实验海洋生物学重点实验室知识库

海洋酸化严重威胁贝类生物的生长和生存，对贝类的钙化作用、遗传繁育、免疫和生理过程均产生不利影响。肝胰腺作为重要的代谢、抗氧化和免疫组织，其对海洋酸化的响应和适应机制越来越引起学者的广泛关注。本研究以长牡蛎Crassostrea gigas为研究对象，综合运用生理学、生物化学、分子生物学和生物信息学等相关技术，系统开展海洋酸化对牡蛎肝胰腺生理功能和能量代谢影响的研究。具体结果如下：

通过对牡蛎肝胰腺细菌组16S rDNA测序与分析，结合抗氧化、免疫和消化等多项生理指标检测，发现酸化处理后肝胰腺细胞内pH（intracullular pH, pHi）显著降低，超氧化物歧化酶和谷胱甘肽在酸化应激过程中发挥重要抗氧化作用，但酸化后总抗氧化能力显著下降，活性氧水平和丙二醛含量上升，说明酸性环境可造成明显的组织氧化损伤；酸化处理后牡蛎5种防御素的变化呈现先上升后降低的趋势，该结果与肝胰腺细菌丰度和多样性在酸化28天明显增加，而随之下降的现象相符，说明防御素在酸化环境可积极响应菌群变化并调节机体免疫能力；长期酸化造成肝胰腺正常菌群结构严重破坏，菌群逐步被厌氧菌或兼性厌氧菌所主导，其中Mycoplasma属和Clostridiales目异常增殖，这些厌氧菌很可能进一步积累酸性物质，从而加剧海洋酸化对肝胰腺生理功能的不利影响；酸化处理后胃蛋白酶活力逐渐降低，脂肪酶和淀粉酶活力呈先降低后恢复的趋势，而纤维素酶活力先升高后恢复，说明酸化可导致肝胰腺能量代谢底物发生明显改变。

为了进一步探讨酸化对肝胰腺能量供给方式的影响，对酸化过程中葡萄糖、糖原和能量代谢中间产物的含量以及酸化前后肝胰腺转录组的变化情况进行了分析。由转录组数据分析可知大量碳代谢相关基因差异表达，其中通过荧光定量PCR技术发现酸化处理后糖异生过程中的关键限速酶PEPCK1、PEPCK2、G6Pase1，G6Pase2基因表达量呈上调趋势，同时G6Pase酶活力显著增强，推测酸化后肝胰腺的糖异生能力增强；再者，半乳糖可转变为糖酵解途径的中间产物葡萄糖-6-磷酸，KEGG富集分析发现半乳糖代谢能力增强，有利于葡萄糖的积累；经GO富集分析发现，酸化7天后cellular amide metabolic process显著富集大量差异表达基因，同时发现酸化后谷丙转氨酶（ALT）和谷草转氨酶（AST）活力呈先上升后恢复的趋势，说明在应激状态下氨基酸代谢逐步增加，在肝胰腺能量供给过程中发挥重要作用；另一方面纤维素酶活力在酸化应激后明显上升，推测肝胰腺很可能通过纤维素酶分解以增强葡萄糖的供给。因此，酸化应激过程中，牡蛎肝胰腺可通过增强氨基酸和半乳糖代谢，促进纤维素酶分解以提高葡萄糖的含量，过量的葡萄糖进而会转变为肝糖原。该分析与葡萄糖和糖原含量的检测结果一致，即酸化处理后肝胰腺葡萄糖和糖原含量呈明显上升趋势，说明酸化应激将导致能量合成和储备的需求增加，以满足机体高能量消耗。葡萄糖分解代谢方面，酸化7天己糖激酶和丙酮酸激酶等糖酵解限速酶基因表达量整体呈上升趋势，同时发现乳酸开始积累，直至酸化14天含量达到峰值；由转录组数据可知在酸化28天后肝胰腺三羧酸循环（TCA）途径中的柠檬酸合酶、异柠檬酸脱氢酶和α-酮戊二酸脱氢酶等调控酶基因表达量显著上调，且酸化42天后丙酮酸含量显著增加，但酸化56天后TCA循环限速酶基因整体下调，说明牡蛎通过增强糖酵解水平快速提供能量以应对酸化应激，随之提升TCA循环以进一步保障高能量需求，但长期酸化后葡萄糖合成和储备降低，TCA循环途径受阻，无法维持高能量输出，从而对生物体的生存产生不利影响。

综上，海洋酸化对肝胰腺产生诸多不利影响，包括酸碱平衡失调，抗氧化和蛋白消化等生理功能损伤，肝胰腺菌群结构及功能严重破坏。酸化后长牡蛎通过改变肝胰腺能量供给策略，如增强氨基酸和半乳糖代谢、促进纤维素酶分解的方式提高葡萄糖的含量，帮助机体更好地应对酸化胁迫，但高能量输出模式不可持续，严重威胁牡蛎的长期生存。

