Dary O, Georghiou G P, Parsons E, Pasteur N
Department of Entomology, University of California, Riverside 92521.
J Econ Entomol. 1991 Feb;84(1):28-33. doi: 10.1093/jee/84.1.28.
A test was developed to detect the presence of insecticide-resistant acetylcholinesterase (AChE) in single insects based on the quasipermanent binding of proteins onto blotting membranes. The method is simple, sensitive, requires inexpensive equipment, and produces a permanent record of results. AChE activity is revealed by the Karnovsky & Roots staining technique in the presence of propoxur, or after exposure of the membrane to paraoxon and rinsing with water. We chose insecticide concentrations that inhibited the sensitive AChE while allowing detectable residual activity of the resistant AChE to remain. By comparing the staining of insecticide-treated and control membranes, susceptible and resistant genotypes for the AChE gene could be distinguished in laboratory strains of mosquitoes (Culex spp. and Anopheles albimanus Wiedemann) and the house fly (Musca domestica L.). Resistant AChE from mosquitoes was less susceptible both to propoxur and paraoxon than the corresponding sensitive AChE, whereas resistant AChE from house fly was less susceptible mainly to paraoxon. The technique worked well for mosquito adults and house fly heads but not for mosquito larvae. Blotted AChE did not show detectable loss of activity during storage of the membranes for 3 wk at 25 degrees C. Storage is an important asset of the technique because transportation of live insect material to the laboratory may not be necessary.
基于蛋白质在印迹膜上的准永久性结合，开发了一种用于检测单个昆虫体内抗杀虫剂乙酰胆碱酯酶（AChE）的方法。该方法简单、灵敏，所需设备便宜，且能产生结果的永久性记录。在残杀威存在的情况下，或在膜暴露于对氧磷并用水冲洗后，通过卡诺夫斯基和罗茨染色技术显示AChE活性。我们选择了能抑制敏感AChE同时使抗性AChE仍具有可检测残留活性的杀虫剂浓度。通过比较经杀虫剂处理的膜和对照膜的染色情况，在蚊子（库蚊属和白纹伊蚊）和家蝇（家蝇）的实验室品系中可以区分AChE基因的敏感和抗性基因型。来自蚊子的抗性AChE比对氧磷和残杀威的敏感性低于相应的敏感AChE，而来自家蝇的抗性AChE主要对对氧磷的敏感性较低。该技术对成年蚊子和家蝇头部效果良好，但对蚊子幼虫无效。在25摄氏度下将膜储存3周期间，印迹的AChE未显示出可检测到的活性损失。储存是该技术的一项重要优势，因为可能无需将活昆虫材料运送到实验室。
