Allele frequency dynamics in a pedigreed natural population.

A central goal of population genetics is to understand how genetic drift, natural selection, and gene flow shape allele frequencies through time. However, the actual processes underlying these changes-variation in individual survival, reproductive success, and movement-are often difficult to quantify. Fully understanding these processes requires the population pedigree, the set of relationships among all individuals in the population through time. Here, we use extensive pedigree and genomic information from a long-studied natural population of Florida Scrub-Jays (Aphelocoma coerulescens) to directly characterize the relative roles of different evolutionary processes in shaping patterns of genetic variation through time. We performed gene dropping simulations to estimate individual genetic contributions to the population and model drift on the known pedigree. We found that observed allele frequency changes are generally well predicted by accounting for the different genetic contributions of founders. Our results show that the genetic contribution of recent immigrants is substantial, with some large allele frequency shifts that otherwise may have been attributed to selection actually due to gene flow. We identified a few SNPs under directional short-term selection after appropriately accounting for gene flow. Using models that account for changes in population size, we partitioned the proportion of variance in allele frequency change through time. Observed allele frequency changes are primarily due to variation in survival and reproductive success, with gene flow making a smaller contribution. This study provides one of the most complete descriptions of short-term evolutionary change in allele frequencies in a natural population to date.

Deconstructing isolation-by-distance: The genomic consequences of limited dispersal.

Geographically limited dispersal can shape genetic population structure and result in a correlation between genetic and geographic distance, commonly called isolation-by-distance. Despite the prevalence of isolation-by-distance in nature, to date few studies have empirically demonstrated the processes that generate this pattern, largely because few populations have direct measures of individual dispersal and pedigree information. Intensive, long-term demographic studies and exhaustive genomic surveys in the Florida Scrub-Jay (Aphelocoma coerulescens) provide an excellent opportunity to investigate the influence of dispersal on genetic structure. Here, we used a panel of genome-wide SNPs and extensive pedigree information to explore the role of limited dispersal in shaping patterns of isolation-by-distance in both sexes, and at an exceedingly fine spatial scale (within ~10 km). Isolation-by-distance patterns were stronger in male-male and male-female comparisons than in female-female comparisons, consistent with observed differences in dispersal propensity between the sexes. Using the pedigree, we demonstrated how various genealogical relationships contribute to fine-scale isolation-by-distance. Simulations using field-observed distributions of male and female natal dispersal distances showed good agreement with the distribution of geographic distances between breeding individuals of different pedigree relationship classes. Furthermore, we built coalescent simulations parameterized by the observed dispersal curve, population density, and immigration rate, and showed how incorporating these extensions to Malécot's theory of isolation-by-distance allows us to accurately reconstruct observed sex-specific isolation-by-distance patterns in autosomal and Z-linked SNPs. Therefore, patterns of fine-scale isolation-by-distance in the Florida Scrub-Jay can be well understood as a result of limited dispersal over contemporary timescales.

Influence of corticosterone treatment on nestling begging in Florida scrub-jays (Aphelocoma coerulescens).

Altricial young are dependent on adults for protection and food, and they display nutritional need by begging to elicit feeding from parents. Begging at high levels can be energetically expensive and attract predators; thus, an individual must balance its nutritional needs with these potential costs. Further, because a parent is limited in the amount of food it can provide, begging can contribute to both parent-offspring conflict and sibling-sibling competition. Many extrinsic and intrinsic factors may contribute to begging behavior. One intrinsic factor of interest is corticosterone (CORT), a metabolic hormone hypothesized to play a role in regulating a nestling's begging behavior. We investigated the hypothesis that increased exposure to CORT influences nestling begging behavior in an altricial species, the Florida scrub-jay (Aphelocoma coerulescens). We treated one nestling per treatment nest with a twice-daily dose of exogenous hormone via a CORT-injected waxworm, whereas a second individual received a vehicle-injected waxworm. We monitored individual nestling and adult behavior at all nests with the use of high-definition video cameras on several days during treatment. We found no difference in begging rate between CORT fed and vehicle fed nestlings within a treatment nest. Further, to determine whether CORT treatment had indirect effects on the entire brood, we monitored additional nests, in which nestlings were not manipulated. When treatment and controls were compared, overall begging rates of nestlings in treatment nests were greater than those in control nests. This result suggests that CORT treatment of an individual altered its behavior, as well as that of its siblings.

