橘小实蝇对甲氨基阿维菌素苯甲酸盐的抗性机制及对其他药剂的交互抗性

入侵害虫橘小实蝇严重为害蔬菜、花卉、水果等经济作物,目前在田间常使用杀虫剂进行防治。为探索甲氨基阿维菌素苯甲酸盐(甲维盐)防控橘小实蝇的抗性风险,采用药膜法测定了橘小实蝇抗甲维盐种群(EB)对8种杀虫剂的交互抗性水平,并测定了橘小实蝇EB种群、金华田间种群(JH)和敏感种群(S)的解毒酶活性。历经33代筛选,橘小实蝇EB种群对甲维盐产生了43.4倍的中等水平抗性,对阿维菌素存在中等水平交互抗性,对吡虫啉、辛硫磷、马拉硫磷存在低水平交互抗性,对噻虫胺、高效氯氟氰菊酯、多杀霉素和虫螨腈未表现出交互抗性。胡椒基丁醚(PBO)、磷酸三苯酯(TPP)和顺丁烯二酸二乙酯(DEM)均可显著提高甲维盐对橘小实蝇EB种群、JH种群和S种群的室内毒力。橘小实蝇EB种群细胞色素P450(3.9倍)和b₅含量(3.3倍)、O-脱甲基酶活性(4.2倍)、谷胱甘肽S-转移酶活性(2.7倍)、羧酸酯酶活性(3.2倍)显著高于S种群。多功能氧化酶、谷胱甘肽S-转移酶、羧酸酯酶活性显著增强可能是橘小实蝇对甲维盐产生高水平抗性的重要机制。     

粘虫抗甲氨基阿维菌素苯甲酸盐种群的交互抗性与生化抗性机制

为指导甲氨基阿维菌素苯甲酸盐（下称甲维盐）在粘虫Mythimna separate Walker防治上的合理使用,测定了粘虫抗甲维盐种群对5种常用杀虫剂的交互抗性及其生化抗性机制。结果表明:粘虫抗甲维盐种群对阿维菌素（抗性倍数RR₅₀=21.80）、毒死蜱（RR₅₀=17.68）和灭多威（RR₅₀=10.85）均具有中等水平的交互抗性,与辛硫磷（RR₅₀=6.00）和氟氯氰菊酯（RR₅₀=5.65）之间交互抗性水平较低。酶抑制剂胡椒基丁醚（PBO）、马来酸二乙酯（DEM）、三丁基三硫磷酸酯（DEF）和磷酸三苯酯（TPP）在粘虫敏感种群和抗性种群生物测定中对甲维盐毒力均有显著的增效作用。粘虫抗甲维盐种群细胞色素P450和b₅含量及O-脱甲基酶、谷胱甘肽S-转移酶和羧酸酯酶活性均显著高于敏感种群,分别为敏感种群的3.23、3.65、3.63、1.64和2.66倍。研究表明,体内解毒代谢酶活性提高可能是粘虫对甲维盐产生抗性的重要原因。     

桃蚜对噻虫嗪代谢抗性机制研究

对桃蚜进行室内噻虫嗪抗性品系筛选,选育至15代后抗性倍数达到75.6倍。对噻虫嗪敏感品系(THI-S)和抗性品系(THI-R)桃蚜的谷胱甘肽S-转移酶(GSTs)、酸性磷酸酯酶(ACP)、碱性磷酸酯酶(ALP)、羧酸酯酶(CarE)、多功能氧化酶(MFO)O-脱甲基活性进行了比较,结果显示:敏感品系(THI-S)和抗性品系(THI-R)的谷胱甘肽S-转移酶比活力分别为3.127 5和3.215 9,差异不显著,桃蚜抗性品系体内酸性磷酸酯酶、碱性磷酸酯酶、羧酸酯酶和多功能氧化酶O-脱甲基活性均显著高于敏感品系,分别达到了1.57、2.10、6.12、2.03倍。表明桃蚜对噻虫嗪抗性的产生与酸性磷酸酯酶、碱性磷酸酯酶、羧酸酯酶和多功能氧化酶O-脱甲基的活性相关。     

