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 steroid 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 g) 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.     

The male red-sided garter snake (Thamnophis sirtalis parietalis): reproductive pattern and behavior

Among the small group of species (e.g., some temperate zone turtles, snakes, and bats) that exhibit a dissociated reproductive pattern, the red-sided garter snake (Thamnophis sirtalis parietalis) is probably the most well studied. For these species, courtship and mating occur immediately upon emergence from winter dormancy; the gonads remain essentially inactive. Male red-sided garter snakes are a particularly informative animal model for examining the role of neuroendocrine factors associated with reproductive physiology and behavior because unlike species that exhibit an associated reproductive pattern, in which sex steroids initiate and control sexual behavior, reproductive behavior in the male garter snake appears to be independent of circulating sex hormone control. In fact, the only factor associated with the initiation of courtship behavior and mating in the male garter snake is an extended period of low temperature dormancy followed by exposure to warm temperatures. Yet the presence of sex steroid-concentrating neurons within the pathways regulating courtship and mating suggests that sex hormones may be involved in the activation of sexual behavior. Although circulating androgens are elevated upon emergence from hibernation, the initiation of courtship behavior and mating appears to be independent of direct androgen control. Thus steroid hormones may have indirect effects on mating behavior in animals that display "dissociated" reproductive behaviors.     

Hormonal control of male courtship behavior and female attractivity in the garter snake (Thamnophis sirtalis sirtalis)

Mating behavior in both intact and gonadectomized garter snakes (Thamnophis sirtalis sirtalis) was measured following hormone administration. Male courtship was androgen-dependent; subcutaneous implants of crystalline testosterone propionate (TP) pellets induced mating behavior within 2 days in both intact, reproductively inactive males and castrated males. Female attractivity, as measured by male courtship of the female, was stimulated by exogenous estrogen; 20 μg/day of estradiol benzoate (EB) was the minimum effective dose for stimulating female attractivity in both intact, reproductively inactive females and ovariectomized females. TP-implanted males selectively courted EB-primed females in both sequential and simultaneous (choice) mating tests. It is probable that males use estrogen-dependent olfactory cues produced by the females to discriminate between hormone- and vehicle-injected females.     

Mating increases plasma levels of prostaglandin F2 alpha in female garter snakes

Plasma levels of prostaglandin F2 alpha (PGF2 alpha) in female red-sided garter snakes (Thamnophis sirtalis parietalis) were measured at intervals after mating or exposure to males. PGF2 alpha 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 PGF2 alpha significantly lower than those of females that mated. These results extend previous findings that unmated female garter snakes injected with PGF2 alpha exhibit sexual behavior characteristic of females that have mated. Together these data indicate that female garter snakes elaborate PGF2 alpha in response to stimuli associated with mating and that PGF2 alpha has a functional role in inducing post-mating declines in sexual behavior of this species.     

The transvestite serpent: why do male garter snakes court (some) other males?

In large mating aggregations of red-sided garter snakes, Thamnophis sirtalis parietalis, in Manitoba, male courtship is directed not only to females, but also to other males with female-like skin lipids ('she-males'). We show that 'she-maleness' is an intrinsic property of a male rather than an artefact of lipid transfer from females, and that male-male courtship is very common in the field. She-males were distinctive in terms of appearance (they were heavier than other males and more often covered with mud), behaviour (they were inactive and rarely courted females) and performance (they were slow crawlers, ineffective courters and easily outcompeted by other males in mating trials). 'She-maleness' was not a characteristic of a particular subset of males, as envisaged in previous work; instead, it was a transitory phase that most (perhaps all) male snakes passed through soon after they first emerged from the winter den. Recently emerged males spent their first day or two relatively inactive, while restoring physiological functions (including locomotor performance and courtship ability). Experimental application of female skin lipids on to males dramatically decreased courtship levels of the recipient snakes. Thus, recently emerged males may derive two kinds of benefit from mimicking female skin lipids. First, female mimicry 'switches off' the male's own (energetically expensive) courtship at a time when that courtship would be unproductive. Second, it may disadvantage his rivals by distracting them from females, and increasing their energy expenditure. Copyright 2000 The Association for the Study of Animal Behaviour.     

Effects of castration and androgen replacement on male courtship behavior in the red-sided garter snake (Thamnophis sirtalis parietalis)

Following artificial hibernation, sexually mature male garter snakes (Thamnophis sirtalis parietalis) exhibited a decline in courtship behavior irrespective of castration, sham operation, or castration with testosterone replacement therapy. Behavior declined more rapidly in castrated animals with testosterone replacement than in castrated or sham-operated animals. In sham-operated animals, the decline in courtship was accompanied by changes in testicular weight and spermatogenic state from small spermatogenically inactive testes to large spermatogenically active testes. Serum androgen levels were more than fourfold greater in sham-operated animals than in castrated animals; cell height of the androgensensitive renal sex segment was greatest in castrated animals with testosterone replacement and least in castrated animals. These findings indicate that following artificial hibernation, male courtship behavior of T.s. parietalis is independent of the presence of the testes.     

