Behaviour-related DRD4 polymorphisms in invasive bird populations.

It has been suggested that individual behavioural traits influence the potential to successfully colonize new areas. Identifying the genetic basis of behavioural variation in invasive species thus represents an important step towards understanding the evolutionary potential of the invader. Here, we sequenced a candidate region for neophilic/neophobic and activity behaviour - the complete exon 3 of the DRD4 gene - in 100 Yellow-crowned bishops (Euplectes afer) from two invasive populations in Spain and Portugal. The same birds were scored twice for activity behaviour while exposed to novel objects (battery or slice of apple) in captivity. Response to novel objects was repeatable (r = 0.41) within individuals. We identified two synonymous DRD4 SNPs that explained on average between 11% and 15% of the phenotypic variance in both populations, indicating a clear genetic component to the neophilic/neophobic/activity personality axis in this species. This consistently high estimated effect size was mainly due to the repeated measurement design, which excludes part of the within-individual nongenetic variance in the response to different novel objects. We suggest that the alternative alleles of these SNPs are likely introduced from the original population and maintained by weak or antagonistic selection during different stages of the invasion process. The identified genetic variants have not only the potential to serve as genetic markers of the neophobic/neophilic/activity personality axis, but may also help to understand the evolution of behaviour in these invasive bird populations.

Population fragmentation leads to isolation by distance but not genetic impoverishment in the philopatric Lesser Kestrel: a comparison with the widespread and sympatric Eurasian Kestrel.

Population fragmentation is a widespread phenomenon usually associated with human activity. As a result of habitat transformation, the philopatric and steppe-specialist Lesser Kestrel Falco naumanni underwent a severe population decline during the last century that increased population fragmentation throughout its breeding range. In contrast, the ubiquitous Eurasian Kestrel Falco tinnunculus did not suffer such adverse effects, its breeding range still remaining rather continuous. Using microsatellites, we tested the effects of population fragmentation on large-scale spatial patterns of genetic differentiation and diversity by comparing these two sympatric and phylogenetically related species. Our results suggest that habitat fragmentation has increased genetic differentiation between Lesser Kestrel populations, following an isolation-by-distance pattern, while the population of Eurasian Kestrels is panmictic. Contrary to expectations, we did not detect significant evidence of reduced genetic variation or increased inbreeding in Lesser Kestrels. Although this study reports genetic differentiation in a species that has potential for long-distance dispersal but philopatry-limited gene flow, large enough effective population sizes and migration may have been sufficient to mitigate genetic depauperation. A serious reduction of genetic diversity in Lesser Kestrels would, therefore, only be expected after severe population bottlenecks following extreme geographic isolation.

Validation of loop-mediated isothermal amplification for fast and portable sex determination across the phylogeny of birds.

PCR is a universal tool for the multiplication of specific DNA sequences. For example, PCR-based sex determination is widely used, and a diversity of primer sets is available. However, this protocol requires thermal cycling and electrophoresis, so results are typically obtained in laboratories and several days after sampling. Loop-mediated isothermal amplification (LAMP) is an alternative to PCR that can take molecular ecology outside the laboratory. Although its application has been successfully probed for sex determination in three species of a single avian Family (raptors, Accipitridae), its generality remains untested and suitable primers across taxa are lacking. We designed and tested the first LAMP-based primer set for sex determination across the modern birds (NEO-W) based on a fragment of the gene chromo-helicase-DNA-binding protein located on the female-specific W chromosome. As nucleotide identity is expected to increase among more related taxa, taxonomically targeted primers were also developed for the Order Falconiformes and Families Psittacidae, Ciconiidae, Estrildidae and Icteridae as examples. NEO-W successfully determined sex in a subset of 21 species within 17 Families and 10 Orders and is therefore a candidate primer for all modern birds. Primer sets designed specifically for the selected taxa correctly assigned sex to the evaluated species. A short troubleshooting guide for new LAMP users is provided to identify false negatives and optimize LAMP reactions. This study represents the crucial next step towards the use of LAMP for molecular sex determination in birds and other applications in molecular ecology.

Offspring body condition and immunocompetence are negatively affected by high breeding densities in a colonial seabird: a multiscale approach.

