我国部分地区28种蚊虫的线粒体COⅠ基因序列分析

摘要/Abstract

摘要： 目的分析我国部分地区28种蚊虫的线粒体细胞色素c氧化酶亚基Ⅰ（mitochondrion cytochrome c oxidase subunit Ⅰ,COⅠ）基因序列,探讨其作为蚊虫分子标记的应用潜力。方法 2015年3月至2016年10月在我国海南、广东、广西、云南、福建、浙江、河南、山西、天津、内蒙古、吉林和黑龙江等12省（自治区、直辖市）的蚊虫孳生地采集幼虫,室内饲养至羽化成蚊,在蚊虫栖息地和蚊虫监测点,采用灯诱法和人诱法采集成蚊。所有成蚊经形态学鉴定后,提取单只蚊虫基因组DNA,采用国际双翅目通用引物PCR扩增并克隆线粒体COⅠ基因5′端片段,并进行测序。所得COⅠ基因序列上传GenBank获取登录号。序列通过Clustal1.83和MEGA5.05软件进行多序列比对,用DNAMAN9.0软件分析同源性。采用Mega5.05软件进行序列特征分析、计算遗传距离。每种蚊虫随机选择1~4条线粒体COⅠ基因序列,采用邻接法（NJ）构建系统发育进化树。结果共采集3亚科6属28种301只蚊虫,分别为伊蚊属（Aedes）的白纹伊蚊（Ae. albopictus）等5种、按蚊属（Anopheles）的中华按蚊（An. sinensis）等6种（亚种）、库蚊属（Culex）的二带喙库蚊（Cx. bitaeniorhynchus）等14种（亚种）、阿蚊属（Armigeres）的骚扰阿蚊（Ar. subalbatus）、曼蚊属（Mansonia）的常型曼蚊（Ma. uniformis）和巨蚊属（Toxorhynchites）的华丽巨蚊（Tx. splendens）。PCR扩增结果显示,28种蚊虫线粒体COⅠ基因的长度约711 bp,经多序列比对排齐后长度为651 bp。不同种别蚊虫的线粒体COⅠ序列同源性范围为97.85%~99.97%。种内K2P遗传距离为0.15%~2.89%,属内种间遗传距离为0.25%~14.50%。其中,按蚊属6种蚊虫COⅠ序列存在153个变异位点,131个简约信息位点,T + A平均含量为67.70%;编码的217个氨基酸残基中保守氨基酸206个（占94.9%）;6种蚊虫的核苷酸同源性为98.31%~99.72%,种内K2P遗传距离为0.41%~1.56%,种间K2P遗传距离为1.07%~14.50%。28种蚊虫（60条）COⅠ序列的系统发育树分析结果显示,种团内近缘种（尖音库蚊、淡色库蚊和致倦库蚊）聚在一起,但近缘种之间未完全隔开;白跗按蚊（An. albitarsis）和迷走按蚊（An. vagus）聚在一起,其余23种蚊虫分别以种为单位聚在一起。结论基于线粒体COⅠ基因的分子标记可以区分捕获蚊虫的部分蚊种,但尚未能有效及准确地区分近缘种.

