Genetic analysis of cercarial release in schistosomes

摘要 鉴定影响传播、毒力和宿主特异性的寄生虫基因是人们最感兴趣的。 以了解宿主/寄生虫的相互作用。这项建议的中心目标是确定两个基因的基础 曼氏血吸虫的这些特征，是一种寄生性吸虫，感染了南美和中东的6700万人 还有非洲。我们将重点关注水生蜗牛寄生虫生命周期的幼虫阶段。在渗透到 蜗牛宿主，血吸虫幼虫克隆繁殖，阉割它们的蜗牛宿主，并成百上千到数万 活动的尾蚴幼虫被释放到水中，在那里它们感染人类或啮齿动物。两个密钥传输- 相关性状表明寄生虫之间存在高度的可遗传遗传变异。第一，释放尾丝虫的时间 蜗牛的接触方式因种群而异，并与其脊椎动物宿主的接触水模式重叠。多数 主要感染人类的曼氏血吸虫种群在上午晚些时候排出尾蚴幼虫，而寄生虫 主要感染啮齿动物的种群在下午晚些时候或夜间排出尾蚴。延迟脱落至少已经演变成 在曼氏血吸虫种群中发生了三次。实验室杂交证明了这一特征的简单遗传基础，但 晚期脱落的遗传结构在已有文献记载的三个种群中是不同的。第二， 从中间水生蜗牛宿主中排出的尾蚴幼虫数量在个体之间差异≥为7倍 血吸虫基因分型。高脱落的寄生虫有更大的传播潜力，但也会阉割和杀死 它们中间的蜗牛比低落的寄生虫更快。实验室实验证明，这一点 性状对选择反应迅速，具有高度的遗传性。 血吸虫寄生虫在人类寄生虫中是不寻常的，因为完整的生命周期很容易 在实验室里维护，这样就可以进行遗传杂交，并分离数以千计的后代，同时完成 基因组序列和不断增长的分子工具包现在可以进行基因组和功能表征。在……里面 初步工作，我们进行了阿曼和阿曼昼夜脱皮寄生虫的遗传杂交。 确定了chr.1上决定尾蚴脱落时间的区域(LOD=6.1)。在目标1中，我们将利用 发展血吸虫分子工具包精细定位和功能分析决定脱落的基因(S) 时间，我们将进行更多的杂交，以确定这一性状在其他 观察到脱落较晚的寄生虫种群。在目标2中，我们将使用RNAseq来检查 阿曼夜间和日间脱落寄生虫在24小时光/暗周期中的表达，以调查 控制尾虫释放的代谢途径。最后，在目标3中，我们将分析遗传杂交 寄生虫之间显示出7倍差异的尾蚴从受感染的钉螺中排出，然后精细作图 并对相关基因进行功能分析。 我们之前已经使用遗传连锁图谱和功能分析来确定潜在的突变 曼氏葡萄球菌对奥沙米昆的耐药性。我们预计这种方法将同样有效地识别 构成可遗传寄生虫特征的基因，这些特征对传播、宿主特异性和致病力至关重要 重要的人类寄生虫。我们的遗传学方法也提供了一种新的方法来探索 血吸虫孢子囊，血吸虫生命周期中被忽视的阶段，是理解传播的关键 从蜗牛到脊椎动物宿主。

SUMMARY Identification of the parasite genes that influence transmission, virulence and host specificity is of central interest for understanding host/parasite interactions. The central aim of this proposal is to identify the genetic basis of two such traits in Schistosoma mansoni, a parasitic fluke that infects 67 million people in South America, Middle-East and Africa. We will focus on the larval stage of the parasite lifecycle in aquatic snails. Following penetration of the snail host, schistosome larvae reproduce clonally, castrating their snail host, and hundreds to tens of thousands of motile cercariae larvae are released into the water where they infect humans or rodents. Two key transmission- related traits show high levels of heritable genetic variation among parasites. First, the timing of cercarial release from the snail varies among populations and overlaps with the water contact patterns of their vertebrate hosts. Most S. mansoni populations that primarily infect humans shed cercariae larvae in late morning, while parasite populations that primarily infect rodents shed cercariae in late afternoon or night. Late shedding has evolved at least three times in S. mansoni populations. Laboratory crosses demonstrate a simple genetic basis for this trait, but the genetic architecture of late shedding is different in the three populations where it has been documented. Second, the number of cercariae larvae shed from the intermediate aquatic snail host varies ≥7-fold among individual schistosome genotypes. High shedding parasites have greater potential for transmission but also castrate and kill their intermediate snails more rapidly than low shedding parasites. Laboratory experiments demonstrate that this trait responds rapidly to selection and is highly heritable. Schistosome parasites are unusual among parasites of humans because the complete lifecycle can be easily maintained in the laboratory, so genetic crosses can be staged and thousands of progeny isolated, while a complete genome sequence and a growing molecular toolkit now allows genomic and functional characterization. In preliminary work, we conducted genetic crosses between diurnal and nocturnal shedding parasites from Oman and identified a region on chr.1 that determines cercarial shedding time (LOD=6.1). In Aim 1, we will exploit the growing schistosome molecular toolkit to fine map and functionally analyze the gene(s) that determine shedding time in the Omani cross, and we will conduct additional crosses to determine the genetic basis of this trait in other parasite populations where late shedding is observed. In Aim 2, we will use RNAseq to examine rhythms in expression of nocturnal and diurnal shedding parasites from Oman across the 24 hr light/dark cycle, to investigate the metabolic pathways underlying control of cercarial release. Finally, in Aim 3, we will analyze genetic crosses between parasites showing 7-fold differences in numbers of cercariae shed from infected snails, and then fine map and functionally analyze the genes involved. We have previously used genetic linkage mapping and functional analysis to identify the mutations underlying oxamniquine resistance in S. mansoni. We anticipate that this approach will be equally powerful for identifying the genes that underlie heritable parasite traits that are critical for transmission, host specificity and virulence in this important human helminth parasite. Our genetic approach also provides a novel way to probe the biology of schistosome sporocysts, a neglected stage in the schistosome lifecycle that is key to understanding transmission from the snail to the vertebrate host.