Phlebotomine sand flies (Diptera: Psychodidae) are the principal vectors of phleboviruses and Leishmania spp., the causative agents of leishmaniases. The Mediterranean sand fly fauna is diverse, and leishmaniasis, mainly caused by Leishmania infantum, is endemic in the Balkan countries. Despite recent entomological surveys, only some districts of Kosovo have been sampled for sand flies, with no proof/confirmation of L. infantum. This study aimed to gain further insights into the species composition of natural sand fly populations in previously unsampled districts and areas in Kosovo without reports of leishmaniasis and to detect Leishmania DNA in sand flies. A sand fly survey was conducted in 2022 in all seven districts of Kosovo. Collected females were screened for Leishmania DNA by PCR. Positive samples were sequenced and subjected to maximum likelihood analysis with reference sequences for further molecular characterization. The trapping activities at 114 different localities resulted in 3272 caught specimens, comprising seven sand fly species of two genera, namely Phlebotomus neglectus, Ph. perfiliewi, Ph. tobbi, Ph. papatasi, Ph. simici, Ph. balcanicus and Sergentomyia minuta. Leishmania infantum DNA was detected in three individual sand flies of Ph. neglectus and Ph. perfiliewi. This study provides the most extensive sand fly survey in Kosovo and reports the first record of L. infantum DNA in sand flies, indicating autochthonous circulation of L. infantum.

Background: Phlebotomus mascittii is one of the most widespread but least studied sand fly species in Europe, occurring from Mediterranean to Central European regions. Despite its broad distribution, its potential role in Leishmania transmission remains unknown, mainly due to the lack of laboratory colonies. This study provides the first experimental assessment of the vector competence of P. mascittii for Leishmania infantum and Leishmania martiniquensis . Methods: Wild-caught P. mascittii females from Styria, Austria, were experimentally infected using membrane feeding with blood containing L. infantum and L. martiniquensis isolates of different geographical origins. Infections were evaluated 7 days post-blood meal (PBM) by microscopy and polymerase chain reaction (PCR). Morphological forms of L. infantum were quantified and compared with infections in Phlebotomus perniciosus , a known competent vector. Results: Fifteen (94% of dissected) P. mascittii females developed L. infantum infections, all showing colonization of the stomodeal valve, whereas L. martiniquensis failed to establish infection. Infection patterns and parasite localization closely resembled those in P. perniciosus . Morphometric analysis revealed a significantly higher proportion of metacyclic and leptomonad forms and fewer nectomonads in P. mascittii than in P. perniciosus . Conclusions: Our findings demonstrate that P. mascittii supports full development of L. infantum to the transmissible metacyclic stage and colonization of the stomodeal valve, confirming its potential vector competence. This study provides the first experimental evidence on P. mascittii vectorial competence and highlights its epidemiological relevance in Europe. Graphical