Does migration constrain glucocorticoid phenotypes? Testing corticosterone levels during breeding in migratory versus resident birds.

Corticosterone, the main glucocorticoid in birds, is a major mediator of the incredible physiological feat of migration. Corticosterone plays important roles in migration, from preparation to in-flight energy mobilization to refueling, and corticosterone levels often show distinct elevations or depressions during certain stages of the migratory process. Here, we ask whether corticosterone's role in migration shapes its modulation during other life history stages, as is the case with some other phenotypically flexible traits involved in migration. Specifically, we use a global dataset of corticosterone measures to test whether birds' migratory status (migrant versus resident) predicts corticosterone levels during breeding. Our results indicate that migratory status predicts neither baseline nor stress-induced corticosterone levels in breeding birds; despite corticosterone's role in migration, we find no evidence that migratory corticosterone phenotypes carry over to breeding. We encourage future studies to continue to explore corticosterone in migrants versus residents across the annual cycle. Additionally, future efforts should aim to disentangle the possible effects of environmental conditions and migratory status on corticosterone phenotypes; potentially fruitful avenues include focusing on regions where migrants and residents overlap during breeding. Overall, insights from work in this area could demonstrate whether migration shapes traits during other important life stages, identify tradeoffs or limitations associated with the migratory lifestyle, and ultimately shed light on the evolution of flexible traits and migration.

Corticosterone exposure is associated with long-term changes in DNA methylation, physiology and breeding decisions in a wild bird.

When facing challenges, vertebrates activate a hormonal stress response that can dramatically alter behaviour and physiology. Although this response can be costly, conceptual models suggest that it can also recalibrate the stress response system, priming more effective responses to future challenges. Little is known about whether this process occurs in wild animals, particularly in adulthood, and if so, how information about prior experience with stressors is encoded. One potential mechanism is hormonally mediated changes in DNA methylation. We simulated the spikes in corticosterone that accompany a stress response using non-invasive dosing in tree swallows (Tachycineta bicolor) and monitored the phenotypic effects 1 year later. In a subset of individuals, we characterized DNA methylation using reduced representation bisulfite sequencing shortly after treatment and a year later. The year after treatment, experimental females had stronger negative feedback and initiated breeding earlier-traits that are associated with stress resilience and reproductive performance in our population-and higher baseline corticosterone. We also found that natural variation in corticosterone predicted patterns of DNA methylation. Finally, corticosterone treatment influenced methylation on short (1-2 weeks) and long (1 year) time scales; however, these changes did not have clear links to functional regulation of the stress response. Taken together, our results are consistent with corticosterone-induced priming of future stress resilience and support DNA methylation as a potential mechanism, but more work is needed to demonstrate functional consequences. Uncovering the mechanisms linking experience with the response to future challenges has implications for understanding the drivers of stress resilience.

Manipulation of a social signal affects DNA methylation of a stress-related gene in a free-living bird.

Social status directly affects the health of humans and other animals. Low status individuals receive more antagonistic encounters, have fewer supportive relationships and have worse health outcomes. However, the physiological and cellular processes that mediate the relationship between the social environment and health are incompletely known. Epigenetic regulation of the hypothalamic-pituitary-adrenal (HPA) axis, the neuroendocrine pathway that activates in response to stressors, may be one process that is sensitive to the social environment. Here, we experimentally manipulated plumage, a key social signal in female tree swallows (Tachycineta bicolor) and quantified methylation of four genes in the HPA axis before and after treatment. We found that dulling the white breast plumage affected methylation in one gene, CRHR1; however, the effect depended on the original brightness of the bird. Methylation in this gene was correlated with baseline corticosterone levels, suggesting that DNA methylation of CRHR1 helps regulate glucocorticoid production in this species. Methylation in two other genes, FKBP5 and GR, changed over the course of the experiment, independent of treatment. These results show that methylation of these genes is labile into adulthood and suggest that epigenetic regulation of the HPA axis could help birds respond to current environmental conditions.

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.

Among-population variation in telomere regulatory proteins and their potential role as hidden drivers of intraspecific variation in life history.