关键词: 蚊虫, 线粒体细胞色素c氧化酶亚基Ⅰ, 分子标记

Abstract: Objective To perform mitochondrial DNA sequence analysis of cytochrome oxidase subunit Ⅰ (COⅠ) gene from 28 species of mosquitoes in China and to explore the potential use of COⅠ as a molecular marker to distinguish mosquito species. Methods Mosquito larvae were collected from mosquito breeding grounds in 12 Provinces/Municipalities/Autonomous Regions including Hainan, Guangdong, Guangxi, Yunnan, Fujian, Zhejiang, Henan, Shanxi, Jilin, Heilongjiang, Tianjin and Inner Mongolia, from April 2015 to October 2016, and were grown into mosquitoes. The human trap and light trap methods were used to collect adult mosquitoes. After morphological identification, genomic DNA was extracted individually from the mosquitoes, and the 5′ fragment of mitochondrial COⅠ gene was amplified by PCR using international Diptera primers and sequenced. Alignment of multiple sequences was performed with Clustal 1.83 and MEGA 5.05 softwares. DNAMAN 9.0 software was used to calculate the identity among sequences and Mega 5.05 software was used to analyze sequence characteristics and calculate the genetic distance. The phylogenetic tree was constructed by Neighbor-Joining (NJ) method based on 1-4 sequences randomly selected for each species. Results A total of 301 mosquitoes were collected, belonging to 28 species, six genera, three subfamilies. The 28 species included 5 species of the Aedes genus, 6 species of the Anopheles genus, 14 species of the Culex genus, as well as species of Armigeres subalbatus, Mansonia uniformis, and Toxorhynchites splendens. The amplification of COⅠ gene in the 28 mosquito species produced bands of ~711 bp, but the final length of sequence for analysis after sequence alignment was 651 bp. The COⅠ sequence identity among different species ranged 97.85%-99.97%. The intraspecies and interspecies K2P genetic distances were 0.15%-2.89% and 0.25%-14.5%, respectively. There were 153 mutational sites and 131 parsimony-informative sites in the COⅠ sequence among the 6 species of the Anopheles genus, with average T + A content of 67.7%, and the encoded 217 amino acids had 206 conserved ones (94.9%). The nucleotide homology among the 6 species was 98.31%-99.72%, and intraspecies and interspecies K2P genetic distances were 0.41%-1.56% and 1.07%-14.5%, respectively. Phylogenetic tree constructed on the 60 COⅠ sequences of 28 species showed clustering of Cx. pipiens, Cx. pipiens pallens and Cx. quinquefasciatus, as well as clustering of An. albitarsis and An. vagus. The other 23 species were clustered within each species. Conclusion COⅠ gene may be used as a molecular marker to distinguish between some mosquito species in some regions of China, but it cannot serve to distinguish between closely-related species.

Key words: Mosquito, Mitochondrial DNA cytochrome oxidase subunit Ⅰ, Molecular marker

中图分类号:

R384.11

郭玉燕, 罗雷, 宋璋瑶, 郑学礼. 我国部分地区28种蚊虫的线粒体COⅠ基因序列分析[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(3): 280-287.

Yu-yan GUO, Lei LUO, Zhang-yao SONG, Xue-li ZHENG. Mitochondrial DNA sequence analysis of cytochrome oxidase subunit Ⅰ gene from 28 mosquito species in China[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(3): 280-287.

