Changes in the rearing environment cause reorganization of molecular networks associated with DNA methylation.

Disentangling the interaction between the genetic basis and environmental context underlying phenotypic variation is critical for understanding organismal evolution. Environmental change, such as increased rates of urbanization, can induce shifts in phenotypic plasticity with some individuals adapting to city life while others are displaced. A key trait that can facilitate adaptation is the degree at which animals respond to stressors. This stress response, which includes elevation of baseline circulating concentrations of glucocorticoids, has a heritable component and exhibits intra- and inter-individual variation. However, the mechanisms behind this variability and whether they might be responsible for adaptation to different environments are not known. Variation in DNA methylation can be a potential mechanism that mediates environmental effects on the stress response, as early-life stressors increase glucocorticoid concentrations and change adult phenotype. We used an inter- and intra-environmental cross-foster experiment to analyse the contribution of DNA methylation to early-life phenotypic variation. We found that at hatching, urban house wren (Troglodytes aedon) offspring had higher methylation frequencies compared with their rural counterparts. We also observed age-related patterns in offspring methylation, indicating the developmental effects of the rearing environment on methylation. At fledgling, differential methylation analyses showed that cellular respiration genes were differentially methylated in broods of different origins and behavioural and metabolism genes were differentially methylated in broods of different rearing environments. Lastly, hyper-methylation of a single gene (CNTNAP2) is associated with decreased glucocorticoid levels and the rearing environment. These differential methylation patterns linked to a specific physiological phenotype suggest that DNA methylation may be a mechanism by which individuals adjust to novel environments during their lifespan. Characterizing genetic and environmental influences on methylation is critical for understanding the role of epigenetic mechanisms in evolutionary adaptation.

Urbanization and maternal hormone transfer: Endocrine and morphological phenotypes across ontogenetic stages.

The phenotypes observed in urban and rural environments are often distinct; however, it remains unclear how these novel urban phenotypes arise. Hormone-mediated maternal effects likely play a key role in shaping developmental trajectories of offspring in different environments. Thus, we measured corticosterone (Cort) and testosterone (T) concentrations in eggs across the laying sequence in addition to Cort concentrations in nestling and adult female house wrens (Troglodytes aedon) at one urban and one rural site. We found that egg T concentrations were not different between birds from urban and rural sites. However, across all life stages (egg, nestling, and adult female), Cort concentrations were higher at the urban site. Additionally, urban nestling Cort concentrations, but not rural, correlated with fine-scale urban density scores. Furthermore, rural egg volume increased over the laying sequence, but urban egg volume leveled off mid-sequence, suggesting either that urban mothers are resource limited or that they are employing a different brood development strategy than rural mothers. Our study is one of the first to show that egg hormone concentrations differ in an urban environment with differences persisting in chick development and adult life stages. We suggest that maternal endocrine programing may shape offspring phenotypes in urban environments and are an overlooked yet important aspect underlying mechanisms of urban evolution.

Natural variation in developmental condition has limited effect on spatial cognition in a wild food-caching bird.

Laboratory studies show that increased physiological burden during development results in cognitive impairment. In the wild, animals experience a wide range of developmental conditions, and it is critical to understand how variation in such conditions affects cognitive abilities later in life, especially in species that strongly depend on such abilities for survival. We tested whether variation in developmental condition is associated with differences in spatial cognitive abilities in wild food-caching mountain chickadees. Using tail feathers grown during development in juvenile birds, we measured feather corticosterone (Cortf) levels and growth rates and tested these birds during their first winter on two spatial learning tasks. In only 1 of the 3 years, higher feather Cortf was negatively associated with memory acquisition. No significant associations between feather Cortf and any other measurement of spatial cognition were detected in the other 2 years of the study or between feather growth rate and any measurement of cognition during the entire study. Our results suggest that in the wild, naturally existing variation in developmental condition has only a limited effect on spatial cognitive abilities, at least in a food-caching species. This suggests that there may be compensatory mechanisms to buffer specialized cognitive abilities against developmental perturbations.

