【作者】 吴精晶；

【导师】 王进军； 豆威；

【作者基本信息】 西南大学 ， 农业昆虫与害虫防治， 2012， 硕士

【摘要】 嗜卷书虱Liposcelis bostrychophila (Badonnel)和嗜虫书虱L. entomophila (Enderlein)隶属于啮虫目Psocoptera、书虱科Liposcelididae,是世界范围内广泛发生并能造成严重经济损失的一类储藏物害虫。谷胱甘肽S-转移酶(Glutathione S-transferases, GSTs; EC 2.5.1.18)是生物体内广泛存在的一类功能多样的超基因家族酶,能催化内源还原性谷胱甘肽(GSH)与各种有害的亲电性底物相结合,增加后者的可溶性从而有利于其从细胞内排出,进而保护生物体内的核酸和蛋白质免受亲电基团攻击。GSTs活性能够被各种亲电子化合物如药剂、杀虫剂、植物次生代谢物质等所诱导。本论文以嗜卷书虱和嗜虫书虱为研究对象,利用生命表方法对利尿酸连续胁迫下嗜卷书虱的生态适合度进行研究,结合生理生化和毒理学方法,明确了利尿酸对嗜卷书虱GSTs的活体胁迫作用；比较分析了利尿酸胁迫30天后嗜卷书虱和嗜虫书虱GSTs的生化毒理学特性差异。主要研究结果如下：1利尿酸对嗜卷书虱生态适合度的影响通过组建生命表的方法,系统观察了不同饲料饲喂下(含0.5和3%利尿酸的人工饲料分别记为0.5%和3% EA,常规人工饲料为对照)嗜卷书虱淮北和北碚种群各世代的存活率、平均发育速率、净增殖率、发育历期等生命表参数,以明确利尿酸连续胁迫对嗜卷书虱生态适合度的影响。结果表明,利尿酸连续胁迫下,嗜卷书虱各代卵的存活率升高而一龄若虫的存活率却显著下降,但对其它各龄若虫的存活率则无显著影响；嗜卷书虱各世代成虫寿命显著延长,其中F,的寿命最长。利尿酸连续胁迫对嗜卷书虱各代的产卵期、寿命、平均产卵量均有影响,淮北种群(3% EA)平均每雌产卵量F2最低(13.21头)；北碚种群各代平均每雌产卵量均高于对照(对照平均每雌产卵量为40.87头)。根据组建的嗜卷书虱实验种群特定时间生命表,计算出利尿酸连续胁迫下书虱各代种群生命表参数。利尿酸连续胁迫后,与对照相比,2个种群(3% EA)内禀增长率rm均降低,其中F2最低；而北碚种群0.5% EA处理组的rm均升高。以R0为基准,相对于对照,淮北种群(0.5%和3% EA)F2生态适合度分别下降到0.72和0.313,其余处理组的相对生态适合度均升高；北碚种群0.5% EA组各代的生态适合度均升高,3% EA处理组F1和F2生态适合度均降低,分别为0.84和0.54。2利尿酸持续胁迫对嗜卷书虱GSTs生化毒理学特性的影响采用药膜法测定了利尿酸处理前后嗜卷书虱各世代成虫对溴氰菊酯的敏感性。实验结果表明,与对照相比,嗜卷书虱淮北种群各世代对溴氰菊酯的敏感性降低；而利尿酸胁迫后北碚种群对溴氰菊酯更敏感。与对照相比,淮北种群各代的比活力升高且达到显著水平；就北碚种群而言,利尿酸处理后F2(0.5%EA)和F1(3%EA)的比活力最低。以GSH为底物时,淮北种群(0.5%和3%EA)F1的Km值最高,北碚种群各代Km值均低于对照；淮北种群(0.5%EA)Vmax值与对照之间的差异不显著,北碚种群(0.5%和3%EA)各代Vmax最高均高于对照。以CDNB为底物时,利尿酸处理组嗜卷书虱的Km值与对照相比差异显著；就Vmax而言,其变化趋势与以GSH为底物时相似。利尿酸处理后,嗜卷书虱GSTs酶促反应的最适温度为37℃,最适pH范围变为pH 7.5-8.5。与对照相比,利尿酸处理后嗜卷书虱各代GSTs对利尿酸、双硫仑、马来酸二乙酯和溴氰菊酯的敏感性降低,但15 mM以上剂量溴氰菊酯对嗜卷书虱(3% EA)的GSTs几乎没有抑制作用。3利尿酸胁迫对两种书虱GSTs纯化产物的影响为明确利尿酸胁迫对嗜卷书虱和嗜虫书虱GSTs纯化产物的影响,比较研究了0.5%和3% EA饲养前和连续饲养30天后两种书虱GSTs的活性差异。结果表明,利尿酸对书虱GSTs的活体胁迫具有明显的剂量效应和种效应关系。利尿酸处理后,嗜卷书虱(0.5%和3% EA)对溴氰菊酯的LC50均降低,而嗜虫书虱(0.5%和3% EA)对溴氰菊酯的LC50均升高。经0.5%和3% EA胁迫后,嗜卷书虱GSTs比活力分别下降了1.87和10.00倍；嗜虫书虱GSTs比活力也明显被抑制,其中0.5%EA的抑制作用最强(下降了11.23倍)。以GSH为底物时,嗜卷书虱处理组Km值显著升高,嗜虫书虱处理组Km值均降低；嗜卷书虱(0.5%和3% EA)Vmax显著低于对照,嗜虫书虱(0.5% EA)Vmax最高。以CDNB为底物时,嗜卷书虱和嗜虫书虱(0.5%和3% EA)Km值均降低；就Vmax而言,其变化趋势与以GSH为底物时Vmax一致。利尿酸处理前后,嗜卷书虱和嗜虫书虱GSTs酶促反应的最适温度均为37℃；最适pH范围为pH 7.0-8.0,而嗜虫书虱(0.5% EA)最适pH范围为pH6.5-7.5。利尿酸处理后,嗜卷书虱对利尿酸、马来酸二乙酯和溴氰菊酯的敏感性降低；嗜虫书虱对利尿酸的敏感性增加,对马来酸二乙酯和溴氰菊酯的敏感性降低。更多还原

【Abstract】 The Liposcelis Bostrychophila and L. Entomophila Belonging to Liposcelididae, Psocoptera, Two Sub-group of Pest Insects, Are Important Causative Agents of Economic Damage to Stored Food Products. Glutathione S-transferase (Gsts, Ec 2.5.1.18) Have Been Implied in A Variety of Functions in Eukaryotic Organisms, Including Cellular Detoxification and Homeostasis. and the Gsts Activity Can Be Induced by Chemicals, Insecticides, Secondary