Demographic modeling to fine-tune conservation targets: importance of pre-adults for the decline of an endangered raptor.

Large, long-lived species with slow life histories and protracted pre-breeding stages are particularly susceptible to declines and extinction, often for unknown causes. Here, we show how demographic modeling of a medium-sized raptor, the Red Kite Milvus milvus, can aid to refocus conservation research and attention on the most likely mechanisms driving its decline. Red Kites' survival and reproduction increased through three sequential stages for 1-2, 3-6, and 7-30 yr of age, mainly corresponding to individuals that are dispersing, attempting to gain a territory, and breeding. As typical of long-lived species, elasticities were highest for adult (≥7 yr old) survival, but this was high, with little scope for improvement. Instead, the declines were driven by an extremely low survival of pre-adults in their first years of life, which weakened the whole demographic system by nullifying the offspring contribution of adults and curtailing their replacement by recruits. For example, 27 pairs were necessary to generate a single prime age adult. Simulation of management scenarios suggested that the decline could be halted most parsimoniously by increasing pre-adult survival to the mean levels recorded for other areas, while only the synergistic, simultaneous improvement of breeding success, adult and pre-adult survival could generate a recovery. We propose three actions to attain such goals through selective supplementary feeding of both breeding and non-breeding individuals, and through mortality improvement by GPS remote-sensing devices employed as surveillance monitoring tools. Our results show how improving demographic models by using real, local vital rates rather than "best guess" vital rates can dramatically improve model realism by refocusing attention on the actual stages and mortality causes in need of manipulation, thus building precious time and resources for conservation management. These results also highlight the frequent key role of pre-adult survival for the management of long-lived species, coherent with the idea of demographic systems as integrated chains only as strong as their weakest link.

Individual improvements and selective mortality shape lifelong migratory performance.

Billions of organisms, from bacteria to humans, migrate each year and research on their migration biology is expanding rapidly through ever more sophisticated remote sensing technologies. However, little is known about how migratory performance develops through life for any organism. To date, age variation has been almost systematically simplified into a dichotomous comparison between recently born juveniles at their first migration versus adults of unknown age. These comparisons have regularly highlighted better migratory performance by adults compared with juveniles, but it is unknown whether such variation is gradual or abrupt and whether it is driven by improvements within the individual, by selective mortality of poor performers, or both. Here we exploit the opportunity offered by long-term monitoring of individuals through Global Positioning System (GPS) satellite tracking to combine within-individual and cross-sectional data on 364 migration episodes from 92 individuals of a raptorial bird, aged 1-27 years old. We show that the development of migratory behaviour follows a consistent trajectory, more gradual and prolonged than previously appreciated, and that this is promoted by both individual improvements and selective mortality, mainly operating in early life and during the pre-breeding migration. Individuals of different age used different travelling tactics and varied in their ability to exploit tailwinds or to cope with wind drift. All individuals seemed aligned along a race with their contemporary peers, whose outcome was largely determined by the ability to depart early, affecting their subsequent recruitment, reproduction and survival. Understanding how climate change and human action can affect the migration of younger animals may be the key to managing and forecasting the declines of many threatened migrants.

Corticosterone implants produce stress-hyporesponsive birds.

In birds, the use of corticosterone (Cort) implants is a frequent tool aimed at simulating systemic elevations of this hormone and studying effects on biological traits (e.g. physiology, morphology, behavior). This manipulation may alter adrenocortical function, potentially changing both baseline (CortBAS) and stress-induced (CortSTRESS) plasma Cort levels. However, implant effects on the latter trait are rarely measured, disregarding downstream consequences of potentially altered stress responses. Here, we analyzed the effects of Cort implants on both CortBAS and CortSTRESS in nestling and adult European white storks, Ciconia ciconia In addition, we performed a review of 50 studies using Cort implants in birds during the last two decades to contextualize stork results, assess researchers' patterns of use and infer current study biases. High and low doses of Cort implants resulted in a decrease of both CortBAS (31-71% below controls) and CortSTRESS (63-79% below controls) in storks. Our literature review revealed that CortBAS generally increases (72% of experiments) whereas CortSTRESS decreases (78% of experiments) following implant treatment in birds. Our results challenge and expand the prevailing assumption that Cort implants increase circulating CortBAS levels because: (i) CortBAS levels show a quadratic association with implant dose across bird species, and decreased levels may occur at both high and low implant doses, and (ii) Cort implants also decrease CortSTRESS levels, thus producing stress-hyporesponsive phenotypes. It is time to work towards a better understanding of the effects of Cort implants on adrenocortical function, before addressing downstream links to variation in other biological traits.

