COMPARATIVE STUDY ON INTESTINAL MICROBIOTA OF CRAYFISH (PROCAMBARUS CLARKII) IN DIFFERENT CULTURE MODELS
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摘要:
为了探究不同养殖模式下克氏原螯虾肠道微生物结构与功能是否存在差异, 研究利用Illumina MiSeq高通量测序技术, 测定了稻虾种养(Rice-crayfish co-culture, RC)、池塘单养(Single-crayfish pond culture, SC)和池塘混养(Mixed-crayfish pond culture, MC)模式下克氏原螯虾肠道微生物16S rDNA基因V3—V4区, 解析了肠道菌群组成、结构与功能, 同时探究了肠道细菌之间及其与水体理化因子之间的关系。结果发现: 肠道微生物物种组成方面, 在门水平上, 不同养殖模式克氏原螯虾肠道细菌组成较为一致, 柔壁菌门(Tenericutes)、变形菌门(Proteobacteria)和厚壁菌门(Firmicutes)是绝对优势类群; 在属水平上, 不同养殖模式之间存在一定差异, 芽孢杆菌属(Bacillus)、乳球菌属(Lactococcus)和气单胞菌属(Aeromonas)细菌相对丰度分别在RC、SC和MC组最高。肠道微生物多样性方面, MC组肠道细菌物种丰富度和多样性最高, 三种养殖模式下克氏原螯虾肠道细菌的群落结构存在显著性差异。肠道微生物功能方面, RC组显著富集维生素生物合成代谢通路, SC组显著富集乳糖降解和半乳糖降解代谢通路, MC组显著富集脂肪酸和脂质降解、CMP-pseudaminate生物合成和脂多糖生物合成的超途径代谢通路。肠道微生物之间及其与水体理化因子之间关系方面, 肠道细菌之间的合作关系占比达83.76%, 丹毒丝菌科(Erysipelotrichaceae)和鞘氨醇单胞菌属(Sphingomonas)细菌是网络主要节点; 肠道中乳杆菌属(Lactobacillus)细菌丰度与水体中TP浓度呈显著正相关关系, 与${\rm{NH}}^+_4 $-N浓度呈显著负相关关系。综上, 克氏原螯虾肠道具有特有的以柔壁菌门、变形菌门和厚壁菌门为主的核心微生物类群, 不因养殖模式改变而变化, 但在不同养殖模式下克氏原螯虾肠道细菌群落结构与代谢功能存在显著差异, 该结果丰富了对克氏原螯虾肠道微生物的认识, 为克氏原螯虾肠道益生菌的筛选及其在生产实践中的应用提供了理论依据。
Abstract:The red swamp crayfish (Procambarus clarkii) is the most extensively farmed crustacean in China. The intestinal microbiota plays a crucial role in host physiological functions. However, it remains unclear whether there are differences in intestinal microbiota structure and function among different culture models of crayfish. In this study, we utilized Illumina MiSeq high-throughput sequencing technology to analyze the intestinal microbiota of crayfish under different cultivation models: rice-crayfish co-culture (RC), single-crayfish pond culture (SC), and mixed-crayfish pond culture (MC). We also analyzed water physicochemical factors and further explored the relationship between intestinal microbiota and physicochemical factors in the water. The results revealed that: Species composition analysis showed that at phylum level, the intestinal microbiota composition was consistent across three culture models, with Tenericutes, Proteobacteria, and Firmicutes as the absolute dominant groups. At genus level, there were some differences among different culture models, with Bacillus having the highest relative abundance in RC group, Lactococcus dominated in SC group, and Aeromonas was most abundant in MC group. Diversity analysis showed that the species richness and diversity of intestinal bacteria in group MC were the highest, and there were significant differences in the community structure of intestinal bacteria of crayfish under three culture models. Functional prediction showed that Vitamin Biosynthetic metabolic pathway was significantly enriched in RC group, while lactose degradation and galactose degradation metabolic pathways were prominent in SC group. In the MC group, pathways related to fatty acid and lipid degradation, CMP-pseudaminate biosynthesis, and superpathway of lipopolysaccharide biosynthesis metabolic pathways were significantly enriched. Network analysis showed that the cooperative relationship accounted for 83.76% of the interactions within the intestinal microbiota, with Erysipelotrichaceae and Sphingomonas bacteria were the main nodes of the network. Furthermore, the abundance of Lactobacillus in the gut was significantly positively correlated with TP concentration and negatively correlated with ${\rm{NH}}^+_4 $ concentration in the water. In conclusion, the intestinal microbiota of crayfish includes core microbial groups mainly composed of Tenericutes, Proteobacteria and Firmicutes in the gut of crayfish, which remain stable across different culture models. However, there are notable variations in both community structure and metabolic function of intestinal bacteria among the three models. These findings enhance our understanding of crayfish intestinal microbiota and provide a theoretical basis for the screening of intestinal probiotics and their application in production practice.
