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Synopsis Comparative analyses in biology rely on the quality of available data. Methodological differences among studies may introduce variation in results that obscure patterns. In the field of eco-immunology, functional immune assays such as antimicrobial capacity assays are widely used for among-species applications. Sample storage time and animal handling time can influence assay results in some species, but how sample holding time prior to freezing influences assay results is unknown. Sample holding time can vary widely in field studies on wild animals, prompting the need to understand the implications of such variation on assay results. We investigated the hypothesis that sample holding time prior to freezing influences assay results in six species (Leiocephalus carinatus, Iguana iguana, Loxodonta africana, Ceratotherium simum, Columba livia, and Buteo swainsoni) by comparing antibacterial capacity of serum with varying processing times prior to snap-freezing. Blood was collected once from each individual and aliquots were placed on ice and assigned different holding times (0, 30, 60, 180, and 240 min), after which each sample was centrifuged, then serum was separated and snap-frozen on dry ice and stored at −80ºC for 60 days prior to assaying. For each aliquot, we conducted antibacterial capacity assays with serial dilutions of serum inoculated with E. coli and extracted the dilution at 50% antibacterial capacity for analysis. We found a decrease in antibacterial capacity with increased holding time in one of the six species tested (B. swainsoni), driven in part by complete loss of antibacterial capacity in some individuals at the 240-min time point. While the majority of species’ antibacterial capacity were not affected, our results demonstrate the need to conduct pilot assays spanning the anticipated variation in sample holding times to develop appropriate field protocols.

It is important to evaluate the role of captivity as a potential stressor. An understanding of stress responses to capture and transition to captivity may inform the limitations of laboratory studies on wild animals, aid in understanding the consequences of introducing animals into captive environments, and help predict which species may be successful invasives. We investigated physiological effects of captivity by comparing at-capture blood variables in wild Burmese pythons (Python bivittatus) in Florida to pythons recently brought into captivity (1-109 days). We conducted an acute restraint test by collecting samples at baseline (immediately at handling) and one hour post-restraint across wild field-sampled (n = 19) and recently-captive (n = 33) pythons to evaluate fluctuations in plasma corticosterone, bacterial killing ability, antibody response, leukogram, and serpentovirus infection. We observed higher baseline plasma corticosterone and monocytes in recently captive compared to wild snakes, which both subsided in snakes held for a longer time in captivity, and a mild decrease in lymphocytes in the middle of the captivity period. Functional immunity and viral infection were not affected by captivity, and pythons maintained restraint-induced responses in corticosterone, heterophil to lymphocyte ratio, and monocyte counts throughout captivity. Prevalence for serpentovirus was 50%, though infection status was related to sampling date rather than captivity, indicating that viral infection may be seasonal. The history of Burmese python as a common captive animal for research and pet trade, as well as its general resilience to effects of capture and short-term captivity, may contribute to its invasion success in Florida.

Glucocorticoid (GC) hormones are important phenotypic mediators across vertebrates, but their circulating concentrations can vary markedly. Here we investigate macroevolutionary patterning in GC levels across tetrapods by testing seven specific hypotheses about GC variation and evaluating whether the supported hypotheses reveal consistent patterns in GC evolution. If selection generally favors the "supportive" role of GCs in responding effectively to challenges, then baseline and/or stress-induced GCs may be higher in challenging contexts. Alternatively, if selection generally favors "protection" from GC-induced costs, GCs may be lower in environments where challenges are more common or severe. The predictors of baseline GCs were all consistent with supportive effects: levels were higher in smaller organisms and in those inhabiting more energetically demanding environments. During breeding, baseline GCs were also higher in populations and species with fewer lifetime opportunities to reproduce. The predictors of stress-induced GCs were instead more consistent with the protection hypothesis: during breeding, levels were lower in organisms with fewer lifetime reproductive opportunities. Overall, these patterns indicate a surprising degree of consistency in how some selective pressures shape GCs across broad taxonomic scales; at the same time, in challenging environments selection appears to operate on baseline and stress-induced GCs in distinct ways.

