A Study On Correlative Enzymes Of Organophosphate Resistance In Oxya Chinensis (Thunberg) From Several Populations

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...