Assessing chronic stress in wild mammals using claw-derived cortisol: a validation using European badgers (Meles meles).

Measuring stress experienced by wild mammals is increasingly important in the context of human-induced rapid environmental change and initiatives to mitigate human-wildlife conflicts. Glucocorticoids (GC), such as cortisol, mediate responses by promoting physiological adjustments during environmental perturbations. Measuring cortisol is a popular technique; however, this often reveals only recent short-term stress such as that incurred by restraining the animal to sample blood, corrupting the veracity of this approach. Here we present a protocol using claw cortisol, compared with hair cortisol, as a long-term stress bio-indicator, which circumvents this constraint, where claw tissue archives the individual's GC concentration over preceding weeks. We then correlate our findings against detailed knowledge of European badger life history stressors. Based on a solid-phase extraction method, we assessed how claw cortisol concentrations related to season and badger sex, age and body-condition using a combination of generalized linear mixed models (GLMM) (n = 668 samples from 273 unique individuals) followed by finer scale mixed models for repeated measures (MMRM) (n = 152 re-captured individuals). Claw and hair cortisol assays achieved high accuracy, precision and repeatability, with similar sensitivity. The top GLMM model for claw cortisol included age, sex, season and the sex*season interaction. Overall, claw cortisol levels were significantly higher among males than females, but strongly influenced by season, where females had higher levels than males in autumn. The top fine scale MMRM model included sex, age and body condition, with claw cortisol significantly higher in males, older and thinner individuals. Hair cortisol was more variable than claw; nevertheless, there was a positive correlation after removing 34 outliers. We discuss strong support for these stress-related claw cortisol patterns from previous studies of badger biology. Given the potential of this technique, we conclude that it has broad application in conservation biology.

Urban bat pups take after their mothers and are bolder and faster learners than rural pups.

BACKGROUND: Urbanization is rapidly changing our planet and animals that live in urban environments must quickly adjust their behavior. One of the most prevalent behavioral characteristics of urban dwelling animals is an increased level of risk-taking. Here, we aimed to reveal how urban fruitbats become risk-takers, and how they differ behaviorally from rural bats, studying both genetic and non-genetic factors that might play a role in the process. We assessed the personality of newborn pups from both rural and urban colonies before they acquired experience outdoors, examining risk-taking, exploration, and learning rates. RESULTS: Urban pups exhibited significantly higher risk-taking levels, they were faster learners, but less exploratory than their rural counterparts. A cross-fostering experiment revealed that pups were more similar to their adoptive mothers, thus suggesting a non-genetic mechanism and pointing towards a maternal effect. We moreover found that lactating urban mothers have higher cortisol levels in their milk, which could potentially explain the transmission of some personality traits from mother to pup. CONCLUSIONS: Young bats seem to acquire environment suitable traits via post-birth non-genetic maternal effects. We offer a potential mechanism for how urban pups can acquire urban-suitable behavioral traits through hormonal transfer from their mothers.

Neuroendocrine Mechanisms Underlying Non-breeding Aggression: Common Strategies Between Birds and Fish.

Aggression is an adaptive behavior that plays an important role in gaining access to limited resources. Aggression may occur uncoupled from reproduction, thus offering a valuable context to further understand its neural and hormonal regulation. This review focuses on the contributions from song sparrows (Melospiza melodia) and the weakly electric banded knifefish (Gymnotus omarorum). Together, these models offer clues about the underlying mechanisms of non-breeding aggression, especially the potential roles of neuropeptide Y (NPY) and brain-derived estrogens. The orexigenic NPY is well-conserved between birds and teleost fish, increases in response to low food intake, and influences sex steroid synthesis. In non-breeding M. melodia, NPY increases in the social behavior network, and NPY-Y1 receptor expression is upregulated in response to a territorial challenge. In G. omarorum, NPY is upregulated in the preoptic area of dominant, but not subordinate, individuals. We hypothesize that NPY may signal a seasonal decrease in food availability and promote non-breeding aggression. In both animal models, non-breeding aggression is estrogen-dependent but gonad-independent. In non-breeding M. melodia, neurosteroid synthesis rapidly increases in response to a territorial challenge. In G. omarorum, brain aromatase is upregulated in dominant but not subordinate fish. In both species, the dramatic decrease in food availability in the non-breeding season may promote non-breeding aggression, via changes in NPY and/or neurosteroid signaling.

