基于宏基因组学分析海南省不同养殖场库蠓共生微生物群落组成

doi:10.11853/j.issn.1003.8280.2023.04.006

摘要/Abstract

摘要：

目的: 探究海南省不同养殖场库蠓共生微生物群落多样性和差异性。方法: 2021年8－9月，在海南省文昌市、定安和乐东县5个不同的养殖场（牛圈、鸡舍、犬场、羊圈和猪圈），用紫外灯诱法采集库蠓，分别使用十六烷基三甲基溴化铵法提取总DNA后，通过宏基因组学测序技术分析其微生物群落结构特征及基因功能分布。结果: 不同养殖场库蠓优势种不同，牛圈、羊圈和猪圈主要是尖喙库蠓，占比分别为92.91%，82.22%和95.85%；鸡舍和犬场均以荒川库蠓为主要优势种，占比分别为66.36%和98.22%。不同养殖场库蠓共生微生物在门水平上，均以变形菌门（33.78%）、子囊菌门（9.09%）和厚壁菌门（8.88%）为主要菌门；在属水平上，均以不动杆菌属（22.48%）和梭菌属（7.25%）为优势菌属。属水平微生物多样性分析发现5个养殖场共有的属最多，有3 357属。微生物基因差异分析显示5个不同的养殖场共有基因数目较少，为1 065个。此外，功能预测结果也显示，29.10%注释基因与新陈代谢相关，如碳水化合物代谢、氨基酸代谢和脂质代谢等。基因数目差异分析结果表明，库蠓种类组成越相似，样品共有基因数目越多。结论: 微生物群落分析发现，海南省不同养殖场库蠓共生微生物的核心菌群相对稳定，库蠓优势种相同的养殖场微生物群落相似度更高。该研究丰富了我国库蠓的微生物群落数据库，对今后库蠓的生物防控有重要意义。

关键词: 库蠓, 共生微生物, 宏基因组学

Abstract:

Objective: To explore the diversity and variability of symbiotic microbial communities in Culicoides on different livestock farms in Hainan Province, China. Methods: From August to September 2021, the ultraviolet lamp-trap method was used to collect Culicoides on five different livestock farms (cattle farms, chicken houses, dog houses, sheep pens, and pigsties) in Wenchang City, Ding'an County, and Ledong County in Hainan Province. After the total DNA was extracted by hexadecyltrimethylammonium bromide method, the structural characteristics and functional distribution of genes in the microbial communities were analyzed using metagenomic sequencing. Results: The dominant species of Culicoides on different livestock farms varied. The dominant species on cattle farms, sheep pens, and pigsties was C. oxystoma, accounting for 92.91%, 82.22%, and 95.85%, respectively; the dominant species in chicken houses and dog houses were C. arakawae, accounting for 66.36% and 98.22%, respectively. On different livestock farms, Proteobacteria (33.78%), Ascomycota (9.09%), and Firmicutes (8.88%) were the dominant phyla of the symbiotic microbial communities in Culicoides, and Acinetobacter (22.48%) and Clostridium (7.25%) were the dominant genera. Microbial diversity analysis at the genus level found that the five livestock farms shared the most microorganisms at the genus level, with 3 357 genera in common. Microbial gene differential analysis revealed that the five livestock farms shared a relatively small number of common genes (1 065). Moreover, the functional prediction results also showed that 29.10% of the annotated genes were related to metabolism, such as carbohydrate metabolism, amino acid metabolism, and lipid metabolism. The analysis of the differences in gene number showed that increased similarity in the species composition of Culicoides was associated with a greater number of genes shared by the samples. Conclusions: Analysis of the microbial communities found that the core symbiotic microflora communities in Culicoides is relatively stable, and livestock farms with the same dominant species of Culicoides have more similarities in microbial communities in Culicoides. This study enriches the microbial community database of Culicoides in China, which is of great significance for the biological prevention and control of Culicoides in the future.

Key words: Culicoides, Symbiotic microorganism, Metagenomics

中图分类号:

R384.5

樊洁丽, 刘焱晖, 殷雅楠, 赵建国, 孙定炜, 廖承红, 韩谦. 基于宏基因组学分析海南省不同养殖场库蠓共生微生物群落组成[J]. 中国媒介生物学及控制杂志, 2023, 34(4): 472-479.