Selection on a behaviour-related gene during the first stages of the biological invasion pathway.

Human-induced biological invasions are common worldwide and often have negative impacts on wildlife and human societies. Several studies have shown evidence for selection on invaders after introduction to the new range. However, selective processes already acting prior to introduction have been largely neglected. Here, we tested whether such early selection acts on known behaviour-related gene variants in the yellow-crowned bishop (Euplectes afer), a pet-traded African songbird. We tested for nonrandom allele frequency changes after trapping, acclimation and survival in captivity. We also compared the native source population with two independent invasive populations. Allele frequencies of two SNPs in the dopamine receptor D4 (DRD4) gene-known to be linked to behavioural activity in response to novelty in this species-significantly changed over all early invasion stages. They also differed between the African native population and the two invading European populations. The two-locus genotype associated with reduced activity declined consistently, but strongest at the trapping stage. Overall genetic diversity did not substantially decrease, and there is little evidence for new alleles in the introduced populations, indicating that selection at the DRD4 gene predominantly worked on the standing genetic variation already present in the native population. Our study demonstrates selection on a behaviour-related gene during the first stages of a biological invasion. Thus, pre-establishment stages of a biological invasion do not only determine the number of propagules that are introduced (their quantity), but also their phenotypic and genetic characteristics (their quality).

Lifetime variation in feather corticosterone levels in a long-lived raptor.

In long-lived animals, the challenges that threaten individual homeostasis, and the way they are dealt with, are expected to vary in an age-related manner, encompassing the progressive selection of superior phenotypes and the acquisition and improvement of key skills (e.g. foraging, breeding and fighting abilities). Since exposure to homeostatic challenges typically elevates circulating glucocorticoid (GC) levels in vertebrates (modulating the behavioural and physiological responses that mediate allostasis), we may expect concomitant age-related changes in these hormones. Here, we investigated whether the level of corticosterone (the main avian GC) deposited in feathers during regular moult reflected the expected lifelong progression of energetic challenges in a long-lived raptor, the black kite (Milvus migrans). Feather corticosterone values were highest in the youngest birds, gradually declined to reach minimum levels in prime age, 7- to 11-year-old birds, and then increased again slightly among the oldest, senescent birds (≥12 years old). This pattern mirrored the age-related changes in reproductive success and survival rates previously reported for this population, suggesting that feather corticosterone levels captured the most vulnerable and challenging periods experienced by these birds as they proceeded through life. Moreover, feather corticosterone levels were negatively related to body size, suggesting that larger birds either experienced fewer homeostatic challenges, or were better able to cope with them. Feather corticosterone measures thus provided a valuable snapshot of how allostatic loads vary along the life of individuals, supporting the idea of a tight, long-term link between cumulative physiological responses to ecological challenges and demographic performance.

White stork movements reveal the ecological connectivity between landfills and different habitats.

