Outcome monitoring of water vole reintroductions in ecological restoration

Growing awareness socially, economically, and politically, of extensive biodiversity loss in the UK is motivating a wide range of ecological restoration targets and projects. These range from targets for tree cover on farmland for environmental schemes to rewilding. Water voles (Arvicola amphibius) in the UK disappeared from 90% of their known range between 1970-2000. Their decline was driven by the introduction of the American mink (Neovison vison), and land use changes, where intensive livestock and dairy production has encroached on riverbank habitat. The restoration of their riparian habitat has included restricting livestock access, and removing the invasive American mink, permitting the reintroduction of captive bred water voles in over 250 sites across the UK since 2003. However, despite this conservation campaign, outcome monitoring of water vole reintroductions is limited, particularly in terms of health and survival rates. Our aim was to fill some of the gaps in post- reintroduction monitoring of water voles, develop a tool for post-release monitoring, and assess the potential of reintroduced species as sentinels of ecosystem health. Given that gut microbiota plays a vital role in host health, influencing immunity and physiology, identifying hypothesised post- reintroduction shifts is therefore important for animal welfare and reintroduction success. We collected faecal and water samples concurrently from two water vole populations: one reintroduced in 2024 and one established in 2014. Samples were collected systematically for three months following the release of the newly reintroduced population. Whole DNA was extracted from each sample and the 16S rRNA gene (V3-4) and ITS2 region were amplified for Illumina amplicon sequencing. Bioinformatic analyses included the estimation and comparison of alpha and beta diversities between populations and over time, temporal changes in bacterial taxa relative abundance, source tracking to assess environmental microbiota influence on water vole microbiota, and inference of the functional roles of taxa associated with post-reintroduction microbial acquisition. The newly reintroduced water vole population showed higher alpha diversity (Shannon index) overall than the established population, with no significant temporal changes observed in either population. Beta diversity (Bray Curtis dissimilarity) in the established population remained stable over time, whereas the reintroduced population experienced an initial rapid shift in community composition during the first two weeks post-release, stabilizing by week five. Temporal beta diversity analysis revealed that the microbiota of the reintroduced population initially converged rapidly towards that of the established population, followed by a relatively stable phase with small fluctuations. We will also discuss the results of source tracking (the influence of environmental microbiota on that of the water voles), and temporal analyses of the microbial community composition at a taxonomic level, including potential pathogens. We conclude that reintroduced water voles experienced a rapid and substantial shift in gut microbiota composition shortly after release, changes not seen in the established population. This highlights the significant impact of reintroduction and adaptation on gut microbial communities and provides a novel timescale for these changes. Our results provide insight into the impact of reintroduction of a captive bred population using a structured comparative study design. Better understanding of gut microbial community composition, sources of acquisition, and predicted functional impacts, will enable advancement and better leverage of microbiota-informed conservation practices.