Sex determination studies in sand flies: a next generation sequencing approach

Sand flies are haematophagous arthropod vectors responsible for the transmission of viruses and protozoa between vertebrate hosts and causing serious diseases as leishmaniasis. The threat represented by these vectors is increasing due to the spread of diseases and inadequacy of existing control measures. Moreover, climate changes have opened new ecological niches in various areas of the world thus putting previously unaffected regions at risk of disease transmission. Hence, novel, sustainable and more effective methods for controlling the natural vector sand fly populations are urgently needed. Phlebotomus perniciosus is a sand fly species widely distributed in the western Mediterranean Basin, where it is a proven vector of Leishmania infantum among dogs and humans. We have applied RNA-seq and digital gene expression analysis to identify genes with sex-specific or sex-biased expression at a genome-wide level and to isolate genes involved in the sex determination pathway of the P. perniciosus. We defined a de novo reference transcriptome of 55,000 transcripts. Functional annotation assigned about 10,000 of the assembled transcripts to records present in the NCBI non-redundant protein database. Within this data set, we have identified well-conserved alternatively spliced transcripts of the Drosophila doublesex (dsx) and fruitless (fru) ortholog genes, which are responsible in the frutifly of somatic sexual differentiation and genetic control of courtship behavior, respectively. An ortholog of the key switch gene of insect female sex determination, the transformer (tra) gene, is apparently absent in P. perniciosus transcriptome while its auxiliary factor, the transformer-2 gene (tra-2), is highly conserved when compared to other dipteran tra-2 orthologs and differently from other Nematocera species such as mosquitoes. Using the publicly available genomic resources of sand fly species Phlebotomus papatasi and the New world species Lutzomyia longipalpis we performed a comparative analysis of the sex determination genes. Our data suggest only a partial conservation of the sex determination cascade in sand flies. These results could contribute to the study of the evolution of sex determination pathway in the under-studied Nematocera dipteran sub-order and to develop new biotechnological control methods for the sand fly vector species.