Nest re‐use in birds has the potential cost of infection by parasites and pathogens but may also be a source of beneficial symbiotic bacteria transmitted horizontally. Eurasian hoopoes Upupa epops host antibiotic‐producing bacteria in their uropygial gland but only while breeding, which suggests that the nest‐hole may be a source of those symbionts. Interestingly, hoopoes do not build nests, thus might prefer for reproduction nest holes with soft materials from previous reproductions. Here, we tested experimentally this preference by installing in the field new nest boxes that were left empty or filled with either sawdust or a mixture of sawdust and hoopoe's nest material from the previous year. We explored the experimental effect on the composition of the uropygial secretion bacterial community, on eggshell bacterial loads, and on several proxies of reproductive success. Hoopoes bred significantly more often in nest boxes with nest material than in empty ones, but the type of nest material did not affect nest box occupancy. Eggs in nest boxes with old‐soft material harbored higher bacterial density on their shells, and the microbiota of the uropygial secretion of nestlings and females in these nest boxes differed from those in nest boxes without old‐soft material. Moreover, although the experiment did not affect breeding success or related proxies, several operational taxonomic units from female uropygial secretions were positively associated with hatching success. This is the first experimental evidence showing that re‐used nest material affects the bacterial community of the uropygial secretions of hoopoe females. This suggests that the nest material can be a source of strains for their incorporation to both the uropygial gland and eggshell communities, highlighting a possible advantage of nest re‐use previously unconsidered.

Avian eggshell colouration might function as a post‐mating sexually selected signal of female quality, influencing male parental investment and, hence, reproductive success. This hypothesis has been tested for intrinsic eggshell pigments as biliverdin (blue‐green colouration) and/or protoporphyrin (brown coloured spots), but never for colourations applied post‐laying. Post‐laying staining colouration due to, for instance, uropygial secretion of the female could reflect its phenotypic properties and, thus, might be a cue for male investment in reproduction. In hoopoes, the uropygial gland of incubating females hosts symbiotic bacteria that are responsible for the brown colour of their uropygial secretion and of the eggshells, as they cover their bluish‐grey eggshells with gland secretion after laying. The secretion protects embryos from pathogenic trans‐shell infections and, thus, egg colouration may function as a cue or even as a post‐mating sexually selected signal of antimicrobial potential in hoopoes. In a wild hoopoe population breeding in nest boxes in Spain, we test this hypothesis by exploring whether egg colour predicts male parental investment. In accordance with the hypothesis, we found that the amount of food provided by males to incubating females was higher in nests with less saturated eggshell colours. This relationship was affected by female body condition. High quality females in terms of body condition and/or in secretion colour obtained better males in terms of provisioning effort during incubation. Given that eggshell saturation is negatively related to density of bacterial symbionts in uropygial secretions, one possibility is that males may regulate their parental investment in accordance to the expected characteristics of mutualistic bacteria hosted in uropygial glands and deposited on eggshells. We discuss alternative explanations for our results, concluding that the post‐mating sexual selection hypothesis is the most likely but experimental modification of egg colour is needed to test it further.

Nestlings of most avian species produce faecal sacs, which facilitate the removal of nestlings’ excrements by parents, thereby reducing proliferation of potentially pathogenic microorganisms and/or detectability by predators and parasites. The nest microbial environment that birds experience during early life might also affect their development and thus, faecal sacs facilitating parental removal may be a strategy to decrease bacterial contamination of nests that could harm developing nestlings. Here, we tested this hypothesis by experimentally broken faecal sacs and spreading them in nests of spotless starlings Sturnus unicolor, thereby avoiding their removal by adults. In accordance with the hypothesis, experimental nests harboured higher bacterial density than control nests. Nestlings in experimental nests were of smaller size (tarsus length) and experienced lower probability of survival (predation) than those in control nests. Moreover, nestlings in experimental nests tended to suffer more from ectoparasites than those in control nests. We discuss the possible pivotal role of bacteria producing chemical volatiles that ectoparasites and predators might use to find avian nests, and that could explain our experimental results in starlings.

Feathers are essential for avian life, and factors affecting their integrity are important to understand their evolution. These factors should depend on, among other traits, species‐specific bacterial environments and life‐history characteristics. However, interspecific variation in feather deterioration, feather susceptibility to degradation by keratinolytic bacteria (degradability), and bacterial environment, have rarely been quantified. Here, we did so by measuring deterioration and degradability of wing feathers of fledglings in 16 bird species, and characterizing the bacterial environment where they developed. We found statistically significant interspecific variation for all considered variables. On average, non‐melanised were more deteriorated than melanised feathers, but differences depended on the species. Moreover, nest bacterial loads were related to feather wear, but the sign of the association depended on the bacterial group considered and on feather pigmentation. We also found a positive association of feather degradability with wear of non‐melanised feathers, and with bacterial loads. These results suggest that bacterial environments determine the integrity of fledgling feathers as well as their resistance to bacterial degradation, which implies a preponderant role of bacteria in driving the evolution of avian feathers.

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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.

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