Ocean acidification seriously threatens the growth and survival of shellfish organisms, and adversely affects the calcification, genetic breeding, immunity and physiological processes. Hepatopancreas, as an important metabolic, antioxidant, and immune tissue, its response and adaptation mechanism to ocean acidification have attracted more and more attention from scholars. In this study, Pacific oysters, Crassostrea gigas, was used as the research object, comprehensively using physiology, biochemistry, molecular biology and bioinformatics and other related technologies to systematically carry out the research on the effect of ocean acidification on the physiological function and energy metabolism of hepatopancreas. The specific results are as follows:

Through the sequencing and analysis of 16S rDNA of the microbiota of hepatopancreas, combined with the detection of multiple physiological indicators such as antioxidant, immunity and digestion, it was found that the intracellular pH (intracullular pH, pHi) of the hepatopancreas was significantly reduced after acidification, and the superoxide dismutase and glutathione plays an important antioxidant role in the process of acidification stress, but the total antioxidant capacity was significantly reduced after acidification, and the level of reactive oxygen species and malondialdehyde content increases, indicating that the acid environment can cause obvious tissue oxidative damage. The changes of the five defensins of oysters showed a trend of increasing first and then decreasing after acidification. The result is consistent with the obvious increase in the abundance and diversity of microbiota at 28 days of acidification, and the subsequent decrease, indicating that the immune capacity of the hepatopancreas after acidification is affected by self-regulation and changes in external bacterial. The long-term acidification has caused serious damage to the normal bacterial community of the hepatopancreas, and the microbiota is gradually dominated by anaerobes or facultative anaerobes. Among them, Mycoplasma and Clostridiales proliferate abnormally. These bacteria are likely to further accumulate acidic substances, thereby exacerbating the adverse effects of ocean acidification on the physiological functions of the hepatopancreas. After acidification, the pepsin activity gradually decreases, and the lipase and amylase activities show a tendency to first decrease and then recover, while the cellulase activity first increased and then recovered, indicating that acidification can cause significant changes in the substrates of energy metabolism.

In order to further explore the influence of acidification on the energy supply mode of the hepatopancreas, the contents of glucose, glycogen and energy metabolism intermediates during the acidification process and the changes of the hepatopancreas transcriptome before and after acidification were analyzed. The analysis of transcriptome data shows that many carbon metabolism-related genes are differentially expressed. Among them, the key rate-limiting enzymes PEPCK1, PEPCK2, G6Pase1, and G6Pase2 gene expression in the process of gluconeogenesis after acidification were found to be up-regulated, while the enzyme activity of G6Pase was significantly enhanced, and it is speculated that the gluconeogenesis ability of the hepatopancreas after acidification is enhanced. In addition, galactose can be converted into glucose-6-phosphate, an intermediate product of the glycolysis pathway, and the KEGG enrichment analysis found that the galactose metabolism ability is enhanced. It is conducive to the accumulation of glucose. GO enrichment analysis found that the cellular amide metabolic process significantly enriched many differentially expressed genes after acidification for 7 days. At the same time, it was found that alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities increased first and then recovered after acidification. The trend indicates that amino acid metabolism gradually increases under stress and plays an important role in the energy supply process of the hepatopancreas. On the other hand, cellulase activity increased significantly in the elevated CO2 concentration. It is speculated that the hepatopancreas may be decomposed by cellulase to enhance the supply of glucose. Therefore, the oyster hepatopancreas can enhance the metabolism of amino acids and galactose, and promote the decomposition of cellulase to increase the content of glucose in the process of acidification stress. The excess glucose will be converted into glycogen. This analysis is consistent with the results of the detection of glucose and glycogen content, that is, the hepatopancreas glucose and glycogen content showed a significant upward trend after acidification, indicating that acidification stress will lead to an increase in the demand for energy synthesis and storage to meet the high energy consumption of oysters. In terms of glucose catabolism, the expression of glycolytic rate-limiting enzyme genes such as hexokinase and pyruvate kinase showed an overall upward trend at 7 days of acidification. At the same time, it was found that lactic acid began to accumulate until the content reached its peak at 14 days of acidification. According to the transcriptome data, the gene expression of citrate synthase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase in the tricarboxylic acid cycle (TCA) pathway was significantly up-regulated 28 days after acidification, and the pyruvate content increased significantly after 42 days of acidification, but the TCA cycle rate-limiting enzyme gene was down-regulated overall after 56 days of acidification, indicating that oysters can quickly provide energy by enhancing glycolysis levels to cope with acidification stress, and then increase the TCA cycle to further ensure high energy demand, but after long-term acidification, glucose synthesis and storage are reduced, the TCA cycle is blocked, and high energy output cannot be maintained, which adversely affects the survival of organisms.

In summary, ocean acidification has many adverse effects on the hepatopancreas, including the imbalance of acid-base balance, damage to physiological functions such as antioxidant and protein digestion, and severe damage to the structure and function of the hepatopancreas microbitoa. After acidification, Pacific oyster improves the glucose content by changing the energy supply strategy of the hepatopancreas, such as enhancing the metabolism of amino acids and galactose, and promoting the decomposition of cellulase to help the oyster better deal with acidification stress, but the high energy output mode is unsustainable and serious threatening the long-term survival of oysters.