基于宏基因组测序技术揭示大泷六线鱼肠道微生物特征

Metagenomic sequencing reveals the intestinal microbial characteristics of Hexagrammos otakii

摘要:

对大泷六线鱼不同生长期肠道微生物进行分析。【目的】基于宏基因组学技术揭示其肠道微生物变化特征及其与营养的联系。【方法】对大泷六线鱼仔鱼、稚鱼和幼鱼肠道微生物样本进行HiSeq高通量测序，分析菌群结构，比较微生物群落的多样性，探究生长过程中肠道微生物的相互演替及功能关系。【结果】门水平上，鱼肠道微生物在生长过程中优势菌门变形菌门(Proteobacteria)递减，而厚壁菌门(Firmcutes)递增；属水平上，仔鱼期的弧菌属(Vibrio)(37.8%)、稚鱼期的发光杆菌属(Photobacterium)(77.8%)、幼鱼期的乳酸菌属(Lactococcus)(42.5%)占优势，微生物群落组成发生显著变化；且仔鱼期的多样性高，幼鱼期的丰富度高；物种差异也呈现着与生长特征的相关性，幼鱼期差异标志物是厚壁菌门、芽孢杆菌纲(Bacilli)、乳酸杆菌目(Lactobacillales)、链球菌科(Streptococcaceae)、乳球菌属(Lactococcus)和乳酸乳球菌(Lactococcus lactis)；稚鱼期差异标志物是发光菌属、托鲁尼发光菌(Photobacterium toruni)；仔鱼期差异标志物是Phaeobacter inhibens、Colwellia aestuarii和Colwellia polaris等。宏基因组功能水平分析显示，KEGG数据统计代谢功能占优势，随生长呈现递增趋势，鱼肠道微生物群在碳水化合物代谢、氨基酸代谢、核苷酸代谢、能量代谢，以及辅助因子和维生素代谢中起着重要作用；从蛋白注释功能上也呈现了类似的结果，碳水化合物代谢(3350)和氨基酸代谢(2424)占代谢通路主要组成部分。功能差异分析表明，随着生长变化，微生物功能逐渐适应机体和环境需求，稚鱼期差异主要体现在能量代谢和糖降解功能；仔鱼期差异显著的是细胞的生长、死亡和凋亡功能，主要体现在光合作用方面；幼鱼期主要体现是二级代谢物的生物合成，其次是糖酵解和糖异生等碳水化合物代谢等。【结论】大泷六线鱼仔、稚、幼不同生长期肠道微生物结构存在显著差异，生长发育改变肠道微生物菌群的功能，采用差异物种和差异功能综合判定生长所需的营养，有助于提高养殖效益，为绿色健康生态养殖提供理论基础。

Abstract:

The intestinal microorganisms in Hexagrammos otakii at different growth stages are analyzed. [Objective] We investigated the changes of intestinal microorganisms in H. otakii and the relationship with the nutrition via metagenomics. [Methods] HiSeq sequencing of the intestinal microbial samples from larvae, juveniles, and young fishes was carried out to analyze the changes in community composition, compare the diversity of microbial community, and explore the interactions between microorganisms and functional relationship in the growth. [Results] The abundance of the dominant Proteobacteria in the intestine of fish decreased, while that of the dominant Firmcutes increased. The dominant genus was Vibrio at larval stage (37.8%), Photobacterium at juvenile stage (77.8%), and Lactococcus at young fish stage (42.5%), suggesting the significant variation of community composition in the growth period. Moreover, the microbial diversity was high at the larval stage and the richness was high at the juvenile stage. Species difference was correlated with the growth. Specifically, at the young fish stage, the differential taxa were Firmcutes, Bacilli, Streptococcaceae, Lactobacillales, Lactococcus and Lactococcus lactis. As for the juvenile stage, the biomarkers were Photobacterium and Photobacterium toroni. At the larval stage, the biomarkers were Phaeobacter inhibens, Colwellia aestuarii, C. polaris, etc. The microbes were mainly involved in the Kyoto encyclopedia of genes and genomes (KEGG) pathway of metabolism and the abundance of this function rose with the growth. To be specific, the intestinal microbiota played an important role in carbohydrate metabolism, amino acid metabolism, nucleoside acid metabolism, energy metabolism, co-factor and vitamin metabolism. Similar results were also presented in terms of protein annotation, which showed that carbohydrate metabolism (3 350) and amino acid metabolism (2 424) dominated the metabolic pathways. The analysis of functional differences demonstrated that microbial functions gradually adapted to the needs of the fish body and environment during the growth. The differential functions were energy metabolism and glucose degradation at the juvenile stage, and the cell growth, death, and apoptosis at the larval stage (mainly in photosynthesis). As for the young fish stage, the major differential function was the biosynthesis of secondary metabolites, followed by carbohydrate metabolism such as glycolysis and gluconeogenesis. [Conclusion] The intestinal microbial structure of H. otakii was significantly different among the larvae, juveniles and young fishes. The functions of the intestinal microbiota changed with the growth and development of the fish. The nutrients for the growth of the fish can be determined based on the differential species and differential functions. This study is expected to improve the breeding efficiency of this fish and lay a theoretical basis for ecological breeding.