Corticosterone in three species of free-ranging watersnakes: Testing for reproductive suppression and an association with body condition.

The potentially suppressive effects of the hypothalamic-pituitary-adrenal (HPA) axis on the hypothalamic-pituitary-gonadal (HPG) axis revolve around the central role that glucocorticoids play in mobilizing energy. As an individual's energy balance becomes negative, the HPA axis helps mobilize energy and shifts energy expenditure away from reproduction toward maintenance and survival. While there is evidence in support of these relationships, substantial species variability exists. Studies in a greater diversity of species promise to enhance our understanding of the interactions between these axes. In this field study we tested for relationships among body condition, corticosterone, and sex steroid concentrations in three species of closely related watersnakes: the common watersnake (Nerodia sipedon), the diamondback watersnake (Nerodia rhombifer) and the queen snake (Regina septemvittata). Snakes were sampled before and after a 30 min acute confinement stressor and body condition was estimated using the scaled mass index. All three species exhibited robust elevations of corticosterone in response to acute stress, but only plasma progesterone was elevated by the acute stressor in queen snakes. There was no evidence for a suppression of sex steroid concentrations in any of the species. Body condition was negatively associated with baseline corticosterone in queen snakes and with post-stressor corticosterone in both queen and common watersnakes. Overall we found fairly strong support for the proposed link between corticosterone and energetics in two of the three watersnake species, but no support for the hypothesis that acute stressors are associated with reproductive suppression, at least as measured by steroid concentrations.

Seasonal changes in baseline corticosterone, association with innate immunity, and effects of confinement in free-ranging Eastern Box Turtles, Terrapene carolina carolina.

Seasonal changes in glucocorticoids and their influence on the immune system are widespread in vertebrates, but whether this occurs in lesser studied taxa like turtles is unclear. The purpose of this study was to test for seasonal changes in baseline corticosterone (CORT), innate immunity, body condition, and metabolic factors (triglycerides, uric acid) in free-ranging Eastern Box Turtles (Terrapene carolina carolina), a species which is in decline across its range. In addition, the effect of handling and confinement on CORT levels was measured. In both years of the seasonal study, baseline CORT concentrations were significantly higher in fall than in spring, but summer CORT levels differed between the years. The annual variation in CORT levels may be related to weather conditions or associated with opportunistic mating. CORT levels of turtles confined for one hour were higher than turtles bled as quickly as possible, and unlike the seasonal study, females had higher baseline and post-stressor CORT levels than males. Baseline CORT levels were positively correlated with hemolysis titer, indicating a possible immunoenhancing effect of CORT. Triglycerides were positively correlated with body condition and were higher in females than males. The higher triglyceride level in females was likely associated with the energetic demands of reproduction and nesting. Males had a lower body condition in the fall than in both spring and summer, while females exhibited no seasonal differences in body condition. Uric acid and innate immunity measures failed to exhibit significant seasonal or sex related differences. Overall our findings of substantial annual, seasonal, and sexual variation in turtle physiology are consistent with the findings reported for other vertebrates and indicate the importance of incorporating these sources of variation into the design of future studies.

Influence of stressor duration on leukocyte and hormonal responses in male Southeastern five-lined skinks (Plestiodon inexpectatus).

Environmental stressors elicit elevations of plasma glucocorticoid hormones. Stressors also result in increases in the circulating heterophil to lymphocyte ratio (H:L ratio), which has been linked to increased glucocorticoids. Recently, there has been debate about the extent to which stress-induced elevations of corticosterone are tied to increases in the H:L ratio and about whether one measure of physiological stress might be used in lieu of the other. Few studies have examined the responses of both variables to standardized stressors in free-living animals, especially reptiles. Stressors may also affect levels of other hormones, such as prostaglandins or testosterone, that may influence leukocytes. Therefore, the H:L ratio, corticosterone, prostaglandin E(2) (PGE(2) ), and testosterone were monitored in free-living male southeastern five-lined skinks, Plestiodon inexpectatus, in response to various durations of confinement stress. Skinks confined for 1 hr had elevated H:L ratios and elevated corticosterone concentrations. In addition, corticosterone was positively correlated with the H:L ratio. In contrast, there were no effects of 2 hr of confinement on the H:L ratio, in spite of maximal corticosterone. Plasma PGE(2) was not affected by 15, 30, or 60 min of confinement but was positively correlated with monocytes and negatively correlated with lymphocytes in the 1 hr of confinement experiment. Consistent with many other studies of reptiles, confinement stress did not result in suppressed testosterone. Overall, the results indicate that, in reptiles at least, the H:L ratio and plasma corticosterone do not covary consistently and one measure of stress cannot be used in lieu of the other.

Effects of breeding season, testosterone and ACTH on the corticosterone response of free-ranging male fence lizards (Sceloporus undulatus).

An attenuated stress response during the breeding season has been reported for several vertebrate species, but the underlying physiological mechanism has received little attention, particularly in reptiles. Modulation could involve changes in the capacity of the adrenal gland to secrete glucocorticoids in addition to upstream changes in the pituitary or hypothalamus. In this study the magnitude of the corticosterone response to capture and confinement was compared between the breeding and postbreeding season in adult male eastern fence lizards, Sceloporus undulatus. Males were captured in both seasons and subjected to the identical stressor of 4h of confinement. Plasma corticosterone levels in response to confinement were significantly lower in the breeding than the postbreeding season. The effect of testosterone on the stress response was tested by experimentally elevating plasma testosterone levels via silastic implants in free-living males during the postbreeding season. Males with experimentally elevated testosterone exhibited significantly weaker corticosterone responses to 1h of confinement than sham-implanted males. Finally the capacity of the adrenal glands to secrete corticosterone during the breeding season was tested by challenging males with adrenocorticotropin (ACTH) injections. In spite of naturally suppressed corticosterone responses during the breeding season, males nonetheless responded robustly to ACTH. Altogether these results suggest that modulation resides upstream of the adrenal gland, as has been shown in some arctic-breeding avian species, and likely involves seasonal changes in testosterone levels.

Effects of acute temperature change, confinement and housing on plasma corticosterone in water snakes, Nerodia sipedon (Colubridae: Natricinae).

Body temperature affects many aspects of reptilian behavior and physiology, but its effect on hormonal secretion has been little studied, especially in snakes. Major objectives of this study were to determine if acute changes in body temperature during confinement influenced plasma corticosterone levels and if initial body temperatures upon capture in the field were related to baseline corticosterone levels in water snakes (Nerodia sipedon). Water snakes were bled upon capture in the field and after one hour of confinement in a cooled, control, or heated incubator. Since little is known about the potential metabolic changes in response to stress in reptiles, plasma triglyceride levels were also measured. Upon completion of the field study, snakes were housed for 5-8 days without food to determine the effect of chronic stress on both corticosterone and triglyceride levels. Plasma corticosterone concentrations were measured using enzyme-linked immunosorbant assay (ELISA) and plasma triglycerides were determined enzymatically. In the field, experimental alterations of body temperature during confinement had no effect on corticosterone levels. Similarly, there was no correlation between initial body temperature and baseline plasma corticosterone concentrations. However, post-confinement corticosterone levels were approximately three-times greater in females than males. Plasma triglyceride levels were not affected by temperature treatment, confinement, or sex. Compared to field values, both baseline and post-confinement corticosterone levels were elevated after the chronic stress of short-term laboratory housing but triglyceride levels decreased. Overall, these results indicate that sex but not body temperature has a major influence on the adrenocortical stress response in Nerodia sipedon.