淡色库蚊不同发育阶段肠道菌群多样性分析

doi:10.12140/j.issn.1000-7423.2022.04.007

摘要/Abstract

摘要：

目的了解淡色库蚊不同发育阶段肠道菌群的组成与多样性。方法取淡色库蚊的Ⅳ龄幼虫、蛹、雌成蚊各20只,分别剖取肠道（每个发育阶段设3个重复）,提取肠道细菌基因组DNA,PCR扩增肠道细菌的16S rRNA高变区序列,采用Illumina NovaSeq6000测序平台进行测序。整理统计测得的序列数目和可操作分类单元（OTU）,采用群落结构柱状图和Veen图分析淡色库蚊不同发育阶段肠道细菌的群落组成与丰度,采用Alpha多样性指数分析微生物群落的丰度和多样性,采用主坐标分析（PCoA）对不同发育阶段肠道细菌群落结构进行差异分析,采用线性判别分析（LEFSe）揭示组间在丰度上有显著差异的物种,采用SPSS 22.0统计学软件对属水平上不同阶段淡色库蚊核心细菌丰度进行单因素方差分析。结果淡色库蚊Ⅳ龄幼虫、蛹、雌成蚊的肠道菌群共有1 972个OTU,归属22门45纲112目178科329属。在属水平上,Ⅳ龄幼虫、蛹和雌成蚊肠道细菌分别由230、233和228个属组成,各发育阶段共有的优势属为拟杆菌属[丰度分别为（16.83 + 3.5）%、（26.98 ± 0.94）%、（20.82 ± 2.63）%]、Muribaculaceae[丰度分别为（12.36 ± 1.94）%、（19.98 ± 1.24）%、（14.90 ± 1.52）%]、另枝菌属[丰度分别为（4.13 ± 0.62）%、（7.29 ± 1.28）%、（5.36 ± 1.00）%]和毛螺菌科_NK4A136组[丰度分别为（3.39 ± 0.39）%、（5.77 ± 0.21）%、（3.93 ± 0.40）%]。在OTU水平,Veen图分析结果显示,Ⅳ龄幼虫、蛹、雌成蚊共有的OTU数目为687, 特有的OTU分别为250、284、309。Alpha多样性显示,不同发育阶段淡色库蚊肠道菌群的组成与多样性存在差异（F = 36.83,P < 0.01;F = 28.91,P < 0.01）,蛹的物种丰度和物种多样性最高（6.58 ± 0.04）。PCoA结果显示,肠道菌群分为3组,各阶段分别归为1组;各阶段肠道菌群组成差异有统计学意义（Df = 2, R² = 0.707 44,P < 0.01）。LEFSe结果显示,共17个分类单元在不同发育阶段具有差异;赖氏菌属、拟杆菌属、Muribaculaceae、沃尔巴克氏体属在淡色库蚊不同发育阶段的丰度差异具有统计学意义（F = 48.331、10.826、17.759、95.139,P < 0.01）,赖氏菌属主要存在于Ⅳ龄幼虫阶段（0.887 ± 0.022）,拟杆菌属（0.270 ± 0.009）和Muribaculaceae（0.200 ± 0.012）主要存在于蛹阶段;沃尔巴克氏体属主要存在于雌成蚊阶段（0.172 ± 0.025）。结论淡色库蚊不同发育阶段肠道优势菌群主要为拟杆菌属和Muribaculaceae,蛹的物种丰度和物种多样性最高。

关键词: 淡色库蚊, 肠道细菌, 多样性, 高通量测序

Abstract:

