rapidly in the fast-growth ecotype, (ii) reproductive failure increases with age in both ecotypes, but the proportion of reproductive failure to total reproductive output remains invariant, and (iii) reproductive effort remains constant in fast-growth individuals with age, but declines in slow-growth individuals. This illustration of increasing fecundity with age, even at the latest ages, deviates from standard expectations for reproductive senescence, as does the lack of increases in reproductive effort. We discuss our findings in light of recent theories regarding the phenomenon of increased reproduction throughout life in organisms with indeterminate growth and its potential to offset theoretical expectations for the ubiquity of senescence.