Why avian colonies vary in size and how food competition among nearby colonies affects offspring quality are still not completely understood. We simultaneously examined the effects of four scales of breeding density on two measures of offspring viability (body condition and T-cell-mediated immunity) in the colonial Magellanic penguin. Body condition of fledglings was inversely correlated with breeding density within 100 m(2) of nests, and decreased with increasing numbers of breeding pairs competing within the parental foraging ranges (100 km), probably as a result of density-dependent food depletion. The T-cell-mediated immune response was positively correlated with body condition, reflecting, to some extent, the previous breeding-density effects, and was negatively correlated with colony size, which may be related to social stress. However, given the effect of protein intake on cell immunity, this result could also indicate a thus far neglected cost of coloniality, namely the consumption of low-protein food to compensate for the depletion of optimal prey. These results were not influenced by other traits, nor by the current exposure of birds to parasites and diseases, as measured by serological variables. Since body condition and the T-cell-mediated immune response of fledgling birds are indicators of their survival and recruitment prospects, the costs we have identified can explain variability in colony size in relation to food competition with surrounding colonies, as well as the skewed distribution toward small colonies in this species.

The T-cell-mediated immune response and return rate of fledgling American kestrels are positively correlated with parental clutch size.

Life-history theory predicts that parents face a trade-off between the number and viability of the progeny they produce. We found evidence for an apparent trade-off in a free-living population of American kestrels (Falco sparverius), as larger clutches produced more but lighter fledglings. However, while the body mass of fledglings has traditionally been used as a measure of survival prospect, offspring immunocompetence should also play an important role. We thus measured the T-cell-mediated immune response of fledgling kestrels in relation to brood traits and nest-rearing conditions through a cross-fostering experiment. The immune response was positively correlated with the body condition of fledglings, but was also higher in those hatched from five-egg than four-egg clutches. These results were not influenced by other brood traits, nor by current exposure to stressors and infectious agents, as measured by serological variables. Such ability to resist pathogens may account for why the probability of offspring returning to the study area in subsequent years, when controlling for brood size, was higher for five-egg than four-egg clutches. These results suggest an optimal clutch size through maternal effects on offspring immunocompetence rather than a trade-off between the number and quality of the offspring.

Genetics, local environment and health as factors influencing plasma carotenoids in wild American kestrels (Falco sparverius).

Carotenoids are important as pigments for bright coloration of animals, and as physiologically active compounds with a wide array of health-related functions. Carotenoid-dependent coloration may have evolved as a signal to conspecifics; however, factors that may limit availability of carotenoids are poorly known. We investigated how the acquisition of carotenoids may be constrained by availability in the environment, diet, genetic make-up and health status of wild American kestrels (Falco sparverius). Plasma concentrations of siblings at the time of fledging showed a high degree of resemblance; however, a cross-fostering experiment revealed that variance was largely explained by nest of rearing, rather than nest of origin, thus indicating a low genetic component. A multivariate analysis of attributes of nestlings (sex, size, plasma proteins, immune function), parental reproduction (laying date, clutch size) and rearing conditions (brood size, size hierarchy, nestling mortality) showed only a small significant effect of leucocyte differentials on carotenoid concentrations of nestlings. A strong environmental effect on plasma carotenoids was demonstrated by levels of adult kestrels being correlated within mated pairs, and having a significant association with the abundance of voles, the primary prey species, per territory.

Environmental and genetic variation in T-cell-mediated immune response of fledgling American kestrels.

We investigated genetic and environmental components of variance in avian T-cell-mediated immune response (CMI) through a cross-fostering experiment conducted on wild American kestrels (Falco sparverius). CMI was evaluated in vivo by an experimental challenge with phytohaemagglutinin, a T-cell mitogen, injected intradermally in fledglings. Additionally, we assessed two measures of nutritional condition (body mass and circulating plasma proteins) which could influence the variance components of CMI. A two-way nested ANOVA indicated that CMI of fledgling kestrels was explained more by the nest where the bird was reared (33% of the explained variance) than by the nest of origin (12%). Body mass was explained equally by familial and environmental components, while plasma proteins were only related to the rearing environment. CMI of fledglings was not related to their circulating plasma proteins, but was positively correlated with their body mass. Fledgling body mass seemed to be influenced by pre-hatching or post-hatching maternal effects prior to manipulation since resemblance in body mass of sibships at the age of manipulation was high (h 2≤0.58), and body mass at this age predicted body mass at fledging. Therefore, pre-manipulation parental effects on body mass, such as investment in egg size, could have inflated the familial effects on body mass of fledglings and then on its correlated CMI. When controlling for body mass, most of the variation in CMI of fledglings was explained by the nest where the bird was reared (36.6%), while the variance explained by the nest of origin (4%) was not significant. This means that environmental influences are major determinants of offspring CMI. The low proportion of variance explained by the familial component may have been due to the high correlation of CMI to fitness.

