嗜人按蚊和中华按蚊相关细菌差异的初步探讨

Primary study on the difference of bacteria borne by two kinds of Anopheles mosquitoes

目的通过对河南省桐柏地区嗜人按蚊和中华按蚊的DNA提取物中细菌DNA的拷贝数和种类是否存在差异进行初步探讨,为进一步深入开展蚊媒中肠细菌与蚊媒传播疟疾能力之间的关系提供理论基础。方法以保存于75%乙醇中的成蚊为研究对象,首先应用聚合酶链反应(PCR)和聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法确定蚊种,在此基础上,用PCR比较蚊虫细菌拷贝数的异同,同时克隆并酶切4只按蚊中细菌的16S rDNA序列,对酶切结果进行统计分析。结果共明确23只按蚊的种类。不同蚊种DNA中所含细菌拷贝数存在差异,但与按蚊的种类没有关系。4只按蚊DNA中共发现19种细菌16S rDNA的酶切类型。不同中华按蚊个体间存在共同的细菌DNA酶切类型;嗜人按蚊仅呈现2种细菌DNA酶切类型,均与中华按蚊不同。结论嗜人按蚊和中华按蚊中含有的细菌种类存在差异,该项研究为我们进一步研究按蚊中肠细菌是否会影响按蚊对疟原虫的感染性奠定了基础。

Objective Bacteria in midguts of adult Anopheles mosquitoes could partially or completely inhibit oocyst formation of Plasmodium. To provide theoretical knowledge and technical information for the study on the relationship between the vector capacity and the bacteria in two kinds of mosquitoes of An. anthropophagus and An. sinensis, quantitative and categorical differences of bacteria in their genome DNA were estimated. Methods The mosquito samples were collected from Tongbai county, Henan province, and stored in 75 % ethanol at -20℃. Their classification was re-identified by PCR and PCR-RFLP of ITS2 gene. Quantitative difference of bacteria in mosquitoes was estimated by PCR products of partial 16S rDNA sequence of bacteria. Complete 16S rDNA sequence of bacteria in 4 mosquitoe~, including three An. sinensis and one An. anthropophagus, were cloned into T-vector and digested by restrictive enzyme Rsa I . The digested patterns were counted and analyzed. Results The category of 23 mosquitoes was confirmed based on PCR and PCR-RFLP. It has difference among bacteria copies of different mosquito DNA but it was not related with mosquitoes species. Totally, 19 digested patterns of bacteria were found in 4 mosquitoes. One common pattern widely existed in An. sinensis. Bacteria in An. anthropophagus only showed two patterns, different from that of An. sinensis. Conclusion Different kinds of bacteria located in An. anthropophagus and An. sinensis. This study supplied useful knowledge and helpful techniques to our further research in the influence of bacteria in midgut of An. sinensis and An. anthropophagus on natural transmission of malaria.