Evidence for fasting induced extra-adrenal steroidogenesis in the male brown anole, Anolis sagrei.

Glucocorticoids (GCs) and dehydroepiandrosterone (DHEA) are steroids secreted by the adrenal glands into circulation to effect distant target tissues and coordinate physiological processes. This classic systemic view of steroids has been challenged by evidence that other tissues can independently synthesize their own steroids. Little is known however regarding circumstances that can promote this extra-adrenal steroidogenesis. Here we tested if fasting can induce tissues to increase GC and DHEA synthesis in the brown anole lizard Anolis sagrei. Lizards fasted for eight days lost body mass and increased fatty acid oxidation. Fasting also increased plasma concentrations of DHEA and corticosterone, but not cortisol. Corticosterone concentration within the adrenals, heart, intestines, lungs and liver exceeded that in plasma, with the latter two increasing with fasting. Levels of DHEA in the adrenals and heart were higher than in plasma, but no significant effect of fasting was observed, expect for a noticeable increase in intestinal DHEA. Two steroidogenic genes, the steroidogenic acute regulatory (Star) protein and Cyp17a1, a cytochrome P450 enzyme, were expressed in several tissues including the liver, lungs and intestines, which were increased with fasting. Continued research should aim to test for expression of additional enzymes further along the steroidogenic pathway. Nonetheless these data document potential extra-adrenal steroidogenesis as a possible mechanism for coping with energy shortages, although much work remains to be done to determine the specific roles of locally synthesized steroids in each tissue.

Fear-based aggression and its relationship to corticosterone responsiveness in three species of python.

It has long been known that even closely related species can vary in their antipredator behavior, and in the last two decades there has been mounting interest in how these differences might relate to the hormonal stress response. We tested the relationship between fear-based aggression, a form of antipredator behavior, and plasma corticosterone levels in three species of python [Children's Python (Antaresia childreni), Ball Python (Python regius), Bismarck Ring Python (Bothrochilus boa)]. We recorded the amount of striking in response to perturbation before and after a controlled, stressful confinement. We also measured plasma corticosterone levels prior to confinement, after confinement, and after confinement plus an adrenocorticotropin hormone (ACTH) injection, the later to induce a maximal corticosterone response. We performed among species analyses using two mixed models, and we determined between individual variance within each species to estimate repeatability. Bismarck Ring Pythons struck more than either Ball Pythons or Children's Pythons, and Ball Pythons had a suppressed corticosterone response compared to Children's and Bismarck Ring Pythons. Thus, mean species fear-based aggression correlated with species level differences in corticosterone profile. We also found evidence suggesting behaviors are repeatable within individuals. Our results point to a need for further exploration of aggression, anti-predator behavior, and corticosterone profile.

Neuropeptide Y and orexin immunoreactivity in the sparrow brain coincide with seasonal changes in energy balance and steroids.

The transition between the breeding and nonbreeding states is often marked by a shift in energy balance. Despite this well-known shift in energy balance, little work has explored seasonal differences in the orexigenic neuropeptides that regulate food intake in wild animals. Here we tested the hypothesis that free-living male song sparrows (Melospiza melodia) show seasonal changes in energetic state, circulating steroids, and both neuropeptide Y (NPY) and orexin (OX) immunoreactivity. Nonbreeding song sparrows had more fat and muscle, as well as a ketone and triglyceride profile suggesting a greater reliance on lipid reserves. Breeding birds had higher plasma androgens; however, nonbreeding birds did maintain androgen precursors in circulation. Nonbreeding birds had more NPY immunoreactivity, specifically in three brain regions: lateral septum, bed nucleus of the stria terminalis, and ventral tegmental area. Furthermore, nonbreeding birds had more OX immunoreactivity in multiple brain regions. Taken together, the data indicate that a natural shift in energy balance is associated with changes in NPY and OX in a region-specific manner.