溴虫氟苯双酰胺对小菜蛾的毒力及相关酶活性的影响

溴虫氟苯双酰胺是一种全新的双酰胺类杀虫剂,主要用于防治鳞翅目、鞘翅目、白蚁以及蚊蝇等害虫。为了明确溴虫氟苯双酰胺对小菜蛾的生物活性及解毒酶活性的影响,本研究选择氯虫苯甲酰胺为对照药剂,采用叶片药膜法测定了溴虫氟苯双酰胺对小菜蛾的生物活性,并测定了亚致死剂量(LC20、LC40)的溴虫氟苯双酰胺和氯虫苯甲酰胺对小菜蛾多功能氧化酶、谷胱甘肽S-转移酶和羧酸酯酶活性的影响。结果表明,溴虫氟苯双酰胺与氯虫苯甲酰胺对小菜蛾的生物活性相当,两者对小菜蛾的LC50分别为0.042和0.056μg/mL。与氯虫苯甲酰胺相似,亚致死剂量的溴虫氟苯双酰胺能够诱导小菜蛾体内羧酸酯酶活性升高,但溴虫氟苯双酰胺对多功能氧化酶和谷胱甘肽S-转移酶活性没有显著影响。     

棉铃虫对甲氧虫酰肼抗性机理的初步研究

在室内通过抗性汰选和敏感性反汰选,获得了对甲氧虫酰肼抗性指数相差为72.61倍的棉铃虫Helicoverpa amigera(Hübner)抗性种群和敏感种群。通过增效试验和离体酶活性测定表明:棉铃虫对甲氧虫酰肼产生的抗药性主要与其多功能氧化酶(MFO)、全酯酶和谷胱甘肽-S-转移酶(GSTs)活性的提高有关。     

绿僵菌与FKBP52联合使用对西花蓟马的毒力

为阐明绿僵菌与昆虫免疫抑制蛋白的增效机理,采用浸渍法测定昆虫免疫抑制蛋白FKBP52与金龟子绿僵菌Metarhizium anisopliae对西花蓟马Frankliniella occidentalis的毒力和其体内羧酸酯酶（carboxylesterase,CarE）、谷胱甘肽-S转移酶（glutathione-S transferase,GST）、酚氧化酶（phenoloxidase,PO）及几丁质酶活性的变化。结果显示,金龟子绿僵菌和FKBP52蛋白联合使用对西花蓟马的致死中时LT₅₀为7.32 d,优于绿僵菌单用（10.87 d）或FKBP52蛋白单用的（14.15 d）。金龟子绿僵菌和FKBP52蛋白联合使用9 d时,西花蓟马的累计死亡率达81.11%,显著高于绿僵菌单用（45.56%）或FKBP52蛋白单用的（36.67%）;金龟子绿僵菌和FKBP52蛋白联合使用的协同毒力指数为24.10,表现为增效作用。金龟子绿僵菌与FKBP52蛋白联合使用1 d后,西花蓟马体内CarE和PO活性小于金龟子绿僵菌单用后的活性;联合使用2 d后,西花蓟马体内几丁质酶活性小于绿僵菌单用后的活性,而CarE和GST活性均大于金龟子绿僵菌单用后的活性;联合使用3 d后,西花蓟马体内几丁质酶、CarE和PO活性均大于绿僵菌单用后的活性。表明与金龟子绿僵菌单用相比,金龟子绿僵菌和FKBP52蛋白联合使用后西花蓟马体内CarE、GST、PO和几丁质酶活性抑制或诱导效应与时间有关,这可能是两者具有增效作用的重要机制。     

桔全爪螨对哒螨灵抗性的选育及其生化机理

模拟田间药剂的选择压力 ,用哒螨灵对桔全爪螨 (Panonychus citri Mc Gregor)逐代处理 ,以选育其抗药性。结果表明 :选育 12代 ,抗性增长到 35.0倍 ;哒螨灵抗性品系对氧乐果、双甲脒、氯氟氰菊酯、水胺硫磷和炔螨特有交互抗性。通过增效剂和离体酶活性测定证明 :桔全爪螨对哒螨灵的抗性主要与谷胱甘肽 S-转移酶、多功能氧化酶和乙酰胆碱酯酶活性的提高有关。     

