基于高通量测序的褐飞虱肠道微生物多样性分析

doi:10.16380/j.kcxb.2019.03.006

摘要/Abstract

摘要：

【目的】探明褐飞虱Nilaparvata lugens成虫肠道微生物群落结构和多样性。【方法】分离褐飞虱成虫完整肠道并提取总DNA，利用Illumina MiSeq(PE300)技术对其肠道细菌16S rRNA的V3-V4变异区和真菌ITS2序列进行测序，统计肠道微生物的操作分类单元(operational taxonomic unit, OTU)数量，分析其物种组成、丰度及Alpha多样性。并通过qPCR技术验证随机挑选注释到的4种肠道菌的高通量测序数据的有效性。【结果】分别获得褐飞虱成虫肠道细菌16S rRNA和真菌ITS2优质序列32 395和24 986条，根据序列相似性进行聚类分析分别获得235和128个OTUs。其中，细菌共注释到7个门, 15个纲, 26个目, 45个科和73个属；真菌共鉴定到3个门, 9个纲, 12个目, 15个科和18个属。在门分类水平上，细菌以变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes)为优势门类；真菌以子囊菌门(Ascomycota)为绝对优势菌门。在属分类水平上，细菌的优势属为不动杆菌属Acinetobacter以及紫单胞菌科(Porphyromonadaceae)未确定属和毛螺菌科(Lachnospiraceae)未确定属，其丰度分别为36.37%, 17.22%和15.01%；真菌的优势属为粪壳菌纲(Sordariomycetes)未确定属，丰度为95.77%。Alpha多样性分析结果显示，褐飞虱肠道细菌(真菌)的观测物种数、Chao1指数、Shannon指数和Simpson 指数分别为235(128), 262.64(165.40), 3.90(0.91)和0.62(0.75)。4种肠道菌的qPCR结果显示高通量测序数据具有较高的有效性。【结论】褐飞虱成虫肠道细菌和真菌群落整体多样性比较丰富。研究结果为从共生微生物角度解析褐飞虱的环境适应性以及开发基于微生物防治的新技术等方面提供了依据。

关键词: 褐飞虱, 肠道微生物, 16S rRNA, ITS2, 高通量测序, 细菌, 真菌, 物种多样性

Abstract: 【Aim】 To reveal the gut microbial community structure and diversity in adults of the brown planthopper (BPH), Nilaparvata lugens. 【Methods】 The total gut DNA was extracted from the adult BPH. The V3-V4 region of the bacterial 16S rRNA gene and the fungal ITS2 fragments were sequenced by Illumina MiSeq (PE300), and then the number of operational taxonomic units (OTUs), species composition, abundance and alpha diversity of gut microbes were analyzed. The high-throughput sequencing data of four randomly selected gut microbes annotated were verified by qPCR. 【Results】 A total of 32 395 valid tags and 235 OTUs were obtained for gut bacteria of adult BPH, while the numbers of valid tags and OTUs for gut fungi were 24 986 and 128, respectively. The total OTUs of gut bacteria were annotated into 7 phyla, 15 classes, 26 orders, 45 families and 73 genera, while the fungal OTUs were annotated into 3 phyla, 9 classes, 12 orders, 15 families and 18 genera. At the phylum levels, the dominant gut bacteria were Proteobacteria, Bacteroidetes and Firmicutes, while the dominant gut fungi with the highest abundance belonged to Ascomycota. Acinetobacter, one indeterminate genus in Porphyromonadaceae and one indeterminate genus in Lachnospiraceae were dominated at the genus level in the bacterial communities, with the abundance of 36.37%, 17.22% and 15.01%, respectively. Fungi belonging to an indeterminate genus in Sordariomycetes were the most dominant, accounting for 95.77%. The alpha diversity analysis revealed that the number of observed species, Chao1 index, Shannon index and Simpson index were 235, 262.64, 3.90 and 0.62 for gut bacteria, and 128, 165.40, 0.91 and 0.75 for gut fungi, respectively. The qPCR results of four gut microbes confirmed that the highthroughput sequencing data had a high validity. 【Conclusion】 The results indicate that the bacteria and fungi are diverse in the gut of adult BPH. The results of this study not only lay the foundation for the further studies on the environmental adaption mechanisms of BPH from a microbial standpoint, but also facilitate the studies on the development of new technology for the biocontrol of BPH.

Key words: Nilaparvata lugens, gut microbiota, 16S rRNA, ITS2, high-throughput sequencing, bacteria, fungi, species diversity

王天召, 王正亮, 朱杭锋, 王紫晔, 俞晓平. 基于高通量测序的褐飞虱肠道微生物多样性分析[J]. 昆虫学报, 2019, 62(3): 323-333.

WANG Tian-Zhao, WANG Zheng-Liang, ZHU Hang-Feng, WANG Zi-Ye, YU Xiao-Ping. Analysis of the gut microbial diversity of the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) by highthroughput sequencing[J]. Acta Entomologica Sinica, 2019, 62(3): 323-333.

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基于高通量测序的褐飞虱肠道微生物多样性分析

Analysis of the gut microbial diversity of the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) by highthroughput sequencing

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