Sources of variation in plasma corticosterone and dehydroepiandrosterone in the male northern cardinal (Cardinalis cardinalis): I. Seasonal patterns and effects of stress and adrenocorticotropic hormone.

The secretion of steroids from the adrenal gland is a classic endocrine response to perturbations that can affect homeostasis. During an acute stress response, glucocorticoids (GC), such as corticosterone (CORT), prepare the metabolic physiology and cognitive abilities of an animal in a manner that promotes survival during changing conditions. Although GC functions during stress are well established, much less is understood concerning how adrenal androgens, namely dehydroepiandrosterone (DHEA) are influenced by stress. I conducted three field studies (one experimental and two descriptive) aimed at identifying how both CORT and DHEA secretion in free-living male northern cardinals (Cardinalis cardinalis), vary during acute stress; across different circulations (brachial vs. jugular); in response to ACTH challenge; and during the annual cycle. As predicted, restraint stress increased plasma CORT, but unexpectedly DHEA levels decreased, but the latter effect was only seen for blood sampled from the jugular vein, and not the brachial. The difference in DHEA between circulations may result from increased neural uptake of DHEA during stress. Injection with exogenous adrenocorticotropic hormone (ACTH) increased CORT concentrations, but failed to alter DHEA levels, thus suggesting ACTH is not a direct regulator of DHEA. Monthly field sampling revealed distinct seasonal patterns to both initial and restraint stress CORT and DHEA levels with distinct differences in the steroid milieu between breeding and non-breeding seasons. These data suggest that the CORT response to stress remains relatively consistent, but DHEA secretion is largely independent of the response by CORT. Although CORT functions have been well-studied in wild animals, little research exists for the role of DHEA and their variable relationship sets the stage for future experimental research addressing steroid stress responses.

Sources of variation in plasma corticosterone and dehydroepiandrosterone in the male northern cardinal (Cardinalis cardinalis): II. Effects of urbanization, food supplementation and social stress.

Perturbations in an organism's environment can induce significant shifts in hormone secretory patterns. In this context, the glucocorticoid (GC) steroids secreted by the adrenal cortex have received much attention from ecologists and behaviorists due to their role in the vertebrate stress response. Adrenal GCs, such as corticosterone (CORT), are highly responsive to instability in environmental and social conditions. However, little is understood about how adrenal dehydroepiandrosterone (DHEA) is influenced by changing conditions. We conducted field experiments to determine how circulating CORT and DHEA vary during restraint stress in the male northern cardinals (Cardinalis cardinalis). Specifically, we examined how four different changes in the physical (urbanization and food availability) and social (territorial conflict, distress of a mate) environment affect CORT and DHEA levels. The majority of cardinals responded to restraint stress by increasing and decreasing CORT and DHEA, respectively, however this depended on sampling context. Cardinals sampled from urban habitats had both lower initial and restraint stress CORT concentrations, but a comparable DHEA pattern to those sampled from a forest. Supplementing food to territorial males did not alter circulating initial DHEA or CORT concentrations nor did it change the response to restraint stress when compared to unsupplemented controls. Exposing cardinals to varying durations of song playback, which mimics a territorial intrusion, did not affect CORT levels, but did attenuate the DHEA response to restraint stress. Examining a larger dataset of males captured before, after or at the same time as their female mate, allowed us to address how the stress of a captured mate affected the male's CORT and DHEA response. Males showed elevated initial and restraint CORT and DHEA when their female mate was captured first. Taken together, these data demonstrate that both CORT and DHEA secretion patterns depends on environmental, and particularly current social conditions.

Eating increases oxidative damage in a reptile.

While eating has substantial benefits in terms of both nutrient and energy acquisition, there are physiological costs associated with digesting and metabolizing a meal. Frequently, these costs have been documented in the context of energy expenditure while other physiological costs have been relatively unexplored. Here, we tested whether the seemingly innocuous act of eating affects either systemic pro-oxidant (reactive oxygen metabolite, ROM) levels or antioxidant capacity of corn snakes (Pantherophis guttatus) by collecting plasma during absorptive (peak increase in metabolic rate due to digestion of a meal) and non-absorptive (baseline) states. When individuals were digesting a meal, there was a minimal increase in antioxidant capacity relative to baseline (4%), but a substantial increase in ROMs (nearly 155%), even when controlling for circulating nutrient levels. We report an oxidative cost of eating that is much greater than that due to long distance flight or mounting an immune response in other taxa. This result demonstrates the importance of investigating non-energetic costs associated with meal processing, and it begs future work to identify the mechanism(s) driving this increase in ROM levels. Because energetic costs associated with eating are taxonomically widespread, identifying the taxonomic breadth of eating-induced ROM increases may provide insights into the interplay between oxidative damage and life history theory.