Stress-responsiveness influences baseline glucocorticoid levels: Revisiting the under 3min sampling rule.

Plasma glucocorticoid (CORT) levels collected within 3min of capture are commonly believed to reflect pre-stressor, baseline CORT levels. Differences in these "baseline" values are often interpreted as reflecting differences in health, or the amount of social and environmental stress recently experienced by an individual. When interpreting "baseline" values it is generally assumed that any effect of capture-and-handling during the initial sampling period is small enough and consistent enough among individuals to not obscure pre-capture differences in CORT levels. However, plasma CORT increases in less than 3min post-capture in many free-living, endothermic species in which timing has been assessed. In addition, the rate of CORT secretion and the maximum level attained (i.e., the degree of stress-responsiveness) during a severe stressor often differs among individuals of the same species. In Florida scrub-jays (Aphelocoma coerulescens), an individual's stress-responsiveness during a 30min post-capture stressor is correlated with CORT levels in samples collected within 1.5min of capture, suggesting there is an intrinsic connection between stress-responsiveness and pre-capture CORT levels. Although differences in stress-responsiveness accounted for just 11% of the variance in these samples, on average, higher stress-responsive jays (top third of individuals) had baseline values twice that of lower stress-responsive jays (bottom third). Further, plasma CORT levels begin to increase around 2min post-capture in this species, but the rate of increase between 2 and 3min differs markedly with CORT increasing more rapidly in jays with higher stress-responsiveness. Together, these data indicate that baseline CORT values can be influenced by an individual's stress response phenotype and the differences due to stress-responsiveness can be exaggerated during sample collection. In some cases, the effects of differences in stress-responsiveness and the increase in CORT during sample collection could obscure, or supersede, differences in pre-capture plasma CORT levels that are caused by extrinsic factors.

Two seconds is all it takes: European starlings (Sturnus vulgaris) increase levels of circulating glucocorticoids after witnessing a brief raptor attack.

Researchers typically study "acute" activation of the hypothalamic-pituitary-adrenal (HPA) axis by measuring levels of circulating glucocorticoids in animals that have been exposed to a predator or a cue from a predator (e.g., odor), or have experienced a standardized capture-and-restraint protocol, all of which are many minutes in duration. However, exposure to predators in the "wild", either as the subject of an attack or as a witness to an attack, is generally much shorter as most depredation attempts upon free-living animals last <5s. Yet, whether a stimulus lasting only seconds can activate the HPA axis is unknown. To determine if a stimulus of a few seconds triggers a glucocorticoid response, we measured levels of corticosterone (CORT; the primary avian glucocorticoid) in wild-caught European starlings (Sturnus vulgaris) after they witnessed a brief (<2-8s) raptor attack upon a conspecific, a human "attack" (i.e., a researcher handling a conspecific), and an undisturbed control. Witnesses of a raptor attack responded with CORT levels comparable to that induced by a standardized capture-and-restraint protocol. Glucocorticoid levels of individuals following the control treatment were similar to baseline levels, and those that witnessed a human "attack" had intermediate levels. Our results demonstrate that witnessing a predator attack of very brief duration triggers a profound adrenocortical stress response. Given the considerable evidence of a role for glucocorticoids in learning and memory, such a response may affect how individuals learn to recognize and appropriately react to predators.

Maternal corticosterone deposition in avian yolk: Influence of laying order and group size in a joint-nesting, cooperatively breeding species.

Glucocorticoid hormones play a key role in day-to-day adjustments to fluctuating metabolic needs. These hormones also mediate physiological and behavioral responses to stressful events, allowing individuals to cope with stressors. Various environmental insults, such as a food shortages, predation attempts, and agonistic encounters often elevate plasma glucocorticoid levels in vertebrates. Because exposure to maternally-derived (via circulation or egg) glucocorticoids may be detrimental to the developing embryo, maternal stress can have negative carryover effects on offspring fitness. We examined corticosterone, the primary avian glucocorticoid, concentrations in egg yolk in a plural-breeding, joint-nesting species, the smooth-billed ani (Crotophaga ani), in which females compete among themselves to lay eggs in the final incubated clutch. We investigated whether yolk corticosterone levels varied with laying order and group size. Because egg-laying competition leads to physiological and social stress that is intensified with group size and laying order, we predicted that yolk corticosterone levels should increase from the early to the late egg-laying period and from single female to multi-female groups. In this two-year field study, we found that yolk corticosterone levels of late-laid eggs within the communal clutch were higher in multi-female groups than in single female groups. Results from this study suggest that laying females experience higher levels of stress in multi-female groups and that this maternal stress influences yolk corticosterone concentrations. This study identifies a novel cost of group-living in plural-breeding cooperatively breeding birds, namely an increase in yolk corticosterone levels with group size that may result in detrimental effects on offspring development.

