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。
【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 16 S 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.
引文
Blaalid R, Kumar S, Nilsson RH, Abarenkov K, Kirk PM, Kauserud H, 2013. ITS1 versus ITS2 as DNA metabarcodes for fungi. Mol. Ecol. Resour., 13(2): 218-224.
Boman HG, Hultmark D, 1987. Cell-free immunity in insects. Annu. Rev. Microbiol., 41: 103-126.
Briones-Roblero CI, Rodríguez-Díaz R, Santiago-Cruz JA, Zúňiga G, Rivera-Orduňa FN, 2017. Degradation capacities of bacteria and yeasts isolated from the gut of Dendroctonus rhizophagus (Curculionidae: Scolytinae). Folia Microbiol., 62(1): 1-9.
Cao W, Ma Z, Yu XP, 2015. Isolation and sensitivity to fungicides of the yeast-like symbiont Pichia anomala (Hemiascomycetes: Saccharomycetaceae) from Laodelphax striatellus (Hemiptera: Delphacidae). Acta Entomol. Sin., 58(3): 271-280. [曹伟, 马正, 俞晓平, 2015. 灰飞虱体内类酵母共生菌异常毕赤酵母的分离培养及其对杀菌剂的敏感性. 昆虫学报, 58(3): 271-280]
Chen JZ, Tu X, Cheng F, Chen JF, Deng ZS, 2014. Isolation, identification and antibacterial activity of intestinal symbiotic fungi from Acrida cinerea. J. China Three Gorges Univ. (Nat. Sci.), 36(3): 108-112. [陈君芝, 涂璇, 程凡, 陈剑锋, 邓张双, 2014. 中华剑角蝗肠道共生真菌的分离鉴定及抑菌活性筛选. 三峡大学学报(自然科学版), 36(3): 108-112]
Dong SZ, Pang K, Bai X, Yu XP, Hao PY, 2011. Identification of two species of yeast-like symbiotes in the brown planthopper, Nilaparvata lugens. Curr. Microbiol., 62(4): 1133-1138.
Douglas AE, 2015. Multiorganismal insects: diversity and function of resident microorganisms. Annu. Rev. Entomol., 60: 17-34.
Engel P, Moran NA, 2013. The gut microbiota of insects-diversity in structure and function. FEMS Microbiol. Rev., 37(5): 699-735.
Frago E, Dicke M, Godfray HC, 2012. Insect symbionts as hidden players in insect-plant interactions. Trends Ecol. Evol., 27(12): 705-711.
Hammer TJ, Bowers MD, 2015. Gut microbes may facilitate insect herbivory of chemically defended plants. Oecologia, 179(1): 1-14.
Hou Y, Ma Z, Dong S, Chen YH, Yu X, 2013. Analysis of yeast-like symbiote diversity in the brown planthopper (BPH), Nilaparvata lugens St?l, using a novel nested PCR-DGGE protocol. Curr. Microbiol., 67(3): 263-270.
Jiang Y, Sun BH, Cao YY, Zhai YN, Wan X, 2018. Diversity of gut bacterial communities in male adults of Odontolabis fallaciosa (Coleoptera: Scarabaeoidea: Lucanidae) with different mandibular forms. Acta Entomol. Sin., 61(3): 322-330. [蒋宇, 孙丙华, 曹玉言, 翟勇宁, 万霞, 2018. 华美奥锹甲不同颚型雄性成虫肠道细菌群落多样性. 昆虫学报, 61(3): 322-330]
Kim JK, Kim NH, Jang HA, Kikuchi Y, Kim CH, Fukatsu T, Lee BL, 2013. Specific midgut region controlling the symbiont population in an insect-microbe gut symbiotic association. Appl. Environ. Microbiol., 79(23): 7229-7233.
Kuraishi T, Hori A, Kurata S, 2013. Host-microbe interactions in the gut of Drosophila melanogaster. Front. Physiol., 4: 375.
