棉铃虫对氰戊菊酯等杀虫剂抗性的选育及其生化机理

从用药水平低的棉田采集棉铃虫Helicover pa armigera(Hubner),在室内恒温条件下以人工饲料饲养,用氰戊菊酯等4种杀虫剂和1种混合剂经点滴法分别逐代处理棉铃虫幼虫,以选育其抗药性。用氰戊菊酯选择15代,抗性达311倍;而用灭多威、甲基对硫磷和辛硫磷分别选择13代、14代及13代,抗性仅分别达10.8倍、3.5倍及5.2倍,抗性发展较慢;甲基对硫磷与辛硫磷的混合剂选择12代,抗性也只有4.8倍。氰戊菊酯和溴氰菊酯、三氟氯氰菊酯间存在明显的交互抗性。用生测法测定酶抑制剂和生化法测定酶活性的结果表明,棉铃虫对氰戊菊酯的抗性主要与幼虫体内多功能氧化酶和羧酸酯酶的活性提高有关。     

棉铃虫P450基因CYP6AE12和CYP9A18的克隆与mRNA表达水平

采用RT-PCR和RACE技术克隆到2个新的棉铃虫细胞色素P450基因：CYP6AE12和CYP9A18。CYP6AE12的cDNA编码区长1 569 bp,编码523个氨基酸;CYP9A18的cDNA编码区长1 590 bp,编码530个氨基酸。用实时定量PCR技术分析了这2个基因在棉铃虫YS敏感品系和YS-FP抗性品系(由氰戊菊酯加辛硫磷混剂筛选YS品系而得) 6龄幼虫脂肪体和中肠中mRNA的表达水平。结果表明：CYP6AE12和CYP9A18的mRNA表达具有组织特异性,CYP6AE12在脂肪体中表达量较高,而CYP9A18在中肠中的表达量较高。与相对敏感品系YS相比,CYP6AE12在YS-FP抗性品系中肠和脂肪体中的mRNA表达量分别为YS品系的3.6倍和1.3倍;CYP9A18在YS-FP品系中肠和脂肪体的mRNA表达量分别为YS品系的0.3倍和1.0倍。CYP6AE12的过量表达与YS-FP品系棉铃虫的抗药性可能有一定关系。     

铃虫对氰戊菊酯抗性和敏感品系的选育

用氰戊菊酯对来自阳谷的棉铃虫（ＹＧ）Ｈｅｌｉｏｔｈｉｓａｒｍｉｇｅｒａ（Ｈｕｂｎｅｒ）进行抗性品系的筛选。在１５代期间经过９代的室内选育,获得抗性品系（Ｆｅｎ－Ｒ）,抗性倍数高达２４６３倍,筛选后Ｆ１,代ＬＤ５０值（２４．１４１２ｇ／头）比筛选前Ｆ１代ＬＤ５０值（０．２０２０　ｇ／头）提高了１１９．５倍。对来自偃师的棉铃虫（ＹＳ）进行了连续两代单对筛选,得到敏感品系（Ｆｅｎ－Ｓ）,敏感品系的ＬＤ５０值为０．０１１６ｇ／头,接近１９８３年东台敏感品系的ＬＤ５０值（０．００９８ｇ／头）。Ｆｅｎ－Ｒ抗性品系筛选前后分别测定了七种杀虫剂的剂量－死亡回归线,发现Ｆｅｎ－Ｒ抗性品系对溴氰菊酯［ＬＤ５０（Ｆｅｎ－Ｒ）ＬＤ５０（ＹＧ）＝５．２Ｘ］和氯氰菊酯（２．５Ｘ）具有一定程度的交互抗性;而对功夫菊酯（０．６６Ｘ）,氯菊酯（０．８７Ｘ）、灭多威（０．７４Ｘ）及久效磷（１．５Ｘ）没有交互抗性。氰戊菊酯加Ｐｂ的增效试验结果表明棉铃虫对氰戊菊酯的抗性主要是由于多功能氧化酶的代谢作用。毒理学资料还暗示抗性为多因子（基因）的。     