Sex steroid profiles and pair-maintenance behavior of captive wild-caught zebra finches (Taeniopygia guttata).

Here, we studied the life-long monogamous zebra finch, to examine the relationship between circulating sex steroid profiles and pair-maintenance behavior in pairs of wild-caught zebra finches (paired in the laboratory for >1 month). We used liquid chromatography-tandem mass spectrometry to examine a total of eight androgens and progestins [pregnenolone, progesterone, dehydroepiandrosterone (DHEA), androstenediol, pregnan-3,17-diol-20-one, androsterone, androstanediol, and testosterone]. In the plasma, only pregnenolone, progesterone, DHEA, and testosterone were above the limit of quantification. Sex steroid profiles were similar between males and females, with only circulating progesterone levels significantly different between the sexes (female > male). Circulating pregnenolone levels were high in both sexes, suggesting that pregnenolone might serve as a circulating prohormone for local steroid synthesis in zebra finches. Furthermore, circulating testosterone levels were extremely low in both sexes. Additionally, we found no correlations between circulating steroid levels and pair-maintenance behavior. Taken together, our data raise several interesting questions about the neuroendocrinology of zebra finches.

A low carbohydrate, high protein diet suppresses intratumoral androgen synthesis and slows castration-resistant prostate tumor growth in mice.

Dietary factors continue to preside as dominant influences in prostate cancer prevalence and progression-free survival following primary treatment. We investigated the influence of a low carbohydrate diet, compared to a typical Western diet, on prostate cancer (PCa) tumor growth in vivo. LNCaP xenograft tumor growth was studied in both intact and castrated mice, representing a more advanced castration resistant PCa (CRPC). No differences in LNCaP tumor progression (total tumor volume) with diet was observed for intact mice (P = 0.471) however, castrated mice on the Low Carb diet saw a statistically significant reduction in tumor growth rate compared with Western diet fed mice (P = 0.017). No correlation with serum PSA was observed. Steroid profiles, alongside serum cholesterol and cholesteryl ester levels, were significantly altered by both diet and castration. Specifically, DHT concentration with the Low Carb diet was 58% that of the CRPC-bearing mice on the Western diet. Enzymes in the steroidogenesis pathway were directly impacted and tumors isolated from intact mice on the Low Carb diet had higher AKR1C3 protein levels and lower HSD17B2 protein levels than intact mice on the Western diet (ARK1C3: P = 0.074; HSD17B2: P = 0.091, with α = 0.1). In contrast, CRPC tumors from mice on Low Carb diets had higher concentrations of both HSD17B2 (P = 0.016) and SRD5A1 (P = 0.058 with α = 0.1) enzymes. There was no correlation between tumor growth in castrated mice for Low Carb diet versus Western diet and (a) serum insulin (b) GH serum levels (c) insulin receptor (IR) or (d) IGF-1R in tumor tissue. Intact mice fed Western diet had higher serum insulin which was associated with significantly higher blood glucose and tumor tissue IR. We conclude that both diet and castration have a significant impact on the endocrinology of mice bearing LNCaP xenograft tumors. The observed effects of diet on cholesterol and steroid regulation impact tumor tissue DHT specifically and are likely to be mechanistic drivers behind the observed tumor growth suppression.

Regulation of local steroidogenesis in the brain and in prostate cancer: lessons learned from interdisciplinary collaboration.