Li DH, Wang Y, Yang H, 2017.The intestinal microbiome of termite that decomposes lignocellulose efficiently. Acta Microbiol. Sin., 57(6): 876-884. [李丹红, 王誉, 杨红, 2017. 高效降解木质纤维素的白蚁肠道微生物组. 微生物学报, 57(6): 876-884]
Li XX, 2011. Diversity of Bacterial in Intestinal Canal of Nilaparvata lugens (St?l). MSc Thesis, Nanjing Agricultural University, Nanjing. [李香香, 2011. 稻飞虱肠道细菌多样性分析. 南京: 南京农业大学硕士学位论文]
Li XX, Yang H, Wang ZW, Su JY, 2011. Diversity of bacterial in intestinal canal of Nilaparvata lugens (St?l). Jiangsu Agric. Sci., (1): 126-129. [李香香, 杨焊, 王志伟, 苏建亚, 2011. 褐飞虱肠道细菌多样性分析. 江苏农业科学, (1): 126-129]
Li Y, Wang G, Tian J, Liu H, Yang H, 2012. Transcriptome analysis of the silkworm (Bombyx mori) by high-throughput RNA sequencing. PLoS ONE, 7(8): e43713.
Liu XG, Yang YJ, Liao QJ, Xu HX, Liu YH, Lu ZX, 2016. Analysis of the bacterial community structure and diversity in the intestine of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae). Acta Entomol. Sin., 59(9): 965-976. [刘小改, 杨亚军, 廖秋菊, 徐红星, 刘映红, 吕仲贤, 2016. 稻纵卷叶螟肠道细菌群落结构与多样性分析. 昆虫学报, 59(9): 965-976]
Lv J, Zhu ZR, Lou YG, Cheng JA, 2013. Review of research into outbreaks and management of rice planthoppers. Chin. J. Appl. Entomol., 50(3): 565-574. [吕进, 祝增荣, 娄永根, 程家安, 2013. 稻飞虱灾变和治理研究透析. 应用昆虫学报, 50(3): 565-574]
Makonde HM, Boga HI, Osiemo Z, Mwirichia R, Mackenzie LM, G?ker M, Klenk HP, 2013. 16S-rRNA-based analysis of bacterial diversity in the gut of fungus-cultivating termites (Microtermes and Odontotermes species). Anton. Leeuw. Int. J. G., 104(5): 869-883.
Mason CJ, Campbell AM, Scully ED, Hoover K, 2019. Bacterial and fungal midgut community dynamics and transfer between mother and brood in the Asian longhorned beetle (Anoplophora glabripennis), an invasive xylophage. Microb. Ecol., 77(1): 230-242.
Morrison M, Pope PB, Denman SE, McSweeney CS, 2009. Plant biomass degradation by gut microbiomes: more of the same or something new? Curr. Opin. Biotech., 20(3): 358-363.
Ohkuma M, Noda S, Hongoh Y, Kudo T, 2002. Diverse bacteria related to the bacteroides subgroup of the CFB phylum within the gut symbiotic communities of various termites. J. Agric. Chem. Soc. Japan, 66(1): 78-84.
Pang K, Dong SZ, Hou Y, Bian YL, Yang K, Yu XP, 2012. Cultivation, identification and quantification of one species of yeast-like symbiotes, Candida, in the rice brown planthopper, Nilaparvata lugens. Insect Sci., 19(4): 477-484.
Pang X, Xiao X, Liu Y, Zhang R, Liu J, Liu Q, Wang P, Cheng G, 2016. Mosquito C-type lectins maintain gut microbiome homeostasis. Nat. Microbiol., 1(5): 16023.
Peterson BF, Scharf ME, 2016. Lower termite associations with microbes: synergy, protection, and interplay. Front. Microbiol., 7: 422.