Increased lead and glucocorticoid concentrations reduce reproductive success in house sparrows along an urban gradient.

Urbanization is increasing at a rapid pace globally. Understanding the links among environmental characteristics, phenotypes, and fitness enables researchers to predict the impact of changing landscapes on individuals and populations. Although avian reproductive output is typically lower in urban compared with natural areas, the underlying reasons for this discrepancy may lie at the intersection of abiotic and biotic environmental and individual differences. Recent advances in urban ecology highlight the effect of heavy metal contamination on stress physiology. As high levels of glucocorticoid hormones decrease parental investment, these hormones might be the link to decreased reproductive success in areas of high environmental pollution. In this study, we aimed to identify which abiotic stressors are linked to avian reproductive output in urban areas and whether this link is mediated by individual hormone levels. We used fine-scaled estimates (2 m2 spatial resolution) of nighttime light, noise, and urban density to assess their impacts on the physiological condition of adult house sparrows (Passer domesticus). We measured circulating levels of lead and glucocorticoid concentrations in 40 breeding pairs of free-living house sparrows and related these physiological traits to reproductive success. Using structural equation modeling, we found that increased urban density levels linked directly to increased plasma corticosterone and lead concentrations that subsequently led to decreased fledgling mass. Sparrows with increased lead concentrations in plasma also had higher corticosterone levels. Although urban areas may be attractive due to decreased natural predators and available nesting sites, they may act as ecological traps that increase physiological damage and decrease fitness. To illustrate, avian development is strongly explained by parental corticosterone levels, which vary significantly in response to urban density and lead pollution. With fine-scale ecological mapping for a species with small home ranges, we demonstrated the presence and impacts of urban stressors in a small city with high human densities.

Infrared thermography is an effective, noninvasive measure of HPA activation.

Infrared thermography (IRT) is increasingly applied as a noninvasive technique for measuring surface body temperature changes related to physiological stress. As a basis for validation of IRT as a tool for diagnostic use, we need to assess its potential to measure hypothalamic-pituitary-adrenal (HPA) axis reactivity. We used experimental manipulations of the HPA axis in house sparrows (Passer domesticus), i.e. adrenal tissue responsiveness to exogenous adrenocorticotropin (ACTH) and the efficacy of negative feedback using the synthetic glucocorticoid dexamethasone (DEX), to test whether IRT is an effective tool for measuring HPA reactivity. Experimental birds showed a pronounced decrease in skin temperatures after ACTH injection and an increase in temperature after DEX injection. However, individual variation in glucocorticoid levels were not related to skin temperatures except after ACTH injection in experimental birds. We show that IRT can be used to measure HPA reactivity but that skin-temperature is not a good index for glucocorticoid secretion at baseline levels. These results suggest that while IRT of skin temperatures is a useful, noninvasive measure of HPA axis reactivity under acute activation, this technique might not be suitable for measuring natural variation of circulating glucocorticoid levels.

Are individuals consistent? Endocrine reaction norms under different ecological challenges.

Quantifying organismal capacity for compensatory mechanisms is essential to forecast responses to environmental change. Despite accumulating evidence for individual variation in physiological plasticity, the causes and consequences of this variation remain unclear. An outstanding question is whether individual reaction norms are consistent across different environmental challenges, i.e. whether an individual that is responsive to one environmental variable will be equally responsive to a different environmental variable. Additionally, are these reaction norms themselves consistent over time, i.e. repeatable? Here, we quantified individual baseline glucocorticoid responses in house sparrows, Passer domesticus, to sequential manipulations of temperature, wind speed and food unpredictability that were repeated in discrete blocks of sampling under both control and stressor-exposed conditions. Individuals significantly decreased their baseline corticosterone levels and increased their mass during treatment exposure. This response was consistent across environmental challenge types. There was high repeatability in the intercept and slope of the baseline corticosterone reaction norm between environmental challenges but broad credible intervals in the repeatability of the reaction norm slope, suggesting that although glucocorticoid levels during baseline conditions are repeatable, among-individual variation in the shape of the glucocorticoid response may be higher than within-individual variation. Within-subject variation in baseline corticosterone levels was mainly explained by within-individual variation in body mass during stressor exposure. Despite the high lability in physiological traits, endocrine plasticity is repeatable across environmental challenges and may be able to evolve as a result of genetic accommodation, in which selection acts on genetic variation of reaction norms.

