基于16S rRNA高通量测序的现场中华按蚊及其孳生地水体菌群结构研究

doi:10.11853/j.issn.1003.8280.2021.05.005

摘要/Abstract

摘要： 目的了解现场中华按蚊与孳生地水体的细菌菌群组成，分析其间的差异性。方法采集上海市嘉定现场的中华按蚊雌蚊，分为未吸血雌蚊（A）和饱血雌蚊（B）2组，以及孳生地水样（W），分别提取宏基因组DNA。应用Illumina Miseq高通量测序技术测定16S rDNA V4变异区。整理统计测得的序列数目和操作分类单元（OTUs），分析细菌菌群的组成和丰度、细菌群落的丰富度和多样性，以及菌群组成在群落间的变化率，并与其幼蚊（L）进行菌群结构的比较。结果获得优质序列数和精简后OTUs数分别为129 056和2 622.5（A）、171 734和3 324.5（B）、225 890.5和2 997（W）。精简后的OTUs隶属20个门，144个科，295个属。Chao1指数从高到低为B>W>A，辛普森指数为B>W>A，香农指数为A>B>W。分析细菌菌群丰度显示，A与B样本中优势菌均隶属变形菌门和厚壁菌门，丰度高的细菌为不动杆菌属（A，41.35%；B，25.71%）和泛菌属（A，10.58%；B，12.70%），B样本的Asaia丰度最高为40.85%。W样本中优势菌均隶属厚壁菌门、拟杆菌门和变形菌门，丰度>10.00%的为Desulfosporosinus（24.19%）、弯杆菌属（19.48%）、产黄菌属（15.27%）、假单胞菌属（12.19%）和Alkalibacter（10.28%）。各样本间细菌菌群差异的t检验结果显示，成蚊A与B样本间差异有统计学意义的门为1个，而W样本与幼蚊、成蚊之间为9个。主成分分析可见同组的样本间细菌多样性相近；热图聚类分析显示W与L聚为一支，B与A聚为另一支。结论获得了现场中华按蚊雌蚊和孳生地稻田水体的细菌菌群组成，雌蚊共生的优势菌群为不动杆菌属、泛菌属和Asaia；W样本中的细菌与其中生活的幼蚊之间、雌蚊A与B样本间的细菌菌群相似程度高。

关键词: 中华按蚊, 细菌菌群, 孳生地水样

Abstract: Objective To investigate the differences in the microbial composition in Anopheles sinensis and water in larvae breeding site. Methods An. sinensis adults were collected from Jiading district, Shanghai, China and divided into unfed (A) and fed (B) groups. Water samples were collected from paddy fields in the same area (W). Metagenomic DNA was extracted and the 16S rDNA V4 region was subjected to Illumina Miseq high-throughput sequencing. The sequence number and operational taxonomic units (OTUs) were used for statistical analysis of the composition and abundance of microflorae, the richness and diversity of bacterial communities, and changes in bacterial composition across communities, which were compared with the microbial composition in An. sinensis larvae (L). Results The numbers of high-quality sequences and clustered OTUs were 129 056/2 622.5 (A), 171 734/3 324.5 (B), and 225 890.5/2 997 (W), respectively. The clustered OTUs were assigned to 20 phyla, 144 families, and 295 genera. The bacterial richness as indicated by Chao1 index was in the order of B>W>A. The bacterial diversity was B>W>A (Simpson index) or A>B>W (Shannon index). The dominant bacteria in fed and unfed adults were Proteobacteria and Firmicutes, and the abundant genera were Acinetobacter (A, 41.35%; B, 25.71%) and Pantoea (A, 10.85%; B, 12.70%). Asaia was the most abundant (40.85%) in fed adults. In paddy field water, the dominant bacteria were Firmicutes, Bacteroides, and Proteobacteria. The genera with >10.00% abundances were Desulfosporosinus (24.19%), Flectobacillus (19.48%), Flavobacterium (15.27%), Pseudomonas (12.19%), and Alkalibacter (10.28%). As shown by the t test, the numbers of phyla with statistical differences between A, B, and L were 1 (B/A), 9 (L/A), and 9 (W/A). Principal component analysis showed similar bacterial diversity among samples in the same group, and heatmap showed that W and L clustered into one clade, while B and A into another clade. Conclusion An. sinensis adults are dominated by Acinetobacter, Pantoea, and Asaia. The composition of bacteria in paddy field water is similar to those in larvae living in the water as well as fed and unfed female mosquitos.

Key words: Anopheles sinensis, Microbial community, Water in larvae breeding site

中图分类号:

R384.1

董昊炜, 南春燕, 周秋明, 袁浩, 李翔宇, 彭恒, 马雅军. 基于16S rRNA高通量测序的现场中华按蚊及其孳生地水体菌群结构研究[J]. 中国媒介生物学及控制杂志, 2021, 32(5): 533-540.

DONG Hao-wei, NAN Chun-yan, ZHOU Qiu-ming, YUAN Hao, LI Xiang-yu, PENG Heng, MA Ya-jun. Composition of microbiota in Anopheles sinensis and water in larvae breeding site: A study based on 16S rRNA high-throughput sequencing[J]. Chinese Journal of Vector Biology and Control, 2021, 32(5): 533-540.

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