实验海洋生物学重点实验室知识库

双壳贝类的夏季大规模死亡事件在世界各地时有报道，给全球水产养殖业造成了重大损失。研究表明，双壳贝类的夏季死亡不仅与病毒和细菌等病原体的感染有关，还与环境因子等非生物因素密切相关。然而，目前对双壳贝类夏季死亡事件的分子机制仍知之甚少。文蛤（Meretrix petechialis）是我国重要海洋经济贝类之一，夏季死亡现象也时有发生。本研究采用转录组学和TMT定量蛋白质组学关联分析的方法对一起2021年7月发生在浙江温州的自然爆发的文蛤夏季死亡事件开展了全方位系统分析，通过与人工感染模拟条件下各组织（鳃、肝胰腺和血细胞）转录组的比较分析，探究了文蛤不同组织和细胞在病原和高温胁迫等条件下分子水平响应的异同，并挖掘了与夏季死亡和机体免疫相关的候选分子标记。另外，从细胞水平研究了弧菌感染对文蛤血细胞不同免疫指标的影响，筛选出评估文蛤生理状态的免疫指标，并将这些候选分子和免疫指标应用到育种实践中。本研究的主要研究内容和结果如下：

1．鳃组织是文蛤最早与水环境中的各种微生物接触的组织，我们首先对自然发病和人工感染条件下的鳃组织进行了多组学分析。通过对自然发病文蛤鳃组织的组学分析发现：与正常组相比，共鉴定出172个差异表达基因（DEGs）和222个差异表达蛋白（DEPs）。免疫相关DEGs/DEPs组间的表达谱的不一致可能是由于发病文蛤的免疫失调所致。值得注意的是，我们在发病组中前20个下调基因中发现了11个溶质载体家族基因（SLCs），提示发病文蛤的跨膜转运能力受到损伤。转录组和蛋白质组的关联分析结果表明，一些代谢过程，如“精氨酸和脯氨酸代谢”和“酪氨酸代谢”在发病组中被抑制，提示文蛤发病过程中出现代谢抑制。通过对自然爆发的夏季死亡和高温弧菌感染的人工模拟实验之间的DEGs表达谱的比较，9/15基因在两种条件下表现出相似的表达趋势，表明夏季死亡可能是由高温和弧菌感染联合作用引起的。另一方面，人工感染条件下的转录组分析同样富集到了多个SLCs，进一步提示SLC家族在文蛤夏季死亡过程中具有重要作用。人工感染条件下的文蛤与自然发病个体的差异基因都与免疫识别和跨膜转运相关，提示鳃组织在激活免疫和物质转运方面发挥重要作用。这些结果将加深我们对夏季死亡事件的认识，并为文蛤抗性育种提供候选分子标记。

2．肝胰腺是文蛤等双壳贝类中重要的免疫器官，我们进而对自然发病和人工感染条件下的肝胰腺进行了多组学的比较分析。对自然发病文蛤肝胰腺的组学分析发现：与鳃组织中的DEGs和DEPs的数目相似，发病文蛤肝胰腺的转录组和蛋白质组中分别鉴定出93个DEGs和227个DEPs。其中，DEGs/DEPs主要涉及免疫效应分子和抗氧化酶等基因/蛋白的变化，但未鉴定到鳃组织中大量SLCs。转录组蛋白组的富集关联分析发现，代谢相关、免疫相关和蛋白质加工等通路具有较好的关联性。其中，免疫通路中的溶酶体和内吞作用通路提示肝胰腺中可能存在浸润的血细胞。另一方面，人工感染条件下的比较转录组分析显示，在高温和弧菌联合作用下（31℃-vibrio），免疫相关的DEGs如补体C1q样蛋白、C型凝集素、大防御素和溶菌酶等基因的数量显著增加，表明高温和弧菌感染的协同作用触发了更强烈的抗菌免疫应答。通过与自然发病条件下的蛋白组比较分析，发现弧菌感染和高温弧菌联合感染与自然发病过程共有信号通路数目相近，主要涉及代谢和免疫相关通路。上述结果表明弧菌感染在自然发病过程中具有重要的作用，弧菌可能是夏季死亡现象更重要的诱因。这些发现提示文蛤肝胰腺在响应感染过程中主要是代谢和免疫通路发挥作用，为揭示夏季死亡的原因和发病过程提供了重要的见解。