Body size influences many traits including those that affect host competence, the propensity to cause new infections. Here, we employ a new framework to reveal that, for at least two infections, West Nile virus and Lyme disease, large hosts should be more competent than small ones, but their lower abundance could mitigate their impacts on local risk. By contrast, for rabies, small hosts will be disproportionately more competent than large ones, an effect amplified by the higher densities of small species. These outcomes differ quite a bit from previous approaches that incorporate allometries into epidemiological models. Subsequently, we advocate for future integrative work to resolve how interspecific variation in body size influences the emergence and spread of infections.

Endocrine systems act as key intermediaries between organisms and their environments. This interaction leads to high variability in hormone levels, but we know little about the ecological factors that influence this variation within and across major vertebrate groups. We study this topic by assessing how various social and environmental dynamics influence testosterone levels across the entire vertebrate tree of life. Our analyses show that breeding season length and mating system are the strongest predictors of average testosterone concentrations, whereas breeding season length, environmental temperature, and variability in precipitation are the strongest predictors of within-population variation in testosterone. Principles from small-scale comparative studies that stress the importance of mating opportunity and competition on the evolution of species differences in testosterone levels, therefore, likely apply to the entire vertebrate lineage. Meanwhile, climatic factors associated with rainfall and ambient temperature appear to influence variability in plasma testosterone, within a given species. These results, therefore, reveal how unique suites of ecological factors differentially explain scales of variation in circulating testosterone across mammals, birds, reptiles, amphibians, and fishes.

Terrestrial mammals span 7 orders of magnitude in body size, ranging from the < 2 g pygmy shrew (Suncus etruscus) to the > 3900 kg African elephant (Loxodonta africana). Although body size has profound effects on the behavior, physiology, ecology, and evolution of animals, how investment in immune defenses changes with body size is unknown. Here, we develop a novel 12-point dilution-curve approach to describe and compare antibacterial capacity against 3 bacterial species among >160 terrestrial species of mammals. We show that antibacterial activity in serum across mammals exhibits isometry, but the serum of large mammals is less hospitable to bacteria than would be predicted by their metabolic rates. Specifically, hypometric metabolic rates would predict that a large species should have disproportionately lower antibacterial capacity than small species, but body size is unrelated to killing capacity across species. Scaling of antibacterial immune defenses provides novel perspectives on the ecology of host-pathogen interactions, and on their co-evolutionary dynamics. These results have direct implications for effectively modeling the evolution of immune defenses and identifying potential reservoir hosts of zoonotic pathogens.

At macroevolutionary scales, stress physiology may have consequences for species diversification and subspecies richness. Populations that exploit new resources or undergo range expansion should cope with new environmental challenges, which could favor higher mean stress responses. Within-species variation in the stress response may also play a role in mediating the speciation process: in species with broad variation, there will always be some individuals that can tolerate an unpredictable environment, whereas in species with narrow variation there will be fewer individuals that are able to thrive in a new ecological niche. We tested for the evolutionary relationship between stress response, speciation rate, and subspecies richness in birds by relying on the HormoneBase repository, from which we calculated within- and among-species variation in baseline (BL) and stress-induced (SI) corticosterone levels. To estimate speciation rates, we applied Bayesian analysis of macroevolutionary mixtures that can account for variation in diversification rate among clades and through time. Contrary to our predictions, lineages with higher diversification rates were not characterized by higher BL or SI levels of corticosterone either at the tips or at the deeper nodes of the phylogeny. We also found no association between mean hormone levels and subspecies richness. Within-species variance in corticosterone levels showed close to zero repeatability, thus it is highly unlikely that this is a species-specific trait that influences diversification rates. These results imply that stress physiology may play a minor, if any, role in determining speciation rates in birds.

It has been well-established that there is variation in the strength and direction of the relationship between circulating glucocorticoids (GCs) and fitness. When studies demonstrate such variation or the direction of the GC-fitness relationship is unexpected, the results are often attributed to context-dependency. However, descriptors of context can be vague (e.g., "environmental context") and few studies explicitly test how the optimal hypothalamic-pituitary-adrenal (HPA) axis response to stressors varies across specific contexts. Although existing hypotheses create a strong foundation for understanding GC-fitness relationships, many do not provide explicit predictions of how, when, and why the relationships will change. Here, we discuss three broad factors which we expect to shape the relationships between HPA axis activity and fitness metrics: (1) whether the HPA axis-mediated response matches the challenge, (2) the intensity and frequency of challenges, and (3) life history. We also make predictions for how these factors might affect GC-fitness relationships and discuss ways to test these predictions. Observational studies, experimental manipulations of context, and large-scale cross-species comparisons will be critical to understanding the observed variation in GC-fitness relationships.

