Summary 1.Stress has pervasive consequences for the well-being of animals. Currently, understanding how individuals cope with stressors is typically accomplished via short-term quantification of blood glucocorticoids released after activation of the hypothalamic-pituitary-adrenal (HPA) axis. 2. We investigated whether the amount of corticosterone (CORT) deposited in growing feathers provides a long-term, integrated measure of HPA activity in birds using captive red-legged partridges Alectoris rufa as a model species. 3. We examined CORT levels in primary feathers induced to grow at the same time as stress series were performed with a capture and restraint protocol. Plasma CORT titres after stress-induced stimulation, but not baseline values, correlated with feather CORT. Feather levels showed the same pattern as plasma of decline across the breeding season, but more severely. 4. For females, CORT in naturally moulted flank feathers was highly and positively correlated with the number of eggs laid in the previous few months, but not clutch size of the following year. For males, the amount of black on a feather, known to be a social signal, was positively correlated with its CORT level. 5. The analysis of feather CORT is a novel methodology that allows for meaningful interpretations of how individuals respond to environmental perturbations and adjust to life-history stages. 6. The analysis of feather hormones has the unique advantages of allowing for experimentation and sampling at any time of the year with minimal investigator-induced impacts and artefacts, and shows the HPA activity of an individual with a flexible time frame from days to months depending on the length of time taken to grow the feather. As this technique can be applied to living or dead birds, or feathers picked up after moult, it provides the ultimate non-invasive physiological measure of considerable benefit in terms of animal welfare and sampling effort.

1. Researchers involved in ecology and toxicology, as well as many other aspects of avian biology, use phytohaemagglutinin (PHA) skin testing as a means of evaluating the immune status of individuals. 2. Immune function, one measure of individual quality, can be used as a sensitive, non‐lethal variable that may be negatively affected in animals exposed to degraded, contaminated or otherwise disturbed ecological zones. 3. Typically this test has been applied by challenging one wing web with the immunostimulant PHA, while the other ‘control’ wing is injected with phosphate buffered saline (PBS). Injection sites on the wing web are measured before and 24 h after injection with PHA or PBS. The immune response is considered to be the difference between the two wings. 4. Results from PHA skin tests conducted on 608 birds in seven studies representing passerines, waterfowl, upland game birds and raptors are examined. 5. Numerous advantages to eliminating the PBS injection as the experimental control are: (i) decrease by half, the time required for testing; (ii) decrease handling‐related stress on the birds (proportional to handling time); (iii) reduce the probability of errors at injection time; (iv) spare the other wing for different tests or uses (e.g. tuberculin DTH testing); and (v) decrease the coefficient of variation that is due to measurement inaccuracies. 6. The only disadvantage identified is that hypersensitive individuals (outliers) could be missed, which in this case represents 2 of 608 individuals.

Short-term elevation of circulating glucocorticosteroids (GCs) in vertebrates facilitates the adoption of a distinct emergency life history state, which allows individuals to cope with perturbations and recover homeostasis at the expense of temporarily suppressing nonessential activities. Although GC responses are viewed as a major evolutionary mechanism to maximize fitness through stress management, phenotypic variability exists within animal populations, and it remains unclear whether interindividual differences in stress physiology can explain variance in unequivocal components of fitness. We show that the magnitude of the adrenocortical response to a standardized perturbation during development is negatively related to survival and recruitment in a wild population of long lived birds. Our results provide empirical evidence for a link between stress response, not exposure to stressors, and fitness in a vertebrate under natural conditions. Recent studies suggest that variability in the adrenocortical response to stress may be maintained if high and low GC responders represent alternative coping strategies, with differential adaptive value depending on environmental conditions. Increased fitness among low GC responders, having a proactive personality, is predicted under elevated population density and availability of food resources, conditions that characterize our study population.animal personality ͉ corticosterone ͉ glucocorticosteroids ͉ reproduction ͉ survival E xposure to environmental perturbations constitutes a major selective force in natural populations. Animals have evolved behavioral and physiological strategies to avoid the deleterious effects of stressors, and among vertebrates, the adrenocortical response is one of the most conserved physiological mechanisms aimed at this end (1-3). In response to modifying factors (e.g., decreased food resources, predation, harsh weather), vertebrates activate the hypothalamous-pituitary-adrenal axis, which triggers a rapid release of glucocorticosteroids (GCs) from the adrenal glands into the bloodstream (4). Elevations of circulating GCs, in turn, redirect individuals into a distinct emergency life history state (3) with changes in physiology and behavior (e.g., increased gluconeogenesis and mobilization of fat stores, suppressed territorial and reproductive behavior, irruptive migration; refs. 2 and 4), aimed at coping with the perturbation and recovering homeostasis at the expense of temporarily suppressing nonessential activities. The quantification of circulating GC titers has become a useful tool in psychology, animal husbandry, and conservation biology because elevations of plasma levels constitute a physiological marker of exposure to stress (e.g., refs. 5-7), and the latter has deleterious effects on fitness. However, even within animal populations exposed to constant environments there is a strong interindividual variability in the adrenocortical response to standardized stressors (8-10), and it remains unknown whether such natural variability exerts ...

