日本血吸虫雌虫在与雄虫合抱后至性成熟过程的关键分子事件研究

Schistosomiasis ranks as the second serious parasitic disease of global priority for humans and animals. In China, it is caused by Schistosoma japonicum and remains a major public health problem. Maturation of female worm is the primary base for the successful establishment of infection. Therefore, to understand the molecular mechanism of male-induced female sexual maturation may help to find ways to interrupt egg laying and thus, the disease transmission. It has been demonstrated that continuous pairing with male is prerequisite for the development of female reproductive organs and egg production. Our recent experiments showed that feeding erythrocytes should be obligatory for the development of female reproductive system. However, the molecular events during the process of female sexual maturation are still unclear. Here we propose a hypothesis for this process: the receptor in the immature female receives the signal released from the male and then activates the downstream signal transduction pathways, which induces the mitoses and cell differentiation in the female leading to the full development of ovary and vitellarium. Meanwhile, the paired female may improve its ability to ingest and digest the red blood cells to store energy and nutrition for the development of gonad and egg production. In this proposal, we will focus on the Schistosoma japonicum female worms at the days from 14dpi to 28dpi (days past infection), which is the key period of female sexual maturation. RNA-seq and MicroRNA-seq techniques will be used to investigate the dynamic developmental mRNA and the mircroRNA expression profiles, and functional network analysis will be applied to identify the critical molecules and pathways involving in this process. The result of this research will be helpful to understand the molecular mechanism of the female sexual maturation, which may provide the target molecules for the development of vaccine and drug through blocking the female maturation and egg laying.

血吸虫病是世界第二大寄生虫病，也是我国重要的公共卫生问题之一。目前认为，雌虫性成熟是血吸虫感染建立的基础，明确雌虫性成熟机制对阻断血吸虫感染建立和血吸虫病传播具有重要意义。已有研究及本课题组前期研究显示，与雄虫合抱和吞食红细胞是雌虫性发育的必要因素，但该过程中的关键分子和机制仍然模糊。我们推测：雌虫接受与之合抱的雄虫传递的某种信号后，激活已知和/或未知信号通路，启动或促进与雌虫性别发育相关的分子事件；同时，雌虫的红细胞摄入和消化能力被提升，为正常发育提供物质和能量保障。本课题拟采集尾蚴感染小鼠后第14-28天（即合抱至发育成熟阶段）的虫体样本，利用RNA-seq、microRNA-seq和生物信息学功能网络分析，描述该过程中基因和microRNA的程序性表达特征，分析期间的关键分子和分子事件。研究成果有助于理解和诠释雌虫性成熟机制，并为开发阻断雌虫性成熟和产卵的疫苗或药物提供靶标。

虫卵是血吸虫致病及传播的主要原因，控制雌虫性成熟和产卵有助于控制血吸虫病的流行和危害。血吸虫雌虫生殖系统的发育及产卵的分子机制仍不清楚。本研究通过二代测序技术完成了日本血吸虫雌虫性成熟和产卵过程中基因转录的动态表达谱（mRNA和miRNA），获得了不同发育阶段和不同性别虫体在合抱至性成熟过程中差异表达基因，分析了该过程中的关键分子和分子事件；初步构建了血吸虫雌虫生殖系统发育调控的功能网络；发掘了一批与日本血吸虫合抱和产卵相关的候选基因，并对其中的关键分子的功能进行了验证。主要研究结果包括：1、构建了日本血吸虫的生殖发育动态mRNA转录组，包含23,099个转录本，其中有5,205条新序列；雌虫和雄虫早期（14-18 d）转录组的差异较小，随着性成熟差异变大，20d是雌虫基因表达巨变的关键时间点；成熟后期雌雄虫上、下调基因互补，显示明显的功能分化；2、发掘了一系列与合抱和生殖发育相关的基因，并通过原位杂交、RNAi以及动物保护性实验初步验证了与血吸虫性成熟相关的重要基因RL-GPCR、与雄虫合抱相关的基因芳香族氨基酸脱羧酶以及性成熟相关转录因子，提出日本血吸虫的雄虫主要通过神经系统及神经递质的兴奋作用来维持合抱；而雌虫在接受雄虫的合抱刺激后，经神经系统传递咽侧体抑制素，进而通过与昆虫类似的激素调控模式来调节生殖系统的发育的理论。3. 完成了血吸虫性成熟过程的miRNA动态表达谱，共获得了79种成熟miRNA和56种miRNA前体，并预测得到127种新的miRNA；虫期和性别是导致测序结果差异的最主要因素；发育阶段对miRNA差异表达的影响大于性别因素对其的影响；不同时期的雌雄虫出现了明显的聚类；早期雄虫（14-20 d）与晚期雄虫(22-24 d)具有明显的基因表达差异，而不同发育期的雌虫miRNA差异表达较小，与mRNA的表达谱正好相反，显示miRNA与mRNA存在广泛的调控关系，提示miRNA（特别是雄虫的miRNA）在血吸虫生殖发育中的重要作用。本研究为深入研究血吸虫发育的分子机理、解析其维持感染的机制提供了重要基础，也为发展新的潜在疫苗和药物靶点提供了重要依据。结果撰写研究论文10篇，其中SCI论文4篇，两篇正式发表，一篇待发表，一篇修回；核心期刊5篇，4篇正式发表，1篇投稿；培养博士研究生3名，硕士研究生3名。