Understanding stress in the healthy animal - potential paths for progress.

Although stress is usually associated with disease, the physiological and behavioral responses to stressors are critical mechanisms of resilience for healthy organisms. A recent workshop comprised of researchers who study healthy humans and both free-living and captive non-human animals identified a number of key roadblocks that are impeding progress in understanding how stress responses integrate into the normal physiology of an animal. These include the lack of: (1) an unambiguous definition of a stress phenotype; (2) a robust biomarker, or suite of biomarkers, to indicate that phenotype; (3) theoretical and quantitative models to predict how humans and other animals will react to stressors; (4) a comprehensive understanding of how individual variability in stress responses arise and (5) an understanding of the transitions between acute and chronic stress responses. Collectively, these deficiencies impair our ability to both assess the physiological status of individuals and develop procedures and techniques to reverse the effects elicited by chronic stress before they become pathological. Workshop participants also identified a number of potential approaches to facilitate progress on these problems. They include: (1) increased use of mathematical models to provide quantitative predictions; (2) use of network theory to expose emergent properties not predicted from traditional approaches; (3) development and deployment of improved sensor technology that will allow long-term, dynamic, non-invasive, multi-factor measurements of suites of stress mediators and (4) the recruitment of scientists with diverse skill sets, such as engineers, bioinformaticians, etc.; and (5) the training of young scientists in the multidisciplinary study of stress. Incorporating these approaches in new research should reinvigorate the study of stress and stimulate progress in understanding both how healthy humans cope with stressors and how other animals, including free-living animals, cope with stressors in a rapidly changing environment.

Hatching asynchrony that maintains egg viability also reduces brood reduction in a subtropical bird.

In birds, hatching failure is pervasive and incurs an energetic and reproductive cost to breeding individuals. The egg viability hypothesis posits that exposure to warm temperatures prior to incubation decreases viability of early laid eggs and predicts that females in warm environments minimize hatching failure by beginning incubation earlier in the laying period, laying smaller clutches, or both. However, beginning incubation prior to clutch completion may incur a cost by increasing hatching asynchrony and possibly brood reduction. We examined whether Florida scrub jays (Aphelocoma coerulescens) began incubation earlier relative to clutch completion when laying larger clutches or when ambient temperatures increased, and whether variation in incubation onset influenced subsequent patterns of hatching asynchrony and brood reduction. We compared these patterns between a suburban and wildland site because site-specific differences in hatching failure match a priori predictions of the egg viability hypothesis. Females at both sites began incubation earlier relative to clutch completion when laying larger clutches and as ambient temperatures increased. Incubation onset was correlated with patterns of hatching asynchrony at both sites; however, brood reduction increased only in the suburbs, where nestling food is limiting, and only during the late nestling period. Hatching asynchrony may be an unintended consequence of beginning incubation early to minimize hatching failure of early laid eggs. Food limitation in the suburbs appears to result in increased brood reduction in large clutches that hatch asynchronously. Therefore, site-specific rates of brood reduction may be a consequence of asynchronous hatching patterns that result from parental effort to minimize hatching failure in first-laid eggs. This illustrates how anthropogenic change, such as urbanization, can lead to loss of fitness when animals use behavioral strategies intended to maximize fitness in natural landscapes.

Allele frequency dynamics under sex-biased demography and sex-specific inheritance in a pedigreed jay population.