Glucocorticoid hormones allow individuals to rapidly adjust their physiology and behavior to meet the challenges of a variable environment. An individual's baseline concentration of glucocorticoids can reflect shifts in life history stage and resource demands while mediating a suite of physiological and behavioral changes that include immune modulation and resource allocation. Thus, glucocorticoids could facilitate a response to parasites that is optimized for an individual's specific challenges and life history stage. We investigated the relationship between endogenous circulating glucocorticoids and measures of resistance and tolerance to Haemosporidian parasites (including those that cause avian malaria) in red-winged blackbirds (Agelaius phoeniceus). We found that higher endogenous concentrations of circulating glucocorticoids were associated with reduced costs of parasite infection, which is indicative of higher tolerance, but were unrelated to parasite burden in free ranging, breeding male birds. Post-breeding, both males and females with higher glucocorticoid concentrations had higher measures of tolerance to Haemosporidian infection. Our findings suggest a potentially adaptive role for glucocorticoids in shifting the response to parasites to align with an individual's current physiological state and the challenges they face.

Glucocorticoid hormones facilitate responses to environmental challenges by mediating diverse physiological and behavioral changes, including resource mobilization and altered reproductive effort. Elevated glucocorticoids might indicate that an individual is facing high levels of environmental challenges and thus, elevated concentrations might be associated with reduced fitness (CORT-fitness hypothesis). Alternatively, the energetic demands of reproduction might be a challenge that requires elevated glucocorticoids to mobilize resources to support reproductive effort, ultimately increasing reproductive investment and fitness (CORT-adaptation hypothesis). Investigations of glucocorticoid-fitness relationships have yielded mixed results. Variation in the direction of this relationship could be caused in part by differences in the contexts in which the relationship was assessed. Incorporating context, such as life history stage, could be key to understanding the role of glucocorticoids in influencing fitness outcomes. We investigated the relationship between corticosterone and reproductive effort and success within a single life history stage: incubation of eggs. In an observational study, we measured baseline corticosterone in incubating female red-winged blackbirds (Agelaius phoeniceus), monitored incubation behavior, and determined hatching success for each nest. Incubating birds with higher baseline corticosterone concentrations had more frequent, shorter incubation bouts and spent less time overall incubating their clutches of eggs than birds with lower corticosterone concentrations. Elevated corticosterone was also associated with lower clutch mass, but neither corticosterone nor incubation effort were correlated with hatching success. Although experimental tests are needed to establish causation, these results suggest that during the incubation period, corticosterone might shift resource investment towards self-maintenance, and away from current reproductive effort.

Introducing Hardy-Weinberg equilibrium into the high school or college classroom can be difficult because many students struggle with the mathematical formalism of the Hardy-Weinberg equations. Despite the potential difficulties, incorporating Hardy-Weinberg into the curriculum can provide students with the opportunity to investigate a scientific theory using data and integrate across the disciplines of biology and mathematics. We present a geometric way to interpret and visualize Hardy-Weinberg equilibrium, allowing students to focus on the core ideas without algebraic baggage. We also introduce interactive applets that draw on the distributive property of mathematics to allow students to experiment in real time. With the applets, students can observe the effects of changing allele frequencies on genotype frequencies in a population at Hardy-Weinberg equilibrium. Anecdotally, we found use of the geometric interpretation led to deeper student understanding of the concepts and improved the students' ability to solve Hardy-Weinberg-related problems. Students can use the ideas and tools provided here to draw connections between the biology and mathematics, as well as between algebra and geometry.

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.