Across time and space: Hormonal variation across temporal and spatial scales in relation to nesting success.

There is a renewed interest in investigating individual variation in hormone levels in relation to fitness metrics, as hormones act as mediators of life-history trade-offs. Hormone concentrations, however, are labile, responding to both internal and external stimuli, so the relationship between hormones and fitness can be non-consistent. One explanation of this inconsistent relationship is that a single hormone sample may not be representative of individual phenotypes in a free-living species. We addressed this issue by repeatedly sampling a free-living population of mountain white-crowned sparrows, Zonotrichia leucophrys oriantha, for baseline and stress-induced corticosterone (cort) and testosterone (T) across different stages of the breeding season. We measured (co)variation using three different methods, taking into account inter- and intra-individual variances, to determine whether hormone levels and the stress response are repeatable. We documented the temporal (over 3 months) and spatial (home-range) variation of individual hormone phenotypes and investigated how these components related to nesting success. At the population level, we found significant repeatability in male stress-induced cort concentrations but no repeatability in male or female baseline cort or male T concentrations. Using a new metric of intra-individual variance focusing on the stress response (profile repeatability), we found a wide range of variance scores, with most individuals showing high variation in their stress response. Similarly, we found a low level of repeatability of the reaction norm intercept and slope for the stress response across different life-history stages. Males with higher concentrations of stress-induced cort had more central home-ranges. Males with higher body condition had larger home-ranges; however, home-range size did not relate to male hormone concentrations or nesting success. We also did not find any significant relationship between variation in hormone levels and nesting success. We recommend that future studies combine both physiological and environmental components to better understand the relationship between hormones and fitness.

Genetic inheritance and environment determine endocrine plasticity to urban living.

As urban areas continue to expand globally, understanding how and why species respond to novel habitats becomes increasingly important. Knowledge of the mechanisms behind observed phenotypic changes in urban animals will enable us to better evaluate the impact of urbanization on current and future generations of wildlife. Physiological changes, such as those involved in the endocrine stress response, may allow individuals to inhabit and thrive in urbanized areas, but it is currently unknown how these changes arise in natural populations. In this study, we performed a four-way cross-foster experiment in free-living house wren chicks, Troglodytes aedon, to disentangle whether differences in baseline corticosterone between urban and rural individuals are a result of genetic and/or plastic mechanisms during development. We found that urban chicks already had higher corticosterone levels than their rural counterparts on the day they hatched, which suggests a possible genetic component to the corticosterone phenotype. However, rural offspring that were moved to an urban environment significantly increased their corticosterone levels, mimicking those of urban offspring. Our findings suggest that, although differences in baseline corticosterone concentrations between urban and rural individuals may have a genetic component, plasticity plays a pivotal role and can modify the corticosterone phenotype in response to the environment experienced in the first two weeks of life.

Hormonally mediated effects of artificial light at night on behavior and fitness: linking endocrine mechanisms with function.

