Immune gene expression and epigenetic potential affect the consumption of risky food by female house sparrows.

When organisms move into new areas, they are likely to encounter novel food resources. Even if they are nutritious, these foods can also be risky, as they might be contaminated by parasites. The behavioural immune system of animals could help them avoid the negative effects of contaminated resources, but our understanding of behavioural immunity is limited, particularly whether and how behavioural immunity interacts with physiological immunity. Here, we asked about the potential for interplay between these two traits, specifically how the propensity of an individual house sparrow (Passer domesticus) to take foraging risks was related to its ability to regulate a key facet of its immune response to bacterial pathogens. Previously, we found that sparrows at expanding geographic range edges were more exploratory and less risk-averse to novel foods; in those same populations, birds tended to over-express Toll-like receptor 4 (TLR4), a pattern-recognition receptor that distinguishes cell-wall components of Gram-negative bacteria, making it the major sensor of potentially lethal gut microbial infections including salmonellosis. When we investigated how birds would respond to a typical diet (i.e., mixed seeds) spiked with domesticated chicken faeces, birds that expressed more TLR4 or had higher epigenetic potential for TLR4 (more CpG dinucleotides in the putative gene promoter) ate more food, spiked or not. Females expressing abundant TLR4 were also willing to take more foraging risks and ate more spiked food. In males, TLR4 expression was not associated with risk-taking. Altogether, our results indicate that behaviour and immunity covary among individual house sparrows, particularly in females where those birds that maintain more immune surveillance also are more disposed to take foraging risks.

Gene expression in the female tree swallow brain is associated with inter- and intra-population variation in glucocorticoid levels.

Studies of the evolutionary causes and consequences of variation in circulating glucocorticoids (GCs) have begun to reveal how they are shaped by selection. Yet the extent to which variation in circulating hormones reflects variation in other important regulators of the hypothalamic-pituitary-adrenal (HPA) axis, and whether these relationships vary among populations inhabiting different environments, remain poorly studied. Here, we compare gene expression in the brain of female tree swallows (Tachycineta bicolor) from populations that breed in environments that differ in their unpredictability. We find evidence of inter-population variation in the expression of glucocorticoid and mineralocorticoid receptors in the hypothalamus, with the highest gene expression in a population from an extreme environment, and lower expression in a population from a more consistent environment as well as in birds breeding at an environmentally variable high-altitude site that are part of a population that inhabits a mixture of high and low altitude habitats. Within some populations, variation in circulating GCs predicted differences in gene expression, particularly in the hypothalamus. However, some patterns were present in all populations, whereas others were not. These results are consistent with the idea that some combination of local adaptation and phenotypic plasticity may modify components of the HPA axis affecting stress resilience. Our results also underscore that a comprehensive understanding of the function and evolution of the stress response cannot be gained from measuring circulating hormones alone, and that future studies that apply a more explicitly evolutionary approach to important regulatory traits are likely to provide significant insights.

High epigenetic potential protects songbirds against pathogenic Salmonella enterica infection.

Animals encounter many novel and unpredictable challenges when moving into new areas, including pathogen exposure. Because effective immune defenses against such threats can be costly, plastic immune responses could be particularly advantageous, as such defenses can be engaged only when context warrants activation. DNA methylation is a key regulator of plasticity via its effects on gene expression. In vertebrates, DNA methylation occurs exclusively at CpG dinucleotides and, typically, high DNA methylation decreases gene expression, particularly when it occurs in promoters. The CpG content of gene regulatory regions may therefore represent one form of epigenetic potential (EP), a genomic means to enable gene expression and hence adaptive phenotypic plasticity. Non-native populations of house sparrows (Passer domesticus) - one of the world's most cosmopolitan species - have high EP in the promoter of a key microbial surveillance gene, Toll-like receptor 4 (TLR4), compared with native populations. We previously hypothesized that high EP may enable sparrows to balance the costs and benefits of inflammatory immune responses well, a trait critical to success in novel environments. In the present study, we found support for this hypothesis: house sparrows with high EP in the TLR4 promoter were better able to resist a pathogenic Salmonella enterica infection than sparrows with low EP. These results support the idea that high EP contributes to invasion and perhaps adaptation in novel environments, but the mechanistic details whereby these organismal effects arise remain obscure.

