Vector Biological Studies in Leishmaniasis

我们之前已经提供了确凿的证据，证明利什曼原虫的无脊椎动物阶段完全有能力在沙蝇媒介的生长和发展过程中进行有性循环。2013年，我们研究了三个主要变量：来自其自然范围内的不同主要乳杆菌菌株与其他主要乳杆菌菌株进行交配的能力；主要乳杆菌与南方乳杆菌跨物种交配的能力；以及天然(Phlebotomus Dubosqi)和非自然(Lutzmyia Longipalpis)沙蝇媒介中杂交品种出现的时间及其发育阶段的相关性。我们已经能够证明起源于这种寄生虫物种的整个地理范围的主要乳杆菌菌株的性能力，以及在自然和非自然沙蝇媒介中的性能力。在涉及主要乳杆菌菌株之间交配的杂交中，在所有测试的配对杂交中，杂交种的恢复效率相似，这表明主要乳杆菌缺乏可检测到的交配类型，限制了自由遗传交换。值得注意的是，梅杰乳杆菌还能够跨越物种障碍与婴儿乳杆菌交配，为已在自然分离株中描述的物种间杂交基因提供了第一个实验支持。在自然载体中，杂交形成的时间与发育阶段的比较表明，蜜腺单胞体是最有可能的有性能力阶段，在感染阶段--超环前鞭毛体第一次出现之前，杂交细胞就出现在中肠的后部。对100多个双耐药杂交种进行了DNA含量分析，并在不同染色体上的多个标记基因座上对亲本等位基因进行了基因分型，以及上颌环DNA。在天然载体中产生的主要杂交种中，97%的核基因座显示两个亲本等位基因，表明它们都是全基因组杂交种；然而，3%的标记基因座只有一个亲本等位基因，这表明可能是由于某种未知的机制造成的杂合性丧失。仅观察到上颌骨kDNA的单亲遗传。这些研究提供了迄今为止最有力的证据，证明性行为是利什曼原虫生殖策略中保守和自然的方面。 我们已经确认正常的中肠微生物群是控制化生的一个重要因素。经青霉素/链霉素处理的大白沙蝇，其支持前鞭毛体正常分化进入后循环期的能力被完全抑制。通过16S rDNA测序，对正常果蝇和抗生素处理果蝇的细菌多样性进行了独立和依赖培养两种方法的研究。在未来的研究中，将研究正常细菌群落在提供寄生虫分化所必需的营养补充剂方面的可能作用，或者促进中肠的营养剥夺或触发化生所需的其他应激条件。

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. In 2013, we have studied three main variables: the ability of diverse L. major strains from across its natural range to undergo mating with other L. major strains; the ability of L. major to mate across species with L. infantum; 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. We have been able to demonstrate the sexual competency of L. major strains originating across the full geographic range of this parasite species, and in both natural and unnatural sand fly vectors. In crosses involving mating between L. major strains, hybrids were recovered at similar efficiencies in all pairwise crosses tested, suggesting that L. major lacks detectable mating types that limit free genetic exchange. Remarkably, L. major was also able to mate across the species barrier with L. infantum, providing the first experimental support for the inter-species hybrid genotypes that have been described amongst natural isolates. 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 in the posterior midgut well before the first appearance of infective stage, metacyclic promastigotes. Over 100 doubly-drug resistant hybrids were analyzed for DNA content and genotyped for parent alleles at a number of marker loci on different chromosomes, as well as the maxicircle DNA. For the L. major hybrids generated in the natural vector, 97% of the nuclear loci showed both parental alleles, suggesting that they are all full genomic hybrids; however, 3% of the marker loci showed only one parental allele, suggesting loss of heterozygosity by some unknown mechanism. Only uniparental inheritance of maxicircle kDNA was observed. These studies provide the strongest evidence to date that sex is a conserved and natural aspect of the reproductive strategy of Leishmania. We have identified the normal midgut microbiome as an essential factor controlling metacyclogenesis. L. major infected P. duboscqi sand flies treated with penicillin/streptomycin were completely inhibited in their ability to support the normal differentiation of promastigotes into the metacyclic stage. 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.