Ben J. Vernasco

Beeswax corticosterone implants produce long-term elevation of plasma corticosterone and influence condition

Glucocorticoids can play a critical role in modulating life-history trade-offs. However, studying the effects of glucocorticoids on life-history often requires experimentally elevating plasma glucocorticoid concentrations for several weeks within normal physiological limits and without repeated handling of the animal. Recently, implants made of beeswax and testosterone (T) were shown to have release dynamics superior to some currently available T implants, and these beeswax implants dissolved, eliminating the need to recapture the animal. We evaluated the utility of beeswax implants containing four different dosages of corticosterone (CORT; the primary glucocorticoid in birds) and their effect on several condition indices in a captive colony of zebra finches (Taeniopygia guttata). The three implants with the greatest CORT doses (0.05, 0.1, and 0.5mg) produced spikes in plasma CORT concentrations 20h after treatment, but were within the limits that zebra finches may normally experience. The 0.5mg CORT implant elevated plasma CORT between typical baseline and restraint stress levels reported in other studies of zebra finches for the entire 35day experiment. Birds in the 0.5mg implant group were heavier, had greater furcular fat scores, and had lower hematocrit than birds in the control and other CORT implant groups. Beeswax CORT implants are a low cost method of elevating plasma CORT for a prolonged time. Furthermore, because there is no need to remove these implants at the end of a study, this method may be amenable to studies of free-ranging animals.

Physiological and behavioral effects of exogenous corticosterone in a free-ranging ectotherm

In the face of global change, free-ranging organisms are expected to experience more unpredictable stressors. An understanding of how organisms with different life history strategies will respond to such changes is an integral part of biodiversity conservation. Corticosterone (CORT) levels are often used as metrics to assess the population health of wild vertebrates, despite the fact that the stress response and its effects on organismal function are highly variable. Our understanding of the stress response is primarily derived from studies on endotherms, leading to some contention on the effects of chronic stress across and within taxa. We assessed the behavioral and hormonal responses to experimentally elevated stress hormone levels in a free-ranging, arid-adapted ectotherm, the Southern Pacific rattlesnake (Crotalus helleri). Plasma CORT was significantly elevated in CORT-implanted snakes 15days after implantation. Implantation with CORT did not affect testosterone (T) levels or defensive behavior. Interestingly, we observed increased defensive behavior in snakes with more stable daily body temperatures and in snakes with higher plasma T during handling (tubing). Regardless of treatment group, those individuals with lower baseline CORT levels and higher body temperatures tended to exhibit greater increases in CORT levels following a standardized stressor. These results suggest that CORT may not mediate physiological and behavioral trait expression in arid-adapted ectotherms such as rattlesnakes.

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.

Good vibrations: Assessing the stability of snake venom composition after researcher-induced disturbance in the laboratory

Abstract Phenotypic plasticity contributes to intraspecific variation in traits of many animal species. Venom is an integral trait to the success and survival of many snake species, and potential plasticity in venom composition is important to account for in the context of basic research as well as in human medicine for treating the various symptoms of snakebite and producing effective anti‐venoms. Researchers may unknowingly induce changes in venom variation by subjecting snakes to novel disturbances and potential stressors. We explored phenotypic plasticity in snake venom composition over time in captive Pacific rattlesnakes (Crotalus oreganus) exposed to vibration treatment, compared to an undisturbed control group. Venom composition did not change significantly in response to vibration, nor was there a detectable effect of overall time in captivity, even though snakes re‐synthesized venom stores while subjected to novel disturbance in the laboratory. This result indicates that venom composition is a highly repeatable phenotype over short time spans and that the composition of venom within adult individuals may be resistant to or unaffected by researcher‐induced disturbance. On the other hand, the change in venom composition, measured as movement along the first principle component of venom phenotype space, was associated with baseline corticosterone (CORT) levels in the snakes. While differential forms of researcher‐induced disturbance may not affect venom composition, significant changes in baseline CORT, or chronic stress, may affect the venom phenotype, and further investigations will be necessary to assess the nature of the relationship between CORT and venom protein expression. HighlightsIndividual snake venoms were highly repeatable in snakes that were exposed to a novel stressor.Initial and final individual snake venoms sampled with a 2 week interval were not significantly different in snakes acclimated to laboratory for at least 10 days.Corticosterone was correlated with slight changes in venom composition after re‐synthesis.

Testosterone drives status-specific patterns of cooperation and transmission of behavior in a social network

Stable social partnerships require physiological mechanisms that maintain a balance between competition and cooperation. Although the endocrine system is known to facilitate competitive behavior, we know very little about how steroid hormones affect cooperation and the stability of partnerships. Here, we examine how testosterone (T) modulates social behavior in wire-tailed manakins (Pipra filicauda), a species in which territorial and non-territorial males form cooperative coalitions within a larger social network. Our approach links repeated hormone sampling and a hormone-manipulation experiment with an automated telemetry system that captured >30,000 male-male social interactions. Using 528 repeated T samples from 209 males, we find that circulating T is repeatable with individual differences accounting for 19% of the total variation, and that among-individual differences in baseline T can explain 17% of the variation in cooperative behavior. Patterns of hormone-mediated behavior were status-specific whereby T significantly increased cooperative behaviors in floaters whereas it tended to decrease the same behaviors in territorial males. Experimental elevation of T caused significant decreases in both the number of partners and frequency of cooperative interactions received by territory-holders, with the strongest effect observed in lower-T individuals. This status-specificity suggests that hormone-mediated behavior is under stabilizing selection and that ontogenetic plasticity in hormone-regulatory networks facilitated the evolution of a stable cooperative society. Our analyses also reveal that one male-s T can indirectly affect the behavior of other individuals with whom he interacts (explaining 1-3% of variation), indicating that hormones are a mechanism underlying the transmission of behavior in social networks.Stable cooperation requires plasticity whereby individuals are able to express competitive or cooperative behaviors depending on social context. To date, however, the physiological mechanisms that underlie behavioral variation in cooperative systems are poorly understood. We studied hormone-mediated behavior in the wire-tailed manakin (Pipra filicauda), a gregarious songbird whose cooperative partnerships are crucial for fitness. We used automated telemetry to monitor > 36,000 cooperative interactions among male manakins over three field seasons, and we examined how circulating testosterone affects cooperation using > 500 hormone samples. Observational data show that in non-territorial floater males, high testosterone is associated with increased cooperative behaviors and subsequent ascension to territorial status. In territory-holding males, however, both observational and experimental evidence demonstrate that high testosterone antagonizes cooperation. Moreover, circulating testosterone explains significant variation (2-8%) in social behavior within each status class. Collectively, our findings show that the hormonal control of cooperation depends on a male’s social status. We propose that the status-dependent reorganization of hormone-regulatory pathways can facilitate stable cooperative partnerships, and thus provide direct fitness benefits for males.