氰戊菊酯和苯巴比妥钠对敏感和抗性棉铃虫中肠多功能氧化酶系的诱导作用

用苯巴比妥钠（2mg/g）和氰戊菊酯（0.2mg/g）拌饲料处理,对敏感品系棉铃虫Helicoverpa armigera中肠的细胞色素P450和细胞色素c还原酶含量均具有明显的诱导作用（两者都使细胞色素P450含量提高了2.24倍,使细胞色素c还原酶的含量分别提高1.33和1.40倍）,但对细胞色素b5诱导作用不显著（仅为对照的1.23和1.15倍）;此外,苯巴比妥钠对敏感棉铃虫中肠的艾氏剂环氧化酶活性和甲氧试卤灵-O-脱甲基酶活性也有显著的诱导作用（分别提高了2.75和2.66倍）,但对7-乙氧香豆素-O-脱乙基酶活性没有诱导作用,而氰戊菊酯对敏感棉铃虫中肠的艾氏剂环氧化酶活性则有2.02倍的诱导作用。同一浓度的苯巴比妥钠和氰戊菊酯使抗性品系棉铃虫中肠的细胞色素P450含量分别提高1.21和1.15倍,使细胞色素c还原酶含量分别提高1.48和1.86倍（差异显著）,但是细胞色素b₅含量没有明显变化（分别为对照的1.15和0.98倍）;此外,氰戊菊酯能使抗性棉铃虫中肠的艾氏剂环氧化酶活性提高1.53倍,但苯巴比妥钠对该酶活性则有明显抑制作用。     

棉铃虫对氰戊菊酯抗性和敏感品系的选育

用氰戊菊酯对来自阳谷的棉铃虫(YG)Heliothis armigera(Hubncr) 进行抗性晶系的筛选。在15代期间经过9代的室内选育,获得抗性品系(Fcn-R),抗性倍数高达2“3倍,筛选后F₁₅代LD₅₀值(24.1412μg/头)比筛选前F1代lD₅₀值(0.2020μg/头)提高了119.5倍。对来自偃师的棉铃虫(YS)进行了连续两代单对筛选,得到敏感品系(Fen-S),敏感晶系的LD50值为0.0116μg/头,接近1983年东台敏感晶系的LD₅₀值(0.0098μg/头)Fcn-R抗性晶系筛选前后分别测定了七种杀虫剂的剂量-死亡回归线,发现Fen-R抗性品系对溴氰菊酯[LD₅₀(Fen-R)/LD₅₀(YG)=5.2X] 和氯氰菊酯(2.5X)具有一定程度的交互抗性;而对功夫菊酯(0.66X),氯菊酯(0.89x)、灭多威(0.74X)及久效磷(1.5x)没有交互抗性。氰戊菊酯加Pb的增效试验结果表明棉铃虫对氰戊菊酯的抗性主要是由于多功能氧化酶的代谢作用。毒理学资料还暗示抗性为多因子(基因)的。     

不同龄期棉铃虫用氰戊菊酯汰选对其抗性发展的影响

利用中抗(15.06倍)品系,室内用氰戊菊酯对棉铃虫Helicoverpa armigera初孵至4龄幼虫分别进行连续汰选,选育9代后,以3龄幼虫汰选的抗性发展最快（31.5倍）, 其次是4龄、2龄幼虫汰选的品系（分别增加25.2倍和14.5倍）,用初孵幼虫汰选的抗性发展最慢（10.2倍）。抗性现实遗传力的测定表明,3龄幼虫汰选的抗性现实遗传力（0.4419）显著大于初孵幼虫的（0.2346）。代谢酶抑制剂的增效实验发现,磷酸三苯酯(TPP)对各品系棉铃虫均无明显增效作用。而增效醚(PBO)对高龄幼虫汰选的品系的增效作用比低龄幼虫汰选的品系增效作用强。测定初孵和3龄幼虫汰选品系试虫的击倒抗性发现,初孵幼虫汰选品系的抗性增加倍数（10.2）与击倒抗性增加的倍数（10.5）相似,而3龄幼虫汰选的抗性增加倍数（31.5）显著高于击倒抗性增加的倍数（19.9）。认为初孵幼虫期多功能氧化酶（MFO）表达不完全,用药主要是筛选击倒抗性,而高龄幼虫期用药则会同时筛选击倒抗性和MFO参与的代谢抗性。因而初孵幼虫期用药抗性发展缓慢。生产上不仅可以提高药剂的防效,同时可以延缓抗性的发展。     

