Female Red-sided Garter Snakes Research Articles

Decreased attractivity in female garter snakes treated with an aromatase inhibitor.

Most experimental studies on sexual signal regulation via hormone manipulation have focused on male signals, yet female signals demonstrate substantial phenotypic variation and hormone-dependent expression. Female red-sided garter snakes (Thamnophis sirtalis parietalis) produce a skin-based sex pheromone used by males in mate selection. The principle female sex steroid, 17 β-estradiol, controls pheromone production in snakes, but studies manipulating female garter snakes have produced conflicting results, relied on behavioral tests with males in the laboratory, and did not quantify pheromone expression. Because aromatase is the terminal enzyme in estradiol biosynthesis, we hypothesized that female garter snakes rely on aromatase to ultimately control pheromone production during the annual cycle of this species. To test this, we used a known pharmacological inhibitor of aromatase, fadrozole (FAD). Wild-caught female garter snakes were chronically treated via subcutaneous injections of either FAD (1.0 mg kg-1 ) or saline (control) for six months in the laboratory during the active period of the annual cycle then hibernated. In two separate field bioassays the next spring at the den site, FAD females received approximately 50% less courtship from wild, sexually active male garter snakes compared to SHAM females. Pheromone analysis revealed that four of the largest, unsaturated methyl ketones were specifically downregulated in FAD females, indicating that aromatase action is a crucial, permissive step in the maintenance of female attractivity.

Energy Metrics of Red-Sided Garter Snakes (Thamnophis sirtalis parietalis) Vary with Sex but Not Life-History Stage

Because reproduction is energetically expensive, an organism's energy stores are likely involved in mediating transitions between reproductive and self-maintenance activities. We investigated whether body condition index, adipocyte follicle size, and liver glycogen differ with the life-history transition from reproduction to migration and foraging in red-sided garter snakes (Thamnophis sirtalis parietalis). Females primarily investing in mating behavior located at the den had a significantly higher body condition index than females migrating to summer feeding grounds. The body condition index of male snakes did not differ between snakes located at the den and those migrating to summer feeding grounds. Neither adipocyte follicle area nor liver glycogen stores differed significantly between snakes performing mating activities at the den and those migrating to summer feeding grounds. We did find a sexual dimorphism in that female red-sided garter snakes had significantly larger adipocyte follicles and higher liver glycogen compared with males. Our findings support the across-species phenomenon of females and males displaying a sexual dimorphism in stored energy substrates. Conversely, we did not find evidence to suggest that red-sided garter snakes primarily utilize fatty acids to fuel the initiation of migration, a finding that is not consistent with other long-distance migrators, such as birds. Because we did not find evidence to suggest that stored energy metrics influence the decision to migrate, a physiological mechanism that induces migration in red-sided garter snakes remains elusive.

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.

Patterns of stress responses shift during seasonal life-history transitions: An analysis comparing baseline, maximal and integrated corticosterone in female red-sided garter snakes (Thamnophis sirtalis parietalis)

Glucocorticoids often rise and fall with a variety of external and internal cues and frequently vary among life-history stages. This suggests that changing glucocorticoids may coordinate life-history transitions. To explore this hypothesis, we asked if the time-course of stress-induced glucocorticoid levels differ between two life-history transitions (i.e., spring and fall migration) in female red-sided garter snakes (Thamnophis sirtalis parietalis). We collected non-migratory females from a communal den and migratory females from a road along the migration route and treated them with 4h of capture stress; plasma corticosterone was measured before, during and after capture stress. During the spring, den-collected females exhibited a stress-induced peak in corticosterone at an earlier sampling time than migrating, road-collected females. Because the pattern of corticosterone responses varied with migratory state, negative feedback on and/or sensitivity of the hypothalamus-pituitaryadrenal (HPA) axis may be linked to spring migration. During the fall, capture stress elicited an increase in corticosterone in den-collected females but not in migrating, road-collected females. Baseline corticosterone was higher and both maximal and integrated corticosterone responses were lower during the fall compared to spring, indicating that stress responses are smaller when baseline corticosterone is elevated, perhaps due to a “ceiling effect”. These data suggest that HPA axis regulation changes during seasonal migration, possibly via altering negative feedback, HPA axis sensitivity, or some other mechanism. This study supports the hypothesis that glucocorticoids coordinate life-history events and suggests that examining a suite of stress response characteristics is most informative for understanding the function of HPA modulation.

