Extra Nestlings That Are Condemned to Die Increase Reproductive Success in Hoopoes.

AbstractThe adaptive value of routinely laying more eggs than can be successfully fledged has intrigued evolutionary biologists for decades. Extra eggs could, for instance, be adaptive as insurance against hatching failures. Moreover, because recent literature demonstrates that sibling cannibalism is frequent in the Eurasian hoopoe (Upupa epops), producing extra offspring that may be cannibalized by older siblings might also be adaptive in birds. Here, directed to explore this possibility in hoopoes, we performed a food supplementation experiment during the laying period and a clutch size manipulation during the hatching stage. We found that females with the food supplement laid on average one more egg than control females and that the addition of a close-to-hatch egg at the end of the hatching period increased the intensity of sibling cannibalism and enhanced fledging success in hoopoe nests. Because none of the extra nestlings from the experimental extra eggs survived until fledging, these results strongly suggest that hoopoes obtain fitness advantages by using temporarily abundant resources to produce additional nestlings that will be cannibalized. These results therefore suppose the first experimental demonstration of the nutritive adaptive function of laying extra eggs in vertebrates with parental care.

Cosmetic coloration of cross-fostered eggs affects paternal investment in the hoopoe (Upupa epops).

The signalling hypothesis suggests that avian eggshell coloration is a sexually selected female signal advertising her quality to its male partner, thereby stimulating his provisioning rate. This hypothesis has been tested for structural eggshell pigments, but not for cosmetic colorations, such as that produced by the uropygial secretion on eggshells. During the breeding season, female hoopoes (Upupa epops) host in their uropygial glands symbiotic bacteria. Females actively smear the eggshells with their secretion, protecting embryos from pathogenic trans-shell infections and changing eggshell coloration. Because the colour of the secretions is related to their antimicrobial potential, cosmetic eggshell coloration may act as a cue or even as a post-mating sexually selected signal if it affects male provisioning rates. To experimentally test this hypothesis, we cross-fostered already-smeared clutches between hoopoe nests, and quantified male feeding behaviour to females before and after the experiment. This approach allows disentanglement of the effects of female quality and of egg coloration on male investment. In accordance with the hypothesis, males adjusted their provisioning rate to the eggshell cosmetic coloration. This is, to our knowledge, the first experimental demonstration that egg colour stained with uropygial secretion could act as a post-mating sexual signal of female quality to males.

Acquisition of Uropygial Gland Microbiome by Hoopoe Nestlings.

Mutualistic symbioses between animals and bacteria depend on acquisition of appropriate symbionts while avoiding exploitation by non-beneficial microbes. The mode of acquisition of symbionts would determine, not only the probability of encountering but also evolutionary outcomes of mutualistic counterparts. The microbiome inhabiting the uropygial gland of the European hoopoe (Upupa epops) includes a variety of bacterial strains, some of them providing antimicrobial benefits. Here, the mode of acquisition and stability of this microbiome is analyzed by means of Automated rRNA Intergenic Spacer Analysis and two different experiments. The first experiment impeded mothers' access to their glands, thus avoiding direct transmission of microorganisms from female to offspring secretions. The second experiment explored the stability of the microbiomes by inoculating glands with secretions from alien nests. The first experiment provoked a reduction in similarity of microbiomes of mother and nestlings. Interestingly, some bacterial strains were more often detected when females had not access to their glands, suggesting antagonistic effects among bacteria from different sources. The second experiment caused an increase in richness of the microbiome of receivers in terms of prevalence of Operational Taxonomic Units (OTUs) that reduced differences in microbiomes of donors and receivers. That occurred because OTUs that were present in donors but not in receivers incorporated to the microbiome of the latter, which provoked that cross-inoculated nestlings got similar final microbiomes that included the most prevalent OTUs. The results are therefore consistent with a central role of vertical transmission in bacterial acquisition by nestling hoopoes and support the idea that the typical composition of the hoopoe gland microbiome is reached by the incorporation of some bacteria during the nestling period. This scenario suggests the existence of a coevolved core microbiome composed by a mix of specialized vertically transmitted strains and facultative symbionts able to coexist with them. The implications of this mixed mode of transmission for the evolution of the mutualism are discussed.

