Mitochondrial DNA sequence analysis of cytochrome oxidase subunit Ⅰ gene from 28 mosquito species in China

Abstract

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

CLC Number:

R384.11

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.

Figures/Tables 5

Table 1

The collection sites, species and number of mosquitoes

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)

Table 1

Fig. 1

Table 2

Fig. 2

Fig. 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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