Aerobic midgut microbiota of sand fly vectors of zoonotic visceral leishmaniasis from northern Iran, a step toward finding potential paratransgenic candidates

BACKGROUND: Leishmaniasis is caused by Leishmania parasites and is transmitted to humans through the bite of infected sand flies. Development of Leishmania to infective metacyclic promastigotes occurs within the sand fly gut where the gut microbiota influences development of the parasite. Paratransgenesis is a new control method in which symbiotic bacteria are isolated, transformed and reintroduced into the gut through their diet to express anti-parasitic molecules. In the present study, the midgut microbiota of three sand fly species from a steppe and a mountainous region of northern Iran, where zoonotic visceral leishmaniasis (ZVL) is endemic, was investigated. METHODS: Briefly, adult female sand flies was collected during summer 2015 and, after dissection, the bacterial composition of the guts were analyzed using a culture-dependent method. Bacterial DNA from purified colonies was extracted to amplify the 16S rRNA gene which was then sequenced. RESULTS: Three ZVL sand fly vectors including Phlebotomus major, P. kandelakii and P. halepensis were found in the highlighted regions. In total, 39 distinct aerobic bacterial species were found in the sand fly midguts. The sand fly microbiota was dominated by Proteobacteria (56.4%) and Firmicutes (43.6%). Bacterial richness was significantly higher in the steppe region than in the mountainous region (32 vs 7 species). Phlebotomus kandelakii, the most important ZVL vector in the study area, had the highest bacterial richness among the three species. Bacillus subtilis and Pantoea agglomerans were isolated from the guts of the sand flies; these are already used for the paratransgenesis of sand flies and mosquitoes, respectively. CONCLUSIONS: The existence of B. subtilis and P. agglomerans in the ZVL vectors and other sand fly species studied so far suggests that these two bacterial species are potential candidates for paratransgenic approach to prevent ZVL transmission. Further research needs to test the possible relationship between the gut microbiome richness and the vector competence of the ZVL vectors.