Biologists aim to explain patterns of growth, reproduction and ageing that characterize life histories, yet we are just beginning to understand the proximate mechanisms that generate this diversity. Existing research in this area has focused on telomeres but has generally overlooked the telomere's most direct mediator, the shelterin protein complex. Shelterin proteins physically interact with the telomere to shape its shortening and repair. They also regulate metabolism and immune function, suggesting a potential role in life history variation in the wild. However, research on shelterin proteins is uncommon outside of biomolecular work. Intraspecific analyses can play an important role in resolving these unknowns because they reveal subtle variation in life history within and among populations. Here, we assessed ecogeographic variation in shelterin protein abundance across eight populations of tree swallow (Tachycineta bicolor) with previously documented variation in environmental and life history traits. Using the blood gene expression of four shelterin proteins in 12-day-old nestlings, we tested the hypothesis that shelterin protein gene expression varies latitudinally and in relation to both telomere length and life history. Shelterin protein gene expression differed among populations and tracked non-linear variation in latitude: nestlings from mid-latitudes expressed nearly double the shelterin mRNA on average than those at more northern and southern sites. However, telomere length was not significantly related to latitude. We next assessed whether telomere length and shelterin protein gene expression correlate with 12-day-old body mass and wing length, two proxies of nestling growth linked to future fecundity and survival. We found that body mass and wing length correlated more strongly (and significantly) with shelterin protein gene expression than with telomere length. These results highlight telomere regulatory shelterin proteins as potential mediators of life history variation among populations. Together with existing research linking shelterin proteins and life history variation within populations, these ecogeographic patterns underscore the need for continued integration of ecology, evolution and telomere biology, which together will advance understanding of the drivers of life history variation in nature.

Neoadjuvant chemo- or chemo-radiation-therapy of pancreatic ductal adenocarcinoma differentially shift ECM composition, complement activation, energy metabolism and ribosomal proteins of the residual tumor mass.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer, often diagnosed at stages that dis-qualify for surgical resection. Neoadjuvant therapies offer potential tumor regression and improved resectability. Although features of the tumor biology (e.g., molecular markers) may guide adjuvant therapy, biological alterations after neoadjuvant therapy remain largely unexplored. We performed mass spectrometry to characterize the proteomes of 67 PDAC resection specimens of patients who received either neoadjuvant chemo (NCT) or chemo-radiation (NCRT) therapy. We employed data-independent acquisition (DIA), yielding a proteome coverage in excess of 3500 proteins. Moreover, we successfully integrated two publicly available proteome datasets of treatment-naïve PDAC to unravel proteome alterations in response to neoadjuvant therapy, highlighting the feasibility of this approach. We found highly distinguishable proteome profiles. Treatment-naïve PDAC was characterized by enrichment of immunoglobulins, complement and extracellular matrix (ECM) proteins. Post-NCT and post-NCRT PDAC presented high abundance of ribosomal and metabolic proteins as compared to treatment-naïve PDAC. Further analyses on patient survival and protein expression identified treatment-specific prognostic candidates. We present the first proteomic characterization of the residual PDAC mass after NCT and NCRT, and potential protein candidate markers associated with overall survival. We conclude that residual PDAC exhibits fundamentally different proteome profiles as compared to treatment-naïve PDAC, influenced by the type of neoadjuvant treatment. These findings may impact adjuvant or targeted therapy options.

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'.

Experimentally elevated corticosterone does not affect bacteria killing ability of breeding female tree swallows (Tachycineta bicolor).

The immune system can be modulated when organisms are exposed to acute or chronic stressors. Glucocorticoids (GCs), the primary hormonal mediators of the physiological stress response, are suspected to play a crucial role in immune modulation. However, most evidence of stress-associated immunomodulation does not separate the effects of glucocorticoid-dependent pathways from those of glucocorticoid-independent mechanisms on immune function. In this study, we experimentally elevated circulating corticosterone, the main avian glucocorticoid, in free-living female tree swallows (Tachycineta bicolor) for one to two weeks to test its effects on immune modulation. Natural variation in bacteria killing ability (BKA), a measure of innate constitutive immunity, was predicted by the interaction between timing of breeding and corticosterone levels. However, experimental elevation of corticosterone had no effect on BKA. Therefore, even when BKA is correlated with natural variation in glucocorticoid levels, this relationship may not be causal. Experiments are necessary to uncover the causal mechanisms of immunomodulation and the consequences of acute and chronic stress on disease vulnerability. Findings in other species indicate that acute increases in GCs can suppress BKA; but our results support the hypothesis that this effect does not persist over longer timescales, during chronic elevations in GCs. Direct comparisons of the effects of acute vs. chronic elevation of GCs on BKA will be important for testing this hypothesis.

Proteometabolomics of initial and recurrent glioblastoma highlights an increased immune cell signature with altered lipid metabolism.

