海洋生态与环境科学重点实验室知识库

硬壳蛤（Mercenaria mercenaria），自然栖息于北美大西洋沿岸，从潮间带至潮下带均有分布。1997年，张福绥院士等人首次从美国引种硬壳蛤至我国，目前已成为我国重要的池塘养殖贝类，养殖面积达数十万亩。受自然潮汐影响，以及在长途运输保存过程中，硬壳蛤经常会遭受干露胁迫。干露会对贝类造成包括缺水、缺氧和饥饿等生理压力，是影响贝类生存的重要胁迫因子。硬壳蛤的干露耐受性极强，在15℃干露胁迫下至少可以存活10天，因此可以作为探索潮间带双壳贝类干露耐受机制的良好模型。

本研究对硬壳蛤血细胞在干露胁迫下的自噬与凋亡过程进行探究。首先，鉴定硬壳蛤血细胞亚群组成并探究其数量占比在干露胁迫下的变化。通过测定硬壳蛤血细胞活性氧（ROS）水平，以及超氧化物歧化酶（SOD）和过氧化氢酶（CAT)的活性，探究硬壳蛤血细胞在干露胁迫下的氧化应激响应。在细胞水平，本研究通过透射电镜观察和流式细胞仪检测等手段，探究了干露胁迫下硬壳蛤血细胞的自噬与凋亡过程。在分子水平，本研究对不同干露胁迫时间下的硬壳蛤血细胞进行转录组测序，使用加权基因共表达网络分析（WGCNA），对硬壳蛤血细胞在干露胁迫下的动态分子响应进行探究，并从血细胞自噬与凋亡角度，为硬壳蛤的干露耐受分子机制提供新的见解。

1、干露胁迫下硬壳蛤血细胞组成变化及面临的氧化应激压力

本研究确定硬壳蛤血细胞主要由颗粒细胞和透明细胞两类细胞亚群组成，二者数量比例约为6:4，并且在干露胁迫过程中，颗粒细胞占总血细胞比例呈下降趋势，而透明细胞占总血细胞比例呈上升趋势。在干露胁迫下，硬壳蛤血细胞内葡萄糖浓度持续下降，表明其处于饥饿状态。干露胁迫早期，硬壳蛤血细胞内ROS大量累积，导致氧化应激发生。在这种情况下，抗氧化酶SOD活性上调以清除ROS。然而，当ROS大量累积时，SOD的抗氧化功能丧失。

2、干露胁迫下硬壳蛤血细胞自噬与凋亡过程

在细胞水平，本研究通过透射电镜和激光共聚焦显微镜观察，以及流式细胞仪检测，发现血细胞自噬在干露第1天时被激活，自噬水平在整个干露胁迫过程中表现出先上升后下降的趋势。该结果表明，在饥饿及ROS水平上升的诱导下，血细胞自噬过程被激活以清除胞内受损的蛋白和细胞器。在干露第20天时，硬壳蛤细胞自噬水平显著下降，凋亡率显著上升。该结果表明，自噬水平的上调具有一定限度。随着干露胁迫强度增加，硬壳蛤通过激活细胞凋亡以加速受损严重细胞的死亡，以维持细胞整体健康。本研究表明，血细胞的自噬与凋亡过程对于硬壳蛤在干露胁迫下内环境稳态的维持具有重要意义。血细胞自噬可能在硬壳蛤抵抗短期干露胁迫（胁迫强度较低）上发挥重要作用，而细胞凋亡对其抵抗长期干露胁迫（胁迫强度较强）具有重要意义。硬壳蛤血细胞凋亡率的大幅上升可能是ROS水平骤降的重要原因。此外，本研究结果表明，颗粒细胞是ROS积累的主要场所，也是自噬与凋亡的主要执行者。

3、干露胁迫下硬壳蛤血细胞自噬与凋亡通路转录响应

在分子水平，本研究结果表明血细胞自噬与凋亡过程是硬壳蛤干露耐受机制的重要组成部分。WGCNA结果显示，干露前20天硬壳蛤血细胞内主要发生的生物学过程包括内质网中错误折叠蛋白的重新折叠、溶酶体介导的磷脂降解和蛋白合成降解过程，这些生物学过程均与细胞自噬密切相关。当干露胁迫达到30天时，大量编码IAPs的基因的表达量显著上调，表明抗凋亡系统激活，该过程有利于延缓细胞大量死亡。此外，本研究结果表明，自噬与凋亡通路上许多重要基因的表达量在干露胁迫下发生显著变化。本研究为针对硬壳蛤的研究提供了宝贵的转录组学资源，并从血细胞自噬与凋亡的角度，为潮间带贝类的抗逆机制提供了新的理解。

在工程实践方面，本研究绘制了硬壳蛤在不同温度（15℃、20℃和25℃）干露胁迫下的存活曲线，确定其开始死亡时间及半数致死时间。另外，在15℃干露胁迫下，本研究结合硬壳蛤血细胞自噬与凋亡相关指标和个体存活率改变，推测硬壳蛤的运输保存时间应控制在5天之内，长途运输保存不宜超过10天。本研究为确定硬壳蛤的干露运输保存条件提供科学依据。

