Neurogenomic landscape associated with status-dependent cooperative behaviour.

The neurogenomic mechanisms mediating male-male reproductive cooperative behaviours remain unknown. We leveraged extensive transcriptomic and behavioural data on a neotropical bird species (Pipra filicauda) that performs cooperative courtship displays to understand these mechanisms. In this species, the cooperative display is modulated by testosterone, which promotes cooperation in non-territorial birds, but suppresses cooperation in territory holders. We sought to understand the neurogenomic underpinnings of three related traits: social status, cooperative display behaviour and testosterone phenotype. To do this, we profiled gene expression in 10 brain nuclei spanning the social decision-making network (SDMN), and two key endocrine tissues that regulate social behaviour. We associated gene expression with each bird's behavioural and endocrine profile derived from 3 years of repeated measures taken from free-living birds in the Ecuadorian Amazon. We found distinct landscapes of constitutive gene expression were associated with social status, testosterone phenotype and cooperation, reflecting the modular organization and engagement of neuroendocrine tissues. Sex-steroid and neuropeptide signalling appeared to be important in mediating status-specific relationships between testosterone and cooperation, suggesting shared regulatory mechanisms with male aggressive and sexual behaviours. We also identified differentially regulated genes involved in cellular activity and synaptic potentiation, suggesting multiple mechanisms underpin these genomic states. Finally, we identified SDMN-wide gene expression differences between territorial and floater males that could form the basis of 'status-specific' neurophysiological phenotypes, potentially mediated by testosterone and growth hormone. Overall, our findings provide new, systems-level insights into the mechanisms of cooperative behaviour and suggest that differences in neurogenomic state are the basis for individual differences in social behaviour.

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.

Systems biology as a framework to understand the physiological and endocrine bases of behavior and its evolution-From concepts to a case study in birds.

Organismal behavior, with its tremendous complexity and diversity, is generated by numerous physiological systems acting in coordination. Understanding how these systems evolve to support differences in behavior within and among species is a longstanding goal in biology that has captured the imagination of researchers who work on a multitude of taxa, including humans. Of particular importance are the physiological determinants of behavioral evolution, which are sometimes overlooked because we lack a robust conceptual framework to study mechanisms underlying adaptation and diversification of behavior. Here, we discuss a framework for such an analysis that applies a "systems view" to our understanding of behavioral control. This approach involves linking separate models that consider behavior and physiology as their own networks into a singular vertically integrated behavioral control system. In doing so, hormones commonly stand out as the links, or edges, among nodes within this system. To ground our discussion, we focus on studies of manakins (Pipridae), a family of Neotropical birds. These species have numerous physiological and endocrine specializations that support their elaborate reproductive displays. As a result, manakins provide a useful example to help imagine and visualize the way systems concepts can inform our appreciation of behavioral evolution. In particular, manakins help clarify how connectedness among physiological systems-which is maintained through endocrine signaling-potentiate and/or constrain the evolution of complex behavior to yield behavioral differences across taxa. Ultimately, we hope this review will continue to stimulate thought, discussion, and the emergence of research focused on integrated phenotypes in behavioral ecology and endocrinology.

Experimental reduction of haemosporidian infection affects maternal reproductive investment, parental behaviour and offspring condition.

When hosts have a long coevolutionary history with their parasites, fitness costs of chronic infection have often been assumed to be negligible. Yet, experimental manipulation of infections sometimes reveals effects of parasites on their hosts, particularly during reproduction. Whether these effects translate into fitness costs remains unclear. Here, we present the results of an experimental study conducted in a free-ranging population of red-winged blackbirds (Agelaius phoeniceus) naturally experiencing a high prevalence of haemosporidian infections, with more than 95% of breeding adults infected with parasites from one or more haemosporidian genus. To assess effects of infection during reproduction, we manipulated adult red-winged blackbird females' parasite burden by administering an anti-haemosporidian medication before onset of egg-laying. Experimental reduction of infection resulted in significant benefits to mothers and their offspring. Medicated females laid heavier clutches, invested more in incubation and provisioning behaviour, and produced more fledglings than control females. Nestlings of medicated females had higher haematocrit, higher blood glucose, and lower reactive oxygen metabolites than nestlings of control females. Overall, our results provide evidence that, even in a species with high prevalence of infection, parasites can lead to decreased maternal investment and offspring quality, substantially reducing fitness.

Environmental and anthropogenic influences on movement and foraging in a critically endangered lemur species, Propithecus tattersalli: implications for habitat conservation planning.

