Effects of cooling on thyroid hormone secretion and growth of eastern bluebird (Sialia sialis) nestlings.

Achieving endothermic homeothermy is a critical aspect of avian development. In pre-homeothermic altricial nestlings, variation in parental brooding behavior results in variable exposure of nestlings to cooling, with consequences for the developing endocrine system. Nestlings facing repeated cooling challenges may benefit from upregulation of thyroid hormone secretion, allowing for earlier onset of thermoregulatory capability to mitigate the potentially negative effects of exposure to non-optimal temperatures during development. We examined the effects of (1) a single cooling challenge on thyroid hormone secretion in pre-homeothermic nestlings, and (2) repeated cooling challenges prior to the onset of homeothermy on nestling growth and thyroid hormone secretion prior to fledging. We found that pre-homeothermic eastern bluebird nestlings exposed to a single cooling challenge increased circulating triiodothyronine (T3), demonstrating that the thyroid system can be activated by cooling early in life. However, we found no consequences of repeated cooling during the first week of life on nestling growth or baseline T3 levels prior to fledging. This work addresses how the nestling hypothalamic-pituitary-thyroid axis responds to acute cooling challenges prior to the development of endothermic homeothermy; future work will confirm whether such responses allow nestlings to hasten the onset of physiological thermoregulation when conditions demand it.

Early-Life Cooling Alters Later Corticosterone Response to Restraint in Prefledging Eastern Bluebirds (Sialia sialis) but Does Not Alter Adrenal Sensitivity to ACTH.

AbstractEnvironmental challenges faced early in life can both activate and shape the development of the hypothalamic-pituitary-adrenal axis. Activation of this axis is characterized in part by elevated levels of glucocorticoids, exposure to which can have profound effects throughout an animal's life. We have demonstrated that in nestling eastern bluebirds (Sialia sialis), bouts of environmentally relevant cooling result in elevations of corticosterone (the primary avian glucocorticoid) very early in life. Nestlings repeatedly exposed to cooling also exhibit dampened corticosterone secretion later in life in response to restraint compared to control nestlings. We explored the mechanistic basis of this phenomenon. Specifically, we asked whether early-life cooling alters adrenal sensitivity to adrenocorticotropic hormone (ACTH), the primary controller of corticosterone synthesis and release. To this end, we subjected nestlings to repeated bouts of cooling (cooled nestlings) or brooding temperatures (control nestlings) early in life and, before fledging, assessed (1) the capacity of the nestlings' adrenals to produce corticosterone following ACTH injection, (2) the effect of cooling on corticosterone responses to restraint, and (3) the effect of cooling on adrenal sensitivity to ACTH. We found that both cooled and control nestlings secreted substantially higher levels of corticosterone following ACTH treatment than they did following restraint. We also confirmed that cooled nestlings had reduced corticosterone secretion in response to restraint compared to control nestlings; however, sensitivity to exogenous ACTH did not differ between temperature treatments. We hypothesize that early-life cooling alters later corticosterone secretion by affecting higher levels of the hypothalamic-pituitary-adrenal axis.

Unpredictable fasting transiently alters corticosterone and feeding behavior but not body mass or later HPA axis function in zebra finches (Taeniopygia guttata).