Alternation between day and night is a predictable environmental fluctuation that organisms use to time their activities. Since the invention of artificial lighting, this predictability has been disrupted and continues to change in a unidirectional fashion with increasing urbanization. As hormones mediate individual responses to changing environments, endocrine systems might be one of the first systems affected, as well as being the first line of defense to ameliorate any negative health impacts. In this Review, we first highlight how light can influence endocrine function in vertebrates. We then focus on four endocrine axes that might be affected by artificial light at night (ALAN): pineal, reproductive, adrenal and thyroid. Throughout, we highlight key findings, rather than performing an exhaustive review, in order to emphasize knowledge gaps that are hindering progress on proposing impactful and concrete plans to ameliorate the negative effects of ALAN. We discuss these findings with respect to impacts on human and animal health, with a focus on the consequences of anthropogenic modification of the night-time environment for non-human organisms. Lastly, we stress the need for the integration of field and lab experiments as well as the need for long-term integrative eco-physiological studies in the rapidly expanding field of light pollution.

Restless roosts: Light pollution affects behavior, sleep, and physiology in a free-living songbird.

The natural nighttime environment is increasingly polluted by artificial light. Several studies have linked artificial light at night to negative impacts on human health. In free-living animals, light pollution is associated with changes in circadian, reproductive, and social behavior, but whether these animals also suffer from physiologic costs remains unknown. To fill this gap, we made use of a unique network of field sites which are either completely unlit (control), or are artificially illuminated with white, green, or red light. We monitored nighttime activity of adult great tits, Parus major, and related this activity to within-individual changes in physiologic indices. Because altered nighttime activity as a result of light pollution may affect health and well-being, we measured oxalic acid concentrations as a biomarker for sleep restriction, acute phase protein concentrations and malaria infection as indices of immune function, and telomere lengths as an overall measure of metabolic costs. Compared to other treatments, individuals roosting in the white light were much more active at night. In these individuals, oxalic acid decreased over the course of the study. We also found that individuals roosting in the white light treatment had a higher probability of malaria infection. Our results indicate that white light at night increases nighttime activity levels and sleep debt and affects disease dynamics in a free-living songbird. Our study offers the first evidence of detrimental effects of light pollution on the health of free-ranging wild animals.

Stressful city sounds: glucocorticoid responses to experimental traffic noise are environmentally dependent.

A major challenge in urban ecology is to identify the environmental factors responsible for phenotypic differences between urban and rural individuals. However, the intercorrelation between the factors that characterize urban environments, combined with a lack of experimental manipulations of these factors in both urban and rural areas, hinder efforts to identify which aspects of urban environments are responsible for phenotypic differences. Among the factors modified by urbanization, anthropogenic sound, particularly traffic noise, is especially detrimental to animals. The mechanisms by which anthropogenic sound affects animals are unclear, but one potential mechanism is through changes in glucocorticoid hormone levels. We exposed adult house wrens, Troglodytes aedon, to either traffic noise or pink noise (a non-traffic noise control). We found that urban wrens had higher initial (pre-restraint) corticosterone than rural wrens before treatment, and that traffic noise elevated initial corticosterone of rural, but not urban, wrens. By contrast, restraint stress-induced corticosterone was not affected by noise treatment. Our results indicate that traffic noise specifically contributes to determining the glucocorticoid phenotype, and suggest that glucocorticoids are a mechanism by which anthropogenic sound causes phenotypic differences between urban and rural animals.

Stressful colours: corticosterone concentrations in a free-living songbird vary with the spectral composition of experimental illumination.

Organisms have evolved under natural daily light/dark cycles for millions of years. These cycles have been disturbed as night-time darkness is increasingly replaced by artificial illumination. Investigating the physiological consequences of free-living organisms in artificially lit environments is crucial to determine whether nocturnal lighting disrupts circadian rhythms, changes behaviour, reduces fitness and ultimately affects population numbers. We make use of a unique, large-scale network of replicated field sites which were experimentally illuminated at night using lampposts emanating either red, green, white or no light to test effect on stress hormone concentrations (corticosterone) in a songbird, the great tit (Parus major). Adults nesting in white-light transects had higher corticosterone concentrations than in the other treatments. We also found a significant interaction between distance to the closest lamppost and treatment type: individuals in red light had higher corticosterone levels when they nested closer to the lamppost than individuals nesting farther away, a decline not observed in the green or dark treatment. Individuals with high corticosterone levels had fewer fledglings, irrespective of treatment. These results show that artificial light can induce changes in individual hormonal phenotype. As these effects vary considerably with light spectrum, it opens the possibility to mitigate these effects by selecting street lighting of specific spectra.

