We have previously provided conclusive evidence that the invertebrate stages of Leishmania are fully capable of a sexual cycle during their growth and development in the sand fly vector. The priority of our studies in 2012 has been to extend the findings of sexual competence to other species and strains of Leishmania and to additional sand fly vector species. We have been able to recover L. major hybrids from Lutzomyia longipalpis, demonstrating that the main genus of sand flies found in the New World are capable of supporting the sexual reproductive cycle of the Leishmania parasites that they transmit. Hybrids were recovered as early as three days post infection, indicating that early developmental stages, e.g. procyclic and/or nectomonad promastigotes, are able to mate. We have also been able to successfully cross L. major sub-strains from West Africa, the Middle East, and Southern Russia, demonstrating that L. major isolates from across the full range of their geographic distribution are sexually competent. Furthermore, we have been able for the first time to generate interspecies hybrids between L. major and L. infantum. The occurrence of intra- and inter-species allelic hybrids amongst natural populations has been substantiated by multi-locus genotyping of over 300 strains. The differentiation of midgut promastigotes into the infectious, metacyclic stage is essential for the development of transmissible infections. We have been able to predict transmission success based on the efficiency of this differentiation process in the anterior midgut. Meta-analysis of multiple transmission experiments allowed us to establish a percent-metacyclic cutoff value that strongly predicted transmission competence. We have also identified the normal midgut microbiome as an essential factor controlling metacyclogenesis. L. major infected P. duboscqi sand flies treated with penicillin/streptomycin were completely inhibited in their ability to support the normal differentiation of promastigotes into the metacyclic stage. The bacterial diversity in normal and antibiotic treated flies was characterized by both culture dependent and independent methods by sequencing of the 16s rDNA. The possible roles of the normal bacterial communities in providing nutritional supplements essential to parasite differentiation, or displacement of the normal midgut microbiota by communities that are harmful to the parasite, will be investigated in future studies.
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