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 involved only mating between parental lines of the same species, L. major. In 2014, we investigated the possibility of genetic exchange between different Leishmania species. A cutaneous strain of L. major and a visceral strain of L. infantum, each bearing a different drug resistant marker, were used to co-infect Lutzomyia longipalpis sand flies. Eleven double drug resistant progeny clones, each the product of an independent mating event, were generated and submitted to genotype and phenotype analyses. The analysis of multiple allelic markers across the genome indicated that each progeny clone inherited at least one full set of chromosomes from each parent, with loss of heterozygosity at some loci, and uniparental inheritance of maxicircle kDNA. Hybrids with DNA contents of 2n, 3n, and 4n were observed. In vivo studies revealed clear differences in the ability of the hybrids to produce pathology in the skin or to disseminate to and grow in the viscera, suggesting polymorphisms and differential inheritance of the gene(s) controlling these traits. The studies represent the first experimental confirmation of cross-species mating in Leishmania, opening the way towards genetic linkage analysis of important traits, and providing strong evidence that genetic exchange is responsible for the generation of the mixed species genotypes observed in natural populations. We have identified the normal midgut microbiota 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. The possible roles of the normal bacterial communities in providing nutritional supplements essential to parasite differentiation, or to promoting nutrient deprivation or other stress conditions in the midgut required to trigger metacyclogenesis, will be investigated in future studies.
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