实验海洋生物学重点实验室知识库

文蛤（Meretrix petechialis）是我国沿海地区一种重要的经济贝类。文蛤体内存在着丰富的共生菌群，这些内生细菌群落的组成和变化对宿主抵御病原体入侵的能力以及机体的抗逆性都具有影响。本研究通过纯培养方式对文蛤鳃组织内共生细菌进行了分离、筛选与鉴定，在此基础上对内共生细菌的种类和数量进行了统计和分析。通过16S rRNA扩增子测序方法，对处于不同健康状态的文蛤鳃及肝胰腺组织的细菌群落结构组成进行了分析，发现文蛤的细菌种群丰度在不同的健康状态下及不同的组织中均有变化，同时发现了不同健康状态下文蛤肝胰腺组织中具有显著性差异的细菌类群。此外，利用前期筛选到的潜在致病菌弓形杆菌MP43对文蛤进行了攻毒实验，评估了对文蛤可能的致病能力及其对免疫的影响。主要结果如下：

1、通过采用纯培养方式，对文蛤鳃组织内共生细菌进行了多批次的分离与筛选。对得到的纯培养细菌进行了16S rRNA基因序列测序及比对分析，共得到了变形菌门（Proteobacteria）、厚壁菌门（Firmicutes）、拟杆菌门（Bacteroidetes）和放线菌门（Actinobacteria）等6个纲、13个目、23个科、47个属的215株细菌。分离细菌较多的属级类群有交替单胞菌属（Alternomonas）、Nautella属、葡萄球菌属（Staphylococcus）和假单胞菌属（Pseudomonas），分别分离到20株、18株、15株以及13株细菌。该研究统计了纯培养方式鉴定得到的文蛤鳃组织中内共生微生物的种类及数量，为后续对文蛤内共生菌群的深入探究提供了参考。

2、采用16S rRNA扩增子测序，对处于不同健康状态的文蛤鳃及肝胰腺组织的细菌群落结构进行了分析。对得到的OTU数据进行聚类分析和物种注释，结果显示文蛤体内的细菌组成在不同的健康状态以及不同的组织中均有差异。健康状态的文蛤鳃及肝胰腺组织中，变形菌门均为丰度最高的类群，肝胰腺中放线菌门、厚壁菌门和拟杆菌门的丰度明显高于鳃组织；在属级单元，文蛤鳃组织中细菌丰度最高的类群为弓形杆菌属（Arcobacter），而肝胰腺中丰度最高的属级类群为红球菌属（Rhodococcus）。发病状态的文蛤鳃组织中的细菌种类在门级单元上远少于肝胰腺组织，在属级单元上鳃组织中Endozoicomonas属占据着极高的丰度，而肝胰腺中丰度最高的类群为不动杆菌属（Acinetobacter）。对两种不同健康状态下的文蛤进行比较，肝胰腺中细菌组成的Alpha多样性和Beta多样性均无显著性差异，但进一步分析发现两组样品中有4个具有显著性差异的细菌类群，可作为文蛤健康状态指示的微生物参考指标。

3、不同温度条件下利用不同浓度的MP43对文蛤的人工感染实验结果表明，致死率与对照组之间均无显著差异，显示该菌株对文蛤不具有直接致病性；通过对攻毒后文蛤肝胰腺组织进行免疫相关基因的表达分析，显示文蛤的免疫系统对于MP43菌株未产生明显响应，肝胰腺弧菌载菌量也未呈现显著变化。研究表明弓形杆菌MP43菌株对文蛤不具有直接致病性，不是引起文蛤爆发性死亡的致病病原。

本研究通过纯培养方法和16S rRNA扩增子测序对文蛤的内共生细菌群落的组成结构进行了初步探究，并对一株文蛤潜在病原菌弓形杆菌的致病性进行了评估。上述研究结果，为文蛤内共生菌群的研究提供了参考，发现了潜在的文蛤健康状态的微生物参考指标，并为贝类致病菌的筛选及鉴定提供了思路。

The clam Meretrix petechialis is one of the important commercial molluscs in the coastal areas of China. There are abundant endosymbiotic bacteria in clams, and the composition and changes of these endosymbiotic bacterial communities have certain effects on the host ability to against pathogens colonization and the resistance to stress. In this study, endosymbiont bacteria in the gill tissue of M. petechialis were isolated, purified and identified by pure culture method, and the species and quantity of these endosymbiont bacteria were counted and analyzed. 16S rRNA amplificon sequencing was performed to analyze the bacterial community structure in the gills and hepatopancreas of M. petechialis under different health states. Our results found the bacterial species abundance variations in different health states and in different tissues, and we also found 4 specific bacterial groups in the clams under different health states. In addition, the earlier screened Arcobacter MP43, a potential pathogen, was used to challenge M. petechialis and to evaluate its potential pathogenic ability and effect on immunity to this clam. The main results of this study are as follows:

1. Pure culture method was used to isolate and purify the endosymbiont bacteria in the gill of M. petechialis. The 16S rRNA genes of all of the obtained strains were sequenced to determine their classification. The total 215 strains were classified into four phyla: Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria. The major genera were Alternomonas, Nautella, Staphylococcus and Pseudomonas, which were isolated up to 20, 18, 15 and 13 strains respectively. The species and abundance of endosymbiont microorganisms in the gill can provide references for the further exploration of endosymbionts in this clam.

2. The bacterial community structures in the gill and hepatopancreas of M. petechialis under different healthy states were analyzed by 16S rRNA amplicon sequencing. The results of cluster analysis and species annotation of the obtained OTU data showed the different bacterial composition for this clam in different health states and in different tissues. Proteobacteria is the most abundant phylum in 4 sample groups. In healthy clams, the abundance of Actinobacteria, Firmicutes and Bacteroidetes were higher in hepatopancreas than their abundance in gill. Arcobacter is the genus group with the highest bacterial abundance in gill tissues, while Rhodococcus is the genus group with the highest bacterial abundance in hepatopancreas. In the diseased clams, the species of bacteria in the gill tissues were far less than that in the hepatopancreas at the genus level, and the genus Endozoicomonas occupied an extremely high abundance in the gill tissues, while the genus group with the highest abundance in the hepatopancreas was Acinetobacter. There was no significant difference in the bacterial Alpha diversity and Beta diversity in the hepatopancreas between healthy and diseased clams, but 4 significantly specific bacterial groups were found between these two groups, which can be used as reference indicators for clam health.

3. Through the artificial infection experiment of Arcobacter MP43 in clams at different temperature and different bacterial concentration, there was no significant difference in the mortality between the control group and the experimental group, indicating that strain MP43 was not directly pathogenic to clams. At the same time, the expression of immune-related genes in the hepatopancreas showed the clams had no obvious immune response and the vibrio load of hepatopancreas had no significant change after MP43 challenge. The results showed that the Arcobacter MP43 strain was not directly pathogenic to M. petechialis, and could not cause the mass mortality of this clam.

In this study, the structure of the endosymbiont bacterial community was studied by pure culture method and 16S rRNA amplification sequencing, and the pathogenicity of a potential pathogen was evaluated. The above research results provide reference for the study of endosymbiosis microflora of clams, discover the potential microbial reference of the healthy state of clams, and provide ideas for the screening and identification of bivalve mollusc pathogenic bacteria.