【作者】 董红刚；

【导师】 王建军；

【作者基本信息】 扬州大学 ， 农药学， 2008， 硕士

【摘要】 斜纹夜蛾Spodoptera litura (Fabricius)是一种世界性分布的重要农业害虫,大量农药的频繁使用,使得斜纹夜蛾抗药性问题日益突出。新型杀虫剂茚虫威具有独特的作用机制、卓越的杀虫活性和对非靶标生物低毒的特点,是目前替代有机磷和拟除虫菊酯类杀虫剂的理想品种。本文以斜纹夜蛾为材料,对茚虫威亚致死效应、交互抗性和抗性机制等有关毒理学问题进行了研究和探讨。通过浸叶法对斜纹夜蛾的生物测定表明,与溴虫腈和氟虫腈这两种常用药剂相比,茚虫威对斜纹夜蛾三龄幼虫具有更高的生物活性。对几种常用生物测定方法的比较研究发现,通过浸虫法测定的茚虫威毒力最低,而浸叶法、人工饲料混毒法与人工饲料浸毒法的结果相似,表明茚虫威对斜纹夜蛾以胃毒作用为主。根据生物测定结果,用对应LC15、LC10和LC5浓度的茚虫威处理斜纹夜蛾三龄幼虫,发现与对照相比,茚虫威处理斜纹夜蛾化蛹率、羽化率和孵化率均明显下降,并且经LC15浓度处理后,成虫寿命以及单雌产卵量皆显著低于对照,表明亚致死浓度茚虫威对斜纹夜蛾当代和下一代的种群增长有一定的抑制作用。研究还发现,亚致死浓度茚虫威处理斜纹夜蛾三龄幼虫后,斜纹夜蛾酯酶和谷胱甘肽-S-转移酶以及多功能氧化酶O-脱甲基活性与对照相比无显著差异,表明亚致死浓度茚虫威对这三种解毒酶活性没有影响。经过10代6次室内抗性选育,获得了斜纹夜蛾对茚虫威抗性品系,与选育前相比,斜纹夜蛾对茚虫威敏感性降低15.57倍。抗性风险评估表明,斜纹夜蛾具有对茚虫威产生高水平抗性的风险。对斜纹夜蛾抗茚虫威品系的适合度研究表明,与敏感品系相比,抗性品系在生长发育和繁殖上具有不利性,抗性品系的相对适合度仅为敏感品系的0.73。交互抗性测定发现,与相对敏感品系相比,茚虫威抗性斜纹夜蛾品系对辛硫磷、高效氯氰菊酯和氟虫腈LC50值分别是敏感品系的1.53、2.42和1.52倍,对溴虫腈和灭多威LC50值分别是敏感品系的0.78和0.96倍,表明茚虫威抗性斜纹夜蛾品系对这几种杀虫剂没有产生明显的交互抗性。对茚虫威抗性和相对敏感品系斜纹夜蛾三龄幼虫解毒酶的活性测定发现,与相对敏感品系相比,抗性品系酯酶活性提高了2.27倍,但谷胱甘肽-S-转移酶和多功能氧化酶O-脱甲基活性与对照相比无显著差异。进一步对抗性选育期间各代斜纹夜蛾三龄幼虫酯酶活性的测定结果发现,随着抗性指数的上升,酯酶活性逐渐提高,表明斜纹夜蛾对茚虫威的抗性与酯酶相关。利用聚合酶链式反应(PCR),通过特异性上游引物和下游引物分别从相对敏感和抗性斜纹夜蛾品系基因组DNA中扩增出了位于钠离子通道基因IIS5-IIS6的341bp DNA片段。序列分析结果表明,与相对敏感品系相比,茚虫威抗性品系斜纹夜蛾钠离子通道基因没有发生突变。害虫抗药性是害虫综合治理中最重要的问题之一,而明确害虫抗药性机制则是害虫抗药性治理的基础。本研究表明,斜纹夜蛾具有对茚虫威产生高水平抗性的风险,并且在国内外首次发现斜纹夜蛾对茚虫威的抗性与酯酶相关,这对制定斜纹夜蛾抗性治理策略、延长茚虫威使用寿命以及更好地发挥茚虫威在害虫可持续治理中的作用具有重要的理论意义和实践价值。更多还原

