氰戊菊酯和苯巴比妥钠(PB)对棉铃虫细胞色素P450含量的影响

用 0 .2 mg/ g氰戊菊酯和 2 mg/ g苯巴比妥钠 (PB)拌饲料处理不同时间 ,对敏感种群 (HDS)和抗性种群 (KQR)棉铃虫不同组织的 P4 50诱导作用不同。两种诱导剂对 HDS种群棉铃虫的中肠、脂肪体和体壁的 P4 50都有明显的诱导作用 ,其中 PB诱导的最高倍数分别达 2 .2 4、2 .0 3和 1.6 0倍 ,氰戊菊酯诱导的最高倍数分别达 2 .14、2 .83和 1.2 8倍 ;两种诱导剂对 KQR种群棉铃虫的部分组织 P4 50也有一定的诱导作用 ,但不及对 HDS棉铃虫的显著 ,最高诱导倍数只有 :中肠 1.2 1倍 (PB)和 1.15倍 (氰戊菊酯 ) ,脂肪体 1.72倍 (PB) ;诱导作用除了表现出种群差异性 ,还表现出诱导剂差异、时间效应和组织特异性。尽管不同组织被诱导的程度不同 ,但两种群中中肠和脂肪体的 P4 50相对更容易被诱导 ,诱导倍数也较高 ;诱导达到最大值所需时间一般为 12～ 4 8h不等 ;两种诱导剂中 ,PB比氰戊菊酯对棉铃虫不同组织的P4 50具有更强的诱导作用。     

棉铃虫微粒体多功能氧化酶系组分含量及酶活性在不同生长发育阶段的变化规律研究

细胞色素 P4 50和细胞色素 b5在 KQR和 HDS种群棉铃虫的不同生长发育阶段含量不同 ,但变化规律大致相似 ,均是在 3龄期含量较低 ,从 3龄到 6龄含量上升并达到最高值 ,然后含量逐渐下降 ,直到蛹期达最低值。单加氧酶 MROD和 ECOD在 KQR种群棉铃虫不同生长发育阶段活性大小不同 ,而且变化规律明显不同。从 3龄到 6龄期间 ,MROD活性呈上升趋势 ,并在 6龄期达到最大 ,之后活性逐渐下降 ,在蛹和成虫阶段活性达最小 ;而 ECOD活性在不同生长发育阶段间的差异则不是很大 ,没有明显的阶段表达特异性     

小菜蛾幼虫对拟除虫菊酯类杀虫剂的抗药性与多功能氧化酶的关系

作者就上海梅陇、广州天河和江西莲塘的小菜蛾对拟除虫菊酯的抗药性及其与微粒体多功能氧化酶(MFO)的关系进行了研究。结果表明,上海种群(R)对溴氰菊酯、氰戊菊酯和氯菊酯的 LD_(50)分别是江西种群(S)的>10414倍、2102倍和245倍。广州种群分别为>10414倍、>3569倍和1533倍。活体增效试验表明,酯酶抑制剂 TBPT 在低剂量(0.208μg/虫)时,对溴氰菊酯和氯菊酯都无增效作用,在高剂量(2.08μg/虫)时分别增效41.7倍和110倍。用 MFO的抑制剂 PB 作同样处理时,低剂量的 PB 对溴氰菊酯和氯菊酯的增效作用分别为5倍和12.9倍;高剂量的 PB 对溴氰菊酯增效562倍以上。~(14)C-氰戊菊酯代谢的离体研究结果表明,R 小菜蛾代谢~(14)C-氰戊菊酯的速度是 S 小菜蛾的2.1倍,TBPT 抑制~(14)C-杀灭菊酯的代谢约20%,而PB 抑制50%以上。由此可以得出结论,MFO 是小菜蛾对拟除虫菊酯的主要机理之一。     

