致倦库蚊成蚊吸食血餐和感染乙型脑炎病毒前后中肠细菌多样性分析  

Diversity analysis of the midgut bacteria in Culex quinquefasciatus(Diptera:Culicidae)adults before and after blood meal or infection with Japanese encephalitis virus

作　　者：徐昌豪 朱茜 和易 刘雨薇 黄裕恬 李睿冬 曹三杰 黄小波 颜其贵 伍锐 文翼平 郎一飞 赵勤 赵珊 王一平 胡雅劼[2] 杜森焱 XU Chang-Hao;ZHU Xi;HE Yi;LIU Yu-Wei;HUANG Yu-Tian;LI Rui-Dong;CAO San-Jie;HUANG Xiao-Bo;YAN Qi-Gui;WU Rui;WEN Yi-Ping;LANG Yi-Fei;ZHAO Qin;ZHAO Shan;WANG Yi-Ping;HU Ya-Jie;DU Sen-Yan(Research Center of Swine Disease,College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China;Sichuan Center for Disease Control and Prevention,Chengdu 610041,China)

机构地区：[1]四川农业大学动物医学院,猪病研究中心,成都611130 [2]四川省疾病预防控制中心,成都610041

出　　处：《昆虫学报》2023年第8期1031-1041,共11页Acta Entomologica Sinica

基　　金：中央引导地方科技发展专项(2021ZYD0086);国家自然科学基金青年科学基金项目(32102748)。

摘　　要：【目的】为了研究致倦库蚊Culex quinquefasciatus成蚊吸食血餐及感染乙型脑炎病毒(Japanese encephalitis virus,JEV)前后的中肠细菌多样性及差异,更深层次地揭示致倦库蚊与其中肠细菌之间的关系,以及血液消化和病毒感染期间中肠细菌发生变化的原因。【方法】提取吸食糖水(10%蔗糖溶液)、不含JEV血餐和含JEV血餐的致倦库蚊成蚊中肠细菌基因组DNA,PCR扩增中肠细菌的16S rDNA高变区序列,采用Illumina NovaSeq6000测序平台进行测序并进行生物信息学分析;提取吸食不含JEV血餐和含JEV血餐的致倦库蚊成蚊中肠总RNA,使用RT-qPCR检测中肠中抗菌肽(antimicrobial peptide,AMP)基因CqAttacin,CqCecropin,CqCecropin A,CqDefensin A和CqDefensin C表达量。【结果】致倦库蚊成蚊吸食血餐前后的中肠细菌共有204个扩增子序列变体(amplicon sequence variants,ASVs),归属于4门9纲17目27科36属;感染JEV前后的致倦库蚊中肠细菌共有120个ASVs,归属于6门13纲23目38科47属。在属水平上,伊丽莎白金菌属Elizabethkingia作为优势菌属普遍存在于各中肠细菌中;肠杆菌属Enterobacter在吸食血餐后第2天时的致倦库蚊成蚊中肠中丰度最高,摩根氏菌属Morganella、根瘤菌属Rhizobium和鲍特氏菌属Bordetella则主要存在于感染了JEV的致倦库蚊成蚊中肠内。吸食糖水与吸食血餐后第2天时的致倦库蚊成蚊中肠细菌代谢通路差异最大,主要差异代谢通路是磷酸转移酶系统、铁载体类非核糖体肽的生物合成、N-聚糖生物合成、脂肪酸生物合成以及一些氨基酸的代谢通路。感染与未感染JEV的致倦库蚊成蚊中肠菌群存在差异,且感染JEV后中肠细菌丰富度更高。吸食血餐使致倦库蚊成蚊中肠中抗菌肽基因CqAttacin,CqCecropin,CqDefensin A,CqDefensin A和CqDefensin C表达量与吸食糖水的对照组的相比上调,而感染JEV使抗菌肽基因CqCecropin A,CqDefensin A和CqDefensin C表达量与吸食不含JEV血【Aim】To study the midgut bacterial diversity and difference in Culex quinquefasciatus adults before and after feeding with blood meal and infection with the Japanese encephalitis virus(JEV),then further reveal the relationship between C.quinquefasciatus and its midgut bacteria,as well as the reasons for the changes of the midgut bacteria during blood digestion and viral infection.【Methods】The bacterial genome DNA of the midguts from C.quinquefasciatus adults fed with sugar water(10%sucrose solution),blood meal without JEV and blood meal with JEV was extracted.The 16S rDNA highly variable region sequence of the midgut bacteria was amplified by PCR,sequenced using the Illumina NovaSeq6000 sequencing platform and analyzed by bioinformatics.The total RNA was extracted from the midguts of C.quinquefasciatus adults fed with blood meal without JEV and blood meal with JEV,and the expression levels of the antimicrobial peptide(AMP)genes CqAttacin,CqCecropin,CqCecropin A,CqDefensin A and CqDefensin C in the midgut were detected by RT-qPCR.【Results】There were 204 amplicon sequence variants(ASVs)in the midgut bacteria of C.quinquefasciatus adults before and after blood meal,belonging to 4 phyla,9 classes,17 orders,27 families and 36 genera,and 120 ASVs in the midgut bacteria before and after infection with JEV,belonging to 6 phyla,13 classes,23 orders,38 families and 47 genera.At the genus level,Elizabethkingia was widely distributed in the midgut bacteria as a dominant genus.The abundance of Enterobacter in the midgut of C.quinquefasciatus adults was the highest on the day 2 after blood meal.Morganella,Rhizobium and Bordetella mainly existed in the midgut of C.quinquefasciatus adults infected with JEV.The difference in the metabolic pathways of the midgut bacteria between C.quinquefasciatus adults on the day 2 after being fed with sugar water and blood meal was the largest.The main differential metabolic pathways were the phosphotransferase system,biosynthesis of siderophore group nonribosomal peptides,N-glycan

关 键 词：致倦库蚊 细菌多样性 16S rDNA 血餐 乙脑病毒 抗菌肽 

分 类 号：R384.1[医药卫生—医学寄生虫学]