西花蓟马抗辛硫磷种群的抗性机制及交互抗性

为明确西花蓟马对辛硫磷的抗性风险,研究了西花蓟马抗辛硫磷种群对其他杀虫剂的交互抗性及其对辛硫磷的抗性机制.交互抗性测定结果表明,西花蓟马抗辛硫磷种群对辛硫磷与毒死蜱、高效氯氟氰菊酯和灭多威存在中等水平的交互抗性,对溴虫腈、吡虫啉、甲维盐和多杀菌素存在低水平交互抗性,对啶虫脒和阿维菌素不存在交互抗性.酶抑制剂与辛硫磷的增效剂测定结果表明,胡椒基丁醚(PBO)、三丁基三硫磷酸酯(DEF)和磷酸三苯酯(TPP)对西花蓟马抗辛硫磷种群(XK)、田间种群(BJ)和敏感种群(S)均起到了显著的增效作用(P＜0.05),马来酸二乙酯对西花蓟马抗辛硫磷种群和敏感种群增效作用均不显著,但对田间种群增效作用显著(P＜0.05).生化测定发现:除田间种群西花蓟马乙酰胆碱酯酶活性提高不显著外,西花蓟马抗辛硫磷种群和田间种群的细胞色素P450含量(2.79和1.48倍)、细胞色素b5含量(2.88和1.88倍)及O-脱甲基酶活性(2.60和1.68倍)、羧酸酯酶活性(2.02和1.61倍)和乙酰胆碱酯酶活性(3.10倍)均显著高于敏感种群(P＜0.05);谷胱甘肽-S-转移酶酶活性也有一定程度提高(1.11和1.20倍),但不显著(P＞0.05).表明其体内解毒代谢酶和靶标酶活性提高是西花蓟马对辛硫磷产生抗性的重要原因.     

甜菜夜蛾对氰戊菊酯和顺式氯氰菊酯的抗性机理

通过对活体增效作用进行测定和生化分析,探讨了甜菜夜蛾对氰戊菊酯和顺式氯氰菊酯的抗性机理.结果表明:增效醚(PBO)、增效磷(SV1)、磷酸三苯酯(TPP)和顺丁烯二酸二乙酯(DEM)对甜菜夜蛾抗氰戊菊酯品系(Fen-R)和敏感品系(S)的增效倍数之比分别为10.2、7.8、12.5和1.1,对抗顺式氯氰菊酯品系(Cyp-R)和敏感品系(S)的增效倍数之比分别为21.6、15.5、8.6和1.2.PBO、SV1和TPP对氰戊菊酯和顺式氯氰菊酯均有显著增效作用,表明多功能氧化酶和羧酸酯酶均参与了甜菜夜蛾对氰戊菊酯和顺式氯氰菊酯的抗性.Fen-R品系和Cyp-R品系4龄幼虫羧酸酯酶的活性分别是S品系的1.9和2.2倍,而谷胱甘肽-S-转移酶活性与S品系差异不显著,表明羧酸酯酶活性的提高是甜菜夜蛾对氰戊菊酯和顺式氯氰菊酯产生抗性的重要原因,谷胱甘肽-S-转移酶与两种药剂的抗性无关.Fen-R品系和Cyp-R品系的Na-K-ATPase活性与S品系均无显著差异,但在相同浓度下氰戊菊酯和顺式氯氰菊酯对S品系Na-K-ATPase的抑制作用显著高于抗性品系,表明抗性品系Na-K-ATPase对杀虫剂的敏感性已明显降低.     