The hypothalamic-pituitary-adrenal (HPA) axis is commonly activated in response to unpredictable conditions, including unstable or inadequate food supply. Extended exposure to unpredictable food resources can alter HPA axis function, with the potential for negative fitness consequences. We addressed the interrelationships of unpredictable food resources, HPA axis activity, and food intake in adult and juvenile zebra finches (Taeniopygia guttata). Finches exposed to prolonged periods of unpredictable food elevate corticosterone (the primary avian glucocorticoid) when food is unavailable; however, whether they experience chronic elevation in baseline corticosterone during periods of unpredictability, even when food is available, is unclear. We subjected adults and juveniles to an extended period of temporally unpredictable food (consisting of a random daily fast) or predictable food supply. We investigated baseline corticosterone under fed conditions and in response to an acute fast (mimicking the daily fasting periods in the unpredictable treatment), and assessed differences in body mass, food intake, and corticosterone responses to restraint. Regardless of sex and age, individuals in both treatment groups elevated corticosterone when fasted, and baseline corticosterone under fed conditions was indistinguishable between groups. Thus, corticosterone levels were not persistently elevated in the unpredictably fed group. Treatment groups did not differ in body mass or corticosterone responses to restraint, but unpredictably fed birds consumed food more rapidly when food was available. Our findings suggest that the unpredictably fed birds experienced repeated, moderate elevations in corticosterone. Such elevations may aid birds in coping with unpredictable food sources, in part by activating compensatory changes in foraging behavior.

Chill out: Environmentally relevant cooling challenge does not increase telomere loss during early life.

In vertebrates, exposure to diverse stressors during early life activates a stress response that can initiate compensatory mechanisms or promote cellular damage with long-term fitness consequences. A growing number of studies associate exposure to stressors during early life with increased damage to telomeres (i.e., promoting the shortening of these highly conserved, repeating sequences of non-coding DNA at chromosome ends). However, some studies show no such relationship, suggesting that the nature, timing, and context of these challenges may determine the degree to which physiological mediators of the stress response act in a damage-mitigating or damage promoting way in relation to telomere dynamics. In free-living eastern bluebirds (Sialia sialis), we have previously demonstrated that bouts of offspring cooling that occur when brooding females leave the nest increase at least one such physiological mediator of the stress response (circulating glucocorticoids), suggesting that variation in patterns of maternal brooding may result in different impacts on telomere dynamics at a young age. Here we experimentally tested whether repeated bouts of ecologically relevant offspring cooling affected telomere dynamics during post-natal development. Rates of telomere shortening during the nestling stage were not affected by experimental cooling, but they were affected by brood size and the rate of growth during the nestling stage. Our data suggest that the effects of developmental stress exposure on offspring telomeres are often context-dependent and that not all challenges that increase physiological mediators of stress result in damage to telomeres. Under some conditions, physiological mediators of stress may instead act as protective regulators, allowing for optimization of fitness outcomes in the face of environmental challenges.

Cooling increases corticosterone deposition in feathers of eastern bluebird chicks.

Exposure to noxious stimuli early in life can both activate and shape the development of the hypothalamic-pituitary-adrenal (HPA) axis in birds and other vertebrates, with the potential for lifelong consequences. Studies assessing early HPA axis activation often rely on collection of blood samples to evaluate circulating glucocorticoid levels. However, blood sampling in small altricial young is invasive, limited by animal size, and not sufficient to provide detailed information about hormone exposure over protracted periods of time. We tested the use of feather corticosterone as an alternative method to assess HPA axis activity early in life in free-living, altricial chicks, for whom timing of growth of first feathers coincides with a period of rapid growth, development of the HPA axis, and reliance on parental care. We investigated (1) whether ecologically relevant bouts of experimental cooling prior to the onset of homeothermy-conditions known to elevate circulating corticosterone-are reflected in changes of feather corticosterone deposition in Eastern bluebird (Sialia sialis) chicks, and (2) whether such changes occurred in a sex-dependent manner. We found that cooling during the first week of life resulted in elevated feather corticosterone in first-grown feathers of experimentally cooled chicks relative to controls. The timing of deposition of corticosterone in feathers in response to temperature treatments was delayed in females compared to males. Results indicate that the hormone deposition in feather tissues of altricial nestlings reflects exposure to environmental stimuli, and can thus provide a minimally invasive tool for assessing HPA activity in early life. The development of the HPA axis, or its activation in response to environmental stimuli early in life, may also occur in a sex-dependent manner in altricial birds.

Assay temperature affects corticosteroid-binding globulin and free corticosterone estimates across species.