Lay Summary Human-induced environmental change can disturb animals, reducing individual and population health. We found no general relationship between urbanization and hormones associated with stressors across birds and reptiles, suggesting hormonal responses to disturbance vary across species and even individuals. Information beyond hormone levels is needed to inform conservation efforts., Rates of human-induced environmental change continue increasing with human population size, potentially altering animal physiology and negatively affecting wildlife. Researchers often use glucocorticoid concentrations (hormones that can be associated with stressors) to gauge the impact of anthropogenic factors (e.g. urbanization, noise and light pollution). Yet, no general relationships between human-induced environmental change and glucocorticoids have emerged. Given the number of recent studies reporting baseline and stress-induced corticosterone (the primary glucocorticoid in birds and reptiles) concentrations worldwide, it is now possible to conduct large-scale comparative analyses to test for general associations between disturbance and baseline and stress-induced corticosterone across species. Additionally, we can control for factors that may influence context, such as life history stage, environmental conditions and urban adaptability of a species. Here, we take a phylogenetically informed approach and use data from HormoneBase to test if baseline and stress-induced corticosterone are valid indicators of exposure to human footprint index, human population density, anthropogenic noise and artificial light at night in birds and reptiles. Our results show a negative relationship between anthropogenic noise and baseline corticosterone for birds characterized as urban avoiders. While our results potentially indicate that urban avoiders are more sensitive to noise than other species, overall our study suggests that the relationship between human-induced environmental change and corticosterone varies across species and contexts; we found no general relationship between human impacts and baseline and stress-induced corticosterone in birds, nor baseline corticosterone in reptiles. Therefore, it should not be assumed that high or low levels of exposure to human-induced environmental change are associated with high or low corticosterone levels, respectively, or that closely related species, or even individuals, will respond similarly. Moving forward, measuring alternative physiological traits alongside reproductive success, health and survival may provide context to better understand the potential negative effects of human-induced environmental change.

Glucocorticoid hormones (GCs) are central mediators of metabolism and the response to challenges. Because circulating levels of GCs increase in response to challenges, within-population variation in GCs could reflect individual variation in condition or experience. At the same time, individual variation in the degree to which GCs increase in response to challenges (which is relatively consistent within individuals over time) could have causal effects on stress coping capacity, and the ability to survive and reproduce. Although a number of studies in vertebrates have tested whether within-population variation in GCs 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 within-population variation in GCs is associated with components of fitness across vertebrates. At the same time, we introduce and test predictions about an overlooked but 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 across vertebrates, 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 negatively associated with survival in organisms that engage in relatively more valuable reproductive attempts. Fecal GCs did not predict survival or reproductive success. We also used a meta-analytic approach to test whether experimental increases in GCs had consistent causal effects on fitness. Experimental increases in GCs reduced both survival and reproductive success, although the latter relationship was not significant when accounting for phylogeny. 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. Future studies that explore the role of other aspects of context (e.g., the nature and frequency of stressors, environmental variation) within and across species could provide important insights how and when variation in GC regulation predicts fitness.

Individual variation in parasite defences, such as resistance and tolerance, can underlie heterogeneity in fitness and could influence disease transmission dynamics. Glucocorticoid hormone concentrations often change in response to fluctuating environmental conditions and mediate changes in immune function, resource allocation and tissue repair. Thus, changes in glucocorticoid hormone concentrations might mediate individual variation in investment in resistance versus tolerance. In this study, we experimentally increased glucocorticoid concentrations in red-winged blackbirds (Agelaius phoeniceus) that were naturally infected with haemosporidian parasites, and assessed changes in resistance and tolerance of infection. Glucocorticoid treatment increased burdens ofPlasmodium, the parasite causing avian malaria, but only in the absence of co-infection with another Haemosporidian,Haemoproteus. Thus, glucocorticoids might reduce resistance to infection, but co-infection can mitigate the negative consequences of increased hormone concentrations. Glucocorticoid treatment also decreased tolerance of infection. We found no evidence that the inflammatory immune response or rate of red blood cell production underlie the effects of glucocorticoids on resistance and tolerance. Our findings suggest that exogenous glucocorticoids can increase the costs of haemosporidian infections by both increasing parasite numbers and reducing an individual's ability to cope with infection. These effects could scale up to impact populations of both host and parasite.