Cold temperatures induce priming of the glucose stress response in tree swallows.

Capricious environments often present wild animals with challenges that coincide or occur in sequence. Conceptual models of the stress response predict that one threat may prime or dampen the response to another. Although evidence has supported this for glucocorticoid responses, much less is known about the effects of previous challenges on energy mobilization. Food limitation may have a particularly important effect, by altering the ability to mobilize energy when faced with a subsequent challenge. We tested the prediction that challenging weather conditions, which reduce food availability, alter the energetic response to a subsequent acute challenge (capture and restraint). Using a three-year dataset from female tree swallows measured during three substages of breeding, we used a model comparison approach to test if weather (temperature, wind speed, and precipitation) over 3- or 72-hour timescales predicted baseline and post-restraint glucose levels, and if so which environmental factors were the strongest predictors. Contrary to our predictions, weather conditions did not affect baseline glucose; however, birds that had experienced lower temperatures over the preceding 72 h tended to have higher stress-induced glucose when faced with an acute stressor. We also saw some support for an effect of rainfall on stress-induced glucose: around the time that eggs hatched, birds that had experienced more rainfall over the preceding 72 h mounted lower responses. Overall, we find support in a wild animal for the idea that the glucose stress response may be primed by exposure to prior challenges.

Glucocorticoid receptor expression as an integrative measure to assess glucocorticoid plasticity and efficiency in evolutionary endocrinology: A perspective.

Organisms have to cope with the changes that take place in their environment in order to keep their physical and psychological stability. In vertebrates, the hypothalamic-pituitary-adrenal (HPA) axis plays a key role in mediating phenotypic adjustments to environmental changes, primarily by regulating glucocorticoids (GCs). Although circulating GCs have widely been used as proxy for individual health and fitness, our understanding of HPA regulation is still very limited, especially in free-living animals. Circulating GCs only exert their actions when they are bound to receptors, and therefore, GC receptors play a pivotal role mediating HPA regulation and GC downstream phenotypic changes. Because under challenging conditions GC actions (as well as negative feedback activation) occur mainly through binding to low-affinity glucocorticoid receptors (GR), we propose that GR activity, and in particular GR expression, may play a crucial role in GC regulation and dynamics, and be ultimately related to organismal capacity to appropriately respond to environmental changes. Thus, we suggest that GR expression will provide more comprehensive information of GC variation and function. To support this idea, we compile previous evidence demonstrating the fundamental role of GR on GC responses and the fine-tuning of circulating GCs. We also make predictions about the phenotypic differences in GC responsiveness - and ultimately HPA regulation capacity - associated with differences in GR expression, focusing on GC plasticity and efficiency. Finally, we discuss current priorities and limitations of integrating measures of GR expression into evolutionary endocrinology and ecology studies, and propose further research directions towards the use of GR expression and the study of the mechanisms regulating GR activity to gather information on coping strategies and stress resilience. Our goals are to provide an integrative perspective that will prompt reconsideration on the ecological and physiological interpretation of current GC measurements, and motivate further research on the role of GR in tuning individual responses to dynamic environments.

Individual variation in natural or manipulated corticosterone does not covary with circulating glucose in a wild bird.

Animals respond to sudden challenges with a coordinated set of physiological and behavioral responses that enhance the ability to cope with stressors. While general characteristics of the vertebrate stress response are well described, it is not as clear how individual components covary between or within individuals. A rapid increase in glucocorticoids coordinates the stress response and one of the primary downstream results is an increase in glucose availability via reduced glucose utilization. Here, we asked whether between- and within-individual variation in corticosterone directly predict variation in glucose. We collected 2673 paired glucose and corticosterone measures from 776 tree swallows (Tachycineta bicolor) from four populations spanning the species range. In adults, glucose and corticosterone both increased during a standardized restraint protocol in all four populations. Moreover, in one population experimentally increasing a precursor that stimulates corticosterone release resulted in a further increase in both measures. In contrast, nestlings did not show a robust glucose response to handling or manipulation. Despite this group-level variation, there was very little evidence in any population that between-individual variation in corticosterone predicted between-individual variation in glucose regulation. Glucose was moderately repeatable within individuals, but within-individual variation in glucose and corticosterone were unrelated. Our results highlight the fact that a strong response in one aspect of the coordinated acute stress response (corticosterone) does not necessarily indicate that specific downstream components, such as glucose, will show similarly strong responses. These results have implications for understanding the evolution of integrated stress response systems.

