Female behaviour and the interaction of male and female genital traits mediate sperm transfer during mating.

Natural selection and post-copulatory sexual selection, including sexual conflict, contribute to genital diversification. Fundamental first steps in understanding how these processes shape the evolution of specific genital traits are to determine their function experimentally and to understand the interactions between female and male genitalia during copulation. Our experimental manipulations of male and female genitalia in red-sided garter snakes (Thamnophis sirtalis parietalis) reveal that copulation duration and copulatory plug deposition, as well as total and oviductal/vaginal sperm counts, are influenced by the interaction between male and female genital traits and female behaviour during copulation. By mating females with anesthetized cloacae to males with spine-ablated hemipenes using a fully factorial design, we identified significant female-male copulatory trait interactions and found that females prevent sperm from entering their oviducts by contracting their vaginal pouch. Furthermore, these muscular contractions limit copulatory plug size, whereas the basal spine of the male hemipene aids in sperm and plug transfer. Our results are consistent with a role of sexual conflict in mating interactions and highlight the evolutionary importance of female resistance to reproductive outcomes.

An airborne sex pheromone in snakes.

Most reptile sex pheromones so far described are lipid molecules too large to diffuse through the air; instead, they are detected via direct contact (tongue-flicking) with another animal's body or substrate-deposited trails, using the vomeronasal system. The only non-lipid pheromone reported in snakes involves courtship termination in red-sided gartersnakes (Thamnophis sirtalis parietalis): males that encounter copulatory fluids cease courtship, presumably reflecting the futility of courting an already-mating female. Our field experiments at a communal den in Manitoba show that this pheromone can work via olfaction: courtship is terminated by exposure to airborne scents from mating conspecifics, and does not require direct contact (tongue-flicking). Hence, the sexual behaviour of reptiles can be affected by airborne as well as substrate-bound pheromones.

The influence of sex steroids on the sexual size dimorphism in the red-spotted garter snake, Thamnophis sirtalis concinnus.

The red-spotted garter snake exhibits adult size dimorphism in which females are the larger sex. To understand which hormones may influence differential growth in this species, growth curves and hormone profiles of estradiol-17beta (E2) and testosterone (T) were constructed in male and female neonates. Growth was manipulated via implantation of exogenous hormones and hormone antagonists. Female neonates are heavier or longer beginning at either 20 or 24 weeks of age, respectively. Although low circulating levels of E2 and T were present in males and females from birth through 15 weeks of age, these levels were not significantly different between the sexes. Differences in the growth curves of the treated and untreated snakes were significant after 24 weeks of age. Antiestrogen produced male-like growth in females but had no effect on males. Antiandrogen had no effect on either males or females. Exogenous T reduced female growth to that observed in males, and E2 reduced male growth. These results suggest that a basal level of either E2 or T is sufficient in males to retain typical male growth patterns. Similar endogenous levels of E2 appear to have growth-promoting effects in females. Endogenous T does not appear to play a role in female growth.

Bumpus in the snake den: effects of sex, size, and body condition on mortality of red-sided garter snakes.

Huge breeding aggregations of red-sided garter snakes (Thamnophis sirtalis parietalis) at overwintering dens in Manitoba provide a unique opportunity to identify sources of mortality and to clarify factors that influence a snake's vulnerability to these factors. Comparisons of sexes, body sizes, and body condition of more than 1000 dead snakes versus live animals sampled at the same time reveal significant biases. Three primary sources of mortality were identified. Predation by crows, Corvus brachyrhynchos (590 snakes killed), was focussed mostly on small snakes of both sexes. Crows generally removed the snake's liver and left the carcass, but very small snakes were sometimes brought back to the nest. Suffocation beneath massive piles of other snakes within the den (301 dead animals) involved mostly small males and (to a lesser extent) large females; snakes in poor body condition were particularly vulnerable. Many emaciated snakes (n = 142, mostly females) also died without overt injuries, probably due to depleted energy reserves. These biases in vulnerability are readily interpretable from information on behavioral ecology of the snakes. For example, sex biases in mortality reflect differences in postemergence behavior and locomotor capacity, the greater attractiveness of larger females to males, and the high energy costs of reproduction for females.