BACKGROUND: Connections between habitats are key to a full understanding of anthropic impacts on ecosystems. Freshwater habitats are especially biodiverse, yet depend on exchange with terrestrial habitats. White storks (Ciconia ciconia) are widespread opportunists that often forage in landfills and then visit wetlands, among other habitats. It is well known that white storks ingest contaminants at landfills (such as plastics and antibiotic resistant bacteria), which can be then deposited in other habitats through their faeces and regurgitated pellets. METHODS: We characterized the role of white storks in habitat connectivity by analyzing GPS data from populations breeding in Germany and wintering from Spain to Morocco. We overlaid GPS tracks on a land-use surface to construct a spatially-explicit network in which nodes were sites, and links were direct flights. We then calculated centrality metrics, identified spatial modules, and quantified overall connections between habitat types. For regional networks in southern Spain and northern Morocco, we built Exponential Random Graph Models (ERGMs) to explain network topologies as a response to node habitat. RESULTS: For Spain and Morocco combined, we built a directed spatial network with 114 nodes and 370 valued links. Landfills were the habitat type most connected to others, as measured by direct flights. The relevance of landfills was confirmed in both ERGMs, with significant positive effects of this habitat as a source of flights. In the ERGM for southern Spain, we found significant positive effects of rice fields and salines (solar saltworks) as sinks for flights. By contrast, in the ERGM for northern Morocco, we found a significant positive effect of marshes as a sink for flights. CONCLUSIONS: These results illustrate how white storks connect landfills with terrestrial and aquatic habitats, some of which are managed for food production. We identified specific interconnected habitat patches across Spain and Morocco that could be used for further studies on biovectoring of pollutants, pathogens and other propagules.

Compensation for wind drift during raptor migration improves with age through mortality selection.

Each year, billions of flying and swimming migrants negotiate the challenging displacement imposed by travelling through a flowing medium. However, little is known about how the ability to cope with drift improves through life and what mechanisms drive its development. We examined 3,140 days of migration by 90 GPS-tagged raptorial black kites (Milvus migrans) aged 1-27 years to show that the ability to compensate for lateral drift develops gradually through many more years than previously appreciated. Drift negotiation was under strong selective pressure, with inferior navigators subject to increased mortality. This progressively selected for adults able to compensate for current cross flows and for previously accumulated drift in a flexible, context-dependent and risk-dependent manner. Displacements accumulated en route carried over to shape the wintering distribution of the population. For many migrants, migratory journeys by younger individuals represent concentrated episodes of trait selection that shape adult populations and mediate their adaptation to climate change.

Hardship at birth alters the impact of climate change on a long-lived predator.

Climate change is increasing the frequency of extreme events, such as droughts or hurricanes, with substantial impacts on human and wildlife communities. Extreme events can affect individuals through two pathways: by altering the fitness of adults encountering a current extreme, and by affecting the development of individuals born during a natal extreme, a largely overlooked process. Here, we show that the impact of natal drought on an avian predator overrode the effect of current drought for decades, so that individuals born during drought were disadvantaged throughout life. Incorporation of natal effects caused a 40% decline in forecasted population size and a 21% shortening of time to extinction. These results imply that climate change may erode populations more quickly and severely than currently appreciated, suggesting the urgency to incorporate "penalties" for natal legacies in the analytical toolkit of impact forecasts. Similar double impacts may apply to other drivers of global change.

Selection on individuals of introduced species starts before the actual introduction.

Biological invasion is a global problem with large negative impacts on ecosystems and human societies. When a species is introduced, individuals will first have to pass through the invasion stages of uptake and transport, before actual introduction in a non-native range. Selection is predicted to act during these earliest stages of biological invasion, potentially influencing the invasiveness and/or impact of introduced populations. Despite this potential impact of pre-introduction selection, empirical tests are virtually lacking. To test the hypothesis of pre-introduction selection, we followed the fate of individuals during capture, initial acclimation, and captivity in two bird species with several invasive populations originating from the international trade in wild-caught pets (the weavers Ploceus melanocephalus and Euplectes afer). We confirm that pre-introduction selection acts on a wide range of physiological, morphological, behavioral, and demographic traits (incl. sex, age, size of body/brain/bill, bill shape, body mass, corticosterone levels, and escape behavior); these are all traits which likely affect invasion success. Our study thus comprehensively demonstrates the existence of hitherto ignored selection acting before the actual introduction into non-native ranges. This could ultimately change the composition and functioning of introduced populations, and therefore warrants greater attention. More knowledge on pre-introduction selection also might provide novel targets for the management of invasive species, if pre-introduction filters can be adjusted to change the quality and/or quantity of individuals passing through such that invasion probability and/or impacts are reduced.