DNA damage in birds after the mining waste spill in southwestern Spain: a Comet assay evaluation.

In April 1998, an ecological disaster resulting from a massive toxic spill of mining acid waste rich in heavy metals posed a serious threat to the Doñana National Park in southwestern Spain. This especially important protected area is the nesting and breeding site for many endangered bird species; white storks (Ciconia ciconia) and black kites (Milvus migrans) are considered the more representative. The suitability of the Comet assay as a biomarker for genotoxic analysis in environmental biomonitoring has been recently validated in studies using different sentinel organisms such as fish, amphibians, rodents, or mollusks. Birds preying on a variety of invertebrate and vertebrate species in the marshlands are appropriate for evaluating the potential deleterious effects of the toxic spill on wildlife of the Dofiana area. Our study on wetland birds high on the aquatic trophic chain sampled within a few months after the toxic spill in the area around Doñana National Park has shown the accumulation of heavy metals. Fourteen months after the mine waste spill, blood samples from white storks and kites collected in the neighborhood of the park and from control birds at reference areas for comparison were examined by fluorescence image analysis after lymphocyte isolation, and by subsequent alkaline single-cell gel (SCG) electrophoresis, known as the Comet assay. Our results indicate that the exposed birds had a significantly increased level of genotoxic damage compared with control animals from noncontaminated locations.

Diet explains interpopulation variation of plasma carotenoids and skin pigmentation in nestling white storks.

Carotenoids have a dietary origin in birds, but mechanisms by which they are absorbed in the gut, transported in the blood, metabolized at various sites, and deposited in the integument remain poorly understood. Variation in both plasma carotenoid levels and external color may reflect different access to dietary carotenoids or individual physiological differences in the uptake and deposition of carotenoids. We compared total plasma carotenoid concentration in nestling white storks (Ciconia ciconia) from 11 Spanish colonies in two consecutive years. The main food item in one of the colonies was the red swamp crayfish (Procambarus clarkii), a recently introduced species. Storks in the remaining colonies ate a variety of foods but no crayfish. Total plasma carotenoid levels in the colony where crayfish were consumed were about five times higher than in any other colony. These differences were maintained after controlling for the significant interyear variability, as well as for sex, age, and body mass of birds. Skin pigmentation also differed, being intensely orange in storks that consumed crayfish but white (unpigmented) in the remaining individuals. With thin-layer chromatography (TLC) and electronic absorption spectroscopy, astaxanthin was confirmed as the major carotenoid in crayfish as well as in the plasma, skin, and body fat of crayfish-eating storks, whereas lutein was the main carotenoid in plasma samples from the other colonies. These results indicate that a newly available carotenoid in the environment, astaxanthin, can be absorbed in large quantities from the gut and be transported in the blood before deposition in different tissues.

Sources of variability in aggregation and sex ratios of Crataerina melbae (Diptera: Hippoboscidae) among adult colonial alpine swifts.

Aggregation of Crataerina melbae flies on breeding adult alpine swifts (Apus melba) was low when compared with other host-parasite systems and varied with sampling date, year, and sex of the flies. Generalized linear models were performed to ascertain which factors, extrinsic and/or intrinsic to the host, explained variability in the number of louse flies present on a single host, i.e., abundance. Overall abundance was unrelated to any host characteristic but varied slightly among years. Abundance of female flies varied among years, but also with date of sampling, the number of females increasing as the breeding season advanced. In contrast, abundance of males decreased as the season progressed, independently of host characteristics. Despite these different patterns, the number of flies of each sex on a given host was strongly intercorrelated. These results suggest that mate attraction may explain aggregation patterns in this louse fly species. Overall sex ratio of louse flies did not differ from unity. However, the proportion of males decreased during the breeding season, as a consequence of the opposite sex-related seasonal patterns in parasite abundance. Sex-ratio variability was not related to host characteristics or to infrapopulation sizes.

