采用MiSeq测序技术分析3种飞虱中肠内容物的菌群结构

doi:10.3969/j.issn.1004-1524.2020.12.10

浙江农业学报 ›› 2020, Vol. 32 ›› Issue (12): 2192-2200.DOI: 10.3969/j.issn.1004-1524.2020.12.10

采用MiSeq测序技术分析3种飞虱中肠内容物的菌群结构

张珏锋¹(), 俞叶飞², 李芳¹, 钟海英¹, 陈建明¹^,^*()

1.浙江省农业科学院植物保护与微生物研究所,浙江杭州 310021
2.浙江大盘山国家级自然保护区管理局,浙江磐安 322300

收稿日期:2020-03-20 出版日期:2020-12-25 发布日期:2020-12-25
通讯作者: 陈建明
作者简介:*陈建明,E-mail: chenjm63@163.com
张珏锋(1978—),女,浙江长兴人,博士,助理研究员,研究方向为水生蔬菜有害生物防控。E-mail: zhangjuefeng@sina.com
基金资助:
浙江省公益性项目(2016C32100)

Diversities of microbial communities in midgut of 3 planthoppers species by MiSeq

ZHANG Juefeng¹(), YU Yefei², LI Fang¹, ZHONG Haiying¹, CHEN Jianming¹^,^*()

1. Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
2. Dapanshan National Nature Reserve Administration of Zhejiang, Pan’an, 322300, China;

Received:2020-03-20 Online:2020-12-25 Published:2020-12-25
Contact: CHEN Jianming

摘要/Abstract

摘要：

运用MiSeq测序技术比较了褐飞虱(Nilaparvata lugens St?l,BPH)、灰飞虱(Laodelphax striatella Fallén,SBPH)和白背飞虱(Sogatella furcifera Horvath,WBPH)体内中肠细菌群落结构的异同。结果显示:3种飞虱共获得可用于分析的有效序列3.44×10⁵条,褐飞虱、灰飞虱和白背飞虱在3%水平和6%水平分别获得76、103、61和49、68、42个操作分类单元(operational taxonomic unit,OTU)。灰飞虱体内约90%的中肠细菌属于F-proteobacteria-unclassified;Arsenophonus是褐飞虱体内的优势菌,约占总量的70%;在白背飞虱中Halomonas、Candidatus_Cardinium和Arsenophonus三种菌含量都比较高,分别占35%、20%和15%。虽然3种飞虱从共同的祖先分化,但其中肠内细菌微生物群落多样性、丰富度仍存在一定程度的差异。

关键词: 褐飞虱, 灰飞虱, 白背飞虱, Miseq, 细菌群落

Abstract:

In this study, we successfully demonstrated that MiSeq was a powerful approach for investigating the bacterial communities in midgut of brown planthopper (BPH) Nilaparvata lugens St?l, white-backed planthopper (WBPH) Sogatella furcifera Horvath and small brown planthopper(SBPH)Laodelphax striatellus(Fallen). For all three planthoppers, 3.44×10 ⁵ effective sequences were selected and utilized for the bacterial diversity and abundance analysis. In total, 76, 103, and 61 operational taxonomic units were obtained at 3% distance cutoff, 49, 68 and 42 at 6% distance cutoff in the BPH, SBPH and WBPH, respectively. Nearly 90% of the sequences in the SBPH were assigned into F-proteobacteria-unclassified. In the other two samples, Arsenophonus was the dominant genus in BPH, accounting for about about 70% sequences, while in WBPH, Halomonas, Candidatus_Cardinium and Arsenophonus were 3 dominant bacterias, accounting for about 35%, 20% and 15%, respectively. Though they were evolved from the common ancestor, the microbial community diversity, richness, dominant bacteria function of endosymbiont in three planthoppers were different.

Key words: brown planthopper (BPH), white-backed planthopper (WBPH), small brown planthopper (SBPH), MiSeq, bacterial communities

中图分类号:

S433.3

张珏锋, 俞叶飞, 李芳, 钟海英, 陈建明. 采用MiSeq测序技术分析3种飞虱中肠内容物的菌群结构[J]. 浙江农业学报, 2020, 32(12): 2192-2200.

ZHANG Juefeng, YU Yefei, LI Fang, ZHONG Haiying, CHEN Jianming. Diversities of microbial communities in midgut of 3 planthoppers species by MiSeq[J]. Acta Agriculturae Zhejiangensis, 2020, 32(12): 2192-2200.

