我国赫坎按蚊种团的分子鉴别及中华按蚊的区系分布研究

doi:10.12140/j.issn.1000-7423.2020.01.009

中国寄生虫学与寄生虫病杂志 ›› 2020, Vol. 38 ›› Issue (1): 58-67.doi: 10.12140/j.issn.1000-7423.2020.01.009

我国赫坎按蚊种团的分子鉴别及中华按蚊的区系分布研究

彭恒¹, 陈翰明², 陈辉莹², 王琰², 李翔宇¹, 李基旭³, 杨振洲⁴, 马雅军^2,*

1中国人民解放军海军军医大学, 病原生物学教研室;
2中国人民解放军海军军医大学, 热带病学教研室,上海 200433;
3 吉林省延边州疾病预防控制中心病媒与寄生虫病防治科,延吉 133001;
4 中国人民解放军疾病预防控制中心,北京 100071

收稿日期:2019-06-13 出版日期:2020-02-28 发布日期:2020-03-19
通讯作者: 马雅军,E-mail: yajun_ma@163.com
作者简介:彭恒（1982-）,男,博士,副教授,主要从事病原生物学与媒介生物学研究。E-mail: pengheng0923@126.com
基金资助:
国家科技重大专项（No. 2017ZX10303404002001）,上海市自然科学基金（No. 19ZR1469600）

Molecular identification of Anopheles hyrcanus group and faunal distribution of Anopheles sinensis (Diptera ∶ Culicidae)in China

PENG Heng¹, CHEN Han-ming², CHEN Hui-ying², WANG Yan², LI Xiang-yu¹, LI Ji-xu³, YANG Zhen-zhou⁴, MA Ya-jun^2,*

1 Department of Medical Microbiology and Parasitology;
2 Department of Tropical Diseases, Navy Medical University, Shanghai 200433, China;
3 Division of Vectors and Parasitic Diseases, Yanbian Center for Disease Control and Prevention, Yanji 133001, China;
4 Institute of Disease Control and Prevention, People’s Liberation Army of China, Beijing 100071, China;

Received:2019-06-13 Online:2020-02-28 Published:2020-03-19
Contact: E-mail: yajun_ma@163.com
Supported by:
Supported by National Science and Technology Major Project (No. 2017ZX10303404002001) and Natural Science Foundation of Shanghai (No. 19ZR1469600)

摘要/Abstract

摘要： 目的改进赫坎按蚊种团部分成员种的多重PCR鉴别方法,鉴别赫坎按蚊种团中的中华按蚊,研究我国中华按蚊的区系分布及其影响因素。方法依据赫坎按蚊种团成员种中华按蚊、八代按蚊、雷氏按蚊、比伦按蚊和克莱按蚊的核糖体DNA内转录间隔区2（rDNA-ITS2）序列差异设计种特异引物,改进多重PCR分子鉴别方法。2013-2018年,在我国8个省（直辖市、自治区）共18个地点,以诱虫灯和人工吸取相结合的方法采集按蚊,依据形态特征初步鉴定为赫坎按蚊种团的成员种。提取单只蚊虫基因组DNA,使用改进的多重PCR方法鉴定种类。对多重PCR法无扩增产物的个体分析rDNA-ITS2序列,在GenBank上进行BLAST比对,确定其种类。查找我国以及韩国和俄罗斯远东地区用分子特征鉴别为中华按蚊的文献,结合本研究结果,汇总中华按蚊采集地的地理位置和气候数据,使用地理探测器模型计算影响决定力q值,分析经度、纬度、年平均气温和年平均降雨量对中华按蚊分布的影响。结果改进的多重PCR法一次扩增即可依据扩增片段大小鉴别赫坎按蚊种团的5个成员种：中华按蚊（490 bp）、雷氏按蚊（313 bp）、八代按蚊（216 bp）、克莱按蚊（386 bp）和比伦按蚊（165 bp）。在我国18个采集点共捕获赫坎按蚊种团按蚊365只,多重PCR鉴别为中华按蚊114只（来自陕西、安徽与山东的采集点）、八代按蚊34只、雷氏按蚊9只、克莱按蚊181只和比伦按蚊5只。22只多重PCR无扩增产物的蚊虫中,经rDNA-ITS2序列分析鉴定为贵阳按蚊2只、类中华按蚊1只、朝鲜按蚊8只、林氏按蚊7只和帕氏按蚊4只。获取用分子特征鉴别为中华按蚊的文献17篇,共汇总了101个采集地信息,其中有中华按蚊分布的采集点80个,无中华按蚊分布的21个地点。地理探测器软件计算获得的q值,从大到小依次为0.592 0（年平均气温）、0.507 2（纬度）、0.351 2（经度）和0.214 4（年平均降雨量）。中华按蚊的分布与年平均气温关系最为密切,其次是纬度。综合分析分布地的纬度和年平均温度等结果显示,年平均气温10 ℃可以作为划分中华按蚊在我国分布北界线的依据。我国中华按蚊的分布范围包括云南、贵州、重庆、河南、山东、天津、江苏、安徽、湖北、浙江、上海、福建、江西、广西、广东、海南等省（直辖市、自治区）及台湾、香港、澳门特别行政区的全境,以及西藏、四川、甘肃、陕西、山西、河北、北京、辽宁等省（直辖市、自治区）的南部部分地区。结论改进的赫坎按蚊种团多重PCR分子鉴定方法快速简单、客观可靠。综合分析显示划分中华按蚊在我国分布北界线的依据是年平均气温10 ℃线,中华按蚊在我国的分布应小于之前记载的范围。