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。     

小菜蛾对阿维菌素的抗性机制及交互抗性研究

用叶片药膜法研究了阿维菌素抗性小菜蛾 Plutella xylostella （L.）品系 对常用药剂的交互抗性谱以及增效醚（PB）和磷酸三苯酯（TPP）的增效作用。小菜蛾对阿 维菌素与高效氯氰菊酯、溴氰菊酯、氰戊菊酯和联苯菊酯等菊酯类药剂间具有比较低的交互 抗性，对后者抗性为3～20倍，对阿维菌素的抗性为575.6倍；对氟虫脲和氟啶脲没有交互抗 性。PB和TPP对阿维菌素分别增效8.2和5.5倍，说明小菜蛾对阿维菌素的抗性可能与多功能 氧化酶（MFO）和羧酸酯酶有关。通过差光谱技术测定了阿维菌素抗性和敏感小菜蛾细胞色 素P450的含量，抗性品系是敏感品系的1.38倍。     

RNAi介导的棉铃虫细胞色素P450酶系几种组分基因沉默对高效氯氰菊酯毒力的影响

为进一步明确细胞色素P450 CYP6B7、细胞色素b5(cytochrome b5,Cyt-b5)和细胞色素P450还原酶(cytochrome P450 reductase,CPR)在棉铃虫Helicoverpa armigera (Hübner)对拟除虫菊酯类药剂抗性中的作用,通过RNA干扰技术沉默了抗氰戊菊酯棉铃虫中3种组分的基因,并测定基因沉默后高效氯氰菊酯对棉铃虫的毒力变化.结果显示,经单个CYP6B7或CYP6 B7与CPR、Cyt-b5基因的双链RNA注射处理后12 ～ 48 h,棉铃虫幼虫死亡率在19.4％ ～ 92.5％之间,明显高于空白对照处理的10.47％ ～ 62.87％;高效氯氰菊酯对基因沉默后幼虫的LD50值随着时间的延长而逐渐下降,12～48 h的LD50值在0.97～2.97 μg/头之间,而空白对照处理为2.27 ～ 3.57μg/头.研究表明,细胞色素P450 CYP6B7,或CYP6B7与CPR、Cyt-b5基因的沉默,可提高高效氯氰菊酯对抗性棉铃虫的毒力,进一步证明了CYP6B7、CPR和Cyt-b5在棉铃虫对菊酯类药剂抗性中起着重要作用.     

增效剂对菜蚜茧蜂杀虫剂敏感性的影响

以田间菜蚜茧蜂为试虫,采用药膜法研究了增效剂胡椒基丁醚(PB)、磷酸三苯酯(TPP)和马来酸二乙酯(DEM)对6种杀虫剂的增效作用。结果表明,三种增效剂对6种杀虫剂均有显著增效作用,其大小依次为PB>DEM>TPP。PB对甲胺磷、阿维菌素、氟虫腈、氰戊菊酯、氯氰菊酯和吡虫啉的增效比达5.0～9.6倍。TPP和DEM对甲胺磷、DEM对氟虫腈的增效比达2.6～3.0倍,但TPP和DEM对阿维菌素、氰戊菊酯和氯氰菊酯、TPP对氟虫腈的增效比均在1.9倍以下。PB、TPP和DEM对吡虫啉的增效比分别高达9.6、6.8和8.2倍。体内抑制试验结果显示,PB、TPP和DEM对菜蚜茧蜂AChE活性无明显抑制作用,而PB和TPP对羧酸酯酶(CarE)、DEM对谷胱甘肽S转移酶(CST)活性有显著抑制作用。由此认为,菜蚜茧蜂对所用的6种杀虫剂的耐药性与多功能氧化酶(MFO)、CarE和GST的解毒作用有关。     

