淡色库蚊溴氰菊酯敏感品系与抗性种群的肠道菌群多样性分析

doi:10.11853/j.issn.1003.8280.2020.05.009

摘要/Abstract

摘要： 目的探讨淡色库蚊溴氰菊酯敏感品系与抗性种群成蚊肠道菌群多样性和组成结构。方法分离淡色库蚊溴氰菊酯敏感品系与抗性种群成蚊肠道并提取总基因组DNA，对敏感品系与抗性种群成蚊肠道菌群16S rDNA基因的V3＋V4区进行测序，分析其物种组成结构、菌群多样性及相对丰度；通过实时荧光定量PCR（RT-qPCR）技术验证细菌相对丰度以检测测序数据的可靠性。结果通过优化剪切得到216 022条有效序列，根据序列相似性进行聚类分析共获取766个操作分类单元，分别注释到31个门，62个纲，132个目，215个科和339个属。样本覆盖度均接近99%，表明测序量基本覆盖所有细菌物种。敏感品系的Chao1指数、香农指数和辛普森指数分别为374.53±40.27、2.56±0.59和0.36±0.11，抗性种群分别为201.58±29.34、1.14±0.13和0.55±0.07。淡色库蚊敏感品系与抗性种群肠道菌群均以变形菌门和厚壁菌门居多；在目分类水平上，敏感品系的优势菌目分别为红螺菌目、肠杆菌目及伯克霍尔德氏菌目，而抗性种群的优势菌目为红螺菌目、气单胞菌目及乳酸杆菌目；在属分类水平上，敏感品系的优势菌属为未鉴定属的醋酸杆菌科，其次是朝井杆菌属与红球菌属，而朝井杆菌属以绝对优势作为抗性种群的优势菌属，其次是未鉴定属的醋酸杆菌科与气单胞菌属。随机挑选细菌的RT-qPCR结果验证了测序数据的可靠性。结论淡色库蚊敏感品系与抗性种群肠道菌群结构和多样性有差异，提示蚊肠道菌群在杀虫剂抗性中发挥重要作用，为蚊媒的综合防制提供新的视角和线索。

关键词: 淡色库蚊, 溴氰菊酯, 肠道微生物, 多样性, 16S rDNA

Abstract: Objective To determine the gut microbial diversity and composition of deltamethrin-susceptible strains and deltamethrin-resistant populations of adult Culex pipiens pallens. Methods The guts of deltamethrin-susceptible strains and deltamethrin-resistant populations of adult Cx. pipiens pallens were isolated, and total genomic DNA was extracted. The V3+V4 region of the 16S rDNA gene from the gut microbiota of deltamethrin-susceptible strains and deltamethrin-resistant populations of adult mosquitoes was sequenced for analysis of their species composition, population diversity, and relative abundance. Quantitative real-time PCR (RT-qPCR) was used to verify the relative bacterial abundance and test the reliability of sequencing data. Results A total of 216 022 valid sequences were obtained by optimized clipping, and 766 operational taxonomic units were obtained by clustering analysis based on sequence similarity, annotated into 31 phyla, 62 classes, 132 orders, 215 families, and 339 genera. The coverage of all samples was close to 99%, which indicated that the sequencing scale was sufficient to cover almost all the bacterial species. The Chao1 index, Shannon index, and Simpson index were 374.53±40.27, 2.56±0.59, and 0.36±0.11, respectively, for deltamethrin-susceptible strains, and 201.58±29.34, 1.14±0.13, and 0.55±0.07, respectively, for deltamethrin-resistant strains. The dominant phyla of gut microbiota of deltamethrin-susceptible strains and deltamethrin-resistant populations were Proteobacteria and Firmicutes. At the taxonomic level of order, the dominant bacteria were Rhodospirillales, Enterobacterales, and Burkholderiales for deltamethrin-susceptible strains and Rhodospirillales, Aeromonadales, and Lactobacillales for deltamethrin-resistant populations. At the taxonomic level of genus, the dominant bacteria for deltamethrin-susceptible strains were an unidentified genus in Acetobacteraceae, followed by Asaia and Rhodococcus; the dominant bacteria for deltamethrin-resistant populations were Asaia (as an absolutely dominant genus), followed by an unidentified genus in Acetobacteraceae and Aeromonas. The reliability of sequencing data was verified by the RT-qPCR results of the randomly selected bacteria. Conclusion There are differences in gut microbial composition and diversity between deltamethrin-susceptible strains and deltamethrin-resistant populations of Cx. pipiens pallens. It indicates the vital role of gut microbiota of mosquitoes in the development of resistance to insecticides, which provides new perspective and clue for the comprehensive prevention and control of mosquito vectors.

Key words: Culex pipiens pallens, Deltamethrin, Gut microbiota, Diversity, 16S rDNA

中图分类号:

刘芷涵, 孙小红, 邢一帆, 周丹, 孙艳, 马磊, 沈波. 淡色库蚊溴氰菊酯敏感品系与抗性种群的肠道菌群多样性分析[J]. 中国媒介生物学及控制杂志, 2020, 31(5): 545-551.

LIU Zhi-han, SUN Xiao-hong, XING Yi-fan, ZHOU Dan, SUN Yan, MA Lei, SHEN Bo. An analysis of gut microbial diversity of deltamethrin-susceptible strains and deltamethrin-resistant populations of Culex pipiens pallens[J]. Chines Journal of Vector Biology and Control, 2020, 31(5): 545-551.

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