DNA sequencing confirms PCR-RFLP identification of wild caught Larroussius sand flies from Crete and Cyprus.

Many Phlebotomine sand fly species (Diptera, Psychodidae) are vectors of the protozoan parasite Leishmania causing a group of diseases called the leishmaniases. The subgenus Larroussius includes sand fly vectors found in South East Mediterranean Basin responsible for Visceral (VL) and Cutaneous human leishmaniasis (CL). It is important to monitor these medically important insects in order to safely predict possible Leishmania transmission cycles. Leishmania infantum is endemic in the islands of Crete and Cyprus with increasing VL cases in humans and dogs and in Cyprus the newly introduced Leishmania donovani causes both VL and CL in humans. The morphological identification of the females of the subgenus Larroussius often presents difficulties. Morphology and COI PCR - RFLP were used to identify wild caught Larroussius sand flies belonging to Phlebotomus tobbi, P. perfiliewi, and P. neglectus species from Crete and Cyprus. The identification results were further confirmed by sequencing (DNA barcoding) and Bayesian phylogenetic analysis. COI PCR - RFLP, when correctly optimized and with respect to geographical origin, can serve as an initial patterning identification tool when large sand fly numbers need to be identified. It could accurately assign Larroussius females and males to their taxa overcoming the difficulties of morphological identification. Finally, DNA barcoding will contribute to a molecular identification database to be used for in-depth species studies.