Objective To determine the composition and diversity of intestinal microflora of Culex pipiens pallens at different developmental stages. Methods The intestinal tracts of 20 Cx. pipiens pallens Ⅳ-instar larvae, pupae and female mosquitoes were dissected to collect intestinal samples for extraction of genomic DNA of intestinal bacteria, with triplicate for each developmental stage. The 16S rRNA highly variable regions of intestinal bacteria was amplified by PCR and sequenced using the Illumina NovaSeq6000 sequencing platform. The number of sequences and operational taxonomic units (OTU) were compiled and statistically analyzed using structural histogram and Veen diagram to reveal the flora composition and abundance of intestinal bacteria at different developmental stages of Cx. pipiens pallens. The abundance and diversity of microbial flora were analyzed by the Alpha diversity index. Principal coordinate analysis (PCoA) was used to examine the variance in the flora structure at different developmental stages. Linear discriminant analysis (LEFSe) was used to differentiate the species with significant differences in abundance between groups. SPSS 22.0 statistical software was used to conduct one-way ANOVA for genus-based analysis of the core bacterial abundance of Cx. pipiens pallens at different stages. Results The intestinal microbiota of Cx. pipiens pallens larvae, pupae and female mosquitoes were 1 972 OTUs, belonging to 22 phyla, 45 classes, 112 orders, 178 families and 329 genera. At the genus level, the fourth instar larvae, pupae and female mosquitoes were composed of 230, 233 and 228 genera, respectively. The dominant genera were Bacteroides with the abundance of (16.83 ± 3.5) %, (26.98 ± 0.94) % and (20.82 ± 2.63) %, respectively; Muribaculaceae, the abundance was (12.36 ± 1.94) %, (19.98 ± 1.24) %, (14.90 ± 1.52) %, respectively; the abundance of Alistipes was (4.13 ± 0.62)%, (7.29 ± 1.28)%, (5.36 ± 1.00)%, respectively; Lachnospiraceae_NK4A136_group were (3.39 ± 0.39)%, (5.77 ± 0.21) % and (3.93 ± 0.40)%, respectively. At the level of OTU, Veen diagram analysis showed that the number of OTU shared by the fourth instar larvae, pupae and female adult was 687, and the unique OTU was 250, 284 and 309, respectively. Alpha diversity showed that the composition and diversity of Cx. pipiens pallens were different at different developmental stages (F = 36.83, P < 0.01; F = 28.91, P < 0.01), the species abundance and diversity of pupae were the highest (6.58 ± 0.04). The results of PCoA showed that the microflora was presented in three groups, with each stage falling into one group, respectively. There were statistically significant differences in the composition of bacteria among different stages (Df = 2, R² = 0.707 44, P < 0.01). The results of LEFSe showed that 17 taxa were different at developmental stages. There were statistically significant differences in the abundance of Leifsonia, Bacteroides, Muribaculaceae and Wolbachia in different developmental stages of Cx. pipiens pallens (F = 48.331, 10.826, 17.759, 95.139, P < 0.01). Leifsonia mainly existed in the fourth instar larval stage (0.887 ± 0.022), Bacteroidetes (0.270 ± 0.009) and Muribaculaceae (0.200 ± 0.012) mainly existed in the pupa stage. Wolbachia mainly existed in the female mosquito stage (0.172 ± 0.025). Conclusion The dominant intestinal flora of Cx. pipiens pallens at different developmental stages are Bacteroides and Muribaculaceae, and the pupa shows the highest species abundance and species diversity.

Key words: Culex pipiens pallens, Intestinal bacteria, Diversity, High throughput sequencing

中图分类号:

R384.112

吕文祥, 程鹏, 彭荟, 王海洋, 刘宏美, 王海防, 郭秀霞, 公茂庆, 刘丽娟. 淡色库蚊不同发育阶段肠道菌群多样性分析[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(4): 460-467.

LV Wen-xiang, CHENG Peng, PENG Hui, WANG Hai-yang, LIU Hong-mei, WANG Hai-fang, GUO Xiu-xia, GONG Mao-qing, LIU Li-juan. Diversity analysis of intestinal bacterial flora of Culex pipiens pallens at different developmental stages[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2022, 40(4): 460-467.