BACKGROUND: Wildlife conservation often focuses on establishing protected areas. However, these conservation zones are frequently established without adequate knowledge of the movement patterns of the species they are designed to protect. Understanding movement and foraging patterns of species in dynamic and diverse habitats can allow managers to develop more effective conservation plans. Threatened lemurs in Madagascar are an example where management plans and protected areas are typically created to encompass large, extant forests rather than consider the overall resource needs of the target species. METHODS: To gain an understanding of golden-crowned sifaka (Propithecus tattersalli) movement patterns, including space use and habitat selection across their range of inhabited forest types, we combined behavior data with Dynamic Brownian Bridge Movement Models and Resource Selection Functions. We also examined the influence of abiotic, biotic, and anthropogenic factors on home range size, movement rates, and foraging patterns. RESULTS: We found that home range size and movement rates differed between seasons, with increased core area size and movement in the rainy season. Forest type also played a role in foraging behavior with sifaka groups in the humid forest avoiding roads in both seasons, groups in the dry deciduous forest avoiding road networks in the rainy season, and groups in the moderate evergreen forest displaying no selection or avoidance of road networks while foraging. CONCLUSION: Our study illustrates the importance of studying primate groups across seasons and forest types, as developing conservation plans from a single snapshot can give an inaccurate assessment of their natural behavior and resources needs of the species. More specifically, by understanding how forest type influences golden-crowned sifaka movement and foraging behavior, conservation management plans can be made to the individual forest types inhabited (dry deciduous, moderate evergreen, humid, littoral, etc.), rather than the region as a whole.

Assessing age, breeding stage, and mating activity as drivers of variation in the reproductive microbiome of female tree swallows.

Sexually transmitted microbes are hypothesized to influence the evolution of reproductive strategies. Though frequently discussed in this context, our understanding of the reproductive microbiome is quite nascent. Indeed, testing this hypothesis first requires establishing a baseline understanding of the temporal dynamics of the reproductive microbiome and of how individual variation in reproductive behavior and age influence the assembly and maintenance of the reproductive microbiome as a whole. Here, we ask how mating activity, breeding stage, and age influence the reproductive microbiome. We use observational and experimental approaches to explain variation in the cloacal microbiome of free-living, female tree swallows (Tachycineta bicolor). Using microsatellite-based parentage analyses, we determined the number of sires per brood (a proxy for female mating activity). We experimentally increased female sexual activity by administering exogenous 17ß-estradiol. Lastly, we used bacterial 16S rRNA amplicon sequencing to characterize the cloacal microbiome. Neither the number of sires per brood nor the increased sexual activity of females significantly influenced female cloacal microbiome richness or community structure. Female age, however, was positively correlated with cloacal microbiome richness and influenced overall community structure. A hypothesis to explain these patterns is that the effect of sexual activity and the number of mates on variation in the cloacal microbiome manifests over an individual's lifetime. Additionally, we found that cloacal microbiome alpha diversity (Shannon Index, Faith's phylogenetic distance) decreased and community structure shifted between breeding stages. This is one of few studies to document within-individual changes and age-related differences in the cloacal microbiome across successive breeding stages. More broadly, our results contribute to our understanding of the role that host life history and behavior play in shaping the cloacal microbiomes of wild birds.

Integrative Studies of Sexual Selection in Manakins, a Clade of Charismatic Tropical Birds.

The neotropical manakins (family Pipridae) provide a great opportunity for integrative studies of sexual selection as nearly all of the 51 species are lek-breeding, an extreme form of polygyny, and highly sexually dimorphic both in appearance and behavior. Male courtship displays are often elaborate and include auditory cues, both vocal and mechanical, as well as visual elements. In addition, the displays are often extremely rapid, highly acrobatic, and, in some species, multiple males perform coordinated displays that form the basis of long-term coalitions. Male manakins also exhibit unique neuroendocrine, physiological, and anatomical adaptations to support the performance of these complex displays and the maintenance of their intricate social systems. The Manakin Genomics Research Coordination Network (Manakin RCN, https://www.manakinsrcn.org) has brought together researchers (many in this symposium and this issue) from across disciplines to address the implications of sexual selection on evolution, ecology, behavior, and physiology in manakins. The objective of this paper is to present some of the most pertinent and integrative manakin research as well as introducing the papers presented in this issue. The results discussed at the manakin symposium, part of the 2021 Society for Integrative and Comparative Biology Conference, highlight the remarkable genomic, behavioral, and physiological adaptations as well as the evolutionary causes and consequences of strong sexual selection pressures that are evident in manakins.

