Study on bacterial community structure in rearing water in small greenhouse of Litopenaeus vannamei
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摘要: 小型温棚养殖模式 (下称小棚模式) 是近年来凡纳滨对虾 (Litopenaeus vannamei) 养殖的热点模式。为阐明小棚模式养殖水体微生物群落的特点，探究该模式高产、高效的原因，基于16S rRNA基因的测序结果，对第2、第44、第69和第96天小棚模式养殖水体的微生物群落组成和功能进行分析。结果表明：凡纳滨对虾小棚养殖后期水体微生物群落的丰富度和多样性显著高于前期；在整个养殖过程中，门水平主要优势菌为变形菌门、拟杆菌门和放线菌门，其中变形菌门丰度在第69天显著增加，放线菌门丰度前期较高，后期减少；属水平优势菌属中Candidatus_Aquiluna的丰度在第2天最高 (28.7%)；海命菌属 (Marivita) 在中期富集，在第69天丰度最高 (9.94%)；黄杆菌属 (Flavobacterium) 的丰度随着养殖时间逐渐增加，在第96天达到最高 (11.63%)。通过PICRUS t2预测微生物群落的功能，丰度前20的代谢功能项在第69和第96天的丰度显著高于第2天，尤其萜类和聚酮类代谢、脂类代谢、异种生物降解和代谢等功能均高度富集，通过FAPROTAX鉴定得出，化能异养类菌的丰度在养殖后期显著增加。环境因子关联分析发现，总氮 (TN) 和化学需氧量 (COD) 对小棚模式水体微生物群落结构的影响最大，海命菌属、Candidatus_Aquiluna、Rhodopirellula等微生物发挥了固碳、降氮、降磷的作用。综上所述，在高密度、高氮磷的环境条件下，小棚模式的水体微生物在保持水环境稳定和增强对虾免疫与抗病能力方面发挥了重要作用。Abstract: In recent years, the small greenhouse has been a hot model of Litopenaeus vannamei culture. In order to elucidate the structure and changes of the microbial community in the rearing water of Litopenaeus vannamei culture in the small greenhouse and explore the reasons for the high yield and high efficiency of this model, we analyzed the composition and function of the microbial community on 2rd, 44th, 69th and 96th day of water in the small greenhouse based on 16S rRNA sequencing results. The results show that the richness and diversity of water microbial community at the late stage of the small greenhouse were significantly higher than those at the early stage. During the whole culture process, the dominant bacteria at the phylum level were Proteobacteria, Bacteroidetes and Actinobacteria, among which the abundance of Proteobacteria increased significantly on 69th day, and the abundance of Actinobacteria increased at the early stage but decreased at the later stage. At the genus level, the abundance of Candidatus_Aquiluna was the highest on 2rd day (28.7%). Marivita was enriched at the middle stage, with the highest abundance on 69th day (9.94%). The abundance of Flavobacterium gradually increased with the culture time, reaching the highest on 96th day (11.63%). PICRUSt2 predicts the function of the microbial communities, and the abundances of metabolic function in the top 20 were significantly higher on 69th and 96th day than on 2rd day. In particular, the functions of terpenoids and polyketides metabolism, lipid metabolism, xenobiotics biodegradation and metabolism were highly enriched. Through FAPROTAX identifying, the abundance of Chemoheterotrophy increased significantly at the late stage. The correlation analysis of environmental factors shows that total nitrogen (TN) and chemical oxygen demand (COD) had the greatest impacts on the microbial community structure in the greenhouse model. Microorganisms such as Marivita, Candidatus_Aquiluna and Rhodopirellula played the role in sequestration of carbon and reduction of nitrogen and phosphorus. In conclusion, under the high density and high nitrogen and phosphorus culture conditions, the water microbial community in the small greenhouse played an important role in maintaining the stability of water environment and enhancing the immunity and disease resistance of prawns.
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图 3 小棚模式水体微生物群落upset维恩图
注：GC1、GC2、GC3 and GC4分别代表养殖第2、第44、第69、第96天的样品，下同。
Figure 3. Upset venn diagram of microbial community in water of small greenhouse
Note: GC1, GC2, GC3 and GC4 represent the samples collected on 2nd, 44th, 69th and 96th day, respectively. The same below.
图 4 小棚模式水体中微生物群落主要微生物物种组成
Figure 4. Relative abundances of major microbial community in water of small greenhouse
图 5 小棚模式水体中微生物群落结构NMDS
Figure 5. UPGMA clustering of microbial community in water of small greenhouse
图 6 小棚模式水体中微生物分类鉴定
注：仅显示养殖水体微生物中线性判别分析 (LDA) 值高于4.0的类群；(a) 柱的长度代表细菌谱系的影响大小；(b) 从门到属的细菌群由中心向外排列；每个圆的直径与细菌分类群的丰度成正比；不同颜色节点表示在对应组别中显著富集，且对组间差异存在显著影响的微生物类群。
Figure 6. Classification and identification of microbial community in water of small greenhouse
Note: Only the taxa whose linear discriminate analysis (LDA) value above 4.0 for rearing water are shown; (a) The length of column represents the effect size of bacterial lineages; (b) The bacterial groups from phylum to genus level are listed from center to outside. Each circle's diameter is proportional to the bacterial taxon's abundance. The nodes in different colors represent the microbial groups which are significantly enriched in the corresponding groups and have a substantial impact on between-group variance.
图 7 小棚模式水体中微生物群落KEGG功能预测
注：(a) 同一行中颜色深浅表示该微生物在不同样品中的功能丰度差异。
Figure 7. KEGG function analysis of microbial community in water of small greenhouse
Note: (a) The difference in the shade of color within the same row indicates the difference in the functional abundance of the microorganism in different samples.
图 8 小棚模式水体中各环境因子的环境贡献度和Pearson分析
注：(a) 环境贡献度分析；(b) x和y轴分别为环境因子和物种，通过计算获得相关性R和P；R在图中以不同颜色展示，右侧图例是不同R的颜色区间；P则用*标出，*. 0.01<P≤0.05，**. 0.001<P≤0.01，***. P≤0.001。
Figure 8. Environmental contribution and Pearson analysis of environmental factors in water of small greenhouse
Note: (a) Environmental contribution analysis; (b) The x-axis and y-axis are environmental factors and species, respectively. The correlation R and P are obtained by calculation. R values are shown in different colors, and the legend on the right is the color range of different R values. P values are marked with *. *. 0.01<P≤0.05; **. 0.001<P≤0.01; ***. P≤0.001.
表 1 小棚模式水体微生物群落Alpha多样性指数表
Table 1. α-diversity indices of microbial community in water of small greenhouse
组别
Group香农指数
Shannon辛普森指数
Simpson丰富度指数
ChaoGC1 5.989±1.071b 0.886±0.047b 1 685.667±377.500ab GC2 6.552±0.204b 0.911±0.027ab 1 368.000±158.547b GC3 9.005±0.105a 0.990±0.001a 2 728.333±194.898a GC4 7.667±1.801ab 0.942±0.072ab 2 065.333±970.462ab 注：同一列数据上标不同字母表示组间差异显著 (P<0.05)。 Note: Different letters within the same column indicate significant difference (P<0.05). 
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