Ophidiomycosis is associated with alterations in the acute glycemic and glucocorticoid stress response in a free-living snake species.

Emerging fungal pathogens are a direct threat to vertebrate biodiversity. Elucidating the mechanisms by which mycoses impact host fitness is an important step towards effective prediction and management of disease outcomes in populations. The vertebrate acute stress response is an adaptive mechanism that allows individuals to meet challenges to homeostasis and survival in dynamic environments. Disease may cause stress, and coping with fungal infections may require shifts in resource allocation that alter the ability of hosts to mount an acute response to other external stressors. We examined the glucocorticoid and glycemic response to acute capture stress in a population of free-living pygmy rattlesnakes, Sistrurus miliarius, afflicted with an emerging mycosis (ophidiomycosis) across seasons. In all combinations of disease status and season, acute capture stress resulted in a significant glucocorticoid and glycemic response. While disease was not associated with elevated baseline or stress-induced corticosterone (CORT), disease was associated with an increased glucocorticoid stress response (post-stress minus baseline) across seasons. Both baseline and stress-induced glucose were lower in snakes with ophidiomycosis compared to uninfected snakes. The relationship between glucose and pre- and post-stress CORT depended on infection status, and positive correlations were only observed in uninfected snakes. The variables which explained CORT and glucose levels were different. The pattern of CORT was highly seasonal (winter high - summer low) and negatively related to body condition. Glucose, on the other hand, did not vary seasonally or with body condition and was strongly related to sex (male high - female low). Our results highlight the fact that circulating CORT and glucose are sensitive to different intrinsic and extrinsic predictor variables and support the hypothesis that disease alters the acute physiological stress response. Whether the effects of ophidiomycosis on the acute stress response result in sublethal effects on fitness should be investigated in future studies.

The metabolic response to an immune challenge in a viviparous snake, Sistrurus miliarius.

Mounting an immune response may be energetically costly and require the diversion of resources away from other physiological processes. Yet, both the metabolic cost of immune responses and the factors that impact investment priorities remain poorly described in many vertebrate groups. For example, although viviparity has evolved many times in vertebrates, the relationship between immune function and pregnancy has been disproportionately studied in placental mammals. To examine the energetic costs of immune activation and the modulation of immune function during pregnancy in a non-mammalian vertebrate, we elicited an immune response in pregnant and non-pregnant pygmy rattlesnakes, Sistrurus miliarius, using lipopolysaccharide (LPS). Resting metabolic rate (RMR) was measured using flow-through respirometry. Immune function was examined using bactericidal assays and leukocyte counts. The RMR of pygmy rattlesnakes increased significantly in response to LPS injection. There was no statistically significant difference in the metabolic response of non-reproductive and pregnant snakes to LPS. Mean metabolic increments for pregnant females, non-reproductive females, and males were 13%, 18% and 26%, respectively. The ratio of heterophils to lymphocytes was elevated in response to LPS across reproductive categories; however, LPS did not impact plasma bactericidal ability in non-reproductive snakes. Although pregnant females had significantly higher plasma bactericidal ability compared with non-reproductive snakes prior to manipulation, their bactericidal ability declined in response to LPS. LPS administration also significantly reduced several litter characteristics, particularly when administrated relatively early in pregnancy. Our results indicate that immune performance is energetically costly and is altered during pregnancy, and that immune activation during pregnancy may result in tradeoffs that affect offspring in a viviparous reptile.

Evidence of Vertical Transmission of the Snake Fungal Pathogen Ophidiomyces ophiodiicola.

Snake fungal disease (ophidiomycosis) is an emerging infection of snakes caused by Ophidiomyces ophiodiicola. Little is known about mechanisms of this pathogen's transmission and its implications for conservation of wild snake populations. We report four cases with evidence of vertical transmission of O. ophiodiicola from dam to offspring.

Seasonal testosterone and corticosterone patterns in relation to body condition and reproduction in a subtropical pitviper, Sistrurus miliarius.

Seasonal constraints on the timing and intensity of reproductive events shape observed variation in life history strategies across latitudes. Selection acts on the endocrine mechanisms that underlie reproductive investment. It is therefore important to examine the seasonal relationship between hormones and reproduction in geographically and phylogenetically diverse taxa. Snakes have proven to be a valuable model in investigations of seasonal hormone production and behavior in field-active vertebrates, but most research has focused on temperate populations from highly seasonal environments. To reduce this bias, we provide a description of the seasonal relationships among testosterone, corticosterone, body condition, and reproductive behavior in a subtropical population of Pygmy Rattlesnakes, Sistrurus miliarius. In central Florida, Sistrurus miliarius exhibits a prolonged breeding season (September-January) compared to most temperate zone snakes. Despite the extended breeding season, the pattern of testosterone in the population was highly seasonal and very similar to temperate pitvipers with a shorter mating season. Testosterone declined steadily through the mating season, but males sampled while engaging in mating behaviors had higher testosterone compared to solitary males throughout the mating season. Testosterone was negatively related to corticosterone throughout the breeding season and during times of year when the gonads were presumed to be quiescent and no mating behavior was observed. Testosterone was positively related to individual body condition both within and outside of the breeding season. A review of the literature reveals no consistent pattern regarding the relationship between corticosterone and testosterone in snakes, but suggests that the condition-dependence of steroid production may be consistent across snake taxa.