Metabolisms of Plant, and So on. Here in This Report, We Through Studied on Ethacrynic Acid (Ea) Continuously Stimulate to L. Bostrychophila Get the Main Parameters of Life Table, in Parallel, Investigate the Influence to Gsts Activity. and the Activity of Gsts from L. Bostrychophila and L. Entamophila After Dietary on 0.5% and 3%Ea for 30 Days Were Also Investigated. the Project Was Lasted for 2 Years, and the Main Results Are As Follows:1 Influence of Ea on Ecological Fitness of L. BostrychophilaDifferent Life Table Parameters, Including Survival Rate, Development Rate, Intrinsic Rate of Increase and Developmental Time of Three Generations of L. Bostrychophila Under the Impact of Continuous Ea Treatment Were Compared Through Constructing the Life Table, to Clarify the Influence of Continuous Ea Treatment on the Ecological Fitness of L. Bostrychophila. the Results Showed That the Mean Survival Rates of Eggs Were Increased Significantly, But the Survival of the First Stadiuman Were Decreased After Dietary on Ea for Three Generations. However on Significant Difference Were Observed for Other Stage. the Adult Longevities of Two Populations After Feed on Ea for Three Generations Were All Longer Than Control, and Fi Showed the Longest Adult Period. Adult Longevity, Oviposition Period and Mean Number of Eggs in Each Generation Were Affected by the Continous Ea Treatment. the Mean Number of Eggs Laid by Per Female Was Lowest in Fi of Huaibei Populations (3%Ea) and the Control of Beibei Population,13.21 and 40.87, Respectively. According to the Life Tables, the Life Table Parameters in Each Generation After Dietary on Ea Were Calculated, Respectively. the Values of Rm Were All Decreased for Two Populations (3%Ea) and Lowest in F2, But Increased for Beibei Populations (0.5%Ea). Using Rvalues, the Fitness for F2 of Huaibei Populations (0.5%Ea and 3%Ea) and F] and F2 of Beibei Populations (3%Ea) Relative to Control Were Calculated As 0.72,0.313, 0.84 and 0.54, Respectively.2 Influence of Continous Ea Impact on Gsts of L. Bostiychophila The susceptibility of deltamethrin against L. bostrychophila after dietary on 0.5% and 3%EA for three generations were assayed by using the pesticide-membrane method. The results indicated that all generations of Huaibei populations were less susceptible to ediethyl maleate. But for Beibei populations, the psocids were more susceptible to it after feed on EA for three generations. Compared to the control, Huaibei population possessed higher specific activity of GSTs, but the F2 (0.5%EA) and F1 (3%EA) shared the lowest GSTs specific