Jie-li FAN, Yan-hui LIU, Ya-nan YIN, Jian-guo ZHAO, Ding-wei SUN, Cheng-hong LIAO, Qian HAN. A metagenomic analysis of the composition of symbiotic microbial communities in Culicoides on different livestock farms in Hainan Province[J]. Chinese Journal of Vector Biology and Control, 2023, 34(4): 472-479.

图/表 9

表 1 2021年海南省3个市（县）5个不同类型养殖场库蠓样品采样信息

Table 1 Sampling information of Culicoides on 5 different livestock farms in 3 cities (counties) in Hainan Province, China, 2021

表 2 2021年海南省3个市（县）5个不同养殖场库蠓的宏基因组测序数据

Table 2 Metagenomic sequencing data of Culicoides on 5 different livestock farms in 3 cities (counties) in Hainan Province, 2021

图 1 2021年海南省3个市（县）5个不同养殖场库蠓门水平上的微生物群落结构注：P1~P5分别代表牛圈、鸡舍、犬场、羊圈和猪圈样本；不同颜色代表门水平不同的物种，堆叠图展示优势门样本占比变化趋势。

Fig. 1 Microbial community structures of Culicoides at the phylum level on 5 different livestock farms in 3 cities (counties) in Hainan Province, 2021

图 2 2021年海南省3个市（县）5个不同养殖场库蠓属水平上的微生物群落结构注：P1~P5分别代表牛圈、鸡舍、犬场、羊圈和猪圈样本；图形最外环左半圆颜色表示各属，右半圆颜色表示各样本；内圈坐标刻度表示对应颜色属的占比（%）；内环的弦由属发出，指向样本；弦粗细表示样本对应属的数值高低。

Fig. 2 Microbial community structures of Culicoides at the genus level on 5 different livestock farms in 3 cities (counties) in Hainan Province, 2021

图 3 2021年海南省3个市（县）5个不同养殖场库蠓基于属水平的微生物多样性分析注：P1~P5分别代表牛圈、鸡舍、犬场、羊圈和猪圈样本；左侧水平柱子表示各样本拥有的属数量，图形下方矩阵中的单个点表示统计某个样本特有的元素，点和点之间的连线表示统计相连样本的交集，右上方竖直柱子表示统计的特有或交集的属数量。

Fig. 3 Microbial diversity analysis of Culicoides at the genus level on 5 different livestock farms in 3 cities (counties) in Hainan Province, 2021

图 4 2021年海南省3个市（县）5个不同养殖场库蠓共生微生物基于属水平的主坐标分析注：P1~P5分别代表牛圈、鸡舍、犬场、羊圈和猪圈样本；基于相异矩阵系数距离进行分析，以样本在二维平面的距离关系反映样本间菌群结构的相似性，样本距离越近，说明样本间属水平物种组成越相似；轴标签括号中的百分比表示各坐标轴占菌群结构总变异的解释度，平面两个轴的解释度越高，说明模型效果越好。

Fig. 4 Principal coordinate analysis of the symbiotic microorganism in Culicoides at the genus level on 5 different livestock farms in 3 cities (counties) in Hainan Province, 2021

图 5 2021年海南省3个市（县）5个不同养殖场库蠓基因差异分析注：P1~P5分别代表牛圈、鸡舍、犬场、羊圈和猪圈样本；A库蠓基于基因数目的韦恩图，花瓣内数字代表不同样本共有或独有的基因数目；B库蠓基于基因数目的样品相关性热图，颜色表示样品的相关性，颜色越深，两样品基因相关性越强。

Fig. 5 Gene differential analysis of Culicoides on 5 different livestock farms in 3 cities (counties) in Hainan Province, 2021

图 6 2021年海南省3个市（县）5个不同养殖场库蠓功能基因注释热图注：P1~P5分别代表牛圈、鸡舍、犬场、羊圈和猪圈样本；横坐标为各样本，纵坐标为6个A级代谢通路的相对丰度；颜色深浅代表相对丰度大小。

Fig. 6 Heatmap of functional gene annotation of Culicoides on 5 different livestock farms in 3 cities (counties) in Hainan Province, 2021

图 7 2021年海南省3个市（县）5个不同养殖场库蠓体内共生微生物代谢通路注释基因数目统计注：P1~P5分别代表牛圈、鸡舍、犬场、羊圈和猪圈样本；纵坐标表示B级水平代谢通路。

Fig. 7 Statistics of the number of annotated genes by metabolic pathways of the symbiotic microorganism in Culicoides on 5 different livestock farms in 3 cities (counties) in Hainan Province, 2021

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