Vector Biological Studies in Leishmaniasis

我们以前提供了确凿的证据，证明利什曼原虫的无脊椎阶段在它们在沙蝇媒介的生长和发育过程中完全能够有性循环。我们之前的研究只涉及同一物种的亲本系之间的交配，L. major。2014年，我们调查了不同利什曼原虫种间基因交换的可能性。采用大乳杆菌皮肤菌株和婴儿乳杆菌内脏菌株，分别携带不同的耐药标记物，共同感染长掌Lutzomyia沙蝇。产生11个双耐药后代克隆，每个克隆都是一个独立交配事件的产物，并提交基因型和表型分析。基因组中多个等位基因标记的分析表明，每个子代克隆至少继承了亲本的全套染色体，但在某些位点存在杂合性缺失，且存在最大环kDNA的单代遗传。观察DNA含量分别为2n、3n和4n的杂种。体内研究显示，杂种在皮肤病理或向内脏扩散和生长的能力上存在明显差异，表明控制这些性状的基因存在多态性和差异遗传。这些研究首次通过实验证实了利什曼原虫的跨物种交配，为重要性状的遗传连锁分析开辟了道路，并提供了强有力的证据，证明遗传交换是在自然种群中观察到的混合物种基因型产生的原因。

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.