Simultaneous analysis of multiple PCR amplicons enhances capillary SSCP discrimination of MHC alleles.

Major histocompatibility complex (MHC) genotyping still remains one of the most challenging issues for evolutionary ecologists. To date, none of the proposed methods have proven to be perfect, and all provide both important pros and cons. Although denaturing capillary electrophoresis has become a popular alternative, allele identification commonly relies upon conformational polymorphisms of two single-stranded DNA molecules at the most. Using the MHC class II (beta chain, exon 2) of the black kite (Aves: Accipitridae) as our model system, we show that the simultaneous analysis of overlapping PCR amplicons from the same target region substantially enhances allele discrimination. To cover this aim, we designed a multiplex PCR capable to generate four differentially sized and labeled amplicons from the same allele. Informative peaks to assist allele calling then fourfold those generated by the analysis of single PCR amplicons. Our approach proved successful to differentiate all the alleles (N=13) isolated from eight unrelated birds at a single optimal run temperature and electrophoretic conditions. In particular, we emphasize that this approach may constitute a straightforward and cost-effective alternative for the genotyping of single or duplicated MHC genes displaying low to moderate sets of divergent alleles.

Proximate and ultimate factors explaining floating behavior in long-lived birds.

Avian populations are composed of reproductive individuals coexisting in space and time with young, non-breeding conspecifics or "floaters". Despite the fact that first breeding can be delayed for years and can exert profound effects on fitness, many aspects of the behavior, ecology and physiology of young floaters remain poorly understood. By means of combining endocrine, behavioral and life history information from a population of black kites (Milvus migrans) monitored long-term, we tested several hypotheses suggesting endocrine function as a determinant of floating status. Sexual function in non-breeding males, estimated through determination of systemic testosterone and progesterone levels, was similar to that in reproductively active conspecifics. Floating females, on the contrary, displayed an endocrine pattern of circulating estrogens and progesterone that was parallel in timing but reduced in magnitude as compared to breeders. Our results suggest that floaters are not physiologically constrained to reproduce, but the cost-benefit balance of attaining complete gonadal function is sexually dependent. While young, unmated males could increase their breeding prospects by attaining sexual maturity regardless of their social environment, natural selection would favor females relying on social cues to mature. Consistent with the sexual roles of socially monogamous species, gonadal recrudescence and testosterone production would allow unmated males to access breeding resources (e.g. through male-male competition and extra-pair fertilizations). Unmated females, on the contrary, would reduce physiological costs by means of delaying ovarian maturation until establishing pair bonds with a male providing access to breeding resources.

Reproductive endocrinology of wild, long-lived raptors.

The last decades have witnessed a surge of studies analyzing the role of sex hormones on the behavior and ecology of wild bird populations, allowing a more integrated view of the evolution of avian physiology and life histories. Despite a marked progress, field studies show a considerable bias towards research on specific phylogenetic groups, neglecting a significant fraction of the class Aves. Here we analysed changes in the circulating levels of sex steroids in relation to reproductive behaviour in wild black kites (Milvus migrans), a long-lived and socially monogamous Accipitridae raptor. Males and females displayed a single seasonal peak of circulating testosterone (males) and estradiol (females) during pre-laying and laying. Absolute male testosterone levels were low even at the seasonal maximum and remained below detection limits in females. The latter results supports the idea that avian species establishing long-term pair bonds require lower amounts of circulating androgens for reproduction. Circulating progesterone showed a single seasonal peak in females and males, but their timing (during Incubation and Post-brooding respectively) did not overlap. The fact that females black kites perform the majority of incubation and males provide the majority of care to fledglings suggests that progesterone is involved in the expression of parental behaviors.

Differences in adrenocortical responses between urban and rural burrowing owls: poorly-known underlying mechanisms and their implications for conservation.