浙江省二化螟不同种群和大螟 对三唑磷的敏感性研究

于2004年用点滴法测定了浙江杭州地区(富阳和余杭)和嘉兴地区(平湖、秀城和秀洲)二化螟、杭州富阳大螟等种群4龄幼虫对三唑磷的敏感性,以及各种群体内与抗药性相关酶系(多功能氧化酶、谷胱甘肽S-转移酶、酯酶和乙酰胆碱酯酶等)的比活力。结果证实上述地区二化螟种群对三唑磷产生了21.1~218.8倍的抗性,其中以富阳和平湖种群的抗性水平较高。二化螟体内多功能氧化酶活性与三唑磷抗性水平呈显著正相关,而乙酰胆碱酯酶活性则呈显著负相关,表明二化螟对三唑磷的抗性可能与多功能氧化酶活性增强和乙酰胆碱酯酶敏感性降低有关。大螟富阳种群对三唑磷的敏感性明显高于二化螟富阳种群,其抗性机制还有待研究。     

斜纹夜蛾对茚虫威的抗性机制

以斜纹夜蛾对茚虫威相对敏感种群和抗性倍数为15.63倍的抗性选育种群为材料,通过解毒酶活性测定与钠离子通道基因片段的克隆、测序,研究了斜纹夜蛾对茚虫威的抗性机制.结果表明,与相对敏感种群相比,抗性种群酯酶活性提高了2.27倍,但抗性和敏感种群谷胱甘肽S-转移酶和多功能氧化酶O-脱甲基活性无显著差异;3龄幼虫酯酶活性随着抗性指数的上升而逐渐提高;从相对敏感和抗性斜纹夜蛾种群3龄幼虫基因组DNA中扩增出位于钠离子通道IIS5-IIS6的341 bp DNA片段;与相对敏感种群相比,茚虫威抗性种群钠离子通道基因没有发生突变.     

西花蓟马取食、机械损伤和外源物质诱导对番茄植株次生物质及西花蓟马解毒酶的影响

为阐明寄主植物番茄受害虫西花蓟马Frankliniella occidentalis取食、机械损伤及外源物质茉莉酸和水杨酸甲酯诱导后次生物质含量的变化,以及害虫如何通过调整体内解毒酶活性适应植物的防御反应,采用生化分析法测定了各诱导处理下次生物质黄酮、总酚和单宁含量及西花蓟马体内解毒酶多功能氧化酶(MFO)、乙酰胆碱酯酶(AchE)、谷胱甘肽S-转移酶(GSTs)和羧酸酯酶(CarE)活性的变化。结果表明:番茄叶片在各诱导处理下黄酮含量在36 h和48 h时均显著高于对照,相应时间段茉莉酸处理与水杨酸甲酯处理的黄酮含量升高最显著,分别达32.07 mg/g和31.76 mg/g;各诱导处理后24 h和36 h时,单宁和总酚含量均有不同程度下降,但总酚含量在48 h时显著升高,其中虫害处理增加最显著,达34.51 mg/g。取食各种诱导处理番茄植株6 h时,西花蓟马体内MFO活性均升高,AchE活性均受到抑制;取食除机械损伤外的其它诱导处理叶片6 h后,西花蓟马体内GSTs活性均被抑制,CarE活性均显著上升,其中取食水杨酸甲酯处理叶片的西花蓟马体内GSTs活性下降幅度最大,而CarE活性升高最显著,分别为3 882.35 U/mg和106.33 U/mg。表明各诱导处理不仅可以使番茄次生物质含量发生变化,诱导植株产生防御反应,也会使西花蓟马改变解毒酶的活性以适应寄主植物的诱导抗性,即寄主植物和害虫通过防御与反防御相互适应。     

Triazophos resistance mechanisms in the rice stem borer (Chilo suppressalis Walker)