Factors influencing paternity in multiply mated female red-sided garter snakes and the persistent use of sperm stored over winter

In some species, sperm is stored within the female reproductive tract for months to years, and yet remains viable to fertilize eggs and produce offspring. Female red-sided garter snakes store sperm for over 7 months of winter dormancy. In previous work, we demonstrated that these stored sperm account for an average of 25 % paternity of a litter when the female mates with a male at spring emergence. Here, we tested whether last-male sperm precedence was prevalent when a female mates with two males during the spring. On average, paternity was shared equally among the first (P1 proportion of paternity of the first male to mate) and second males (P2) to mate in the spring, and stored sperm (Pss), but the variance in paternity was high. Thus, last male sperm precedence may diminish when a female has more than two mates. Male size did not affect paternity, but, as the interval between matings increased, P1 increased at the expense of Pss. Interestingly, as the second spring male’s copulation duration increased, P1 also increased at the expense of P2. This result suggests that female influence over sperm and/or copulatory plug transfer during matings may also affect which male fathers her offspring in response to coercive matings as we assisted females to mate for their second mating. Finally, all females were spring “virgins”; consequently, sperm stored from autumn matings (and/or previous spring matings) remain competitive even when faced with two rivals in sperm competition and is likely the driver of the evolution of sperm longevity.

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 4h 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 (15μg) dose of exogenous CORT, while all females responded to a pharmacological (60μg) 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.

Low Temperature Dormancy Affects the Quantity and Quality of the Female Sexual Attractiveness Pheromone in Red-sided Garter Snakes

Low temperature dormancy is a necessary requirement of the annual cycle of most nonmigratory, temperate vertebrates. The red-sided garter snake, Thamnophis sirtalis parietalis, overwinters in communal dens during its prolonged winter dormancy (8 mo), and upon emergence, reproductive behavior of both sexes is maximal. Previous work on this species showed that male courtship behavior is maximally induced after simulated low temperature dormancy. The purpose of this study was to determine whether low temperature dormancy affects the pheromone profiles of individual female red-sided garter snakes. We collected females in the fall at den sites in Manitoba, Canada, and extracted pheromones from individuals at three different time points: fall (field), winter (lab), and spring (lab). Total skin lipid and pheromone fraction masses increased from fall to spring, and pheromone profiles were distinctly different in the fall and spring. Pheromone profiles became dominated by the long-chain, unsaturated methyl ketone components of the blend by the time snakes emerged in the spring. Further, the amounts of both saturated and unsaturated components increased from fall to spring, suggesting significant sex pheromone synthesis was induced by low temperature dormancy.

Mating is Correlated with a Reduced Risk of Predation in Female Red-sided Garter Snakes, Thamnophis sirtalis parietalis

We took advantage of unique aspects of the red-sided garter snake's breeding biology at northern latitudes to investigate whether mating was correlated with a reduced risk of predation. We scored the number of mated vs. unmated and predated vs. surviving female snakes at four dens in Manitoba, Canada. Predation intensity did not significantly vary through time during the course of the study, nor did it vary among dens. Female mating significantly decreased the odds that a female would be the victim of predation. This is likely a result of reduced courtship of females after mating, which reduces female conspicuousness and facilitates the ability to escape. These results provide support for the hypothesis that the copulatory pheromone, which communicates female mating status to males and thus reduces male courtship, benefits females by indirectly reducing their risk of predation after mating.

Minimal overwintering temperatures of red-sided garter snakes (Thamnophis sirtalis parietalis): a possible cue for emergence?

Red-sided garter snakes ( Thamnophis sirtalis parietalis (Say in James, 1823)) in Manitoba, Canada, undergo 8 months of continuous winter dormancy prior to spring emergence. As in other ectothermic species, increases in ground temperature may be the cue for emergence from winter dormancy in these populations. To test this hypothesis, we measured body temperatures during winter dormancy by surgically implanting small temperature loggers into 32 female red-sided garter snakes before they entered their native hibernaculum. The following spring, we recaptured seven of the snakes implanted with temperature loggers. Body temperature declined gradually from mid-September (14.7 ± 0.24 °C, mean ± SE) to early April (1.1 ± 0.16 °C, mean ± SE) during winter dormancy, reaching minimal values approximately 1 month prior to spring emergence. Body temperatures of emerging snakes ranged from 0.5 °C during early spring to 6.3 °C during late spring (3.4 ± 0.84 °C, mean ± SE). These results do not support the hypothesis that an increase in ground temperature (and hence body temperature) is necessary for emergence from winter dormancy. We suggest that critically low temperatures (i.e., 0.5–1 °C) are a Zeitgeber entraining an endogenous circannual cycle that regulates snake emergence. These results offer new insight into the mechanisms regulating seasonal emergence from winter dormancy.

