First Detection of Human- and Dog-Associated Demodex Mites (Acari, Arachnida) in Southern European Wolves (Canis lupus)

<i>Demodex</i> mites are common commensals of mammalian skin, but under certain conditions, they can cause severe skin diseases. This study analyzed the presence, diversity, and phylogenetic relationships of <i>Demodex</i> species in two wolf subspecies from southern Europe to determine whether species-level differences exist between wild and domestic canids after thousands of years of divergence. A total of 1400 hair samples from 140 wolves were analyzed using a real-time PCR (qPCR) targeting mitochondrial <i>16S rRNA</i> and nuclear <i>18S rRNA</i> genes. Overall, 37.1% (52/140; 95% CI: 29.0-45.9%) of wolves were positive for <i>Demodex</i> DNA, with a higher prevalence in Italian (46%) than in Iberian (36%) wolves. The lip and chin areas were the most reliable sampling sites. Four <i>Demodex</i> species were identified in wolves: <i>D. injai</i> and <i>D. canis</i> (associated with dogs), and <i>D. folliculorum</i> and <i>D. brevis</i> (associated with humans). Co-infestations involving multiple <i>Demodex</i> species were recorded for the first time in wild canids. These results challenge the long-held belief of strict host specificity in <i>Demodex</i> mites. The discovery of <i>Demodex</i> species associated with both humans and dogs in wolves supports the idea that host-switching and ecological interactions have occurred throughout the evolution of canids and humans. Such cross-species transfers may have taken place during the early domestication of dogs, representing a plausible scenario compatible with our data. However, given the isolated history of the two southern wolf populations, it is more probable that these findings result from recent interspecific transmission events, likely facilitated by ecological overlap with domestic animals and human environments. Future genomic studies will be essential for clarifying the evolutionary relationships within the genus <i>Demodex</i> and its host associations.