海洋酸化对泥蚶多精受精影响的机理研究

Huge amount of CO2 emissions are absorbed by ocean, which leads to ocean acidification and poses great threat to aquaculture industry of marine bivalves. Most marine bivalves release their gametes into the water column, where the fertilization occurs. Therefore, ocean acidification, which changes the physical and chemical characteristics of seawater, has significant impact on the fertilization of marine bivalves. Due to the recent discovery of ocean acidification and the knowledge paucity in polyspermy blocking mechanism of marine organisms, the impact and the underlying affecting mechanism of ocean acidification on polyspermy of marine invertebrates still remain poorly understood. To date, little is known about the triggering mechanism, genes and pathways involved in polyspermy blocking of marine bivalves, which significantly constrain the solving of polyspermy problem in artificial breeding of commercial bivalves. Previous study showed that ocean acidification significantly increased polyspermy and slowed down the process of superfluous detachment in blood clam (Tegillarca granosa), suggesting that ocean acidification may hamper oocytes membrane depolarization and fertilization membrane formation result from cortical granule exocytosis. Several genes may related to polyspermy blocking in blood clam has been cloned by applicants through RNA-seq and RACE. However, a further verification of their function in polyspermy blocking is needed. Therefore, in the present study, the cationic demands and the timeliness of oocytes membrane depolarization, the function of chs-1, EGG-4/5, GLD, etc. in cortical granule exocytosis, and the impacts of ocean acidification on oocytes membrane depolarization and cortical granule exocytosis will be investigated in a typical marine mudflat bivalve species, the blood clam, T. granosa. The results of this work will help to expend the research scope of both ocean acidification and polyspermy blocking mechanism, and subsequently provide experimental data for aquaculture industry to cope with ocean acidification and solve polyspermy problem in artificial breeding of commercial marine bivalves.

随着人源CO2排放的持续增加，由CO2驱动的海洋酸化对海洋贝类的多个生活史阶段构成了威胁。目前其对贝类多精受精影响的研究刚刚起步，且由于海洋贝类多精受精阻止机制的触发机理、参与的基因与通路仍不明晰，尚未见海洋酸化影响贝类多精受精的作用机理报道。前期研究表明，海洋酸化使泥蚶的多精受精概率显著增加，说明海洋酸化可能影响了泥蚶的多精受精阻止机制（卵膜去极化、皮层反应）。申请人团队已筛选得到了可能参与泥蚶多精受精阻止机制的多个基因，但其具体功能及海洋酸化如何对其产生影响，仍有待进一步深入研究。因此，本研究以泥蚶为研究对象，分析其卵膜去极化的离子需求与时效性，探讨chs-1、EGG-4/5、GLD等基因在多精受精阻止机制中的作用，并研究海洋酸化影响泥蚶卵膜去极化与皮层反应的作用机理。本研究的开展有助于深化对海洋酸化生态效应的理解，并对海洋贝类养殖产业解决多精受精问题和应对海洋酸化影响具有指导意义。

化石燃料的大量使用，造成CO2排放的持续增加。排放到大气中的CO2有约1/3被海洋吸收，导致海洋酸化（Ocean Acidification）问题，进而对包括贝类在内的多种海洋生物的多个生命过程构成了潜在威胁。近期的研究表明，海洋酸化可能导致体外受精的海洋无脊椎动物多精受精率显著升高，但其作用机理仍不明晰。针对此问题，本研究以典型经济贝类泥蚶为研究对象，在探明泥蚶受精卵卵膜去极化离子需求、关键离子通道以及Chs-1、CaM等基因在阻止多精受精中的生物功能的基础上，分析了海洋酸化对泥蚶受精卵卵膜去极化和皮层反应的影响。研究结果表明，钙离子、钙离子通道、钙调蛋白在泥蚶受精卵的卵膜去极化中具有重要的生物功能。此外钙调蛋白和细胞骨架直接调控或参与了受精卵的皮层颗粒外排过程。研究发现海洋酸化不仅显著干扰了泥蚶受精卵的卵膜去极化过程，导致卵膜电位变化强度显著降低、卵膜去极化持续时间变短，而且显著阻碍了皮层反应进程，并可能在快反应（卵膜去极化）与慢反应（皮层反应）之间造成无保护的空窗期，进而导致泥蚶多精受精现象显著升高。上述研究结果不仅阐明了海洋酸化导致多精受精的作用机理，也有助于丰富目前海产双壳贝类多精受精阻止机制知识体系，对海洋贝类育苗产业解决多精受精问题和科学应对海洋酸化影响具有重要的指导意义。