Exogenous leptin promotes reproductive behavior during aphagia in red-sided garter snakes (Thamnophis sirtalis parietalis).

Despite the established dichotomy between investment in either reproduction or self-maintenance, a hormonal mechanism that influences an organism's decision to prioritize these behaviors remains elusive. The protein hormone leptin is a likely candidate because it is secreted from adipocytes in proportion to the amount of stored fat in numerous species. Although the majority of studies suggest that leptin stimulates reproduction, the actions of leptin can be context-dependent. Leptin increases sexual behavior in fed individuals, but inhibits sexual behavior in food-restricted individuals. We investigated if exogenous leptin influences sexual behavior in red-sided garter snakes (Thamnophis sirtalis parietalis) experiencing a predictable bout of aphagia during the mating season. We tested two doses of recombinant murine leptin injected for three days. Males were subjected to three mating trials, one on each day of injections, while females were subjected to one mating trial on the last day of injections. Leptin affects male and female snakes similarly by increasing both appetitive (i.e., mating behavior score) and consummatory (i.e., number of copulations, proportion of individuals copulated) sex behavior. We found no evidence to suggest that leptin influenced latency to copulate or duration of copulation. Because leptin promotes reproductive behavior in non-feeding garter snakes, these findings do not align with research on food-restricted mammals. Further investigations into how leptin affects sexual behavior in snakes exposed to food-restriction manipulations would clarify if the role of leptin is evolutionarily divergent.

Physiological correlates of reproductive decisions: Relationships among body condition, reproductive status, and the hypothalamus-pituitary-adrenal axis in a reptile.

When opportunities to feed and reproduce are limited, females are often unable to recover sufficient energy stores to reproduce in consecutive years. Body condition has been used as a proxy for recent reproductive history in such species. We previously found that glucocorticoid responses to capture stress vary with body condition in female red-sided garter snakes (Thamnophis sirtalis parietalis), a species with limited seasonal breeding opportunities. Because variation in glucocorticoid receptor (GR) protein in the brain could explain these differences, we first assessed GR protein content in females in different body conditions. To investigate if body condition during the spring mating season accurately reflects recent reproductive history, we measured glucocorticoid responses to stress in females with different body conditions, assessed their mating behavior and brought mated females to our lab to determine which females would give birth during the summer (i.e., were parturient). Female red-sided garter snakes reproduce biennially, and therefore mated females that did not give birth were deemed non-parturient. In this study, glucocorticoid stress responses and mating behavior did not vary with body condition, nor was body condition related to brain GR or reproductive condition (parturient vs non-parturient). Only unreceptive females showed a significant stress-induced increase in glucocorticoids, suggesting that reduced stress responsiveness is associated with receptivity. Parturient females mated faster (were more proceptive) than non-parturient females. These data suggest that HPA axis activity modulates receptivity, while proceptivity is related primarily to reproductive condition.

Pheromonal mediation of intraseasonal declines in the attractivity of female red-sided garter snakes, Thamnophis sirtalis parietalis.

During the breeding season, female red-sided garter snakes (Thamnophis sirtalis parietalis) produce and express a sexual attractiveness pheromone that elicits male courtship behavior. Composed of a homologous series of saturated and monounsaturated methyl ketones, this pheromone is expressed in female skin lipids. Recent studies have shown that the sexual attractivity of unmated female garter snakes declines as the breeding season progresses. Here, we investigated whether temporal changes in the quantity and/or quality of the female sexual attractiveness pheromone are responsible for the observed loss of attractivity. Female red-sided garter snakes were collected immediately following spring emergence and held under natural conditions for the duration of the breeding season. Behavioral experiments confirmed that unmated females become significantly less attractive to males within two weeks of emergence from hibernation. Additionally, these females had lower estradiol concentrations at two weeks post-emergence. Subsequent chemical analyses revealed qualitative variation between the pheromone profiles of newly emerged females and those of females at two weeks post-emergence. Together, these results support the hypothesis that changes in the female sexual attractiveness pheromone are responsible for declining post-emergence female attractivity in garter snakes.