Glucocorticoid hormones are often measured to assess how organisms physiologically respond to challenges in their environment. In plasma, glucocorticoids circulate in two forms: bound to corticosteroid-binding globulins (CBG) or unbound (free). Measuring CBG allows us to estimate the amount of free glucocorticoids present in a plasma sample. However, free glucocorticoid estimates are affected by the assay temperature used when measuring CBG, with colder temperatures maximizing specific binding but likely underestimating glucocorticoid's affinity for CBG. Here, we test how a biologically relevant incubation temperature (41 °C) changes the disassociation constant (Kd; used to estimate free glucocorticoid levels) when compared to the traditional 4 °C incubation temperature, across four commonly studied avian species. We then apply the new Kd's calculated at 41 °C to existing data sets to examine how the change in Kd affects free corticosterone estimates and data interpretation. Kd's were generally higher (lower affinity for CORT) at warmer incubation temperatures which resulted in higher levels of estimated free CORT in all four species but differed among subspecies. This increase in free CORT levels did not qualitatively change previously reported statistical relationships, but did affect variance and alpha (P) values. We suggest that future assays be run at biologically relevant temperatures for more accurate estimates of free CORT levels in vivo and to increase the chances of detecting biological patterns of free-CORT that may not be revealed with the classic methodology that tends to underestimate free CORT levels.

Interactions of body temperature and nutritional status on hypothalamo-pituitary-adrenal axis activity in pre-thermoregulatory eastern bluebird chicks (Sialia sialis).

Early life experiences can affect the function of the hypothalamo-pituitary-adrenal (HPA) axis of vertebrates, with potential fitness consequences later in life. In altricial species, for example, variation in parental behavior, e.g. brooding or feeding, can modify the activity of the HPA axis of the young by altering their exposure to noxious stimuli as the young develop in the nest. We have shown that a drop in the body temperature of eastern bluebird (Sialia sialis) chicks, such as occurs when females are away from the nest, elevates their blood corticosterone levels. If repeated during the early days of their development, cooling bouts also reduce the chicks' later corticosterone secretion in response to handling. Thus, variation in maternal behavior has the capacity to shape the function of the chicks' HPA axis. To better understand how maternal absence from the nest activates the HPA axis of bluebird chicks, we experimentally mimicked the cooling that occurs when the female is away from the nest, and investigated a) the age at which the HPA axis becomes capable of responding to cooling by increasing corticosterone secretion, b) whether corticosterone secretion remains elevated throughout long periods of cooling, and c) whether fasting (also potentially associated with maternal absence) interacts with cooling to affect corticosterone secretion. Cooling for 18 min significantly elevated circulating corticosterone levels of chicks as young as 4 days post-hatch, indicating that their HPA axis is sensitive to cooling very early in life. Corticosterone levels remained elevated throughout longer bouts of cooling. However, a 1-hr period of fasting had no effect on corticosterone secretion, regardless of whether chicks were cooled or not. Collectively, these data demonstrate that variation in maternal brooding behavior can substantially modify the corticosterone profiles of chicks during early postnatal development, and that chick temperature is likely the main driver of this.

Neural Versus Gonadal GnIH: Are they Independent Systems? A Mini-Review.

Based on research in protochordates and basal vertebrates, we know that communication across the first endocrine axes likely relied on diffusion. Because diffusion is relatively slow, rapid responses to some cues, including stress-related cues, may have required further local control of axis outputs (e.g., steroid hormone production by the gonads). Despite the evolution of much more efficient circulatory systems and complex nervous systems in vertebrates, production of many "neuro"transmitters has been identified outside of the hypothalamus across the vertebrate phylogeny and these neurotransmitters are known to locally regulate endocrine function. Our understanding of tissue-specific neuropeptide expression and their role coordinating physiological/behavioral responses of the whole organism remains limited, in part, due to nomenclature and historic dogma that ignores local regulation of axis output. Here, we review regulation of gonadotropin-inhibitory hormone (GnIH) across the reproductive axis in birds and mammals to bring further attention to context-dependent disparities and similarities in neuropeptide production by the brain and gonads. We find that GnIH responsiveness to cues of stress appears conserved across species, but that the response of specific tissues and the direction of GnIH regulation varies. The implications of differential regulation across tissues remain unclear in most studies, but further work that manipulates and contrasts function in different tissues has the potential to inform us about both organism-specific function and endocrine axis evolution.