Development of the renal sexual segment in immature snakes: effect of sex steroid hormones

The renal sexual segment (RSS) of immature Northern and Diamondback Water Snakes and Red-Sided Garter Snakes exhibited varying responses to testosterone or 17β-estradiol. In both male and female water snakes, kidney mass was not a reliable indicator of hormone treatment, whereas tubule diameter, epithelial height and number of sexual granules responded to hormone treatment. In male water snakes, either hormone initiated granule development by day 16; by day 23, only testosterone increased granule density. Female water snakes receiving either hormone exhibited a small number of granules by day 16; by day 23, granules increased only in Diamondback Water Snakes receiving testosterone. Hormones did not initiate RSS hypertrophy in female Red-Sided Garter Snakes. Tubule diameter and epithelial height of testosterone-treated males exhibited significant hypertrophy, while 17β-estradiol initiated significant increases in tubule diameter. Garter snakes initiated sexual granule development in response to hormone treatment with males exhibiting a greater response than females and testosterone stimulating a greater response than 17β-estradiol. Sex steroids appear to mimic sexual maturity in immature snakes initiating RSS development. Whereas the RSS of adult males respond to testosterone, our data suggest specific changes in the RSS of females during maturation effectively negates the effect of 17β-estradiol evident in immature female RSS.

Male red-sided garter snakes, Thamnophis sirtalis parietalis, determine female mating status from pheromone trails

Recently mated females of some species experience a refractory period following mating during which a physiological change or a mating plug prevents remating. Males that determine female mating status from a distance will minimize time spent searching for mates by avoiding unavailable females. Female red-sided garter snakes experience a refractory period after mating due to a mating plug. Female garter snakes leave pheromone trails, which males detect and follow to find potential mates. We investigated the ability of male garter snakes to determine the mating status of females based solely on these pheromone trails. Males were given a choice between following two trails on a Y maze to examine whether they discriminate between mated and unmated females, whether this discrimination is based on changes in the sex-attractiveness pheromone or the copulatory fluids, and whether they continue to discriminate after the mated females have lost their mating plug. We found that male garter snakes discriminate between the trails of mated and unmated females. This discrimination is based on the presence of a copulatory pheromone, rather than changes in the sex-attractiveness pheromone. The duration of the copulatory pheromone coincides with the duration of the mating plug. Thus, male garter snakes are able to minimize time spent searching for unavailable females by determining female mating status from a distance.

Differential effects of courtship and mating on receptivity and brain metabolism in female red-sided garter snakes (Thamnophis sirtalis parietalis).

In the female red-sided garter snake (Thamnophis sirtalis parietalis), the loss of receptivity following intromission during mating can be prevented by injection of a local anesthetic (tetracaine) in the cloacal region prior to courtship and mating. Females that were courted and then mated had significantly higher uptake of radio-labeled [14C]2-fluoro-2-deoxyglucose (2-DG) in the preoptic area (25%) and significantly lower uptake in the ventromedial hypothalamus (-20%) compared with females that were courted but not mated. Tetracaine-treated females had accumulation patterns similar to courted but unmated females and to females exposed only to other females. These results suggest that in the female red-sided garter snake, sensory input from the cloaca during mating alters patterns of metabolism in those brain areas most often associated with female sexual behavior.

Differential effects of courtship and mating on receptivity and brain metabolism in female red-sided garter snakes (Thamnophis sirtalis pareitalis).

Differential effects of courtship and mating on receptivity and brain metabolism in female red-sided garter snakes (Thamnophis sirtalis pareitalis).