Behavioral and hormonal responses to corticosterone in the male red-sided garter snake, Thamnophis sirtalis parietalis.

Stress and glucocorticoids are generally thought to suppress reproductive function at multiple levels. We tested the hypotheses that exogenous corticosterone would suppress sexual behavior in a dose-dependent manner, as well as drive a decrease in plasma testosterone levels in the male red-sided garter snake, Thamnophis sirtalis parietalis. We examined this by challenging individual males with intraperitoneal injections of exogenous corticosterone, and subsequently exposing them to sexually attractive females or taking a blood sample. Previous work has demonstrated a hormonal but no behavioral response to stress in this species. In this study, increasing concentrations of exogenous corticosterone rapidly suppressed mating behavior in a threshold manner. However, exogenous corticosterone had no effect on plasma levels of testosterone. Thus, these data suggest that the mechanism is in place for corticosterone to suppress mating behavior in this species and that these effects do not occur because of an indirect effect on plasma levels of testosterone but rather are the direct effect of the hormone itself. In addition, the negative relationship observed previously between plasma levels of corticosterone and testosterone in this species was probably not the direct result of corticosterone acting on the hypothalamic-pituitary-gonadal (HPG) axis. Rather, our results seem to indicate that the negative associations between the hypothalamic-pituitary-adrenal axis (HPA) and the HPG axis occur at other levels of these neuroendocrine pathways.

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.

Pheromone trailing behavior of the brown tree snake, Boiga irregularis.

The ability of snakes to follow pheromone trails has significant consequences for survival and reproduction. Of particular importance is the ability of snakes to locate conspecifics during the breeding season via the detection of pheromone trails. In this study, the ability of male brown tree snakes (Boiga irregularis), a tropical, rear-fanged colubrid, to follow pheromone trails produced by reproductively active conspecifics was tested in the laboratory by using a Y maze. Males displayed a trailing response to both female and male pheromone trails over blank controls. As males of this species display ritualized combat behavior, these responses likely represent both direct and indirect mechanisms, respectively, for the location of potential mates in the wild. Males did not, however, discriminate between male and female trails when given a choice on the Y maze.

Conflicts between courtship and thermoregulation: the thermal ecology of amorous male garter snakes (Thamnophis sirtalis parietalis, colubridae).

Thermoregulatory behavior is an important component of daily activities for many reptiles, especially for small heliothermic (sun-basking) species that inhabit cold climates. However, the relative costs and benefits of thermoregulation depend on numerous factors, such that reptiles may sometimes accord a low priority to precise control of body temperatures. We observed and radio tracked garter snakes (Thamnophis sirtalis parietalis) in central Manitoba during the mating season (spring). Previous studies on this species have documented precise behavioral regulation of body temperatures during summer. In contrast, the courting snakes that we studied in springtime spent little time in overt thermoregulatory behavior. Body temperatures were extremely variable (both in outdoor enclosures and in the field) despite abundant opportunities for more precise thermal control. These small elongate reptiles cool so quickly (relative to the time periods needed for effective courtship) that any benefit to higher body temperatures would be transitory at best. Experiments show that hotter males are no better at obtaining matings or at detecting predators. Thus, male garter snakes concentrate on courtship rather than on basking. In the face of conflicting priorities, reptiles may often forgo precise thermoregulation because its benefits are too low, and its costs too high, compared with alternative behaviors.

Relationships between annual cycles of testosterone, corticosterone, and body condition in male red-spotted garter snakes, Thamnophis sirtalis concinnus.

