Maternal glucocorticoid levels during incubation predict breeding success, but not reproductive investment, in a free-ranging bird.

The hormone corticosterone (CORT) has been hypothesized to be linked with fitness, but the directionality of the relationship is unclear. The 'CORT-fitness hypothesis' proposes that high levels of CORT arise from challenging environmental conditions, resulting in lower reproductive success (a negative relationship). In contrast, the CORT-adaptation hypothesis suggests that, during energetically demanding periods, CORT will mediate physiological or behavioral changes that result in increased reproductive investment and success (a positive relationship). During two breeding seasons, we experimentally manipulated circulating CORT levels in female tree swallows (Tachycineta bicolor) prior to egg laying, and measured subsequent reproductive effort, breeding success, and maternal survival. When females were recaptured during egg incubation and again during the nestling stage, the CORT levels were similar among individuals in each treatment group, and maternal treatment had no effect on indices of fitness. By considering variation among females, we found support for the CORT-adaptation hypothesis; there was a significant positive relationship between CORT levels during incubation and hatching and fledging success. During the nestling stage CORT levels were unrelated to any measure of investment or success. Within the environmental context of our study, relationships between maternal glucocorticoid levels and indices of fitness vary across reproductive stages.

Maternal immunization increases nestling energy expenditure, immune function, and fledging success in a passerine bird.

Female birds transfer maternally derived antibodies (matAb) to their nestlings, via the egg yolk. These antibodies are thought to provide passive protection, and allow nestlings to avoid the costs associated with mounting an innate immune response. To test whether there is an energetic benefit to nestlings from receiving matAb, we challenged adult female tree swallows (Tachycineta bicolor) prior to clutch initiation with either lipopolysaccharide (LPS) or saline (Control). Following hatching, one half of each female's nestlings were immunized on day 8 post-hatch with LPS or saline, and the 4-h post-immunization nestling metabolic rate (MR) was measured. There was no difference in either LPS-reactive antibodies or total Ig levels between offspring of immunized and non-immunized mothers on day 6 or 14 post-hatch, possibly reflecting a relatively short half-life of matAbs in altricial birds. Additionally, we found no evidence that nestlings from LPS-immunized mothers could avoid the growth suppression that may result from activation of an inflammatory response. Unexpectedly, we found that control nestlings from LPS mothers had higher resting MR than control nestlings of control mothers. We attribute the increased MR to the costs associated with a general non-specific enhancement of immune function in nestlings from LPS-immunized mothers. Consistent with enhanced immune function, nestlings of immunized mothers had a more robust inflammatory response to phytohaemagglutinin and higher fledging success. Our results suggest that maternal antigen exposure pre-laying can result in increased fitness for both mothers and offspring, depending on food availability.

A maternal high-fat, high-sucrose diet has sex-specific effects on fetal glucocorticoids with little consequence for offspring metabolism and voluntary locomotor activity in mice.

Maternal overnutrition and obesity during pregnancy can have long-term effects on offspring physiology and behaviour. These developmental programming effects may be mediated by fetal exposure to glucocorticoids, which is regulated in part by placental 11ß-hydroxysteroid dehydrogenase (11ß-HSD) type 1 and 2. We tested whether a maternal high-fat, high-sucrose diet would alter expression of placental 11ß-HSD1 and 2, thereby increasing fetal exposure to maternal glucocorticoids, with downstream effects on offspring physiology and behaviour. C57BL/6J mice were fed a high-fat, high-sucrose (HFHS) diet or a nutrient-matched low-fat, no-sucrose control diet prior to and during pregnancy and lactation. At day 17 of gestation, HFHS dams had ~20% lower circulating corticosterone levels than controls. Furthermore, there was a significant interaction between maternal diet and fetal sex for circulating corticosterone levels in the fetuses, whereby HFHS males tended to have higher corticosterone than control males, with no effect in female fetuses. However, placental 11ß-HSD1 or 11ß-HSD2 expression did not differ between diets or show an interaction between diet and sex. To assess potential long-term consequences of this sex-specific effect on fetal corticosterone, we studied locomotor activity and metabolic traits in adult offspring. Despite a sex-specific effect of maternal diet on fetal glucocorticoids, there was little evidence of sex-specific effects on offspring physiology or behaviour, although HFHS offspring of both sexes had higher circulating corticosterone at 9 weeks of age. Our results suggest the existence of as yet unknown mechanisms that mitigate the effects of altered glucocorticoid exposure early in development, making offspring resilient to the potentially negative effects of a HFHS maternal diet.

