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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.

Abstract Sandflies (Diptera; Psychodidae) are medical and veterinary vectors that transmit diverse parasitic, viral, and bacterial pathogens. Their identification has always been challenging, particularly at the specific and sub-specific levels, because it relies on examining minute and mostly internal structures. Here, to circumvent such limitations, we have evaluated the accuracy and reliability of Wing Interferential Patterns (WIPs) generated on the surface of sandfly wings in conjunction with deep learning (DL) procedures to assign specimens at various taxonomic levels. Our dataset proves that the method can accurately identify sandflies over other dipteran insects at the family, genus, subgenus, and species level with an accuracy higher than 77.0%, regardless of the taxonomic level challenged. This approach does not require inspection of internal organs to address identification, does not rely on identification keys, and can be implemented under field or near-field conditions, showing promise for sandfly pro-active and passive entomological surveys in an era of scarcity in medical entomologists.

Sand flies (Diptera: Psychodidae: Phlebotominae) are blood-feeding insects that transmit the protozoan parasites Leishmania spp. and various arthropod-borne (arbo) viruses. While in Mediterranean parts of Europe the sand fly fauna is diverse, in Central European countries including Austria mainly Phlebotomus mascittii is found, an assumed but unproven vector of Leishmania infantum. To update the currently understudied sand fly distribution in Austria, a sand fly survey was performed and other entomological catches were screened for sand flies. Seven new trapping locations of Ph. mascittii are reported including the first record in Vienna, representing also one of the first findings of this species in a city. Morphological identification, supported by fluorescence microscopy, was confirmed by two molecular approaches, including sequencing and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) protein profiling. Sand fly occurrence and activity were evaluated based on surveyed locations, habitat requirements and climatic parameters. Moreover, a first comparison of European Ph. mascittii populations was made by two marker genes, cytochrome c oxidase subunit 1 (COI), and cytochrome b (cytb), as well as MALDI-TOF mass spectra. Our study provides new important records of Ph. mascittii in Austria and valuable data for prospective entomological surveys. MALDI-TOF MS protein profiling was shown to be a reliable tool for differentiation between sand fly species. Rising temperatures and globalization demand for regular entomological surveys to monitor changes in species distribution and composition. This is also important with respect to the possible vector competence of Ph. mascittii.

Transmission of Leishmania tropica was studied in 2 adjacent foci in Israel where vector populations differ. Only Phlebotomus sergenti was found infected with L. tropica in the southern focus; P. arabicus was the main vector in the northern focus. Rock hyraxes (Procavia capensis) were incriminated as reservoir hosts in both foci. L. tropica strains from the northern focus isolated from sand flies, cutaneous leishmaniasis cases, and rock hyraxes were antigenically similar to L. major, and strains from the southern focus were typically L. tropica. Laboratory studies showed that P. arabicus is a competent vector of L. tropica, and P. sergenti is essentially refractory to L. tropica from the northern focus. Susceptibility of P. arabicus may be mediated by O glycoproteins on the luminal surface of its midgut. The 2 foci differ with respect to parasites and vectors, but increasing peridomestic rock hyrax populations are probably responsible for emergence of cutaneous leishmaniasis in both foci.