Over a 2-yr period, we investigated the annual cycles of plasma testosterone and corticosterone and the relationships between these hormones and body condition in a wild population of male red-spotted garter snakes, Thamnophis sirtalis concinnus. In the 10 mo that were sampled, a peak in testosterone was observed in late summer during gametogenesis and declining through the spring breeding period. Corticosterone and testosterone cycles were positively correlated, in contrast to many vertebrates, suggesting the lack of a direct negative interaction between the two hormones. Body condition, defined as the residual of the regression of mass on snout-vent length, also cycled annually, with individuals being more robust during the summer than during the spring or fall. Individuals with a positive body condition had significantly lower plasma levels of corticosterone than did individuals with a negative body condition, supporting the energetic role of glucocorticoids. There was no relationship between body condition and testosterone. This study suggests that annual cycles of testosterone, corticosterone, and body condition can be associated with one another, and considering all three simultaneously is necessary to understand their control and function.

Fatal mycotic dermatitis in captive brown tree snakes (Boiga irregularis).

Cutaneous fungal infections occurred in four captive brown tree snakes (Boiga irregularis). The ventral scales were most commonly affected, and lesions began as areas of erythema and edema with vesicle formation, followed by development of caseous brown plaques. Lesions usually started where ventral scales overlapped and spread rapidly. All snakes died within 14 days after clinical signs were first noted. The deaths of three of the snakes were directly attributable to the cutaneous disease; the other snake died from renal failure and visceral gout, most likely induced by gentamicin therapy. Histologically, lesions consisted of epidermal hyperplasia and hyperkeratosis, with foci of epidermal necrosis, intraepidermal vesicle formation, and subacute inflammation of the underlying dermis. These lesions were associated with bacteria and numerous septate, branched fungal hyphae within the epidermis and overlying serocelluar crusts. Hyphae that penetrated through the superficial surface of the epidermis often formed terminal arthroconidia. The same species of fungus was isolated in pure culture from the skin of three snakes, but fungal cultures were not performed on samples from the fourth snake. The fungus has been identified as the Chrysosporium anamorph of Nannizziopsis vriesii based on its formation of solitary dermatophytelike aleurioconidia and alternate and fission arthroconidia. The source of the fungus in this outbreak was not determined; however, the warm, moist conditions under which the snakes were housed likely predisposed them to opportunistic cutaneous fungal infections.

Gonadotropin antagonist modulates courtship behavior in male red-sided garter snakes, Thamnophis sirtalis parietalis.

Behavioral studies were used to investigate the central effects of chicken-I GnRH, chicken-II GnRH, and D-Phe2,6,Pro3-GnRH, a GnRH antagonist, on the courtship behavior of male red-sided garter snakes, Thamnophis sirtalis parietalis. Intracerebroventricular (i.c.v.) injections of chicken-I or chicken-II GnRH had no effect on time spent courting or latency to court when experimental males were exposed to unmated females, or when experimental males were exposed to the female sex attractiveness pheromone. I.c.v. injections of D-Phe2,6,Pro3-GnRH caused a significant decrease in latency to court when experimental males were exposed to unmated females. When males injected with D-Phe2,6,Pro3-GnRH were exposed to the female sex attractiveness pheromone, it caused a significant increase in time spent courting compared to that in saline-injected controls. D-Phe2,6,Pro3-GnRH was not able to initiate courtship behavior during the nonbreeding season, indicating that courtship behavior is dependent on the interaction of multiple components. This study does demonstrate that a hormone or neuropeptide can modulate sexual behavior in garter snakes.

Neuroanatomical distribution of chicken-I gonadotropin-releasing hormone (cGnRH-I) in the brain of the male red-sided garter snake.