Diverse migration strategies in hoopoes (Upupa epops) lead to weak spatial but strong temporal connectivity.

The annual cycle of migrating birds is shaped by their seasonal movements between breeding and non-breeding sites. Studying how migratory populations are linked throughout the annual cycle-migratory connectivity, is crucial to understanding the population dynamics of migrating bird species. This requires the consideration not only of spatial scales as has been the main focus to date but also of temporal scales: only when both aspects are taken into account, the degree of migratory connectivity can be properly defined. We investigated the migration behaviour of hoopoes (Upupa epops) from four breeding populations across Europe and characterised migration routes to and from the breeding grounds, location of non-breeding sites and the timing of key migration events. Migration behaviour was found to vary both within and amongst populations, and even though the spatial migratory connectivity amongst the populations was weak, temporal connectivity was strong with differences in timing amongst populations, but consistent timing within populations. The combination of diverse migration routes within populations and co-occurrence on the non-breeding grounds between populations might promote exchange between breeding populations. As a result, it might make hoopoes and other migrating bird species with similar strategies more resilient to future habitat or climatic changes and stabilise population trends.

The Microbiome of the Uropygial Secretion in Hoopoes Is Shaped Along the Nesting Phase.

Microbial symbiont acquisition by hosts may determine the effectiveness of the mutualistic relationships. A mix of vertical and horizontal transmission may be advantageous for hosts by allowing plastic changes of microbial communities depending on environmental conditions. Plasticity is well known for gut microbiota but is poorly understood for other symbionts of wild animals. We here explore the importance of environmental conditions experienced by nestling hoopoes (Upupa epops) during the late nesting phase determining microbiota in their uropygial gland. In cross-fostering experiments of 8 days old nestlings, "sibling-sibling" and "mother-offspring" comparisons were used to explore whether the bacterial community naturally established in the uropygial gland of nestlings could change depending on experimental environmental conditions (i.e., new nest environment). We found that the final microbiome of nestlings was mainly explained by nest of origin. Moreover, cross-fostered nestlings were more similar to their siblings and mothers than to their stepsiblings and stepmothers. We also detected a significant effect of nest of rearing, suggesting that nestling hoopoes acquire most bacterial symbionts during the first days of life but that the microbiome is dynamic and can be modified along the nestling period depending on environmental conditions. Estimated effects of nest of rearing, but also most of those of nest of origin are associated to environmental characteristics of nests, which are extended phenotypes of parents. Thus, natural selection may favor the acquisition of appropriated microbial symbionts for particular environmental conditions found in nests.

Preening as a Vehicle for Key Bacteria in Hoopoes.

Oily secretions produced in the uropygial gland of incubating female hoopoes contain antimicrobial-producing bacteria that prevent feathers from degradation and eggs from pathogenic infection. Using the beak, females collect the uropygial gland secretion and smear it directly on the eggshells and brood patch. Thus, some bacterial strains detected in the secretion should also be present on the eggshell, beak, and brood patch. To characterize these bacterial communities, we used Automatic Ribosomal Intergenic Spacer Analysis (ARISA), which distinguishes between taxonomically different bacterial strains (i.e. different operational taxonomic units [OTUs]) by the size of the sequence amplified. We identified a total of 146 different OTUs with sizes between 139 and 999 bp. Of these OTUs, 124 were detected in the uropygial oil, 106 on the beak surface, 97 on the brood patch, and 98 on the eggshell. The highest richness of OTUs appeared in the uropygial oil samples. Moreover, the detection of some OTUs on the beak, brood patch, and eggshells of particular nests depended on these OTUs being present in the uropygial oil of the female. These results agree with the hypothesis that symbiotic bacteria are transmitted from the uropygial gland to beak, brood patch, and eggshell surfaces, opening the possibility that the bacterial community of the secretion plays a central role in determining the communities of special hoopoe eggshell structures (i.e., crypts) that, soon after hatching, are filled with uropygial oil, thereby protecting embryos from pathogens.