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.     

Courtship tactics in garter snakes: how do a male's morphology and behaviour influence his mating success?

The behavioural determinants of male mating success play a pivotal role in sexual selection, but remain poorly known for most kinds of organisms, including most reptiles. In Manitoba, Canada, large numbers of red-sided garter snakes, Thamnophis sirtalis parietalis, court and mate in early spring near communal overwintering dens. To understand how a male's morphology and behaviour might influence his mating success, we videotaped 21 groups of snakes each comprising four males of varying body sizes plus a female. All males engaged in courtship, and mating occurred in all groups. Males with better body condition courted more vigorously and successfully than their smaller rivals did. The males that obtained matings were those that maintained their own cloaca closest to that of the female, aligned most of their body with the female, showed high rates of caudocephalic waving, chin pressing and tail searching, and did not allow other males to insert their body beside the female's. These behavioural differences between ‘winners’ and ‘losers’ were apparent from the beginning of each trial. Thus, male mating success in this ‘scramble’ system is determined not by chaotic, stochastic struggle (as has often been inferred) but instead is strongly linked to male courtship tactics. Energy stores (as evidenced by good body condition) may be critical determinants of mating success in these vigorous courtship episodes.     

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.     

Seasonal aromatase activity in the brain of the male red-sided garter snake

We investigated regional and seasonal variations in neural aromatase activity (AA), the enzyme that converts androgens into estrogens, to examine a possible indirect role of testosterone (T) in mediating spring reproductive behavior of red-sided garter snakes, a species exhibiting a dissociated reproductive pattern. Neural AA in male snakes varied significantly among brain regions. Additionally, there were significant interactions between brain region and season. In the spring, actively courting males had greater AA in the olfactory region (O) compared to the septum/anterior-hypothalamus preoptic area (S/AHPOA), nucleus sphericus (NS) and midbrain (Mb). Fall animals collected as they returned to the den prior to winter dormancy had significantly greater AA in the S/AHPOA compared to all other regions. These findings were consistent using either regional (gross) dissection or punch microdissection, which allowed us to separate the S and AHPOA. There were no significant differences in AA production between the S and AHPOA. This study provides the first documentation of seasonal and regional variations in AA in a snake brain and suggests that aromatization of androgens may play a role in regulating reproduction in red-sided garter snakes. During spring mating, elevated AA in the O may activate pathways essential for detection of courtship pheromones, while increased AA in the S and AHPOA of fall animals suggests that circulating androgens play an indirect role in programming critical neural pathways involved in reproduction. Thus, as in many other vertebrates, estrogenic metabolites of testosterone may be a critical hormonal component regulating reproductive behavior in this dissociated breeder.     

Behavioral and physiological control of yolk synthesis and deposition in the female red-sided garter snake (Thamnophis sirtalis parietalis)

Ovarian recrudescence in female garter snakes, Thamnophis sirtalis parietalis, follows spring emergence from hibernation and mating. In the laboratory, courtship and mating stimuli significantly increased the proportion of female garter snakes becoming pregnant, although some noncourted nonmated controls also became pregnant. Females given artificial mating stimuli under anesthetic, without courtship stimuli, were no more likely than either noncourted, nonmated or anesthetized controls to become pregnant. Hormonal changes and yolk synthesis rapidly followed mating in both laboratory and field females; serum estradiol increased more than 10-fold in 2 days and serum calcium, a measure of yolk precursor lipoprotein (vitellogenin) concentration, increased more than two times in 10 days. Administration of exogenous estradiol stimulated yolk synthesis, but did not result in yolk deposition into ovarian follicles. However, administration of ovine follicle-stimulating hormone induced both hepatic yolk synthesis and yolk deposition. Our results are consistent with the hypotheses that courtship and copulation are facilitatory to ovarian recrudescence but neither alone nor in combination is necessary nor sufficient, and in this species yolk synthesis and yolk deposition are separately regulated.     

