吉林省延边地区图们江流域按蚊形态学及基因特征分析

Species identification of Anopheles mosquitoes from the Tumen river basin in Yanbian area of Jilin province based on morphological and molecular characteristics

目的了解吉林省延边朝鲜族自治州(延边州)图们江流域按蚊种类,掌握常见按蚊形态特征和基因特征。方法于2012-2013年5-10月选择吉林省延边州图们市月晴镇、曲水镇和龙井市开山屯镇3个边境村,利用诱蚊灯法采集当地按蚊雌成虫,提取其基因组DNA,利用其核糖体核酸内转录间隔2区段(r DNA ITS2)基因特征,应用PCR基因鉴别技术,确定其种类。对按蚊基因组DNA扩增产物进行测序,与GenBank中按蚊序列进行同源性分析,进一步确定延边地区图们江流域按蚊基因特征。根据分子生物学鉴定结果,反向分析其形态特征。结果在延边州图们江流域共发现赫坎按蚊种团(Anopheles hycranus group)类中华按蚊、克莱按蚊、雷氏按蚊、暗灰按蚊(近黑按蚊)和比伦按蚊,其蚊种基因组DNA扩增序列与GenBank中已发表相应按蚊序列的同源性达97%~100%。克莱按蚊、暗灰(近黑)按蚊和雷氏按蚊的亚缘脉白斑、亚尖端白斑、尖端白斑和翅5.2纵脉末端繸斑(fringe)在形态学上存在明显种特异性。结论在延边州图们江流域未发现中华按蚊和朝鲜按蚊。比伦按蚊为该地首次发现,与已发现的克莱按蚊同为该地首次纪录种。将原纪录种嗜人按蚊订正为雷氏按蚊,原八代按蚊订正为暗灰按蚊。利用按蚊雌成虫翅白斑种特异性可对克莱按蚊、暗灰(近黑)按蚊和雷氏按蚊等新蚊种和近缘种快速、便捷地进行形态学鉴定。

Objective To understand the species of Anopheles mosquitoes in the Tumen river basin in Yanbian area of Jilinprovince, and master the morphological and gene characteristics of common Anopheles mosquitoes. Methods The threeborder towns were chosen in the Tumen river basin in Yanbian area, Yueqing, Qushui, Kaishan in 2012-2013. From May toOctober every year, the black light trap was used to collect the female adult of Anopheles monthly, from which the genomeDNA was extracted. The PCR gene identification technique was used to identify the Anopheles species according to thecharacterization of its ribosomal nucleic acid internal transcribed spacer 2 segment（rDNA ITS2）gene. The genome DNAamplification products of Anopheles were sequenced, and homology analysis with gene sequence of Anopheles in GenBank ofNCBI was carried out to further determine the genetic characteristics of Anopheles mosquitoes in Tumen river basin ofYanbian area. Finally, according to the molecular identification results, reverse analyses was conducted to provide theconfirmation for morphological classification. Results Five Anopheles species including An. hycranus group An. sineroides,An. kleini, An. lesteri, An. pullus, and An. belenrae were found in the study area, and the homology between their genomicDNA amplification sequences and that of Anopheles published in GenBank reached 97%-100%. The subcostal, preapical,apical fringe and fringe had obvious species specificity in morphology among An. kleini, An. lesteri, and An. pullus.Conclusion Anopheles sinensis and An. koreicus were not found in Tumen river basin of Yanbian area, An. belenrae was found for the first time in this area and became the new recorded species with An. kleini. The initial recorded species of An. anthropophagus was revised to An. lesteri. The An. yatsushiroensis was revised to An. pullus. According to the white asspots of wings as species specificity of female adults Anopheles mosquitoes, we can identify the morphology of new andrelated species rapidly and conveniently, such as An. kleini, An. lesteri, and An. pullus.