格林库蠓的形态学和分子生物学鉴定

Morphological and molecular biological identification of Culicoides kelinensis

目的采用形态学和分子生物学鉴定的方法,对云南省贡山县采集的格林库蠓雌虫、雄虫进行系统鉴定。方法2017年8月在贡山县用灯诱法采集蠓虫标本,在体式显微镜下挑选出格林库蠓,无损伤法提取DNA,扩增COI基因、ITS-1基因进行序列分析;再将虫体制作玻片标本进行形态学鉴定。结果在贡山县采集到6只格林库蠓,其中雌虫5只,雄虫1只。鉴别特征:两复眼连接,小眼面间无柔毛,AR 1.04,PR 4.50,翅径2室全部淡色,翅面有由淡斑形成形状不甚规则的3条淡色横带,有2个受精囊,略不等大;雄虫第9背板后缘有1对小而尖的侧突,阳茎中叶中部宽大,端部呈细长的棒形,阳基侧突分离,端部有细分枝。在COI基因和ITS-1基因进化树上,格林库蠓均位于独立的进化分支,COI基因核苷酸同源性为100.0%,ITS-1基因核苷酸同源性99.2%~100.0%。结论格林库蠓雌虫形态学鉴定结果与原描述相同,补充了雄虫尾器第9背板后缘有1对钝突的鉴别特征,同时获得了格林库蠓雌虫、雄虫COI基因和ITS-1基因序列信息,为格林库蠓的准确鉴定提供了依据。

Objective To systematically identify Culicoides kelinensis collected in Gongshan county of Yunnan province by using morphological and molecular biological methods.Methods In August 2017,the midges were collected by using the trap lamp in Gongshan county.Culicoides kelinensis was selected by morphological identification,DNA was extracted by non-destructive tissue digestion,COI gene and ITS-1 gene were amplified for sequence analysis.Culicoides kelinensis tablet was made for identification based on morphology.Results A total of 5 females and 1 male Culicoides kelinensis were collected from Gongshan county in Yunnan province.Diagnosis:the female Culicoides kelinensis was eyes continguous,bare,AR 1.04,PR 4.50 and cell R2 with pale,the wing surface had 3 transverse bands of light color formed by pale spots,spermathecae 2,unequal.The male hypopygium Culicoides kelinensis with 1 pair lateral process of small and pointed,the adeagus with broad in the middle,a slender rod at the end,parameresis separatedand end with small branches.The phylogenetic analysis based on COI gene and ITS-1 gene sequences showed that Culicoides kelinensis was located in an independent evolutionary branch.The nucleotide homology of COI gene was 100.0%,and the nucleotide homology of ITS-1 gene was 99.2%-100.0%between them.Conclusion The results of morphological identification of Culicoides kelinensis were the same as those of types specimens.The sequence information of COI gene and ITS-1 gene of female and male Culicoides kelinensis were obtained,which will provide the basis for identification of Culicoides kelinensis in the future.