A novel mechanism regulating a sexual signal: The testosterone-based inhibition of female sex pheromone expression in garter snakes

Vertebrates communicate their sex to conspecifics through the use of sexually dimorphic signals, such as ornaments, behaviors and scents. Furthermore, the physiological connection between hormones and secondary sexual signal expression is key to understanding their dimorphism, seasonality and evolution. The red-sided garter snake (Thamnophis sirtalis parietalis) is the only reptile for which a described pheromone currently exists, and because garter snakes rely completely on the sexual attractiveness pheromone for species identification and mate choice, they constitute a unique model species for exploring the relationship between pheromones and the endocrine system. We recently demonstrated that estrogen can activate female pheromone production in male garter snakes. The purpose of this study was to determine the mechanism(s) acting to prevent female pheromone production in males. We found that castrated males (GX) are courted by wild males in the field and produce appreciable amounts of female sex pheromone. Furthermore, pheromone production is inhibited in castrates given testosterone implants (GX + T), suggesting that pheromone production is actively inhibited by the presence of testosterone. Lastly, testosterone supplementation alone (T) increased the production of several saturated methyl ketones in the pheromone but not the unsaturated ketones; this may indicate that saturated ketones are testosterone-activated components of the garter snake's skin lipid milieu. Collectively, our research has shown that pheromone expression in snakes results from two processes: activation by the feminizing steroid estradiol and inhibition by testosterone. We suggest that basal birds and garter snakes share common pathways of activation that modulate crucial intraspecific signals that originate from skin.     

Sexual conflict over mating in red-sided garter snakes (Thamnophis sirtalis) as indicated by experimental manipulation of genitalia

Sexual conflict over mating can result in sex-specific morphologies and behaviours that allow each sex to exert control over the outcome of reproduction. Genital traits, in particular, are often directly involved in conflict interactions. Via genital manipulation, we experimentally investigated whether genital traits in red-sided garter snakes influence copulation duration and formation of a copulatory plug. The hemipenes of male red-sided garter snakes have a large basal spine that inserts into the female cloaca during mating. We ablated the spine and found that males were still capable of copulation but copulation duration was much shorter and copulatory plugs were smaller than those produced by intact males. We also anaesthetized the female cloacal region and found that anaesthetized females copulated longer than control females, suggesting that female cloacal and vaginal contractions play a role in controlling copulation duration. Both results, combined with known aspects of the breeding biology of red-sided garter snakes, strongly support the idea that sexual conflict is involved in mating interactions in this species. Our results demonstrate the complex interactions among male and female traits generated by coevolutionary processes in a wild population. Such complexity highlights the importance of simultaneous examination of male and female traits.     

Role of the anterior hypothalamus-preoptic area in the regulation of courtship behavior in the male Canadian red-sided garter snake (Thamnophis sirtalis parietalis): intracranial implantation experiments

Testosterone (T) was administered intracranially to intact adult male Canadian red-sided garter snakes (Thamnophis sirtalis parietalis) in the fall and in the summer. Implants in the anterior hypothalamus-preoptic area (AH-POA) as well as other regions of the brain, including the medial and basal hypothalamus, thalamus, medial cortex, and third ventricle, failed to elicit courtship behavior. The spermatogenic stage was more advanced, and the circulating level of androgens was significantly higher, in animals that received implants of T in the AH-POA. These findings suggest the sex steroid-concentrating sites in the AH-POA of the garter snake are involved in feedback regulation of pituitary gonadotropin secretion and not in the control of courtship behavior. Hematocrit was found to be higher in animals that received implants of T, regardless of location, a response that may be related to changes in blood chemistry prior to hibernation. These findings support previous research indicating that in the adult male red-sided garter snake, a species exhibiting a dissociated reproductive tactic, courtship behavior is independent of testicular androgens.     

Mating increases plasma levels of prostaglandin F2α in female garter snakes

Plasma levels of prostaglandin F_2α (PGF_2α) in female red-sided garter snakes (

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) 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.     

Species specificity of methyl ketone profiles in the skin lipids of female garter snakes,genus Thamnophis

One of the relatively few vertebrate pheromones to be chemically identified, the female sex pheromone of the red-sided garter snake (Thamnophis sirtalis parietalis) is a series of saturated and monounsaturated methyl ketones contained within female skin lipids. During the breeding season, this pheromone is responsible for eliciting male courtship behaviors and males are able to utilize pheromonal variation to discriminate among females. While the pheromone system of the red-sided garter snake has been the subject of many studies, relatively little is known about the pheromone systems of other garter snakes. Through chemical analyses, we demonstrate that female skin lipids of the red-spotted garter snake (Thamnophis sirtalis concinnus), northwestern garter snake (Thamnophis ordinoides), and plains garter snake (Thamnophis radix) contain similar methyl ketones. The methyl ketone profiles of these snakes differ qualitatively from one another and from the methyl ketone profiles of red-sided garter snakes with differences particularly pronounced between sympatric species. Our results provide evidence that the use of methyl ketones in sexual signaling may be ubiquitous for Thamnophis species and suggest that these compounds could play a role in reproductive isolation between species in this genus.     