Acute changes in whole body corticosterone in response to perceived predation risk: A mechanism for anti-predator behavior in anurans?

Anuran larvae exhibit behavioral and morphological plasticity in response to perceived predation risk, although response type and magnitude varies through ontogeny. Increased baseline corticosterone is related to morphological response to predation risk, whereas the mechanism behind behavioral plasticity remains enigmatic. Since tadpoles alter behavioral responses to risk immediately upon exposure to predator cues, we characterized changes in whole body corticosterone at an acute (<1h post-exposure) timescale. Tadpoles (Lithobates sylvaticus) at Gosner stage (GS) 25 (free-swimming, feeding larvae) increased corticosterone levels to a peak at 10-20min post-exposure to predator cues, paralleling the acute stress response observed among other taxa. Tadpoles reared for 3weeks (mean GS29) with predation risk (caged, fed Aeshnid dragonfly nymph) had lower corticosterone levels at 10-20min post-exposure to dragonfly cues than predator-naïve controls, suggesting habituation, although the magnitude of increase was markedly diminished when compared to younger tadpoles (GS25). These experiments represent the first assessment of tadpole hormonal responses to predation risk at the acute timescale. Further research is required to establish causality between hormonal responses and behavioral changes, and to examine how and why responsiveness changes over ontogeny and with chronic exposure to risk.

Adult Ovenbirds (Seiurus aurocapilla) show increased stress-responsiveness in logged forests.

Forest harvesting is a form of anthropogenic disturbance, yet the effects of such disturbance on the endocrine physiology of wildlife have been infrequently studied. We investigated the effect of two methods of forest harvesting ('intensive' and 'typical' group-selection silviculture) and un-harvested control sites on the glucocorticoid levels of adult Ovenbirds (Seiurus aurocapilla), a forest interior bird species. We collected blood samples from adult males immediately after capture to examine baseline corticosterone, and then following a standardized capture and restraint protocol, to examine stress-induced levels. There was no significant repeatability in either baseline or stress-induced corticosterone levels for eleven individuals measured in both years of study. Despite no differences across harvesting treatments in male body mass or baseline corticosterone levels, males captured in sites subjected to intensive harvesting had significantly higher stress-induced corticosterone levels than males in other treatments. Currently, the mechanism driving differences in stress-reactivity is unknown although we hypothesize that the size of gaps resulting from intensive group-selection silviculture may increase perceived predation risk. In comparison to our previous work on nestling Ovenbirds, adults respond differently to stress from group-selection silviculture.

Acute stress during ontogeny suppresses innate, but not acquired immunity in a semi-precocial bird (Larus delawarensis).

Wild animals often encounter adverse conditions, and in response, activate their hypothalamic-pituitary-adrenal (HPA) axis. To date, work examining the development of the stress response has focused on altricial species, with little work focusing on species with other developmental patterns. Additionally, the effects of acute stress on indices of innate and adaptive immunity have been little studied in birds, particularly during development. We examined the ontogeny of the stress response in the semi-precocial ring-billed gull (Larus delawarensis). At hatch, 10, and 20days post-hatching, chicks underwent a standardized handling stress protocol, with blood samples taken within 3min of, and 30min after, initial disturbance. Levels of corticosterone (CORT), natural antibodies (NAb), complement activity, and immunoglobulins (IgY) were assessed in plasma samples. In contrast with altricial species, ring-billed gull chicks had detectible CORT levels at hatch, and were able to mount a stress response. At all ages, acute handling stress depressed NAb levels and complement-mediated lysis, but not IgY levels. IgY levels were higher in two chick broods than three chick broods, suggesting levels are determined in part by resource dependence. Our data provide insight into the development of the stress response and immune function in a colonial waterbird species, in which chicks are mobile shortly post hatch, and subject to aggression and possible injury from nearby adults.