BACKGROUND: There is an urgent need to better understand the mechanisms associated with the development, progression, and onset of recurrence after initial surgery in glioblastoma (GBM). The use of integrative phenotype-focused -omics technologies such as proteomics and lipidomics provides an unbiased approach to explore the molecular evolution of the tumor and its associated environment. METHODS: We assembled a cohort of patient-matched initial (iGBM) and recurrent (rGBM) specimens of resected GBM. Proteome and metabolome composition were determined by mass spectrometry-based techniques. We performed neutrophil-GBM cell coculture experiments to evaluate the behavior of rGBM-enriched proteins in the tumor microenvironment. ELISA-based quantitation of candidate proteins was performed to test the association of their plasma concentrations in iGBM with the onset of recurrence. RESULTS: Proteomic profiles reflect increased immune cell infiltration and extracellular matrix reorganization in rGBM. ASAH1, SYMN, and GPNMB were highly enriched proteins in rGBM. Lipidomics indicates the downregulation of ceramides in rGBM. Cell analyses suggest a role for ASAH1 in neutrophils and its localization in extracellular traps. Plasma concentrations of ASAH1 and SYNM show an association with time to recurrence. CONCLUSIONS: We describe the potential importance of ASAH1 in tumor progression and development of rGBM via metabolic rearrangement and showcase the feedback from the tumor microenvironment to plasma proteome profiles. We report the potential of ASAH1 and SYNM as plasma markers of rGBM progression. The published datasets can be considered as a resource for further functional and biomarker studies involving additional -omics technologies.

Social signal manipulation and environmental challenges have independent effects on physiology, internal microbiome, and reproductive performance in tree swallows (Tachycineta bicolor).

The social environment that individuals experience appears to be a particularly salient mediator of stress resilience, as the nature and valence of social interactions are often related to subsequent health, physiology, microbiota, and overall stress resilience. Relatively few studies have simultaneously manipulated the social environment and ecological challenges under natural conditions. Here, we report the results of experiments in wild tree swallows (Tachycineta bicolor) in which we manipulated both ecological challenges (predator encounters and flight efficiency reduction) and social interactions (by experimental dulling of a social signal). In two experiments conducted in separate years, we reversed the order of these treatments so that females experienced either an altered social signal followed by a challenge or vice-versa. Before, during, and after treatments were applied, we tracked breeding success, morphology and physiology (mass, corticosterone, and glucose), nest box visits via an RFID sensor network, cloacal microbiome diversity, and fledging success. Overall, we found that predator exposure during the nestling period reduced the likelihood of fledging and that signal manipulation sometimes altered nest box visitation patterns, but little evidence that the two categories of treatment interacted with each other. We discuss the implications of our results for understanding what types of challenges and what conditions are most likely to result in interactions between the social environment and ecological challenges.

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.

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.

The relative speed of the glucocorticoid stress response varies independently of scope and is predicted by environmental variability and longevity across birds.

The acute glucocorticoid response is a key mediator of the coordinated vertebrate response to unpredictable challenges. Rapid glucocorticoid increases initiate changes that allow animals to cope with stressors. The scope of the glucocorticoid response - defined here as the absolute increase in glucocorticoids - is associated with individual differences in performance and varies across species with environment and life history. In addition to varying in scope, responses can differ enormously in speed; however, relatively little is known about whether speed and absolute glucocorticoid levels covary, how selection shapes speed, or what aspects of speed are important. We used corticosterone samples collected at 5 time points from 1750 individuals of 60 species of birds to ask i) how the speed and scope of the glucocorticoid response covary and ii) whether variation in absolute or relative speed is predicted by environmental context or life history. Among species, faster absolute glucocorticoid responses were strongly associated with a larger scope. Despite this covariation, the relative speed of the glucocorticoid response (standardized within species) varied independently of absolute scope, suggesting that selection could operate on both features independently. Species with faster relative glucocorticoid responses lived in locations with more variable temperature and had shorter lifespans. Our results suggest that rapid changes associated with the speed of the glucocorticoid response, such as those occurring through non-genomic receptors, might be an important determinant of coping ability and we emphasize the need for studies designed to measure speed independently of absolute glucocorticoid levels.

Gut Microbiome as a Mediator of Stress Resilience: A Reactive Scope Model Framework.

Stress resilience is defined as the ability to rebound to a homeostatic state after exposure to a perturbation. Organisms modulate various physiological mediators to respond to unpredictable changes in their environment. The gut microbiome is a key example of a physiological mediator that coordinates a myriad of host functions including counteracting stressors. Here, we highlight the gut microbiome as a mediator of host stress resilience in the framework of the reactive scope model. The reactive scope model integrates physiological mediators with unpredictable environmental changes to predict how animals respond to stressors. We provide examples of how the gut microbiome responds to stressors within the four ranges of the reactive scope model (i.e., predictive homeostasis, reactive homeostasis, homeostatic overload, and homeostatic failure). We identify measurable metrics of the gut microbiome that could be used to infer the degree to which the host is experiencing chronic stress, including microbial diversity, flexibility, and gene richness. The goal of this perspective piece is to highlight the underutilized potential of measuring the gut microbiome as a mediator of stress resilience in wild animal hosts.

Timing of Breeding Reveals a Trade-Off between Immune Investment and Life History in Tree Swallows.