The hard clam (Mercenaria mercenaria), naturally inhabits on the west coast of the Atlantic Ocean of North America, ranging from the intertidal zone to subtidal zone. In 1997, academician Zhang Fusui et al. first introduced the hard shell clam from the United States to China, and it has become an important shellfish pond culture in China, with the cultivation area of hundreds of thousands of mu. Hard clams are often subjected to air exposure due to natural tides and during long distance transportation and preservation. Exposure to air can result in physiological stress on shellfish, including desiccation, hypoxia and starvation. Therefore, air exposure is considered as one of the key stress factors influencing the survival of shellfish. The hard clams exhibit remarkable tolerance ability to air exposure and can survive for at least 10 days under air exposure at 15℃. Consequently, hard clam can serve as a suitable model to explore the molecular tolerance mechanism of intertidal shellfish to air exposure.

In this study, the process of autophagy and apoptosis of hemocytes in hard clams exposed to air were investigated. Firstly, the sub-populations of hemocytes and the change of their number ratio under air exposure were identified. Additionally, the reactive oxygen species (ROS) level and the activities of superoxide dismutase (SOD) and catalase (CAT) were measured to investigate the oxidative stress response of hemocytes in hard clams exposed to air. At the cellular level, the level of autophagy and apoptosis of hemocytes in hard clams exposed to air were investigated through transmission electron microscopy and flow cytometry. At the molecular level, the transcriptome sequencing was performed on the hemocytes of hard clams under different durations of air exposure and the weighted correlation network analysis (WGCNA) was used to reveal the dynamic molecular response of hemocytes in hard clams exposed to air. This study also provided new insights on the molecular tolerance mechanism to air exposure in hard clams from the perspectives of autophagy and apoptosis of hemocytes.

1. Identification of the sub-population of hemocytes in hard clams and oxidative stress response under air exposure

In this study, it was determined that the hemocytes of hard clams were composed of granulocytes and hyalinocyte, and the number ratio was about 6:4. The proportion of granulocytes in the total hemocytes decreased, while the proportion of hyalinocyte in the total hemocytes increased during air exposure. During air exposure, the glucose concentration in the hemocytes of hard clams decreased continuously, indicating the state of starvation. At the early stage of air exposure, ROS significantly accumulated in the hemocytes, resulting in oxidative stress. In this case, SOD activity was up-regulated to remove ROS. However, when the ROS accumulation reaches high level, the antioxidant effect of SOD was lost.

2. Autophagy and apoptosis of hematocyte in hard clams under air exposure

At the cellular level, this study found that the autophagy process of hemocytes was activated on the first day after air exposure detected by transmission electron microscopy, confocal laser scanning microscope and flow cytometry. The autophagy level increased first and then decreased during the whole process of air exposure. This result suggested that autophagy was activated to remove damaged intracellular proteins and organelles under the induction of starvation and ROS accumulation. On the 20th day of air exposure, the autophagy level of hemocytes decreased significantly, whereas apoptosis rate increased significantly. This results indicated that the up-regulation of autophagy level was limited. With the increase of stress intensity, hard clams could accelerate the death of severely damaged cells by activating apoptosis, with the aim to maintain the overall health of cells. Our results indicated that autophagy and apoptosis of hemocytes were important for maintaining homeostasis of hard clams under air exposure. The autophagic process of hemocytes may play an important role in the tolerance of hard clams to short-stages air exposure (low stress intensity), while apoptotic process plays an important role in the tolerance of hard clams to long-stage air exposure (high stress intensity).The significant increase in the rate of apoptotic hemocytes may be an important reason for the sudden decrease of ROS level. In addition, the results of this study suggested that granulocytes are the main site of ROS accumulation, and the main performers of autophagy and apoptosis.

3. Transcriptional response of autophagy and apoptosis pathways in hemocytes of hard clams under air exposure

At the molecular level, our results suggested that autophagic and apoptotic process consist the important components of the tolerance mechanism to air exposure in hard clams. WGCNA results exhibited that the main biological processes occurred in the hemocytes during the first 20 under air exposure were the refolding of misfolded proteins in endoplasmic reticulum, lysosomal-mediated phospholipid degradation and protein synthesis and degradation. Notably, These biological processes were all closely related to autophagy. On the 20th day of air exposure, the expression levels of a large number of genes encoding IAPs were significantly up-regulated, indicating that the anti-apoptotic system was activated, with the aim to delay the mass cell death. Additionally, our results showed that the expression levels of many important genes enriched in the autophagy and apoptosis pathways were significantly changed under air exposure. This study provides the valuable transcriptome resource for researches on hard clams, and adds a new understanding on the tolerance mechanism of intertidal shellfish from the perspective of autophagy and apoptosis of hemocytes.

In terms of engineering practice, this study provided the survival curves of hard clam under air exposure at different temperatures (15℃, 20℃ and 25℃), and determined the first death time and the lethal exposure time 50%. In addition, this study combined with the changes of autophagy and apoptosis related index of hemocytes and individual survival rate of hard clams, it was speculated that the transportation and preservation time of clam should be controlled within 5 days, and the long-distance transportation and preservation should not exceed 10 days. This study provides a scientific basis for the determination of transportation and preservation conditions of hard clams.