Effects of melatonin on the behavioral and hormonal responses of red-sided garter snakes (Thamnophis sirtalis parietalis) to exogenous corticosterone.

We investigated possible interactions between melatonin and corticosterone in modulating the reproductive behavior of male red-sided garter snakes (Thamnophis sirtalis parietalis) following spring emergence. We also examined whether melatonin's modulatory actions could be explained by its potential properties as a serotonin receptor antagonist. Exogenous corticosterone significantly reduced courtship behavior of male snakes in a dose-dependent manner. Melatonin also significantly reduced courtship behavior of male garter snakes. Pretreatment with melatonin before administering corticosterone treatments further suppressed courtship behavior of red-sided garter snakes. These results indicate additive inhibitory effects of melatonin and corticosterone in modulating reproductive behavior. Snakes receiving ketanserin, a serotonergic type 2A receptor antagonist, followed by corticosterone also showed reduced courtship behavior; this serotonin receptor antagonist followed by treatment with vehicle did not significantly influence courtship behavior of male snakes. Neither melatonin nor corticosterone treatments significantly influenced testosterone + 5-alpha-dihydrotestosterone concentrations of male garter snakes, supporting a direct effect of melatonin and corticosterone on courtship behavior that is independent of any effect on androgen concentrations. We propose that a serotonin system is involved in the modulation of male courtship behavior by melatonin and corticosterone. In addition, our data support the hypothesis that melatonin may function as a serotonin receptor antagonist. Further research is necessary to discern whether the actions of melatonin and corticosterone are converging on the same pathway or if their effects on different pathways are having additive inhibitory effects on courtship behavior.

Pheromonally mediated sexual isolation among denning populations of red-sided garter snakes, Thamnophis sirtalis parietalis.

Utilizing behavioral experiments and chemical analyses, we examined whether pheromonally mediated sexual isolation exists between denning populations of red-sided garter snakes (Thamtnophis sirtalis parietalis) in Manitoba, Canada. Simultaneous choice tests conducted during the breeding season revealed that adult males from a hibernaculum in central Manitoba displayed a strong courtship preference for females from their own population over females from a hibernaculum in western Manitoba, whereas males from the western Manitoba hibernaculum showed no such preference. In addition. trailing experiments testing the response of males from the two hibernacula to familiar and unfamiliar female trails showed similar results, demonstrating that the observed male preference is mediated through chemical cues. Subsequent chemical analysis of the female sexual attractiveness pheromone. a homologous series of long-chain saturated and (omega-9 cis-unsaturated methyl ketones responsible for eliciting male courtship behavior and trailing behavior in garter snakes, showed significant variation in the composition of the pheromone between the two populations. Specifically, the two populations varied in the relative concentrations of individual unsaturated methyl ketones expressed by females. These results suggest that sexual isolation exists to a degree among denning populations of red-sided garter snakes due to variation in the expression of the female sexual attractiveness pheromone.

Variation in a female sexual attractiveness pheromone controls male mate choice in garter snakes.

Male red-sided garter snakes (Thamnophis sirtalis parietalis) display a courtship preference for larger females during the breeding season. Utilizing behavioral experiments and chemical analyses, we tested the hypothesis that males can discriminate among females of varying size solely by means of the sexual attractiveness pheromone, a previously characterized sex pheromone composed of a homologous series of long-chain saturated and omega-9 cis-unsaturated methyl ketones contained in the skin lipids of females. When presented with skin lipid extracts from large and small females, a greater proportion of males displayed courtship behaviors to large female extracts. This demonstrates that there is an intrinsic property of the female skin lipids that allows males to differentiate among large and small females. Analysis of the sexual attractiveness pheromone revealed that the necessary variation exists for this pheromone to function as a reliable indicator to males of female body size. Specifically, we observed a strong correlation between female snout-vent length and the relative concentration of saturated and omega-9 cis-unsaturated methyl ketones composing the pheromone; smaller females expressed pheromone profiles higher in saturated methyl ketones. while larger females expressed pheromone profiles dominated by unsaturated methyl ketones. The results of this study suggest that male red-sided garter snakes utilize compositional variation in the female sexual attractiveness pheromone to differentiate among potential mates of varying size.