Tracking Outcomes of Snake Fungal Disease in Free-ranging Pygmy Rattlesnakes ( Sistrurus miliarius).

Snake fungal disease (SFD) is caused by the fungus Ophidiomyces ophiodiicola and its documentation in wild snake populations has risen sharply in the past decade. Little is known regarding the fate of individual, free-ranging snakes afflicted with SFD. We monitored an afflicted population of pygmy rattlesnakes ( Sistrurus miliarius) at Lake Woodruff National Wildlife Refuge in Central Florida, US for 2 yr. The severity of SFD in individual snakes was unrelated to the probability of their recapture later in the study, and half of the snakes diagnosed as severely infected at the onset of the study were recaptured later with no clinical signs of SFD. The clinical progress of 12 serially recaptured individuals also showed that individuals cleared the infection and fluctuated between no or low and severe clinical signs over the 2-yr study.

Patterns of Circulating Corticosterone in a Population of Rattlesnakes Afflicted with Snake Fungal Disease: Stress Hormones as a Potential Mediator of Seasonal Cycles in Disease Severity and Outcomes.

Snake fungal disease (SFD) is an emerging threat to snake populations in the United States. Fungal pathogens are often associated with a physiological stress response mediated by the hypothalamo-pituitary-adrenal axis (HPA), and afflicted individuals may incur steep coping costs. The severity of SFD can vary seasonally; however, little is known regarding (1) how SFD infection relates to HPA activity and (2) how seasonal shifts in environment, life history, or HPA activity may interact to drive seasonal patterns of infection severity and outcomes. To test the hypothesis that SFD is associated with increased HPA activity and to identify potential environmental or physiological drivers of seasonal infection, we monitored baseline corticosterone, SFD infection severity, foraging success, body condition, and reproductive status in a field-active population of pigmy rattlesnakes. Both plasma corticosterone and the severity of clinical signs of SFD peaked in the winter. Corticosterone levels were also elevated in the fall before the seasonal rise in SFD severity. Severely symptomatic snakes were in low body condition and had elevated corticosterone levels compared to moderately infected and uninfected snakes. The monthly mean severity of SFD in the population was negatively related to population-wide estimates of body condition and temperature measured in the precedent month and positively correlated with corticosterone levels measured in the precedent month. Symptomatic females were less likely to enter reproductive bouts compared to asymptomatic females. We propose the hypothesis that the seasonal interplay among environment, host energetics, and HPA activity initiates trade-offs in the fall that drive the increase in SFD prevalence, symptom severity, and decline in condition observed in the population through winter.

Environmental and physiological correlates of the severity of clinical signs of snake fungal disease in a population of pigmy rattlesnakes, Sistrurus miliarius.

In the past decade, snake fungal disease (SFD) has been identified as an emerging threat to snake populations throughout the eastern USA. Snake fungal disease is caused by the fungus Ophidiomyces ophiodiicola. Little is known regarding the environmental or physiological variables that affect host vulnerability and O. ophiodiicola virulence in wild snake populations. Understanding the intrinsic and extrinsic factors that correlate with infection severity is a key first step in understanding host-pathogen dynamics. Host vulnerability may vary seasonally as a result of thermal conditions or energetic trade-offs, and pathogen growth rates or dispersal may be tied to seasonal trends in climate. To determine whether season, environmental temperature or energetic trade-offs associated with life-history stage influence an individual's susceptibility to infection, we monitored the severity of clinical signs of SFD, surface air temperature, reproductive status, body condition and serum complement activity (plasma bactericidal ability) in free-ranging pigmy rattlesnakes, Sistrurus miliarius, over the course of 18 months. Seasonal increases in the severity of clinical signs of SFD were correlated negatively with monthly air surface temperature and the mean body condition of the population. Bactericidal ability varied seasonally, but pigmy rattlesnakes suffering from active SFD infections did not exhibit deficits in innate immune function. Infected snakes were in significantly lower body condition when compared with the general population, but seasonal patterns in the mean body condition of the population were not driven by seasonal patterns of infection severity. Our results highlight the potential importance of the thermal environment and energetic status in determining infection severity and outcomes and the need for managers and researchers to consider seasonality of symptom presentation when the goal is to identify the prevalence or incidence of SFD in populations.

