Deborah I. Lutterschmidt

Responses to capture stress and exogenous corticosterone vary with body condition in female red-sided garter snakes (Thamnophis sirtalis parietalis).

This study examined whether hormonal and behavioral responses to capture stress and exogenous corticosterone (CORT) vary with body condition in female red-sided garter snakes (Thamnophis sirtalis parietalis). Female snakes were collected during the spring mating season and treated with 4 h of capture stress. We measured plasma CORT and estradiol before, during and after capture stress treatment followed by latency to copulate, a measure of female receptivity. Body condition was determined as the residual from a regression of body mass on snout-vent-length. Baseline CORT did not differ between females in positive and negative body condition, but females in negative body condition showed a significantly larger increase in plasma CORT in response to capture stress. Estradiol, which is generally low during the mating season in this population, did not change in response to capture stress. Body condition, but not capture stress, influenced latency to copulate, suggesting that females are resistant to the behavioral effects of capture stress during the spring mating season. In a second experiment, only females in negative body condition increased latency to copulate in response to injection of a physiological (15 μg) dose of exogenous CORT, while all females responded to a pharmacological (60 μg) dose. These results indicate that behavioral responses to exogenous CORT vary with female body condition during the short mating season. Taken together, our data suggest that variation in body condition may be associated with differences in HPA axis sensitivity and/or glucocorticoid receptor density in the brain.

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.

Seasonal and sex differences in responsiveness to adrenocorticotropic hormone contribute to stress response plasticity in red-sided garter snakes (Thamnophis sirtalis parietalis)

ABSTRACT As in many vertebrates, hormonal responses to stress vary seasonally in red-sided garter snakes (Thamnophis sirtalis parietalis). For example, males generally exhibit reduced glucocorticoid responses to a standard stressor during the spring mating season. We asked whether variation in adrenal sensitivity to adrenocorticotropic hormone (ACTH) explains why glucocorticoid responses to capture stress vary with sex, season and body condition in red-sided garter snakes. We measured glucocorticoids at 0, 1 and 4 h after injection with ACTH (0.1 IU g−1 body mass) or vehicle in males and females during the spring mating season and autumn pre-hibernation period. Because elevated glucocorticoids can influence sex steroids, we also examined androgen and estradiol responses to ACTH. ACTH treatment increased glucocorticoids in both sexes and seasons. Spring-collected males had a smaller integrated glucocorticoid response to ACTH than autumn-collected males. The integrated glucocorticoid response to ACTH differed with sex during the spring, with males having a smaller glucocorticoid response than females. Although integrated glucocorticoid responses to ACTH did not vary with body condition, we observed an interaction among season, sex and body condition. In males, ACTH treatment did not alter androgen levels in either season, but androgen levels decreased during the sampling period. Similar to previous studies, plasma estradiol was low or undetectable during the spring and autumn, and therefore any effect of ACTH treatment on estradiol could not be determined. These data provide support for a mechanism that partly explains how the hypothalamus–pituitary–adrenal (HPA) axis integrates information about season, sex and body condition: namely, variation in adrenal responsiveness to ACTH. Summary: The adrenal glucocorticoid response to ACTH varies with season and sex in garter snakes; decreased responsiveness to ACTH partly explains reduced stress sensitivity in males during the spring breeding season.

Chronobiology of reproduction in garter snakes: Neuroendocrine mechanisms and geographic variation

