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

Latrofa MS1, Dantas-Torres F1, Weigl S1, Tarallo VD1, Parisi A2, Traversa D3, Otranto D1.1 Dipartimento di Sanità Pubblica e Zootecnia, Università degli Studi di Bari, Strada Provinciale per Casamassima km 3, 70010 Valenzano (Bari), Italy2 Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Contrada S. Pietro Pitumo, 70017 Putignano, Italy3 Dipartimento di Scienze Biomediche Comparate, Università degli Studi di Teramo, Piazza Aldo Moro 45, 64100 Teramo, ItalyActa Tropica 2011 May 25. [Epub ahead of print]This study reports a combined analysis of mitochondrial and ribosomal DNA target regions of phlebotomine sand flies (Diptera: Psychodidae) from the Mediterranean region. A ~900bp long fragment of the mitochondrial DNA encompassing regions within cytb and nd1 gene and the complete ITS2 ribosomal region (~500bp) were sequenced and characterized for Phlebotomus perniciosus, P. perfiliewi, P. neglectus, P. 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.

VBORNET comment, 26/8/2010: Despite their potential utility in providing an unequivocal identification of sand flies and ulterior information on their ecology and vector capacity, no molecular datasets for mitochondrial and ribosomal DNA gene sequences have been generated for the simultaneous comparison of the most representative phlebotomine sand fly species of the Mediterranean region. The main goal of this study is to assess the genetic diversity within and among phlebotomine sand fly species collected in southern Italy and to compare them with those available from Mediterranean region, together with the analysis of their phylogenetic relationships. Latrofa et al. obtained and demonstrated the usable completed data set according to two DNA target regions. Such molecular data will also be useful for further investigations on the association between phlebotomine sand fly species genetic variability and their role as vectors of L. infantum.