Vasotocin receptor blockade disrupts maternal care of offspring in a viviparous snake, Sistrurus miliarius.

Parental care is a complex social behavior that is widespread among vertebrates. The neuroendocrine regulation of parent-offspring social behavior has been well-described in mammals, and to a lesser extent, in birds and fish. However, little is known regarding the underlying mechanisms that mediate the expression of care behaviors in squamate reptiles. In mammalian model species and humans, posterior pituitary hormones of the oxytocin and vasopressin families mediate parental care behaviors. To test the hypothesis that the regulatory role of posterior pituitary neuropeptides is conserved in a viviparous squamate reptile, we pharmacologically blocked the vasotocin receptor in post-parturient pigmy rattlesnakes, Sistrurus miliarius, and monitored the spatial relationship between mothers and offspring relative to controls. Mothers in the control group demonstrated spatial aggregation with offspring, with mothers having greater post-parturient energy stores aggregating more closely with their offspring. Blockade of vasotocin receptors eliminated evidence of spatial aggregation between mothers and offspring and eliminated the relationship between maternal energetic status and spatial aggregation. Our results are the first to implicate posterior pituitary neuropeptides in the regulation of maternal behavior in a squamate reptile and are consistent with the hypothesis that the neuroendocrine mechanisms underlying social behaviors are broadly conserved among vertebrates.

Mechanisms underlying maternal care in a viviparous snake, Sistrurus miliarius: Does maternal body condition, corticosterone, or arginine vasotocin mediate post-birth maternal investment?

The level of parental investment in free-living offspring varies greatly within and among vertebrate taxa, and the mechanisms that mediate investment are likely targets of selection in the evolution of reproductive strategies. In mammalian and avian systems, individual investment is adjusted according to parental energetic status, and regulatory mechanisms have been described. Parental care is also a component of the life history of several groups of squamates and archosaurs, but little to nothing is known regarding the mechanisms that underlie individual variation in parental investment in these groups. Therefore, we examined the relationships among maternal body condition, corticosterone, arginine vasotocin (AVT), and maternal care in postparturient pigmy rattlesnakes. Energetic status was manipulated in pregnant females by supplemental feeding, and AVT was manipulated in postparturient mothers by intraperitoneal injection. Circulating corticosterone and body condition were recorded in postparturient mothers. Mother-offspring spatial relationships were analyzed to determine the closeness of mother-offspring associations. No significant relationship between maternal body condition and baseline corticosterone was observed in mothers, and no significant relationship was found between corticosterone and the level of maternal association with offspring. Manipulation of AVT did not stimulate maternal care. There was a strong and repeatable positive relationship between maternal postparturient body condition and the level of maternal association with offspring. The lack of a significant correlation between body condition and corticosterone does not support the hypothesis that a postparturient glucocorticoid response drives the observed condition-dependence of maternal offspring association.

Male Snakes Allocate Time and Energy according to Individual Energetic Status: Body Condition, Steroid Hormones, and Reproductive Behavior in Timber Rattlesnakes, Crotalus horridus.

Life-history theory predicts that organisms will hedge current reproductive investment against potential costs in terms of survivorship and future fecundity. However, little is known regarding the endocrine mechanisms underlying bet-hedging strategies in free-ranging male vertebrates. We examined the relationships among individual energetic status, steroid hormones, mate search, and reproductive behavior in free-ranging male timber rattlesnakes. Snakes were monitored over four active seasons in order to test two hypotheses: (1) males adjust the amount of time and energy allocated toward reproduction according to the level of individual energy stores, and (2) observed condition-dependent reproductive allocation is associated with circulating concentrations of steroid hormones (testosterone and corticosterone) thought to regulate reproductive behaviors in vertebrates. A positive relationship between body condition and testosterone was observed in both the field and the laboratory. Male mate search effort was positively correlated with both body condition and testosterone. Body condition and testosterone concentrations were negatively related to time allocated toward foraging during the breeding season. A strong effect of year was observed in the analysis of testosterone and search effort, suggesting that multiple environmental factors impact hormone production and reproductive investment. Corticosterone was not related to any measured variable. Therefore, our results did not indicate a clear role of corticosterone in mediating observed relationships between energetic status and behavior. Observed relationships are consistent with the hypothesis that males allocate time and energy toward reproduction according to individual energetic status and that testosterone plays a role in mediating the trade-off between current reproductive investment and residual reproductive value.

Natural variation in steroid hormone profiles of male Timber Rattlesnakes, Crotalus horridus, in northwest Arkansas.