Ecologically Relevant Cooling Early in Life Alters Prefledging Adrenocortical Response in Free-Living Songbirds.

In vertebrates, exposure to stressful stimuli early in development may alter the activity of the hypothalamo-pituitary-adrenal (HPA) axis, with the potential for fitness consequences later in life. For altricial species, whose young rely on their parents for food, warmth, and protection from predators, adult behavior can modify the impact of some stressors on their offspring after birth or hatching. We have shown that single bouts of cooling that normally occur when brooding females leave the nest elevate corticosterone secretion in very young free-living eastern bluebird (Sialia sialis) chicks. Thus, natural variation in maternal brooding patterns can result in differential exposure of offspring to cooling, and also to glucocorticoids, very early in development. We tested the hypothesis that exposure to repeated bouts of cooling (mimicking those that occur normally when females leave the nest) would alter the activity of the chicks' HPA axis later in life. We exposed free-living chicks to either four 18-min bouts of cooling or brooding temperatures (control) during the first week after hatching. Then, just before fledging (i.e., at least 7 d after the cooling treatments had ceased), we assessed their corticosterone responses to restraint. Repeatedly cooled chicks had a significantly lower corticosterone response to restraint than did control chicks but did not differ from controls in other measures of growth and development. Our data suggest that natural variation in maternal brooding patterns, and hence natural variation in the chicks' body temperature, can alter the activity of the HPA axis well beyond the brooding period.

Differential response of GnIH in the brain and gonads following acute stress in a songbird.

Gonadotropin-inhibitory hormone (GnIH) acts to inhibit reproduction at all levels of the hypothalamo-pituitary-gonad axis. GnIH expression and/or immunoreactivity in the hypothalamus increase with acute stress in some birds and mammals, and thus may be involved in stress-induced reproductive inhibition. Much is known about GnIH and stress in seasonal and continuous breeders, but far less is known about these interactions in opportunistic breeders. For opportunistically breeding animals, reproductive readiness is closely associated with unpredictable environmental cues, and thus the GnIH system may be more sensitive to stress. To test this, we collected tissues from zebra finches immediately following capture or after 60 min of restraint. Restraint significantly increased plasma corticosterone in males and females but, contrary to studies on other species, restrained birds had significantly fewer GnIH immunoreactive (GnIH-ir) cell bodies than control birds. GnIH-ir cell number did not differ between the sexes. Stressed females had lower mRNA expression of the beta subunit of follicle stimulating hormone (FSHß) in the pituitary, suggesting that the reduction in observed GnIH immunoreactivity in females may have been due to increased GnIH release in response to acute stress. GnIH expression increased in the testes, but not the ovaries, of restrained animals. Our data suggest that although GnIH responsiveness to stress appears to be conserved across species, specific tissue response and direction of GnIH regulation is not. Variation in the GnIH response to stress between species might be the result of ecological adaptations or other species differences in the response of the GnIH system to stress.

Endocrine and neuroendocrine regulation of fathering behavior in birds.