The allocation of limited resources among life history traits creates trade-offs that constrain the range of possible phenotypes of organisms. In animals, the cost of maintaining an effective immune response may reduce the ability to invest in reproduction, resulting in altered susceptibility to disease. However, not all members of a population face identical constraints because differences in an individual's environmental context or physiological state can influence the degree to which traits are negatively associated. Here, we evaluated how variation in timing of breeding, a correlate of fitness, may result in different patterns of trait associations between immunity and reproduction. We measured constitutive immunity in breeding female tree swallows (Tachycineta bicolor) using a bacteria killing assay with blood plasma to assess the relationships between bacteria killing ability (BKA), reproductive effort, and reproductive success. We found that timing of breeding can influence the association between BKA and reproductive effort, but its effects are not homogeneous among all traits. Late-breeding tree swallows with stronger BKA laid smaller clutches, a pattern that was not apparent in early breeders. Regardless of the timing of breeding, birds with stronger BKA fed their nestlings less. Despite a negative association with reproductive effort, we found no association between immunity and reproductive success. We provide evidence that individual tree swallows do not experience some trade-offs equally, and that timing of breeding likely plays a role in how costs of immunity are weighed. To understand how investment in immunity can limit life history traits, we must consider how a variation among individuals influences the relative costs of immunity.

Light at night disrupts trophic interactions and population growth of lady beetles and pea aphids.

Natural variation in light has historically correlated with seasonality, providing an honest cue to organisms with seasonal life history cycles. However, with the onset of widespread light at night (LAN), the reliability of light as a cue has decreased in polluted areas, making its timing or intensity potentially clash with temperature trends. These clashing cues may influence biological systems on multiple levels. Yet, a few studies have connected behavioral underpinnings and larger community-level processes, resulting in a knowledge gap bridging individual-, population-, and community-level responses to mismatched cues. We experimentally investigated impacts of cool temperature and LAN on a lady beetle-aphid-fava system to test how light and temperature influenced aphid population growth and their underlying behavioral drivers. We used Coccinella septempunctata and Coleomegilla maculata beetles to understand the interaction of the environment and predation on pea aphid (Acyrthosiphon pisum) population growth. Aphids and their predators reacted differently to variation in light and temperature, influencing the strength of aphid-driven and predator-driven dynamics in the different conditions. We observed evidence of aphid-driven dynamics in the cool, light conditions where aphids excel and exhibited strong anti-predator behavior. In contrast, we found stronger predator-driven dynamics in warm conditions where lady beetle predatory success was higher. Overall, we found that LAN has context-dependent effects on insect communities due to the varied responses each player has to its environment.

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.

Methods-Motivational Interviewing Approach for Enhanced Retention and Attendance.

INTRODUCTION: Suboptimal and differential participant engagement in randomized trials-including retention at primary outcome assessments and attendance at intervention sessions-undermines rigor, internal validity, and trial conclusions. METHODS: First, this study describes Methods-Motivational Interviewing approach and strategies for implementation. This approach engages potential participants before randomization through interactive, prerequisite orientation sessions that illustrate the scientific rationale behind trial methods in accessible language and use motivational interviewing to diffuse ambivalence about participation. Then, this study examines the potential improvements in retention (proportion of participants assessed at follow-up visits) and attendance (e.g., mean percentage of intervention sessions attended, percentage of participants who attended 0 sessions) in 3 randomized weight-management trials that quickly added prerequisite orientations to their protocols following early signs of suboptimal or differential participant engagement (Supporting Health by Integrating Nutrition and Exercise [2009-2013, n=194]; Get Social [2016-2020, n=217]; GestationaL Weight Gain and Optimal Wellness [2014-2018, n=389]). Using a pre-post analytical design, adjusted estimates from regression models controlling for condition and assessment timepoint (analyses from 2020) are reported. RESULTS: After adding prerequisite orientations, all 3 trials attained higher participant engagement. Retention at assessments was 11.4% and 17.3% higher (Get Social and Supporting Health by Integrating Nutrition and Exercise, respectively). Mean percentage of attendance at intervention sessions was 8.8% higher (GestationaL Weight Gain and Optimal Wellness), and 10.1% fewer participants attended 0 intervention sessions (Get Social). Descriptively, all the remaining retention and attendance outcomes were consistently higher but were nonsignificant. Across the trials, adding prerequisite orientations did not impact the proportion of eligible participants enrolled or the baseline demographics. CONCLUSIONS: The Methods-Motivational Interviewing approach shows promise for increasing the rigor of randomized trials and is readily adaptable to in-person, webinar, and conference call formats. TRIAL REGISTRATION: All 3 trials are registered at www.clinicaltrials.gov (Supporting Health by Integrating Nutrition and Exercise: NCT00960414; Get Social: NCT02646618; and GestationaL Weight Gain and Optimal Wellness: NCT02130232).