图/表 6

表1 三种飞虱基因序列参数比较

Table 1 Comparison of parameters from gene sequences among the three planthopper species

样本名称 Sample	序列数量 Sequence number/10⁴	碱基数量 Base number/ (10⁷ bp)	平均长度 Average length/bp
BPH	3.53±0.18	1.59±0.83	449.63±0.08
SBPH	4.05±0.31	1.81±0.14	448.23±0.40
WBPH	3.87±0.39	1.72±0.17	445.66±0.20

表2 三种飞虱种群中肠细菌群落丰富度和多样性指数比较

Table 2 Comparisons of bacterial community richness and diversity indices in midguts among three planthopper species

样本 Sample	水平 Distance/%	读数 Reads	OTU	菌群丰富度 Richness		测序深度 Coverage/%	菌群多样性指数 Diversity indices
样本 Sample	水平 Distance/%	读数 Reads	OTU	Ace	Chao	测序深度 Coverage/%	Shannon	Simpson
褐飞虱BPH	3	102 952	76	78.53 (76.55,87.78)	76.50 (76.05,81.47)	99.99	1.19 (1.18,1.20)	0.49 (0.49,0.50)
灰飞虱SBPH	3	117 697	61	66.33 (62.39,81.35)	68 (62.45,94.75)	99.98	0.55 (0.54,0.56)	0.81 (0.81,0.81)
白背飞虱WBPH	3	112 189	103	103.35 (103.03,107.62)	103.00 (103,0)	99.98	2.27 (2.26,2.28)	0.19 (0.19,0.19)
褐飞虱BPH	6	104 882	49	52.67 (49.76,66.82)	50.00 (49.11,57.97)	99.99	1.12 (1.12,1.13)	0.49 (0.49,0.50)
灰飞虱SBPH	6	119 570	42	43.33 (42.18,51.90)	43.50 (42.15,57.08)	99.98	0.54 (0.53,0.55)	0.81 (0.80,0.81)
白背飞虱WBPH	6	114 492	68	68.71 (68.06,75.93)	68.00 (68.00,0)	99.98	2.21 (2.21,2.22)	0.19 (0.18,0.19)

图1 三种飞虱在3%水平的稀释性曲线图中1,2,3表示每种飞虱的生物学重复数。

Fig.1 Rarefaction curves of three samples at cutoff levels of 3 % The number 1, 2 and 3 represent biological duplications of each planthopper species.

图2 不同飞虱中肠细菌群落结构 Gammaproteobacteria-unclassified,γ-变形菌纲(未确定);Arsenophonus,杀雄菌属;Halomonas,盐单胞菌属;Acinetobacter,不动杆菌属;Candidatus_Cardinium,Caidinium属;Wolbachia,沃巴克氏体;Aeromonas,气单胞属;Serratia,沙雷氏菌;Pelagibacterium,海洋杆菌属;Aliihoeflea,化能自养砷氧化菌;Pantoea,泛菌属;Sphingobacterium,鞘氨醇杆菌属;Stenotrophomonas,寡养单胞菌;Rickettsia,立克次氏体;Herbaspirillum,草螺菌属;Phyllobacteriaceaen,叶瘤杆菌科;Massilia,马赛菌属;Pseudomonas,假单胞菌菌;Sphingobacterium,鞘氨醇杆菌属;Others,其他。

Fig.2 Bacterial community structure of midgut in three different planthoppers

图3 三种飞虱中肠内主要细菌种类的相对含量 Arsenophonus,杀雄菌属;Acinetobacter,不动杆菌属;Serratia,沙雷氏菌;Sphingobacterium,鞘氨醇杆菌属;Stenotrophomonas,寡养单胞菌;Pantoea,泛菌属;Enterobacteriaceae_Unclassified,肠杆菌科(未确定);Leucobacter,白色杆菌属;Halomonas,盐单胞菌属;Pseudoclavibacter,假棒状杆菌属;Gammaproteobacteria-unclassified,γ-变形菌纲(未确定);Wolbachia,沃巴克氏体;Rickettsia,立克次氏体;Herbaspirillum,草螺菌属;Staphylococcus,葡萄球菌属;Kocuria,考克氏菌属;Yersinia,耶尔森菌属;Corynebacterium,棒杆菌属;Candidatus_Cardinium,Caidinium属;Acinetobacter,不动杆菌属;Aeromonas,气单胞菌属;Pelagibacterium,海洋杆菌属;Aliihoeflea,化能自养砷氧化菌;Phyllobacteriaceae_Unclassified,叶杆菌科(未确定);Massilia,马赛菌属。

Fig.3 Comparison of relative contents of main bacterias in three planthopper species

图4 三种飞虱体内细菌类微生物群落结构主成分分析

Fig.4 Principal component analysis of microbial community structure in three planthoppers

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采用MiSeq测序技术分析3种飞虱中肠内容物的菌群结构

Diversities of microbial communities in midgut of 3 planthoppers species by MiSeq

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