Qu LY, Lou YH, Huang HJ, Ding ZJ, Ye YX,Zhang CX, 2013. Molecular detection of the endosymbiont Wolbachia in different Asian populations of the brown planthopper. Chin. J. Appl. Entomol., 50(5): 1320-1327. [屈吕宇, 楼怡寒, 黄海剑, 丁张军, 叶雨轩, 张传溪, 2015. 亚洲不同地理种群褐飞虱内共生菌Wolbachia的分子检测. 应用昆虫学报, 50(5): 1320-1327]
Robacker DC, Lauzon CR, 2002. Purine metabolizing capability of Enterobacter agglomerans affects volatiles production and attractiveness to Mexican fruit fly. J. Chem. Ecol., 28(8): 1549-1563.
Schloss PD, Delalibera I, Handelsman J, Raffa KF, 2006. Bacteria associated with the guts of two wood-boring beetles: Anoplophora glabripennis and Saperda vestita (Cerambycidae). Environ. Entomol., 35(3): 625-629.
Shentu XP, Li DT, Xu JF, She L, Yu XP, 2016. Effects of fungicides on the yeast-like symbiotes and their host, Nilaparvata lugens St?l (Hemiptera: Delphacidae). Pestic. Biochem. Physiol., 128: 16-21.
Shi WB, Syrenne R, Sun JZ, Yuan JS, 2010. Molecular approaches to study the insect gut symbiotic microbiota at the ‘omics’ age. Insect Sci., 17(3): 199-219.
Shi WJ, Cheng JA, Zhu ZR, Jiang MX, Lou YY, 2002. Progress in the studies of insect symbiont Wolbachia. Acta Ecol. Sin., 22(3): 409-419. [施婉君, 程家安, 祝增荣, 蒋明星, 娄永根, 2002. 昆虫共生细菌Wolbachia的研究进展. 生态学报, 22(3): 409-419]
Su MM, Guo L, Tao YL, Zhang YJ, Wan FH, Chu D, 2016. Effects of host plant factors on the bacterial communities associated with two whitefly sibling species. PLoS ONE, 11(3): e0152183.
Tian XY, Song FP, Zhang J, Liu RM, Zhang XP, Duan JY, Shu CL, 2017. Diversity of gut bacteria in larval Protaetia brevitarsis (Coleoptera: Scarabaedia) fed on corn stalk. Acta Entomol. Sin., 60(6): 632-641. [田小燕, 宋福平, 张杰, 刘荣梅, 张兴鹏, 段江燕, 束长龙, 2017. 饲喂玉米秸秆的白星花金龟幼虫肠道细菌多样性. 昆虫学报, 60(6): 632-641]
Wang SB, Ghosh AK, Bongio N, Stebbings KA, Lampe DJ, Jacobs-Lorena M, 2012. Fighting malaria with engineered symbiotic bacteria from vector mosquitoes. Proc. Natl. Acad. Sci. USA, 109(31): 12734-12739.
Wang SB, Qu S, 2017. Insect symbionts and their potential application in pest and vector-borne disease control. Bull. Chin. Acad. Sci., 32(8): 863-872. [王四宝, 曲爽, 2017. 昆虫共生菌及其在病虫害防控中的应用前景. 中国科学院院刊, 32(8): 863-872]
Wang WX, Zhu TH, Lai FX, Fu Q, 2015. Diversity and infection frequency of symbiotic bacteria in different populations of the rice brown planthopper in China. J. Entomol. Sci., 50(1): 47-66.
Xia X, Zheng D, Zhong H, Qin B, Gurr GM, Vasseur L, Lin H, Bai J, He W, You M, 2013. DNA sequencing reveals the midgut microbiota of diamondback moth, Plutella xylostella (L.) and a possible relationship with insecticide resistance. PLoS ONE, 8(7): e68852.