Laying date, incubation and egg breakage as determinants of bacterial load on bird eggshells: experimental evidence.

Exploring factors guiding interactions of bacterial communities with animals has become of primary importance for ecologists and evolutionary biologists during the last years because of their likely central role in the evolution of animal life history traits. We explored the association between laying date and eggshell bacterial load (mesophilic bacteria, Enterobacteriaceae, Staphylococci, and Enterococci) in natural and artificial magpie (Pica pica) nests containing fresh commercial quail (Coturnix coturnix) eggs. We manipulated hygiene conditions by spilling egg contents on magpie and artificial nests and explored experimental effects during the breeding season. Egg breakage is a common outcome of brood parasitism by great spotted cuckoos (Clamator glandarius) on the nests of magpie, one of its main hosts. We found that the treatment increased eggshell bacterial load in artificial nests, but not in magpie nests with incubating females, which suggests that parental activity prevents the proliferation of bacteria on the eggshells in relation to egg breakage. Moreover, laying date was positively related to eggshell bacterial load in active magpie nests, but negatively in artificial nests. The results suggest that variation in parental characteristics of magpies rather than climatic variation during the breeding season explained the detected positive association. Because the eggshell bacterial load is a proxy of hatching success, the detected positive association between eggshell bacterial loads and laying date in natural, but not in artificial nests, suggests that the generalized negative association between laying date and avian breeding success can be, at least partially, explained by differential bacterial effects.

Environmental factors shape the community of symbionts in the hoopoe uropygial gland more than genetic factors.

Exploring processes of coevolution of microorganisms and their hosts is a new imperative for life sciences. If bacteria protect hosts against pathogens, mechanisms facilitating the intergenerational transmission of such bacteria will be strongly selected by evolution. By disentangling the diversity of bacterial strains from the uropygium of hoopoes (Upupa epops) due to genetic relatedness or to a common environment, we explored the importance of horizontal (from the environment) and vertical (from parents) acquisition of antimicrobial-producing symbionts in this species. For this purpose, we compared bacterial communities among individuals in nonmanipulated nests; we also performed a cross-fostering experiment using recently hatched nestlings before uropygial gland development and some nestlings that were reared outside hoopoe nests. The capacity of individuals to acquire microbial symbionts horizontally during their development was supported by our results, since cross-fostered nestlings share bacterial strains with foster siblings and nestlings that were not in contact with hoopoe adults or nests also developed the symbiosis. Moreover, nestlings could change some bacterial strains over the course of their stay in the nest, and adult females changed their bacterial community in different years. However, a low rate of vertical transmission was inferred, since genetic siblings reared in different nests shared more bacterial strains than they shared with unrelated nestlings raised in different nests. In conclusion, hoopoes are able to incorporate new symbionts from the environment during the development of the uropygium, which could be a selective advantage if strains with higher antimicrobial capacity are incorporated into the gland and could aid hosts in fighting against pathogenic and disease-causing microbes.

Special structures of hoopoe eggshells enhance the adhesion of symbiont-carrying uropygial secretion that increase hatching success.

Animals live in a bacterial world, and detecting and exploring adaptations favouring mutualistic relationships with antibiotic-producing bacteria as a strategy to fight pathogens are of prime importance for evolutionary ecologists. Uropygial secretion of European hoopoes (Upupa epops, Linnaeus) contains antimicrobials from mutualistic bacteria that may be used to prevent embryo infection. Here, we investigated the microscopic structure of hoopoe eggshells looking for special features favouring the adhesion of antimicrobial uropygial secretions. We impeded female access to the uropygial gland and compared microscopic characteristics of eggshells, bacterial loads of eggs and of uropygial secretion, and hatching success of experimental and control females. Then, we explored the link between microbiological characteristics of uropygial secretion and these of eggs of hoopoes, as well as possible fitness benefits. The microscopic study revealed special structures in hoopoes' eggshells (crypts). The experimental prevention of females' gland access demonstrated that crypts are filled with uropygial secretion and that symbiotic enterococci bacteria on the eggshells come, at least partially, from those in the female's uropygial gland. Moreover, the experiment resulted in a higher permeability of eggshells by several groups of bacteria and in elimination of the positive relationships detected for control nests between hatching success and density of symbiotic bacteria, either in the uropygial secretion of females or on the eggshell. The findings of specialized crypts on the eggshells of hoopoes, and of video-recorded females smearing secretion containing symbiotic bacteria at a high density onto the eggshells strongly support a link between secretion and bacteria on eggs. Moreover, the detected associations between bacteria and hatching success suggest that crypts enhancing the adhesion of symbiont-carrying uropygial secretion likely protect embryos against infections.

