【作者】 宋锋林；

【导师】 赵彤言；

【作者基本信息】 中国人民解放军军事医学科学院 ， 病原生物学， 2004， 博士

【摘要】 淡色库蚊(Culex pipiens pallens)是我国北方重要的媒介蚊种。随着化学杀虫剂的广泛使用，蚊虫逐渐产生了抗药性，影响到杀虫剂的使用效果，需要加以治理。抗药性机制是蚊虫抗药性监测和治理的基础，由于击倒抗性(Knockdownresistance，kdr)体现了杀虫剂(拟除虫菊酯)与其靶标(钠通道)之间直接的作用，对kdr的研究有助于更深入地揭示蚊虫抗药性产生和发展的过程。本研究的主要结果如下：1 通过抗药性品系选育、RT-PCR、基因序列分析等，初步明确了钠通道ⅡS6节段L1014F型突变是淡色库蚊kdr产生的分子基础。2 建立了检测钠通道基因突变的分子生物学方法(AS-PCR，ASO-PCR)。两种检测方法符合率较高(总符合率为97． 2％)。序列测定结果证实两种方法具有较高的特异性和敏感性。3 分别使用溴氰菊酯和Es-生物丙烯菊酯对淡色库蚊进行逐代筛选，在实验室建立了两种抗药性品系(分别为DEL品系和EBT品系)。4 实验证实DEL品系对拟除虫菊酯抗药性发展为线性增长方式，EBT品系抗药性发展为对数增长方式，前者抗药性发展速率显著快于后者。5 通过增效实验、逐代kdr等位基因频率测定，提示两种品系的抗药性机理有差别。随着抗药性发展(本实验至F8) ，DEL品系以P450介导的代谢抗性为主，EBT品系以击倒抗性为主。6 两种品系的抗性现实遗传力(H2) 比较接近，分别为0． 3804和0． 4192。但DEL品系抗药性表型标准差(0． 42) 高于EBT品系(0． 19) 。表明EBT品系的抗药性已处于相对稳定状态，继续施加筛选压抗药性变化不大；而DEL品系的抗药性则处于不稳定状态，在筛选压力作用下抗药性会继续上升。7统计分析表明，淡色库蚊实验室种群及自然种群对拟除虫菊酷，特别是对Es-生物丙烯菊酷(抗药性以击倒抗性为主)的抗药性与七必.等位基因频率之间有显著的正相关。提示拓介等位基因可作为淡色库蚊对拟除虫菊酷抗药性的分子遗传标记，用于从分子水平上对蚊虫抗药性进行监测。同时也进一步明确了钠通道L1014F型突变是淡色库蚊击倒抗性产生的分子基础。8从686个重组质粒中筛选到8条淡色库蚊微卫星(Mierosatellite)DNA序列，已在GenBank数据库中注册(Aeeession number:AY573280.AY573287)。用其中的4个微卫星DNA作为分子遗传标记，研究淡色库蚊自然种群遗传分化对无动月等位基因扩散的影响。结果表明各自然种群之间有较高的迁移率(平均刀功值为6.11)，提示种群之间的基因流在一定程度上会影响种群拓介等位基因的扩散，进而影响种群对拟除虫菊醋抗药性的发展。更多还原

【Abstract】 Culex pipiens pollens is a principal vector mosquito in northern China. Because of intensive usage of pyrethroid in pest control, the resistance of Cx. pipiens pallens to pyrethroid developed rapidly and widely in many area of China. It’s necessary to reveal the mechanism for surveillance and management of mosquito resistance. Knockdown resistance (kdr) is characterized by the direct interaction between pyrethroid and the target site of insects, which will be well done for displaying the occurrence and development of pyrethroid resistance . It was studied the molecular ecological characteristics of the pyrethroid resistance of lab and natural populations of Cx. pipiens pallens in this research, the results were as followed: 1. The substitution of L1014F in IIS6 segment of sodium channel is the molecular basis of kdr resistance of Cx pipiens pallens through resistance selection and on mosquito populations , RT-PCR, and sequence analysis.2. Allele specific PCR (AS-PCR) protocol was developed for the estimation of kdr allele and another new approach polymerase chain reaction followed by labeled allele-specific oligonucleotide hybridization (ASO-PCR) was also developed for checking the accuracy of AS-PCR. The accordance percentage between two protocols was up to 97.2%. Sequencing alignment proved the two methods had both identity and sensitivity.3. A deltamethrin resistance strain and an esbiothrin resistance strain was selected respectively on a natural population by deltamethrin and esbiothrin, called DEL strain and EBT strain.4. It was found that the increasing style of resistance in DEL strain was linear but was logarithmic in EBT strain during selection. The increasing speed in DEL strain was faster than that in EBT strain.5. It was metabolic resistance that acted an important role in DEL strain but was kdr resistance in EBT strain, based on PBO synergic experiments and kdr allele frequency detection.6. The realized population heritability of resistance in DEL strain and EBT strain was 0.3804 and 0.4192 respectively. The conclusion of resistance balance state in DEL strain but conversely in EBT strain was based upon that phenotypic variance of DEL strain (0.42) was higher than EBT strain (0.19).7. It was showed that the correlation of the prevalence of kdr frequency was significant with the pyrethroid resistance level in natural populations, which suggested that the kdr frequency can be a molecular marker served for resistance monitor on mosquito population.8. It was revealed that there was higher migration frequency between compared populations (total Nm was 6.11 across all populations) based on the genetic differentiation among natural populations with the microsatellite loci isolated from the genome pool. It was implied that the gene flow among populations will affect the kdr gene expanding, which will bring on the change of the resistance development among population.更多还原