Antimicrobial Activity and Genetic Profile of Enteroccoci Isolated from Hoopoes Uropygial Gland

Symbiotic microorganisms may be directly transferred from parents to offspring or acquired from a particular environment that animals may be able to select. If benefits for hosts vary among microbial strains, natural selection may favour hosts holding the most beneficial one. Enterococci symbionts living in the hoopoe (Upupa epops) uropygial gland are able to synthesise bacteriocins (antimicrobial peptides that inhibit the growth of competitor bacteria). We explored variability in genetic profile (through RAPD-PCR analyses) and antimicrobial properties (by performing antagonistic tests against ten bacterial indicator strains) of the different isolates obtained from the uropygial glands of hoopoe females and nestlings. We found that the genetic profile of bacterial isolates was related to antimicrobial activity, as well as to individual host identity and the nest from which samples were obtained. This association suggest that variation in the inhibitory capacity of Enterococci symbionts should be under selection.This work was financed by Ministerio de Ciencia e Innovación (Spanish National Government) and FEDER founds (projects CGL2010-19233-C03-01, and CGL2010-19233-C03-03), and Junta de Andalucía (P09-RNM-4557)

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 discussedMinisterio de Ciencia e Innovación (España)Junta de Andalucí

The Hoopoe's Uropygial Gland Hosts a Bacterial Community Influenced by the Living Conditions of the Bird

Molecular methods have revealed that symbiotic systems involving bacteria are mostly based on whole bacterial communities. Bacterial diversity in hoopoe uropygial gland secretion is known to be mainly composed of certain strains of enterococci, but this conclusion is based solely on culture-dependent techniques. This study, by using culture-independent techniques (based on the 16S rDNA and the ribosomal intergenic spacer region) shows that the bacterial community in the uropygial gland secretion is more complex than previously thought and its composition is affected by the living conditions of the bird. Besides the known enterococci, the uropygial gland hosts other facultative anaerobic species and several obligated anaerobic species (mostly clostridia). The bacterial assemblage of this community was largely invariable among study individuals, although differences were detected between captive and wild female hoopoes, with some strains showing significantly higher prevalence in wild birds. These results alter previous views on the hoopoe-bacteria symbiosis and open a new window to further explore this system, delving into the possible sources of symbiotic bacteria (e.g. nest environments, digestive tract, winter quarters) or the possible functions of different bacterial groups in different contexts of parasitism or predation of their hoopoe host.This work was supported by the Ministerio de Ciencia y Tecnología (projects CGL2005-06975/BOSFEDER; CGL2007-61251/BOSFEDER), the Ministerio de Ciencia e Innovación (projects CGL2009-14006/BOSFEDER; CGL2010-19233-C03-01/BOSFEDER; CGL2010-19233-C03-03/BOSFEDER), the Ministerio de Economía y Competitividad (projects CGL2013-48193-C3-1-P/BOSFEDER; CGL2013-48193-C3-2-P/BOSFEDER), and the Junta de Andalucía (RNM 345, P09-RNM-4557). SMRR received a grant from the Ministerio de Ciencia e Innovación (FPI program, BES-2011-047677)

Ectoparasite activity during incubation increases microbial growth on avian eggs

We thank Estefanía López for lab work, and Tomás Pérez-Contreras and Emilio Pagani-Núñez for facilitating collection of some of the flies used in manipulations. We also thank Ángela Martínez-García for help with management of ARISA data and Natalia Juárez and Deseada Parejo for the pictures of owls and roller clutches, respectively. We appreciate the comments provided by Dr. Adèle Mennerat and five anonymous referees on earlier versions of the manuscript.All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.While direct detrimental effects of parasites on hosts are relatively well documented, other more subtle but potentially important effects of parasitism are yet unexplored. Biological activity of ectoparasites, apart from skin injuries and blood-feeding, often results in blood remains, or parasite faeces that accumulate and modify the host environment. In this way, ectoparasite activities and remains may increase nutrient availability that may favour colonization and growth of microorganisms including potential pathogens. Here, by the experimental addition of hematophagous flies (Carnus hemapterus, a common ectoparasite of birds) to nests of spotless starlings Sturnus unicolor during incubation, we explore this possible side effect of parasitism which has rarely, if ever, been investigated. Results show that faeces and blood remains from parasitic flies on spotless starling eggshells at the end of incubation were more abundant in experimental than in control nests. Moreover, eggshell bacterial loads of different groups of cultivable bacteria including potential pathogens, as well as species richness of bacteria in terms of Operational Taxonomic Units (OTUs), were also higher in experimental nests. Finally, we also found evidence of a link between eggshell bacterial loads and increased embryo mortality, which provides indirect support for a bacterial-mediated negative effect of ectoparasitism on host offspring. Trans-shell bacterial infection might be one of the main causes of embryo death and, consequently, this hitherto unnoticed indirect effect of ectoparasitism might be widespread in nature and could affect our understanding of ecology and evolution of host-parasite interactionsFinancial support was provided by Spanish Ministerio de Economía y Competitividad and FEDER (CGL2013-48193-C3-1-P, CGL2013-48193-C3-2-P), by JAE programme to DMG and MRR, and by Juan de la Cierva and Ramón y Cajal programmes to GT. All procedures were conducted under licence from the Environmental Department of the Regional Government of Andalucía, Spain (reference SGYB/FOA/AFR)

Protection and consolidation of stone heritage by self-inoculation with indigenous carbonatogenic bacterial communities

Enhanced salt weathering resulting from global warming and increasing environmental pollution is endangering the survival of stone monuments and artworks. To mitigate the effects of these deleterious processes, numerous conservation treatments have been applied that, however, show limited efficacy. Here we present a novel, environmentally friendly, bacterial self-inoculation approach for the conservation of stone, based on the isolation of an indigenous community of carbonatogenic bacteria from salt damaged stone, followed by their culture and re-application back onto the same stone. This method results in an effective consolidation and protection due to the formation of an abundant and exceptionally strong hybrid cement consisting of nanostructured bacterial CaCO3 and bacterially derived organics, and the passivating effect of bacterial exopolymeric substances (EPS) covering the substrate. The fact that the isolated and identified bacterial community is common to many stone artworks may enable worldwide application of this novel conservation methodology.This work was supported by the Spanish Government (Grants MAT2012-37584, CGL2012-35992 and CGL2015-70642-R), the Junta de Andalucía through Proyecto de excelencia RNM-3493 and Project P11-RNM-7550, the Research Groups BIO 103 and RNM-179, and the University of Granada (Unidad Científica de Excelencia UCE-PP2016-05). Additional funds were provided by the Molecular Foundry (Lawrence Berkeley National Laboratory, LBNL, University of California, Berkeley, CA) for a research stay of M.S. (project #1451; User Agreement No. NPUSR009206)