Sex steroids play critical roles in the regulation of the brain and many other organs. Traditionally, researchers have focused on sex steroid signaling that involves travel from the gonads via the circulation to intracellular receptors in target tissues. This classic concept has been challenged, however, by the growing number of cases in which steroids are synthesized locally and act locally within diverse tissues. For example, the brain and prostate carcinoma were previously considered targets of gonadal sex steroids, but under certain circumstances, these tissues can upregulate their steroidogenic potential, particularly when circulating sex steroid concentrations are low. We review some of the similarities and differences between local sex steroid synthesis in the brain and prostate cancer. We also share five lessons that we have learned during the course of our interdisciplinary collaboration, which brought together neuroendocrinologists and cancer biologists. These lessons have important implications for future research in both fields.

Fasting increases aggression and differentially modulates local and systemic steroid levels in male zebra finches.

Aggression enables individuals to obtain and retain limited resources. Studies of the neuroendocrine regulation of aggression have focused on territorial and reproductive contexts. By contrast, little is understood concerning the neuroendocrine regulation of aggression over other resources, such as food. Here, we developed a paradigm to examine the role of steroids in food-related aggression. In groups of male zebra finches, a 6-hour fast decreased body mass and increased aggressive interactions among subjects that competed for a point source feeder. Fasting also dramatically altered circulating steroid levels by decreasing plasma testosterone but not estradiol (E2). By contrast, both plasma corticosterone and dehydroepiandrosterone (DHEA) concentrations were elevated with fasting. Interestingly, short-term access to food (15 minutes) after fasting normalized circulating steroid levels. Fasting increased corticosterone levels in a wide range of peripheral tissues but increased DHEA levels specifically in adrenal glands and liver; these effects were quickly normalized with refeeding. DHEA can be metabolized within specific brain regions to testosterone and E2, which promote the expression of aggression. We measured E2 in microdissected brain regions and found that fasting specifically increased local E2 levels in 3 regions: the periaqueductal gray, ventral tegmental area, and ventromedial nucleus of the hypothalamus. These regions are part of the vertebrate social behavior network and regulate the expression of aggression. Together, these data suggest that fasting stimulates secretion of DHEA from the adrenals and liver and subsequent conversion of DHEA to E2 within specific brain regions, to enable individuals to compete for limited food resources.

Unpredictable food availability induces metabolic and hormonal changes independent of food intake in a sedentary songbird.

Environments often vary with regard to their temporal resource availability, but little is understood concerning how resource predictability impacts animals. The adaptive regulation hypothesis suggests that organisms act to conserve their current energetic state during periods of diminished food access and recuperate their energetic reserves (fat and muscle) during periods of greater food availability. In contrast, the chronic stress hypothesis suggests that variation in access to food can induce a prolonged stress response, resulting in maladaptive usage of energy reserves and increased behavioral activity. To distinguish between these hypotheses we compared the behavioral, hormonal and metabolic responses of captive curve-billed thrashers, Toxostoma curvirostre, fed varying amounts each day (variable group) with those of birds fed a constant amount every day (constant feeding group). Birds of both groups consumed, on average, a similar total amount of food during the course of the study, but birds in the variable feeding group lost mass and increased their circulating initial levels of the stress hormone corticosterone, showed evidence for increased secretion of a hypothalamic stress peptide, vasotocin, used greater amounts of fat and protein energy reserves, and were more behaviorally active than birds in the constant feeding group. Overall, these findings support the chronic stress hypothesis and suggest that birds such as thrashers may be particularly susceptible to the perception of unpredictable variation in food supplies independent of actual energetic constraints.

Brain arginine vasotocin immunoreactivity differs between urban and desert curve-billed thrashers, Toxostoma curvirostre: relationships with territoriality and stress physiology.

