Comparison of the pyloric caecum microbiota community structure between the wild and farmed Larimichthys crocea in Sansha Bay
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摘要: 大黄鱼（Larimichthys crocea）幽门盲囊呈半封闭结构，保留了部分早期孵化阶段的微生物特征，是用于野生和养殖群体溯源的理想部位。本研究采用高通量测序技术，对三沙湾大黄鱼野生和养殖群体的幽门盲囊微生物的α多样性、核心菌群相对丰度、网络关系等进行分析，构建随机森林模型进行群体溯源分析。结果显示，养殖群体的幽门盲囊微生物有着更多特有操作分类单元（Operational Taxonomic Unit, OTU），其α多样性显著高于野生群体。幽门盲囊微生物的优势菌群有变形菌门（Proteobacteria）、厚壁菌门（Firmicutes）、拟杆菌门（Bacteroidota）、放线菌门（Actinobacteria）及酸杆菌门（Acidobacteria），且各菌群在野生和养殖群体中的相对丰度差异显著。微生物网络分析结果显示，野生和养殖群体的幽门盲囊微生物群落结构差异明显，野生群体拥有更高的负边缘/正边缘比率和模块性以及更少的节点数和连接数。基于此，我们构建了随机森林分类群体溯源预测模型，其准确率（Accuracy, ACC）达92.31%，Kappa系数为0.845 2，ROC曲线下面积为0.952 4，利用此模型识别三沙湾大黄鱼野生和养殖群体的准确率分别可达91.67%和92.86%。综上所述，三沙湾大黄鱼野生和养殖群体的幽门盲囊微生物群落结构差异显著，利用幽门盲囊微生物追踪鱼类来源的方法是可行且有效的，研究结果为区分三沙湾大黄鱼野生和养殖群体提供了新思路。Abstract: The semienclosed pyloric caecum of Larimichthys crocea is an ideal organ to perform the host source tracking, as it containes several local bacteria colonized during early development. The alpha diversity, relative abundance of core bacteria and network relationship of the pyloric caecum microbiota in L. crocea from Sansha Bay were analyzed using the Illumina high-throughput sequencing. Furthermore, the random forest model was used to predict the population source (the wild population or the farmed population). The results showed that the farmed fish had more unique OUT and higher alpha diversity than the wild one. The wild and farmed fish significantly differed in the relative abundance of dominant bacteria (Proteobacteria, Firmicutes, Bacteroidota, Actinobacteria and Acidobacteria) (p < 0.05). The result of network analysis showed that the wild fish had higher ratio of negative to positive edges and modularity, but fewer nodes and edges than the farmed fish. Furthermore, a random forest classification prediction model with the accuracy of 92.31%, the Kappa coefficient of 0.8452, and an area under the ROC curve of 0.952 4 was constructed. Using this prediction model, the accuracy of source identification for the wild and farmed fish was 91.67% and 92.86%, respectively. Overall, the structure of microbial communities in the pyloric caecum of L. crocea was different between the wild and farmed populations, making it a potential marker for host source tracking. Our findings provide new insights into distinguishing wild and farmed L. crocea.
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图 1 三沙湾大黄鱼野生群体（YS）和养殖群体（YZ）幽门盲囊微生物的Venn图
不同颜色代表不同组样本，图中数字表示OTU数量
Fig. 1 Venn diagram of the pyloric caecum microbiota of the wild population (YS) and farmed population (YZ) of Larimichthys crocea in Sansha Bay
Different colors indicate different groups, and the numbers within cycles indicate the number of OTU
图 2 三沙湾大黄鱼野生群体（YS）和养殖群体（YZ）幽门盲囊微生物的Alpha多样性分析
Fig. 2 Alpha diversity of the pyloric caecum microbiota of the wild population (YS) and farmed population (YZ) of Larimichthys crocea in Sansha Bay
图 3 三沙湾大黄鱼野生群体（YS）和养殖群体（YZ）幽门盲囊微生物的PCoA分析
图中每个点代表一个样本，点的颜色代表样本的分组
Fig. 3 PCoA analysis of the pyloric caecum microbiota of the wild population (YS) and farmed population (YZ) of Larimichthys crocea in Sansha Bay
Each point represents a sample, and different colors indicate different groups
图 4 三沙湾大黄鱼野生群体（YS）和养殖群体（YZ）幽门盲囊微生物在门水平的相对丰度分布
*代表显著性差异（p < 0.05）
Fig. 4 Relative abundance of the dominant bacterial phyla of the pyloric caecum microbiota in the wild population (YS) and farmed population (YZ) of Larimichthys crocea in Sansha Bay
* Indicates significant difference (p < 0.05)
图 5 三沙湾大黄鱼野生群体（YS）和养殖群体（YZ）幽门盲囊微生物在属水平的相对丰度分布
*代表显著性差异（p < 0.05）
Fig. 5 Relative abundance of dominant bacterial genera in the wild (YS) and farmed (YZ) of Larimichthys crocea in Sansha Bay
* Indicates significant difference (p < 0.05)
图 6 三沙湾大黄鱼野生群体（YS）和养殖群体（YZ）幽门盲囊微生物的LEfSe分析
Fig. 6 LEfSe analysis of the pyloric caecum microbiota of the wild population (YS) and farmed population (YZ) of Larimichthys crocea in Sansha Bay
图 7 三沙湾大黄鱼野生群体（YS）和养殖群体（YZ）幽门盲囊微生物的网络关系
图中点的不同颜色代表不同门，点的大小表示连接到该节点边缘数的多少；各点之间相互连线的粗细表示相关性系数绝对值的大小（p < 0.05, r > 0.6）
Fig. 7 The co-occurrence networks of the pyloric caecum microbiota of the wild population (YS) and farmed population (YZ) of Larimichthys crocea in Sansha Bay
Different colors of nodes indicate different bacterial phyla, and the size of nodes indicates the number of connected edges; the thickness of the connecting line between each point indicates the absolute value of correlation coefficient (p < 0.05, r > 0.6)
图 8 随机森林规模与误差率的关系
图中黑线、红线和绿线分别代表所有个体、养殖群体和野生群体的误差率
Fig. 8 The relationship between random forest size and error rate
Black line, red line and green line indicate the error rate of all individuals, farmed population and wild population, respectively
表 1 三沙湾大黄鱼野生群体（YS）和养殖群体（YZ）幽门盲囊微生物的网络图特征参数
Tab. 1 Characteristic parameters of network diagram of the pyloric caecum microbiota of the wild population (YS) and farmed population (YZ) of Larimichthys crocea in Sansha Bay
网络图特征参数 YS YZ 节点数 163 306 连接数 1 561 12 309 正连接数 1 525 12 125 负连接数 36 184 负/正连接数比 0.023 0.015 平均度 5.423 52.118 平均聚集系数 0.461 0.631 模块性 0.695 0.292 
下载: 导出CSV
表 2 随机森林模型参数
Tab. 2 The parameters of the random forest model
模型参数 数值 准确率 92.31% Kappa系数 0.8452 敏感性 92.86% 特异性 91.67% 正预测值 92.86% 负预测值 91.67% 平衡精度 92.26% ROC 曲线下面积 0.952 4
下载: 导出CSV
表 3 测试集中随机森林预测模型分类结果
Tab. 3 Classification results of the test set using the random forest prediction model
预测分类 实际分类 准确率/% YS YZ YS 11 1 91.67 YZ 1 13 92.86
下载: 导出CSV
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