The hypothalamus-pituitary-adrenal/interrenal (HPA) axis of vertebrates integrates external information and orchestrates responses to cope with energy-demanding and stressful events through changes in circulating glucocorticoid levels. Urbanization exposes animals to a wide variety of ever-changing stimuli caused by human activities that may affect local wildlife populations. Here, we empirically tested the hypothesis that urban and rural owls (Athene cunicularia) show different adrenocortical responses to stress, with urban individuals showing a reduced HPA-axis response compared to rural counterparts to cope with the high levels of human disturbance typical of urban areas. We applied a standard capture-restraint protocol to measure baseline levels and stress-induced corticosterone (CORT) responses. Urban and rural owls showed similar circulating baseline CORT levels. However, maximum CORT levels were attained earlier and were of lower magnitude in urban compared to rural owls, which showed a more pronounced and long-lasting response. Variability in CORT responses was also greater in rural owls and contained the narrower variability displayed by urban ones. These results suggest that only individuals expressing low-HPA-axis responses can thrive in cities, a pattern potentially mediated by three alternative and non-exclusive hypotheses: phenotypic plasticity, natural selection and matching habitat choice. Due to their different conservation implications, we recommend further research to properly understand wildlife responses to humans in an increasingly urbanized world.

Overland and oversea migration of white storks through the water barriers of the straits of Gibraltar.

Soaring landbirds typically exploit atmospheric uplift as they fly overland, displaying a highly effective energy-saving locomotion. However, large water bodies lack thermal updrafts, potentially becoming ecological barriers that hamper migration. Here we assessed the effects of a sea surface on the migratory performance of GPS-tagged white storks (Ciconia ciconia) before, during and after they crossed the straits of Gibraltar. Oversea movements involved only flapping and gliding and were faster, traversed in straighter, descending trajectories and resulted in higher movement-related energy expenditure levels than overland, supporting the water barrier hypothesis. Overland movements at both sides of the sea straits resulted in tortuous routes and ascending trajectories with pre-crossing flights showing higher elevations and more tortuous routes than post-crossing, thus supporting the barrier negotiation hypothesis. Individual positions at both ends of the sea narrow were predicted by zonal winds and storks´ location at entry in the European hinterland, and birds did not show compensational movements overland in anticipation to subsequent wind displacements oversea. The length of the water narrow at departure shore, the elevation therein and the winds on route affected major components of sea crossing performance (such as distances and times overwater, minimum elevations, climb angles, speeds and energy expenditure), supporting the departure position and oversea winds hypotheses. In summary, our study provides a prime example at high temporal resolution of how birds adjust their behavior and physiology as they interact with the changing conditions of the travelling medium, reallocating resources and modifying their movement to overcome an ecological barrier.

Foraging at Solid Urban Waste Disposal Sites as Risk Factor for Cephalosporin and Colistin Resistant Escherichia coli Carriage in White Storks (Ciconia ciconia).

White stork (Ciconia ciconia) may act as a reservoir and vehicle of cephalosporin resistant (CR) Escherichia coli. Between 2011 and 2014, we sampled white storks from colonies exposed to different degrees of anthropic pressure across the major areas of natural distribution of white storks in Spain. Cloacal swab samples (n = 467) were obtained from individuals belonging to 12 different colonies from six different regions. Additionally, 70 samples were collected from recently deposited droppings at the base of nesting platforms. We phenotypically characterized E. coli isolates, confirmed presence of CR genes and classified plasmids. Risk factors for acquiring these genes were assessed. Overall, 8.8% (41 out of 467) storks carried CR E. coli in their cloaca and five (7.1%) were identified from recently deposited droppings; therefore, 46 isolates were further characterized. Of them, 20 contained bla CTX-M- 1, nine bla CMY- 2, six bla CTX-M- 14, four bla SHV- 12, three bla CTX-M- 15, two bla CTX-M- 32, one bla CTX-M- 1 together with bla CMY- 2, and one bla CTX-M- 1 together with bla SHV- 12. All were multidrug-resistant, and four harbored the plasmid-mediated colistin resistance mcr-1 gene. CR genes were associated with the presence of IncI1, IncFIB, and IncN replicon families. XbaI-macrorestriction analysis revealed a great diversity among most of the XbaI-PFGE types, but indistinguishable types were also seen with isolates obtained from different locations. Clonal complex 10 was the most common among CR E. coli and two bla CTX-M- 15 positive isolates were identified as B2-ST131. Carriage of CR E. coli was significantly higher in colonies located close to solid urban waste disposal sites in which foraging on human waste was more likely and in one case to cattle grazing. The co-occurrence of bla CMY- 2 and mcr-1 on plasmids of E. coli isolated from wild birds as early as 2011 is of note, as the earliest previous report of mcr-1 in wild birds is from 2016. Our study shows that foraging at landfills and in association with cattle grazing are important risk factors for the acquisition of CR E. coli in white storks.

