Comparisons of Properties of Acetylcholinesterase from Two Field-Collected Populations of Oxya chinensis Thunberg (Orthoptera: Acrididae) and the Role of Acetylcholinesterase in the Susceptibility to Malathion

In this study, acetylcholinesterase (AChE) was extracted from two field-collected populations of Oxya chinensis (Xinxiang City, Henan Province and Changzhi City, Shanxi Province). AChE activities were decreased when concentrations of ATC increased, showing a characteristic phenomenon of substrate inhibition at high concentration in both populations. Such inhibition occurred at relatively low concentration for AChE from Xinxiang population but relatively high for AChE from Changzhi population. The kinetic study showed that there were no significant differences between the two populations in the Km values. The Km value in Changzhi population was only 1.09-fold higher than that in Xinxiang population. However,significant differences were observed between the two populations in Vmax values. The Vmaxvalue in Changzhi population was 1.32-fold higher than that in Xinxiang population. The inhibition study in vitro showed that the AChE from both populations exhibited similar rank order in sensitivity to inhibition by three OPs, as determined by comparison of their bimolecular rate constants (ki), from the most potent inhibition to the least was chlopyrifos-oxon ＞ paraoxon ＞demeton-s-methyl for AChE from the two populations and that the ki values in Xinxiang population were lower than those in Changzhi population. The I50 values of AChE from Xinxiang population were 4.84-, 2.66-, and 1.92-fold less sensitive to inhibition by paraoxon, chlopyrifos-oxon, and demeton-s-methyl. These results were consistent with the results in bioassay. It is inferred that AChE insensitivity to OP insecticides plays an important role in the differences of insusceptibility of Oxya chinensis to malathion between the two populations.     

The Susceptibilities of Oxya chinensis （Orthoptera： Acridoidea） to Malathion and Comparison of the Esterase Properties from Three Collected Populations in Tianjin Area,China

The susceptibilities of Oxya chinensis to malathion were studied in three populations collected from outskirt of Tianjin, China, using bioassays and biochemical analysis. Populations were chosen to represent different exposure to insecticides： BDG （Beidagang; low exposure）, BD （Baodi; high exposure previously but low exposure now）, and JN （Jinnan; high exposure）. The results showed that the LD50 values of BD and JN populations were 3.95- and 12.02-fold and 3.64- and 10.07- fold higher than that of BDG population in females and males, respectively. The LD50 values in females were higher than those in males. The results of biochemical analysis indicated that the esterase （EST） activities in JN population were higher than those in BD and BDG populations. They showed that when α-NA, α-NB, and α-NA were used as substrates, females＇ EST activities of JN population were 1.11-, 1.30-, and 1.14-fold and 1.39-, 1.59-, and 1.54-fold higher than those of BD and BDG populations, respectively. When α-NA, α-NB, and β-NA were used as substrates, males＇ EST activities of JN population were 1.13-, 1.12-, and 1,00-fold and 1.20-, 1.14-, and 1.07-fold higher than those of BD and BDG populations, respectively. The results also showed that the specific activities of the females were higher than those of the males in the BD and JN populations, whereas the specific activities of the males were higher than those of the females in the BDG population. The results of bioassay were consistent with those of biochemical analysis. Thus, it was inferred that the elevated ESTs activities might play an important role in conferring the differences of susceptibility of O. chinensis to malathion in the three collected populations. Enzyme kinetic studies indicated that the Km and Vmax values were different among the three collected populations and between the females and the males. The observed changes in the kinetic parameters might be explained by differential expression patterns of isozymes so that the insect esterases have different affinities and maximum velocities toward the same substrate.     