棉蚜啶虫脒抗性种群交互抗性和增效剂增效作用的研究

【目的】明确棉蚜Aphis gossypii Glover啶虫脒抗性品系与其它杀虫剂的交互抗性现状以及增效剂的增效作用,为延缓和治理棉蚜对啶虫脒的抗性提供依据。【方法】采用单头反选育和群体汰选的方式,获得了棉蚜啶虫脒敏感和抗性品系;采用叶片药膜法测定了13种杀虫剂对啶虫脒的交互抗性以及增效剂对啶虫脒的增效作用。【结果】经过室内棉蚜敏感和抗性品系的筛选,获得了相对抗性倍数为82.33倍的棉蚜啶虫脒抗性品系。棉蚜啶虫脒抗性品系的交互抗性谱的研究表明,交互抗性倍数小于5的药剂为:吡蚜酮,甲基阿维菌素;交互抗性倍数在5~10倍的药剂为:噻虫嗪,联苯菊酯,毒死蜱,马拉硫磷,丙溴磷,辛硫磷;交互抗性倍数在10~15倍的药剂为:硫丹,阿维菌素,高效氯氰菊酯,三唑磷,氧化乐果;交互抗性倍数大于1 5倍的药剂为:吡虫啉。增效剂实验表明,TPP和PBO在啶虫脒敏感品系中增效作用不明显,但在抗性品系中增效作用显著。在啶虫脒抗性品系中的增效比为1.77、1.61,在啶虫脒敏感品系中的增效比为1.02、1.03。DEM在啶虫脒抗性、敏感品系中的增效作用均不明显,增效比为1.04、1.02。TPP和PBO对啶虫脒有很好的增效作用。以室内棉蚜敏感品系(LC_(50)为0.180 mg/L)为基础,对新疆各主要棉区的棉蚜种群进行了啶虫脒药剂的抗性调查,结果表明新疆各主要棉区棉蚜对啶虫脒的相对抗性倍数为6.1~22.0倍。【结论】由此说明新疆主要棉区棉蚜对啶虫脒具有一定的抗性风险,生产中可以利用无交互抗性的吡蚜酮和甲基阿维菌素来治理抗性棉蚜种群。     

亚洲玉米螟对丁硫克百威的抗性筛选及交互抗性

采用点滴法测定丁硫克百威对亚洲玉米螟Ostrinia furnacalis(Guenée)幼虫的致死中量,并采用亚致死剂量筛选亚洲玉米螟10代。结果表明,丁硫克百威处理亚洲玉米螟5代后,抗性即显著上升,抗性倍数为3.23倍,到10代时,抗性达到了5.43倍。对高效氯氟氰菊酯、辛硫磷和丁硫克百威3种玉米螟抗性品系进行交互抗性测定,发现丁硫克百威与其它2种农药存在交互抗性。建议在害虫防治谨慎使用丁硫克百威。     

Relative contribution of detoxifying enzymes to pyrethroid resistance in a resistant strain of Helicoverpa armigera

Abstract:  The relative contribution of oxidases and esterases to pyrethroid resistance was studied in a YS-FP strain of Helicoverpa armigera from China. The YS-FP strain was derived from a field-collected strain (YS) by 16 generations of selection with a mixture of fenvalerate and phoxim. Compared with the YS strain, the YS-FP strain showed 1850- to >7140-fold resistance to four ester-bonded phenoxybenzyl alcohol pyrethroids (fenvalerate, deltamethrin, cypermethrin and cyhalothrin), >205-fold resistance to a non-ester phenoxybenzyl alcohol pyrethroid (etofenprox) and only 19-fold resistance to an ester-bonded methylated biphenyl alcohol pyrethroid (bifenthrin). The oxidase inhibitor piperonyl butoxide eliminated most the of resistance to fenvalerate, deltamethrin, cypermethrin, cyhalothrin and etofenprox, whereas the esterase inhibitor S,S,S -tributylphosphorothioate had a small synergistic effect for fenvalerate and cyhalothrin only. This suggests that the resistance to these pyrethroids in the YS-FP strain was mainly because of enhanced oxidative detoxification. The monooxygenase activities of the midguts of sixth-instar larvae of the YS-FP strain to substrates p -nitroanisole, ethoxycoumarin and methoxycoumarin were 3.7-, 4.7- and 10-fold, respectively, compared with that of the YS strain. Glutathione S -transferase activity and esterase activity were not significantly altered in the YS-FP strain. This confirms that enhanced oxidative detoxification was a major mechanism contributing to pyrethroid resistance in the YS-FP strain.     

