They are what they eat: effects of diet on the gut microbiota of the gray wolf (Canis lupus)

The grey wolf (Canis lupus L.) is undergoing a notable increase in population size and range across Europe, and the recolonization of peri-urban and agricultural areas may lead to dietary changes due to increased predation on livestock. Food intake allows the provision of resources necessary for the survival of both the host and its gut microbiota (e.g. bacteria, fungi, virus, helminths). Microorganisms adapted to live in the mammalian gut often have a mutualistic relationship with their host and contribute to the host‘s health and adaptive potential to environmental changes. Although well-studied in humans, the effect of diet changes on the gut microbiota of wild animals (including the wolf) living in human-impacted environments remains largely unknown. In this study, we investigated the effects of dietary shifts on wolf gut microbiota by analysing faecal samples collected with non-invasive methods from a pack of nine healthy wolves hosted by the BELPARK – Parco Faunistico Spormaggiore (Italy). Over a three-month period, wolves were sequentially fed a diet based on either whole sheep (Ovis aries) or roadkill (whole red deer, Cervus elephas) before returning to a conventional mixed diet. Faecal samples were collected daily from the wolf enclosure to capture short-term (i.e. within 48 hours) and mid-term (i.e. one to three weeks) responses of gut microbial diversity to each dietary change. Individual genetic identification of each sample was performed using a standard set of 10 microsatellite markers. Prokaryotic and fungal communities of 70 faecal samples representing the entire pack throughout the experiment were characterized using metataxonomy and targeting the prokaryotic 16S rRNA gene and fungal ITS2 region. To link taxonomic changes to changes in gut function, the gut microbial transcriptional profile of a subset of 20 samples was investigated using metatranscriptomics. The wolf prokaryotic microbiota was rich in Bacillota, Bacteroidota and Fusobacteriota, phyla commonly found in carnivores gut microbiota, while fungal communities were dominated by Ascomycota. Longitudinal comparisons among individuals revealed a small core microbiota of dominant bacteria and fungi shared by all animals (109 bacteria out of 6,248 and 30 fungi out of 2,365), alongside a large number of low-abundance or individual-specific microorganisms (604 bacteria and 1131 fungi). Dietary shifts were associated with significant changes in the taxonomy of bacterial and fungal taxa. Notably, the transition from a domestic to a wildlife-based diet elicited contrasting responses: bacterial richness increased rapidly but transiently following dietary change, whereas fungal communities exhibited a continuous increase in richness and greater inter-individual variability. While fungal community composition was strongly associated with diet and differed significantly among dietary regimes, changes in bacterial community composition were only marginally linked to diet, a pattern increasingly observed in wildlife microbiome studies. Our results delve deeper into the extent of natural variation through time of the gut microbiota hosted by related animals living in captivity and thus sharing a limited and controlled home range. These findings have direct implications for wildlife health and will contribute to the development of dietary regimes that preserve gut microbial diversity and functional potential, thereby supporting the welfare of animals housed in wildlife parks.