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Bacteria associated with the uropygial gland secretions have recently been detected in two different species of Coraciformes, the hoopoe Upupa epops (Martín-Platero et al. 2006) and in the closely related red-billed woodhoopoe Phoeniculus purpureus (Law-Brown & Meyers 2003). For the former, a bacterial strain of the species Enterococcus faecalis that produced at least two different kinds of peptides with strong antibiotic properties (bacteriocins) has been isolated (Martín-Platero et al. 2006). For the latter, a new bacterial species (E. phoeniculicola) was isolated in their uropygial gland secretions (Law-Brown & Meyers 2003), where some chemical components with known antimicrobial activity also exist (Burger et al. 2004). The secretions of the woodhoopoes, as well as those of nestling and female hoopoes (but not those of males), during the breeding season are very similar and peculiar. Both species secrete brown and malodorous oils that are believed to function as predator repellents (Cramp 1985; Ligon 2001). The secretions may also prevent infections given the presence of antimicrobial chemical components and/or bacteriocin-like inhibitory substances-producing bacteria.
Here, we study the antimicrobial properties of the brown, malodorous uropygial gland secretion of nestling and breeding female European hoopoes, but also of the white and odourless secretion of males and non-breeding females. Soon after laying hoopoe eggs become completely smeared with the secretion of females, changing the colour of eggshells from pale blue to completely brown (Cramp 1998), suggesting a potential antimicrobial function. We experimentally deactivate antimicrobial peptides from the uropygial gland secretion by adding protease to the eggshells and study the effect on bacteria growth on eggshell, and on hatching success. We also identify bacteria isolated from secretions and explore the presence of bacteria within both the organ and the secretion.
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- Material and methods
The main findings of this study were that (i) the brown, but not the white uropygial secretions of hoopoes demonstrated antibiotic properties against pathogenic bacteria; (ii) there was an association between antibiotic properties of the secretion and the presence of bacteria at high density; (iii) colonies isolated from brown secretions were in most cases identified as E. faecalis, and such isolates demonstrated antibiotic properties, likely mediated by bacteriocin production; and (iv) microscopic observation of brown secretions and uropygial glands revealed high-density aggregations of bacteria within the papilla of the gland. All these results taken together strongly suggest a symbiotic association between hoopoes and bacteria that live in their uropygial gland during the nesting phase. Furthermore, (v) the inactivation of antimicrobial peptides in nests of hoopoes resulted in a relatively higher egg bacterial infection and hatching failures than in control nests where only water was added. We will briefly discuss these findings and their possible implications in the symbiotic association between bacteria and hoopoes. It should be noted here that, although the term ‘symbiont’ is typically applied to mutualistic microorganisms, we use this term in its broad sense, which includes associates for which the full spectrum of effects on hosts is not known (see, Moran 2006).
It is well known that the uropygial secretions of birds may contain antibiotic substances (Jacob & Ziswiler 1982; Shawkey, Pillai & Hill 2003) and, in accordance, we here found that brown but not white secretion of hoopoes had antimicrobial properties. In addition, in contraposition to the scarce white secretions, brown secretions are abundant, malodorous, and contain bacteria. The differences in antimicrobial properties of brown and white secretions of hoopoes are not due to differences in volume of secretion since tests of antagonism were performed with similar volumes of white and brown secretions. The presence of bacteria is likely an important factor explaining properties of brown secretions since the experimental injection of antibiotics affected properties of the uropygial gland (i.e. size) of hoopoes as well as the colour, stored volume, and odour of the secretion (Martin-Vivaldi et al. submitted). The injection of antibiotic did not clarify whether bacteria are responsible of the antimicrobial property of the secretion because obviously the exogenous commercial antibiotic used in the experiment conferred antimicrobial properties to the uropygial secretion of experimental individuals.
Most colonies isolated from brown secretions in this study were identified as E. faecalis, and only one analysed strain was not within the Genus Enteroccocus. We have however analysed only bacteria that grew aerobically in trypticase soy agar, the most usual general medium for growing bacteria. However, it is known from molecular methodologies that some others bacteria cannot be detected in general media (Pace 1997; Bisson et al. 2007) and, thus, we cannot discount the possibility that others anaerobic and/or unculturable bacteria were also present within the uropygial gland of hoopoes. In any case, the very high prevalence of E. faecalis in particular and of the genus Enterococus in general suggests a symbiotic relationship between hoopoes and enteroccoci. Enterococci are facultatively aerobic and, thus, can and do grow under anaerobic conditions (e.g. the uropygial gland of hoopoes).
Could the apparent association between enterococci and the uropygial gland of hoopoes be the result of contamination of the secretion after extraction from the uropygial gland of hoopoes? Several results contradict this interpretation. First, we only detected bacteria in the brown secretion samples and the risk of contamination should have been equal for both brown and white. Second, we observed at the microscope bacterial aggregations at a high density in secretions fixed immediately after extraction (Fig. 1) as well as inside the uropygial gland of a female hoopoe (Fig. 2). Finally, accidental contamination of the uropygial gland or its secretions would predict for a low prevalence, but we detected this association in most brown samples collected during the breeding season.