Predictors of floater status in a long-lived bird: a cross-sectional and longitudinal test of hypotheses.

1. Few studies have been capable of monitoring the nonterritorial sector of a population because of the typically secretive behaviour of floating individuals, despite the existing consensus over the demographic importance of floating. Furthermore, there is almost no information on floating behaviour for migratory species. 2. The factors that determine whether an individual will be a floater or a territory owner have been framed into five, non-mutually exclusive hypotheses: (i) territory holders are morphologically superior to floaters (resource-holding potential hypothesis); (ii) age confers skills and fighting motivation which lead to social dominance and territory ownership (age hypothesis); (iii) occupancy time of a site determines asymmetries in its knowledge, familiarity and value for potential contenders (site-dominance hypothesis); (iv) contenders use an arbitrary rule to settle contests leading to pre-defined cut-off points for a biologically meaningful trait (e.g. age, body size) separating floaters from territory holders (arbitrary convention hypothesis); and (v) floaters set up a 'war of attrition' at arbitrarily chosen territories (arbitrary attrition hypothesis). 3. We tested these hypotheses using long-term data on a long-lived, migratory raptor, the black kite Milvus migrans Boddaert. 4. Floating status was best explained by the concerted action of mechanisms consistent with the age and site-dominance hypotheses. 5. In both cross-sectional and longitudinal analyses, acquisition of a territory was determined by a complex interaction between age and early arrival from migration, suggesting: (i) a progressive incorporation of early arriving individuals in the territorial contingent of the population, and (ii) the existence of an alternative restraint strategy of delayed territoriality mediated by long-term acquisition of social dominance. 6. Such results suggested that territory acquisition was mediated by the establishment of site dominance through pre-emption and, secondarily, despotism. In this population, age and arrival date aligned individuals along a demographic continuum ranging from successful breeders monopolizing high-quality resources to floaters with no resources, consistent with the notion of floating as an extreme form of breeding failure.

Short- and long-term consequences of individual and territory quality in a long-lived bird.

Site-quality is a major determinant of fitness but its effect can be confounded by individual quality, a relationship that has been little studied in large, long-lived vertebrates. The fitness effects of quality estimates depend on the assumption of co-variation between individual and territory quality and can be framed as five working hypotheses: no effect on fitness, exclusive effect of individual quality, exclusive effect of site quality, and independent or interactive effects of the two. We explored such a framework using a medium-sized raptor, the black kite Milvus migrans, as a model species. Individual and territory quality co-varied, but the strength of the relationship varied across different estimates of individual quality (age, body size, or mass residuals). Short-term production of fledglings was related to the independent effects of both individual and territory quality. However, longer-term production of recruits was related solely to territory quality. The disappearance of individual quality effects over the long-term may be caused by antagonistic selective pressures acting during different stages of the life cycle. Our results contribute to a growing appreciation of the long-term fitness-benefits of advantages experienced in early life and highlight the importance of a long-term perspective in studies assessing the effects of individual and territory quality. In our case study, prioritizing sites for conservation on the basis of territory quality may be a feasible pathway to maintain the viability of the population. However, scenarios where such a method could be inefficient have been previously reported, suggesting caution in its application. More studies are needed to understand the generality of the efficiency of priority-setting approaches based on site quality.

When and where mortality occurs throughout the annual cycle changes with age in a migratory bird: individual vs population implications.

