【作者】 杨美玲；

【导师】 马恩波；

【作者基本信息】 山西大学 ， 动物学， 2005， 硕士

【摘要】 中华稻蝗是我国主要农业害虫之一,20世纪90年代以来,受异常气候及生态环境恶化等因素的影响,蝗虫发生逐年加重,对农业生产为害极其严重。我国目前控制蝗虫仍以化学防治为主,由于农药品种及数量无限制地使用,中华稻蝗对常用有机磷杀虫剂产生了抗药性,这个问题已经引起了相关植保部门的注意。 害虫对杀虫剂的抗性机制主要有代谢抗性和靶标抗性。代谢抗性是指对杀虫剂起水解或隔离作用的酶发生改变,从而对杀虫剂起到解毒作用,相关解毒酶系有酯酶、谷胱甘肽-S-转移酶等;靶标抗性是指由于杀虫剂作用靶标敏感度降低而产生的抗性。如杀虫剂的作用靶标—乙酰胆碱酯酶。这两种抗性机理在果蝇、家蝇、棉铃虫、小菜蛾、蚊类等害虫中已经有深入的研究。 本文选取我国中华稻蝗江苏徐州、山西临猗、山西代县和山西繁峙四个杀虫剂选择压力不同的中华稻蝗自然种群进行有机磷抗性相关酶的生化特性研究。江苏徐州种群采自水库边的玉米田及附近草丛中,这里炎热湿润的气候具备中华稻蝗生存的生态条件,适合中华稻蝗的发育、生长、繁殖,多年以来这里一直是重点化学防治区,有机磷作为一种有效的杀虫剂而被广泛使用。而临猗种群采自山西临猗县黄河边的荒滩上,这里的土壤沙化严重,该地的蝗虫治理以监测防治为主较少使用化学药剂;代县种群采自稻田中,近几年这里连续使用过有机磷农药。繁峙种群采自水沟边的草丛中,马拉硫磷在这里的使用频率较低。通过马拉硫磷对这四个中华稻蝗种群的生物测定,结果表明,山西临猗和山西繁峙种群对马拉硫磷的敏感性较高,而山西代县和江苏徐州种群的较低。江苏徐州、山西临猗及山西代县三种群对马拉硫磷的LD₅₀值分别是山西繁峙种群的3.06、1.24及2.02倍,这说明,我国不同地区杀虫剂选择压力的差异已经造成当地中华稻蝗种群对常用杀虫剂的敏感性下降程度的不同。 对上述四个中华稻蝗种群酯酶的生化特性进行了比较研究。酯酶动更多还原