关键词: 赫坎按蚊种团, 中华按蚊复合体, 分子鉴别, 区系分布

Abstract: Objective To improve the multiplex PCR molecular methods to identify species of Anopheles hyrcanus group, and investigate the faunal distribution of An. sinensis in China and the factors influencing its distribution. Methods The species-specific primers were designed based on rDNA-ITS2 sequences of Anopheles (An. sinensis, An. lesteri, An. yatsushiroensis, An. kleini and An. belenrae) to improve the specificity and sensitivity of the multiplex PCR method to identify the Anopheles mosquito species. Anopheles mosquitoes were collected from 18 locations in 8 provinces (municipalities or autonomous regions) in China using light trap and entomological aspirator from 2013 to 2018. Those mosquitoes identified as members of An. hyrcanus group based on morphological characteristics were used for further analysis. The genomic DNA of individual mosquito was extracted, and the multiplex PCR assay was used to determine the species of An. hyrcanus group. For those mosquitoes without multiplex PCR products, the rDNA-ITS2 was amplified and sequenced, and the sequences were subjected to BLAST of the NCBI GenBank database to determine the species. Based on the results of this study and the review of 17 publications that identified An. sinensis using molecular biological methods in China, South Korea and the Far East of Russian, as well as the collected geographical location and climate data, the influence q value was calculated using the geo-detector software. The effects of geographical longitude, latitude, annual average temperature and rainfall on the distribution of An. sinensis were analyzed. Results The improved multiplex PCR method was able to simultaneously identify five members of the An. hyrcanus group based on the size of the amplified fragments as An. sinensis (490 bp), An. lesteri (313 bp), An. yatsushiroensis (216 bp), An. kleini (386 bp) and An. belenrae (165 bp). In this study, the species of 365 Anopheles mosquitoes were identified by multiplex PCR, including An. sinensis (n = 114, collected from Shaanxi, Anhui and Shandong), An. yatsushiroensis (n = 34), An. lesteri (n = 9), An. kleini (n = 181), An. belenrae (n = 5). The 22 individuals without acquiring amplified products were identified as An. kweiyangensis (n = 2), An. sineroides (n = 1), An. koreicus (n = 8), An. lindesayi (n = 7) and An. pattoni (n = 4) by rDNA-ITS2 sequencing. The geographic and ecological data were collected from 101 sites, including 80 sites with An. sinensis and 21 sites without An. sinensis distribution. The calculated q values were 0.592 0 for annual average temperature, 0.507 2 for latitude, 0.351 2 for longitude and 0.214 4 for annual average rainfall. The results indicate that the most significant factor affecting the distribution of An. sinensis is temperature, followed by latitude. Comprehensive analysis of the impact of latitude and annual average temperature on the distribution of Anopheles revealed that the annual average temperature of 10 ℃ could be used as the reference to determine the northern boundary of An. sinensis distribution in China. An. sinensis was distributed in the entire provinces (municipalities or autonomous regions) of Yunnan, Guizhou, Chongqing, Henan, Shandong, Tianjin, Jiangsu, Anhui, Hubei, Zhejiang, Shanghai, Fujian, Jiangxi, Guangxi, Guangdong, Hainan, Taiwan, Hong Kong and Macao, and the southern parts of the Tibet, Sichuan, Gansu, Shaanxi, Shanxi, Hebei, Beijing and Liaoning. Conclusion The improved multiplex PCR assay used to identify An. hyrcanus group is fast, simple and reliable. The annual average temperature of 10 ℃ could be used as reference to determine the northern boundary for the geographical distribution of An. sinensis in China, and the distribution range is smaller than that in the previously recorded document.

Key words: Hyrcanus group, Anopheles sinensis complex, Molecular identification, Faunal distribution

中图分类号:

R384.111

彭恒, 陈翰明, 陈辉莹, 王琰, 李翔宇, 李基旭, 杨振洲, 马雅军. 我国赫坎按蚊种团的分子鉴别及中华按蚊的区系分布研究[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(1): 58-67.

PENG Heng, CHEN Han-ming, CHEN Hui-ying, WANG Yan, LI Xiang-yu, LI Ji-xu, YANG Zhen-zhou, MA Ya-jun. Molecular identification of Anopheles hyrcanus group and faunal distribution of Anopheles sinensis (Diptera ∶ Culicidae)in China[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2020, 38(1): 58-67.

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我国赫坎按蚊种团的分子鉴别及中华按蚊的区系分布研究

Molecular identification of Anopheles hyrcanus group and faunal distribution of Anopheles sinensis (Diptera ∶ Culicidae)in China

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[1]	方圆，施文琦，张仪*. 赫坎按蚊种团（双翅目∶蚊科）分类研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2016, 34(6): 16-565-570.
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[3]	马雅军;瞿逢伊;曹毓存;杨宝金. 我国雷氏按蚊和嗜人按蚊的分子鉴别和分类地位的探讨[J]. 中国寄生虫学与寄生虫病杂志, 2000, 18(6): 2-328.
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