A field population of the rice stem borer (Chilo suppressalis Walker) with 203.3-fold resistance to triazophos was collected. After 8-generation of continuous selection with triazophos in laboratory, resistance increased to 787.2-fold, and at the same time, the resistance to isocarbophos and methamidophos was also enhanced by 1.9- and 1.4-fold, respectively, implying some cross-resistance between triazophos and these two organophosphate insecticides. Resistance to abamectin was slightly enhanced by triazophos selection, and fipronil and methomyl decreased. Synergism experiments in vivo with TPP, PBO, and DEM were performed to gain a potential indication of roles of detoxicating enzymes in triazophos resistance. The synergism results revealed that TPP (SR, 1.92) and PBO (SR 1.63) had significant synergistic effects on triazophos in resistant rice borers. While DEM (SR 0.83) showed no effects. Assays of enzyme activity in vitro demonstrated that the resistant strain had higher activity of esterase and microsomal O-demethylase than the susceptible strain (1.20- and 1.30-fold, respectively). For glutathione S-transferase activity, no difference was found between the resistant and the susceptible strain when DCNB was used as substrate. However, 1.28-fold higher activity was observed in the resistant strain when CDNB was used. These results showed that esterase and microsomal-O-demethylase play some roles in the resistance. Some iso-enzyme of glutathione S-transferase may involve in the resistance to other insecticides, for this resistant strain was selected from a field population with multiple resistance background. Acetylcholinesterase as the triazophos target was also compared. The results revealed significant differences between the resistant and susceptible strain. The V_max and K_m of the enzyme in resistant strain was only 32 and 65% that in the susceptible strain, respectively. Inhibition tests in vitro showed that I₅₀ of triazophos on AChE of the resistant strain was 2.52-fold higher. Therefore, insensitive AChE may also involved in triazophos resistance mechanism of rice stem borer.     

Monitoring of beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae) resistance to chlorantraniliprole in China

Beet armyworm, Spodoptera exigua is a major insect pest of vegetables in China, and has been reported to develop resistance to many broad-spectrum insecticides. Recently registered chlorantraniliprole provides a novel option for control of this pest resistant to other conventional insecticides. The susceptibilities of field collected populations were measured by diet incorporation assay with neonate, obvious variation of susceptibility was observed among the 18 field populations with LC₅₀ values varying from 0.039 to 0.240 mg/liter. Moderate resistant level was discovered in 8 of 18 field populations, other 8 populations had become low level tolerance to chlorantraniliprole, and only one population in all the field colonies remained susceptible. Biochemical assays were performed to determine the potential mechanisms involved in tolerance variation. Field populations displayed varied detoxification enzyme activities, but the regression analysis between chlorantraniliprole toxicities and enzyme activities demonstrated each field population might have specific biochemical mechanisms for tolerance. Artificial selection in laboratory with chlorantraniliprole was carried out, 23 generations of continuous selections resulted in 11.8-fold increase in resistance to chlorantraniliprole, and 3.0-fold and 3.7-fold increases in mixed function oxidase and esterase, respectively. Compared with the susceptible strain kept in laboratory the selection strain had developed 128.6-fold resistance to this insecticide. Synergism assays showed the detoxification enzymes might not involved in the resistance observed in field collected populations and the selected strain.     

Fipronil resistance mechanisms in the rice stem borer, Chilo suppressalis Walker

Fipronil resistance mechanisms were studied between the laboratory susceptible strain and the selective field population of rice stem borer, Chilo suppressalis Walker in the laboratory. The borer population was collected from Wenzhou county, Zhejiang province. After five generations of selection, fipronil resistance ratio was 45.3-fold compared to the susceptible strain. Synergism experiments showed that the synergistic ratios of PBO, TPP and DEF on fipronil in susceptible and resistant strains of C. suppressalis were 7.55-, 1.93- and 2.91-fold, respectively, and DEM showed no obvious synergistic action on fipronil. Activities of carboxylesterase and microsomal-O-demethylase in the resistant strain were 1.89- and 1.36-fold higher that in susceptible strain, and no significant difference of glutathione-S-transferase activity was found between the resistant and susceptible strains. The K_m and V_max experiments also demonstrated that fipronil resistance of C. suppressalis was closely relative to the enhanced activities of carboxylesterase and microsomal-O-demethylase. Moreover, cross-resistance between fipronil and other conventional insecticides and the multiple resistant properties of the original Wenzhou’s population were also discussed.     

Selection and characterization of spinosad resistance in Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae)

The cross-resistance and biochemical mechanism of the beet armyworm, Spodoptera exigua (Hübner), to spinosad was studied in the laboratory. S. exigua population were collected from Shanghai suburb. After five generations of selection, the resistance of S. exigua to spinosad increased 345.4 times compared with the susceptible strain. There was no cross-resistance between spinosad and fenvalerate, phoxim, methomyl, abamectin, and cyfluthrin. When the inhibitors, PBO, TPP, DEF, and DEM were used as synergist in the susceptible strain and resistant strain, the synergistic ratio was 0.7-, 0.5-, 1.0-, and 0.6- fold for the susceptible strain, and 9.8-, 1.5-, 2.6-, and 1.5-fold for the resistant strain, respectively. The results revealed that PBO had significant synergistic effect on the resistant strain. The activity in vitro of microsomal-O-demethylase and glutathione S-transferase in the resistant strain was 5.2- and 1.0-fold of the susceptible strain, respectively. The results implied that microsomal-O-demethylase might be important in conferring spinosad resistance in the S. exigua population.     