【Abstract】 The grasshopper Oxya chinensis （Thunberg） （Orthoptera: Acridoidea） is a major insect pest in China, which seriously threatens the agriculture production. Due to the unwonted climate and the deteriorated ecological environment, the destructive outbreaks of locust had been increasing in China since 1990s. So far the chemical controls are still the major measures taken in locust management programs in China. The resistance to organophosphate compounds has been found in the Oxya chinensis because of the frequent use of insecticides both in quantity and variety, which has been paid attention to by the Chinese plant protection agencies.There are two major mechanisms in resistance development, which are enhanced metabolic detoxification primarily via sequestration or hydrolyzing of insecticides by large amounts of enzymes present such as metabolic enzymes of esterases and glutathione-S-transferases （GST）, and reduced sensitivity of insecticide target-sites such as acetylcholinesterase （AChE）. The resistance mechanisms have been widely studied in fruit fly, housefly, cotton bollworm, diamond back moth and mosquitoes.This dissertation studied biochemical characteristics of enzymes associated to OP resistance in field populations of Oxya chinensis （Thunberg） from Xuzhou-Jiangsu, Linyi-Shanxi, Daixian-Shanxi and Fanshi-Shanxi, where the insecticide selection pressures are considered different. The Xuzhou population is distributed in the reservoir-shore area which consist of a maize field and a weed field, where O. chinensis is feeding mainly on maize and gramineous plants and insecticides mainly organophosphates were applied in recent years for protecting crop. Furthermore, the higher temperature and humidity in Xuzhou are favorable to the survival of O. chinensis. However, the Linyi population belongs to a river flood area, which is a desolate sand, and some weed including primarily gramineous plants are abundant food supply for O. chinensis, insecticides were seldom used for O. chinensis control. Daixian population belongs to the rice field, and OPs havebeen used continuously in recent years. Fanshi population is distributed in the area covered with thick grasses nearby a ditch, and there were little insecticides used for O. chinensis management. Comparison of malathion susceptibility of O. chinensis in four populations was presented that the Xuzhou and Daixian populations were less susceptible to malathion than the Linyi and Fanshi populations. LD₅0 of the Xuzhou population, the Linyi population and the Daixian population were 3.06, 1.24 and 2.02-fold higher than that of the Fanshi population, which shows that different populations have varies susceptibility to malathion due to different insecticide pressure.General esterases of Oxya chinensis （Thunberg） collected from the four locations were compared. Kinetic studies demonstrated that general esterases in four populations of O. chinensis hydrolyze a-NA, a-NB and |3-NA as substrate. Among the four substrates, a-NA seemed to be the most favorable substrate for general esterases of the O. chinensis. Inhibition studies of the esterases using four inhibitors （i. e., paraoxon, malaoxon, eserine, and carbaryl） indicated that most of general esterases in the four populations were B-type. Moreover, significant difference in general esterases activities was observed among four populations. Fanshi-Shanxi population showed the lowest general esterase activities with substrates of a-NA, a-NB and (3-NA. For the samples from Xuzhou-Jiangsu, Linyi-Shanxi and Daixian-Shanxi, the esterases activities were 2.01-, 1.07-, and 1.70-fold higher for females and 2.68-, 1.13-, and 2.18-fold higher for males using a-NA as substrate; and 1.80-, 1.25-, and 1.10-fold higher for females and 1.70-, 1.29-, and 1.10-fold higher for males using a-NB as substrate; and 1.96-, 1.60-, and 1.91-fold higher for females and 1.80-, 1.48-, and 1.79-fold higher for males using （3-NA as substrate than those from Fanshi-Shanxi population. The distributing of general esterases activities in O. chinensis individuals showed significant difference among four populations. There were more individuals with low esterase activity in Linyi-Shanxi population and Fanshi-Shanxi population, and more individuals with high esterase activity in Xuzhou-Jiangsupopulation. However, there were so many individuals with acitivity of the same span in the Daixian-Shanxi population. Non-denaturing PAGE analyses of general esterases from the two populations of Xuzhou-Jiangsu and Dianxian-Shanxi displayed similar band patterns of esterases, which were characterized by the presence of three slowly migrating bands with deep staining in both populations. However, only one band was present with weak staining in both Linyi-Shanxi and Fanshi-Shanxi populations. In addition, there were rapidly migrating bands with deeper staining in Xuzhou-Jiangsu population than those in the other populations. It is suggested that the three slow esterase bands as shown in non-denaturing PAGE be responsible for the higher esterase activities in Xuzhou-Jiangsu and Daixian-Shanxi populations and the large amount of esterases observed in the rapidly migrating bands in Xuzhou-Jiangsu population might be related to the higher esterase activities. We proposed that increased general esterases activity might attribute to the decreased sensitive to malathion both in Xuzhou-Jiangsu and Daixian-Shanxi populations. The difference of general esterases activity was correlated with the insecticides application background in these four O. chinensis breeding areas. It was proposed that esterases activity might be useful to assess the resistance of O. chinensis in fields.Among the four Oxya chinensis populations, Linyi-Shanxi population showed the lowest glutathione-S-transferase activities. The activities of glutathione-S-transferase were found 1.78-, 1.22-, and 1.3 3-fold higher for females from Xuzhou-Jiangsu, Daixian-Shanxi and Fanshi-Shanxi populations; and 1.32-, 1.25-, and 1.44-fold higher for males from Xuzhou-Jiangsu, Daixian-Shanxi and Fanshi-Shanxi than those from Linyi-Shanxi population. The distributing of glutathione-S-transferase activities in O. chinensis individuals showed that more individuals were concentrated in the middle activity scope, and there were some individuals with low GST activities in Linyi-Shanxi population but some with high GST activities in Xuzhou-Jiangsu population. The averaged activities ofglutathione-S-transferase among the other three populations were not associate with their malathion sensitivity except for the Xuzhou-Jiangsu population. This showed that the varied resistance mechanism might have been present in different populations due to the changeable habitat environment in which some plants act as inducers. It indicated that the high glutathione-S’-transferase activities and esterases activities might contribute to the decreasing sensitivity to malathion in Xuzhou-Jiangsu population. However, there were high glutathione-iS-transferase activities and low esterases activities in Fanshi-Shanxi population, which was speculated that a certain degree of insensitivity to malathion may be existed in Fanshi-Shanxi population and glutathione-S-transferase activities may play important role in the resistance.The activities of acetylcholinesterase （AChE） extracted using Triton X-100 were inhibited by high substrate （ATC） concentration in the four Oxya chinensis populations. There were not significant differences in Km value and ^max value in the four populations. The sensitivity of AChE to three OPs inhibitions including paraoxon, chlorpyrifos oxon, and dementon-S-methyl from four populations was showed that AChE of the Fanshi-Shanxi and Linyi-Shanxi populations were higher sensitive to three inhibitions than that of the Xuzhou-Jiangsu and Daixian-Shanxi populations. AChE from Xuzhou-Jiangsu, Linyi-Shanxi and Fanshi-Shanxi populations were 1.91-, 2.17-, 2.56-fold sensitive to inhibition by paraoxon, 1.26-, 1.91-, 2.80-fold sensitive to inhibition by chlorpyrifos oxon, and 1.25-, 1.35-, 2.29-fold sensitive to inhibition by dementon-S-methyl than those from Daixian-Shanxi population, respectively. It was speculated that the changeable structure of AChE might be responsible for high resistance to many OPs in Xuzhou-Jiangsu and Daixian-Shanxi populations and the lower OPs selective pressure might contribute to the higher sensitive to OPs inhibitions in Fanshi-Shanxi and Linyi-Shanxi populations.In conclusion, this dissertation demonstrated that: 1）. The increasing更多还原