This article is part of a Special Issue "Parental Care". Although paternal care is generally rare among vertebrates, care of eggs and young by male birds is extremely common and may take on a variety of forms across species. Thus, birds provide ample opportunities for investigating both the evolution of and the proximate mechanisms underpinning diverse aspects of fathering behavior. However, significant gaps remain in our understanding of the endocrine and neuroendocrine influences on paternal care in this vertebrate group. In this review, I focus on proximate mechanisms of paternal care in birds. I place an emphasis on specific hormones that vary predictably and/or unpredictably during the parental phase in both captive and wild birds: prolactin and progesterone are generally assumed to enhance paternal care, whereas testosterone and corticosterone are commonly-though not always correctly-assumed to inhibit paternal care. In addition, because endocrine secretions are not the sole mechanistic influence on paternal behavior, I also explore potential roles for certain neuropeptide systems (specifically the oxytocin-vasopressin nonapeptides and gonadotropin inhibitory hormone) and social and experiential factors in influencing paternal behavior in birds. Ultimately, mechanistic control of fathering behavior in birds is complex, and I suggest specific avenues for future research with the goal of narrowing gaps in our understanding of this complexity. Such avenues include (1) experimental studies that carefully consider not only endocrine and neuroendocrine mechanisms of paternal behavior, but also the ecology, phylogenetic history, and social context of focal species; (2) investigations that focus on individual variation in both hormonal and behavioral responses during the parental phase; (3) studies that investigate mechanisms of maternal and paternal care independently, rather than assuming that the mechanistic foundations of care are similar between the sexes; (4) expansion of work on interactions of the neuroendocrine system and fathering behavior to a wider array of paternal behaviors and taxa (e.g., currently, studies of the interactions of testosterone and paternal care largely focus on songbirds, whereas studies of the interactions of corticosterone, prolactin, and paternal care in times of stress focus primarily on seabirds); and (5) more deliberate study of exceptions to commonly held assumptions about hormone-paternal behavior interactions (such as the prevailing assumptions that elevations in androgens and glucocorticoids are universally disruptive to paternal care). Ultimately, investigations that take an intentionally integrative approach to understanding the social, evolutionary, and physiological influences on fathering behavior will make great strides toward refining our understanding of the complex nature by which paternal behavior in birds is regulated.

Food, stress, and circulating testosterone: Cue integration by the testes, not the brain, in male zebra finches (Taeniopygia guttata).

Food abundance is closely associated with reproductive readiness in vertebrates. Food scarcity can activate the hypothalamo-pituitary-adrenal axis, decrease sex steroid secretion, and dampen reproductive behavior. However, the mechanisms underlying these transient effects are unclear. Gonadotropin inhibitory hormone (GnIH), a neuropeptide present in the brain and gonads, is also influenced by glucocorticoids and fasting in some species. We investigated whether fasting stress activated the GnIH system in zebra finches (Taeniopygia guttata), with the potential for downstream effects on reproductive physiology and behavior. We fasted or fed males ad libitum for 10h. Fasting increased corticosterone and decreased testosterone in circulation. To assess whether the decrease in testosterone was mediated by changes in the hypothalamus and/or the gonads, we (1) quantified GnRH- and GnIH-positive neurons in the hypothalamus, (2) assessed hypothalamic gene expression for GnRH and GnIH, and (3) examined gene expression for proteins involved in testosterone synthesis in fasted and control birds. No measure of hypothalamic neuropeptides was related to treatment or circulating steroids. However, birds with higher corticosterone had higher testicular GnIH expression and lower testosterone. StAR and LHR expression were lower in the testes of fasted birds than controls. Thus, the decrease in testosterone was not likely mediated by hypothalamic GnIH, but rather by direct actions of fasting and/or corticosterone on the testes, indicating that the testes can integrate and respond to cues of stress directly. Such local inhibition of testosterone synthesis may allow for rapid and reversible changes in physiology and behavior when conditions are inappropriate for breeding.

Island life shapes the physiology and life history of eastern bluebirds (Sialia sialis).