Glucocorticoids can play a critical role in modulating life-history trade-offs. However, studying the effects of glucocorticoids on life-history often requires experimentally elevating plasma glucocorticoid concentrations for several weeks within normal physiological limits and without repeated handling of the animal. Recently, implants made of beeswax and testosterone (T) were shown to have release dynamics superior to some currently available T implants, and these beeswax implants dissolved, eliminating the need to recapture the animal. We evaluated the utility of beeswax implants containing four different dosages of corticosterone (CORT; the primary glucocorticoid in birds) and their effect on several condition indices in a captive colony of zebra finches ( Taeniopygia guttata ). The three implants with the greatest CORT doses (0.05, 0.1, and 0.5 mg) produced spikes in plasma CORT concentrations 20 h after treatment, but were within the limits that zebra finches may normally experience. The 0.5 mg CORT implant elevated plasma CORT between typical baseline and restraint stress levels reported in other studies of zebra finches for the entire 35 day experiment. Birds in the 0.5 mg implant group were heavier, had greater furcular fat scores, and had lower hematocrit than birds in the control and other CORT implant groups. Beeswax CORT implants are a low cost method of elevating plasma CORT for a prolonged time. Furthermore, because there is no need to remove these implants at the end of a study, this method may be amenable to studies of free-ranging animals.

Glucocorticoids (GCs) are stress hormones that can strongly influence physiology, behavior, and an organism's ability to cope with environmental change. Despite their importance, and the wealth of studies that have sought to understand how and why GC concentrations vary within species, we do not have a clear understanding of how circulating GC levels vary within and across the major vertebrate clades. New research has proposed that much interspecific variation in GC concentrations can be explained by variation in metabolism and body mass. Specifically, GC concentrations should vary proportionally with mass-specific metabolic rates and, given known scaling relationships between body mass and metabolic rate, GC concentrations should scale to the -1/4 power of body mass and to the power of 1 with mass-specific metabolic rate. Here, we use HormoneBase, the newly compiled database that includes plasma GC concentrations from free-living and unmanipulated vertebrates, to evaluate this hypothesis. Specifically, we explored the relationships between body mass or mass-specific metabolic rate and either baseline or stress-induced GC (cortisol or corticosterone) concentrations in tetrapods. Our phylogenetically-informed models suggest that, whereas the relationship between GC concentrations and body mass across tetrapods and among mammals is close to -1/4 power, this relationship does not exist in amphibians, reptiles, and birds. Moreover, with the exception of a positive association between stress-induced GC concentrations and mass-specific metabolic rate in birds, we found little evidence that GC concentrations are linked to metabolic rate, although the number of species sampled was quite limited for amphibians and somewhat so for reptiles and mammals. Nevertheless, these results stand in contrast to the generally accepted association between the two and suggest that our observed positive association between body mass and GC concentrations may not be due to the well-established link between mass and metabolism. Large-scale comparative approaches can come with drawbacks, such as pooling and pairing observations from separate sources. However, these broad analyses provide an important counterbalance to the majority of studies examining variation in GC concentrations at the population or species level, and can be a powerful approach to testing both long-standing and new questions in biology.

Ecoimmunology is the study of the causes and consequences of variation in immunity. This integrative field builds on and complements comparative immunology by evaluating immunity across levels of biological organization using an evolutionary and ecological framework. One major accomplishment of ecoimmunology has been to demonstrate that immune defenses have costs, and these costs can be context-dependent. As a result, researchers assess immunity not only in terms of specific immune pathways but also as functional, emergent traits, such as resistance against and tolerance of parasites. Ecoimmunology also highlights the importance of integration across physiological systems, including the immune, endocrine, and nervous systems, in the production of immune defenses. The field has demonstrated the importance of genetics, developmental environment, and current conditions to individual variation in immune function and emphasized the traits of species, such as body size, that could be integral to immune phenotypes. Furthermore, ecoimmunological studies have shown that heterogeneity in immunity at the individual and species levels can have important consequences for disease dynamics within populations and communities as well as population cycling. Herein we discuss these key research areas in ecoimmunology as they relate to three central themes: (1) immunity in the context of the whole organism, (2) heterogeneity in immunity, and (3) the broad consequences of individual variation in immunity. We conclude by reviewing future directions for ecoimmunology, focusing on those that could have important implications for public health, conservation, and wildlife management.