Comparative Studies of Substrate and Inhibitor Specificity of Glutathione S-Transferases in Six Tissues of Oxya chinensis (Thunberg) (Orthoptera: Acrididae)

Specific activity, substrate specificity, and kinetic parameters (Km and Vmax) of glutathione S-transferases (GSTs) towards three substrates, 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), and p-nitrobenzene chloride (pNBC) were investigated in six tissues (foregut, midgut, hindgut, fat body, hemolymph, and muscle) of Oxya chinensis. In addition, the inhibition in vitro (ethacrynic acid, and Cibacron Blue 3GA) of Oxya chinensis in the six tissues was also investigated. Glutathione S-transferase activity was detected in all the six tissues examined. The rank order of GST activities towards CDNB was fat body > midgut > hindgut > muscle > foregut > hemolymph both in females and males. Glutathione 5-transferase activities in the fat body in females and males were 1.3- to 10.4-fold and 1.1- to 10.0-fold higher than those in the other tissues. The rank order of GST activities towards the other substrates changed slightly. From these results, it was inferred that GSTs in the fat body and midgut played important roles in detoxifying xenobiotics including insecticides and plant allelochemicals in O. chinensis. In the three substrates examined, CDNB seemed to be the best substrate, followed by pNBC and DCNB. The kinetic parameters of GSTs were different among the six tissues. This suggested that GSTs in different tissues have various affinities and catalytic efficiency to substrates. In vitro inhibition study showed that the median inhibition concentration (IC50) values of the two inhibitors to GSTs from the six tissues were different. The results suggested that the two inhibitors have different inhibition potency to GSTs from the different tissues. The observed changes in kinetic parameters and inhibition in vitro among the six tissues of the insect might suggest that the number and structure of isoenzymes and their rate of expression varied for the different tissues.     

中华稻蝗田间种群对马拉硫磷和溴氰菊酯的敏感性及其毒理机制的研究

采用生物和生化测定方法,研究了3个中华稻蝗种群对马拉硫磷和溴氰菊酯的敏感性及相关酶系的作用。生物测定结果表明,汉中种群对马拉硫磷和溴氰菊酯的LD50值均最高,临猗种群次之,繁峙种群最低。繁峙和汉中种群对溴氰菊酯比对马拉硫磷敏感,临猗种群则相反。结合生物测定结果和各种群的防治背景,认为杀虫剂的选择压力是引起这3个种群对杀虫剂敏感性差异的主要原因。在无杀虫剂选择压力的作用下,抗性会逐渐消退。生化测定结果表明,汉中种群的酯酶活性最高,繁峙种群次之,临猗种群最低。当以α-NA为底物时,汉中种群酯酶活性分别是临猗和繁峙种群的2.54和1.57倍;当以α-NB为底物时,汉中种群酯酶活性分别是临猗和繁峙种群的1.61和1.53倍;当以β-NA为底物时,汉中种群酯酶活性分别是临猗和繁峙种群的1.84和1.59倍。繁峙种群的谷胱甘肽S-转移酶活性最高,临猗和汉中种群的谷胱甘肽S-转移酶活性相近。此外,繁峙种群的乙酰胆碱酯酶活性最高,临猗种群次之,汉中种群最低。3种氧化型有机磷抑制剂对3个种群乙酰胆碱酯酶体外抑制结果表明,用对氧磷作为抑制剂时,汉中种群的双分子速率常数ki值比临猗和繁峙种群分别降低了7.20和8.50倍;当以氧化毒死蜱作为抑制剂时,汉中种群的ki值比临猗和繁峙种群分别降低了1.21和1.77倍;当以甲基内吸磷为抑制剂时,汉中种群的ki值比临猗和繁峙种群分别降低了7.91和13.43倍。从生物测定和生化测定的结果可以推断,酯酶活性的提高和乙酰胆碱酯酶对抑制剂的敏感性降低是引起3个种群对杀虫剂敏感性差异的主要原因,而谷胱甘肽S-转移酶和乙酰胆碱酯酶活性的变化是次要原因。     