Abstract Island organisms face a range of extrinsic threats to their characteristically small populations. Certain biological differences between island and continental organisms have the potential to exacerbate these threats. Understanding how island birds differ from their continental relatives may provide insight into population viability and serve as a predictive tool for conservation efforts. We compared an eastern bluebird population in Ohio with a threatened population in Bermuda in terms of the birds' development, morphology, immunology, and reproduction. These comparisons revealed that island nestlings had shorter wings and island adults had longer wings than their continental analogs. Island nestlings also had shorter tarsi than continental nestlings at day 8 posthatch, but this difference was absent at day 15 and in adults. Adults weighed less in Bermuda than in Ohio, and both nestlings and adults in Bermuda exhibited higher levels of two immunological indexes (concentrations of an acute-phase protein and titers of nonspecific antibodies). Clutch sizes and hatch rates did not differ between the island and continental populations; however, as the breeding season progressed, brood sizes declined in Bermuda, whereas no such decline occurred in Ohio. Despite these differences and differences in nestling development, island and continental parents fed their nestlings at equal rates. Overall, our results suggest that the Bermuda phenotype may be adjusted to certain aspects of the island environment but not to others. Efforts to conserve the bluebirds of Bermuda may be improved by focusing on the intraseasonal patterns in nestling mortality and, more generally, the survival probabilities of different age classes.

Environmentally relevant bouts of cooling stimulate corticosterone secretion in free-living eastern bluebird (Sialia sialis) nestlings: potential links between maternal behavior and corticosterone exposure in offspring.

In vertebrates, exposure to stressful stimuli or to elevated glucocorticoids early in development can contribute to phenotypic variation that may have significant fitness consequences. In species with altricial young, offspring may be partially buffered from elevations in glucocorticoids by a period of low glucocorticoid responsiveness to stressors coupled with high levels of parental care. Because altricial young depend heavily on their parents for warmth, parental brooding behavior could buffer offspring from glucocorticoid exposure associated with cooling. We studied eastern bluebirds (Sialia sialis) with two goals: (1) to determine whether an experimental drop in body temperature such as that which might occur when a brooding female is off the nest was sufficient to stimulate glucocorticoid secretion in young chicks, and (2) to examine the extent to which chicks experienced such bouts of cooling in the field. We subjected chicks to treatments simulating nest temperatures while females were brooding or absent from the nest. We also recorded chick surface temperatures and ambient temperatures at nests during the first week of the brood period. Reductions of surface temperature of less than 10°C significantly elevated corticosterone secretion in chicks as young as 5days old, and thermal and hormonal responses of chicks to cooling increased in an age-dependent manner. One quarter of broods experienced repeated, natural bouts of cooling of this magnitude or greater in the nest. Our data suggest that natural variations in maternal brooding patterns can result in differential exposure of offspring to glucocorticoids, and this may have important phenotypic consequences later in life.

Neonatal handling alters the development of the adrenocortical response to stress in a wild songbird (eastern bluebird, Sialia sialis).

Neonatal handling of captive vertebrates can shape the development of their hypothalamo-pituitary-adrenal (HPA) axis and alter their ability to respond to stressful stimuli later in life. However, the long-term effects of such handling on this endocrine axis in free-living species are not well understood. We investigated the effects of age and neonatal handling on corticosterone secretion in response to restraint in eastern bluebird (Sialia sialis) chicks. We found that unhandled ("naïve") and handled ("experienced") chicks exhibited no corticosterone response to handling early in development. Thereafter, naïve individuals exhibited the progressive development of a corticosterone response with age, and by day 12 post-hatch, the response resembled that of adult bluebirds. Experienced nestlings, which were handled every other day from the day of hatch, showed a similar pattern of HPA development until day 12 post-hatch, when their corticosterone response was significantly reduced compared to that of naïve nestlings. In contrast, chicks that were handled only once, when 10days old, did not show a reduced corticosterone response at 12days old. Taken together, our data suggest that a certain threshold of accumulated neonatal handling episodes is necessary to depress corticosterone secretion, and/or that the cumulative effects of several handling episodes only manifest themselves once the HPA axis is fully developed. Our findings, in concert with studies on two other wild species, indicate that routine handling of nestlings in the field can alter their responses to stress in a species-specific manner, potentially leading to important fitness consequences.

