The existence of hybrid genotypes among natural isolates of Leishmania has been known for years, and hybrids recovered from sand flies experimentally co-infected with different strains or species of Leishmania formally demonstrated that members of the genus possess the machinery for genetic exchange. As neither chromosome condensation, gamete stages, nor cell fusion events have been directly observed in the vector, the main approach to determine if Leishmania has a true sexual cycle is a classical genetic analysis. Here, we used whole genome sequencing to follow the chromosomal inheritance patterns of 44 experimental hybrids generated between and within Leishmania species, including L. major, L. infantum, and L. tropica. We found that the parental somy and allele contributions matched the inheritance patterns expected under meiosis 97-99% of the time, depending on the experiment. The hybrids generated between largely homozygous parents were heterozygous throughout most of the genome for the markers that were homozygous and different between the parents. Analysis of 18 hybrids generated from crosses involving a largely heterozygous strain of L. tropica revealed frequent and genome wide patterns of recombinations, demonstrating that classical crossing over occurs at meiosis, and allowed us to construct the first genetic map in Leishmania. The present studies are also the first to directly explore hybrid fertility. In comparison to the parents used for their generation, the intraspecies F1 hybrids in L. major and L. tropica had substantially reduced fertility, while the L. major x L. infantum interspecies hybrids were sterile. Decreasing fertility correlated with the degree of sequence divergence between the hybridizing genomes. Our findings provide unequivocal evidence that meiotic sex is an inherent source of genetic variation in Leishmania, with hybrids likely experiencing a period of post-zygotic isolation permitting adaptive genotypes to clonally thrive, while aneuploid mechanisms and inbreeding gradually restore homozygosity, fertility, and new opportunities for genetic exchange. Visceral leishmaniasis (VL), which is endemic in the northeast Indian state of Bihar, is thought to have an anthroponotic transmission cycle as no mammalian host other than humans has ever been shown to harbor the etiologic agent, L. donovani. However, which infected humans can act as important reservoirs for transmission to the vector, Phlebotomus argentipes, remains poorly studied. The possibilities include active VL cases, clinically cured cases, patients with post-kala-azar dermal leishmaniasis (PKDL), and infected but asymptomatic individuals. Understanding the dynamics and epidemiology of anthroponotic transmission holds clear importance for the development of control strategies. Direct xenodiagnosis, the use of live, uninfected insects to detect viable disease organisms in individuals with presumptive infections, has been historically employed for the diagnosis of American trypanosomiasis caused by Trypanosoma cruzi. For xenodiagnostic studies aimed at defining the ability of specific human-subject groups across the infection spectrum to transmit viable L. donovani organisms to sand flies, an on-site, self-sustaining sand fly colony, has been established in Bihar, India, and is the first and only self-sustaining laboratory-based colony of sand flies in the Indian sub-continent. Once self-sustaining, the colony was closed to infusion with wild-caught material and certified free of specific human pathogens and as safe to use in human xenodiagnostic studies. In this endemic region, patients with active disease are considered to be important reservoirs based on the clustering of cases around households with a history of VL. Completion of the xenodiagnostic studies on a cohort of 76 active cases of VL has revealed that patients in the early stage of disease transmit poorly, or not at all, to the vector, and none of the VL patients transmitted following treatment. This is crucial data to indicate that early diagnosis and treatment will prevent human VL cases from contributing to the transmission cycle of the disease.
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