海水酸化和缺氧及其昼夜节律变化对东海两种贻贝复合携带效应的比较研究

The significant positive correlation between subsurface water pH and oxygen concentration has been reported, and sometimes they represent a diel-cycling type, namely low in night and high in daytime. In recent years, the trends of ocean acidification and hypoxia of the Changjiang Estuary waters are obvious. Both of them occur frequently in the shellfish aquaculture waters around the Zhoushan Island. Most studies on ocean acidification and hypoxia focus on the biological effects of single factor, i.e., dissolved oxygen or acidification, and their diel-cycling types in the natural environment are less considered as well as the interspecific variation. Moreover, the combined latent (carry-over) effects of the seawater acidification and hypoxia have not been studied well. In this project, we will choose the typical economic bivalves, the thick shell mussel Mytilus coruscus and the blue mussel Mytilus edulis as target species, and use factorial design to study the carry-over effects of ocean acidification and hypoxia under both laboratory and field conditions on the two mussel species from molecular, cellular and individual levels, respectively. Also the response differences caused by interspecific variation will be compared and elucidated between the two species. We will study the early development, calcification and proteomic response of larvae hatched from the adults exposed to hypoxia and acidification; growth, metabolic physiology, immune responses, acid-base balance and proteomic response of haemocytes of juveniles developed from the individuals experienced hypoxia and acidification as well as their diel-cycling variation for the two mussel species from the East China Sea, and accordingly reveal the ecophysilogical carry-over effects of ocean acidification and hypoxia on two mussel species and their interspecific difference of adaptation. This study will illuminate the interactive effects of pH and DO (including diel-cycling type) on mussels, and provide theoretical basis for evaluating the ecophysiological effects of ocean acidification and hypoxia on mussels. Comparison of ecophysiology between two species will serve as a useful guideline for aquaculture in mussels.

研究证实水体pH和溶氧呈现显著正相关，且两者在天然环境中呈现一定的昼夜节律变化，即夜间低白天高的特点。长江口海域近年来海洋酸化和缺氧趋势明显，二者在舟山贝类养殖水域常同步发生。近年关于二者大多是研究酸化或溶氧的单因素生物学效应，且很少考虑实际环境中的昼夜节律性，以及同属物种间的差异性，而关于酸化和缺氧对海洋生物后代及发育后期的携带效应更鲜有报道。本研究以东海厚壳贻贝和紫贻贝为研究对象，基于实验室和野外试验研究海水酸化和缺氧及昼夜变化模式的复合胁迫对两种贻贝的携带效应，并比较两种贻贝的适应性差异。项目研究两种贻贝亲本暴露于酸化和缺氧条件下产生的后代早期发育、钙化及蛋白质组变化；浮游期暴露于复合胁迫下成功附着后稚贝的生长、代谢生理、血淋巴酸碱平衡、免疫功能及蛋白质表达差异，以揭示酸化和缺氧(含昼夜循环性)对两种贻贝的生态生理携带效应及种间适应性差异，为评估二者对贝类生态生理影响提供理论支持。

在过去的几十年中，由于人为活动，一些沿海水域同时发生酸化和低氧。在自然环境中，pH和溶解氧可能会波动并表现出昼夜循环模式。因此阐明典型海洋贝类在全球气候变化下的生态生理适应机制对预测物种的种群变化及贝类养殖具有重要意义，本项目按照既定计划超额完成了全部任务。第一，短期暴露于酸化和缺氧对紫贻贝和厚壳贻贝性腺发育及早期发育性状的跨代影响。将两种贻贝放到两种恒定水平的溶解氧DO（2，8mg/L）和pH（7.3，8.1），酸化对两种贻贝的受精率，胚胎畸形率和D型幼体壳长有负面影响，但是，低氧对受精率没有影响。进一步研究发现酸化可以导致厚壳贻贝的性腺指数的降低，与生殖相关基因的表达也受到低 pH的影响，而缺氧没有显著影响。第二，基于整合生理学方法研究昼夜节律的缺氧和酸化对贝类适应性的影响。厚壳贻贝暴露于上述实验条件中，结果表明，酸化和低氧显着降低了贻贝的细胞外pH（pHe）和状态指数（CI），并显着提高了血淋巴HCO3-，pCO2和贻贝的标准代谢率（SMR）。昼夜波动的缺氧和酸化作用还显著降低了pHe和CI，并显着增加了pCO2和SMR，但对HCO3-没有显著影响。第三，运用行为生理学方法调查在贻贝捕食者—蟹存在的条件下，酸化和温度对紫贻贝和厚壳贻贝的反捕食行为响应。两种贻贝（厚壳贻贝和紫贻贝）暴露于酸化（-0.4 pH单位），变暖（+5°C）及捕食者存在的条件下。结果表明酸化和变暖对足丝的数量和重量，足丝附着力和足丝盘面积具有特异的独立负面影响。对于贻贝聚群行为，观察到了类似的负面影响。第四，运用流式细胞术，原子吸收光谱法及分光光度法等研究两种贻贝暴露于不同pH和溶氧条件下的血淋巴的免疫功能、酸碱平衡和抗氧化功能。以厚壳贻贝为研究对象，于两个pH，溶氧水平和两个温度水平下暴露30 天，研究发现处于应激条件下的贻贝可能会诱导或抑制抗氧化酶的活性，抗氧化酶活性的降低可能导致脂质，蛋白质和细胞膜的损伤，低pH，低DO和高温会对厚壳贻贝抗氧化参数产生不利影响。第五，采用高覆盖定量代谢组学技术研究海洋酸化下贻贝生物能量的变化。综合生化和代谢组学分析在细胞水平上证明了酸化对能量代谢的负面影响，并揭示了氨基酸生物合成和代谢的改变是酸化引起贻贝代谢紊乱的一种机制。