Hormones are central regulators of organismal function and flexibility that mediate a diversity of phenotypic traits from early development through senescence. Yet despite these important roles, basic questions about how and why hormone systems vary within and across species remain unanswered. Here we describe HormoneBase, a database of circulating steroid hormone levels and their variation across vertebrates. This database aims to provide all available data on the mean, variation, and range of plasma glucocorticoids (both baseline and stress-induced) and androgens in free-living and un-manipulated adult vertebrates. HormoneBase ( www.HormoneBase.org ) currently includes >6,580 entries from 476 species, reported in 648 publications from 1967 to 2015, and unpublished datasets. Entries are associated with data on the species and population, sex, year and month of study, geographic coordinates, life history stage, method and latency of hormone sampling, and analysis technique. This novel resource could be used for analyses of the function and evolution of hormone systems, and the relationships between hormonal variation and a variety of processes including phenotypic variation, fitness, and species distributions. Machine-accessible metadata file describing the reported data (ISA-Tab format)

Animals go through different life history stages such as reproduction, moult, or migration, of which some are more energy-demanding than others. Baseline concentrations of glucocorticoid hormones increase during moderate, predictable challenges and thus are expected to be higher when seasonal energy demands increase, such as during reproduction. By contrast, stress-induced glucocorticoids prioritize a survival mode that includes reproductive inhibition. Thus, many species down-regulate stress-induced glucocorticoid concentrations during the breeding season. Interspecific variation in glucocorticoid levels during reproduction has been successfully mapped onto reproductive investment, with species investing strongly in current reproduction (fast pace of life) showing higher baseline and lower stress-induced glucocorticoid concentrations than species that prioritize future reproduction over current attempts (slow pace of life). Here we test the "glucocorticoid seasonal plasticity hypothesis", in which we propose that interspecific variation in seasonal changes in glucocorticoid concentrations from the non-breeding to the breeding season will be related to the degree of reproductive investment (and thus pace of life). We extracted population means for baseline (for 54 species) and stress-induced glucocorticoids (for 32 species) for the breeding and the non-breeding seasons from the database "HormoneBase", also calculating seasonal glucocorticoid changes. We focused on birds because this group offered the largest sample size. Using phylogenetic comparative methods, we first showed that species differed consistently in both average glucocorticoid concentrations and their changes between the two seasons, while controlling for sex, latitude, and hemisphere. Second, as predicted seasonal changes in baseline glucocorticoids were explained by clutch size (our proxy for reproductive investment), with species laying larger clutches showing a greater increase during the breeding season-especially in passerine species. In contrast, changes in seasonal stress-induced levels were not explained by clutch size, but sample sizes were more limited. Our findings highlight that seasonal changes in baseline glucocorticoids are associated with a species' reproductive investment, representing an overlooked physiological trait that may underlie the pace of life.

Glucocorticoids are highly conserved hormones that mediate a suite of responses to changing conditions in vertebrates. Recent work has focused on understanding how selection operates on glucocorticoid secretion in natural populations. Because heritability is rarely estimated and difficult to measure in the wild, many studies report within-individual repeatability as an estimate of stable between individual differences in glucocorticoid secretion. We conducted a systematic review and meta-analysis on estimates of within-individual glucocorticoid repeatability to elucidate general patterns of repeatability, and to test for relationships between covariates and estimates of repeatability. To this end, we collected 203 estimates of within-individual glucocorticoid repeatability drawn from 71 separate studies and 55 species. Overall, we found moderate levels of repeatability (0.29). We also found that repeatability varied by sample type. Long-term measures (e.g., fecal and feather samples) and acute stress-induced plasma glucocorticoids had higher repeatability (long-term: 0.44, stress-induced: 0.38), than baseline glucocorticoid levels (0.18). Repeatability also decreased with increasing time between repeated sampling events. Despite significant overall repeatability, there was substantial heterogeneity in estimates from different studies, suggesting that repeatability of glucocorticoid secretion varies substantially across systems and conditions. We discuss the implications of our results for understanding selection on glucocorticoid traits and suggest that continuing work should focus on evaluating the repeatability of within-individual glucocorticoid reaction norms.