4种杀虫剂对棉铃虫抗药性种群幼虫呼吸代谢的影响

利用 L D50 及 L D95剂量的氰戊菊酯、灭多威、甲基对硫磷和硫丹 4种杀虫剂 ,分别处理棉铃虫 H elicoverp a armigra Hübner敏感和抗药性种群的 5龄幼虫 ,测定各药剂对幼虫呼吸代谢的影响。结果表明 ,以 L D50 计 ,氰戊菊酯和灭多威对抗药性种群的处理剂量分别是敏感种群的 15.8和 14.1倍 ,但其呼吸抑制率却显著降低 ;用甲基对硫磷、硫丹处理 30 0～144 0 min对抗药性种群的呼吸抑制率也低于对敏感种群。在 L D95剂量下 ,用氰戊菊酯、甲基对硫磷处理 10～ 6 0 min对抗性种群呼吸抑制率分别为 14.7%～ 2 7.3%和 2 7.2 %～ 15.5% ,而对敏感种群则分别达 4 2 .0 %～ 4 8.6 %和 4 5.8%～ 50 .4 %。     

棉铃虫抗溴氰菊酯品系选育、生化机理及遗传方式研究

用溴氰菊酯选育抗性水平中等的河北冀县棉铃虫,经5代4次筛选获得一抗性品系(Del—R),抗性倍数由筛选前F_1代的52.8倍上升到筛选后F_5代的585.9倍(与1983年东台敏感品系相比)。利用此抗性品系进行增效剂(PBo、TPP、DEF)的活体增效作用测定和离体的酶活力研究,无论是对田间品系还是实验室选育的抗性品系,PBo对多功能氧化酶(MFO)的抑制作用是明显的,增效比从3.0—16.5不等,DEF、TPP的增效比分别为2.7和1.0。Del—R品系的多功能氧化酶环氧化活力(AE)是敏感品系的1.5倍,而Del—R品系的酯酶活力是敏感品系的1.01倍,因此多功能氧化酶活力增强是棉铃虫对溴氰菊酯产生抗性的原因之一,酯酶和羧酸酯酶在棉铃虫对溴氰菊酯的抗性中无多大作用。采用剂量对数-死亡机率值(LD—P线)分析法,将棉铃虫对溴氰菊酯的抗性品系和室内培育的敏感品系进行正交、反交和回交试验,得到的抗性遗传研究结果表明,棉铃虫对溴氰菊酯的抗性为常染色体多基因不完全显性遗传。     

北京、山东和湖南地区烟粉虱抗药性及CYP4v2和CYP6CX1 mRNA水平表达量分析

于2011年采集北京、山东和湖南三地的烟粉虱,进行B、Q隐种鉴定,并测定4种杀虫剂的抗药性,同时通过荧光定量PCR分析CYP4v2和CYP6CX1两个基因的mRNA水平的表达量。结果表明,北京、湖南和长沙烟粉虱均为Q隐种。抗药性监测表明,北京和湖南种群对阿维菌素敏感,山东种群抗性水平较低,而对烟碱类药剂噻虫嗪出现不同程度的抗药性,其中湖南地区烟粉虱对噻虫嗪的抗药性达到49.08倍的高抗水平,北京和山东地区也达到中抗水平。另外,这3个地区的种群对毒死蜱和联苯菊酯抗性水平都较低。通过qRT-PCR分析三地的CYP4v2和CYP6CX1基因表达量,发现相对于敏感种群CYP4v2基因在北京、山东和湖南3个地理种群中分别过量表达3.85倍、19.57倍和10.78倍,而CYP6CX1基因在北京种群中过量表达20.55倍。结果提示田间烟粉虱的细胞色素P450基因CYP4v2和CYP6CX1过量表达可能会是烟粉虱抗药性的形成机制之一。     

Cross-resistance and biochemical mechanisms of abamectin resistance in the western flower thrips, Frankliniella occidentalis