3．血细胞作为先天免疫系统的关键组分，不仅是细胞免疫的执行者，也是体液免疫分子的合成者。为建立一个响应夏季死亡的全面分子表达谱，我们研究了弧菌感染不同时间后文蛤血细胞的动态响应过程。弧菌感染组相较于对照组，在攻毒1dpi和3dpi的血细胞中分别鉴定出342和1861个差异表达基因，表明血细胞的免疫响应具有时间动态性。对差异表达基因富集发现，DNA复制和多个免疫相关通路如溶酶体、吞噬体、内吞作用、自噬和过氧化物酶体等在攻毒后的两个时间点均有富集。DEGs的蛋白网络互作分析显示形成了以参与细胞增殖和存活过程的PCNA、CDK1/2、MCM3/7基因为中心的互作网络。通过qPCR验证了增殖相关基因的表达，发现弧菌感染后抑制了细胞的增殖过程。另外，对感染1dpi和3dpi的DEGs的表达量进行比较发现，有89个DEGs在攻毒3dpi的表达相较于1dpi发生显著变化。其中，抗凋亡蛋白Bcl2基因在攻毒3dpi的表达水平显著降低。进一步通过蛋白印迹检测了其在1dpi和3dpi的蛋白表达量，发现Bcl2在弧菌感染3dpi后的表达量被显著抑制，这可能诱导了细胞凋亡的发生。上述结果为了解血细胞在病原感染过程中的细胞防御机制提供了分子依据，有助于全面的认识夏季死亡过程伴随的分子水平变化及宿主的免疫应答机制。

4．为进一步验证上述多组学数据提示的结果并探究不同类型血细胞的功能异质性，我们通过流式细胞术和显微观察对文蛤血细胞进行了分类，并研究了不同类型细胞在副溶血弧菌感染后的功能差异。结果显示，文蛤中主要存在三种类型的血细胞，即颗粒细胞（GC）、半颗粒细胞（SGC）和透明细胞（HC）。通过检测血细胞的多种免疫指标发现，颗粒细胞是主要的吞噬细胞，而半颗粒细胞是溶酶体、活性氧（ROS）和一氧化氮（NO）的主要生产者。上述研究结果表明，每个血细胞亚群在抗菌防御中的参与程度不同。在弧菌感染3dpi时，弧菌感染组文蛤的血细胞总数（THC）显著增加；而在10dpi时，THC和存活率显著降低。值得注意的是，感染早期血细胞表现出高水平的吞噬活力、细胞凋亡水平、溶酶体和ROS含量及低含量NO，表明血细胞积极启动免疫防御以应对和抵御弧菌感染。此外，Spearman相关分析表明，细胞凋亡与THC和溶酶体含量呈显著正相关，吞噬作用与ROS和NO水平相关。因此，细胞凋亡水平和吞噬活性可作为评估文蛤健康状况的可靠指标。研究结果为不同血细胞亚群的免疫功能分析及双壳贝类健康状态评估提供了新认识。

5．最后，我们对文蛤不同育种材料进行了副溶血弧菌感染实验，通过生存曲线和组织载菌量反映不同群体或家系的抗性与易感程度，并结合获得的候选分子和血细胞指标进行了分析比较。通过对不同壳色的3个群体的比较发现，群体间的累积死亡率无显著差异，同时鳃和肝胰腺组织中的弧菌量、组织损伤和血细胞免疫指标呈现相同的变化趋势，群体间未检测出显著差异；对6个免疫分子（Bax、Big-defensin、Duox、Lysozyme、MITF和TLR）的表达量分析发现，不同群体免疫基因的表达量呈现出差异。对5个家系进行副溶血弧菌感染，发现5个家系的死亡率从96%到61.33%不等，家系F1和F11分别为敏感家系和抗性家系；家系间鳃和肝胰腺中的载菌量无显著差异，但不同家系弧菌量随着感染时间的变化呈现不同的趋势；进一步比较了6个免疫基因在F1和F11家系之间表达的差异，根据其表达量高低和时间变化趋势确定了感染不同阶段的分子标记，并尝试评价了这些标记用于双壳贝类抗病选育的可行性。