Correlation between fenvalerate resistance and cytochrome P450-mediated O-demethylation activity in Helicoverpa armigera (Lepidoptera: Noctuidae)

Cytochrome P450 monooxygenases are a major metabolic mechanism responsible for pyrethroid resistance in Helicoverpa armigera (Hübner) from Asia. Cytochrome P450-mediated O-demethylation activity toward p-nitroanisole (PNOD) of individual fourth instars was determined in five strains of H. armigera by using a microplate reader. The four resistant strains of YS, HD, YGF, and YG59 had 6-, 71-, 2540-, and 11,800-fold resistance, respectively, to fenvalerate in comparison with the susceptible BK77 strain. Their mean PNOD activity was 4-, 10-, 24-, and 60-fold, respectively, compared with the BK77 strain. A strong positive correlation (correlation coefficient r = 0.98) between PNOD activity and fenvalerate resistance was found. Of 48 larvae from each strain, only 4% larvae of the susceptible BK77 strain had detectable PNOD activity, whereas 25, 33, 79, and 96% of larvae from the resistant strains YS, HD, YGF, and YG59 exhibited PNOD activity, respectively. There was a clear discrimination of patterns of PNOD frequency distribution between H. armigera strains and their magnitudes of fenvalerate resistance. The PNOD activity can be used as a biochemical marker for monooxygenase-mediated pyrethroid resistance in field populations of H. armigera.     

The involvement of microsomal oxidases in pyrethroid resistance in Helicoverpa armigera from Asia

Five contemporary strains of the bollworm Helicoverpa armigera Hübner from China, Pakistan and India, all with high resistance to pyrethroids, were compared with a standard susceptible strain that originated from the Cote D'Ivoire in the 1970s ('SCD'). Two of the Chinese strains ('YGF' and 'YGFP') were derived by laboratory selection from a third, field collected strain ('YG'). The strain 'YG' exhibited 7-, 14- and 21-fold resistance to fenvalerate, cypermethrin and deltamethrin, respectively. After selection with fenvalerate for 14 generations ('YGF'), this increased to 1690-, 540- and 73-fold. Selection with a mixture of fenvalerate and piperonyl butoxide (PBO) for 14 generations ('YGFP') resulted in resistance ratios of 2510, 2920 and 286. The synergistic ratios to fenvalerate that resulted from pre-treatment of PBO were 5-, 462- and 12-fold in YG, YGF and YGFP strains, respectively. Resistance ratios for a Pakistani strain (PAK) were 2320-, 4100- and 223-fold to fenvalerate, cypermethrin and deltamethrin, respectively. The synergistic ratio of PBO to these pyrethroids was 450-, 950- and 11-fold. The strong synergism of pyrethroids by PBO implied that an oxidative metabolism could be involved in pyrethroid resistance in these resistant strains. The activities of cytochrome P450 monooxygenases from midguts of final instar larvae to p-nitroanisole (PNOD), ethoxycoumarin (ECOD), methoxyresorufin (MROD) significantly increased in all the resistant strains when compared with the susceptible strain. This further implies that cytochrome P450 monooxygenases are involved in pyrethroid resistance in Asian H. armigera. Comparative in vitro studies of the metabolism of 14C-deltamethrin by midgut microsomes of the resistant PAK and susceptible SCD strains showed that the resistant strain had a much greater capacity than the susceptible strain for the metabolic degradation of deltamethrin. This enhanced metabolic degradation occurred in the presence of NADPH which suggested an oxidative detoxification. In the resistant strains, minor increases in glutathione S-transferase activity (to the substrates CDNB and DCNB), and esterase activity (to the substrate alpha-naphthyl acetate) further suggested that, of the putative metabolic mechanisms, oxidases are the most important. This study provides the first evidence that cytochrome P450 monooxygenases are a major metabolic mechanism responsible for pyrethroid resistance in H. armigera from Asia.     