Experimental Manipulation of Corticosterone Does Not Affect Venom Composition or Functional Activity in Free-Ranging Rattlesnakes.

AbstractVenom is an integral feeding trait in many animal species. Although venom often varies ontogenetically, little is known about the proximate physiological mediators of venom variation within individuals. The glucocorticoid hormone corticosterone (CORT) can alter the transcription and activation of proteins, including homologues of snake venom components such as snake venom metalloproteinases (SVMPs) and phospholipase A2 (PLA2). CORT is endogenously produced by snakes, varies seasonally and also in response to stress, and is a candidate endogenous mediator of changes in venom composition and functional activity. Here, we tested the hypothesis that CORT induces changes in snake venom by sampling the venom of wild adult rattlesnakes before and after they were treated with either empty (control) or CORT-filled (treatment) Silastic implants. We measured longitudinal changes in whole-venom composition, whole-venom total protein content, and enzymatic activity of SVMP and PLA2 components of venom. We also assessed the within-individual repeatability of venom components. Despite successfully elevating plasma CORT in the treatment group, we found no effect of CORT treatment or average plasma CORT level on any venom variables measured. Except for total protein content, venom components were highly repeatable within individuals ([Formula: see text]). Our results indicate that the effects of CORT, a hormone commonly associated with stress and metabolic functions, in adult rattlesnake venom are negligible. Our findings bode well for venom researchers and biomedical applications that rely on the consistency of venoms produced from potentially stressed individuals and provide an experimental framework for future studies of proximate mediators of venom variation across an individual's life span.

Testosterone-mediated behaviour shapes the emergent properties of social networks.

Social networks can vary in their organization and dynamics, with implications for ecological and evolutionary processes. Understanding the mechanisms that drive social network dynamics requires integrating individual-level biology with comparisons across multiple social networks. Testosterone is a key mediator of vertebrate social behaviour and can influence how individuals interact with social partners. Although the effects of testosterone on individual behaviour are well established, no study has examined whether hormone-mediated behaviour can scale up to shape the emergent properties of social networks. We investigated the relationship between testosterone and social network dynamics in the wire-tailed manakin, a lekking bird species in which male-male social interactions form complex social networks. We used an automated proximity system to longitudinally monitor several leks and we quantified the social network structure at each lek. Our analysis examines three emergent properties of the networks-social specialization (the extent to which a network is partitioned into exclusive partnerships), network stability (the overall persistence of partnerships through time) and behavioural assortment (the tendency for like to associate with like). All three properties are expected to promote the evolution of cooperation. As the predictor, we analysed the collective testosterone of males within each social network. Social networks that were composed of high-testosterone dominant males were less specialized, less stable and had more negative behavioural assortment, after accounting for other factors. These results support our main hypothesis that individual-level hormone physiology can predict group-level network dynamics. We also observed that larger leks with more interacting individuals had more positive behavioural assortment, suggesting that small groups may constrain the processes of homophily and behaviour-matching. Overall, these results provide evidence that hormone-mediated behaviour can shape the broader architecture of social groups. Groups with high average testosterone exhibit social network properties that are predicted to impede the evolution of cooperation.

Prolactin is related to incubation constancy and egg temperature following a disturbance in a precocial bird.

To maximize fitness, parents may trade-off time and energy between parental care and self-maintenance. In vertebrates, prolactin and corticosterone are two important hormones that regulate parental investment because they stimulate parental care and mobilize energy, respectively. Further, concentrations of both hormones change in response to disturbances. One of the most important parental care behaviors in birds is incubation, since small changes in egg temperature have large effects on offspring. We investigated how prolactin and corticosterone may mediate parental incubation constancy (i.e., the daily amount of time spent incubating eggs) and regulation of egg temperature. We collected blood samples from female wood ducks (Aix sponsa) near the start and end of the incubation period to measure baseline and stress-induced (30 min after capture and restraint) hormone concentrations. We also quantified incubation constancy and egg temperature using artificial egg temperature loggers. As expected, prolactin decreased and corticosterone increased after 30 min of capture and restraint. Corticosterone concentrations (baseline and stress-induced) were negatively related to body mass, but were not related to incubation constancy. In contrast, prolactin concentrations (baseline and stress-induced) were higher at the end than the start of the incubation period, and stress-induced prolactin concentrations were positively related to incubation constancy following a nest disturbance (i.e., capture). Further, prolactin (baseline and stress-induced) concentrations were positively related to egg temperatures, but only after the disturbance. These results suggest that prolactin may be associated with the regulation of parental incubation constancy and resulting heat-transfer after a disturbance, which may ultimately affect offspring development.

