中国科学院海洋研究所机构知识库

文蛤（Meretrix petechialis）是我国一种常见的海水养殖经济贝类，随着文蛤集约化养殖规模的扩大和滩涂环境的恶化，由弧菌感染导致的文蛤大规模爆发性死亡制约着文蛤养殖业的发展。副溶血弧菌( Vibrio parahaemolyticus)是一种嗜盐嗜温，兼性厌氧的革兰氏阴性菌，通常在夏季高温时期导致文蛤大量死亡。本研究分析了弧菌感染后，同一攻毒浓度下文蛤不同个体的载菌量；分析了不同感染强度下文蛤肝胰腺的载菌量与水体含菌量的相关关系。通过16s扩增子测序分析了弧菌感染后文蛤肝胰腺微生物群落的结构变化，发现了攻毒后微生物类群对弧菌不同感染阶段的指示作用。另外，研究分析了弧菌感染后文蛤免疫凋亡相关基因表达量的变化。本研究的主要结果如下：

1. 采用TCBS平板涂布法，检测了实验水体及文蛤肝胰腺中副溶血弧菌的含量。在对攻毒后水体含菌量日变化的监测中，发现菌含量呈现出一个升高、维持、降低的趋势，且实际攻毒浓度能够符合预期的浓度要求。在对宿主的感染中，检测到了在浸泡攻毒过程中，弧菌在文蛤肝胰腺组织中大量增殖的过程，攻毒后第1天弧菌载量明显增高，在感染后第3天体内载菌量急剧下降，并随后一直呈现较低水平。通过改变水体弧菌的感染浓度，我们发现感染的早期阶段，随着水体中弧菌浓度的升高，相应的文蛤体内载菌量也更高。而在感染的中后期，早期具有不同弧菌载量的文蛤体内的弧菌载量无显著差异，都呈现出一个较低水平。另外，高浓度的弧菌刺激会导致更高的死亡率。该研究为开展文蛤感染发病过程中弧菌载量和免疫抗性评价相关研究提供了参考。

2. 采用16S rDNA扩增子测序技术，对连续攻毒后不同天数文蛤肝胰腺中的微生物群落结构和多样性的变化进行了分析。结果显示未感染弧菌的健康文蛤和感染弧菌的文蛤之间，肝胰腺微生物群落结构发生了剧烈的变化。感染后第1天弧菌相对丰度（14.9%±8.8%）与未攻毒对照组（0.2-1.8%）比出现了显著升高，其绝对含量（文蛤肝胰腺弧菌载量）也显著增高，但随后弧菌绝对载量和相对丰度出现明显的降低，一直处于较低的水平。感染后第3天文蛤肝胰腺微生物群落种群多样性显著升高，而到了感染后第6天微生物种群丰度及多样性显著降低，同时伴随着宿主死亡高峰的发生。以上结果表明菌群的变化与宿主的健康可能存在一定的关系，并筛选了一系列可用于指示不同感染阶段的微生物类群。

3. 初步分析了文蛤Bcl-2家族中促凋亡和抑制凋亡不同类型的基因在弧菌感染后的表达变化特征。其中促凋亡基因MpBax的cDNA全长序列为1309bp，包括615bp的开放阅读框，编码204个氨基酸，其蛋白序列与泥蚶的相似度最高（66%），在文蛤鳃组织中表达量最高。抑制凋亡基因MpBcl-xL（Bcl-2-like 1 or Bcl2L1）的cDNA包含558bp的开放阅读框，编码185个氨基酸，与其他物种相比，MpBcl-xL蛋白与菲律宾蛤仔的相似度最高达到70%。MpBcl-w（Bcl-2-like 2 or Bcl2L2）的cDNA包含698bp的开放阅读框，编码252个氨基酸, MpBcl-w蛋白与菲律宾蛤仔相似度最高达到87%。副溶血弧菌攻毒后，MpBax表达下调，在攻毒过程中呈现先下调后上升的趋势，在第8天表达量最低。抑凋亡基因MpBcl-xL、MpBcl-w在弧菌刺激后表达上调。结果显示弧菌感染后，文蛤体内的免疫凋亡相关因子的表达模式与其功能是相吻合的。

小结：本文研究了副溶血弧菌攻毒后，文蛤体内载菌量和环境水体含菌量变化的相关关系；分析了攻毒后文蛤体内微生物群落结构组成的变化，发现了微生物群落对感染后疾病爆发过程的指示作用；克隆了文蛤凋亡相关基因并检测了其在弧菌感染后的表达量变化。上述研究结果，从一个侧面揭示了文蛤对弧菌感染的免疫响应机制，可以为文蛤养殖中疾病防控和抗性育种提供参考。

The clam Meretrix petechialis is one of the important economic aquaculture shellfish in China. With the expansion of high-density farming mode and the deterioration of the ecological environment, mass mortality events often occurred during the process of clam culture, which have become the primary restriction for the development and sustainability of the clam culture industry. Vibrio parahaemolyticus is an opportunistic pathogen causing mass mortality of cultured clams in summer.