Tales of testosterone: Advancing our understanding of environmental endocrinology through studies of neotropical birds

Studies of birds have greatly advanced our understanding of how testosterone modulates complex phenotypes, specifically its role in mediating male reproductive and associated behaviors. Yet most of the foundational studies have been limited to northern latitude breeding species despite the fact that they represent only a small fraction of worldwide avian diversity. In contrast, phylogenetic, life-history, and mating system diversity all reach their apex in neotropical avifauna and yet these birds, along with more southern latitude species, remain very poorly understood from an endocrine perspective. Despite the relatively limited previous work on taxa breeding in Central and South America, empirical findings have had a disproportionately large impact on our understanding of testosterones role in everything from geographic variation to behavioral roles and neuroplasticity. Here, we synthesize how studies of neotropical breeding avifauna have advanced our understanding of how testosterones actions can and are associated with the broad patterns of phenotypic diversity that we see in birds. In addition, we outline how these studies can be used individually or in a comparative context to address fundamental questions about the environmental endocrinology of testosterone and to understand the diversity of roles that testosterone plays in mediating behavioral variation, reproductive strategies, and associated life-history trade-offs.

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.

Sampling baseline androgens in free-living passerines: Methodological considerations and solutions

Obtaining baseline hormone samples can be challenging because circulating hormone levels often change rapidly due to the acute stress of capture. Although field protocols are established for accurately sampling baseline glucocorticoid concentrations, fewer studies have examined how common sampling techniques affect androgens levels. Indeed, many studies focused on understanding the functional significance of baseline androgen levels use sampling methods known to activate the endocrine responses to stress. To understand how different field sampling protocols affect plasma androgen levels, we measured the androgen response to two types of capture stressors in a free-living tropical bird, the wire-tailed manakin (Pipra filicauda). First, we subjected males to a standardized capture and restraint protocol lasting either 15 or 30 min. Second, males were passively captured in nets that were filmed (to establish exact duration of time between capture and blood sampling) and checked every 30 min. The first study showed that circulating plasma androgen levels decreased significantly following both 15 and 30 min of restraint in a cloth bag, with a trend for the 30 min samples to be lower than the 15 min samples. Further, the change in androgen levels was dependent on an individuals initial androgen levels, with the individuals with the highest initial levels registering the largest declines. The results of the second study suggest that hanging in a mist net for extended periods of time also leads to a decrease in circulating androgen levels, but this effect was weaker than that of capture and restraint in a cloth bag. Our findings demonstrate that, overall, circulating androgen levels decrease in response to common sampling techniques; a finding that has important implications for studies measuring baseline androgen levels in free-living birds. Future studies should prioritize sampling individuals immediately upon removal from the mist net, as handling and restraint have a strong negative effect on circulating androgen levels. When constant monitoring of the mist net is not possible, investigators should use video cameras to record the amount of time an individual spends in the net prior to blood sampling and then statistically control for the effect of this variable in analyses.

Sex and seasonal differences in mRNA expression of estrogen receptor α (ESR1) in red-sided garter snakes (Thamnophis sirtalis parietalis)

Estrogens are important regulators of reproductive physiology including sexual signal expression and vitellogenesis. For the regulation to occur, the hormone must bind and activate receptors in target tissues, and expression of the receptors can vary by sex and/or season. By simultaneously comparing circulating hormone levels with receptor expression, a more complete understanding of hormone action can be gained. Our study species, the red-sided garter snake (Thamnophis sirtalis parietalis), provides an excellent opportunity to study the interaction between sex steroid hormones and receptor expression in addition to sexual dimorphism and seasonality. During the spring mating season, male garter snakes rely exclusively on the females skin-based, estrogen-dependent sex pheromone to direct courtship. Males can be stimulated to produce this sexual attractiveness pheromone by treatment with estradiol (E2), which also induces male vitellogenesis. Estrogen receptors (ESRs) are required to transduce the effects of estrogens, thus we used quantitative RT-PCR to analyze expression of ESR alpha (ERα; gene ESR1) mRNA in the skin and liver of wild caught male and female garter snakes across simulated spring and fall conditions in the laboratory. While ESR1 was present in the skin of both sexes, there were no sex or seasonal differences in expression levels. Liver expression of ESR1, however, was sexually dimorphic, with females showing greatest expression in fall when circulating E2 concentrations were lowest. There were no statistically significant correlations between E2 and ESR1 expression. Our data suggest that the skin of both sexes is sensitive to estrogen signaling and thus the production of sex pheromone is dependent on bioavailable levels of E2. Female expression of ESR1 in the liver may increase in the fall to prime energy storage mechanisms required for vitellogenesis the following year.

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.