图/表 5

表1

蚊虫的采集地、种类和数量

采集地 Collection sites			蚊种（数量/只） Species (Number)
广东省Guangdong Province	广州Guangzhou	23°07′N 113°15′E	白纹伊蚊 Ae. albopictus (10),致倦库蚊 Cx. quinquefasciatus (2),褐尾库蚊 Cx. fuscanus (1)
	深圳Shenzhen	22°32′N 113°55′E	白纹伊蚊 Ae. albopictus (10),致倦库蚊 Cx. quinquefasciatus (2)
	东莞Dongguan	22°28′N 111°24′E	白纹伊蚊 Ae. albopictus (3),致倦库蚊 Cx. quinquefasciatus (1)
	汕头Shantou	23°21′N 116°40′E	白纹伊蚊 Ae. albopictus (10),刺扰伊蚊 Ae. vexans (3),三带喙库蚊 Cx. tritaeniorhynchus (1)
	韶关Shaoguan	24°11′N 113°24′E	白纹伊蚊 Ae. albopictus (1),三带喙库蚊 Cx. tritaeniorhynchus (1),致倦库蚊Cx. quinquefasciatus (1)
	实验室Lab	23°07′N 113°15′E	埃及伊蚊 Ae. aegypti (6)
广西壮族自治区 Guangxi Zhuang Autonomous Region	梧州Wuzhou	23°53′N 110°32′E	白纹伊蚊 Ae. albopictus (10),中华按蚊 An. sinensis (2),三带喙库蚊 Cx. tritaeniorhynchus (1),致倦库蚊 Cx. quinquefasciatus (1),尖音库蚊 Cx. pipiens (1)
海南省Hainan Province	海口Haikou	20°02′N 110°11′E	刺扰伊蚊 Ae. vexans(1),白纹伊蚊 Ae. albopictus (10),致倦库蚊 Cx.quinquefasciatus (1),骚扰阿蚊 Ar. subalbatus (1)
	澄迈Chengmai	19°44′N 110°00′E	刺扰伊蚊 Ae. vexans (2),白纹伊蚊 Ae. albopictus (10),嗜人按蚊 An. anthropophagus (1),须喙按蚊 An. barbirostis (1),中华按蚊 An. sinensis (1),棋斑按蚊 An. tessellatus (1),迷走按蚊 An. vagus(1),褐尾库蚊 Cx. fuscanus (1),三带喙库蚊 Cx. tritaeniorhynchus(1),致倦库蚊 Cx.quinquefasciatus (1),骚扰阿蚊 Ar. subalbatus (1),华丽巨蚊 Tx. splendens (1),杂鳞库蚊 Cx. vishnui (3),常型曼蚊 Ma. uniformis (1)
	儋州Danzhou	19°31′N 109°34′E	白纹伊蚊 Ae. albopictus (10),三带喙库蚊 Cx. tritaeniorhynchus(1),致倦库蚊 Cx. quinquefasciatus (1),白雪库蚊 Cx. gelidus (1)
	琼海Qionghai	19°10′N 110°22′E	白纹伊蚊 Ae. albopictus(8),白跗按蚊 An. albitarsis(1),中华按蚊 An. sinensis (1),棋斑按蚊 An. tessellatus (2),迷走按蚊 An. vagus (1),三带喙库蚊 Cx. tritaeniorhynchus(1),棕头库蚊 Cx. fuscocehala(1),白胸库蚊 Cx. pallidothorax (2),致倦库蚊 Cx. quinquefasciatus (1)
	白沙Baisha	19°13′N 109°26′E	刺扰伊蚊 Ae. vexans (1),窄翅伊蚊 Ae. lineatopennis (1),白纹伊蚊 Ae. albopictus (10),嗜人按蚊 An. anthropophagus (2),须喙按蚊 An. barbirostis (2),中华按蚊 An. sinensis (1),棋斑库蚊 An. tessellatus(1),三带喙库蚊 Cx. tritaeniorhynchus (2),带纹库蚊 Cx. cinctellus (1),白雪库蚊 Cx. gelidus (1),幼小库蚊 Cx. infantulus (1),薛氏库蚊 Cx. shebbearei (2),骚扰阿蚊 Ar.subalbatus (1)
	琼中Qiongzhong	19°02′N 109°50′E	窄翅伊蚊 Ae. lineatopennis (2),白纹伊蚊 Ae. albopictus (10),白胸库蚊 Cx. pallidothorax (3),骚扰阿蚊 Ar. subalbatus (1)
	昌江Changjiang	19°17′N 109°03′E	刺扰伊蚊 Ae. vexans (1),白纹伊蚊 Ae. albopictus (10),棋斑按蚊 An. tessellatus (1),三带喙库蚊 Cx. tritaeniorhynchus (5),致倦库蚊 Cx.quinquefasciatus(1),杂鳞库蚊 Cx. vishnui (1),骚扰阿蚊 Ar. subalbatus (2)
