Genetic analysis of cercarial release in schistosomes

总结 鉴定影响寄生虫传播、毒力和宿主特异性的基因是人们感兴趣的中心 来理解宿主和寄生虫之间的相互作用这项建议的主要目的是确定两种遗传基础， 曼氏血吸虫的这种特征，这种寄生吸虫感染了南美、中东和非洲的6700万人， 和非洲我们将集中在水生蜗牛寄生虫生命周期的幼虫阶段。在渗透了 蜗牛宿主，寄生虫幼虫克隆繁殖，阉割他们的蜗牛宿主，和数百至数万 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。两个关键的传输- 相关性状显示寄生虫之间存在高度可遗传的遗传变异。首先，尾蚴释放的时间 不同种群之间的蜗牛，并与脊椎动物宿主的水接触模式重叠。最 S.主要感染人类的曼氏线虫种群在上午晚些时候释放尾蚴幼虫， 主要感染啮齿动物的种群在下午晚些时候或晚上排出尾蚴。晚期脱落至少进化出了 在S. mansoni人口。实验室杂交证明了这种性状的简单遗传基础，但 晚脱落的遗传结构在已记录的三个种群中是不同的。二是 从中间水生螺宿主上脱落的尾蚴幼虫数个体间差异≥7倍 多基因型。高脱落寄生虫具有更大的传播潜力，但也会阉割和杀死 它们的中间蜗牛比低脱落寄生虫更快。实验室实验表明， 性状对选择反应迅速，具有高度遗传性。 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可以很容易地 保持在实验室中，因此可以进行遗传杂交，并分离出数千个后代，而一个完整的 基因组序列和不断增长的分子工具包现在允许基因组和功能表征。在 初步工作，我们进行了遗传杂交昼夜脱落寄生虫从阿曼和 确定了一个决定尾蚴脱落时间的区域（LOD=6.1）。在目标1中，我们将利用 不断发展的复杂分子工具包，用于精细绘制和功能分析决定脱落的基因 时间在阿曼交叉，我们将进行额外的杂交，以确定这种性状的遗传基础，在其他 观察到晚期脱落的寄生虫种群。在目标2中，我们将使用RNAseq检查 阿曼夜间和昼夜脱落寄生虫在24小时光/暗周期中的表达，以研究 控制尾蚴释放的代谢途径。最后，在目标3中，我们将分析遗传杂交 寄生虫之间显示7倍的差异，从受感染的蜗牛脱落的尾蚴数量，然后精细地图 并对相关基因进行功能分析。 我们以前使用遗传连锁图谱和功能分析来确定潜在的突变， S. mansoni我们预计，这种方法将同样强大的识别 这些基因是可遗传的寄生虫特征的基础，这些特征对传播、宿主特异性和毒力至关重要， 重要人体蠕虫寄生虫。我们的遗传学方法也提供了一种新的方法来探索生物学， 寄生虫孢子囊，寄生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.