Sex-biased demography, including sex-biased survival or migration, can alter allele frequency changes across the genome. In particular, we can expect different patterns of genetic variation on autosomes and sex chromosomes due to sex-specific differences in life histories, as well as differences in effective population size, transmission modes, and the strength and mode of selection. Here, we demonstrate the role that sex differences in life history played in shaping short-term evolutionary dynamics across the genome. We used a 25-year pedigree and genomic dataset from a long-studied population of Florida Scrub-Jays (Aphelocoma coerulescens) to directly characterize the relative roles of sex-biased demography and inheritance in shaping genome-wide allele frequency trajectories. We used gene dropping simulations to estimate individual genetic contributions to future generations and to model drift and immigration on the known pedigree. We quantified differential expected genetic contributions of males and females over time, showing the impact of sex-biased dispersal in a monogamous system. Due to female-biased dispersal, more autosomal variation is introduced by female immigrants. However, due to male-biased transmission, more Z variation is introduced by male immigrants. Finally, we partitioned the proportion of variance in allele frequency change through time due to male and female contributions. Overall, most allele frequency change is due to variance in survival and births. Males and females make similar contributions to autosomal allele frequency change, but males make higher contributions to allele frequency change on the Z chromosome. Our work shows the importance of understanding sex-specific demographic processes in characterizing genome-wide allele frequency change in wild populations.

Physiology of reproductive senescence in Florida scrub-jays: results from a long-term study and GnRH challenge.

In most vertebrates, production of reproductive hormones wanes with age, co-occurring with a decline in reproductive output. Measurement of these hormones can serve as a key marker of the onset of reproductive senescence. Longitudinal studies of physiological parameters in populations of free-living animals are relatively uncommon; however, we have monitored baseline concentrations of hormones for nine years in a population of Florida scrub-jays (Aphelocoma coerulescens). We hypothesized that concentrations of circulating reproductive hormones change with age, and predicted declines in reproductive hormones in the oldest jays. We found that baseline levels of luteinizing hormone (LH) and testosterone (T) were relatively low in both young and old male breeders and reach their highest levels in birds aged 4-7years. Conversely, we found no age-related patterns in baseline levels of LH or estradiol in female jays. In males we determined which component of the hypothalamo-pituitary-gonadal (HPG) axis is responsible for observed age-based differences, by challenging males of different ages with gonadotropin-releasing hormone (GnRH); thereby allowing assessment of pituitary and gonadal responsiveness by measuring plasma concentrations of luteinizing hormone and testosterone, respectively. The magnitude of increase in levels of both LH and T in response to GnRH challenge decreased with age in male breeders. Combined with the baseline levels, the results from the GnRH challenge suggest that younger birds have the capability to produce higher levels of reproductive hormone, whereas the old birds may be constrained by senescence in their ability to produce these hormones.

Repeatability of baseline and stress-induced corticosterone levels across early life stages in the Florida scrub-jay (Aphelocoma coerulescens).

Recent studies have posited that the pattern of glucocorticoid secretion within an individual represents a stable, fixed physiological trait. To test this hypothesis, we assessed the repeatability of baseline and stress-induced corticosterone (CORT) secretion across developmental stages and years in Florida scrub-jays. We sampled individuals from multiple cohorts repeatedly from the age of 11 days post-hatch up to 4 years of age. We found a significant degree of repeatability within individuals in stress-induced corticosterone levels, i.e., the amount of hormone secreted during a standardized stress protocol (corrected integrated corticosterone). However, baseline corticosterone levels were not statistically repeatable, although there was some indication that nestling levels predicted levels at 1 year of age. The results of this study indicate that stress-induced CORT levels are consistent within individual scrub-jays, and the degree to which a young jay mounts an acute stress response appears to be somewhat "set" by the age of nutritional independence. Thus stress-induced corticosterone secretion appears to be a stable, repeatable trait within individuals and as such may be subject to natural selection.

Corticosterone, brood size, and hatch order in free-living Florida scrub-jay (Aphelocoma coerulescens) nestlings.

It is well known that variation in developmental conditions can have profound effects upon lifetime fitness. In altricial avian species, nestlings undergo a substantial portion of development in the nest after hatching, often in the presence of nest-mates. This can result in the formation of brood hierarchies based on age, size, and competitive ability. Measurement of baseline corticosterone (CORT) levels in developing birds may provide a means to assess whether individuals within a brood experience stress due to sibling competition or nutritional state. However, few studies have attempted to correlate corticosterone concentrations with brood hierarchies in free-living, developing birds. We investigated the degree to which corticosterone levels in nestling Florida scrub-jays (Aphelocoma coerulescens) were predicted by brood size, hatching order, and body mass. Nestling corticosterone levels were inversely correlated with body mass but did not differ among brood sizes. Within broods of two, second-hatched nestlings had lower CORT than first-hatched nestlings, although there was no effect of hatch order in broods of three or four nestlings. The results of this study suggest that conditions within the nest, particularly those associated with within-brood hierarchies, are manifested through differential body condition and nestling corticosterone secretion. The consequences of this variation in nestling corticosterone are unknown, but development of the adult phenotype, as well as life-long survival, may be impacted.

