In Work Package 1 (Captures and Personality), 108 squirrels belonging to two species were trapped in various study areas characterised by different levels of urbanisation. At each site, seasonal trapping sessions were carried out to obtain longitudinal data at the individual level. We collected faecal samples from each squirrel at each trapping for later metagenomic and parasitological analyses (WPs 2 and 3). We also conducted behavioural tests (e.g. the Open Field Test and Mirror Image Stimulation Test) in a field arena to generate personality estimates for 101 squirrels. In WP2 (Microbiome and Diet), we processed 190 faecal samples using the standardised Earth Hologenome Initiative (EHI) workflow, including DNA extraction, library preparation, and shotgun sequencing. The raw sequencing data were then processed using the EHI bioinformatics pipeline to reconstruct microbial genomes using an assembly-based approach. This resulted in the reconstruction of almost 1,700 bacterial genomes, generating a catalogue of squirrel bacteria and archaea complete with taxonomic and functional annotations. We also generated dietary data by mapping our raw reads to taxonomically annotated marker-gene sequences. The raw sequencing data and bacterial genomes have been made available in public repositories and an accompanying data article has been published. In WP3 (Parasites), we analysed 125 fresh faecal samples from red and grey squirrels using copromicroscopy (i.e. faecal flotation, MacMaster egg counts and tape tests) to determine infection status. To retrieve adult helminths, we processed the intestines of 37 grey squirrels that had been culled during control activities at the end of WP1. We also quantified the fat content of the bone marrow of these animals as a proxy for body condition. Finally, in WP4 (Synthesis), we collated the available literature on microbiomes and animal invasions, publishing a review to promote the adoption of new approaches and common standards in microbiome-invasion studies. We then analysed microbiota and diet data in order to answer questions Q1 and Q2. Briefly, we characterised the microbiomes of the two host species in terms of alpha and beta diversity, as well as the factors affecting these diversity estimates. We then identified microbial taxa and functions that were exclusive to or enriched in each squirrel species. Finally, we used joint-species distribution models to elucidate microbiome dynamics, examining specifically how urbanisation and seasonality affect the composition and functionality of the gut microbial community in each species. Additionally, we tested specific hypotheses concerning the dietary preferences and genetic diversity of the two squirrel species. The full R code used for the analysis is publicly available on GitHub, and the results will be published in an original article currently at an advanced draft stage. Parasitological and behavioural data generated within Work Packages 1 and 3 will soon be integrated with the metagenomic data and analysed in a similar way to address Q3 and Q4.