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Keywords:
- Intestinal microbiota /
- Culture mode /
- Physicochemical factor /
- Crayfish
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图 1 不同养殖模式克氏原螯虾肠道细菌门水平组成
每根柱子代表一个池塘/稻田, 门水平上相对丰度取每个池塘/稻田中5个重复的平均值; RC. 稻虾种养模式; SC. 池塘单养模式; MC. 池塘混养模式; 下同
Figure 1. Intestinal bacteria composition of Procambarus clarkii in different culture models at phylum level
Each column represents a pond/paddy, the relative abundance at phylum level is taken as the mean of the 5 replicates in each pond/paddy; RC. rice-crayfish co-culture; SC. single-crayfish pond culture; MC. mixed-crayfish pond culture; The same applies below
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图 2 不同养殖模式克氏原螯虾肠道细菌属水平组成
Figure 2. Intestinal bacteria composition of Procambarus clarkii in different culture models at genus level
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图 3 不同养殖模式克氏原螯虾肠道中希瓦氏菌属和气单胞菌属细菌相对丰度
不同字母表示各组之间在P<0.05水平上有显著性差异, 含有相同字母则差异不显著
Figure 3. Relative abundance of Shewanella and Aeromonas in intestinal of Procambarus clarkii in different culture models
Different letters indicate significant difference at the P<0.05 level, while the same letters show no significant difference
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图 4 不同养殖模式克氏原螯虾肠道细菌主坐标分析
Figure 4. Principal coordinate analysis of intestinal bacteria of Procambarus clarkii in different culture models
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图 5 显著差异物种LDA值柱状图(a)和分类学分支图(b)
Figure 5. Histogram of LDA values (a) and taxonomic cladistics (b) of significantly different species
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图 6 不同养殖模式克氏原螯虾肠道细菌功能预测分析
Figure 6. Predictive analysis of intestinal bacterial function in Procambarus clarkii in different culture models
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图 7 克氏原螯虾肠道细菌之间关联网络分析
节点之间, 连线为红色, 代表显著正相关; 连线为绿色, 代表显著负相关
Figure 7. Analysis of association network between intestinal bacteria in Procambarus clarkii
Between nodes, the red lines represent significant positive correlation; the green lines represent significant negative correlation
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图 8 克氏原螯虾肠道细菌与环境理化因子的相关关系分析
Figure 8. Analysis of the correlation between intestinal bacteria and environmental physicochemical factors in Procambarus clarkii
表 1 不同养殖模式克氏原螯虾肠道细菌Alpha多样性指数
Table 1 Intestinal bacterial Alpha diversity index of Procambarus clarkii in different culture models
组别
Group物种丰富度
Species richness物种多样性
Species diversityChao1指数
Chao1 indexObserved
species指数
Observed species indexShannon指数
Shannon index稻虾种养RC 571.73±247.96 505.56±234.43 3.81±1.34 池塘单养SC 627.20±288.52 560.72±282.00 3.95±1.31 池塘混养MC 698.47±244.55 619.56±230.90 4.01±1.18 注: 表中数据为15个重复的平均值, 以平均值±标准差表示; 同一列数据右上角无上标字母表示无显著性差异(P≥0.05)Note: Data in the table are means of 15 replicates, present as the mean±standard deviation; No marked letter in the upper right corner of the same column indicates no significant difference (P≥0.05) 
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