Phylogenetic insights into Wolbachia natural infections in sand flies of Northern Italy

Background Sand flies (Diptera: Phlebotominae) are important vectors of several pathogens, exposing over 1 billion people to infection in endemic areas. In the absence of effective vaccines, alternative vector control strategies are increasingly being explored. Among these, the endosymbiont Wolbachia spp. has attracted particular interest due to its ability to manipulate host reproduction and reduce vector competence. However, information on its occurrence in natural sand fly populations remains scarce. This study investigates the presence, prevalence, and genetic diversity of Wolbachia in wild-caught sand flies from Northern Italy. Methods Sand flies were collected from 24 sites across Northern Italy using CO₂-baited and CDC light traps between 2023 and 2025. Specimens were pooled according to sex, site, and collection date. Males were identified morphologically, while females were screened for Wolbachia via PCR. Positive samples were further analyzed by amplifying five multi-locus sequence typing (MLST) loci ( coxA , fbpA , ftsZ , gatB , and hcpA ), along with the cytochrome oxidase I ( COI ) gene for barcoding, and, for four samples, a partial wsp gene. Samples for which all genes were successfully amplified were subjected to nanopore sequencing. Results During the sampling period, 1158 males were morphologically identified, resulting in the detection of 7 sand fly species, with Phlebotomus perniciosus being the most abundant ( N = 751), followed by P. perfiliewi , P. neglectus , P. ariasi , Sergentomyia minuta , P. mascittii , and P. papatasi . Moreover, a total of 1,937 sand flies were divided into 124 pools and screened for Wolbachia , including 10 male pools that were not morphologically identified. Overall Wolbachia prevalence was 38.7%, varying by year (44.9% in 2023, 21.3% in 2024, and 57.1% in 2025). Complete MLST profiles were obtained for 11 samples, all assigned to Wolbachia Supergroup B and identified as P. perniciosus by COI barcoding. Remarkably, all 11 sequenced Wolbachia shared an identical profile despite being collected across different years and habitats. No exact matches were found in public databases, with the closest profile originating from Tribolium confusum . Conclusions This study presents the first MLST characterization of Wolbachia in P. perniciosus . These findings expand our understanding of Wolbachia diversity in sand flies and provide a foundation for future studies on its effects on reproduction, survival, and vector competence, supporting potential biological control strategies against sand fly-borne pathogens.