Control of Attractivity and Receptivity in Female Red-Sided Garter Snakes

Female red-sided garter snakes emerge from their hibernacula in the spring attractive and receptive to males. Attractivity is communicated by a pheromone released through the female's skin and is a consequence of ovarian recrudescence the previous summer. Receptivity, on the other hand, is stimulated by ovarian estrogen secretion during emergence itself. Mating renders females both unattractive and unreceptive. Another “mating” pheromone of male origin is important in making females unattractive after mating. To investigate the role of cloacal stimulation in the loss of attractivity and receptivity we injected a local anesthetic (lidocaine or tetracaine) in the cloacal region of females before mating. This does not prevent mating, although it blocks neural transmission of copulatory sensory stimuli. The time course of transition from attractive and receptive states was then observed. Females treated with local anesthetic as well as control females were unattractive within 15 min of mating. However, when retested 2–3 and 24 h after mating, a significantly higher proportion of treated females regained their attractivity, while mated control females remained unattractive. This restorative effect was transient, though, as treated females retested 48 h after mating were as unattractive as the controls. Both anesthetized and control females were unreceptive when tested following mating and did not regain receptivity with time. Last, the mating-induced surge in circulating concentrations of prostaglandin was diminished in females that received a local anesthetic prior to mating. Taken together these results indicate that the loss of attractivity and receptivity following mating in the red-sided garter snake is due to combined effects of a mating pheromone and a physiological, neurally mediated response to the sensation of stimuli associated with the act of mating.

Effects of ovariectomy and estrogen replacement on attractivity and receptivity in the red-sided garter snake (Thamnophis sirtalis parietalis).

Activation of courtship behavior in male red-sided garter snakes, Thamnophis sirtalis parietalis, is independent of the presence of sex steroids. The only consistent treatment that stimulates courtship behavior in males is prolonged exposure to low temperature followed by subsequent warming, mimicking the emergence from hibernation. We investigated whether attractivity and receptivity in female red-sided garter snakes is similarly steriod independent. Female red-sided garter snakes are attractive when they emerge from hibernation and are courted by males; most mate within an hour of emergence. In a series of experiments, groups of females were either ovariectomized (OVEX) in the late spring, fall or while in hibernation. They were tested for attractivity and receptivity upon emergence from hibernation. Females OVEX in the spring were unattractive whereas those OVEX in fall or while in hibernation were attractive. Thus, attractivity appears determined the year before emergence and is dependent on the presence of the ovaries. All OVEX females were unreceptive upon emergence. OVEX females were also given replacement estradiol (E) treatment (either in Silastic capsules or single injections) at various points of their annual cycle. The only treatment that resulted in reinstating receptivity in OVEX females was the injection of E (20 micrograms) one hour prior to emergence. The effectiveness of E in reinstating receptivity was time dependent: the longer the period between emergence and injection, the less effective the same dosage was in stimulating receptive behavior. These experiments suggest that sexual behavior in female red-sided garter snakes is, unlike males, dependent on the presence sex steroid hormones. Although E is naturally at its lowest seasonal level upon emergence, the concentration is sufficient to stimulate receptivity. However, it appears that temperature regulates a time-limited window of sensitivity to E.

Effect of fall mating on ovarian development in the red-sided garter snake.

Although spring mating is an important factor in initiating vitellogenesis in female red-sided garter snakes (Thamnophis sirtalis parietalis), some females can become vitellogenic without having mated in the spring. Two hypotheses have been proposed to explain this phenomenon: 1) long-term storage of copulatory stimuli from mating the previous fall, or 2) additional cue(s) overcoming the lack of mating to stimulate vitellogenesis. Through oviductal biopsy, the presence of sperm in females returning to the hibernaculum in the fall was assessed. Laparotomies performed just before and 6 wk after hibernation indicated that although fall mating may stimulate slight ovarian development in the fall, there appears to be no effect of fall mating on spring ovarian recrudescence. Spring mating seems to be the most important factor in determining ovarian maturation. Therefore, it appears that there is no long-term storage of copulatory cues and that other as yet unknown cues are responsible for initiating vitellogenesis in spring unmated females.