Age-related differences in baseline and stress-induced corticosterone in Florida scrub-jays.

In physiological studies of free-living species, it is essential to consider the context of the life history stage at which an individual was observed in order to link measures of physiology with ecological parameters. One such measure that is important to consider is the age of an individual. We tested whether baseline or stress-induced corticosterone levels vary with age in free-living Florida scrub-jays (Aphelocoma coerulescens) during the pre-breeding period. Corticosterone (CORT), the primary avian stress hormone, is released in response to stressful stimuli, and stimulates gluconeogenesis; however, it also serves as a chemical messenger that can influence other physiological processes, reproduction, and behavior. We monitored both baseline CORT levels longitudinally throughout a five-year period and stress-induced CORT responses over a shorter two-year period. We predicted that older jays would have lower baseline CORT levels and a dampened stress response compared to younger birds, as has been shown in other avian species. We found no significant differences in baseline CORT levels with age. We found a decrease in total corticosterone responses to a stressor with age, however, the oldest birds in the population showed greater total corticosterone responses to a stressor. These results may be a product of age-related changes in physiological processes related to the stress response or a result of selection acting on the population, resulting in only the most responsive individuals surviving to old age.

The influence of nest attendance and provisioning on nestling stress physiology in the Florida scrub-jay.

Stressful conditions during development may have lasting consequences for an animal's lifetime fitness. We investigated the degree to which parental provisioning and nest attendance influenced baseline levels of the stress hormone corticosterone in nestling Florida scrub-jays. Provisioning rates of male and female breeders and nest attendance of female breeders were recorded during focal watches conducted between days 3 and 5 post-hatch. A small blood sample was taken from each nestling on day 11 post-hatch and used to quantify levels of baseline corticosterone. The proportion of time spent by female breeders at a considerable distance from the nest was positively related to nestling corticosterone levels. Nestling corticosterone was also negatively related to parental provisioning rate, although this effect seemed to be secondary to the effect of the female's time away from the nest. These results indicate that parental behavior contributes to nestling stress physiology, which may in turn direct the formation of the adult phenotype and influence an individual's chances of survival.

Selection on innate immunity and body condition in Florida scrub-jays throughout an epidemic.

Opportunities to investigate selection in free-living species during a naturally occurring epidemic are rare; however, we assessed innate immunocompetence in Florida scrub-jays before the population suffered the greatest over-winter mortality in 20 years of study. Propitiously, three months prior to the epidemic, we had sampled a number of male breeders to evaluate a suite of physiological measures that are commonly used to estimate the overall health-state of an individual. There was a significant, positive selection gradient for both Escherichia coli bacterial killing capability and body condition, suggesting that directional selection had occurred upon each of these traits during the disease epidemic.

Development of the adrenal stress response in the Florida scrub-jay (Aphelocoma coerulescens).

Nestlings of altricial species undergo a period of substantial growth and development in the nest after hatching. The hypothalamic-pituitary-adrenal axis regulates the release of stress hormones such as corticosterone, which in adults is critical in allowing an animal to respond to a stressor. However, activation of this axis in young birds may be detrimental to growth and possibly survival. The developmental hypothesis predicts that altricial nestlings should display a dampened corticosterone response to stress as a means of protection against the potentially harmful effects of elevated corticosterone. We examined this hypothesis in Florida scrub-jays, a cooperatively breeding species with altricial young. Blood samples were collected from nestlings, nutritionally independent young, and yearlings for measurement of corticosterone levels. Baseline corticosterone levels did not differ between age-classes; however, stress-induced corticosterone levels were highest in yearlings, intermediate in independent young, and lowest in nestlings. The nestling stress response was also of a shorter duration than the response in independent young and yearlings. This variation in stress responsiveness across ages may be an adaptive mechanism to protect the developing bird from the negative effects of corticosterone on growth and cognitive development.

Environment, glucocorticoids, and the timing of reproduction.