Glucocorticoids and land cover: a largescale comparative approach to assess a physiological biomarker for avian conservation.

As humans alter landscapes worldwide, land and wildlife managers need reliable tools to assess and monitor responses of wildlife populations. Glucocorticoid (GC) hormone levels are one common physiological metric used to quantify how populations are coping in the context of their environments. Understanding whether GC levels can reflect broad landscape characteristics, using data that are free and commonplace to diverse stakeholders, is an important step towards physiological biomarkers having practical application in management and conservation. We conducted a phylogenetic comparative analysis using publicly available datasets to test the efficacy of GCs as a biomarker for large spatial-scale avian population monitoring. We used hormone data from HormoneBase (51 species), natural history information and US national land cover data to determine if baseline or stress-induced corticosterone varies with the amount of usable land cover types within each species' home range. We found that stress-induced levels, but not baseline, positively correlated with per cent usable land cover both within and across species. Our results indicate that GC concentrations may be a useful biomarker for characterizing populations across a range of habitat availability, and we advocate for more physiological studies on non-traditional species in less studied populations to build on this framework. This article is part of the theme issue 'Endocrine responses to environmental variation: conceptual approaches and recent developments'.

Endocrine flexibility can facilitate or constrain the ability to cope with global change.

Global climate change has increased average environmental temperatures world-wide, simultaneously intensifying temperature variability and extremes. Growing numbers of studies have documented phenological, behavioural and morphological responses to climate change in wild populations. As systemic signals, hormones can contribute to orchestrating many of these phenotypic changes. Yet little is known about whether mechanisms like hormonal flexibility (reversible changes in hormone concentrations) facilitate or limit the ability of individuals, populations and species to cope with a changing climate. In this perspective, we discuss different mechanisms by which hormonal flexibility, primarily in glucocorticoids, could promote versus hinder evolutionary adaptation to changing temperature regimes. We focus on temperature because it is a key gradient influenced by climate change, it is easy to quantify, and its links to hormones are well established. We argue that reaction norm studies that connect individual responses to population-level and species-wide patterns will be critical for making progress in this field. We also develop a case study on urban heat islands, where several key questions regarding hormonal flexibility and adaptation to climate change can be addressed. Understanding the mechanisms that allow animals to cope when conditions become more challenging will help in predicting which populations are vulnerable to ongoing climate change. This article is part of the theme issue 'Endocrine responses to environmental variation: conceptual approaches and recent developments'.

What happens when the lights are left on? Transcriptomic and phenotypic habituation to light pollution.

Artificial light at night (ALAN) is a ubiquitous pollutant worldwide. Exposure can induce immediate behavioral and physiological changes in animals, sometimes leading to severe health consequences. Nevertheless, many organisms persist in light-polluted environments and may have mechanisms of habituating, reducing responses to repeated exposure over time, but this has yet to be tested experimentally. Here, we tested whether zebra finches (Taeniopygia guttata) can habituate to dim (0.3 lux) ALAN, measuring behavior, physiology (oxidative stress and telomere attrition), and gene expression in a repeated measures design, over 6 months. We present evidence of tolerance to chronic exposure, persistent behavioral responses lasting 8 weeks post-exposure, and attenuation of responses to re-exposure. Oxidative stress decreased under chronic ALAN. Changes in the blood transcriptome revealed unique responses to past exposure and re-exposure. Results demonstrate organismal resilience to chronic stressors and shed light on the capacity of birds to persist in an increasingly light-polluted world.

The great tit HapMap project: A continental-scale analysis of genomic variation in a songbird.