Mass mortalities of bivalves have been reported worldwide, resulting in significant losses for the aquaculture industry. While previous research has highlighted the connection between bivalve summer mortality and pathogen infections, such as viruses and bacteria, it has also emphasized the impact of abiotic factors such as environmental factors. However, there was limited information concerning the molecular responses to various stressors leading to summer mortality. Meretrix petechialis, one of the important marine economic mollusks in China, frequently suffers from summer mortality. In this study, RNA sequencing (RNA-seq) and tandem mass tagging (TMT)-based quantitative proteomics were used to conduct a comprehensive analysis of the natural outbreak of summer mortality in the clam M. petechialis in Wenzhou, Zhejiang Province, in July 2021. We explored the molecular responses in various tissues (gill, hepatopancreas and hemocytes) in clams between the natural summer mortality and artificial infection involving heat stress and Vibrio infection through multi-omics analysis, and explored candidate molecular markers related to summer mortality and host immunity. In addition, we delved into the effects of Vibrio infection on the immune parameters of hemocytes at the cellular level, identifying reliable immune indicators for evaluating the physiological condition of clams. These candidate molecules and immune indicators were applied to breeding practices. The main contents and results of this study are as follows:

1. Initially, a comparative analysis was performed on the gill tissues of clams experiencing natural summer mortality and those subjected to artificial infection conditions, given that the gill is the primary tissue exposed to various microorganisms in the aquatic environment. Analysis of gill tissues of naturally diseased clams showed that a total of 172 differentially expressed genes (DEGs) and 222 differentially expressed proteins (DEPs) were identified in comparison to the normal group. The inconsistent expression profiles of immune-related DEGs/DEPs may be due to the immune dysregulation in the diseased clams. Notably, 11 solute carrier family genes (SLCs) were found among the top 20 down-regulated genes in the diseased group, indicating that weakened transmembrane transport ability might occur in the diseased clams. Integration analysis of transcriptomic and proteomic results showed that some metabolic processes such as “arginine and proline metabolism” and “tyrosine metabolism” were inhibited in the diseased group, suggesting metabolic inhibition occurred during the disease process of clams. The comparison of DEGs expression between the natural summer mortality event and an artificial challenge experiment involving both heat stress and Vibrio infection revealed 9/15 genes showing similar expression trends between the two conditions, suggesting that the summer mortality might be caused by a combination of high temperature and Vibrio infection. On the other hand, transcriptome analysis under artificial infection conditions also enriched many SLCs, emphasizing the crucial role of the SLC family in response to summer mortality in clams. The DEGs in both artificially infected clams and naturally diseased clams are connected to immune recognition and transmembrane transport, suggesting that gill tissue may play an important role in immune activation and substance transport. These results would deepen our understanding of summer mortality and provide candidate molecular markers for clam resistance breeding.

2. The hepatopancreas is an essential immune organ in bivalves such as M. petechialis, and we then conducted a multi-omics comparative analysis of the hepatopancreas under natural summer mortality and artificial infection conditions. A similar number of DEGs and DEPs in hepatopancreas and gill tissues in naturally diseased clams, with 93 DEGs and 227 DEPs identified in the transcriptome and proteome of the hepatopancreas of naturally diseased clams respectively. Among them, DEGs/DEPs are primarily associated with changes in genes/proteins such as immune effector molecules and antioxidant enzymes, but a large number of SLCs in gill tissue have not been identified. The enrichment correlation analysis between transcriptome and proteome indicated a strong relationship between metabolic, immune, and protein processing pathways. Notably, the lysosomal and endocytosis in transcriptome and proteome suggested the possible presence of infiltrating hemocytes in the hepatopancreas. On the other hand, comparative transcriptome analysis revealed a significant increase of immune-related DEGs such as complement C1q-like protein, C-type lectin, big defensin, and lysozyme in the 31°C-vibrio group, suggesting that the synergistic effect of high temperature and Vibrio infection triggers a stronger antibacterial immune response. It was found that 27°C-vibrio infection and 31°C-vibrio group share similar numbers of signaling pathways with the proteome under natural disease processes, mainly involving metabolic and immune related pathways. The above results suggested that Vibrio infection may play an important role in the natural course of disease and may be a more important cause of summer mortality. Overall, our findings emphasize the involvement of the hepatopancreas in metabolic and immune pathways in response to disease, offering valuable insights into the causes and pathogenesis of summer mortality.