图/表 6

表1

图1

图2

表2

图3

图4

参考文献 34

[1]	Engel P, Moran NA. The gut microbiota of insects-diversity in structure and function[J]. FEMS Microbiol Rev, 2013, 37(5): 699-735.
[2]	Hu ZY, Xia Q. Advances in the histology study, function and application of insect intestinal flora[J]. Biotechnol Bull, 2021, 37(1): 102-112. (in Chinese)
	( 胡紫媛, 夏嫱. 昆虫肠道菌群组学研究及功能和应用进展[J]. 生物技术通报, 2021, 37(1): 102-112.)
[3]	Yun JH, Roh SW, Whon TW, et al. Insect gut bacterial diversity determined by environmental habitat, diet, developmental stage, and phylogeny of host[J]. Appl Environ Microbiol, 2014, 80(17): 5254-5264.
[4]	Wang WW, He C, Cui J, et al. Comparative analysis of the composition of intestinal bacterial communities in Dastarcus helophoroides fed different diets[J]. J Insect Sci, 2014, 14: 111.
[5]	Coon KL, Brown MR, Strand MR. Mosquitoes host communities of bacteria that are essential for development but vary greatly between local habitats[J]. Mol Ecol, 2016, 25(22): 5806-5826.
[6]	Gimonneau G, Tchioffo MT, Abate L, et al. Composition of Anopheles coluzzii and Anopheles gambiae microbiota from larval to adult stages[J]. Infect Genet Evol, 2014, 28: 715-724.
[7]	Feng LZ, Liu WD. Studies on the morphological differences of the adults of Chinese Culex pipiens var. pallens and Culex fatigans[J]. Acta Entomol Sin, 1954(2): 103-114, 196. (in Chinese)
	( 冯兰洲, 刘维德. 中国尖音库蚊淡色变种与乏倦库蚊成虫在形态上的区别的研究[J]. 昆虫学报, 1954(2): 103-114, 196.)
[8]	Yan DM, Wang WZ, Wang XS, et al. Investigation of mosquitoes and mosquito-borne viruses in some regions of Guizhou Province, China, 2019[J]. Chin J Vector Biol Control, 2021, 32(4): 422-427. (in Chinese)
	( 闫冬明, 王文周, 王雪霜, 等. 贵州省部分地区2019年蚊虫及蚊媒病毒调查研究[J]. 中国媒介生物学及控制杂志, 2021, 32(4): 422-427.)
[9]	Nan XW, Xie XX, Yu HM, et al. An investigation of the vector mosquitoes and arboviruses during a Japanese encephalitis epidemic in Baotou of Inner Mongolia Autonomous Region, China, 2018[J]. Chin J Vector Biol Control, 2020, 31(6): 652-656. (in Chinese)
	( 南晓伟, 解新霞, 于红敏, 等. 内蒙古包头市2018年一起流行性乙型脑炎疫情的媒介蚊虫及感染虫媒病毒调查[J]. 中国媒介生物学及控制杂志, 2020, 31(6): 652-656.)
[10]	Liu LJ, Zhang BG, Cheng P, et al. Overwintering of Culex pipiens pallens (Diptera : Culicidae) in Shandong, China[J]. J Entomol Sci, 2016, 51(4): 314-320.
[11]	Wang Y, Gilbreath TM 3rd, Kukutla P, et al. Dynamic gut microbiome across life history of the malaria mosquito Anopheles gambiae in Kenya[J]. PLoS One, 2011, 6(9): e24767.
[12]	Wang XM, Liu T, Wu Y, et al. Bacterial microbiota assemblage in Aedes albopictus mosquitoes and its impacts on larval development[J]. Mol Ecol, 2018, 27(14): 2972-2985.
[13]	Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data[J]. Bioinformatics, 2014, 30(15): 2114-2120.
[14]	Reyon D, Tsai SQ, Khayter C, et al. FLASH assembly of TALENs for high-throughput genome editing[J]. Nat Biotechnol, 2012, 30(5): 460-465.
[15]	Rognes T, Flouri T, Nichols B, et al. VSEARCH: a versatile open source tool for metagenomics[J]. PeerJ, 2016, 4: e2584.
[16]	Caporaso JG, Kuczynski J, Stombaugh J, et al. QIIME allows analysis of high-throughput community sequencing data[J]. Nat Methods, 2010, 7(5): 335-336.
[17]	Wang Q, Garrity GM, Tiedje JM, et al. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy[J]. Appl Environ Microbiol, 2007, 73(16): 5261-5267.
[18]	Yadav KK, Datta S, Naglot A, et al. Diversity of cultivable midgut microbiota at different stages of the Asian tiger mosquito, Aedes albopictus from Tezpur, India[J]. PLoS One, 2016, 11(12): e0167409.
[19]	Tandina F, Almeras L, Koné AK, et al. Use of MALDI-TOF MS and culturomics to identify mosquitoes and their midgut microbiota[J]. Parasit Vectors, 2016, 9(1): 495.
[20]	Liu XG, Yang YJ, Liao QJ, et al. Analysis of the bacterial community structure and diversity in the intestine of Cnaphalocrocis medinalis (Lepidoptera : Pyralidae)[J]. Acta Entomol Sin, 2016, 59(9): 965-976. (in Chinese)
	( 刘小改, 杨亚军, 廖秋菊, 等. 稻纵卷叶螟肠道细菌群落结构与多样性分析[J]. 昆虫学报, 2016, 59(9): 965-976.)
[21]	Ya YK, Xia M, Li J, et al. Diversity analysis of intestinal flora in the third and fourth-instar larvae of Solenopsis invicta[J]. J Environ Entomol, 2021, 43(2): 420-429. (in Chinese)
	( 押玉柯, 夏敏, 李军, 等. 红火蚁三龄、四龄幼虫肠道菌菌群多样性分析[J]. 环境昆虫学报, 2021, 43(2): 420-429.)
[22]	Liu J, Chen D, Zhuang GF, et al. Isolation and identification of cultivable symbiotic bacteria from the intestinal tract of Musca domestica during development[J]. Chin J Parasitol Parasit Dis, 2017, 35(2): 120-124. (in Chinese)
	( 刘婧, 陈丹, 庄桂芬, 等. 家蝇发育过程中肠道可培养共生细菌的分离与鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(2): 120-124.)
[23]	Chen HJ, Hao DJ, Wei ZQ, et al. Bacterial communities associated with the pine wilt disease insect vector Monochamus alternatus (Coleoptera : Cerambycidae) during the larvae and pupae stages[J]. Insects, 2020, 11(6): 376.
[24]	Ma SL, Yang Y, Jack CJ, et al. Effects of Tropilaelaps mercedesae on midgut bacterial diversity of Apis mellifera[J]. Exp Appl Acarol, 2019, 79(2): 169-186.
[25]	Zhang ZQ, Jiao S, Li XH, et al. Bacterial and fungal gut communities of Agrilus mali at different developmental stages and fed different diets[J]. Sci Rep, 2018, 8(1): 15634.
[26]	Vera-Ponce de León A, Jahnes BC, Duan J, et al. Cultivable, host-specific bacteroidetes symbionts exhibit diverse polysaccharolytic strategies[J]. Appl Environ Microbiol, 2020, 86(8): e00091-20.
[27]	Sibai M, Altunta ş E, Yldrm B, et al. Microbiome and longevity: high abundance of longevity-linked Muribaculaceae in the gut of the long-living rodent Spalax leucodon[J]. OMICS, 2020, 24(10): 592-601.
[28]	Dickson LB, Jiolle D, Minard G, et al. Carryover effects of larval exposure to different environmental bacteria drive adult trait variation in a mosquito vector[J]. Sci Adv, 2017, 3(8): e1700585.
[29]	Minard G, Mavingui P, Moro CV. Diversity and function of bacterial microbiota in the mosquito holobiont[J]. Parasit Vectors, 2013, 6: 146.
[30]	Kittayapong P, Baisley KJ, Sharpe RG, et al. Maternal transmission efficiency of Wolbachia superinfections in Aedes albopictus populations in Thailand[J]. Am J Trop Med Hyg, 2002, 66(1): 103-107.
[31]	Wu JR, Zhang QJ, Xiao YJ. Research on Wolbachia technique for control of mosquito-borne diseases[J]. Biol Teach, 2017, 42(7): 13-14. (in Chinese)
	( 吴姣榕, 张秋金, 肖义军. 沃尔巴克氏体技术控制蚊媒病研究概述[J]. 生物学教学, 2017, 42(7): 13-14.)
[32]	Yang C, Xi ZY, Hu ZY. Blocking transmission of mosquito-borne diseases through population suppression using Wolbachia[J]. Chin J Vector Biol Control, 2020, 31(1): 113-116. (in Chinese)
	( 杨翠, 奚志勇, 胡志勇. 应用沃尔巴克氏体通过种群压制阻断蚊媒病传播的研究进展[J]. 中国媒介生物学及控制杂志, 2020, 31(1): 113-116.)
[33]	Pan XL, Pike A, Joshi D, et al. The bacterium Wolbachia exploits host innate immunity to establish a symbiotic relationship with the dengue vector mosquito Aedes aegypti[J]. ISME J, 2018, 12(1): 277-288.
[34]	González-Serrano F, Pérez-Cobas AE, Rosas T, et al. The gut microbiota composition of the moth Brithys crini reflects insect metamorphosis[J]. Microb Ecol, 2020, 79(4): 960-970.

