The life cycle of the Leishmania parasite in the sand fly vector involves differentiation into several distinctive forms, each thought to represent an adaptation to specific microenvironments in the midgut of the fly. Based on transcriptome sequencing (RNA-Seq) results, we describe the first high-resolution analysis of the transcriptome dynamics of four distinct stages of Leishmania major as they develop in a natural vector, Phlebotomus duboscqi. The early transformation from tissue amastigotes to procyclic promastigotes in the blood-fed midgut was accompanied by the greatest number of differentially expressed genes, including the downregulation of amastins, and upregulation of multiple cell surface proteins, sugar and amino acid transporters, and genes related to glucose metabolism and cell cycle progression. The global changes accompanying post-blood meal differentiation of procyclic promastigotes to the nectomonad and metacyclic stages were less extensive, though each displayed a unique signature. The transcriptome of nectomonads, which has not been studied previously, revealed changes consistent with cell cycle arrest and the upregulation of genes associated with starvation and stress, including autophagic pathways of protein recycling. Maturation to the infective, metacyclic stage was accompanied by changes suggesting preadaptation to the intracellular environment of the mammalian host, demonstrated by the amastigote-like profiles of surface proteins and metabolism-related genes. Finally, a direct comparison between sand fly-derived and culture-derived metacyclics revealed a reassuring similarity between the two forms, with the in vivo forms distinguished mainly by a stronger upregulation of transcripts associated with nutrient stress.

Invertebrate stages of Leishmania are capable of genetic exchange during their extracellular growth and development in the sand fly vector. Here we explore two variables: the ability of diverse L. major strains from across its natural range to undergo mating in pairwise tests; and the timing of the appearance of hybrids and their developmental stage associations within both natural (Phlebotomus duboscqi) and unnatural (Lutzomyia longipalpis) sand fly vectors. Following co-infection of flies with parental lines bearing independent drug markers, doubly-drug resistant hybrid progeny were selected, from which 96 clonal lines were analyzed for DNA content and genotyped for parent alleles at 4–6 unlinked nuclear loci as well as the maxicircle DNA. As seen previously, the majority of hybrids showed ‘2n’ DNA contents, but with a significant number of ‘3n’ and one ‘4n’ offspring. In the natural vector, 97% of the nuclear loci showed both parental alleles; however, 3% (4/150) showed only one parental allele. In the unnatural vector, the frequency of uniparental inheritance rose to 10% (27/275). We attribute this to loss of heterozygosity after mating, most likely arising from aneuploidy which is both common and temporally variable in Leishmania. As seen previously, only uniparental inheritance of maxicircle kDNA was observed. Hybrids were recovered at similar efficiencies in all pairwise crosses tested, suggesting that L. major lacks detectable ‘mating types’ that limit free genetic exchange. In the natural vector, comparisons of the timing of hybrid formation with the presence of developmental stages suggest nectomonads as the most likely sexually competent stage, with hybrids emerging well before the first appearance of metacyclic promastigotes. These studies provide an important perspective on the prevalence of genetic exchange in natural populations of L. major and a guide for experimental studies to understand the biology of mating.

Hybrid genotypes have been repeatedly described among natural isolates of Leishmania , and the recovery of experimental hybrids from sand flies co-infected with different strains or species of Leishmania has formally demonstrated that members of the genus possess the machinery for genetic exchange. As neither gamete stages nor cell fusion events have been directly observed during parasite development in the vector, we have relied on a classical genetic analysis to determine if Leishmania has a true sexual cycle. Here, we used whole genome sequencing to follow the chromosomal inheritance patterns of experimental hybrids generated within and between different strains of L . major and L . infantum . We also generated and sequenced the first experimental hybrids in L . tropica . We found that in each case the parental somy and allele contributions matched the inheritance patterns expected under meiosis 97–99% of the time. The hybrids were equivalent to F1 progeny, heterozygous throughout most of the genome for the markers that were homozygous and different between the parents. Rare, non-Mendelian patterns of chromosomal inheritance were observed, including a gain or loss of somy, and loss of heterozygosity, that likely arose during meiosis or during mitotic divisions of the progeny clones in the fly or culture. While the interspecies hybrids appeared to be sterile, the intraspecies hybrids were able to produce backcross and outcross progeny. Analysis of 5 backcross and outcross progeny clones generated from an L . major F1 hybrid, as well as 17 progeny clones generated from backcrosses involving a natural hybrid of L . tropica , revealed genome wide patterns of recombination, demonstrating that classical crossing over occurs at meiosis, and allowed us to construct the first physical and genetic maps in Leishmania . Altogether, the findings provide strong evidence for meiosis-like sexual recombination in Leishmania , presenting clear opportunities for forward genetic analysis and positional cloning of important genes.