3. Hemocytes, as a key component of the innate immune system, are not only the executors of cellular immunity but also the synthesizers of humoral immune molecules. To establish a comprehensive molecular expression profile in response to summer mortality, we investigated the dynamic response process of hemocytes after Vibrio infection at different times. Compared with the control group, 342 and 1861 DEGs were identified in the hemocytes of the Vibrio infection at 1-day post-infection (dpi) and 3dpi, respectively, indicating that the immune response of hemocytes has temporal dynamics. Enrichment of DEGs revealed that DNA replication and several immune-related pathways such as lysosome, phagosome, endocytosis, autophagy, and peroxisomes were enriched both at 1dpi and 3dpi. The protein network interaction analysis of DEGs further highlighted the formation of an interaction network centered around PCNA, CDK1/2, and MCM3/7, which play crucial roles in cell proliferation and survival. The expression of proliferation-related genes was verified through qPCR, revealing that Vibrio infection hindered the cell proliferation process. Notably, 89 DEGs significantly changed at 3dpi compared to 1dpi, with the Bcl2 gene showing a marked reduction at 3dpi. Consequently, the protein expression levels of Bcl2 were detected at 1dpi and 3dpi using Western blotting, demonstrating a significant suppression of Bcl2 expression post-Vibrio infection at 3dpi, potentially leading to cell apoptosis. These findings help us to elucidate the molecular foundations underlying the cellular defense mechanisms of hemocytes during pathogen infection, and contribute to a holistic comprehension and the molecular alterations linked to summer mortality.

4. To further validate the results indicated by the aforementioned omics data and explore the functional heterogeneity of different hemocyte types. We classified the clam hemocytes and investigated the functional differences among various cell types under V. parahaemolyticus infection via flow cytometry and microscopic observation. Three major types of hemocytes were identified in the M. petechialis, including granulocyte (GC), semi-granulocyte (SGC) and hyalinocyte (HC). Our results from the detection of different immune parameters of hemocytes found that granulocytes are the main phagocytes, whereas semi-granulocytes primarily act as the main producers of lysosomes, reactive oxygen species (ROS), and nitric oxide (NO). These suggest a distinct involvement of each hemocyte subpopulation in antibacterial defense. Specifically, the total hemocyte count (THC) of the Vibrio-infected clams exhibited a significant increase at 3dpi, followed by a significant decrease in THC and survival rate at 10dpi. Remarkably, hemocytes exhibit high levels of phagocytosis, apoptosis, lysosomal content, and ROS levels to eliminate Vibrio in the early stages of infection with the low production of NO, suggesting hemocytes actively initiate immune defense to cope with Vibrio infection. Furthermore, Spearman’s correlation analysis further revealed a significant positive correlation between apoptosis and THC as well as lysosomal content, while phagocytosis showed a correlation with ROS and NO levels. Accordingly, apoptosis levels and phagocytic activity may serve as reliable indicators for evaluating the health status of clams. In conclusion, these results provide new insights into the immune function analysis of different hemocyte subpopulations and the health status evaluation of bivalves.

5. Finally, different breeding strains were subjected to the Vibrio infection experiment to assess the resistance and susceptibility of various populations or families. The evaluation was conducted by examining the survival curve and bacterial load in tissues, as well as comparing the candidate molecules and hemocyte indicators. There was no significant difference in the cumulative mortality among the three populations with different shell colors. Furthermore, the Vibrio load and tissue damage in gill and hepatopancreas, and immune indicators in hemocytes exhibited a similar pattern among the populations, although no significant difference was observed. Analysis of the expression levels of six immune genes (Bax, Big-defensin, Duox, Lysozyme, MITF and TLR) revealed different expression levels in different populations. On the other hand, Vibrio infection was carried out in five families, resulting in mortality rates ranging from 96% to 61.33%. Family 1 (F1) was identified as susceptible, while family 11 (F11) exhibited resistance. Although no significant variation in Vibrio presence was observed in the gill and hepatopancreas across the five families, distinct trends emerged in the Vibrio amount across different families throughout the infection. We further compared the expression difference of six immune genes between F1 and F11, determined molecular markers at different infection stages based on their expression levels and temporal trends, and attempted to evaluate the potential utility of these markers for disease-resistance breeding in bivalves.