【Abstract】 The common cutworm, Spodoptera litura (Fabricius), was a worldwide distributed agricultural pest. Frequent application of chemical insecticides against Spodoptera litura had led to the development of pesticide resistance. Indoxacarb was a novel insecticide belonging to the oxadiazine class with unique action mechanism, excellent insecticidal activity and low toxicity to nontarget organisms, it was therefore an ideal candidate to substitute organophosphate and pyrethroid insecticides. In this paper, the toxicological characteristics of indoxacarb against Spodoptera litura had been studied, including sublethal effects, cross-resistance and resistance mechanisms.Toxicity of insecticides including indoxacarb against Spodoptera litura were tested at the laboratory using leaf dipping method, and the indoxacarb showed the highest insecticidal activity when compared with chlorfennapyr and fipronil, which were commonly used in the control of Spodoptera litura at present. Furthermore, indoxacarb showed lower insecticidal activity when exposed to Spodoptera litura by larvae dipping method, while similar toxicity against Spodoptera litura was found using leaf dipping, artificial diet incorporation and artificial diet dipping methods, suggesting that indoxacarb is more effective as a stomach poison than as a contact insecticide.Based on the bioassay results, 3rd instar larvae of Spodoptera litura were treated with indoxacarb at concentrations corresponding to LC15、LC10 and LC5 by artificial diet dipping methods. Lower pupation rate, eclosion rate and egg hatchability were observed when compared with control, and adult longevity and eggs laid per female decreased significantly when exposed to indoxacarb at LC15 concentrations. These results indicated that indoxacarb at sublethal concentrations had a negative effect on population development of Spodoptera litura. Furthermore, the activities of esterase ( EST) , glutathione S-transferase ( GST) and mixed function oxidase ( MFO) were compared between indoxacarb treated larvae and control, and no significant difference was observed, suggested that indoxacarb at sublethal concentrations had no obvious effect on activities of these three detoxification enzymes.After selection with indoxacarb 6 times during 10 generations, a resistant strain of S. litura was achieved with resistance ratio of 15.57 compared with unselected parent strain, suggesting that S. litura had high capability of developing resistance to indoxacarb, as was confirmed by resistance risk assessment. Developmental and reproductive disadvantages in resistant strain were observed when compared with susceptible strain, and the resistant strain exhibited a fitness value of 0.73 relative to the susceptible strain. Bioassay showed that the LC50 values of phoxim, betacypermethrin, fipronil were 1.53, 2.42 and 1.52 folds higher in resistant strain than that in susceptible stain, respectively, while the LC50 values of chlorfennapyr and methomyl were 0.78 and 0.96 folds lower in resistant strain than that in susceptible strain, suggesting that indoxacarb-resistant Spodoptera litura had little cross-resistance to these tested insecticides.The activities of detoxification enzymes were analyzed in both resistant and susceptible strains. The results showed that the activity of esterase increased by 2.27-fold compared with the susceptible strain, while the activities of mixed function oxidase and glutathione S-transferase had no significant difference. Furthermore, positive correlation was observed between esterase activity in 3rd larvae of every selected generation and resistance factor. These results suggested that the esterase played an important role in the resistance development of Spodoptera litura to indoxacarb.DNA fragments of 341 bp in length in the IIS5–IIS6 region of sodium channel para-orthologous gene in both resistant and susceptible strain were amplified with a pair of specific forward primer and reverse primer by using PCR method. The result showed that there was no sodium channel mutation in the resistant strain compared with the susceptible strain.The pest resistance was one of the most important problems in integrated pest management (IPM), and understanding the resistance mechanisms was the basis of insect resistance management (IRM). The present study showed that S. litura had high capability of developing resistance to indoxacarb, and esterase played an important role in the resistance development of Spodoptera litura to indoxacarb. These results will be helpful in establishing the scientific resistance management strategies, prolonging the lifetime of indoxacarb, and making full use of indoxacarb in sustainable pests management.更多还原