Mechanisms explaining colonization of the gland by the bacteria may include immune depression of hoopoes during reproduction. The elevated energetic costs of reproduction and the production of sexual hormones at this stage implies a physiological stress that may have immune suppressive effects (Alonso-Alvarez et al. 2004). Enteroccocus sp. are abundant in the digestive tract and faeces of birds and, contrary to most hole-nesters, hoopoes do not keep their nest clean of faeces. Thus, the faecal contamination of nests of hoopoes, together with a possible worsened immune system during the nesting phase, would facilitate the colonization of the gland of hoopoes by Enterococcus sp. However, immune responses of females toward injections of the mitogenic phytohaemagglutinin (PHA) were significantly larger during the nesting phase (incubation (N = 3): mean PHA response (SE) = 1·18 (0·16); brooding (N = 6): mean PHA response (SE) = 1·34 (0·11)) than when UGS of females were of white colour (pairing (N = 12): mean PHA response (SE) = 0·43 (0·08); after abandoning the nest for feeding nestlings (N = 19): mean PHA response (SE) = 0·68 (0·06)) (Martin-Vivaldi, Soler & Ruiz-Rodriguez unpublished data), which suggests that females have an enhanced immune system during the nesting phase of reproduction instead of suffering immunosuppression.
Colonies of Enterococcus isolated from hoopoe glands, as well as the brown secretions, demonstrated clear growth-inhibition activity against different bacteria including B. licheniformis, a common feather-degrading bacterium (Burtt & Ichida 1999). Moreover, the addition of protease inhibited the antimicrobial activity of colonies, which suggests that the antimicrobial properties of Enterococcus isolated from hoopoes are mediated by production of peptide substances. Therefore, although the brown secretion of hoopoes may contain some other antibiotic substances not directly related to the associated enterococci, due to the antimicrobial properties of the bacteriocin-like inhibitor substances produced (Martín-Platero et al. 2006), it is likely that the antimicrobial properties of the uropygial secretion of hoopoes were, at least partially, the product of symbiotic bacteria.
The beneficial antimicrobial-effect of enterococci, due to the production of bacteriocin-like inhibitor substances, is known from research on poultry and food conservation (Foulquié Moreno et al. 2006). Among the two species of enterococci that have been intensively studied, E. faecium and E. faecalis, the beneficial effects are clearer for the former than for the latter. For instance, although strains of both species have been applied in human and veterinary probiotic supplements, nine different strains of E. faecium, but none of E. faecalis, are authorized by the European Union as additive to feeds (see, Foulquié Moreno et al. 2006). For E. faecalis, although it is considered an opportunistic pathogen responsible of nosocomial infections (Franz, Holzapfel & Stiles 1999), some clear benefits due to competitive exclusion of more pathogenic bacteria have been detected (Wagner, Holland & Cerniglia 2002). Moreover, in a wild population of flycatchers, prevalence of both enteroccoci species in the cloaca of nestlings were negatively associated and that of E. faecium was strongly and positively associated with fledging body size and mass (Moreno et al. 2003).
To test the hypothetical role of bacteriocin-like inhibitor substances produced by symbiotic bacteria, by means of protease experiments, we deactivated possible antimicrobial peptides on the eggshells of hoopoes and explored the effect on bacterial growth and hatching success. In accordance with the hypothesis we found that, in comparisons with control nests, eggs in experimental nests experienced higher bacterial infection and hatching failures. These effects were not due to protease directly affecting both bacterial infection and hatching failure because avian eggs contain natural inhibitors of proteases (Board et al. 1994; Mine 2007), and the experiment had no effect in nests of spotless starling where bacteriocin-like inhibitor substances have not been detected. These results suggest a mutualistic association between hoopoes and E. faecalis living in the uropygial gland that provide hosts with substances that inhibit growth of pathogenic bacteria. Alternatively, our experiments might have also deactivated antimicrobial peptides that are directly produced by hoopoes. However, chemical composition of the uropygial secretions of birds do not usually included peptides (Burger et al. 2004; Gebauer et al. 2004; Montalti et al. 2005) and, thus, this explanation is unlikely. In any case, and whatever the origin of deactivated peptides, the association between the experimental effects on bacterial growth and hatching failures detected in hoopoes, at least indicated that protease on the eggshell provoked an increased probability of bacterial infection that might be the cause of the increase in hatching failures.
The use of the spotless starling as a control species for the effect of the protease experiment has also some caveat. Different effects for different species could be explained by interspecific differences not only in the production of bacteriocin-like inhibitor substances by associated bacteria, but also in nest's environmental conditions and eggshell characteristics. However, that problem would occur independently of the species used. Moreover, sample size of starlings is quite low and, thus, the absence of significant effects of the experiment in this species should be cautiously interpreted. Thus, the use of starlings as a control species should be interpreted as a first approach trying to evaluating the effect of protease in eggs or embryos of species other than hoopoes.
Summarizing, we present strong evidence of an association between the presence of enterococci and characteristics of the uropygial gland and its secretion, including the antimicrobial properties, which suggests a symbiotic relationship between hoopoes and these bacteria. Furthermore, the experimental deactivation of peptides on the eggshells of hoopoes resulted in increased hatching failure. Although those results suggest a mutualistic relationship, more studies of potential benefits and costs associated with this symbiotic association are needed. Furthermore, questions related to the acquisition of the bacteria and its functioning within the uropygial gland of hoopoes are necessary for a better understanding of the symbiotic association and, therefore, for concluding that bacteria are beneficial for hoopoes.