第 1 章 绪论.......................................................................................... 1
1.1 文蛤的生物学特征及养殖现状......................................................................... 1
1.1.1 文蛤的分类地位和经济价值...................................................................... 1
1.1.2 文蛤的养殖现状.......................................................................................... 1
1.2 双壳贝类夏季大规模死亡现象......................................................................... 2
1.2.1 夏季大规模死亡背景及原因...................................................................... 2
1.2.2 夏季大规模死亡现状及研究进展.............................................................. 3
1.2.3 与细菌性疾病相关的大规模死亡现象...................................................... 4
1.3 双壳贝类免疫系统............................................................................................. 5
1.3.1 体液免疫分子.............................................................................................. 6
1.3.2 体液免疫屏障.............................................................................................. 8
1.3.3 血细胞和细胞免疫反应............................................................................ 10
1.4 组学技术在双壳贝类夏季死亡现象的应用................................................... 14
1.4.1 基因组........................................................................................................ 15
1.4.2 转录组........................................................................................................ 15
1.4.3 蛋白组........................................................................................................ 16
1.4.4 代谢组........................................................................................................ 17
1.5 本研究的内容和意义....................................................................................... 18
第 2 章 自然发病与人工感染条件下鳃组织的多组学分析 ............ 19
2.1 研究背景........................................................................................................... 19
2.2 材料方法........................................................................................................... 20
2.2.1 自然发病样品收集.................................................................................... 20
2.2.2 弧菌感染与样品收集................................................................................ 20
2.2.3 转录组测序及生物信息学分析................................................................ 21
2.2.4 蛋白组测序及生物信息学分析................................................................ 21
2.2.5 转录组蛋白组关联分析............................................................................ 22
2.2.6 RNA 提取、cDNA 合成与 qPCR 验证.................................................... 22
2.3 实验结果........................................................................................................... 24
2.3.1 自然发病条件下鳃组织的转录组和蛋白组数据特征............................ 24
2.3.2 自然发病条件下鳃组织的转录组分析.................................................... 25
2.3.3 自然发病条件下鳃组织的蛋白组分析.................................................... 26
2.3.4 鳃组织的转录组与蛋白组关联分析........................................................ 27
2.3.5 鳃组织转录组 DEG 表达量验证 ............................................................. 28
2.3.6 人工感染条件下鳃组织的转录组分析.................................................... 29
2.3.7 自然发病与人工感染条件下鳃组织的组学比较.................................... 31