样品Sample	原始标签数No. raw tag	有效标签数 No. effective tag	平均片段长度/bp Average length/bp	不同分类水平物种数量 Statistics of the number of classify distribution
样品Sample	原始标签数No. raw tag	有效标签数 No. effective tag	平均片段长度/bp Average length/bp	门Phyla	纲Class	目Order	科Family	属Genus
Ⅳ龄幼虫The fourth instar larva
L1	67 114	58 096	415.40	11	19	55	90	162
L2	72 466	64 056	415.25	12	21	58	93	171
L3	71 951	62 972	414.63	15	25	72	105	187
蛹Pupa
P1	73 525	63 673	415.01	12	23	61	95	164
P2	69 258	60 714	415.03	14	23	58	89	160
P3	69 857	60 684	414.74	16	27	64	102	179
雌成蚊 Female mosquito
A1	67 119	59 037	412.94	11	19	55	81	141
A2	74 434	66 009	414.13	12	23	60	93	162
A3	70 069	62 294	414.62	13	25	60	94	166
总计Total				22	45	112	178	329

样品 Sample	Chao1指数 Chao1 index	香农指数Shannon index	辛普森指数Simpson index	覆盖度Good’s coverage
Ⅳ龄幼虫The fourth instar larva	1 035.17 ± 41.71^a	6.05 ± 0.16^a	0.95 ± 0.00^a	0.99
蛹Pupa	1 038.76 ± 31.56^a	6.58 ± 0.04^a	0.95 ± 0.00^a	0.99
雌成蚊Female mosquito	963.24 ± 71.00^a	5.93 ± 0.05^b	0.93 ± 0.01^b	0.99