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.     

Resistance to the Whitefly Aleurotrachelus socialis（Hemiptera：Aleyrodidae） and SSR Marker Identification in Advanced Populations of the Hybrid Manihot esculenta subsp.Manihot flabellifolia

Genes resistant to Aleurotrachelus socialis were transferred to the F 1 from the interspecific hybrid wild species of Manihot flabellifolia to M.esculenta and two advanced generations of backcrosses（BC 1 and BC 2）.We characterized the resistance of A.socialis transferred to BC 2 parents（CW67-160,CW67-130,CW67-44）,MTAI-8（BC 1）,resistant（CMB9B-73） and susceptible（CMB9B-104） genotypes from contrasting pools,and resistant（MEcu-72） and susceptible（CMC-40） genotypes.Whitefly demography and biology were evaluated.SSR molecular markers associated with a phenotypic response of plant resistance were detected in segregating populations（BC 2）.Results showed that although female survival time was similar on all hosts,the lowest averages of longevity,fecundity and oviposition rate were observed in the resistant control MEcu- 72,only being significantly similar to the parent CW67-130.When the BC 1 and BC 2 populations were compared,it was found that A.socialis fecundity was eight times lower on CMB9B-73 progeny than on CW67-130,expressing the highest levels of resistance to the whitefly.Ten genotypes of CMB9A and CMB9B family had the best segregation.A total of 486 microsatellite primers were evaluated using bulked segregant analysis（BSA）,11 showed polymorphism between the contrasting pools and only one showed significant differences between resistant and susceptible individuals.In conclusion,fecundity was the parameter that impacted most on the intrinsic rate of A.socialis population growth.     

中华稻蝗两地理种群乙酰胆碱酯酶生化特性研究

采用生物测定方法,对采自山西省临猗县和江苏省徐州市郊中华稻蝗五龄若虫的乙酰胆碱酯酶(AChE)特性进行了比较研究,同时测定了2种群五龄若虫对马拉硫磷的敏感性。用对氧磷、氧化毒死蜱和甲基内吸磷3种有机磷抑制剂对2种群中华稻蝗五龄若虫AChE的体外抑制进行的研究表明,该2种群AChE对对氧磷、氧化毒死蜱、甲基内吸磷的敏感性没有显著差异,临猗种群比徐州种群分别高1.24、1.13和0.82倍;乙酰胆碱酯酶动力学研究结果表明,临猗种群的动力学参数米氏常数(Km值)和最大反应速度(Vmax值)均较徐州种群为高;用乙酰硫代胆碱(ATC)做底物测定AChE活性,2种群AChE活性无显著差异,临猗种群是徐州种群的1.24倍。而2种群生物测定结果表明,临猗种群和徐州种群五龄若虫对马拉硫磷的敏感度有差异,徐州种群五龄若虫的LD50值(13.00μg·g-1虫质量)是临猗种群(4.64μg·g-1虫质量)的2.8倍。据此推测,中华稻蝗徐州种群马拉硫磷敏感性下降与AChE无相关性,可能存在其他影响因素。     

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.     

Comparative Study of Malathion Toxicity and General Esterases in Larvae and Adults from a Field Population of Oxya chinensis (Thunberg)(Orthoptera:Acridoidea)

The susceptibility of Oxya chinensis to malathion was compared in larvae and adults from a field population, collected from Jinyuan outskirt, Shanxi Province. The results showed that Oxya chinensis was more susceptible to malathion in the adult stage than in the larval stage. The LD50 values for malathion susceptibility of Oxya chinensis were 4.94 and 2.44 mg g-1 body weight in the larvae and adults respectively. The results indicated that the larvae were 2.02-fold less susceptible to malathion than the adults. The general esterases and the kinetics were characterized and compared between the two life stages and between females and males. Larval preparations of Oxya chinensis were more active than adult preparations in females and males. The larvae showed 1.18-, 1.49-, and 1.17-fold higher specific activities than the adults in females with α -NA, α -NB and β -NA respectively. In males, the ratios were 1.34-, 1.70-, and 1.06-fold. Female preparations were more active than those of males in the adults. The reverse results were observed in the larvae where male preparations were more active than female preparations. Kinetic studies showed that Km values of general esterases hydrolyzing α -NA, α -NB, and β -NA in the adult stage were 1.36-, 1.32- and 1.39-fold respectively, higher than those in the larval stage in females. In males, the ratios were 1.24-, 2.14-, and 1.20-fold. The esterase from male insects had a higher affinity (lower Km value) to the substrate than those from females. The results also showed that the Vmax values of general esterase hydrolyzing α -NA, α -NB, and β -NA in the two stages were similar. From the results of bioassays and biochemical analyses, it has been inferred that a higher level of resistance to malathion in larvae than in adults would appear to result from differences in the expression of resistance mechanisms in these two life stages. Enhanced esterase activities appeared to play a major role in resistance to malathion in both larvae and adults. From the analysis of inhibition in vitro, the esterases in the two life stages were B-type,and carboxylesterases were predominant enzymes in the composition of the esterases in the two stages.     