Food, stress, and reproduction: short-term fasting alters endocrine physiology and reproductive behavior in the zebra finch.

Stress is thought to be a potent suppressor of reproduction. However, the vast majority of studies focus on the relationship between chronic stress and reproductive suppression, despite the fact that chronic stress is rare in the wild. We investigated the role of fasting in altering acute stress physiology, reproductive physiology, and reproductive behavior of male zebra finches (Taeniopygia guttata) with several goals in mind. First, we wanted to determine if acute fasting could stimulate an increase in plasma corticosterone and a decrease in corticosteroid binding globulin (CBG) and testosterone. We then investigated whether fasting could alter expression of undirected song and courtship behavior. After subjecting males to fasting periods ranging from 1 to 10h, we collected plasma to measure corticosterone, CBG, and testosterone. We found that plasma corticosterone was elevated, and testosterone was decreased after 4, 6, and 10h of fasting periods compared with samples collected from the same males during nonfasted (control) periods. CBG was lower than control levels only after 10h of fasting. We also found that, coincident with these endocrine changes, males sang less and courted females less vigorously following short-term fasting relative to control conditions. Our data demonstrate that acute fasting resulted in rapid changes in endocrine physiology consistent with hypothalamo-pituitary-adrenal axis activation and hypothalamo-pituitary-gonadal axis deactivation. Fasting also inhibited reproductive behavior. We suggest that zebra finches exhibit physiological and behavioral flexibility that makes them an excellent model system for studying interactions of acute stress and reproduction.

A single exposure to an acute stressor has lasting consequences for the hypothalamo-pituitary-adrenal response to stress in free-living birds.

In vertebrates, activation of the hypothalamo-pituitary-adrenal (HPA) axis in response to unpredictable events results in elevated glucocorticoid secretion. Repeated exposure to stressors alters subsequent glucocorticoid secretion, either by inducing chronic stress or as a result of habituation. However, most studies of repeated stress focus on the impacts of multiple and frequent exposures to acute stressors, and few have been carried out in free-living animals. We investigated whether a single exposure to a novel stressor was sufficient to produce long-lasting alterations in HPA function in free-living eastern bluebirds (Sialia sialis). We subjected adult females to a capture/restraint protocol in which we collected serial blood samples over an hour of restraint to be analyzed for corticosterone. We administered this protocol to three groups of females during the nestling phase of their first and/or second brood of the season: Repeaters (sampled during brood 1 and brood 2), Naïve-Brood 1 (sampled only during brood 1), and Naïve-Brood 2 (sampled only during brood 2). Repeaters had attenuated corticosterone responses to the second restraint bout compared to the first, and in brood 2, Repeaters had lower responses than Naïve-Brood 2 females. However, Naïve-Brood 1 and Naïve-Brood 2 birds did not differ in their responses to restraint. Thus, as little as one prior experience with an acute stressor was sufficient to alter subsequent HPA responsiveness, and this effect was not due to a natural change in HPA responsiveness as the breeding season progressed. These data may have important implications for understanding how acute stressors can alter a free-living animal's ability to cope in the face of subsequent stressors, and for longitudinal field studies in which individuals are repeatedly sampled for glucocorticoid responsiveness.

Supplementary testosterone inhibits paternal care in a tropically breeding sparrow, Zonotrichia capensis.

