bioRxiv preprint doi: https://doi.org/10.1101/211490; this version posted October 30, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Protective role of the vulture facial and gut microbiomes aid adaptation to 2 scavenging 3 M. Lisandra Zepeda Mendoza1*, Gary R. Graves2, Michael Roggenbuck3, Karla Manzano Vargas1,4, 4 Lars Hestbjerg Hansen5, Søren Brunak6, M. Thomas P. Gilbert1, 7*, Thomas Sicheritz-Pontén6* 5 6 1 Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen. Øster 7 Voldgade 5-7, 1350 Copenhagen K, Denmark. 8 2 Department of Vertebrate Zoology, National Museum of Natural History, Smithsonian Institution, 9 Division of Birds, 20013 Washington, DC, USA. 10 3 Department for Bioinformatics and Microbe Technology, Novozymes A/S, 2880 Bagsværd, 11 Denmark. 12 4 Undergraduate Program on Genomic Sciences, Center for Genomic Sciences, National 13 Autonomous University of Mexico, Av. Universidad s/n Col. Chamilpa, 62210 Cuernavaca, Morelos, 14 Mexico. 15 5 Section for Microbiology and Biotechnology, Department of Environmental Science, Aarhus 16 University, Frederiksborgvej 399, 4000 Roskilde, Denmark. 17 6 Center for Biological Sequence Analysis, Department of Bio and Health Informatics, Technical 18 University of Denmark, Anker Engelunds Vej 1 Bygning 101A, 2800 Kgs. Lyngby, Denmark. 19 7 Norwegian University of Science and Technology, University Museum, 7491 Trondheim, Norway. 20 *Correspondence to: Thomas Sicheritz-Pontén, e-mail: [email protected], M. Thomas P. Gilbert, 21 e-mail: [email protected], and M. Lisandra Zepeda Mendoza, e-mail: [email protected] 22 23 24 1 bioRxiv preprint doi: https://doi.org/10.1101/211490; this version posted October 30, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 25 Abstract 26 Background: Vultures have adapted the remarkable ability to feed on carcasses that may contain 27 microorganisms that would be pathogenic to most other animals. The holobiont concept suggests that 28 the genetic basis of such adaptation may not only lie within their genomes, but additionally in their 29 associated microbes. To explore this, we generated shotgun DNA sequencing datasets of the facial 30 and gut microbiomes from the black and turkey vultures. We characterized i) the functional potential 31 and taxonomic diversity of their microbiomes, ii) the potential pathogenic challenges they face, and 32 iii) elements in the microbiome that could play a protective role to the vulture’s face and gut. 33 Results: We found elements involved in diseases, such as periodontitis and pneumonia (more 34 abundant in the face), and gas gangrene and food poisoning (more abundant in the gut). Interestingly, 35 we found taxa and functions with potential for playing health beneficial roles, such as antilisterial 36 bacteria in the gut, and genes for the production of antiparasites and antiinsectisides in the face. Based 37 on the identified phages, we suggest that phages aid in the control, and possibly elimination as in 38 phage therapy, of microbes reported as pathogenic to a variety of species. Interestingly, we also 39 identified Adineta vaga in the gut, an invertebrate that feeds on dead bacteria and protozoans, 40 suggesting a defensive predatory mechanism. Finally, we suggest a colonization resistance role 41 though biofilm formation played by Fusobacteria and Clostridia in the gut. 42 Conclusions: Our results highlight the importance of complementing genomic analyses with 43 metagenomics in order to obtain a clearer understanding of the host-microbial alliance and show the 44 importance of microbiome-mediated health protection for adaptation to extreme diets, such as 45 scavenging. 46 47 Keywords: Microbiome, diet specialization, scavenging, colonization resistance, pathogens, vulture, 48 metagenomics. 2 bioRxiv preprint doi: https://doi.org/10.1101/211490; this version posted October 30, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 49 50 Background 51 Vultures are scavengers whose global populations are under serious threats, and a better 52 understanding of various aspects for their biology are necessary [1,2]. Vultures are known as nature’s 53 clean-up crew as they feed on muscles and viscera from carcasses of animals that have died mainly 54 from malnutrition, accidents, and natural or infectious diseases, thus are expected to carry pathogens 55 such as those causing anthrax, tuberculosis, brucellosis, etc. [3]. Carcasses are a very nutrient-rich 56 resource and it has been speculated that the release of toxins and pathogenicity genes in the carcass 57 microbiome are part of a microbial strategy for outcompeting other microbes [4,5]. The main 58 colonizers of a carcass are microbes originating from the normal flora of the dead animal, which 59 might turn