Cryptic forcible insemination: male snakes exploit female physiology, anatomy, and behavior to obtain coercive matings

Whether males can inseminate uncooperative females is a central determinant of mating system evolution that profoundly affects the interpretation of phenomena such as multiple mating by females, mate choice, reproductive seasonality, and courtship tactics. Forcible insemination is usually inferred from direct physical battles between the sexes and has been dismissed on intuitive grounds for many kinds of animals. For example, snakes have elongate flexible bodies (making it difficult for a male to restrain a female physically), males are typically smaller than females, and copulation requires female cloacal gaping to enable intromission. Male garter snakes (Thamnophis sirtalis) do not display any overt aggression during courtship and simply lie over the female and exhibit rhythmic pulsating caudocephalic waves of muscular contraction; previous studies have interpreted this behavior as a mechanism for eliciting female receptivity. In contrast, we show that male garter snakes forcibly inseminate females. They do so by taking advantage of specific features of snake physiology, respiratory anatomy, and antipredator behavior. The snake lung extends along most of the body, with the large posterior section (the saccular lung) lacking any respiratory exchange surface. Rhythmic caudocephalic waves by courting male garter snakes push anoxic air from the saccular lung forward and across the respiratory surfaces such that females cannot obtain oxygen. Their stress response involves cloacal gaping, which functions in other contexts to repel predators by extruding feces and musk but in this situation permits male intromission. Thus, superficially benign courtship behaviors may involve cryptic coercion even in species for which intuition dismisses any possibility of forcible insemination.     

Effects of male age on the mating behavior of both sexes in the sorghum plant bug, Stenotus rubrovittatus (Hemiptera: Miridae)

In the sorghum plant bug Stenotus rubrovittatus (Matsumura) (Hemiptera: Miridae), mating behavior consists of male courtship and female mating receptivity. Previous studies have found that female age is correlated with ovary development and that mature females with developed ovaries are more receptive to male courtship. Thus, we examined whether male age affects the mating behavior of both sexes and male accessory gland development. Unmated males 0–9 days after emergence and 3-day-old virgin females receptive to male courtship were studied. Immediately after emergence, only 20 % of the males courted females (n = 25). At 3 days old, 68 % of the males courted females (n = 25), the most active age. In contrast, more than 75 % of the courted females were receptive to the male courtship regardless of male age. These results indicate that only male courtship behavior is affected by male age, although it is not enhanced in proportion to male age. Male accessory glands developed with male age. However, no clear relationship was detected between male courtship behavior and accessory gland development. In S. rubrovittatus, it is difficult to explain male courtship behavior solely from male age and accessory gland development.     

Do female garter snakes evade males to avoid harassment or to enhance mate quality?

Females of many species behave in ways that make it difficult for males to locate, court, and inseminate them. Two hypotheses have been advanced to explain such behavior: either a female thereby minimizes costs of harassment (sexual conflict model) or by playing "hard to get" she discourages inferior suitors (indirect mate choice model). Our studies of garter snakes (Thamnophis sirtalis parietalis) at a communal den in Manitoba support an interpretation of sexual conflict rather than indirect mate choice. Female snakes dispersed rapidly from the den through areas with relatively few males rather than waiting for additional courtship. Many females dispersed without mating. Experimental (pheromonal) manipulation of the intensity of courtship accelerated rates of female dispersal rather than delaying dispersal, as would be predicted if females wait to obtain matings. The behaviors of females escaping from courting groups were maximally effective in losing their suitors regardless of the number of courting males or whether or not the female was capable of mating (recently mated females cannot mate again because of a mating plug). In total, our data are most consistent with the hypothesis that female garter snakes at communal dens evade males to escape harassment rather than to enhance mate quality.     

Environmental and seasonal adaptations of the adrenocortical and gonadal responses to capture stress in two populations of the male garter snake, Thamnophis sirtalis

Stress and reproduction are generally thought to work in opposition to one another. This is often manifested as reciprocal relationships between glucocorticoid stress hormones and sex steroid hormones. However, seasonal differences in how animals respond to stressors have been described in extreme environments. We tested the hypothesis that garter snakes, Thamnophis sirtalis, with limited reproductive opportunities will suppress their hormonal stress response during the breeding season relative to conspecifics with an extended breeding season. The red-sided garter snake, T.s. parietalis, of Manitoba, Canada, has a brief breeding season during which males displayed no change in either plasma levels of testosterone or corticosterone, which were both elevated above basal levels, in response to capture stress. During the summer, capture stress resulted in increased plasma corticosterone and decreased testosterone. During the fall, when mating can also occur, males exhibited a significant decrease in testosterone but no increase in corticosterone in response to capture stress. The red-spotted garter snake, T.s. concinnus, of western Oregon, has an extended breeding season during which males displayed a stress response of increased plasma corticosterone and decreased testosterone levels. The corticosterone response to capture stress was similar during the spring, summer, and fall. In contrast, the testosterone response was suppressed during the summer and fall when gametogenesis was occurring. These data suggest that male garter snakes, in both populations, seasonally adapt their stress response but for different reasons and by potentially different mechanisms. J. Exp. Zool. 289:99-108, 2001.