Beta-cypermethrin resistance associated with high carboxylesterase activities in a strain of house fly, Musca domestica (Diptera: Muscidae)

A housefly strain, originally collected in 1998 from a dump in Beijing, was selected with beta-cypermethrin to generate a resistant strain (CRR) in order to characterize the resistance and identify the possible mechanisms involved in the pyrethroid resistance. The resistance was increased from 2.56- to 4419.07-fold in the CRR strain after 25 consecutive generations of selection compared to a laboratory susceptible strain (CSS). The CRR strain also developed different levels of cross-resistance to various insecticides within and outside the pyrethroid group such as abamectin. Synergists, piperonyl butoxide (PBO) and S,S,S-tributyl phosphorotrithioate (DEF), increased beta-cypermethrin toxicity 21.88- and 364.29-fold in the CRR strain as compared to 15.33- and 2.35-fold in the CSS strain, respectively. Results of biochemical assays revealed that carboxylesterase activities and maximal velocities to five naphthyl-substituted substrates in the CRR strain were significantly higher than that in the CSS strain, however, there was no significant difference in glutathione S-transferase activity and the level of total cytochrome P450 between the CRR and CSS strains. Therefore, our studies suggested that carboxylesterase play an important role in beta-cypermethrin resistance in the CRR strain.     

Toxicity of chlorantraniliprole to Cry1Ac-susceptible and resistant strains of Helicoverpa armigera

Transgenic Bt cotton expressing Cry1Ac is important in controlling various agricultural pests, including Helicoverpa armigera. Especially for transgenic crops that are cultivated in large expanses, avoiding resistance development is a key for ensuring sustainability of Bt technologies. Integrated pest management, in which transgenic crops are strategically combined with rational pesticide use, may help to prevent H. armigera resistance acquisition in Bt cotton. In this study, we evaluated the toxicity of a novel insecticide (chlorantraniliprole) on Cry1Ac-susceptible and resistant individuals of H. armigera. More specifically, we assessed the effect of chlorantraniliprole on the activity of two enzymes and conducted laboratory bioassays to determine its toxicity on H. armigera larvae. Chlorantraniliprole increased esterase and glutathione-S-transferase activities in Cry1Ac susceptible and resistant populations of H. armigera. Cry1Ac resistant populations XJ-F (Cry1Ac resistance ratio 21.8-fold), XJ-10.0 (95.8-fold) and BTR (3536.5-fold) did not show cross-resistance to chlorantraniliprole, with LC₅₀ values of 0.0733 (μg/mL) in XJ-F, 0.0545 (μg/ml) in XJ-10.0 and 0.0731 (μg/mL) in BTR, which were close to that in the susceptible strain 96S (0.0954 μg/mL). Our work shows that chlorantraniliprole could be considered to be integrated in Bt cotton management schemes to delay the H. armigera resistance development.     

Molecular basis of resistance to sulfonylureas in Papaver rhoeas

A Papaver rhoeas population resistant to several acetolactate synthase (ALS) inhibiting herbicides, called 25/98, was found in Catalonia (Northeastern of Spain). This population has an altered form of the enzyme that showed cross-resistance to several herbicides of this group. The highest resistance was found with tribenuron-methyl and sulfometuron-methyl. Studies were conducted to define the molecular basis of this resistance. Two regions of the ALS gene were amplified using degenerated universal primers and sequenced. Population 25/98 contained a single nucleotide substitution in domain A changing Pro₁₉₇ by Ser (using the nomenclature of Arabidopsis thaliana) that confers sulfonylurea resistance. Another change was detected in a region located outside of any conserved domains described to date, but its implication in the resistance remains unclear. We analyze the putative role of the found mutations in relation to the observed resistance using a putative three-dimensional model of the Papaver ALS enzyme.