Abamectin resistance was selected in the western flower thrips [Frankliniella occidentalis (Pergande)] under the laboratory conditions, and cross-resistance patterns and possible resistance mechanisms in the abamectin-resistant strain (ABA-R) were investigated. Compared with the susceptible strain (ABA-S), the ABA-R strain displayed 45.5-fold resistance to abamectin after 15 selection cycles during 18 generations. Rapid reversion of abamectin resistance was observed in the ABA-R strain in the absence of the insecticide selection pressure. Moderate and low levels of cross-resistance to chlorpyrifos (RR 11.4) and lambda-cyhalothrin (3.98) were observed in the ABA-R strain, but no significant cross-resistance was found to spinosad (2.00), acetamiprid (1.47) and chlorfenapyr (0.26). Our studies also showed that the esterase inhibitor S,S,S-tributyl phosphorotrithioate (DEF) and glutathione S-transferase inhibitor diethyl maleate (DEM) were not able to synergize the toxicity of abamectin, whereas the oxidase inhibitor piperonyl butoxide (PBO) conferred a significant synergism on abamectin in the ABA-R strain (SR 3.00). Biochemical analysis showed that cytochrome P450 monooxygenase activity of the ABA-R strain was 6.66-fold higher than that of the ABA-S strain. It appears that enhanced oxidative metabolism mediated by cytochrome P450 monooxygenases was a major mechanism for abamectin resistance in the western flower thrips.     

抗阿维菌素小菜蛾的细胞色素P450酶系研究

通过对不同发育时期敏感和抗阿维菌素小菜蛾品系细胞色素P450含量的测定,以及使用不同模式底物对P450单加氧酶活性的比较研究发现:除成虫期外,不同发育时期抗性品系小菜蛾中P450和细胞色素b5的含量都高于敏感品系;抗性品系还原型辅酶Ⅱ(NADPH)-细胞色素P450还原酶活性是敏感品系的1.97倍;同时发现抗性品系中甲氧试卤灵-O-脱甲基酶(MROD)、乙氧试卤灵-O-脱乙基酶(EROD)、乙氧基香豆素-O-脱乙基酶(ECOD)以及对硝基苯甲醚-O-脱甲基酶(PNOD)的活性均明显高于敏感品系,分别为敏感品系的9.41、4.15、1.67和2.94倍。研究结果表明,细胞色素P450含量和单加氧酶活性的增高是小菜蛾对阿维菌素产生抗性的一个重要机制。     

Cytochrome P450 monooxygenase-mediated neonicotinoid resistance in the house fly Musca domestica L

Neonicotinoids play an essential role in the control of house flies Musca domestica. The development of neonicotinoid resistance was found in two field populations. 766b was 130- and 140-fold resistant to imidacloprid and 17- and 28-fold resistant to thiamethoxam in males and females, respectively. 791a was 22- and 20-fold resistant to imidacloprid and 9- and 23-fold resistant to thiamethoxam in males and females, respectively. Imidacloprid selection of 791a increased imidacloprid resistance to 75- and 150-fold in males and females, respectively, whereas selection with thiamethoxam had minimum impact. Neonicotinoid resistance was in all cases suppressed by PBO. The cytochrome P450 genes CYP6A1, CYP6D1 and CYP6D3 were constitutively over-expressed in resistant strains and CYP6D1 and CYP6D3 differentially expressed between sexes. The highest level of CYP6A1 expression was observed in both gender of the imidacloprid-selected strain after neonicotinoid exposure. CYP6D1 expression was increased after neonicotinoid exposure in resistant males. CYP6D3 expression was induced in both sexes upon neonicotinoid exposure but significantly higher in females.     

Identification of cytochrome P450 differentiated expression related to developmental stages in bromadiolone resistance in rats (Rattus norvegicus)