Are hippoboscid flies a major mode of transmission of feather mites?

Feather mites (Astigmata) are distributed around the world, living on the feathers of birds, but their mechanisms for transmission among hosts are not fully understood. There is anecdotal evidence of feather mites attached to louseflies (Diptera: Hippoboscidae), suggesting that feather mites may use these flies as a mode of phoretic transmission among birds. Two bird-lousefly associations (alpine swift Apus melba-Crataerina melbae and feral pigeon Columba livia-Pseudolynchia canariensis) were inspected to test the hypothesis that feather mites use hippoboscid flies as major mode of transmission. Both bird species showed a high prevalence and abundance of feather mites and louseflies. However, no feather mites were found attached to the 405 louseflies inspected, although skin mites (Epidermoptidae and Cheyletiellidae) were found on louseflies collected from feral pigeons. This study suggests that feather mites do not use hippoboscid flies as a major mode of transmission among birds.

High host specificity of Crataerina melbae (Diptera: Hippoboscidae) in a mixed colony of birds.

We looked for louse flies (Diptera, Hippoboscidae) in a mixed colony of 9 species of birds from 1991 to 1997. Alpine swifts (Apus melba) exhibited an unusually high prevalence (85.9%) by Crataerina melbae (Rondani, 1879). No birds from the other 8 breeding bird species were parasitized by this louse fly. We suggest that the number of potential swiftlike hosts as well as the size of hosts may account for the differential prevalence of the louse flies within the colony.

Louse flies on birds of Baja California.

Louse flies were collected from 401 birds of 32 species captured in autumn of 1996 in Baja California Sur (Mexico). Only one louse fly species (Microlynchia pusilla) was found. It occurred in four of the 164 common ground doves (Columbina passerina) collected. This is a new a host species for this louse fly.

Hematozoa in two populations of the threatened red-billed chough in Spain.

The prevalence of hematozoa in two populations of red-billed choughs (Pyrrhocorax pyrrhocorax) was sampled in 1992 and 1994 in Spain. Two blood parasites infected red-billed choughs. A species of Plasmodium, possibly Plasmodium relictum, and the piroplasm Babesia frugilegica, are described for the first time from this host. Low prevalence (1/178, < 1%) of hematozoa in these populations, was evidence for a lack of effects of blood parasites on the life history and conservation of this threatened species in at least the two populations studied.

Stress response during development predicts fitness in a wild, long lived vertebrate.

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.

An epizootic of avian pox in endemic short-toed larks (Calandrella rufescens) and Berthelot's pipits (Anthus berthelotti) in the Canary Islands, Spain.

Between January 2002 and November 2003, 50% (n = 395) of short-toed larks (Calandrella rufescens) and 28% (n = 139) of Berthelot's pipits (Anthus berthelotti) examined on the islands of Fuerteventura and Lanzarote, Canary Islands, had gross lesions compatible with avian pox. However, Spanish sparrows (Passer hispaniolensis, n = 128) and trumpeter finches (Bucanetes githagineus, n = 228), which inhabit the same steppe habitats associated with goat husbandry, did not have poxlike lesions. Histopathology and electron microscopy confirmed poxvirus in the lesions, whereas serology using standard, fowl poxvirus-and pigeon poxvirus-based diagnostic agar gel immunodiffusion techniques was negative, likely because of the limited (74.6% pipit; 74.9% lark) similarity between the viruses in our species and fowlpox virus on which the serologic tests rely. On the basis of polymerase chain reaction analyses, the virus isolated from dried lesions of C. rufescens has 80.5% similarity with the virus isolated from A. berthelotti and 91.3% similarity with canarypox, whereas A. berthelotti poxvirus has only 80% similarity with canarypox. We have two distinct and possibly new avian poxviruses. Both poultry and the wild birds on the farms were heavily infested by fleas, which may have acted as vectors in transmission of poxvirus. Disease prevalence in these Canary Island passerines is higher than that described in song birds in Hawaii that are now threatened, endangered, or extinct. Environmental and biological factors contributing to increased disease susceptibility of these isolated populations must be investigated.