Chronic, low-intensity parasite infections can reduce host fitness through negative impacts on reproduction and survival, even if they produce few overt symptoms. As a result, these parasites can influence the evolution of host morphology, behaviour and physiology. The physiological consequences of chronic infection can provide insight into the processes underlying parasite-driven natural selection. Here, we evaluate the physiological consequences of natural, low-intensity infection in an avian host-parasite system: adult male red-winged blackbirds (Agelaius phoeniceus) infected with haemosporidian parasites. Chronic haemosporidian infection has previously been shown to reduce both reproductive success and survival in several avian species. We used antimalarial medications to experimentally reduce haemosporidian parasitaemia (the proportion of blood cells infected with haemosporidian parasites) and measured the effect of treatment on body condition, haematology, immune function, physiological stress and oxidative state. Treatment with an antimalarial medication reduced parasitaemia for the most prevalent haemosporidian parasites from the genus Plasmodium. Treatment also increased haemoglobin and haematocrit, and decreased red blood cell production rates. We detected no effect of treatment on body condition, immune metrics, plasma corticosterone concentrations, total antioxidant capacity or reactive oxygen metabolites. Our results suggest that the damage and replacement of red blood cells during infection could be important costs of chronic haemosporidian infection. Strong links between parasitaemia and the physiological consequences of infection indicate that even for relatively low-intensity infections, measuring parasitaemia rather than only presence/absence could be important when evaluating the role of infection in influencing hosts' behaviour, physiology or fitness.

Highly plastic endocrine traits are thought to play a central role in allowing organisms to respond rapidly to environmental change. Yet, not all individuals display the same degree of plasticity in these traits, and the costs of this individual variation in plasticity are unknown. We studied individual differences in corticosterone levels under varying conditions to test whether there are consistent individual differences in (1) baseline corticosterone levels; (2) plasticity in the hormonal response to an ecologically relevant stressor (food restriction); and (3) whether individual differences in plasticity are related to fitness costs, as estimated by oxidative stress levels. We took 25 wild-caught house sparrows into captivity and assigned them to repeated food restricted and control treatments (60% and 110% of their daily food intake), such that each individual experienced both food restricted and control diets twice. We found significant individual variation in baseline corticosterone levels and stress responsiveness, even after controlling for changes in body mass. However, these individual differences in hormonal responsiveness were not related to measures of oxidative stress. These results have implications for how corticosterone levels may evolve in natural populations and raise questions about what we can conclude from phenotypic correlations between hormone levels and fitness measures. Published version