Volatiles of symbiotic bacterial origin explain ectoparasitism and fledging success of hoopoes.

BACKGROUND: Some parasites use olfactory cues to detect their hosts and, since bacterial symbionts are partially responsible for animal odours, they could influence host parasitism. By autoclaving nest materials of hoopoe (Upupa epops) nests before reproduction started, we explored the hypothetical links between host-associated bacteria, volatiles and parasitism. During the nestling stage, we (i) estimated the level of ectoparasitism by chewing lice (Suborder Mallophaga) in adult hoopoe females and by Carnus haemapterus flies in nestlings, and (ii) characterized microbial communities and volatile profiles of nest environments (nest material and nest cavity, respectively) and uropygial secretions. RESULTS: Experimental nests had less diverse bacterial communities and more diverse volatile profiles than control nests, while occupants experienced lower intensity of parasitism in experimental than in control nests. The experiment also affected beta diversity of the microbial communities of nest material and of the volatiles of the nestling uropygial secretions. Moreover, microbial communities of uropygial secretions and of nest materials covaried with their volatile profiles, while the volatile profile of the bird secretions explained nest volatile profile. Finally, a subset of the volatiles and bacteria detected in the nest material and uropygial secretions were associated with the ectoparasitism intensity of both adult females and nestlings, and with fledging success. CONCLUSIONS: These results show that a component of animal odours is linked with the microbial communities of the host and its reproductive environment, and emphasize that the associations between bacteria, ectoparasitism and reproductive success are partially mediated by volatiles of bacterial origin. Future work should focus on mechanisms underlying the detected patterns.

Social environment influences microbiota and potentially pathogenic bacterial communities on the skin of developing birds.

BACKGROUND: Animal bacterial symbionts are established early in life, either through vertical transmission and/or by horizontal transmission from both the physical and the social environment, such as direct contact with con- or heterospecifics. The social environment particularly can influence the acquisition of both mutualistic and pathogenic bacteria, with consequences for the stability of symbiotic communities. However, segregating the effects of the shared physical environment from those of the social interactions is challenging, limiting our current knowledge on the role of the social environment in structuring bacterial communities in wild animals. Here, we take advantage of the avian brood-parasite system of Eurasian magpies (Pica pica) and great spotted cuckoos (Clamator glandarius) to explore how the interspecific social environment (magpie nestlings developing with or without heterospecifics) affects bacterial communities on uropygial gland skin. RESULTS: We demonstrated interspecific differences in bacterial community compositions in members of the two species when growing up in monospecific nests. However, the bacterial community of magpies in heterospecific nests was richer, more diverse, and more similar to their cuckoo nest-mates than when growing up in monospecific nests. These patterns were alike for the subset of microbes that could be considered core, but when looking at the subset of potentially pathogenic bacterial genera, cuckoo presence reduced the relative abundance of potentially pathogenic bacterial genera on magpies. CONCLUSIONS: Our findings highlight the role of social interactions in shaping the assembly of the avian skin bacterial communities during the nestling period, as exemplified in a brood parasite-host system.

Microbiota and the volatile profile of avian nests are associated with each other and with the intensity of parasitism.