Mating increases plasma levels of prostaglandin F 2α in female garter snakes

Plasma levels of prostaglandin F 2α (PGF 2α) in female red-sided garter snakes ( Thamnophis sirtalis parietalis ) were measured at intervals after mating or exposure to males. PGF 2α levels increased significantly within 15 minutes of mating and peaked 6–24 hr after mating. Females that did not mate, but received similar amounts of male courtship, had levels of PGF 2α significantly lower than those of females that mated. These results extend previous findings that unmated female garter snakes injected with PGF 2α exhibit sexual behavior characteristics of females that have mated. Together these data indicate that female garter snakes elaborate PGF 2α in response to stimuli associated with mating and that PGF 2α has a functional role in inducing post-mating declines in sexual behavior of this species.

Role of light and temperature in the regulation of reproduction in the red-sided garter snake, Thamnophis sirtalis parietalis

The influence of photoperiodic manipulation on sexual behavior and ovarian recrudescence of male and female red-sided garter snakes (Thamnophis sirtalis parietalis) was examined over a 4-year period. Snakes were exposed to photoperiodic manipulations before, during, and after a 17-week cold temperature dormancy; sexual behavior of males and females and ovarian recrudescence were observed after emergence from cold temperature dormancy. In the 1st year (1982), males were exposed to two conditions representing minimum and maximum exposure to daylight: (i) 6 weeks of a short-day prehibernation period (10L:14D, 28:18 °C), followed by 17 weeks of hibernation in complete darkness (0L:24D, 4 °C) and emergence into warm dark conditions (0L:24D, 28:18 °C); and (ii) 6 weeks of a long-day prehibernation period (14L:10D, 28:18 °C), followed by 17 weeks of hibernation with exposure to light (12L:12D, 4 °C) and emergence into warm, long days (14L:10D, 28:18 °C). Males in both conditions exhibited intense courtship behavior on emergence from hibernation. Females in 1982 were significantly influenced only by long-day prehibernation conditions (14L:10D, 28:18 °C); under these conditions, ovarian recrudescence on emergence was inhibited. Long prehibernation photoperiod did not significantly influence female receptive behavior on emergence, indicating that neuroendocrine control of ovarian activation and sexual behavior may be separate in this species. In three subsequent years (1983, 1984, 1985) none of the photoperiodic conditions significantly influenced male or female sexual behavior or ovarian recrudescence. Slight differences in experimental protocol in these subsequent years that may account for differences in results from 1982 are discussed. Finally, ovarian development was found to be clearly tied to the duration of cold temperature dormancy in this species. Females receiving 7 or 17 weeks of exposure to cold (4 °C) underwent vitellogenesis at similar frequencies. Most females receiving 0 or only 4 weeks of exposure to cold (4 °C) did not become vitellogenic. Mating on emergence was not a requirement for the initiation of vitellogenesis in this study.

Plasma steroid hormone levels of female red-sided garter snakes, Thamnophis sirtalis parietalis: Relationship to mating and gestation

Plasma levels of progesterone (P), testosterone (T), estradiol (E 2), and corticosterone (B) of female red-sided garter snakes were measured during the period of ovarian development. Differences in hormone levels were analyzed with respect to three factors: (1) whether the female mated in the spring, (2) ovarian condition, and (3) time after emergence from hibernation. The influence of these three factors on steroid hormone levels of two groups of females were then compared. In experiment I, females were obtained in the fall, subjected to an artificial dormancy period, and placed on warm, summer-like conditions in the laboratory. In experiment II, females were collected in the spring and sampled in the field. They were held in the field on fluctuating conditions for several weeks and then returned to the laboratory for sampling during early vitellogenesis. Females in experiment I had a shortened but otherwise normal ovarian and gestational cycle, whereas females in experiment II had an ovarian and gestational cycle typical of females in the field. In spite of these differences, the steroid hormone levels in relation to the ovarian cycle were remarkably similar for the two groups of females. We confirmed that mating in the spring induces a surge in E 2; E 2 also was elevated in a single sample obtained from animals collected in the fall. This elevation in plasma levels of E 2 in the fall occurs at a time when the majority of females have recently deposited sperm in their oviducts. Plasma levels of T, P, and B were not significantly influenced by mating. Unlike previous reports of other viviparous snakes, plasma levels of P were low and mostly nondetectable, even during late gestation. Plasma T was significantly elevated around the time of late vitellogenesis and ovulation, and there was a tendency for E 2 levels to be elevated at this time. In the field, plasma B levels were initially high immediately after capture and declined with time. Plasma B was significantly elevated in all females several weeks after emergence, suggesting that levels of B may vary with other annual cycles.