FKBP5 expression is related to HPA flexibility and the capacity to cope with stressors in female and male house sparrows.

The hypothalamic-pituitary-adrenal (HPA) axis and its end products, the glucocorticoids, are critical to responding appropriately to stressors. Subsequently, many studies have sought relationships between glucocorticoids and measures of health or fitness, but such relationships are at best highly context dependent. Recently, some endocrinologists have started to suggest that a focus on HPA flexibility, the ability of an individual to mount appropriate responses to different stressors, could be useful. Here, we tested the hypothesis that expression of FKBP5, a cochaperone in the glucocorticoid receptor complex, is a simple and reliable proxy of HPA flexibility in a wild songbird, the house sparrow (Passer domesticus). We quantified HPA flexibility in a novel way, using guidance from research on heart rhythm regulation. As predicted, we found that adult sparrows with low stress-induced FKBP5 expression in the hypothalamus exhibited high HPA flexibility. Moreover, low FKBP5 expression was associated with greater exploratory disposition and were better at maintaining body mass under stressful conditions. Altogether, these results suggest that FKBP5 may be important in the regulation of HPA flexibility, potentially affecting how individuals cope with natural and anthropogenic adversity.

Epigenetic potential affects immune gene expression in house sparrows.

Epigenetic mechanisms may play a central role in mediating phenotypic plasticity, especially during range expansions, when populations face a suite of novel environmental conditions. Individuals may differ in their epigenetic potential (EP; their capacity for epigenetic modifications of gene expression), which may affect their ability to colonize new areas. One form of EP, the number of CpG sites, is higher in introduced house sparrows (Passer domesticus) than in native birds in the promoter region of a microbial surveillance gene, Toll-like Receptor 4 (TLR4), which may allow invading birds to fine-tune their immune responses to unfamiliar parasites. Here, we compared TLR4 gene expression from whole blood, liver and spleen in house sparrows with different EP, first challenging some birds with lipopolysaccharide (LPS), to increase gene expression by simulating a natural infection. We expected that high EP would predict high inducibility and reversibility of TLR4 expression in the blood of birds treated with LPS, but we did not make directional predictions regarding organs, as we could not repeatedly sample these tissues. We found that EP was predictive of TLR4 expression in all tissues. Birds with high EP expressed more TLR4 in the blood than individuals with low EP, regardless of treatment with LPS. Only females with high EP exhibited reversibility in gene expression. Further, the effect of EP varied between sexes and among tissues. Together, these data support EP as one regulator of TLR4 expression.

Does variation in glucocorticoid concentrations predict fitness? A phylogenetic meta-analysis.

Glucocorticoid hormones (GCs) are central mediators of metabolism and the response to challenges. Because circulating GC levels increase in response to challenges, within-population variation in GCs could reflect among-individual variation in condition or experience. At the same time, individual variation in GC regulation could have causal effects on energetic balance or stress coping capacity in ways that influence fitness. Although a number of studies in vertebrates have tested whether variation in GCs among individuals predicts components of fitness, it is not clear whether there are consistent patterns across taxa. Here we present the first phylogenetic meta-analysis testing whether variation in GCs is associated with survival and reproductive success across vertebrates. At the same time, we introduce and test predictions about a potentially important mediator of GC-fitness relationships: life history context. We suggest that strong context-dependence in the fitness benefit of maintaining elevated GCs could obscure consistent patterns between GCs and fitness across taxa. Meta-analyses revealed that baseline and stress-induced GCs were consistently negatively correlated with reproductive success. This relationship did not differ depending on life history context. In contrast, the relationships between GCs and survival were highly context dependent, differing according to life history strategy. Both baseline and stress-induced GCs were more strongly negatively associated with survival in longer-lived populations and species. Stress-induced GCs were also more positively associated with survival in organisms that engage in relatively more valuable reproductive attempts. Fecal GCs did not correlate with survival or reproductive success. We also found that experimental increases in GCs reduced both survival and reproductive success; however, evidence of publication bias and the small sample size suggest that more data is required to confirm this conclusion. Overall, these results support the prediction that GC-fitness relationships can be strongly context dependent, and suggest that incorporating life history may be particularly important for understanding GC-survival relationships.

