基于16S rRNA基因扩增子测序分析日本囊对虾肠道菌群结构与功能的特征

The structural and functional characteristics of the gut microbiota of Marsupenaeus japonicus as revealed by 16S rRNA gene amplicon sequencing

摘要:

【背景】肠道菌群在对虾的生理活动中起关键作用。日本囊对虾是我国海水养殖虾类中的主要品种之一，迄今为止有关其肠道菌群结构与功能的研究还鲜有报道。【目的】利用高通量测序技术探究日本囊对虾肠道菌群的组成结构与功能作用，揭示虾体肠道菌群与外源菌群结构间的相关性。【方法】60 d的养殖周期结束后，分别采集日本囊对虾肠道样品(归为虾肠组，n=3)、养殖水体样品(归为水体组，n=3)和对虾饲料样品(归为饲料组，n=3)，提取各样品总DNA进行16S rRNA基因扩增子测序，基于生物信息学方法分析与比较样品间的菌群结构特征，并使用PICRUSt软件预测日本囊对虾肠道菌群功能。【结果】3组样品测序共获得822 713条有效序列，抽平处理后可聚类为3 416个OTU。虾肠组样品中有28.49%、59.30%的OTU可以依次在水体组、饲料组样品中检测到。门水平上，虾肠组样品中的优势菌门为变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)和梭杆菌门(Fusobacteria)。水体组、饲料组与虾肠组样品中的优势菌门结构不尽相同，但均由变形菌门和拟杆菌门组成。属水平上，虾肠组样品中的优势菌属包括弧菌属(Vibrio)、另类弧菌属(Aliivibrio)、假交替单胞菌属(Pseudoalteromonas)、假黄棕杆菌属(Pseudofulvibacter)、科尔韦尔氏菌属(Colwellia)、小纺锤状菌属(Fusibacter)、发光杆菌属(Photobacterium)、脱硫弧菌属(Desulfovibrio)、嗜冷杆菌属(Psychrobacter)以及弓形杆菌属(Arcobacter)。水体组和饲料组中检出的核心菌属结构与虾肠组相比有明显差异，其中海命菌属(Marivita)和假单胞菌属(Pseudomonas)分别为养殖水体及对虾饲料样品中的最优势菌属。PICRUSt预测结果显示，日本囊对虾肠道菌群的基因功能主要与新陈代谢类功能有关，包含氨基酸代谢、碳水化合物代谢与能量代谢等。【结论】日本囊对虾肠道菌群与其他种类对虾肠道菌群的结构间存在共性，其形成在一定程度上受到了外源菌群的干预，并在虾体的日常代谢活动中发挥了一定的作用。

Abstract:

[Background] Gut microbiota plays a pivotal role in the physiological activities of prawns. The Kuruma prawn (Marsupenaeus japonicus) is one of the most important seawater prawn species for aquaculture in China. However, until now, little is known about the structure and function of gut microbiota in M. japonicus. [Objective] To explore the bacterial structure and function in the intestines of M. japonicus and to investigate the impact of external (water and feed) microbes on the structure of M. japonicus gut microbiota by using high-throughput sequencing technology. [Methods] After 60 days of culture period, the M. japonicus intestines (n=3), pond water (n=3), and prawn feed (n=3) samples were collected respectively, and then total genome DNA was extracted from each sample for 16S rRNA gene amplicon sequencing. The comparative analysis of bacterial composition of all samples was based on bioinformatics methods, and PICRUSt software was performed to predict the functional profiles of M. japonicus gut microbiota. [Results] A total of 822 713 valid reads were obtained from the sequencing data, resulting in 3 416 OTUs after data rarefying, and there were 28.49%, 59.30% of OTUs observed in prawn gut samples could also be found in water and diet samples, respectively. The predominant phyla observed in prawn gut samples were Proteobacteria, Bacteroidetes, Firmicutes, and Fusobacteria, which were different from those present in water and diet samples, expect for Proteobacteria and Bacteroidetes. The major genera identified in prawn gut samples were Vibrio, Aliivibrio, Pseudoalteromonas, Pseudofulvibacter, Colwellia, Fusibacter, Photobacterium, Desulfovibrio, Psychrobacter, and Arcobacter. Moreover, culture-associated environmental microbes were distinct from those present in prawn intestines, and included Marivita (the most abundant genus in water samples) and Pseudomonas (the most abundant genus in diet samples). PICRUSt analysis revealed that the core functional profile of gut bacterial community in M. japonicus was metabolism, including amino acid metabolism, carbohydrate metabolism, energy metabolism, and etc. [Conclusion] There existed some similarities in gut microbiota structure between M. japonicus and other pawn species. The structure of M. japonicus gut microbiota, which played a certain role in daily metabolic activities of the host, was partly influenced by the external microbes.