pathogenic in the carcass environment [6], as well as soil-dwelling bacteria, nematodes, 60 fungi and insects [7]. In spite of the potentially serious health implications posed by their 61 consumption, the pathogenic repertoire of the gut of these species of birds has not been fully 62 characterized in relation to their possible implications in the environment. Thus, one of the most 63 intriguing aspects of vulture biology is how they protect themselves against the health challenges 64 posed by their dietary source. Physiologic, genetic and genomic analyses of different species of 65 vultures have explored this aspect and identified genes associated to respiration, immunity and gastric 66 secretion as candidates for the adaptation to its scavenging diet [8,9]. 67 With the genomic revolution it has become apparent that besides genomic changes, host-associated 68 microbiota play an important role in diet specialization across vertebrates [10] and that the gut 69 microbiome may play highly relevant yet unexplored role in diet-driven speciation [11]. The gut 70 microbiome is related to digestion traits, such as energy harvest, nutrient acquisition, and intestinal 71 homeostasis [12], among other phenotypes related to the immune and neuroendocrine systems [13– 72 15]. Furthermore, the role of the microbiome in health protection to the host has also been 3 bioRxiv preprint doi: https://doi.org/10.1101/211490; this version posted October 30, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 73 demonstrated [16,17], and disorders in it lead to diseases such as irritable bowel syndrome, 74 inflammatory bowel disease, obesity and diabetes [18–20]. In light of the key roles that host- 75 microbiome relationships can play in adaptation, it has been acknowledged that genomic adaptations 76 alone may not provide the full answer to the vulture adaptations for scavenging [8]. However, neither 77 the complete microbial taxonomic diversity (including non-bacterial microbes) nor the gene 78 catalogue of any vulture species’ face and gut microbiomes have been examined for their protective 79 role against microbes that would normally pose serious health risks for other non-scavenging 80 vertebrate species. 81 In order to evaluate the protective role of the vulture’s facial and gut microbiome, we generated 82 metagenomic datasets from facial swabs and gut samples for two species of New World vultures, the 83 black vulture (Coragyps atratus) and the turkey vulture (Cathartes aura), and performed 84 metagenomic taxonomic and functional analyses. We identified a wide variety of taxa and genes that 85 would cause serious diseases, e.g. sepsis, gangrene and food poisoning, to animals without protection 86 provided by a specialized scavenging microbiome. Such protection could consist of health defence 87 elements not only from bacterial probiotics, but also from phages and predatory eukaryotes. Our 88 results suggest that the vulture’s microbiota plays a significant health-protective role in its adaptation 89 to a scavenging diet. 90 91 Methods 92 Sampling method and DNA sequencing 93 We analysed a subset of the sample set used by Roggenbuck et al. [21]. Briefly, the vultures were 94 collected over a period of several days in Tenessee, USA. Coragyps atratus were live-trapped at deer 95 carcasses and then transported to a central facility within a couple of hours of trapping. They were 96 then euthanized with CO2, necropsied, and sampled within 30-45 minutes of death. Cathartes aura 4 bioRxiv preprint doi: https://doi.org/10.1101/211490; this version posted October 30, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 97 vultures were shot at roosts, bagged individually, and transported to the processing facility where 98 they were refrigerated 2-6 hours before necropsy and sampling. The samples were kept in RNAlater 99 and DNA was extracted and the shotgun libraries for HiSeq PE 100 prepared using the Nextera library 100 building kit following the manufacturer’s instructions, as in Roggenbuck et al. [21]. We used a total 101 of 22 turkey vulture (Cathartes aura) and 25 black vulture (Coragyps atratus) large intestine samples, 102 and 16 turkey vulture and 17 black vulture facial samples. 103 Data processing 104 Two pipelines were used for the processing of the raw reads. In the first approach, we removed 105 adapter sequences and bases with quality <15 using Trimmomatic v0.32 [22]. Afterwards, in order to 106 filter out non-bacterial reads of avian, human, and Phi phage origin, the datasets were mapped against 107 the bird genomes dataset of the avian phylogenomic project [23] (which includes the turkey vulture 108 genome), the human (hg19) and the Phi phage genomes, and only the non-mapping reads were kept.