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.

1 It has been recently showed that one bacterial strain isolated from the uropygial gland of a nestling hoopoe Upupa epops produced antimicrobial peptides active against a broad spectrum of pathogenic bacteria. These bacteria might thus mediate antimicrobial properties of the uropygial secretions as a consequence of the symbiotic association with hoopoes. 2 We study antimicrobial properties of white (from males and non-breeding females) and brown (from nestlings and breeding females) uropygial gland secretions of hoopoes Upupa epops, as well as the association with the presence of bacteria living inside their uropygial gland. 3 We found that brown, but not white secretions contained bacteria and showed antimicrobial activity against the feather degrading bacterium Bacillus licheniformis. The antagonistic activity of bacterial colonies was mediated by antimicrobial peptides because protease inhibited antimicrobial properties. 4 All except one identified bacterium in aerobic cultures were of the genus Enterococcus, and the microscopic study of uropygial secretions and glands confirmed a high density of bacteria within the gland. 5 Furthermore, we studied potential benefits of antimicrobial peptides produced by symbiotic bacteria of hoopoes by adding protease to incubating nests. 6 The experiment increased bacterial growth and hatching failures in hoopoes but not in spotless starlings Sturnus unicolor, a species that does not harbour bacteria in its uropygial gland. 7 Thus, microbiological, anatomical and ecological results suggest a tight symbiotic interaction between bacteria that produce antibiotic substances and the hoopoes.

Summary: 1. The use of feathers as nest lining material has traditionally been explained by the thermoregulatory properties of feathers. Feather nest lining could additionally affect nest detectability by predators, or play a role in a sexually selected context. Furthermore, feather nest lining harbours microorganisms that may influence environmental conditions where eggs and nestlings develop. 2. Microorganisms growing on nest lining feathers could affect the bacterial load of eggshells because they occupy space and/or produce antimicrobial substances against other bacteria, including egg pathogens. Feathers of different colours are known to differ in their bacterial community (i.e. feather degrading bacteria) and, thus, colour composition of nest lining feather could also affect the bacterial environment of avian nests. 3. Here we tested this hypothesis in the barn shallow (Hirundo rustica) by exploring the relationship between eggshell bacterial loads and number of feathers, and the effect of experimentally modified colour composition of nest lining feathers on eggshell bacterial load. 4. In agreement with the hypothesis we found that, before treatment, the number of nest lining feathers (mainly that of unpigmented‐white colour) predicted eggshell bacterial load, and that, at the end of the incubation period, eggshells of experimental nests with white feathers had a lower bacterial density than those in experimental nests with black feathers. 5. We failed to detect a relationship between bacterial load and hatching success. However, since evidence of that relationship exists for other species, these results would explain the previously detected experimental effect of colour composition of nest lining feathers on hatching success of swallows. 6. Nest design in general, and the use of nest‐lining white feathers in particular, may therefore have important consequences for reproductive success of birds. The reduced eggshell bacterial loads of experimental white nests would explain preferences by barn swallows for feathers of white colour.

Summary: 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.