The majority of studies on reproductive neuroendocrinology in snakes have focused on one particular snake population in Manitoba, Canada, the red-sided garter snake (Thamnophis sirtalis parietalis). Although traditionally these studies have emphasized its unusual temporal dissociation between mating behavior and peak gonadal activity, current evidence suggests that reproductive regulation in this population may be more similar to the norm than previously thought. Like other ectotherms, temperature plays a critical role in activating reproductive behavior in red-sided garter snakes. Diel melatonin and corticosterone rhythms appear to be important in transducing temperature cues, and it is clear that both hormones regulate courtship behavior during spring. Current evidence also suggests that sex steroid hormones are in fact central to reproductive regulation in males, although the timing of their action occurs during winter dormancy. Whether this is also true for female T. sirtalis parietalis requires further study, but it should be noted that patterns of sex steroid hormones are sexually dimorphic during winter dormancy, as are melatonin rhythms during spring emergence. While continuing to advance our understanding of reproductive regulation in this extremely well-studied population is prudent, future comparative studies are critical for understanding if and how reproductive regulatory mechanisms differ across environments, populations, and phylogenies. For example, melatonin and corticosterone responses to environmental cues vary significantly among populations of T. sirtalis in a common garden, as do male courtship behavior and androgen concentrations. These data support the hypothesis that neuroendocrine-mediated responses to environmental cues underlie phenotypic plasticity in reproductive life history traits.

Brain nuclei in actively courting red-sided garter snakes: a paradigm of neural trimorphism.

During the breeding season, two distinct male phenotypes are exhibited by red-sided garter snakes (Thamnophis sirtalis parietalis), with courtship behavior being directed not only toward females, but also toward a sub-population of males called she-males. She-males are morphologically identical to other males except for a circulating androgen level three times that of normal males and their ability to produce a female-like pheromone. As in other vertebrates, limbic nuclei in the red-sided garter snake brain are involved in the control of sexual behaviors. For example, an intact anterior hypothalamus pre-optic area (AHPOA) is essential for the initiation and maintenance of reproduction. To determine if brain morphology varies among the three behavioral phenotypes (i.e., males, she-males, and females) during the breeding season, we examined the volume, cell size and cell density of the AHPOA as well as a control region, the external nucleus of the optic tract (ENOT). We used Luxol Fast Blue and Ziehls Fuchsin to visualize neurons and glial cells, respectively. No significant differences were observed among the three behavioral phenotypes in the volume, cell size or density in the control region. In contrast, the volume, cell size and density of the AHPOA of she-males were significantly greater than those of both male and female snakes. While the volume of the AHPOA was significantly greater in females compared to males, no differences were observed in cell size or density. These differences in brain morphology suggest a possible underlying mechanism for phenotypic-specific behavioral patterns.

Arginine Vasotocin and Neuropeptide Y Vary with Seasonal Life-History Transitions in Garter Snakes

Transitions between life-history stages are often accompanied by dramatic behavioral switches that result from a shift in motivation to pursue one resource over another. While the neuroendocrine mechanisms that regulate such behavioral transitions are poorly understood, arginine vasotocin (AVT) and neuropeptide Y (NPY) are excellent candidates because they modulate reproductive and feeding behavior, respectively. We asked if seasonal changes in AVT and NPY are concomitant with the seasonal migration to and from the feeding grounds in red-sided garter snakes (Thamnophis sirtalis parietalis). Male and female snakes were collected in different migratory states during both the spring and fall. The total number of AVT- and NPY-immunoreactive (ir) cells was then quantified in each brain region of interest. To correct for potential variation in region volume related to sexually dimorphic body size in this species, we first determined that snout-vent length is an accurate proxy for regional brain volume. We then corrected each individuals ir cell number by its SVL to directly compare seasonal changes in AVT and NPY between males and females. Within the supraoptic nucleus, both males and females had more AVT-ir cells during the fall compared with spring. As predicted, males had significantly more AVT-ir cells during the spring mating season in the hypothalamus (HYP) and bed nucleus of the stria terminalis, brain regions important in regulating reproductive behavior. Females also had significantly more AVT-ir cells in the HYP during the spring, as well as a significantly higher number of hypothalamic AVT cells than males. During the fall, males had significantly more NPY-ir cells in the cortex and posterior HYP compared with spring, possibly reflecting increased feeding behavior during summer foraging. Females did not exhibit significant main effects of season on NPY-ir cell number in any region. Neither AVT- nor NPY-ir cell number varied significantly with migratory status, but we did observe significant changes related to seasonal transitions in reproductive state. Our results indicate that changes in brain AVT and NPY are associated with seasonal transitions in reproductive and foraging behaviors, and may be involved in mediating sex differences in the timing of life-history events.

