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. Our prior studies demonstrated mating both within and between different Leishmania species. In 2015, we generated hybrids between two genetically distant species: L. major from the Middle East and L. amazonensis from Brazil. Hybrid progeny were obtained at frequencies similar to those reported for L. major intra-species crosses, indicating that genetic distance is not a barrier to mating. Altogether, the outcrosses generated experimentally support the conclusion that the accumulating examples of hybrid genotypes observed in natural populations may have arisen by genetic exchange. In addition to outcrosses, we have studied the self-mating competency in Leishmania. Self-mating events were significantly less frequent than outcrosses and interestingly, increased in the presence of an outcross. This suggests that Leishmania parasites can distinguish between self and non-self cells for the purpose of mating. Preliminary results suggest that self-mating in Leishmania can produce alterations in DNA content and in phenotypic diversity. Since opportunities for outcrossing in the sand fly vector maybe extremely rare, self-mating may be the more important reproduction strategy contributing to the remarkable diversity in Leishmania. Finally, we assessed the fertility of F1 L. major hybrids by backcross and outcross mating attempts. Several hybrids were generated, though at significantly lower frequencies than those involving parental crosses, indicating that the F1 generation Leishmania hybrids have reduced mating competency, but they are not sterile. Whole genome sequencing of a backcross line provided the first direct evidence for recombination events and a Mendelian pattern of inheritance in Leishmania. A potential barrier to Leishmania development in the sand fly vector is the natural gut microbiota, which is acquired by adult sand flies during feeding on their respective animal and plant sources of blood and sugar, or from re-colonization of the gut by the microbes ingested by the terrestrial dwelling larval stages and that survive pupation. For many arthropod vectors, the diverse bacteria and fungi that inhabit the gut can negatively impact pathogen colonization, either by a direct microbial interaction, by activation of immune mediators, or by competing for essential nutrients. To the contrary, we have identified the normal midgut microbiota as essential to the developmentof transmissible infections in the fly. Specifically, the normal microbial flora was identified as an essential factor controlling metacyclogenesis - the development of Leishmania promastigotes into their infective stage. L. major infected P. duboscqi sand flies treated with penicillin/streptomycin were completely inhibited in their ability to support the normal growth and differentiation of promastigotes into the metacyclic stage. Metacyclogenesis in antibiotic treated flies could be rescued by reconstituting the sand fly midguts with defined bacterial species isolated from the midguts. The bacterial diversity in normal and antibiotic treated flies was characterized by both culture dependent and independent methods by sequencing of the 16s rDNA. We could find no role for the microbiota in providing nutritional supplements essential to parasite differentiation. Instead, the microbiota appears to compete for essential nutrients producing the stress conditions required to signal metacyclogenesis to proceed.
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