Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic.
Cytometry, Faculty of Science, Charles University, Prague, Czech Republic.
Parasit Vectors. 2018 Jan 15;11(1):37. doi: 10.1186/s13071-018-2613-2.
Leishmania development in sand flies is confined to the alimentary tract and is closely connected with blood meal digestion. Previously, it has been published that activities of sand fly midgut proteases are harmful to Leishmania, especially to amastigote-promastigote transition forms. However, our experiments with various Leishmania-sand fly pairs gave quite opposite results.
We evaluated the effect of semi-digested midgut content on different life stages of Leishmania donovani and Leishmania major in vitro. Various morphological forms of parasites, including macrophage-derived amastigotes and transition forms, were incubated 2 h with midguts dissected at various intervals (6-72 h) post-blood meal or with commercially available proteinase, and their viability was determined using flow cytometry. In parallel, using amastigote-initiated experimental infections, we compared development of L. donovani in sand flies that are either susceptible (Phlebotomus argentipes and P. orientalis) or refractory (P. papatasi and Sergentomyia schwetzi) to this parasite.
In vitro, sand fly midgut homogenates affected L. major and L. donovani in a similar way; in all sand fly species, the most significant mortality effect was observed by the end of the blood meal digestion process. Surprisingly, the most susceptible Leishmania stages were promastigotes, while mortality of transforming parasites and amastigotes was significantly lower. Parasites were also susceptible to killing by rabbit blood in combination with proteinase, but resistant to proteinase itself. In vivo, L. donovani developed late-stage infections in both natural vectors; in P. argentipes the development was much faster than in P. orientalis. On the other hand, in refractory species P. papatasi and S. schwetzi, promastigotes survived activity of digestive enzymes but were lost during defecation.
We demonstrated that Leishmania transition forms are more resistant to the killing effect of semi-digested blood meal than 24 h-old promastigotes. Data suggest that Leishmania mortality is not caused directly by sand fly proteases, we assume that this mortality results from toxic products of blood meal digestion. Survival of L. donovani promastigotes in refractory sand flies until blood meal defecation, together with similar mortality of Leishmania parasites incubated in vitro with midgut homogenates of susceptible as well as refractory species, contradict the previously raised hypotheses about the role of midgut proteases in sand fly vector competence to Leishmania.
在沙蝇中，利什曼原虫的发育仅限于消化道，并与血餐消化密切相关。此前，已有研究表明沙蝇中肠蛋白酶的活性对利什曼原虫有害，尤其是对前鞭毛体-无鞭毛体转化形式。然而，我们用各种利什曼原虫-沙蝇对进行实验的结果却恰恰相反。
我们评估了半消化中肠内容物对不同生活阶段的利什曼原虫 Donovan 和利什曼原虫 major 在体外的影响。用商业蛋白酶或不同间隔（6-72 小时）剖取的消化道处理后 2 小时，孵育各种寄生虫形态，包括巨噬细胞衍生的无鞭毛体和转化形式，并使用流式细胞术确定其存活率。同时，利用无鞭毛体引发的实验感染，我们比较了在易感染（银足血蝇和东方血蝇）和不易感染（白蛉扇头蝇和斯氏按蚊）该寄生虫的沙蝇中，利什曼原虫 Donovan 的发育情况。
在体外，沙蝇中肠匀浆对利什曼原虫 major 和利什曼原虫 Donovan 的影响相似；在所有沙蝇物种中，在血餐消化过程结束时观察到最显著的死亡率效应。令人惊讶的是，最易受影响的利什曼原虫阶段是前鞭毛体，而转化寄生虫和无鞭毛体的死亡率明显较低。寄生虫也容易被兔血和蛋白酶联合杀死，但对蛋白酶本身有抵抗力。在体内，利什曼原虫 Donovan 在两种自然载体中均发展为晚期感染；在银足血蝇中，其发育速度明显快于东方血蝇。另一方面，在不易感染的物种白蛉扇头蝇和斯氏按蚊中，前鞭毛体在消化酶的活性下存活，但在排粪时丢失。
我们证明了利什曼原虫转化形式比 24 小时龄的前鞭毛体更能抵抗半消化血餐的杀伤作用。数据表明，利什曼原虫的死亡率不是由沙蝇蛋白酶直接引起的，我们假设这种死亡率是由血餐消化的有毒产物引起的。在不易感染的沙蝇中，利什曼原虫 Donovan 前鞭毛体一直存活到血餐排粪，同时在体外与易感染和不易感染的物种的中肠匀浆孵育的利什曼原虫寄生虫也有类似的死亡率，这与先前提出的关于中肠蛋白酶在沙蝇媒介对利什曼原虫的易感性中的作用的假说相矛盾。