Cloacal bacterial communities of tree swallows (Tachycineta bicolor): Similarity within a population, but not between pair-bonded social partners.

Host-associated microbial communities can influence the overall health of their animal hosts, and many factors, including behavior and physiology, can impact the formation of these complex communities. Bacteria within these communities can be transmitted socially between individuals via indirect (e.g., shared environments) or direct (e.g., physical contact) pathways. Limited research has been done to investigate how social interactions that occur in the context of mating shape host-associated microbial communities. To gain a better understanding of these interactions and, more specifically, to assess how mating behavior shapes an animal's microbiome, we studied the cloacal bacterial communities of a socially monogamous yet genetically polygynous songbird, the North American tree swallow (Tachycineta bicolor). We address two questions: (1) do the cloacal bacterial communities differ between female and male tree swallows within a population? and (2) do pair-bonded social partners exhibit more similar cloacal bacterial communities than expected by chance? To answer these questions, we sampled the cloacal microbiome of adults during the breeding season and then used culture-independent, 16S rRNA gene amplicon sequencing to assess bacterial communities. Overall, we found that the cloacal bacterial communities of females and males were similar, and that the communities of pair-bonded social partners were not more similar than expected by chance. Our results suggest that social monogamy does not correlate with an increased similarity in cloacal bacterial community diversity or structure. As social partners were not assessed at the same time, it is possible that breeding stage differences masked social effects on bacterial community diversity and structure. Further, given that tree swallows exhibit high variation in rates of extra-pair activity, considering extra-pair activity when assessing cloacal microbial communities may be important for understanding how these bacterial communities are shaped. Further insight into how bacterial communities are shaped will ultimately shed light on potential tradeoffs associated with alternative behavioral strategies and socially-transmitted microbes.

Gene expression in the social behavior network of the wire-tailed manakin (Pipra filicauda) brain.

The vertebrate basal forebrain and midbrain contain a set of interconnected nuclei that control social behavior. Conserved anatomical structures and functions of these nuclei have now been documented among fish, amphibians, reptiles, birds and mammals, and these brain regions have come to be known as the vertebrate social behavior network (SBN). While it is known that nuclei (nodes) of the SBN are rich in steroid and neuropeptide activity linked to behavior, simultaneous variation in the expression of neuroendocrine genes among several SBN nuclei has not yet been described in detail. In this study, we use RNA-seq to profile gene expression across seven brain regions representing five nodes of the vertebrate SBN in a passerine bird, the wire-tailed manakin Pipra filicauda. Using weighted gene co-expression network analysis, we reconstructed sets of coregulated genes, showing striking patterns of variation in neuroendocrine gene expression across the SBN. We describe regional variation in gene networks comprising a broad set of hormone receptors, neuropeptides, steroidogenic enzymes, catecholamines and other neuroendocrine signaling molecules. Our findings show heterogeneous patterns of brain gene expression across nodes of the avian SBN and provide a foundation for future analyses of how the regulation of gene networks may mediate social behavior. These results highlight the importance of region-specific sampling in studies of the mechanisms of behavior.

Who rises to the challenge? Testing the Challenge Hypothesis in fish, amphibians, reptiles, and mammals.

According to the Challenge Hypothesis, social interactions, particularly among males, have a strong influence on circulating androgen levels. Specifically, males should respond to social challenges from conspecific males with a rapid increase in plasma androgen levels which support and stimulate further aggression. This basic tenet of the Challenge Hypothesis, an androgen increase in response to a social challenge from another male, has been tested in all vertebrate classes. While early studies generally supported the Challenge Hypothesis, more recent work has noted numerous exceptions, particularly in birds. Here, we conduct a meta-analysis of studies in fish, amphibians, non-avian reptiles, and mammals that test the prediction that circulating androgen levels of males should increase in response to an experimental challenge from another male. We found that teleost fish often increase androgens during such challenges, but other vertebrate groups show more mixed results. Why should fish be different from the other taxa? In fish with paternal care of young, the potential conflict between mating, being aggressive towards other males, and taking care of offspring is alleviated, because females typically choose males based on their defense of an already existing nest. Hence, rather than regulating the trade-off between mating, aggression, and parenting, androgens may have been co-opted to promote all three behaviors. For other taxa, increasing androgen levels only makes sense when the increase directly enhances reproductive success. Thus, the increase in androgen levels is a response to mating opportunities rather than a response to challenge from another male. To further our understanding of the role of a change in androgen levels in mediating behavioral decision-making between mating, aggression, and parenting, we need studies that address the behavioral consequences of an increase in androgens after male-male encounters and studies that test the androgen responsiveness of species that differ in the degree of paternal care.

