Targeted Molecular Screening of Leishmania infantum in Sergentomyia minuta Populations from Southern Italy.

Sergentomyia minuta is a widespread Mediterranean sand fly traditionally regarded as herpetophilic and of limited relevance for human leishmaniasis. Occasional detection of mammalian Leishmania DNA in Sergentomyia species has challenged this assumption. This study aimed to investigate the abundance, host-feeding behaviour, and molecular detection of Leishmania infantum DNA in S. minuta in peri-urban areas of southern Italy. Sand flies were collected using sticky traps at three sites in the Catania area (Italy). Unengorged females were screened for Leishmania spp. DNA using ITS1 and SSU rRNA markers. ITS1 PCR-RFLP was used exclusively to discriminate L. infantum-compatible from non-L. infantum molecular profiles. Engorged females were analysed for blood-meal origin using mitochondrial cytochrome b markers (CytB1 and CytB2) and a nuclear human-specific transthyretin (TTR) marker through PCR-RFLP and multiplex PCR. A total of 1361 S. minuta specimens were collected, with a strongly female-biased sex ratio. Leishmania spp. DNA was detected in a substantial proportion of pooled samples. ITS1 PCR-RFLP profiles revealed heterogeneous molecular patterns that consistently excluded L. infantum and were compatible with non-L. infantum trypanosomatid DNA. Blood-meal analyses suggested feeding on multiple vertebrate hosts, including occasional detection of human mitochondrial DNA, whereas the nuclear TTR marker was consistently negative, likely reflecting DNA degradation during blood digestion. S. minuta populations from southern Italy display high abundance, ecological plasticity, and a strong association with peri-urban habitats. Our findings refine, rather than overturn, the current understanding of the epidemiological role of S. minuta in Mediterranean ecosystems. Multilocus molecular screening revealed heterogeneous non-L. infantum trypanosomatid DNA patterns, while no evidence of Leishmania infantum was detected. Occasional human blood meals were detected, but the epidemiological relevance of S. minuta for human leishmaniasis remains unproven. These findings highlight the importance of integrated entomological and molecular approaches for careful interpretation of vector-parasite-host relationships Mediterranean ecosystems.