淡色库蚊不同发育阶段肠道菌群多样性分析  

Diversity analysis of intestinal bacterial flora of Culex pipiens pallens at different developmental stages

作　　者：吕文祥 程鹏[1] 彭荟 王海洋 刘宏美[1] 王海防[1] 郭秀霞[1] 公茂庆[1] 刘丽娟[1] 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(Shandong Institute of Parasitic Diseases,Shandong First Medical University&Shandong Academy of Medical Sciences,Jining 272033,China)

机构地区：[1]山东省寄生虫病防治研究所,山东第一医科大学(山东省医学科学院),济宁272033

出　　处：《中国寄生虫学与寄生虫病杂志》2022年第4期460-467,共8页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(81871685,81902096)。

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1972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,R^(2)=0.70744,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 pallensⅣ-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 1972 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

关 键 词：淡色库蚊 肠道细菌 多样性 高通量测序 

分 类 号：R384.112[医药卫生—医学寄生虫学]