	陵水Lingshui	18°30′N 110°01′E	窄翅伊蚊 Ae. lineatopennis (1),白纹伊蚊 Ae. albopictus (10),白跗按蚊 An. albitarsis (1),嗜人按蚊 An. anthropophagus (1),棋斑按蚊 An. tessellatus (1),迷走按蚊 An. vagus (5),三带喙库蚊 Cx. tritaeniorhynchus (3),致倦库蚊 Cx. quinquefasciatus (1),海滨库蚊 Cx. sitiens (2),骚扰阿蚊 Ar. subalbatus (1),常型曼蚊 Ma. uniformis (1)
	保亭Baoting	18°38′N 109°41′E	白纹伊蚊 Ae.albopictus (10),三带喙库蚊 Cx. tritaeniorhynchus (1),白胸库蚊Cx. pallidothorax (2),尖音库蚊 Cx. pipiens (1),骚扰阿蚊 Ar. subalbatus (3)
	三亚Sanya	19°16′N 111°47′E	中华按蚊 An. sinensis (1),三带喙库蚊 Cx. tritaeniorhynchus (1),致倦库蚊 Cx. quinquefasciatus (1)
	乐东Ledong	18°45′N 109°14′E	白纹伊蚊 Ae. albopictus (1),三带喙库蚊 Cx. tritaeniorhynchus (2)
福建省 Fujian Province	古田Gutian	25°13′N 118°15′E	白纹伊蚊 Ae. albopictus (4),致倦库蚊 Cx. quinquefasciatus (1)
浙江省Zhejiang Province	金华Jinhua	28°56′N 119°13′E	白纹伊蚊 Ae. albopictus (3),中华按蚊 An. sinensis (1),二带喙库蚊 Cx. bitaeniorhynchus (1),致倦库蚊 Cx. quinquefasciatus (1),骚扰阿蚊 Ar. subalbatus (1)
山西省Shanxi Province	吕梁Lvliang	34°29′N 113°15′E	淡色库蚊 Cx. pipiens pallens (1)
河南省Henan Province	郑州Zhengzhou	34°38′N 113°43′E	淡色库蚊 Cx. pipiens pallens (3)
河南省Henan Province	开封Kaifeng	34°47′N 114°18′E	窄翅伊蚊 Ae. lineatopennis (1),白纹伊蚊 Ae. albopictus (11),中华按蚊 An. sinensis (2),三带喙库蚊 Cx. tritaeniorhynchus (2)
天津 Tianjin	天津Tianjin	38°56′N 113°12′E	淡色库蚊 Cx. pipiens pallens (1)
内蒙古自治区 Neimenggu Autonomous Region	呼伦贝尔 Hulunbeier	38°39′N 104°04′E	背点伊蚊 Ae. dorsalis (1)
吉林省Jilin Province	吉林Jilin	42°50′N 128°22′E	淡色库蚊 Cx. pipiens pallens (1)
黑龙江Heilongjiang Province	齐齐哈尔Qiqihaer	47°21′N 124°11′E	淡色库蚊 Cx. pipiens pallens (1)
云南省Yunnan Province	昆明Kunming	25°17′N 102°58′E	三带喙库蚊 Cx. tritaeniorhynchus (1),致倦库蚊 Cx. quinquefasciatus (1)
	景洪Jinghong	22°00′N 100°46′E	白纹伊蚊 Ae. albopictus (10)
	勐海Menghai	21°49′N 103°03′E	三带喙库蚊 Cx. tritaeniorhynchus (1)

表1

图1

表2

图2

图3

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蚊种Species	白跗按蚊 An. albitarsis	嗜人按蚊 An. anthropophagus	须喙按蚊 An. barbirostis	中华按蚊 An. sinensis	迷走按蚊 An. vagus	棋斑按蚊 An. tessellatus
白跗按蚊 An. albitarsis	0.015 6	0.010 6	0.004 1	0.011 7	0.012 6	0.005 5
嗜人按蚊An. anthropophagus	0.145 0
须喙按蚊An. barbirostis	0.138 8	0.105 6
中华按蚊An. sinensis	0.143 2	0.010 7	0.103 7
迷走按蚊An. vagus	0.015 8	0.145 1	0.139 9	0.142 4
棋斑按蚊An. tessellatus	0.131 0	0.107 2	0.090 4	0.106 4	0.130 5

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