Immunocytochemistry was used to investigate the neuroanatomical distribution of the chicken-I form of gonadotropin-releasing hormone (cGnRH-I) in reproductively active, male, red-sided garter snakes (Thamnophis sirtalis parietalis). Cell bodies with cGnRH-I immunoreactivity (ir) were found in the terminal nerve ganglion, nucleus of the diagonal band of Broca, medial preoptic area, and the hypothalamus. Fibers containing cGnRH-Iir were distributed in the following brain areas. Within the olfactory bulb, fibers were found in the internal plexiform, mitral and glomerular cell layers, as well as in the terminal nerve; within the forebrain, fibers were observed in the diagonal band of Broca, rostral and lateral septum, lateral pallium, retrobulbar region pars dorsomedialis, nucleus accumbens, medial preoptic area, hippocampal commissure, amygdala, posterior dorsal ventricular ridge, hypothalamus, median eminence, and the thalamus; within the midbrain, fibers were found in the interpeduncular nucleus and the stratum album periventricular of the optic tectum. This study shows that the distribution of cell bodies for cGnRH-Iir in this reptile is consistent with the distribution of immunoreactivity for cGnRH-I in birds and mammalian GnRH in amphibians and mammals. Using antiserum specific to cGnRH-I, the endogenous form of GnRH, this is the first study to show that the terminal nerve in a reptile contains GnRH immunoreactivity.

Plasma triglyceride and beta-hydroxybutyric acid levels in red-sided garter snakes (Thamnophis sirtalis parietalis) at emergence from hibernation.

Measurement of plasma levels of triglycerides and beta-hydroxybutyric acid in females and males of the red-sided garter snake (Thamnophis sirtalis parietalis) suggest that the former may provide a useful physiological marker of condition. Levels of triglycerides at emergence from hibernation during a month of natural aphagia were significantly greater in females than in males and she-males, a subset of the male population that mimics females. Higher levels of triglycerides in the females may be attributed to their greater body mass per unit length, which was correlated with the level of triglycerides. Plasma triglyceride levels declined in females within one month of emergence, at the onset of feeding, and were unrelated to mating.

On insect attractants from pitcher plants of the genusHeliamphora (sarraceniaceae).

Examination ofHeliamphora heterodoxa andH. tatei from the Guayana Highlands of Venezuela reveals that the enol diacetal monoterpene, sarracenin, is the major volatile compound present in the spoon-shaped structures of leaves of the pitchers. In addition, erucamide, phenol, cinerone, phenylacetaldehyde, and a series of methyl esters also occur in extracts of the spoon-shaped appendages of pitchers at the time during which they attract insects.

Chemical recognition of kingsnakes by crotalines: effects of size on the ophiophage defensive response.

When confronted by an ophiophagous (snake-eating) kingsnake, venomous snakes of the subfamily Crotalinae exhibit a suite of defensive responses including head hiding, thrashing, and an unusual response termed 'body bridging'. Other responses observed, such as biting and 'freezing', are more general in nature and can occur in a variety of contexts. Various crotalines of differing sizes were tested for their responses to kingsnakes (Lampropeltis getulus). Responses of individuals were recorded for up to 18 months. The results indicate that, if habituation can be overcome by periodically allowing a kingsnake to confront but not harm the crotaline, the response is dependent on the size of the crotaline, in that smaller specimens (< 0.9 m) respond readily, while larger snakes (> 1.0 m) tend not to respond. The size of the kingsnake apparently does not have an effect on the crotaline response. These data appear to resolve apparent conflicts in the literature regarding whether certain species respond to ophidian ophiophages. In addition, hexane extracts of kingsnake skin were fractionated using an alumina column. The various fractions obtained were tested to determine which elicited the defensive response. Activity was found only in the most non-polar fraction. Preliminary analysis by gas chromatography/mass spectrometry indicated that this fraction contained straight and branched, saturated and polyunsaturated long-chain hydrocarbons.

Chemical ecology of the red-sided garter snake, Thamnophis sirtalis parietalis.

The red-sided garter snake, Thamnophis sirtalis parietalis is an ideal model for the study of chemical or pheromonal communication in vertebrates. Results of long-term field and laboratory investigations of the chemical ecology of these unusual animals is summarized. These studies include a description of the characterization of the female attractiveness pheromone that serves to induce male courtship behavior. The male sex recognition pheromone system that identifies males as inappropriate individuals to court is also described. She-males, individual males that are courted as if they were females, seem to possess semiochemical components intermediate to both males and females. Finally, investigations of the species-specific nature of these pheromones indicate that chemical differences exist in the methyl ketones of all species examined to date.