In most male birds that exhibit paternal care, elevation in testosterone above the breeding baseline reduces nestling provisioning, which can be detrimental to offspring survival. Mechanisms that may allow some males to avoid this detrimental effect of elevated testosterone include (1) decreased sensitivity to testosterone's effects on behavior and (2) uncoupling of testosterone secretion from territorial challenges (thus reducing the number of transient elevations in testosterone above the breeding baseline). Both of these "cost-avoidance" mechanisms have been documented, but whether selection for these mechanisms is correlated or independent is unknown. We investigated the relationship between elevated testosterone and paternal care in a tropical bird, the rufous-collared sparrow (Zonotrichia capensis). Zonotrichia capensis males exhibit an uncoupling of testosterone secretion from territorial aggression, and this species has a flexible breeding season and a clutch size smaller than those of temperate congeners. We implanted males with testosterone or empty implants and observed paternal behavior 2-3 and 6-7 d posthatch. During both observation periods, 100% of control males fed chicks, whereas 22% and 0% of testosterone-implanted males fed chicks on days 2-3 and 6-7, respectively. Chicks of testosterone-implanted males weighed less than control chicks, but tarsus growth, wing growth, and fledging success did not differ. Thus, we demonstrate a robust negative effect of testosterone on nestling provisioning that may not ultimately affect reproductive success. We suggest that these results relate to extreme flexibility in breeding schedule and the small clutch size in this tropical species. Our data also suggest that selection for the two mechanisms to avoid deleterious effects of elevations in testosterone above the breeding baseline likely occurs independently.

Behavioral insensitivity to testosterone: why and how does testosterone alter paternal and aggressive behavior in some avian species but not others?

Considerable research has been conducted on the interrelationships of the steroid hormone testosterone and reproductive behavior in seasonally breeding birds. In species in which males provide paternal care, males experience a peak in testosterone secretion that coincides with territory establishment and pair bonding, and then drops to a breeding baseline as nests are initiated. A large body of evidence suggests that a male's seasonal profile of testosterone reflects a trade-off between expression of sexual and territorial behavior and expression of paternal behavior. For example, studies utilizing testosterone implants to extend the early season peak in plasma testosterone have demonstrated that testosterone tends to increase sexual behavior as well as intensity and persistence of male-male aggression, but also decreases paternal care of offspring. However, recent studies demonstrate that in some species, males do not respond to experimentally elevated testosterone with alterations in aggression and/or paternal care. This phenomenon of "behavioral insensitivity" to testosterone may relate to a variety of environmental factors, particularly those that necessitate high levels of paternal investment. This review explores both ultimate and proximate explanations for behavioral insensitivity to testosterone, and explores hypotheses to explain how this phenomenon may relate to androgen responses to social interactions during breeding. Further research into behavioral insensitivity to testosterone in a variety of vertebrates may provide additional insights into the complex patterns of sex steroid secretion and its behavioral consequences.

Dissociation of testosterone and aggressive behavior during the breeding season in male chestnut-collared longspurs, Calcarius ornatus.

In many species of birds, expression of male-male aggression during breeding elicits increased secretion of testosterone. Elevations in testosterone in turn enhance expression of aggressive behavior in appropriate contexts. However, in other species, the relationship between elevated plasma testosterone and aggressive behavior is subtle or nonexistent. We examined the relationship between high testosterone and male-male aggression in the chestnut-collared longspur, a species in which males exhibit a peak in testosterone early in the breeding season that declines to a breeding baseline after nests are initiated. Elevated testosterone enhances sexual behavior after nests are initiated, but does not affect care of nestlings. We used simulated territorial intrusions (STIs) to test (1) if males increased testosterone secretion after an aggressive interaction early in the breeding season (2) if expression of aggressive behavior declined as testosterone levels declined to the breeding baseline, and (3) if experimentally elevated testosterone enhanced aggression when testosterone levels were at the breeding baseline. Plasma testosterone was unrelated to duration of STI before nests were initiated. In addition, although males were significantly more aggression prior to nest initiation than during incubation, elevation of plasma testosterone to pre-nesting levels did not enhance aggression during incubation. Thus, although elevated plasma testosterone and expression of aggressive behavior appear to overlap temporally, high plasma testosterone and male-male aggression may not be fully coupled in this species. We suggest that high plasma testosterone may be more important in eliciting and maintaining sexual behavior than aggressive behavior in this species.