Many species of Thryothorus wrens are renowned for their highly coordinated male-female song duets; the banded wren (T. pleurostictus) is one of the few Thryothorus that does not duet, with this loss a likely derived trait (Mann et al., submitted). Freedom from the constraints of duetting is associated with vigorous immediate-variety singing by the male banded wren, in contrast to the repeat-mode singing of all duetting relatives (Molles & Vehrencamp 1999). Markers for investigating the species’ genetic mating system will help distinguish among adaptive scenarios for the loss of duetting, including selection on the male to increase song versatility for the purposes of attracting extra-pair copulations, selection on the female to relinquish joint territorial defense, release from male mate-guarding, or female preference for versatile male singers (Hall 2004; Mennill & Vehrencamp 2005). Many of these scenarios predict that extra-pair paternity should be higher in the banded wren compared to duetting congeners. Banded wren DNA was isolated from whole blood in lysis buffer (Hoelzel 1992), using DNeasy™ Tissue Kits (Qiagen Inc., Valencia, CA). A microsatellite library from genomic DNA was constructed following the enrichment procedure described in Stenzler and Fitzpatrick (2002) with the modifications described in Stenzler et al. (2004). One hundred positive plasmid clones were sequenced. PCR primers were designed and tested for the 30 microsatellite-containing sequences. Of those, 11 microsatellite loci appeared suitable for further development and were tested for variability on a panel of 11 banded wrens (Table 1). Table 1 Characteristics of microsatellite loci in Thryothororus pleurostictus and cross-species allele size ranges and number of alleles in Troglodytes aedon and Thryothorus rufalbus Polymerase chain reactions (PCRs) were performed in eight-strip 0.2 mL tubes or 96-well plates using a DYAD® thermal cycler (MJ Research). The cycling profile was 1 cycle at 95° C for three min, 35 cycles of 1 min at 95° C, 1 minute at the locus-specific annealing temperature (see Table 1), and 1 min at 72° C, followed by a final extension cycle of 5 min at 72° C. Reactions (10 μL) contained 10–100 ng of genomic DNA, 0.25 units of Jumpstart™ Taq Polymerase (Sigma, St. Louis, MO), 10 mM Tris-HCL (pH 8.3), 50 mM KCl, MgCl2 specific to each locus (Table 1), 200 μM of dNTPs (Invitrogen), and 2.0 pmol each of forward and reverse primers, one of which was modified at the 5′ end by addition of a fluorescent label (PET, 6-FAM, VIC, or NED -Applied Biosystems). Labeled PCR products were analyzed on an ABI Prism 3100 Genetic Analyser (Applied Biosystems), and allele sizes were estimated using GENEMAPPER™ version 3.0 (Applied Biosystems). During preliminary screening of microsatellite loci isolated from the banded wren we noted that an unusually large proportion of loci were invariant, and in subsequent screening with primers developed for other wren taxa (song wren (Cyphorhinus phaeocephalus; Hughes and Robinson 2001) and house wren (Troglodytes aedon; Cabe and Marshall 2001), we similarly found little to no variation at the subset of those loci that amplified robustly in the banded wren. To explore whether this pattern of low variation was taxon-specific, we used the same protocols to test amplification and variability of the banded wren markers in panels of individuals of two additional wren taxa: the rufous-and-white wren (T. rufalbus), which occurs sympatrically with Thryothorus pleurostictus at our Costa Rica study site (Mennill & Vehrencamp 2005), and the southern house wren (Troglodytes aedon bonariae) from southern Buenos Aires Province, Argentina. A total of 11 banded wrens, 15 rufous-and-white wrens, and 15 house wrens were genotyped at each of the 11 banded wren-specific loci (Table 1). Of the primers that produced a PCR product, the number of variable loci in each species was: banded wren, 3 (2–3 alleles); rufous-and-white wren, 6 (2–7 alleles); and house wren, 3 (2–11 alleles). While allelic variation was present at 27% of banded wren loci in banded wrens, multiple alleles were found at 60% and 50% of loci that amplified in the rufousand-white and house wrens, respectively. Given the higher variability of these markers in related species, we suspect that the low microsatellite variability in this banded wren population stems from past demographic bottlenecks, despite the relative abundance of this species at the present time. The banded wren is a habitat specialist of lowland tropical dry-forests, whereas the rufous-and-white wren is a semi-humid and gallery forest specialist and the southern house wren is a generalist of semi-open habitat ranging from arid to humid and from lowland to montane areas. During the Last Glacial Maximum and subsequent Early Holocene, the Pacific lowlands of southern Central America experienced multiple rapid and extreme changes in climate and vegetation (Piperno and Jones 2003). During this period, the expansion of the humid lowland forest and concomitant fragmentation of dry forest habitats may have reduced and fragmented the banded wren’s populations more strongly than the populations of the other two wren species. In subsequent screening, we genotyped 40 complete banded wren families (246 individuals) with ThPl-14, ThPl-27, and ThPl-37. The numbers of alleles at these loci were 3, 6, and 3, respectively, in this larger pool of individuals. All loci were in Hardy-Weinberg equilibrium and there was no evidence of linkage among the loci (all P > 0.05, n = 70 adult males and females – offspring excluded; tests done in GENEPOP (Raymond & Rousset 1999).

We describe 11 microsatellite loci isolated from the American crow (Corvus brachyrhynchos), the most wide-spread cooperatively breeding bird in North America. Polymorphism at these loci ranged from four to 43 alleles, and observed heterozygosities from 0.53 to 0.92. Genetic parentage analyses using these markers will allow us to describe the mating system of this common but socially complex species, and to interpret their behavioural interactions in light of relatedness.