All feathers were prepared by first removing the calamus (i.e. the proximal, vaneless portion of the quill) and the length of the remaining portion, or segments of it, was measured and weighed. None of the samples were washed prior to hormone analysis as natural substances such as preen oils were found not to influence the results (Bortolotti et al., 2008). Feathers were stored in ordinary paper envelopes between collection (various years, see below) and analysis (2004)(2005)(2006)(2007)(2008). Understanding what environmental perturbations are perceived as stressors, and quantifying how they are responded to, how often they occur and the negative consequences of exposure to glucocorticoids, has been problematic and limited to short-term physiological measures. By contrast, the quantification of corticosterone (CORT) in feathers represents a long-term, integrated measure of hypothalamic-pituitary-adrenal activity. In the present study, we show that by understanding how the hormone is deposited in feathers, in combination with specific sampling protocols, one can identify localised patterns of CORT deposition that reveal different temporal patterns of a bird's response to stressors. CORT in feathers appears to be stable over time, is resistant to heat exposure and is useful in determining both the overall exposure of the bird to the hormone over days or weeks, as well as identifying discrete, punctuated, stressful events. Variation in feather CORT can also be examined among individuals of a population at one point in time, as well as over years by using museum specimens. The ability to track stress over time allows for new questions to be asked about the health and ecology of birds and their environment.

Androgens and carotenoids play a fundamental role in the expression of secondary sex traits in animals that communicate information on individual quality. In birds, androgens regulate song, aggression, and a variety of sexual ornaments and displays, whereas carotenoids are responsible for the red, yellow, and orange colors of the integument. Parallel, but independent, research lines suggest that the evolutionary stability of each signaling system stems from tradeoffs with immune function: androgens can be immunosuppressive, and carotenoids diverted to coloration prevent their use as immunostimulants. Despite strong similarities in the patterns of sex, age and seasonal variation, social function, and proximate control, there has been little success at integrating potential links between the two signaling systems. These parallel patterns led us to hypothesize that testosterone increases the bioavailability of circulating carotenoids. To test this hypothesis, we manipulated testosterone levels of red-legged partridges Alectoris rufa while monitoring carotenoids, color, and immune function. Testosterone treatment increased the concentration of carotenoids in plasma and liver by >20%. Plasma carotenoids were in turn responsible for individual differences in coloration and immune response. Our results provide experimental evidence for a link between testosterone levels and immunoenhancing carotenoids that (i) reconciles conflicting evidence for the immunosuppressive nature of androgens, (ii) provides physiological grounds for a connection between two of the main signaling systems in animals, (iii) explains how these signaling systems can be evolutionary stable and honest, and (iv) may explain the high prevalence of sexual dimorphism in carotenoid-based coloration in animals.honest signaling ͉ immune function ͉ immunocompetence handicap hypothesis ͉ coloration P arallel, but independent, lines of research indicate that both androgens and carotenoids play a major role in the development and expression of honest signals of individual quality among vertebrates, relevant in sexual and social contexts (1-14). In birds, androgens control song, aggression, and a variety of sexual ornaments and displays (1-4), and carotenoids are bright red and yellow pigments of showy integument (6-12). In addition to signaling functions, testosterone regulates male sexual maturation and sperm production (15), and carotenoids have antioxidant and immunoenhancing activity (8,9,12,16,17). Signaling theory posits that the stability of traits conveying information on individual quality relies on the costs associated with signal production (18, 19), which prevent cheating in poor-quality individuals (they are ''honest'' signals) (19). Following this reasoning, the immunocompetence handicap hypothesis (ICHH) (20) posits that the honesty of androgen-dependent sexual traits relies on the immunosuppressive action of testosterone (21,22). Recent studies emphasize that carotenoid allocation to color displays imply diversion away from the immune and deto...