中华稻蝗黄酮的提取及抗氧化活性研究

目的:测定了中华稻蝗体内总黄酮的含量并研究了黄酮粗提物的抗氧化作用,为进一步开发利用动物黄酮资源提供科学依据。方法:正交设计优选中华稻蝗体内总黄酮的提取条件;用SOD、CAT、GSH-Px、MDA试剂盒测定了抗氧化指标。结果:中华稻蝗黄酮最佳提取条件是:90%甲醇、65℃、料液比1∶30、提取时间2h,提取量可达到1124mg/100g(干物质);黄酮粗提物能显著增强小鼠体内SOD、CAT和GSH-Px活性,降低MDA的含量。结论:优选的提取方法效率高,含量测定方法操作简便;黄酮粗提物具有明显的抗氧化作用。     

中华稻蝗几丁质合成酶1基因的mRNA表达及功能

【目的】研究中华稻蝗（Oxya chinensis（Thunberg））几丁质合成酶1（CHS1）基因的表达特性及其在生长发育过程中的作用,从而为实现基于RNAi的蝗虫有效控制提供理论依据。【方法】根据已知中华稻蝗几丁质合成酶1基因（OcCHS1）保守区域部分cDNA序列（GenBank登录号：HM214491）设计特异性表达引物,运用RT-PCR和qPCR研究时空表达特性;采用RNA干扰技术研究其生物学功能。【结果】OcCHS1在中华稻蝗各龄期都有表达,其体表为高表达,其次是气管,其它组织部位表达量低。RNA干扰试验发现,4龄第4天若虫注射OcCHS1的双链RNA（dsRNA）后,与对照组相比,OcCHS1 mRNA表达量被沉默了70.8%,稻蝗出现蜕皮时间延迟、不能完成蜕皮或腹部皱缩死亡等现象,注射dsOcCHS1组死亡率为85.2%,与对照组（7.4%）相比差异显著。【结论】几丁质合成酶1对中华稻蝗的生长发育具有重要作用,其主要参与体表和气管几丁质的合成。采用RNA干扰技术使该基因沉默后可导致中华稻蝗死亡。     

Comparative Studies of Substrate and Inhibitor Specificity of Glutathione S-Transferases in Six Tissues of Oxya chinensis （Thunberg） （Orthoptera： Acrididae）

Specific activity, substrate specificity, and kinetic parameters （Km and Vmax） of glutathione S-transferases （GSTs） towards three substrates, 1-chloro-2,4-dinitrobenzene （CDNB）, 1,2-dichloro-4-nitrobenzene （DCNB）, and p-nitrobenzene chloride （pNBC） were investigated in six tissues （foregut, midgut, hindgut, fat body, hemolymph, and muscle） of Oxya chinensis. In addition, the inhibition in vitro （ethacrynic acid, and Cibacron Blue 3GA） of Oxya chinensis in the six tissues was also investigated. Glutathione S-transferase activity was detected in all the six tissues examined. The rank order of GST activities towards CDNB was fat body 〉 midgut 〉 hindgut 〉 muscle 〉 foregut 〉 hemolymph both in females and males. Glutathione S-transferase activities in the fat body in females and males were 1.3- to 10.4-fold and 1.1- to 10.0- fold higher than those in the other tissues. The rank order of GST activities towards the other substrates changed slightly. From these results, it was inferred that GSTs in the fat body and midgut played important roles in detoxifying xenobiotics including insecticides and plant allelochemicals in O. chinensis. In the three substrates examined, CDNB seemed to be the best substrate, followed by pNBC and DCNB. The kinetic parameters of GSTs were different among the six tissues. This suggested that GSTs in different tissues have various affinities and catalytic efficiency to substrates. In vitro inhibition study showed that the median inhibition concentration （IC50） values of the two inhibitors to GSTs from the six tissues were different. The results suggested that the two inhibitors have different inhibition potency to GSTs from the different tissues. The observed changes in kinetic parameters and inhibition in vitro among the six tissues of the insect might suggest that the number and structure of isoenzymes and their rate of expression varied for the different tissues.