Multilocus molecular and phylogenetic analysis of phlebotomine sand flies (Diptera: Psychodidae) from southern Italy

This study reports a combined analysis of mitochondrial and ribosomal DNA target regions of phlebotomine sand flies (Diptera: Psychodidae) from the Mediterranean region. A ∼900 bp long fragment of the mitochondrial DNA encompassing regions within cytb and nd1 gene and the complete ITS2 ribosomal region (∼500 bp) were sequenced and characterized for Phlebotomus perniciosus, Phlebotomus perfiliewi, Phlebotomus neglectus, Phlebotomus papatasi, and Sergentomyia minuta, captured in two sites of southern Italy. From one to eight mitochondrial haplotypes and from one to three ITS2 sequence types were found for the examined specimens according to the different sand fly species. The mean interspecific difference in the mitochondrial sequences was of 16.1%, with an overall intraspecific nucleotide variation from 0.1 to 2.8%. A higher interspecific difference (mean 25.1%) was recorded for the ITS2 sequence, with an overall intraspecific nucleotide variation up to 4.9%. The sequence types alignment of ITS2 region showed that all phlebotomine specimens possessed a split 5.8S rRNA, consisting of a mature 5.8S rRNA and a 2S rRNA separated by a short transcribed spacer. Phylogenetic analysis of the Phlebotomus spp. sequences, herein determined and of those available in GenBank™ were concordant in clustering P. neglectus, P. perfiliewi and P. papatasi with the same species collected from different geographic areas of the Mediterranean basin in four main clades for mtDNA and ITS2, respectively. This study demonstrates the utility of multilocus sequencing, provides a dataset for the molecular identification of the most prevalent phlebotomine sand flies in southern Europe and defines the phylogenetic relationships among species examined.