Bacteria have been suggested as being partially responsible for avian nest odours and, thus, volatiles from their metabolism could influence the intensity of selection pressures due to parasites detecting olfactory cues of their hosts. Here, we tested this hypothesis by exploring intraspecific and interspecific variability in microbial environments, volatile profiles and intensity of ectoparasitism by Carnus hemapterus in the nests of 10 avian species. As expected, we found that (i) alpha and beta diversity of microbial and volatile profiles were associated with each other. Moreover, (ii) alpha diversity of bacteria and volatiles of the nest environment, as well as some particular bacteria and volatiles, was associated with the intensity of parasitism at early and late stages of the nestling period. Finally, (iii) alpha diversity of the nest microbiota, as well as some particular bacteria and volatiles, was correlated with fledging success. When considering them together, the results support the expected links between the microbial environment and nest odours in different bird species, and between the microbial environment and both ectoparasitism intensity and fledging success. Relative abundances of particular volatiles and bacteria predicted ectoparasitism and/or fledging success. Future research should prioritise experimental approaches directed to determine the role of bacteria and volatiles in the outcomes of host-ectoparasite interactions.

Conservation through connectivity: can isotopic gradients in Africa reveal winter quarters of a migratory bird?

Conservation of migratory wildlife requires knowledge of migratory connectivity between breeding and non-breeding locations. Stable isotopes in combination with geographical isotopic patterns (isoscapes) can provide inferences about migratory connectivity. This study examines whether such an approach can be used to infer wintering areas in sub-Saharan Africa, where we lack such knowledge for many species, but where this method has not been used widely. We measured δ (2)H, δ (13)C and δ (15)N in winter-grown feathers of a breeding Swiss and Spanish population of European hoopoe Upupa epops--a typical Palaearctic-Afrotropical migrant. δ (2)H values predicted that ~70 % of the hoopoes spent the non-breeding season in the western portion of their potential winter range. This was corroborated by a shallow east-west gradient in feather-δ (2)H values of museum specimens from known African origin across the potential winter range and by the recovery of Swiss hoopoes marked with geolocators. Hoopoes categorized as from eastern versus western regions of the wintering range were further delineated spatially using feather δ (13)C and δ (15)N. δ (15)N showed no trend, whereas adults were more enriched in (13)C in the western portion of the range, with eastern adults being in addition more depleted in (13)C than eastern juveniles. This suggests that eastern juveniles may have occupied more xeric habitats than sympatric adults. We demonstrated that stable isotopes, especially δ (2)H, could only very roughly delineate the winter distribution of a trans-Saharan Palaearctic migrant restricted primarily to the Sahelian and savanna belt south of the Sahara. Further refinements of precipitation isoscapes for Africa as well the development of isoscapes for δ (13)C and δ (15)N may improve assignment of this and other migrants.

Food supply and provisioning behavior of parents: Are small hoopoe nestlings condemned to die?

Parents might use signals of need or of quality to decide food provisioning among their offspring, while the use of one or another signal might depend on food availability. Begging success of nestlings of different quality (i.e., body size) would also depend on food availability, and we here explore the effect of experimental food supply in begging success of nestlings and in provisioning of female hoopoes (Upupa epops), a species with extreme hatching asynchrony and nestlings size hierarchy. We video-recorded food allocation of females, begging success of nestlings of different size, and the social context (i.e., the size category of the other nestlings that were begging for food) during periods when experimental food supply was or was not available in the same nests. We found that when experimental food supplementation was present, begging success of the intermediate, but not that of large or small-sized nestlings, increased. The experiment, however, did not affect the feeding preferences of females toward nestlings of different size. Moreover, when small nestlings were the only ones that were begging for food, their begging success decreased in the experimental period, and females used supplemented prey to feed themselves. Those results, on one hand, confirm the importance of food availability for the begging success of nestlings of particular sizes and, on the other, indicate that females prefer to use extra food for their own rather than for the smallest nestlings. We discuss possible mechanisms explaining the detected experimental effects and the adaptive and nonadaptive explanations of mothers ignoring the small nestlings.

Cognitive skills and bacterial load: comparative evidence of costs of cognitive proficiency in birds.