Linking sex differences in corticosterone with individual reproductive behaviour and hatch success in two species of uniparental shorebirds.

In birds, corticosterone (CORT) appears to facilitate reproductive activity because baseline and stress-induced CORT levels are elevated in breeding individuals compared with other times of the year. In particular, CORT is lower in the sex providing most of the parental care (i.e., incubation), which could be an important adaptation to tolerate stressors that result in abandoning reproduction. Therefore, one explanation for sex differences in CORT is that lower levels are favoured during the incubation/parental phase of reproduction. Using two species of uniparental shorebird - polyandrous red phalaropes (Phalaropus fulicarius) and polygynous white-rumped sandpipers (Calidris fuscicollis) - we predicted that the incubating sex would have lower baseline and stress-induced CORT, and incubating individuals with lower CORT would more effectively defend nests against a simulated intrusion, would return more quickly afterwards, and would ultimately have higher hatch success. We found that phalaropes followed the predicted pattern: incubating individuals (males) had lower baseline and stress-induced CORT than females but for baseline CORT these differences existed prior to males commencing incubation. Incubating male phalaropes with lower baseline and stress-induced CORT returned to incubate more quickly after a disturbance and there was non-significant tendency for baseline CORT to be lower in successful nests. In sandpipers, we observed no sex differences and no significant relationships between individual CORT levels and nest defence behaviours or hatch success. Our results demonstrate that in phalaropes at least, selection favours lower baseline and stress-induced CORT during the nesting period. These results can explain sex differences in stress-induced levels of CORT, however sex differences in baseline CORT were present prior to incubation.

Catch-up growth in Japanese quail (Coturnix Japonica): relationships with food intake, metabolic rate and sex.

The effects of early environmental conditions can profoundly affect individual development and adult phenotype. In birds, limiting resources can affect growth as nestlings, but also fitness and survival as adults. Following periods of food restriction, individuals may accelerate development, undergoing a period of rapid "catch-up" growth, in an attempt to reach the appropriate size at adulthood. Previous studies of altricial birds have shown that catch-up growth can have negative consequences in adulthood, although this has not been explored in species with different developmental strategies. Here, we investigated the effects of resource limitation and the subsequent period of catch-up growth, on the morphological and metabolic phenotype of adult Japanese quail (Coturnix japonica), a species with a precocial developmental strategy. Because males and females differ in adult body size, we also test whether food restriction had sex-specific effects. Birds that underwent food restriction early in development had muscles of similar size and functional maturity, but lower adult body mass than controls. There was no evidence of sex-specific sensitivity of food restriction on adult body mass; however, there was evidence for body size. Females fed ad lib were larger than males fed ad lib, while females subjected to food restriction were of similar size to males. Adults that had previously experienced food restriction did not have an elevated metabolic rate, suggesting that in contrast to altricial nestlings, there was no metabolic carry-over effect of catch-up growth into adulthood. While Japanese quail can undergo accelerated growth after re-feeding, timing of food restriction may be important to adult size, particularly in females. However, greater developmental flexibility compared to altricial birds may contribute to the lack of metabolic carryover effects at adulthood.

DHEA and estradiol levels in brain, gonads, adrenal glands, and plasma of developing male and female European starlings.