In present study, we analyzed the bacterial load variation in different individuals in the same vibrio dose challenge using the hepatopancreas tissue. The correlation between the host bacterial load and the artificial vibrio dose of the environment seawater was investigated. We also study the diversity and dynamics of hepatopancreas microbiota during the infection process by 16s amplicon sequencing. Our results revealed several stage-specific indicator species by LEfSe analysis during vibrio disease progression. In addition, the changes of the immune related genes expression were analyzed after vibrio infection.

The main results are as follows:

1. The vibrio load changes both in host hepatopancreas tissue and environment seawater under vibrio challenge was investigated by TCBS plate account. The bacterial growth and reproduction in the seawater showed a trend of increase, maintenance and decrease process during 24 hours. The maintenance time of vibrio effective concentration in seawater account for over 1/3 of the time in the 24-hour cycle, which was able to meet the vibrio concentration requirements in experiment design. Then, vibrio immersion experiments were carried out to simulate vibrio challenge in natural environment. It was found that bacterial load in clam hepatopancreas was sharply increased in day 1 and then decreased rapidly in day 3 after challenge, and then kept on a low level. In the different vibrio dose challenge experiments, a significant positive correlation was found between the bacterial load and the vibrio dose in the seawater (Spearman’s ρ=0.899， P=0.000) in the initially phase, and no significant difference in bacterial load among different groups in the middle-late stage with a lower level of 0-205 CFU/mg. In addition, high concentrations of vibrio stimulation can lead to higher clam mortality. The results provide references for the study on vibrio load during infection and resistance evaluation in the clam M. petechialis.

2. We used 16S rDNA amplicon sequencing technology to analyze the microbial community structure and diversity in the hepatopancreas microbiota changes to vibrio challenge. It revealed that there were significant microbial diversity composition differences between healthy and infected clam. During the course of the vibrio challenge, the highest relative abundance of vibrio (14.9%±8.8%) was found at 1 DPI, which was in agreement with the vibrio colony counts. However, the relative abundance of vibrio decreased at the subsequent days and kept on a low level. A significant increase in the Alpha diversity was observed at 3 DPI (P < 0.05), followed by a distinct decline at 4 and 6 DPI (P < 0.05). The low abundance and diversity microbial population at 6 DPI has been found to coincide with the outbreak of death event. It has also been found that the stability of these microflora may have crucially affected the health status of clams during vibrio infection, and our survey further revealed stage-specific indicator species by LEfSe analysis.

3. Bcl-2 family proteins play key roles in apoptosis, which proteins can be divided into two types pro-apoptotic and anti-apoptotic proteins. Here one pro- and two anti-apoptotic genes expression pattern were investigated under vibrio infection. The pro-apoptotic gene MpBax was cloned. The full-length of MpBax cDNA sequence is 1309bp, containing a 615bp open reading frame (ORF), coding a protein of 204 amino acids, and has highest similarity with Tegillarca granosa（66%）. MpBax was expressed at the highest level in the gill, followed by the foot and hepatopancreas. The cDNA of the anti-apoptotic gene Bcl-xL contains a 558 bp open reading frame, encoding 185 amino acids, and has a similarity to the Philippine clams up to 70%. The cDNA of the anti-apoptotic gene Bcl-w contains a 698 bp open reading frame, encoding 252 amino acids, and has a similarity to the Philippine clams up to 70%. MpBax expression was down-regulated, then gradually recover to baseline, and the low point of expression appeared at 8 DPI under vibrio infection. On the contrary, the expression of MpBcl-xL and Bcl-w gene was up-regulated after stimulation. The expression pattern of these pro- and anti-apoptotic genes is consistent with its potential function.

Conclusion: In this paper, the relationship between bacteria load in clam hepatopancreas tissue and in environment seawater was investigated under the challenge of vibrio. Meanwhile, the microbial community structure and diversity in the hepatopancreas microbiota changes to vibrio challenge was studied, and the stage-specific indicator species during the infection process was discovered. Finally, the expression pattern of one pro- and two anti-apoptotic genes response to infection was understood. Taken together, our results provide references for better understand the immune mechanism of clam response to bacterial challenge, and provide useful information for the diagnosis, prediction and prevention of disease outbreak in clam aquaculture.