Testosterone as a mediator of the tradeoff between cooperation and competition in the context of cooperative reproductive behaviors.

Behavioral tradeoffs occur when the expression of one behavior detracts from the expression of another. Understanding the proximate mediators of behavioral tradeoffs is important as these tradeoffs can act as potential constraints on evolutionary responses to selection. Here, we describe the tradeoff between cooperation and competition faced by species that exhibit cooperative reproductive behaviors and propose that testosterone is a key hormonal mediator of the tradeoff. Cooperative reproductive behaviors occur when multiple individuals coordinate their efforts to gain a reproductive advantage over other individuals and/or those individuals attempting to reproduce in absence of cooperation. We propose that testosterone, a sex steroid known to mediate a number of physiological and behavioral actions associated with reproductive competition, is involved in mediating the tradeoff between cooperation and competition. To support this proposition, we first describe the importance of individual variation in behavior to the evolution of cooperative behaviors. We then describe how proximate mechanisms represent a prominent source of individual variation in social behaviors and highlight evidence suggesting testosterone mediates variation in cooperative behaviors. Two case studies in which the relationship between testosterone and cooperative behaviors have been investigated in detail are then summarized. Throughout we highlight the importance of studying individual variation to understand the mechanistic basis of behaviors, behavioral tradeoffs, and the evolution of cooperative reproductive behaviors more broadly.

Testosterone Modulates Status-Specific Patterns of Cooperation in a Social Network.

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 and competition for status are both 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 nonterritorial 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.

Exogenous glucocorticoids amplify the costs of infection by reducing resistance and tolerance, but effects are mitigated by co-infection.

Individual variation in parasite defences, such as resistance and tolerance, can underlie heterogeneity in fitness and could influence disease transmission dynamics. Glucocorticoid hormone concentrations often change in response to fluctuating environmental conditions and mediate changes in immune function, resource allocation and tissue repair. Thus, changes in glucocorticoid hormone concentrations might mediate individual variation in investment in resistance versus tolerance. In this study, we experimentally increased glucocorticoid concentrations in red-winged blackbirds ( Agelaius phoeniceus) that were naturally infected with haemosporidian parasites, and assessed changes in resistance and tolerance of infection. Glucocorticoid treatment increased burdens of Plasmodium, the parasite causing avian malaria, but only in the absence of co-infection with another Haemosporidian, Haemoproteus. Thus, glucocorticoids might reduce resistance to infection, but co-infection can mitigate the negative consequences of increased hormone concentrations. Glucocorticoid treatment also decreased tolerance of infection. We found no evidence that the inflammatory immune response or rate of red blood cell production underlie the effects of glucocorticoids on resistance and tolerance. Our findings suggest that exogenous glucocorticoids can increase the costs of haemosporidian infections by both increasing parasite numbers and reducing an individual's ability to cope with infection. These effects could scale up to impact populations of both host and parasite.

Water-borne and plasma corticosterone are not correlated in spotted salamanders.

Water-borne hormone measurement is a noninvasive method suitable for amphibians of all sizes that are otherwise difficult to sample. For this method, containment-water is assayed for hormones released by the animal. Originally developed in fish, the method has expanded to amphibians, but requires additional species-specific validations. We wanted to determine physiological relevance of water-borne corticosterone in spotted salamanders (Ambystoma maculatum) by comparing concentrations to those taken using established corticosterone sampling methods, such as plasma. Using a mixture of field and laboratory studies, we compared water-borne corticosterone levels to other traditional methods of sampling corticosterone for spotted salamander larvae, metamorphs, and adults. Despite multiple attempts, and detecting differences between age groups, we found no correlations between water-borne and plasma corticosterone levels in any age group. Water-borne sampling measures a rate of release; whereas plasma is the concentration circulating in the blood. The unique units of measurement may inherently prevent correlations between the two. These two methods may also require different interpretations of the data and the physiological meaning. We also note caveats with the method, including how to account for differences in body size and life history stages. Collectively, our results illustrate the importance of careful validation of water-borne hormone levels in each species in order to understand its physiological significance.

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 individual's 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 female's 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.