Traditionally, sexual differentiation of the brain was thought to be driven by gonadal hormones, particularly testosterone (T). However, recent studies in songbirds suggest that other steroids may also be important. For example, dehydroepiandrosterone (DHEA) can be synthesized by the gonads, adrenal glands, and/or brain and locally metabolized into T and 17ß-estradiol (E(2)). Here, we examined DHEA and E(2) levels in the brain, peripheral tissues, and plasma of wild European starlings (Sturnus vulgaris). In Study 1, samples were collected from males and females at P0 (day of hatch), P6, and P8. In Study 2, samples were collected at P4. At P0, DHEA levels in the diencephalon were higher in males than females. DHEA levels were generally high in the gonads and adrenals, and they were higher in testes than ovaries at P8. Further, E(2) levels were non-detectable in most brain samples, suggesting that DHEA was not metabolized to E(2) or that locally produced E(2) was rapidly inactivated. At P4, DHEA levels in telencephalic regions were lower in males than females. Taken together, these data suggest that sex differences in peripheral DHEA secretion and neural DHEA metabolism at specific ages during development might play a role in sexual differentiation of the songbird brain.

Cortisol and corticosterone in immune organs and brain of European starlings: developmental changes, effects of restraint stress, comparison with zebra finches.

Glucocorticoids (GCs) are produced in the adrenal glands and also in extra-adrenal sites, including immune organs and brain. Here, we examined regulation of systemic GC levels in plasma and local GC levels in immune organs and brain during development. We conducted two studies and examined a total of 462 samples from 70 subjects. In study 1, we determined corticosterone and cortisol levels in the plasma, immune organs, and brain of wild European starlings on posthatch day 0 (P0) and P10 (at baseline and after 45 min of restraint). Baseline corticosterone and cortisol levels were low in the immune organs and brain at P0 and P10, providing little evidence for local GC synthesis in starlings. At P0, restraint had no significant effects on corticosterone or cortisol levels in the plasma or tissues; however, there was a trend for restraint to increase both corticosterone and cortisol in the immune organs. At P10, restraint increased corticosterone levels in the plasma and all tissues, but restraint increased cortisol levels in the plasma, thymus, and diencephalon only. In study 2, we directly compared GC levels in European starlings and zebra finches at P4. In zebra finches but not starlings, cortisol levels were higher in the immune organs than in plasma. This difference in immune GC levels might be due to evolutionary lineage, life history strategy, or experiential factors, such as parasite exposure. This is the first study to measure immune GC levels in wild animals and one of the first studies to measure local GC levels after restraint stress.

Juveniles exposed to embryonic corticosterone have enhanced flight performance.

Exposure to maternally derived glucocorticoids during embryonic development impacts offspring phenotype. Although many of these effects appear to be transiently 'negative', embryonic exposure to maternally derived stress hormones is hypothesized to induce preparative responses that increase survival prospects for offspring in low-quality environments; however, little is known about how maternal stress influences longer-term survival-related performance traits in free-living individuals. Using an experimental elevation of yolk corticosterone (embryonic signal of low maternal quality), we examined potential impacts of embryonic exposure to maternally derived stress on flight performance, wing loading, muscle morphology and muscle physiology in juvenile European starlings (Sturnus vulgaris). Here we report that fledglings exposed to experimentally increased corticosterone in ovo performed better during flight performance trials than control fledglings. Consistent with differences in performance, individuals exposed to elevated embryonic corticosterone fledged with lower wing loading and had heavier and more functionally mature flight muscles compared with control fledglings. Our results indicate that the positive effects on a survival-related trait in response to embryonic exposure to maternally derived stress hormones may balance some of the associated negative developmental costs that have recently been reported. Moreover, if embryonic experience is a good predictor of the quality or risk of future environments, a preparative phenotype associated with exposure to apparently negative stimuli during development may be adaptive.

Neurosteroids, immunosteroids, and the Balkanization of endocrinology.