Biological Rhythms: Melatonin Shapes the Space–Time Continuum of Social Communication

A new study shows that nocturnal courtship vocalization is regulated by a circadian rhythm and potentiated by melatonin at multiple timescales. These findings are in contrast to those in diurnal vocalizers and provide a striking example of melatonins niche-specific functions.

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

ABSTRACT 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. HIGHLIGHTSWe asked if body condition, HPA activity, and reproductive outcome are related.Neither plasma glucocorticoids nor brain GR varied with female body condition.Stress responses varied with female receptivity, but not reproductive status.Proceptivity was highest in parturient snakes, but was unrelated to body condition.Sensitivity of the HPA axis may influence female reproductive decisions.

Sexually Dimorphic Patterns of Cell Proliferation in the Brain Are Linked to Seasonal Life-History Transitions in Red-Sided Garter Snakes

Seasonal rhythms in physiology and behavior are widespread across diverse taxonomic groups and may be mediated by seasonal changes in neurogenesis, including cell proliferation, migration, and differentiation. We examined if cell proliferation in the brain is associated with the seasonal life-history transition from spring breeding to migration and summer foraging in a free-ranging population of red-sided garter snakes (Thamnophis sirtalis) in Manitoba, Canada. We used the thymidine analog 5-bromo-2′-deoxyuridine (BrdU) to label newly proliferated cells within the brain of adult snakes collected from the den during the mating season or from a road located along their migratory route. To assess rates of cell migration, we further categorized BrdU-labeled cells according to their location within the ventricular zone or parenchymal region of the nucleus sphericus (homolog of the amygdala), preoptic area/hypothalamus, septal nucleus, and cortex (homolog of the hippocampus). We found that cell proliferation and cell migration varied significantly with sex, the migratory status of snakes, and reproductive behavior in males. In most regions of interest, patterns of cell proliferation were sexually dimorphic, with males having significantly more BrdU-labeled cells than females prior to migration. However, during the initial stages of migration, females exhibited a significant increase in cell proliferation within the nucleus sphericus, hypothalamus, and septal nucleus, but not in any subregion of the cortex. In contrast, migrating males exhibited a significant increase in cell proliferation within the medial cortex but no other brain region. Because it is unlikely that the medial cortex plays a sexually dimorphic role in spatial memory during spring migration, we speculate that cell proliferation within the male medial cortex is associated with regulation of the hypothalamus-pituitary-adrenal axis. Finally, the only brain region where cell migration into the parenchymal region varied significantly with sex or migratory status was the hypothalamus. These results suggest that the migration of newly proliferated cells and/or the continued division of undifferentiated cells are activated earlier or to a greater extent in the hypothalamus. Our data suggest that sexually dimorphic changes in cell proliferation and cell migration in the adult brain may mediate sex differences in the timing of seasonal life-history transitions.

Methyl Ketone Production in Juvenile Red-Sided Garter Snakes

The skin lipids of female red-sided garter snakes contain a series of methyl ketones which function as a sexual attractiveness pheromone. The pheromone is likely regulated by oestrogen. As previous studies have focused mainly on adult females, it is unknown whether juvenile garter snakes produce methyl ketones. The current study was designed to determine whether (1) juvenile female garter snakes are physiologically capable of producing methyl ketones and (2) oestrogen initiates or enhances methyl ketone production in juveniles. Snakes were injected with either oestradiol or an oil vehicle, or received no injection. Subsequent chemical analyses of skin lipid samples clearly demonstrated that juveniles were able to produce methyl ketones regardless of treatment. However, we were unable to demonstrate that oestradiol affected methyl ketone production at this stage in the female life cycle.