Xu HX, Zheng XS, Yang YJ, Wang X, Fu Q, Ye GY, Lü ZX, 2014. PCR-DGGE analysis of the bacterial community in different populations of rice brown planthopper, Nilaparvata lugens St?l. Chin. J. Rice Sci., 28(2): 217-222. [徐红星, 郑许松, 杨亚军, 王新, 傅强, 叶恭银, 吕仲贤, 2014. 褐飞虱体内细菌群落的PCR-DGGE分析. 中国水稻科学, 28(2): 217-222]
Yang LP, Chang HH, Li J, Zhang ZB, Huang Y, 2017. Study of the biodiversity in intestinal symbiotic fungi in grasshoppers species by using DNA meta-barcoding. Acta Ecol. Sin., 37(20): 6905-6913. [杨丽平, 常会会, 李杰, 张智斌, 黄原, 2017. 基于DNA复合条形码技术的蝗虫肠道共生真菌多样性研究. 生态学报, 37(20): 6905-6913]
Yang XQ, Wang ZL, Wang TZ, Yu XP, 2018. Analysis of the bacterial community structure and diversity in the small brown planthopper, Laodelphax striatellus (Hemiptera: Delphacidae) by 16S rRNA high-throughput sequencing. Acta Entomol. Sin., 61(2): 200-208. [杨晓晴, 王正亮, 王天召, 俞晓平, 2018. 基于16S rRNA高通量测序的灰飞虱体内细菌群落结构及多样性分析. 昆虫学报, 61(2): 200-208]
Yun JH, Roh SW, Whon TW, Jung MJ, Kim MS, Park DS, Yoon C, Nam YD, Kim YJ, Choi JH, Kim JY, Shin NR, Kim SH, Lee WJ, Bae JW, 2014. Insects gut bacterial diversity determined by host environmental habitat, diet, developmental stage and phylogeny. Appl. Environ. Microb., 13(6): 5254-5264.
Zhang JY, Zhu BC, Xu C, Ding X, Li JF, Zhang XG, Lu ZH, 2015. Strategy of selecting 16S rRNA hypervariable regions for matagenome-phylogentic marker genes based analysis. Chin. J. Appl. Entomol., 26(11): 3545-3553. [张军毅, 朱冰川, 徐超, 丁啸, 李俊锋, 张学工, 陆祖宏, 2015. 基于分子标记的宏基因组16S rRNA基因高变区选择策略. 应用生态学报, 26(11): 3545-3553]
Zhang Y, Meng XC, 2017. Stress responses and impact of carbonhydrate metabolism in lactic acid bacteria. J. Chin. Inst. Food Sci. Tech., 17(6): 145-151. [张筠, 孟祥晨, 2017. 乳酸菌的胁迫应答及其对碳水化合物代谢的影响. 中国食品学报, 17(6): 145-151]
Zhang YF, Wu H, Chen JM, Zheng XS, Chen LZ, Yu XP, 2007. A strain isolated from brown planthopper and its molecular identification. Chin. J. Rice Sci., 21(5): 551-554. [张珏锋, 吴鸿, 陈建明, 郑许松, 陈列忠, 俞晓平, 2007. 一株褐飞虱内共生菌的分离及分子鉴定. 中国水稻科学, 21(5): 551-554]
Zhang YL, Ge HM, Zhao W, Dong H, Xu Q, Li SH, Li J, Zhang J, Song YC, Tan RX, 2008. Unprecedented immunosuppressive polyketides from Daldinia eschscholzii, a mantis-associated fungus. Angew. Chem. Int. Ed. Engl., 47(31): 5823-5826.
Zhou HY, Sun B, Wu HL, Hu XM, Hao Y, Ye JM, 2015. Research progress of intestinal microbial function of insect and silkworm. North Seric., 36(4): 1-4. [周洪英, 孙波, 吴洪丽, 胡兴明, 郝瑜, 叶建美, 2015. 昆虫肠道微生物功能及家蚕肠道微生物研究进展. 北方蚕业, 36(4): 1-4]