Traditionally, the production and regulation of steroid hormones has been viewed as a multi-organ process involving the hypothalamic-pituitary-gonadal (HPG) axis for sex steroids and the hypothalamic-pituitary-adrenal (HPA) axis for glucocorticoids. However, active steroids can also be synthesized locally in target tissues, either from circulating inactive precursors or de novo from cholesterol. Here, we review recent work demonstrating local steroid synthesis, with an emphasis on steroids synthesized in the brain (neurosteroids) and steroids synthesized in the immune system (immunosteroids). Furthermore, recent evidence suggests that other components of the HPG axis (luteinizing hormone and gonadotropin-releasing hormone) and HPA axis (adrenocorticotropic hormone and corticotropin-releasing hormone) are expressed locally in target tissues, potentially providing a mechanism for local regulation of neurosteroid and immunosteroid synthesis. The balance between systemic and local steroid signals depends critically on life history stage, species adaptations, and the costs of systemic signals. During particular life history stages, there can be a shift from systemic to local steroid signals. We propose that the shift to local synthesis and regulation of steroids within target tissues represents a "Balkanization" of the endocrine system, whereby individual tissues and organs may become capable of autonomously synthesizing and modulating local steroid signals, perhaps independently of the HPG and HPA axes.

Sex differences in DHEA and estradiol during development in a wild songbird: Jugular versus brachial plasma.

Sexual differentiation of the brain has traditionally been thought to be driven by gonadal hormones, particularly testosterone (T). Recent studies in songbirds and other species have indicated that non-gonadal sex steroids may also be important. For example, dehydroepiandrosterone (DHEA)--a sex steroid precursor that can be synthesized in the adrenal glands and/or brain--can be converted into active sex steroids, such as 17beta-estradiol (E(2)), within the brain. Here, we examine plasma DHEA and E(2) levels in wild developing European starlings (Sturnus vulgaris), from hatch (P0) to fledging (P20). Blood samples were collected from either the brachial vein (n=143) or the jugular vein (n=129). In songbirds, jugular plasma is enriched with neurally-synthesized steroids and, therefore, jugular plasma is an indirect measure of the neural steroidal milieu. Interestingly, brachial DHEA levels were higher in males than females at P4. In contrast, jugular DHEA levels were higher in females than males at P0 and P10. Brachial E(2) levels were higher in males than females at P6. Surprisingly, jugular E(2) levels were not high and showed no sex differences. Also, we calculated the difference between brachial and jugular steroid levels. At several ages, jugular steroid levels were lower than brachial levels, particularly in males, suggesting greater neural metabolism of circulating DHEA and E(2) in males than females. At a few ages, jugular steroid levels were higher than brachial levels, suggesting neural secretion of DHEA or E(2) into the general circulation. Taken together, these data suggest that DHEA may play a role in brain sexual differentiation in songbirds.

Stress hormones: a link between maternal condition and sex-biased reproductive investment.

In species where offspring fitness is sex-specifically influenced by maternal reproductive condition, sex allocation theory predicts that poor-quality mothers should invest in the evolutionarily less expensive sex. Despite an accumulation of evidence that mothers can sex-specifically modulate investment in offspring in relation to maternal quality, few mechanisms have been proposed as to how this is achieved. We explored a hormonal mechanism for sex-biased maternal investment by measuring and experimentally manipulating baseline levels of the stress hormone corticosterone in laying wild female European starlings (Sturnus vulgaris) and examining effects on sex ratio and sex-specific offspring phenotype adjustment. Here we show that baseline plasma corticosterone is negatively correlated with energetic body condition in laying starlings, and subsequent experimental elevation of maternal baseline plasma corticosterone increased yolk corticosterone without altering maternal condition or egg quality per se. Hormonal elevation resulted in the following: female-biased hatching sex ratios (caused by elevated male embryonic mortality), lighter male offspring at hatching (which subsequently grew more slowly during postnatal development), and lower cell-mediated immune (phytohemagglutinin) responses in males compared with control-born males; female offspring were unaffected by the manipulation in both years of the study. Elevated maternal corticosterone therefore resulted in a sex-biased adjustment of offspring quality favorable to female offspring via both a sex ratio bias and a modulation of male phenotype at hatching. In birds, deposition of yolk corticosterone may benefit mothers by acting as a bet-hedging strategy in stochastic environments where the correlation between environmental cues at laying (and therefore potentially maternal condition) and conditions during chick-rearing might be low and unpredictable. Together with recent studies in other vertebrate taxa, these results suggest that maternal stress hormones provide a mechanistic link between maternal quality and sex-biased maternal investment in offspring.