Early-life adversity programs long-term cytokine and microglia expression within the HPA axis in female Japanese quail.

Stress exposure during prenatal and postnatal development can have persistent and often dysfunctional effects on several physiological systems, including immune function, affecting the ability to combat infection. The neuroimmune response is inextricably linked to the action of the hypothalamic-pituitary-adrenal (HPA) axis. Cytokines released from neuroimmune cells, including microglia, activate the HPA axis, while glucocorticoids in turn regulate cytokine release from microglia. Because of the close links between these two physiological systems, coupled with potential for persistent changes to HPA axis activity following developmental stress, components of the neuroimmune system could be targets for developmental programming. However, little is known of any programming effects of developmental stress on neuroimmune function. We investigated whether developmental stress exposure via elevated prenatal corticosterone (CORT) or postnatal unpredictable food availability had long-term effects on pro- (IL-1ß) and anti-inflammatory (IL-10) cytokine and microglia-dependent gene (CSF1R) expression within HPA axis tissues in a precocial bird, the Japanese quail (Coturnix japonica). Following postnatal stress, we observed increased IL-1ß expression in the pituitary gland, reduced IL-10 expression in the amygdala and hypothalamus, and reduced CSF1R expression within the hypothalamus and pituitary gland. Postnatal stress disrupted the ratio of IL-1ß:IL-10 expression within the hippocampus and hypothalamus. Prenatal stress only increased IL-1ß expression in the pituitary gland. We found no evidence for interactive or cumulative effects across life stages on basal cytokine and glia expression in adulthood. We show that postnatal stress may have a larger impact than elevated prenatal CORT on basal immunity in HPA-axis-specific brain regions, with changes in cytokine homeostasis and microglia abundance. These results provide evidence for postnatal programming of a pro-inflammatory neuroimmune phenotype at the expense of reduced microglia, which could have implications for central nervous system health and subsequent neuroimmune responses.

The lingering impact of stress: brief acute glucocorticoid exposure has sustained, dose-dependent effects on reproduction.

Acutely stressful experiences can have profound and persistent effects on phenotype. Across taxa, individuals differ remarkably in their susceptibility to stress. However, the mechanistic causes of enduring stress effects, and of individual differences in stress susceptibility, are poorly understood. Here, we tested whether brief, acute increases in glucocorticoid hormones have persistent effects on phenotype, and whether effects differ according to the magnitude or duration of elevation. We used a novel method to non-invasively manipulate hormone levels on short time scales: the application of corticosterone gel to a model egg secured in the nest. Free-living female tree swallows (Tachycineta bicolor) exposed to several brief corticosterone increases during incubation showed dose-dependent differences in behaviour throughout the reproductive period. Birds receiving treatments that simulated higher or longer acute stress responses later provisioned larger broods at lower rates; the resulting offspring were smaller in size. Treatment did not influence female body condition, oxidative stress, reproductive success or inter-annual survival, but exposed females maintained higher baseline corticosterone after treatments ceased. Overall, these results indicate that brief, acute elevations in glucocorticoids in adulthood can have long-term consequences. Furthermore, individuals that mount a greater or longer acute stress response may be more likely to experience lingering effects of stress.

Efficacy of negative feedback in the HPA axis predicts recovery from acute challenges.

The glucocorticoid stress response mediates a suite of physiological and behavioural changes that allow vertebrates to cope with transient stressors. Chronically elevated glucocorticoid levels are known to result in a variety of organismal costs, but relatively little is known about the downstream effects of mounting a series of brief, acute spikes in circulating glucocorticoids. Conceptual models of stress suggest that repeated acute stressors might produce 'wear-and-tear' on the stress-response system when encountered in sequence. We used a novel technique to experimentally induce acute corticosterone spikes on either three or six consecutive days in incubating tree swallows. Consistent with the 'wear-and-tear' hypothesis, we found that (i) a sequence of corticosterone spikes produced cumulative effects on corticosterone regulation, (ii) treatment frequency predicted the severity of consequences, and (iii) individual variation in the ability to terminate the stress response through negative feedback predicted the duration of physiological disruption in the group that experienced the most frequent challenges. Our results illustrate the importance of assessing multiple aspects of the hormonal stress response and have implications for understanding both individual and population resilience to repeated transient stressors.