Glucocorticoids mediate glucose availability under stressful and non-stressful conditions and, therefore, are essential for life. However, data across taxa demonstrate that chronic or elevated secretion of corticosterone or cortisol (CORT) can have negative effects at many levels and can trigger physiological or behavioral responses that may delay or, even halt reproduction. We present a brief overview of the effects that glucocorticoids, primarily the avian form, corticosterone, can have on the reproductive axis. Considerable data have demonstrated that environmental perturbations can result in elevated CORT levels that alter a bird's investment in current reproduction. Studies in our laboratory have shown a link between CORT and timing of reproduction in Florida scrub-jays: in "bad" years, clutch initiation dates are positively correlated with baseline CORT levels of female breeders. Also, population-level differences in CORT levels may explain timing of reproduction as lower CORT levels in suburban-dwelling jays are coupled with early breeding. Most research on stress and CORT concentrates on transient effects of CORT secretion. However, developmental CORT exposure, either from the yolk or embryo, may have long-term effects upon adult phenotype. For example, CORT levels in nestling scrub-jays predicts later 'personality,' as levels were highly correlated (r(2)=0.84) with fearfulness at 7 months of age. One can imagine that such 'personality' traits might also translate into differential success in gaining a territory or a mate. While speculative, it may be that early CORT exposure effectively programs adult behaviors that have wide ranging effects, including upon reproduction.

Adult Provisioning Influences Nestling Corticosterone Levels in Florida Scrub Jays (Aphelocoma coerulescens).

We studied Florida scrub jay (Aphelocoma coerulescens) nestlings to examine the relationship between parental feeding rates and levels of corticosterone (CORT), a metabolic and stress-related steroid hormone hypothesized to play a role in mediating begging behavior. It has been documented that nutritional deficiency results in increased glucocorticoid levels in nestling birds. Further, previous studies have found that CORT levels of Florida scrub jay nestlings are negatively correlated with parental nest attendance and provisioning rates; however, the behavioral observations were made several days before the collection of samples to assess CORT levels. Few studies have investigated whether experience immediately before sampling impacts nestling glucocorticoid levels, especially in a free-living species. By monitoring parental activity at the nest before sample collection, we found that nestling CORT levels varied as a function of parental provisioning rate and the time since their last feed. However, counter to our predictions, higher provisioning rates and more recent feedings were associated with higher CORT levels in nestlings rather than lower CORT levels. These results suggest that some aspect of parental provisioning results in increased CORT levels in nestling Florida scrub jays.

The rate of telomere loss is related to maximum lifespan in birds.

Telomeres are highly conserved regions of DNA that protect the ends of linear chromosomes. The loss of telomeres can signal an irreversible change to a cell's state, including cellular senescence. Senescent cells no longer divide and can damage nearby healthy cells, thus potentially placing them at the crossroads of cancer and ageing. While the epidemiology, cellular and molecular biology of telomeres are well studied, a newer field exploring telomere biology in the context of ecology and evolution is just emerging. With work to date focusing on how telomere shortening relates to individual mortality, less is known about how telomeres relate to ageing rates across species. Here, we investigated telomere length in cross-sectional samples from 19 bird species to determine how rates of telomere loss relate to interspecific variation in maximum lifespan. We found that bird species with longer lifespans lose fewer telomeric repeats each year compared with species with shorter lifespans. In addition, phylogenetic analysis revealed that the rate of telomere loss is evolutionarily conserved within bird families. This suggests that the physiological causes of telomere shortening, or the ability to maintain telomeres, are features that may be responsible for, or co-evolved with, different lifespans observed across species.This article is part of the theme issue 'Understanding diversity in telomere dynamics'.

Hormone levels and infection of Haemoproteus danilewskyi in free-ranging blue jays (Cyanocitta cristata).

Annual spring relapse of blood parasite infections in birds is believed to be the result of hormonal changes associated with breeding. As part of a larger study on the epizootiology of Haemoproteus danilewskyi in blue jays in south-central Florida, we studied the relationship between H. danilewskyi infections and levels of luteinizing hormone, prolactin, progesterone, testosterone, estradiol, and corticosterone. We found a positive association between intensity of H. danilewskyi infection and corticosterone levels in females but not in males. We also found no association between infection and levels of prolactin, luteinizing hormone, progesterone, testosterone, or estradiol in males or females. In addition, we found a positive relationship between levels of corticosterone and handling time and between corticosterone and testosterone levels. We suggest a possible influence of corticosterone on spring relapse of Haemoproteus spp. infections in birds but provide no support for the influence of breeding hormones on relapse of these parasites.