The neuropeptide arginine vasotocin (AVT: the avian homolog of vasopressin) has numerous functional roles including mediating social behaviors, coregulating the adrenocortical stress response and maintaining water balance. These functions of AVT make it susceptible to environmental influence, yet little is understood concerning the variation in the AVT system across habitats. In this study, AVT immunoreactivity was compared between male curve-billed thrashers, Toxostoma curvirostre, from native Sonoran Desert locations and those within the city of Phoenix, Ariz. Previous research found that urban thrashers are more responsive to territorial intrusion, secrete more corticosterone (CORT) during capture stress, and they may also have greater access to water than desert counterparts. Variation in AVT immunoreactivity was also related to levels of plasma CORT and osmolality, and with behavioral responses to a simulated territorial intrusion. Birds from these two habitats showed different AVT immunoreactive patterns in two brain regions: the paraventricular nucleus of the hypothalamus and the medial bed nucleus of the stria terminalis (BSTM), a part of the limbic system. Immunoreactive AVT within the paraventricular nucleus was associated with plasma CORT levels in urban, but not desert, birds, but no such association with osmolality was observed in birds from either habitat. The total number of BSTM AVT-immunoreactive cells was related to a decreased responsiveness to territorial intrusion. These data suggest that divergence in the AVT system between urban and desert thrashers may help explain observed differences in both the adrenocortical stress response and territorial behavior between populations. Whether differences in water availability between habitats contribute to population differences in the brain AVT system is unknown.

Effects of captivity and body condition on plasma corticosterone, locomotor behavior, and plasma metabolites in curve-billed thrashers.

The acute stress response involves the secretion of catabolic glucocorticoids, such as corticosterone (CORT) in birds, that mobilize intrinsic energy stores primarily through a gluconeogenic pathway involving fat breakdown, thus linking body condition and stress. We measured changes in CORT and gluconeogenic metabolites (triglycerides, free glycerols, glucose) during handling stress in curve-billed thrashers Toxostoma curvirostre from two habitats (urban vs. desert) that may differ in food abundance in the wild, in captivity, and in response to both food restriction and subsequent recovery. Urban thrashers were heavier and secreted more CORT than desert birds in the field, but differences did not persist in captivity. Decreased access to food resulted in decreased body mass and a diminished ability to elevate plasma CORT in response to handling stress. However, the opposite effect was observed as these birds recovered from food restriction. Plasma levels of glucose and triglycerides did not change with stress. Food restriction also increased locomotor activity, which likely further exacerbated energy loss. These observations suggest that body condition and stress differences between urban and desert birds may be related to differences in their relative energetic states, possibly due to food availability. Body condition may affect the extent to which an individual can elevate CORT and use free glycerol as energy during acute stress.

Plasma corticosterone of city and desert Curve-billed Thrashers, Toxostoma curvirostre, in response to stress-related peptide administration.

We compared the activity and responsiveness of the hypothalamo-pituitary-adrenal (HPA) axis of an urban (Phoenix, Arizona) and desert population of a male songbird species (Curve-billed Thrasher, Toxostoma curvirostre), by measuring plasma corticosterone in response to acute administration of corticotropin-releasing factor, arginine vasotocin, or adrenocorticotropin hormone. Urban adult male thrashers showed greater responsiveness than desert birds to an injection of arginine vasotocin or adrenocorticotropin hormone, suggesting a population difference in pituitary and adrenal gland sensitivity. Plasma corticosterone in response to corticotropin-releasing factor injection did, however, not differ between populations. The differential corticosterone response to arginine vasotocin and corticotropin-releasing factor may reflect effects of chronic stress or habituation, which are known to favor arginine vasotocin over corticotropin-releasing factor sensitivity. Efficacy of HPA negative feedback by glucocorticoids was determined by measuring plasma corticosterone in response to acute administration of the synthetic glucocorticoid dexamethasone. This administration decreased plasma corticosterone similarly in urban and desert thrashers, suggesting that the negative feedback of glucocorticoids on the HPA axis in the two populations was equally effective. The higher sensitivity of urban than desert thrashers to adrenocorticotropin hormone and arginine vasotocin may result from up-regulation of the HPA axis in urban birds. This up-regulation may in turn make it easier for city birds to cope with urban environment-associated stressors.

Context-specific territorial behavior in urban birds: no evidence for involvement of testosterone or corticosterone.