Gender and viability selection on morphology in fledgling pied flycatchers.

Until recently, analyses of gender-dependent differences in viability selection and the ontogeny of sexual size dimorphism have been plagued by difficulties in determining the sex of nestling birds on the basis of morphology. Recently, this problem was overcome using molecular sex identification to report for the first time body-size-mediated antagonistic selection on the viability of male and female collared flycatchers. We used molecular sex identification to analyse natural selection on fledgling viability, sexual size dimorphism and effects of parasites in relation to gender in a Mediterranean population of the related pied flycatcher Ficedula hypoleuca. There was directional positive selection on fledgling weight but no selection on tarsus length. Fledgling weight was the most important determinant of fledgling survival, with heavier fledglings having increased viability. Although selective trends were of the same sign for both sexes, only among female fledglings were selection differentials and gradients statistically significant. Therefore, similar trends in selection were revealed in analyses of a data set where sex was ignored and in separate analyses using same-sex sibship trait means. Mite nest ectoparasites negatively affected fledgling weight, and the effects were stronger in female than male fledglings. There was no effect of parasitism on the tarsus length in males, as previously reported in retrospective analyses performed without knowledge of sex until recruitment. Overall, selection on fledgling viability on the basis of morphological traits and hatching date was not confounded by an individual's gender.

Assessment of genotoxic damage by the comet assay in white storks (Ciconia ciconia) after the Doñana Ecological Disaster.

Single cell gel electrophoresis, the so-called "Comet" assay, was performed as a genotoxicity test in white storks sampled in an area heavily contaminated after the ecological disaster in south western Spain. This disaster occurred as a consequence of a massive toxic spillage of acid waste rich in heavy metals that impacted on the Doñana National Park. The importance of this protected area as a breeding and wintering site for many endangered bird species makes this analysis of DNA damage of special interest. Our results clearly show that white storks born in the contaminated area 1 year after the toxic spill bear a high burden of genetic damage as compared with control individuals. The possible implications for future survival as well as reproductive rate are discussed.

Habitat, world geographic range, and embryonic development of hosts explain the prevalence of avian hematozoa at small spatial and phylogenetic scales.

The factors explaining interspecific differences in prevalences of blood parasites in birds are poorly known. We simultaneously assessed 20 social, ecological, life history, and sampling-related variables that could influence hemoparasite prevalences among diurnal birds of prey in Spain. Our results show that multiple factors are responsible for the studied host-parasite association. We confirmed for the first time that prevalence is inversely correlated to the embryonic development period, and thus probably to immune performance, even among closely related birds. Macrohabitat features related to vector availability are also important, prevalences being higher in species breeding in forested habitats. Finally, prevalence is positively correlated with the host's world geographic range. We hypothesize that larger geographic ranges offered more opportunities for host-vector-hemoparasite associations to become established. The results from our multivariate analyses differ from those obtained through univariate ones, showing that all potential factors should be assessed jointly when testing any ecological or evolutionary hypothesis dealing with parasites.

Ecological, morphological and phylogenetic correlates of interspecific variation in plasma carotenoid concentration in birds.

Carotenoids are important as pigments for bright coloration of animals, and as physiologically active compounds with a wide array of health-related benefits. However, the causes of variation in carotenoid acquisition and physiology among species are poorly known. We measured the concentration of carotenoids in the blood of 80 wild bird species differing in diet, body size and the extent of carotenoid-based traits. Preliminary analyses showed that diet significantly explains interspecific variability in plasma carotenoids. However, dietary influences were apparently overridden by phylogenetic relationships among species, which explained most (65%) of this variability. This phylogenetic effect could be due partly to its covariation with diet, but may also be caused by interspecific differences in carotenoid absorption from food to the blood stream, mediated, for example by endothelial carriers or gut parasites. Carotenoid concentrations also decreased with body size (which may be explained by the allometric relationship between ingestion rate and body mass), and correlated positively with the extent of carotenoid-dependent coloration of plumage and bare parts. Therefore, the acquisition of carotenoids from the diet and their use for both health and display functions seem to be constrained by ecological and physiological aspects linked to the phylogeny and size of the species.