应用高通量测序技术分析拟穴青蟹肠道及其养殖环境菌群结构

采用基于Illumina Hi Seq测序平台的高通量测序技术,对拟穴青蟹(Scylla paramamosain)肠道及其养殖池塘水体、底泥中细菌种类及丰度进行了研究。测序结果显示,3个样品共获得有效序列234575条,可聚类于2812个分类操作单元(OTUs),归属于拟穴青蟹肠道、养殖水体、池塘底泥样品的操作分类单元(OTU)个数分别为453、706和2547,其中有184个OTU均能在3个样品中检测到,在青蟹肠道和养殖水体、青蟹肠道和池塘底泥中分别检测到197和309个共有OTU。物种注释结果显示,拟穴青蟹肠道中优势细菌种类为变形菌门(Proteobacteria)(39.96%)、柔膜菌门(Tenericutes)(23.09%)和厚壁菌门(Firmicutes)(16.58%);养殖水体中优势细菌种类为变形菌门(63.02%)、放线菌门(Actinobacteria)(24.96%)和拟杆菌门(Bacteroidetes)(8.41%);池塘底泥中优势细菌种类为变形菌门(75.23%)、拟杆菌门(5.72%)和放线菌门(3.83%)。此外,对各样品中丰度最高的前10位OTU分析显示,不同样品中占优势地位的10种细菌在数据库(SILVA)缺乏相关已知序列,并且各样品中的优势细菌种类完全不同。实验结果表明拟穴青蟹肠道与其池塘养殖环境中菌群结构存在着密切的相关性,但肠道菌群同时具有一定的独立性,其优势细菌种类与养殖环境中优势细菌种类无关。本研究旨在为拟穴青蟹健康养殖和微生态调控提供实验依据。

Application of high-throughput sequencing techniques for analyzing bacterial communities in pond-raised mud crab (Scylla paramamosain) intestine and its aquaculture environment

Mud crab (Scylla paramamosain), widely cultured in brackish and seawater ponds along the coast of southern China, has become the most productive seawater crab species because of its strong adaptive abilities and high economic value. Bacteria play an important role in pond aquaculture ecosystems; however, little is known about the bacterial composition and diversity in earthen aquaculture pond microbial communities of S. parama-mosain. Accordingly, we applied Illumina HiSeq high-throughput sequencing to investigate bacterial community structure and diversity in pond-raised S. paramamosain intestinal and its aquacultural environment. The results showed that 234575 effective sequences were detected in three samples, and they could be classified into 2812 operational taxonomic units (OTUs), among which 453, 706, and 2547 OTUs belong to the gut of S. paramamo-sain, the aquaculture water, and the sediment, respectively. 184 OTUs were shared among all samples, 197 OTUs between S. paramamosain intestine and aquaculture water, 309 OTUs between S. paramamosain intestine and sediment, and 572 OTUs between the aquaculture water and sediment. This indicates that bacterial richness and diversity were lower in S. paramamosain intestine than in water and sediment. In total, 48 phyla were identified, among them Proteobacteria (39.96%), Tenericutes (23.09%), Firmicutes (16.58%), CKC4 (6.55%), and Bacter-oidetes (4.96%), which constituted the predominant components of the S. paramamosain intestine; Proteobacteria (63.02%), Actinobacteria (24.96%), Bacteroidetes (8.41%), Cyanobacteria (1.19%), and Firmicutes (0.83%) were the predominant phyla in the water; and the predominant phyla in the sediment were Proteobacteria (75.23%), Bacteroidetes (5.72%), Actinobacteria (3.83%), Chloroflex I (2.65%), and Acidobacteria (1.80%). Analysis of the 10 most abundant bacterial OTUs in different samples revealed that the most dominant bacteria in S. paramamo-sain intestine and its aquacultural environment were totally different, and there were only four kinds of dominant bacteria that have close sequences in the SILVA database. These results suggest that there exists a close correlation between bacterial species composition in the S. paramamosain intestine and its aquacultural environment. Mean-while, intestinal microflora may be a relatively independent microbial ecosystem and the micro-ecosystem could hardly be influenced by the bacteria in the culture environment.