activity. GSTs in F1 of Huaibei population possessed a significantly lower affinity to the substrate GSH, but all generations of Beibei populations exhibited higher affinity to it. Meanwhile, GSTs of Beibei populations had higher catalytic activities for GSH, but no difference of catalytic activities for GSH were observed in Huaibei populations (0.5%EA) comparing with the control. The optimal pH of GSTs activity assay for each generation of two psocids was pH 7.5-8 and 37℃was considered to be optimal for the assay. The in vitro inhibition studies showed GSTs of two psocids after dietary 0.5%and 3%EA for three generations were less susceptible to ethacrynic acid, disulfiram and ediethyl maleate, and 15 mM eltamethrin exhibited poor inhibitory effects on GSTs of L. bostrychophila after dietary on 3%EA.3 Influence of EA on purified GSTs of L. bostrychophila and L. entamophilaIn order to definite the influence of ethacrynic acid on purified glutathione S-transferase from booklice. The activities of GSTs from L. bostrychophila and L. entamophila after dietary on 0.5%and 3%EA for 30 days and without EA were investigated. The results showed that the influence of EA on glutathione S-transferases from booklice exsert dose-and populations-dependent manners. After dietary of on 0.5%and 3%EA for 30 days, the LC50 values of deltamethrin form L. bostrychophila were all decreased, while for L. entamophila, the LC50 values were increased. The GSTs specific activities were decreased 1.87-and 10.00-fold (L. bostrychophila), and the GSTs specific activities of L. entamophila were also decreased after reared on the food contain 0.5%and 3%EA, respectively. GSTs of L. bostrychophila exhibited lower Km values (CDNB) and Vmax values (GSH and CDNB), but higher Km values (GSH) in comparing with control. The Km values of GSH and CDNB in L. entamophila after dietary on 0.5%and 3%EA were all deceaed respectively. The GSTs activities of L. bostrychophila and L. entamophila showed same temperature (37℃), and same pH stabilities (pH 7.0-8.0), excepting the psocids of L. entamophila (0.5%EA), who reached its activity peak at pH 6.5-7.5. The in vitro inhibition studies of GSTs indicated That the Populations of L. Bostrychophila and L. Entamophila After Dietary Ea for 30 Days Were Less Susceptible to Ediethyl Maleate and Deltamethrin Compared with Control Groups. Furthermore, As to Ethacrynic Acid, L. Bostrychophila Were Less Susceptible to Ethacrynic Acid, But L. Entamophila Were More Susceptible to It in Comparing with Control. Sds-page Revealed One Band at 23 Kda for the Psocids After Dietary on Ea.更多还原