Reduced resistance to oxidative stress during reproduction as a cost of early-life stress.

Stress exposure during early-life development can have long-term consequences for a variety of biological functions including oxidative stress. The link between early-life stress and oxidative balance is beginning to be explored and previous studies have focused on this link in adult non-breeding or immature individuals. However, as oxidative stress is considered as the main physiological mechanism underlying the trade-off between self-maintenance and investment in reproduction, it is necessary to look at the consequences of early-life stress on oxidative status during reproduction. Here, we investigated the effects of exposure to pre- and/or post-natal stress on oxidative balance during reproduction under benign or stressful environmental conditions in an avian model species, the Japanese quail. We determined total antioxidant status (TAS), total oxidant status (TOS) and resistance to a free-radical attack in individual exposed to pre-natal stress, post-natal stress or both and in control individuals exposed to none of the stressors. TAS levels decreased over time in all females that reproduced under stressful conditions. TOS decreased between the beginning and the end of reproductive period in pre-natal control females. In all females, resistance to a free-radical attack decreased over the reproductive event but this decrease was more pronounced in females from a pre-natal stress development. Our results suggest that pre-natal stress may be associated with a higher cost of reproduction in terms of oxidative stress. These results also confirm that early-life stress can be associated with both benefits and costs depending of the life-history stage or environmental context.

Early life stress shapes female reproductive strategy through eggshell pigmentation in Japanese quail.

Physiological constraints on colouration have been widely reported; especially in birds, which trade-off antioxidant responses against colourful costly signals. One female extended phenotypic trait, which might also highlight important physiological trade-offs, is the pigmentation of their eggshells. In ground-nesting species, producing eggs that are visually undetectable by predators is the best camouflage strategy. However, the condition-dependence of eggshell pigmentation, and the pigments role in oxidative stress, may constrain females to trade-off between their antioxidant capacity and maximising the camouflage of their eggs when they deposit eggshell pigments. Developmental stress is one factor that influences female antioxidant capacity, and could lead to variations in eggshell pigmentation that might have crucial consequences on individual fitness if egg crypsis is compromised especially under stressful conditions. We investigated the interaction between developmental and breeding conditions with respect to eggshell pigmentation in Japanese quail. We studied 30 females that bred under both control and stressful conditions, and were exposed to pre- and/or post-natal stress, or neither. Pre- and post-natal stress independently influenced eggshell pigmentation strategies under stressful breeding conditions. Under stressful reproduction, eggshell protoporphyrin concentration and maculation were affected by pre-natal stress, whereas eggshell reflectance and biliverdin concentration were influenced by post-natal stress. These changes may reflect potential adaptive strategies shaped by developmental stress, but additional data on the benefit of egg crypsis in quail, combined with studies on the role of both pigments on chick survival, will help to clarify whether early life stress can enhance fitness through eggshell pigmentation when developmental and reproductive environments match.

HPA flexibility and FKBP5: promising physiological targets for conservation.

Hypothalamic-pituitary-adrenal axis (HPA) flexibility is an emerging concept recognizing that individuals that will cope best with stressors will probably be those using their hormones in the most adaptive way. The HPA flexibility concept considers glucocorticoids as molecules that convey information about the environment from the brain to the body so that the organismal phenotype comes to complement prevailing conditions. In this context, FKBP5 protein appears to set the extent to which circulating glucocorticoid concentrations can vary within and across stressors. Thus, FKBP5 expression, and the HPA flexibility it causes, seem to represent an individual's ability to regulate its hormones to orchestrate organismal responses to stressors. As FKBP5 expression can also be easily measured in blood, it could be a worthy target of conservation-oriented research attention. We first review the known and likely roles of HPA flexibility and FKBP5 in wildlife. We then describe putative genetic, environmental and epigenetic causes of variation in HPA flexibility and FKBP5 expression among and within individuals. Finally, we hypothesize how HPA flexibility and FKBP5 expression should affect organismal fitness and hence population viability in response to human-induced rapid environmental changes, particularly urbanization. This article is part of the theme issue 'Endocrine responses to environmental variation: conceptual approaches and recent developments'.

