云南省连斑库蠓形态学和分子生物学鉴定

Morphological and molecular biological identification of Culicoides jacobsoni collected in Yunnan province

目的采用形态学和分子生物学方法,系统鉴定在云南省采集的连斑库蠓。方法2020—2021年在云南省江城、勐腊、广南、双江县用灯诱法采集蠓虫,根据形态特征在显微镜下挑选出初步鉴定为连斑库蠓的蠓虫,采用无损伤形态的方法提取库蠓基因组DNA,扩增COI基因部分序列和ITS-1基因全长序列,使用Mega 6和MegAlign软件进行分析,同时用压片法对库蠓进行系统形态学鉴定。结果共采集到连斑库蠓5只,其中雌虫4只、雄虫1只。鉴别特征:雌虫翅径5室有1个淡斑,通常呈“V”型,凹口朝远端,臀室的3个淡斑由1条淡色带相连接;雄虫第9腹板后缘中部凹陷宽而深,阳茎中叶近锥形,抱器基节的背、腹踝较细而长,抱器端节细长向内侧弯曲。形态特征符合连斑库蠓鉴别特征。5只连斑库蠓COI基因序列375 bp,ITS-1基因全长序列396 bp,在COI基因和ITS-1基因的进化树上,云南省的连斑库蠓与中国和其他国家的连斑库蠓处于同一进化分支,COI基因核苷酸同源性为97.6%~100.0%,ITS-1基因核苷酸同源性99.0%~100.0%。COI基因和ITS-1基因种内平均遗传距离分别为0.005(0.000~0.021)和0.004(0.000~0.012)。结论通过形态学鉴定和COI基因、ITS-1基因鉴定相结合的方法对连斑库蠓雌虫、雄虫进行了全面鉴定,3种鉴定方法互为佐证,为今后连斑库蠓的鉴定提供了依据。

Objective To identify Culicoides jacobsoni collected in Yunnan province by using morphological and molecular biology identification methods.Methods The midges were collected by lamp-trapping method in Jiangcheng,Mengla,Guangnan and Shuangjiang counties of Yunnan Province from 2020 to 2021.The genome DNA of Culicoides was extracted by non-invasive method,and COI,ITS-1 genes were anoplified by PCR.The sequence analysis were performed by using Mega6 and MegAlign softwares,simultaneously,the systematic morphological identification of Culicoides was carried out by tableting method.Results A total of 5 C.jacobsoni were trapped,including 4 females and 1 male.The female C.jacobsoni:distal pale spot in cell R5 more or less V-shaped,distal side usually concave,three light spots in the cell M2 connected by a pale streak in basal portion.The male C.jacobsoni:ninth sternum with moderately deep caudomedian excavation,which was wide and deep aedeagus approximately tapered,Basistyle with dorsal and ventral roots slender,dististyle slender and curved.The morphological characteristics were consistent with the identification characteristics of C.jacobsoni.The COI gene sequence of C.jacobsoni was 375 bp,and the fulllength ITS-1 gene sequence was 396 bp.Phylogenetic analysis based on COI and ITS-1 genes showed that C.jacobsoni collected in Yunnan province and other countries were all located in an independent evolutionary branch.The nucleotide homology of COI genes were 97.6%-100.0%,ITS-1 genes were 99.0%-100.0%.The average genetic distances within species of COI and ITS-1 gene were 0.005(0.000-0.021)and 0.004(0.000-0.012),respectively.Conclusion The female and male C.jacobsoni was comprehensively,identified by the combination of morphological identification and COI,ITS-1 gene identification.The three identification methods proved each other,which provided a basis for the future identification of C.jacobsoni.