Visualisation of Leishmania donovani Fluorescent Hybrids during Early Stage Development in the Sand Fly Vector
Visualisation of Leishmania donovani Fluorescent Hybrids during Early Stage Development in the Sand Fly Vector
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DOI：
10.1371/journal.pone.0019851
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发表时间：
2011-05-27
期刊：
影响因子：
3.7
通讯作者：
Miles, Michael A.
中科院分区：
文献类型：
--
作者：
Sadlova, Jovana;Yeo, Matthew;Miles, Michael A.
背景：利什曼原虫寄生虫导致毁灭性的人类疾病。利什曼原虫被认为是克隆复制，没有遗传交换。然而，越来越多的证据表明，在自然种群中存在种间和种内杂种。第一个也是迄今为止唯一一个遗传交换的实验证据是在2009年获得的，当时通过用携带不同耐药标记的两种菌株共感染白蛉产生了双重耐药的大型利什曼原虫杂交种。然而，在沙蝇的杂交事件的位置和时间没有被描述。方法/主要调查结果：在这里，我们有共同感染Phlebotomus perniciosus和Lutzomyia longipalpis与杜氏利什曼原虫菌株携带潮霉素或新霉素抗性基因和红色或绿色荧光标记的转基因前鞭毛体。在血餐（PBM）后不同时间解剖进食雌性动物，并通过荧光显微镜或荧光激活细胞分选（FACS），然后通过共聚焦显微镜进行检查。在混合感染中，菌株LEM 3804和Gebre-1比菌株LEM 4265和LV 9更快地到达贲门和吻合口瓣膜。明确表达红色和绿色荧光的杂交体在用LEM 4265和Gebre-1共感染的两种测试载体的单蝇中观察到。在内围食间隙的半消化血液中，杂种在PBM 2天时作为短（前环）前鞭毛体存在。还通过FACS实现了明显共表达的杂交体的回收。然而，杂交体在体外无法维持生长。结论/意义：我们首次观察到L。donovani杂种在白蛉媒介中的PBM 2天，并描述了所涉及的形态学阶段。荧光显微镜与FACS的组合允许可视化和恢复的后代的实验杂交，但在这种情况下，杂交体在体外是不能生存的。然而，L. donovani具有深远的流行病学意义，因为它促进了新的表型特征的出现和传播。
Background: The Leishmania protozoan parasites cause devastating human diseases. Leishmania have been considered to replicate clonally, without genetic exchange. However, an accumulation of evidence indicates that there are inter-specific and intra-specific hybrids among natural populations. The first and so far only experimental proof of genetic exchange was obtained in 2009 when double drug resistant Leishmania major hybrids were produced by co-infecting sand flies with two strains carrying different drug resistance markers. However, the location and timing of hybridisation events in sand flies has not been described.Methodology/Principal Findings: Here we have co-infected Phlebotomus perniciosus and Lutzomyia longipalpis with transgenic promastigotes of Leishmania donovani strains carrying hygromycin or neomycin resistance genes and red or green fluorescent markers. Fed females were dissected at different times post bloodmeal (PBM) and examined by fluorescent microscopy or fluorescent activated cell sorting (FACS) followed by confocal microscopy. In mixed infections strains LEM3804 and Gebre-1 reached the cardia and stomodeal valves more rapidly than strains LEM4265 and LV9. Hybrids unequivocally expressing both red and green fluorescence were seen in single flies of both vectors tested, co-infected with LEM4265 and Gebre-1. The hybrids were present as short (procyclic) promastigotes 2 days PBM in the semi-digested blood in the endoperitrophic space. Recovery of a clearly co-expressing hybrid was also achieved by FACS. However, hybrids could not sustain growth in vitro.Conclusions/Significance: For the first time, we observed L. donovani hybrids in the sand fly vector, 2 days PBM and described the morphological stages involved. Fluorescence microscopy in combination with FACS allows visualisation and recovery of the progeny of experimental crosses but on this occasion the hybrids were not viable in vitro. Nevertheless, genetic exchange in L. donovani has profound epidemiological significance, because it facilitates the emergence and spread of new phenotypic traits.