贵州白纹伊蚊感染沃尔巴克氏体以及WO噬菌体的初步调查

A preliminary investigation of symbiotic bacteria(Wolbachia)and The WO phage infecting Aedes albopictus in Guizhou Province

目的了解贵州白纹伊蚊种群中沃尔巴克氏体(Wolbachia)以及WO噬菌体的感染情况。方法于2017年在贵州省贵阳市、都匀市、铜仁市、凯里市、遵义市、兴义市、贵安新区、毕节市采用勺舀法采集白纹伊蚊幼虫标本,实验室饲养至成蚊。提取雌蚊基因组DNA,PCR扩增沃尔巴克氏体表面蛋白基因(Wolbachia surface protein,wsp)和WO噬菌体衣壳蛋白的核糖体S7基因(Ribosomal S7 gene,orf7)。分别从8个地区的阳性检测标本中随机选取10个样品进行测序,使用DNAStar 7.0软件分析基因的基本特征;采用MEGA 4.0软件对所获基因序列进行分析比对,并构建系统进化树;采用SPSS 20.0软件对WO噬菌体侵染wAlbA、wAlbB株沃尔巴克氏体的检测结果进行关联性分析。结果贵州省8个地区的白纹伊蚊wAlbA和wAlbB株沃尔巴克氏体超感染率为84.30%(322/382),wAlbA株、wAlbB株沃尔巴克氏体单感染率分别为4.97%(19/382)和3.66%(14/382),WO噬菌体的感染率为76.44%(292/382)。通过测序获得沃尔巴克氏体的wsp序列,其中wAlbA株(379 bp)80条,wAlbB株(501 bp)78条;获得WO噬菌体的orf7序列(263 bp)73条。比对分析显示贵州省8个地区获得所有wAlbA株、wAlbB株的wsp序列、orf7序列的同源性均为100%。确切概率法分析WO噬菌体侵染与wAlbA和wAlbB株沃尔巴克氏体超感染有关联性(P<0.05)。结论贵州白纹伊蚊种群普遍感染WO噬菌体和沃尔巴克氏体,且以超感染wAlbA和wAlbB株沃尔巴克氏体为主。WO噬菌体主要侵染的对象是超感染wAlbA和wAlbB株的沃尔巴克氏体。

Objective To investigate the infection of Aedes albopictus by symbiotic bacteria(Wolbachia) and the WO phage in Guizhou Province. Methods In 2017, Ae. albopictus larvae from 8 regions of Guizhou Province(Guiyang, Duyun, Tongren, Kaili, Zunyi, Xingyi, Guian, and Bijie) were collected with a dipper. Genomic DNA was extracted from female mosquitoes, and the Wolbachia surface protein gene(wsp) and the ribosomal S7 gene(orf7) of the WO phage were amplified with PCR. Ten samples were randomly selected from the positive test samples from the 8 regions for sequencing, and the basic characteristics of the genes were analyzed with the package DNAStar 7.0. Phylogenetic trees were constructed with the software MEGA 4.0. The correlation between infection with the WO page and detection of the wAlbA and wAlbB strains was analyzed using the software SPSS 20.0. Results The rate at which Ae. albopictus was infected with the wAlbA and wAlbB strains of Wolbachia was 84.30%(322/382) in 8 regions of Guizhou Province. Ae. albopictus was infected with only the wAlbA strain of Wolbachia at a rate of 4.97%(19/382) and with only the wAlbB strain at a rate of 3.66%(14/382). The rate of infection with the WO phage was 76.44%(292/382). The wsp sequences of Wolbachia were obtained by sequencing, including 80 strains of wAlbA(379 bp) and 78 strains of wAlbB(501 bp);73 orf7 gene sequences from the WO phage(263 bp) were obtained. A comparison of sequences indicated that the orf7 sequences and the wsp sequences of all wAlbA and wAlbB strains in 8 regions of Guizhou Province were 100% similar. The exact probability method was used to analyze the association between WO phage infection and super-infection with the wAlbA and wAlbB strains of Wolbachia(P<0.05). Conclusion The population of Ae. albopictus in Guizhou was generally infected with the WO phage and Wolbachia, the main strains of which were wAlbA and wAlbB. The WO phage mainly targets mosquitoes super-infected with the wAlbA and wAlbB strains of Wolbachia.