Testosterone produced by the gonads is a primary mediator of seasonal patterns of territoriality and may directly facilitate territorial behavior during an encounter with a potential intruder. Costs and benefits associated with territoriality can vary as a function of habitat, for example through differences in resource distribution between areas occupied by different individuals. We investigated behaviors in response to simulated territorial intrusions (hereafter territorial behaviors) in urban (Phoenix, Arizona) and nearby desert populations of two Sonoran Desert birds (Curve-billed Thrasher and Abert's Towhee). We also examined the degree to which these behaviors are mediated by testosterone (T) and the adrenal steroid, corticosterone (CORT), which can interact with T in territorial contexts. In both species, urban birds displayed more territorial behaviors than their desert conspecifics, but this difference was not associated with variation in either plasma total or in plasma free (i.e., unbound to binding globulins) T or CORT. In addition, neither plasma T nor plasma CORT changed as a function of duration of the simulated territorial intrusion. Urban Abert's Towhees displayed more territorial behaviors in areas where their population densities were high than in areas of low population densities. Urban Curve-billed Thrashers displayed more territorial behaviors in areas with a high proportion of desert-type vegetation, particularly in areas that differed in vegetation composition from nearby randomly sampled areas, than in areas with a high proportion of exotic or non-desert type vegetation. Associations between territorial behavior and habitat characteristics were not related to plasma T or CORT. Understanding the hormonal processes underlying these associations between behavior and habitat may provide insight into how free-ranging animals assess territorial quality and alter their defensive behavior accordingly.

Acute stress rapidly decreases plasma testosterone in a free-ranging male songbird: potential site of action and mechanism.

We used a free-ranging, seasonally breeding adult male songbird, the rufous-winged sparrow, Aimophila carpalis, to investigate the effects of acute stress-induced by capture followed by restraint, on the hypothalamo-pituitary-testicular axis. Intra- and interindividual comparisons revealed that males decreased their plasma testosterone (T) by 37-52% in response to acute stress. The decrease occurred within 15 min of capture and persisted for at least another 15 min. Within 15 min, the decrease in plasma T was not associated with a reduction in plasma luteinizing hormone (LH). Thirty minutes after capture and restraint, the decrease in plasma T either was likewise not associated with decreased plasma LH (intraindividual comparison) or concurred with a reduction in plasma LH (interindividual comparison). These observations indicate that effects of stress may have been mediated at the pituitary gland and also directly at the testicular levels. To address this question, we measured the hormonal response to an injection of the glutamate receptor agonist N-methyl-d,l-aspartate (NMA) to stimulate to stimulate the release of gonadotropin-releasing hormone (GnRH) or of GnRH to stimulate the release of LH. Treatment with NMA did not change plasma LH, presumably because the birds were in breeding condition and already secreting GnRH at a maximum rate. Administration of GnRH increased plasma LH equally in birds that were or were not stressed before the treatment. An injection of purified ovine LH (oLH) increased plasma T equally in birds that were or were not acutely stressed before the hormone injection. Thus, the observed acute stress-induced decrease in plasma T was apparently not mediated by decreased responsiveness of the pituitary gland to GnRH or of the testes to LH. Decreased plasma T following stress may involve a direct impairment of the testicular endocrine function.

Corticosterone and corticosteroid binding globulin in birds: relation to urbanization in a desert city.

As cities expand worldwide, understanding how species adapt to novel urban habitats will become increasingly important to conservation. The adrenocortical stress response enables vertebrates to cope with novel environmental challenges to homeostasis. We examined total and estimates of free baseline and stress-induced corticosterone (CORT) concentrations and CORT binding globulin (CBG) levels in five passerine species within and around Phoenix, Arizona. We tested whether baseline and stress-induced CORT patterns differed among species living at varying densities in Phoenix and tested the hypothesis that, for species capable of successfully colonizing cities, individuals living in urban areas have a decreased acute stress response compared to individuals living in native desert. Baseline total CORT levels were generally similar in urban and rural birds. Capture and handling stress typically produced greater total CORT responses in urban birds than in rural birds, although these responses differed as a function of the life history stage (non-breeding, breeding or molt). CBG binding capacity did not change with life history stage or locality. Estimated free CORT concentrations differed less between groups than total CORT concentrations. Urban birds showed less variability in stress responses across life history stages than rural birds. We propose that more predictable resources in the city than in rural areas may decrease the need to vary stress responsiveness across life history stages. The results highlight the species-specific effects of urbanization on stress physiology and the difficulty to predict how urbanization impacts organisms.