Parasite-mediated selection may affect the evolution of cognitive abilities because parasites may influence development of the brain, but also learning capacity. Here, we tested some predictions of this hypothesis by analyzing the relationship between complex behaviours (feeding innovations (as a measure of behavioural flexibility) and ability to detect foreign eggs in their nests (i.e. a measure of discriminatory ability)) and abundance of microorganisms in different species of birds. A positive relationship would be predicted if these cognitive abilities implied a larger number of visited environments, while if these skills favoured detection and avoidance of risky environments, a negative relationship would be the prediction. Bacterial loads of eggshells, estimated for mesophilic and potentially pathogenic bacteria (i.e. Enterococcus, Staphylococcus and Enterobacteriaceae), were used as a surrogate of probability of contact with pathogenic bacteria. We found that bird species with higher feeding innovation rates and rejection rates of experimental brood parasitic eggs had higher density of bacteria on their eggshells than the average species. Since the analysed groups of microorganisms include pathogenic bacteria, these results suggest that both feeding innovation and ability to recognize foreign eggs are costly and highlight the importance of parasite-mediated selection in explaining the evolution of cognitive abilities in animals.

Made-up mouths with preen oil reveal genetic and phenotypic conditions of starling nestlings.

Animal coloration results from pigments, nanostructures, or the cosmetic use of natural products, and plays a central role in social communication. The role of cosmetic coloration has traditionally been focused in scenarios of sexual selection, but it could also take place in other contexts. Here, by using spotless starlings (Sturnus unicolor) as a model system, we explore the possibility that nestlings cosmetically use their intensely yellow-colored uropygial secretion to signal their genetic and/or phenotypic quality. In agreement with the hypothetical cosmetic use of the uropygial secretion, (i) video recorded nestlings collected secretion with the bill at the age of feathering, (ii) cotton swabs turned to the color of secretion after rubbing with them nestlings' gape, and (iii) gape and skin colorations correlated positively with that of secretion. Furthermore, we found that (iv) secretion coloration has a genetic component, and (v) associated positively with Vitamin E supplementation and (vi) with plasma carotenoid concentration, which highlights the informative value of nestling secretion. Finally, (vii) coloration of begging-related traits and of secretion of nestlings predicted parental feeding preferences. Consequently, all these results strongly suggest that the cosmetic use of colored uropygial secretion might also play a role in parent-offspring communication, complementing or amplifying information provided by the flamboyant colored gapes and skin of nestlings. The use of makeups by offspring for communication with relatives has been scarcely explored and we hope that these results will encourage further investigations in birds and other taxa with parental care.

Avian sibling cannibalism: Hoopoe mothers regularly use their last hatched nestlings to feed older siblings.

Sibling cannibalism is relatively common in nature, but its evolution in birds and certain other vertebrates with extended parental care had been discarded. Here, however, we demonstrate its regular occurrence in two European populations of the Eurasian hoopoe (Upupa epops) and explore possible adaptive and non-adaptive explanations. Results showed that sibling cannibalism was more frequently detected in Spain (51.7%) than in Austria (5.9%). In these two populations, the hoopoes laid similar clutch sizes, resulting in similar fledging production, but hatching failures were more frequent in the northern population. Consequently, having more nestlings condemned to die in the southern population may explain the higher incidence of sibling cannibalism. In accordance with this interpretation, hatching span and failure, but not breeding date, explained the probability of sibling cannibalism in the Spanish hoopoes, while all three variables predicted brood reduction intensity. Furthermore, experimental food supply reduced the probability of sibling cannibalism, but not the intensity of brood reduction. Finally, females allocated fewer resources to the smallest nestlings when they were going to starve, but not necessarily when they were going to be used as food for their siblings. These results suggest that hoopoes produce extra eggs that, in the case of reduced hatching failure and food scarcity, produce nestlings that are used to feed older siblings. These findings provide the first evidence that sibling cannibalism occurs regularly in a bird species, thus expanding our evolutionary understanding of clutch size, hatching asynchrony, parent-offspring conflict, infanticide, and sibling cannibalism in the animal kingdom.

Microbial infection risk predicts antimicrobial potential of avian symbionts.