The annual cycle of most animals is structured into discrete stages, such as breeding, migration and dispersal. While there is growing appreciation of the importance of different stages of an organism's annual cycle for its fitness and population dynamics, almost nothing is known about if and how such seasonal effects can change through a species lifespan. Here, we take advantage of the opportunity offered by a long-term satellite/GPS-tracking study and a reliable method of remote death-detection to show that certain stages of both the annual and life cycle of a migratory long-lived raptor, the Black kite Milvus migrans, may represent sensitive bottlenecks for survival. In particular, migratory journeys caused bursts of concentrated-mortality throughout life, but the relative importance of stage-specific survival changed with age. On the other hand, the balance between short-stages of high mortality and long-stages of low mortality made population-growth similarly dependent on all portions of the annual cycle. Our results illustrate how the population dynamics of migratory organisms can be inextricably linked to ecological pressures balanced over multiple stages of the annual cycle and thus multiple areas of the globe, suggesting the frequent need for challenging conservation strategies targeting all portions of a species year-round range.

Adrenocortical response to stress and thyroid hormone status in free-living nestling white storks (Ciconia ciconia) exposed to heavy metal and arsenic contamination.

BACKGROUND/OBJECTIVE: Endocrine parameters have proven useful in the detection of early or low-level responses to pollutants. Although most of the studies on endocrine modulation have been focused on processes involving gonadal steroids, contaminants may target other parts of the endocrine system as well. In this study we examined the adrenocortical stress response and thyroid hormone status in free-living nestling white storks (Ciconia ciconia) in relation to heavy metals (zinc, lead, copper, cadmium) and arsenic levels in blood. METHODS: Fieldwork was conducted in an area polluted by the Aznalcóllar mine accident (southwestern Spain) and in a reference site. We used a standardized capture, handling, and restraint protocol to determine both baseline and maximum plasma corticosterone. Circulating levels of thyroxine (T4) and triiodothyronine (T3) were also measured. RESULTS: No effects of metals or As were found on baseline corticosterone, but maximum levels of corticosterone were positively related to Pb in both locations. This relationship was stronger in single nestlings than in birds from multiple-chick broods, which suggests a greater impact of Pb on more stressed individuals. Metal pollution did not affect plasma T4 or T3 levels, although thyroid status differed with location. CONCLUSIONS: Because a compromised hypothalamus-pituitary-adrenal (HPA) function can have far-reaching consequences in terms of altered behavioral and metabolic processes necessary for survival, our results suggest that birds exposed to sublethal Pb levels may be at risk through an altered adrenocortical stress response, and further support the idea that HPA axis-related end points might be useful indicators of metal exposure and potential toxicity in wild animals.

Effects of Ontogeny, Diel Rhythms, and Environmental Variation on the Adrenocortical Physiology of Semialtricial Black Kites (Milvus migrans).

We examined whether hypothalamic-pituitary-adrenal axis activity in the nestlings of a semialtricial raptor, the black kite (Milvus migrans), varied with advancing age, throughout the day, and in response to a number of socioecological factors presumed to affect allostatic load. Both baseline corticosterone (CORT) titers and maximum CORT levels during 30 min of handling and restraint augmented across all sampled ages, suggesting that nestlings' energetic demands and capacity to respond to perturbations increase progressively throughout development. CORT secretion also peaked in the early morning, reached minimum levels in the central hours of the day, and increased again before dusk, suggesting a possible role of CORT in the regulation of activity-inactivity patterns. Finally, nestlings raised in a year of low marsh inundation, implying lower food availability and heightened allostatic loads, exhibited higher adrenocortical responsiveness to stress than nestlings raised in years of intermediate or high flooding. The nondetectable effect of other socioecological variables, such as territory quality, temperature, or brood order, suggests that parents may be able to buffer their nestlings from adverse environmental conditions or that the effect of such factors may have been obscured by selective mortality operating before sampling. We propose that future studies increase the simultaneous use of complementary techniques (fecal sampling, feather analysis) to reach firmer and more comprehensive conclusions, especially for planning the management and conservation of sensitive species.