Background Gastrointestinal nematodes are ubiquitous for both domestic and wild ungulates and have varying consequences for health and fitness. They exist as complex communities of multiple co-infecting species, and we have a limited understanding of how these communities vary in different hosts, regions and circumstances or of how this affects their impacts. Methods We have undertaken ITS2 rDNA nemabiome metabarcoding with next-generation sequencing on populations of nematode larvae isolated from 149 fecal samples of roe deer of different sex and age classes in the two isolated populations of Chizé and Trois Fontaines in France not co-grazing with any domestic ungulate species. Results We identified 100 amplified sequence variants (ASVs) that were assigned to 14 gastrointestinal nematode taxa overall at either genus (29%) or species (71%) level. These taxa were dominated by parasites classically found in cervids—e.g. Ostertagia leptospicularis, Spiculopteragia spp. Higher parasite species diversity was present in the Trois Fontaines population than in the Chizé population including the presence of species more typically seen in domestic livestock (Haemonchus contortus, Bunostomum sp., Cooperia punctata, Teladorsagia circumcincta). No differences in parasite species diversity or community composition were seen in the samples collected from three zones of differing habitat quality within the Chizé study area. Young roe deer hosted the highest diversity of gastrointestinal nematodes, with more pronounced effects of age apparent in Trois Fontaines. The effect of host age differed between gastrointestinal nematode species, e.g. there was little effect on O. leptospicularis but a large effect on Trichostrongylus spp. No effect of host sex was detected in either site. Conclusions The presence of some livestock parasite species in the Trois Fontaines roe deer population was unexpected given the isolation of this population away from grazing domestic livestock since decades. Overall, our results illustrate the influence of host traits and the local environment on roe deer nemabiome and demonstrate the power of the nemabiome metabarcoding approach to elucidate the composition of gastrointestinal nematode communities in wildlife. Graphical Abstract

Background: Marker gene surveys have a wide variety of applications in species identification, population genetics, and molecular epidemiology. As these methods expand to new types of organisms and additional markers beyond 16S and 18S rRNA genes, comprehensive databases are a critical requirement for proper analysis of these data. Results: Here we present an ITS2 rDNA database for marker gene surveys of both free-living and parasitic nematode populations and the software used to build the database. This is currently the most complete and up-to-date ITS2 database for nematodes and is able to reproduce previous analysis that used a smaller database. Conclusions: This database is an important resource for researchers working on nematodes and also provides a tool to create ITS2 databases for any given taxonomy.

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Marker gene surveys have a wide variety of applications in species identification, population genetics, and molecular epidemiology . As these methods expand to new types of organisms and additional markers beyond 16S and 18S rRNA genes, comprehensive databases are a critical requirement for proper analysis of these data. Here we present an ITS2 rDNA database for maker gene surveys of both free-living and parasitic nematode populations and the software used to build the database. This is an important resource for researchers working on nematodes and also provides a tool to create ITS2 databases for any given taxonomy. Availability and Implementation: The database is available as an interactive web app at

BackgroundGastrointestinal nematodes are ubiquitous for both domestic and wild ungulates which have varying consequences to health and fitness. They exist as complex communities of multiple co-infecting species and we have a very limited understanding of how these communities vary in different hosts, regions and circumstances or on how this affects their impacts. MethodsWe have undertaken ITS-2 rDNA nemabiome metabarcoding on populations of nematode larvae isolated from fecal samples of roe deer of different sex and age classes in the two isolated populations of Chizé and Trois Fontaines in France not co-grazing with any domestic ungulate species. ResultsWe identified 100 Amplified Sequence Variants (ASVs) that assigned to 14 gastrointestinal nematode taxa overall either at genus (38%), or species (62%) level. These taxa were dominated by parasites classically found in cervids - e.g., Ostertagia leptospicularis, Spiculopteragia spp. Higher parasite species diversity was present in the Trois Fontaine than in the Chize populations including the presence of some species more typically seen in domestic livestock, (Haemonchus contortus, Bunostomum trigonocephalum, Cooperia punctata, Teladorsagia circumcincta). No differences in parasite species diversity or community composition was seen in the samples collected from three zones of differing habitat quality within the Chize region. Young roe deer hosted the highest diversity of gastrointestinal nematodes, with more pronounced effects of age apparent in Trois Fontaines. The effect of host age differed between gastrointestinal nematode species, e.g. there was little effect on Ostertagia leptospicularis but a high effect on Trichostrongylus spp. No effect of host sex was detected in either site. ConclusionsThe presence of some livestock parasite species in the Trois Fontaine roe deer popualtions was surprising given the isolation of this population away from grazing domestic livestock. However, it should be noted that a small group of sheep were resident in the region, albeit separately fenced, which raises some interesting questions regarding the introduction of non-native parasite species into wildlife populations. Overall, our results suggest complex interactions between hosts, environments and parasites and illustrate the power of the nemabiome metabarcoding approach to elucidate the composition of gastrointestinal nematode communities in wildlife.