We describe the seasonal profile of circulating steroid hormones (testosterone and corticosterone) in relation to the breeding season in free ranging male Timber Rattlesnakes, Crotalus horridus, over the course of three active seasons. In addition, we examine variation in steroid concentrations across years and in relation to body condition. We found that seasonal profiles of plasma testosterone were different compared to other crotalines with similar mating patterns. Concentrations of testosterone were elevated above baseline in the three months leading up to the single late summer breeding season. Testosterone peaked in July at the onset of the breeding season and dropped to baseline during the peak months of breeding (August and September). Testosterone concentrations also varied annually. Although the exact cause of annual variation could not be established, our results indicate that weather patterns may have driven observed differences. Testosterone concentrations were positively related to body condition, indicating that testosterone production is modulated according to energetic status (particularly in the two months prior to the breeding season). Corticosterone did not vary seasonally or with any measured variable, a result similar to other studied crotalines. Our results highlight the importance of long-term descriptive studies of the regulatory mechanisms that underlie behavior and physiology in diverse taxa, as these mechanisms can vary greatly within and among populations and are valuable in elucidating the intrinsic and extrinsic sources of such variation.

Variability in mRNA expression of fms-like tyrosine kinase-1 variants in normal and preeclamptic placenta.

BACKGROUND: Preeclampsia is a complication of pregnancy characterised by gestational hypertension and proteinuria and is a leading cause of morbidity and mortality in both mothers and infants. Certain anti-angiogenic factors have long been implicated in the pathogenesis of preeclampsia and the placental expression of factors such as soluble fms-like tyrosine kinase-1 (sFLT-1) are often reported in studies of normal and diseased placentae. Despite evidence showing significant differences in placental gene expression by collection site, many studies fail to provide sufficient details on sample selection and collection. FINDINGS: With ourselves and others investigating and reporting on the expression of FLT-1 variants and other genes in the placenta of normotensive and preeclamptic patients, we felt it prudent to examine the variation in expression of FLT-1 variants across human placenta. We examined the differential expression of FLT-1 variants in samples obtained from 12 sites on normal and preeclamptic placentae and found expression to be highly variable between sites. We therefore developed an algorithim to calculate the mean expression for any number of these sites collected and in any combination. The coefficient of variation for all combinations of sites was then used to determine the minimum number of sites required to reduce coefficient of variation to below an acceptable 10%. We found that 10 and 11 sites had to be sampled in the normal and preeclamptic placentae respectively to ensure a representative expression pattern for all FLT-1 variants for an individual placenta. CONCLUSIONS: These findings demonstrate significant variation in expression levels of several commonly investigated genes across sites in both normal and preeclamptic placenta. This highlights both the importance of adequate sampling of human placenta for expression studies and the effective communication of sample selection and collection methods, for data interpretation and to ensure the reproducibility and reliability of results and conclusions drawn.

The relationship between plasma steroid hormone concentrations and the reproductive cycle in the Northern Pacific rattlesnake, Crotalus oreganus.

We describe the reproductive cycle of Northern Pacific rattlesnakes (Crotalus oreganus) by quantifying steroid hormone concentrations and observing reproductive behaviors in free-ranging individuals. Additionally, we examined reproductive tissues from museum specimens. Plasma steroid hormone concentrations were quantified for both male and female snakes throughout the active season (March-October). We measured testosterone (T), 5alpha-dihydrotestosterone (DHT), and corticosterone (B) concentrations in both sexes and 17beta-estradiol (E2) and progesterone (P) in females only. We observed reproductive behaviors (e.g., consortship, courtship, and copulation) in the field and measured testis and follicle size in male and female snakes from museum collections to relate steroid hormone concentrations to the timing of reproductive events. Our study revealed that C. oreganus in central California exhibits a bimodal pattern of breeding, with most mating behavior occurring in the spring and some incidences of mating behavior observed in late summer/fall. Each breeding period corresponded with elevated androgen (T or DHT) levels in males. Testes were regressed in the spring when the majority of reproductive behavior was observed in this population, and they reached peak volume in August and September during spermatogenesis. Although we did not detect seasonal variation in female hormone concentrations, some females had high E2 in the spring and fall, coincident with mating and with increased follicle size (indicating vitellogenesis) in museum specimens. Females with high E2 concentrations also had high T and DHT concentrations. Corticosterone concentrations in males and females were not related either to time of year or to concentrations of any other hormones quantified. Progesterone concentrations in females also did not vary seasonally, but this likely reflected sampling bias as females tended to be underground, and thus unobtainable, in summer months when P would be expected to be elevated during gestation. In females, P was positively correlated with T and DHT, and E2 was positively correlated with T.