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。     

马拉硫磷锐劲特和氯氟氰菊酯对中华稻蝗五龄若虫的急性毒性

采用室内生物测定方法,研究了山西晋源、阳明堡和陕西杨桥镇3个不同地理种群中华稻蝗五龄若虫对马拉硫磷的急性毒性。结果表明,马拉硫磷对3个不同地理种群中华稻蝗的LD50值由低到高依次为:8.14(山西晋源种群)、14.02(陕西杨桥镇种群)和17.35(山西阳明堡种群),且山西晋源种群的毒力回归线斜率b值(4.07113)显著高于山西阳明堡种群(2.62049)和陕西杨桥镇种群(2.86311)的b值。此外,以山西晋源种群中华稻蝗为试验对象,测定并比较了马拉硫磷、锐劲特和氯氟氰菊酯3种农药的急性毒性。3种农药的LD50值由小到大依次为锐劲特(0.12μg·g-1)、氯氟氰菊酯(0.53μg·g-1)和马拉硫磷(8.14μg·g-1),且锐劲特和马拉硫磷的斜率b值(5.19182和4.07113)显著高于氯氟氰菊酯(2.19430)。     

不同中华稻蝗种群对马拉硫磷的敏感性及乙酰胆碱酯酶特性的比较

采用生物测定方法．对采自山西省晋源区和陕西省长安县的中华稻蝗种群对马拉硫磷的敏感性及其乙酰胆碱酯酶（ACHE）进行了研究。结果表明,长安种群比晋源种群对马拉硫磷的敏感性低4．79倍。动力学研究表明,晋源种群的动力学参数米氏常数（Km值）和最大反应速度（Vmax值）均比长安种群的高。体外抑制结果表明,两个种群中华稻蝗对所选用的3种有机磷杀虫剂的敏感性从高到低依次为：氧化毒死蜱〉对氧磷〉甲基内吸磷,且长安种群的双分子速率常数（k值）比晋源种群的低。体外抑制结果与生物测定的结果相一致,但动力学研究结果与生物测定存在差异。由此可见,两种群对马拉硫磷的敏感性差异的原因之一可能是乙酰胆碱酯酶的不敏感性,而与酶活性的升高没有明显相关性。     

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.     

中华稻蝗几丁质酶基因10（OcCht10）的分子特性及功能

【目的】获得中华稻蝗（Oxya chinensis）几丁质酶基因10（OcCht10）的cDNA序列,预测其功能域,并做聚类分析明确该基因的系统发育关系。绘制OcCht10在5龄若虫不同组织部位和发育时间的表达图谱,研究其在中华稻蝗蜕皮过程中的生物学功能,为害虫防治提供高效安全的候选基因。【方法】从中华稻蝗转录组数据库搜索OcCht10 cDNA片段,经过blast分析、比对、拼接后,翻译为氨基酸序列,预测其功能域,并与其他昆虫几丁质酶家族基因进行聚类分析,进一步确认该基因的系统发育关系并进行准确命名;选取中华稻蝗5龄第6天不同组织部位及5龄若虫不同天数表皮样本,总RNA提取后反转录为cDNA模板,采用Real-time quantitative PCR（qPCR）方法获得OcCht10在5龄稻蝗若虫不同组织部位和不同发育阶段的表达谱;设计OcCht10 dsRNA引物,用试剂盒体外合成dsOcCht10,采用注射法进行RNA干扰;取注射24 h稻蝗表皮样本,用qPCR方法检测OcCht10基因沉默效率;并仔细观察dsRNA注射后稻蝗表型,统计其死亡率。【结果】经对中华稻蝗转录组数据库的搜索,获得OcCht10 cDNA片段长度为9 318 bp,开放阅读框为8 613 bp,编码2 870个氨基酸;其3′非编码区为705 bp,推测OcCht10 5'端有500多碱基尚未获得;预测其氨基酸序列具有多个结构域,包含有5个几丁质酶催化域和6个几丁质结合域;与其他昆虫几丁质酶基因聚类分析结果显示OcCht10属于昆虫几丁质酶家族基因Ⅱ组,该组基因与昆虫蜕皮相关;5龄若虫组织特异性分析表明该基因主要在表皮、前肠和后肠等外胚层发育而成的组织器官中表达,提示OcCht10可能参与表皮几丁质代谢;发育时间表达图谱表明该基因在5龄第6-7天,即蜕皮前的表皮中高表达,表明OcCht10可能负责蜕皮时表皮几丁质降解;5龄若虫第2天注射该基因的dsRNA,24 h后取表皮检测其干扰效率,结果显示该基因被沉默70%;与注射dsGFP的对照组相比,注射dsOcCht10 5龄若虫表现为龄期延长,旧表皮无法开裂,蜕皮受阻,昆虫无法正常活动导致死亡,死亡率达到100%。【结论】获得OcCht10的部分cDNA序列,该基因在昆虫蜕皮前表皮中高表达;OcCht10参与中华稻蝗的蜕皮发育,注射dsOcCht10可有效沉默靶基因,并导致试虫无法完成正常蜕皮而死亡。     

中华稻蝗（Oxya chinensis）对重金属镉的累积及排泄

为了研究重金属镉(Cd)在植食性昆虫体内的累积和排泄规律,通过Cd慢性染毒的方法,用不同浓度Cd溶液培养的麦苗饲喂中华稻蝗(从4龄若虫至成虫),采用原子吸收法测定Cd在中华稻蝗(Oxyachinensis)整虫和中肠的蓄积及Cd在其粪便和蜕中的排泄量。研究结果表明,Cd在中华稻蝗整虫和中肠中的累积规律相似,即随着麦苗中Cd含量的增加,整虫和中肠中Cd的累积量明显升高,且各处理间存在显著差异(P<0.05)。部分Cd可以通过粪便的排泄和蜕皮过程而排出体外。用不同Cd浓度的麦苗饲喂中华稻蝗后,其粪便和蜕中的Cd排泄量与对照组相比显著升高(P<0.05),且各处理间差异显著(P<0.05)。中华稻蝗整虫、中肠、粪便和蜕中的Cd含量与食物(麦苗叶片)中的Cd含量存在显著的剂量-反应关系,其相关系数分别为:整虫0.977、中肠0.920、粪便0.967、蜕0.840。研究结果可为进一步研究Cd在中华稻蝗体内积累和排泄的动态变化规律提供理论和实验依据。