2.4 讨论................................................................................................................... 32
2.5 小结................................................................................................................... 34
第 3 章 自然发病与人工感染条件下肝胰腺组织的多组学分析 .... 35
3.1 研究背景........................................................................................................... 35
3.2 材料方法........................................................................................................... 36
3.2.1 自然发病样品收集.................................................................................... 36
3.2.2 弧菌感染与样品收集................................................................................ 36
3.2.3 转录组测序及生物信息学分析................................................................ 36
3.2.4 蛋白组测序及生物信息学分析................................................................ 36
3.2.5 转录组蛋白组关联分析............................................................................ 37
3.3 实验结果........................................................................................................... 37
3.3.1 自然发病条件下肝胰腺转录组与蛋白组数据的特征............................ 37
3.3.2 自然发病条件下肝胰腺组织的转录组分析............................................ 38
3.3.3 自然发病条件下肝胰腺组织的蛋白组分析............................................ 39
3.3.4 肝胰腺组织的转录组与蛋白组关联分析................................................ 41
3.3.5 人工感染条件下肝胰腺组织的转录组分析............................................ 42
3.3.6 自然发病与人工感染条件下的组学比较................................................ 45
3.4 讨论................................................................................................................... 47
3.5 小结................................................................................................................... 49
第 4 章 弧菌感染条件下血细胞转录组分析.................................... 50
4.1 研究背景........................................................................................................... 50
4.2 材料方法........................................................................................................... 51
4.2.1 弧菌感染与血细胞收集............................................................................ 51
4.2.2 血细胞转录组测序与生物信息学分析.................................................... 51
4.2.3 RNA 提取、cDNA 合成与 qPCR 验证.................................................... 51
4.2.4 血细胞总蛋白提取及定量........................................................................ 52
4.2.5 Western Blot 检测 Bcl2 表达..................................................................... 52
4.3 实验结果........................................................................................................... 53
4.3.1 文蛤生存曲线............................................................................................ 53
4.3.2 血细胞转录组的数据表征........................................................................ 54
4.3.3 弧菌感染后血细胞差异表达基因鉴定.................................................... 54
4.3.4 弧菌感染后血细胞富集分析.................................................................... 55
4.3.5 弧菌感染后血细胞 DEGs 蛋白互作分析................................................ 57
4.3.6 弧菌感染不同时间后血细胞转录组的比较............................................ 58
4.3.7 血细胞增殖相关基因表达量验证............................................................ 59
4.3.8 弧菌感染后对血细胞凋亡过程的影响.................................................... 60
4.4 讨论................................................................................................................... 61
4.5 小结................................................................................................................... 62
第 5 章 文蛤血细胞的鉴定和弧菌感染后免疫指标分析 ................ 63
5.1 研究背景........................................................................................................... 63
5.2 材料方法........................................................................................................... 64
5.2.1 血细胞收集与分选.................................................................................... 64
5.2.2 弧菌感染实验与血细胞收集.................................................................... 64
5.2.3 血细胞计数与存活率统计........................................................................ 64
5.2.4 血细胞增殖能力检测................................................................................ 65
5.2.5 流式细胞仪检测免疫相关指标................................................................ 65
5.2.6 激光共聚焦显微镜观察免疫相关指标.................................................... 65
5.2.7 斯皮尔曼相关性检验................................................................................ 65
5.2.8 数据分析.................................................................................................... 66
5.3 实验结果........................................................................................................... 66
5.3.1 文蛤血细胞类型........................................................................................ 66
5.3.2 弧菌感染后文蛤生存曲线........................................................................ 67
5.3.3 弧菌感染后血细胞总数、细胞类型及细胞存活率变化........................ 67
5.3.4 弧菌感染后血细胞吞噬活力变化............................................................ 68
5.3.5 弧菌感染后血细胞增殖能力变化............................................................ 70
5.3.6 弧菌感染后血细胞凋亡水平变化............................................................ 70
5.3.7 弧菌感染后血细胞溶酶体含量变化........................................................ 71
5.3.8 弧菌感染后血细胞 ROS 水平变化.......................................................... 73
5.3.9 弧菌感染后血细胞 NO 水平变化............................................................ 74
5.3.10 各免疫指标相关性分析.......................................................................... 75
5.4 讨论................................................................................................................... 75
5.5 小结................................................................................................................... 78
第 6 章 弧菌感染条件下不同群体和家系的抗性比较 .................... 79
6.1 研究背景........................................................................................................... 79
6.2 材料方法........................................................................................................... 80
6.2.1 不同群体和家系弧菌感染实验及取样.................................................... 80
6.2.2 TCBS 涂板计数.......................................................................................... 80
6.2.3 Vp-ompK 免疫组化实验 ........................................................................... 80
6.2.4 RNA 提取、cDNA 合成与 qPCR 验证.................................................... 81
6.2.5 血细胞吞噬、溶酶体和 ROS 含量检测.................................................. 82
6.3 实验结果........................................................................................................... 82
6.3.1 弧菌感染后不同壳色群体的生存曲线.................................................... 82
6.3.2 Vp-ompK 在不同壳色群体文蛤鳃和肝胰腺组织定位 ........................... 82
6.3.3 不同壳色群体免疫基因表达的比较........................................................ 84
6.3.4 不同壳色群体文蛤血细胞吞噬、溶酶体和 ROS 含量的比较.............. 85
6.3.5 弧菌感染后不同家系的生存曲线............................................................ 88
6.3.6 不同家系文蛤鳃和肝胰腺组织载菌量的比较分析................................ 89
6.3.7 抗性家系和敏感家系文蛤免疫基因表达比较........................................ 91
6.4 讨论................................................................................................................... 92

6.5 小结................................................................................................................... 93
第 7 章 总结与展望............................................................................ 95
参考文献................................................................................................. 97
附录....................................................................................................... 117
致谢....................................................................................................... 133
作者简历及攻读学位期间发表的学术论文与其他相关学术成果 ... 135