A major aim of evolutionary biology is to understand why patterns of genomic diversity vary within taxa and space. Large-scale genomic studies of widespread species are useful for studying how environment and demography shape patterns of genomic divergence. Here, we describe one of the most geographically comprehensive surveys of genomic variation in a wild vertebrate to date; the great tit (Parus major) HapMap project. We screened ca 500,000 SNP markers across 647 individuals from 29 populations, spanning ~30 degrees of latitude and 40 degrees of longitude - almost the entire geographical range of the European subspecies. Genome-wide variation was consistent with a recent colonisation across Europe from a South-East European refugium, with bottlenecks and reduced genetic diversity in island populations. Differentiation across the genome was highly heterogeneous, with clear 'islands of differentiation', even among populations with very low levels of genome-wide differentiation. Low local recombination rates were a strong predictor of high local genomic differentiation (FST), especially in island and peripheral mainland populations, suggesting that the interplay between genetic drift and recombination causes highly heterogeneous differentiation landscapes. We also detected genomic outlier regions that were confined to one or more peripheral great tit populations, probably as a result of recent directional selection at the species' range edges. Haplotype-based measures of selection were related to recombination rate, albeit less strongly, and highlighted population-specific sweeps that likely resulted from positive selection. Our study highlights how comprehensive screens of genomic variation in wild organisms can provide unique insights into spatio-temporal evolutionary dynamics.

Small increases in corticosterone before the breeding season increase parental investment but not fitness in a wild passerine bird.

Correlative evidence from field studies has suggested that baseline concentrations of corticosterone, the main avian glucocorticoid hormone, affect reproductive strategies in vertebrate species. Such a role is conceivable in light of corticosterone's function as a metabolic hormone in regulating glucose and fat metabolism. From such correlational studies, however, the question has remained open whether glucocorticoid concentrations change in advance of reproductive activities or whether corticosterone concentrations vary passively as a consequence of the individual's reproductive investment and workload. To test such causal relationships, we manipulated corticosterone concentrations prior to the breeding season in adult great tits (Parus major) and quantified reproductive investment and success. Two weeks before egg-laying, we administered subcutaneous silastic implants filled with corticosterone that elevated circulating levels within the baseline range for approximately 30 days to adult males and females. Corticosterone manipulation did not affect lay date or yearly offspring production. However, reproductive behaviors were affected by corticosterone treatment: males fed their mates more often during incubation, and females increased incubation of eggs and brooding of nestlings compared to control individuals. Other behaviors during the nestling stage, when the implants were no longer effective, did not differ between the two treatment groups. Our findings do not support the view that baseline corticosterone concentrations, at least at the time of year when we administered implants, change reproductive strategies per se. The current data suggest that baseline corticosterone levels represent internal signals that causally mediate reproductive effort in individuals of a wild bird species. By increasing reproductive investment, baseline corticosterone concentrations may have functions during the breeding season that diverge from the suppressive effects of stress-induced concentrations.

Dim artificial light at night alters immediate early gene expression throughout the avian brain.

Artificial light at night (ALAN) is a pervasive pollutant that alters physiology and behavior. However, the underlying mechanisms triggering these alterations are unknown, as previous work shows that dim levels of ALAN may have a masking effect, bypassing the central clock. Light stimulates neuronal activity in numerous brain regions which could in turn activate downstream effectors regulating physiological response. In the present study, taking advantage of immediate early gene (IEG) expression as a proxy for neuronal activity, we determined the brain regions activated in response to ALAN. We exposed zebra finches to dim ALAN (1.5 lux) and analyzed 24 regions throughout the brain. We found that the overall expression of two different IEGs, cFos and ZENK, in birds exposed to ALAN were significantly different from birds inactive at night. Additionally, we found that ALAN-exposed birds had significantly different IEG expression from birds inactive at night and active during the day in several brain areas associated with vision, movement, learning and memory, pain processing, and hormone regulation. These results give insight into the mechanistic pathways responding to ALAN that underlie downstream, well-documented behavioral and physiological changes.