Effects of three insecticides on the expression of cytochrome P450 CYP6B7 in Helicoverpa armigera

Cytochrome P450 genes can be induced by xenobiotics, which may contribute to insect's adaptability to the environments and resistance to insecticides. Previous studies indicated that cytochrome P450 CYP6B7 played a vital role in the resistance of Helicoverpa armigera to fenvalerate. However, effects of different insecticides on the expression of CYP6B7 in H. armigera are still unclear. In this study, resistance level of H. armigera to six insecticides was determined by topical application method, and effects of fenvalerate, phoxim and indoxacarb on the expression of CYP6B7 in susceptible (HDS) and fenvalerate-resistant (BJR) strains of H. armigera were evaluated by RT-qPCR. The results showed that BJR strain had an extremely high level of resistance to fenvalerate (1990.57-fold), and the induction of CYP6B7 in different tissues of BJR strain was significantly higher than that of HDS strain after exposure to fenvalerate for 24 and 48 hr. The highest induction level by fenvalerate was observed in the midgut, which were 13.7-fold in HDS strain and 127.9-fold in BJR strain at 24 and 48 hr, respectively. After exposure to phoxim, the expression level of CYP6B7 in HDS and BJR strains was induced by 2.3- and 316.8-fold at 24 hr, respectively. It is worth to note that CYP6B7 could be induced by phoxim at different time points in BJR strain, but only induced at 24 and 72 hr in HDS strain. After indoxacarb exposure, the expression of CYP6B7 was induced by 1.6-fold at 72 hr in BJR strain, whereas it was induced at 24 and 48 hr in HDS strain. These results demonstrated that the expression level of CYP6B7 could be induced by fenvalerate, phoxim and indoxacarb, but the induction time and levels varied; moreover, the induction in BJR strain was markedly higher than that in HDS strain after exposure to fenvalerate and phoxim.     

棉铃虫对辛硫磷抗性的风险评估与预报

在室内用辛硫磷对采自江苏东台的棉铃虫Helicoverpa armigera连续筛选了12代,平均成活率为37.6%,抗性上升了4.9倍。据Tabashnik介绍的方法,估计了抗性现实遗传力为0.0865。并预报了棉铃虫对辛硫磷的抗性发展速率。还对抗性风险评估、影响抗性发展速率的因素及抗性治理进行了讨论。     

棉铃虫对氰戊菊酯抗性遗传分析

本文采用剂量对数-死亡机率值回归线(LD-P线)分析法, 利用室内选育的棉铃虫 Heliccverpa armitera(Hubner)对氰戊菊酯抗性晶系和敏感品系,研究了棉铃虫对氰戊菊酯的抗性遗传规律。结果表明,棉铃虫对氰戊菊酯的抗性为多基因遗传,其主要基因为不完全显性,抗性不属性连锁遗传。利用增效剂对抗性机理进行的研究表明,多功能氧化酶(MFO)是棉铃虫对氰戊菊酯产生抗药性的主要因子,因此,可以推断多功能氧化酶基因为不完全显性。     

In vitro metabolism of pyriproxyfen by microsomes from susceptible and resistant housefly larvae

Levels of cytochrome P450 and b₅ were investigated in microsomal enzymes of houseflies from the gut and fat body of the third instar larvae of a pyriproxyfen-resistant strain (YPPF) and two pyriproxyfen-susceptible strains (YS and SRS). In comparison to the YS and SRS strains, YPPF microsomes had higher levels of total cytochrome P450s in both the gut and fat body. Furthermore, microsomes from the gut and fat body of YPPF larvae were found to have a much greater ability to hydroxylate aniline than YS larvae. In vitro metabolism studies of pyriproxyfen indicated that the metabolic rates were much higher in both the gut and fat body of YPPF larvae than of YS and SRS larvae. The major metabolites of pyriproxyfen in houseflies were identified to be 4′-OH-pyriproxyfen and 5′-OH-pyriproxyfen. Cytochrome P450 inhibitors, piperonyl butoxide (PB) and 2-propynyl 2,3,6-trichlorophenyl ether (PTPE), decreased the metabolic rates significantly in all three strains. This study confirmed that microsomal cytochrome P450 monooxygenases play an important role in the pyriproxyfen resistance of the housefly. Furthermore, it suggests that the fat body must be as important as the gut for the metabolism of pyriproxyfen in resistant housefly larvae. Arch. Insect Biochem. Physiol. 37:215–224, 1998. © 1998 Wiley-Liss, Inc.