Symbiotic bacteria on animal hosts can prevent pathogenic bacterial infections by several mechanisms. Among them, symbiotic bacteria can indirectly enhance host's immune responses or, directly, produce antimicrobial substances against pathogens. Due to differences in life-style, different host species are under different risks of microbial infections. Consequently, if symbiotic bacteria are somewhat selected by genetically determined host characteristics, we would expect the antimicrobial properties of bacterial symbionts to vary among host species and to be distributed according to risk of infection. Here we have tested this hypothesis by measuring the antimicrobial ability of the bacterial strains isolated from the uropygial-gland skin of 19 bird species differing in nesting habits, and, therefore, in risk of microbial infection. In accordance with our predictions, intensity and range of antimicrobial effects against the indicator strains assayed varied among bird species, with hole-and open-nesters showing the highest and the lowest values, respectively. Since it is broadly accepted that hole-nesters have higher risks of microbial infection than open nesters, our results suggest that the risk of infection is a strong driver of natural selection to enhance immunocompetence of animals through selecting for antibiotic-producing symbionts. Future research should focus on characterizing symbiotic bacterial communities and detecting coevolutionary processes with particular antibiotic-producing bacteria within-host species.

Antimicrobial chemicals in hoopoe preen secretions are produced by symbiotic bacteria.

Animals frequently use metabolites produced by symbiotic bacteria as agents against pathogens and parasites. Secretions from the preen gland of birds are used for this purpose, although its chemicals apparently are produced by the birds themselves. European hoopoes Upupa epops and green woodhoopoes Phoeniculus purpureus harbour symbiotic bacteria in the uropygial gland that might be partly responsible for the chemical composition of secretions. Here we investigate the antimicrobial activity of the volatile fraction of chemicals in hoopoe preen secretions, and, by means of experimental antibiotic injections, test whether symbiotic bacteria living within the uropygial gland are responsible for their production. Hoopoes produce two different kinds of secretions that differ drastically in their chemical composition. While the malodorous dark secretions produced by nestlings included a complex mix of volatiles, these chemicals did not appear in white secretions produced by non-nesting birds. All volatiles detected showed strong antibacterial activity, and a mixture of the chemicals at the concentrations measured in nestling glands inhibited the growth of all bacterial strains assayed. We found support for the hypothesized role of bacteria in the production of such antimicrobial chemicals because experimental clearance of bacteria from glands of nestlings with antibiotics resulted in secretions without most of the volatiles detected in control individuals. Thus, the presence of symbiotic bacteria in the uropygial gland provides hoopoes with potent antimicrobials for topical use.

Autoclaving Nest-Material Remains Influences the Probability of Ectoparasitism of Nestling Hoopoes (Upupa epops).

Nest bacterial environment influences avian reproduction directly because it might include pathogenic- or antibiotic-producing bacteria or indirectly because predators or ectoparasites can use volatile compounds from nest bacterial metabolism to detect nests of their avian hosts. Hoopoes (Upupa epops) do not build nests. They rather reuse holes or nest-boxes that contain remains of nest-materials from previous breeding seasons. Interestingly, it has been recently described that the nest's bacterial environment partly affects the uropygial gland microbiota of hoopoe females and eggshells. Blood-sucking ectoparasites use chemical cues to find host nests, so we experimentally tested the hypothetical effects of microorganisms inhabiting nest-material remains before reproduction regarding the intensity of ectoparasitism suffered by 8-day-old nestling hoopoes. In accordance with the hypothesis, nestlings hatched in nest-boxes with autoclaved nest-material remains from the previous reproductive seasons suffered less from ectoparasites than those hatched in the control nest-boxes with nonautoclaved nest-material. Moreover, we found a positive association between the bacterial density of nest-material during the nestling phase and ectoparasitism intensity that was only apparent in nest-boxes with autoclaved nest-material. However, contrary to our expectations, nest bacterial load was positively associated with fledgling success. These results suggest a link between the community of microorganisms of nest-material remains and the intensity of ectoparasitism, and, on the other hand, that the nest bacterial environment during reproduction is related to fledging success. Here, we discuss possible mechanisms explaining the experimental and correlative results, including the possibility that the experimental autoclaving of nest material affected the microbiota of females and nestlings' secretion and/or nest volatiles that attracted ectoparasites, therefore indirectly affecting both the nest bacterial environment at the nestling stage and fledging success.