Developmental stage-dependent effects of perceived predation risk on nestling tree swallows (Tachycineta bicolor).

The risk of predation directly affects the physiology, behavior, and fitness of wild birds. Strong social connections with conspecifics could help individuals recover from a stressful experience such as a predation event; however, competitive interactions also have the potential to exacerbate stress. Few studies have investigated the interaction between environmental stressors and the social landscape in wild bird populations. In 2 years of field studies, we experimentally simulated predation attempts on breeding female tree swallows (Tachicyneta bicolor). At the same time, we manipulated female breast plumage color, a key social signal. Simulated predation events on tree swallows early in the nestling period reduced young nestlings' mass by approximately 20% and shortened telomere lengths. Ultimately, only 31% of nestlings in the predation group fledged compared with 70% of control nestlings. However, the effects of experimental manipulations were timing dependent: the following year when we swapped the order of the experimental manipulations and simulated predation during incubation, there were no significant effects of predation on nestling condition or fledging success. Contrary to our expectations, manipulation of the social environment did not affect the response of tree swallows to simulated predation. However, manipulating female plumage during the nestling period did reduce nestling skeletal size and mass, although the effects depended on original plumage brightness. Our data demonstrate that transient stressors on female birds can have carry-over effects on their nestlings if they occur during critical periods in the breeding season.

Developmental programming: cumulative effects of increased pre-hatching corticosterone levels and post-hatching unpredictable food availability on physiology and behaviour in adulthood.

Prolonged exposure to stress during development can have long-term detrimental effects on health and wellbeing. However, the environmental matching hypothesis proposes that developmental stress programs physiology and behaviour in an adaptive way that can enhance fitness if early environments match those experienced later in life. Most research has focused on the harmful effects that stress during a single period in early life may exert in adulthood. In this study, we tested the potential additive and beneficial effects that stress experienced during both pre- and post-hatching development may have on adult physiology and behaviour. Japanese quail experienced different stress-related treatments across two developmental life stages: pre-hatching corticosterone (CORT) injection, post-hatching unpredictable food availability, both pre- and post-hatching treatments, or control. In adulthood, we determined quails' acute stress response, neophobia and novel environment exploration. The pre-hatching CORT treatment resulted in attenuated physiological responses to an acute stressor, increased activity levels and exploration in a novel environment. Post-hatching unpredictable food availability decreased adults' latency to feed. Furthermore, there were cumulative effects of these treatments across the two developmental stages: quail subjected to both pre- and post-hatching treatments were the most explorative and risk-taking of all treatment groups. Such responses to novel environments could enhance survival in unpredictable environments in later life. Our data also suggest that these behavioural responses may have been mediated by long-term physiological programming of the adrenocortical stress response, creating phenotypes that could exhibit fitness-enhancing behaviours in a changing environment.

Pre- and post-natal stress have opposing effects on social information use.

Theoretical models of social learning predict that animals should copy others in variable environments where resource availability is relatively unpredictable. Although short-term exposure to unpredictable conditions in adulthood has been shown to encourage social learning, virtually nothing is known concerning whether and how developmental conditions affect social information use. Unpredictable food availability increases levels of the stress hormone corticosterone (CORT). In birds, CORT can be transferred from the mother to her eggs, and have downstream behavioural effects. We tested how pre-natal CORT elevation through egg injection, and chick post-natal development in unpredictable food conditions, affected social information use in adult Japanese quail (Coturnix japonica). Pre-natal CORT exposure encouraged quail to copy the foraging decisions of demonstrators in video playbacks, whereas post-natal food unpredictability led individuals to avoid the demonstrated food source. An individual's exposure to stress and uncertainty during development can thus affect its use of social foraging information in adulthood. However, the stressor's nature and developmental timing determine whether an adult will tend to copy conspecifics or do the opposite. Developmental effects on social information use might thus help explain individual differences in social foraging tactics and leadership.