Adult, 20-week-old, rats from a Danish bromadiolone-resistant strain of rats (Rattus norvegicus) over-express the cytochrome P450 genes Cyp2e1, Cyp3a2 and Cyp3a3 upon bromadiolone exposure. Furthermore, adult female rats of this strain over-express the Cyp2c13 gene and suppress Cyp2c12, while males over-express the Cyp2a1 gene. The altered gene expression has been suggested to be involved in the bromadiolone resistance by facilitating enhanced anticoagulant metabolism. To investigate the gene expression of these cytochrome P450 genes in rats of different developmental stages we compared expression profiles from 8-, 12- and 20-week-old resistant rats of the Danish strain to profiles of anticoagulant-susceptible rats of same ages. The three age-groups were selected to represent a group of pre-pubertal, pubertal and adult rats. We found expression profiles of the pre-pubertal and pubertal resistant rats to concur with profiles of the adults suggesting that cytochrome P450 enzymes are involved in the Danish bromadiolone resistance regardless of developmental stage. We also investigated the relative importance of the six cytochrome P450s in the different development stages of the resistant rats. The P450-3a2 and -3a3 isoforms were proposed to be of higher importance in adult male resistance than in pre-pubertal resistance. In contrast, the P450-2c13 and -3a2 isoforms were proposed to be more important in sexual immature female resistance, while the P450-2e1 and -3a3 isoforms were suggested to play a more significant role in adult female resistance.     

Biochemical mechanisms conferring cross-resistance between tebufenozide and abamectin in Plutella xylostella

Experiments have been carried out to confirm the cross-resistance between abamectin and tebufenozide in Plutella xylostella and demonstrate its mechanism. The results showed that the resistant strain of P. xylostella selected by tebufenozide (RF 99.38) really showed high cross-resistance to abamectin (RF 29.25). When this strain was subjected to resistance decaying treatment, breeding without contacting any insecticides, and abamectin resistance selection for 20 generations, the former resulted in decrease of its resistance to both tebufenozide and abamectin to about one third of the original (RF 35.03 and 11.67, respectively), and the later enhanced its resistance to abamectin dramatically (RF 303.77), but not to tebufenozide(RF 50.04). PBO showed high synergism to abamectin (SR 2.11-12.23), and the synergism ratio positively related to the resistance level among different strains. Enzyme analysis also proved that the activity of cytochrome P450 monooxygenase (MFO) was notable enhanced in the strains resistant to both tebufenozide and abamectin (1.71- to 3.01-fold). Based on discussion, it was concluded that tebufenozide selection could resulted in significant cross-resistance of P. xylostella to abamectin. The major mechanism for the cross-resistance should be the enhancement of MFO activity. For resistance management, tebufenozide and abamectin would not recommend for rotational use.     

Biochemical characteristics of insecticide resistance in the fall armyworm, Spodoptera frugiperda (J.E. Smith)

A strain of the fall armyworm, Spodoptera frugiperda (J.E. Smith), collected from corn in Citra, Florida, showed high resistance to carbaryl (562-fold) and methyl parathion (354-fold). Biochemical studies revealed that various detoxification enzyme activities were higher in the field strain than in the susceptible strain. In larval midguts, activities of microsomal oxidases (epoxidases, hydroxylase, sulfoxidase, N-demethylase, and O-demethylase) and hydrolases (general esterase, carboxylesterase, β-glucosidase) were 1.2- to 1.9-fold higher in the field strain than in the susceptible strain. In larval fat bodies, various activities of microsomal oxidases (epoxidases, hydroxylase, N-demethylase, O-demethylases, and S-demethylase), glutathione S-transferases (CDNB, DCNB, and p-nitrophenyl acetate conjugation), hydrolases (general esterase, carboxylesterase, β-glucosidase, and carboxylamidase) and reductases (juglone reductase and cytochrome c reductase) were 1.3- to 7.7-fold higher in the field strain than in the susceptible strain. Cytochrome P450 level was 2.5-fold higher in the field strain than in the susceptible strain. In adult abdomens, their detoxification enzyme activities were generally lower than those in larval midguts or fat bodies; this is especially true when microsomal oxidases are considered. However, activities of microsomal oxidases (S-demethylase), hydrolases (general esterase and permethrin esterase) and reductases (juglone reductase and cytochrome c reductase) were 1.5- to 3.0-fold higher in the field strain than in the susceptible strain. Levels of cytochrome P450 and cytochrome b₅ were 2.1 and 1.9-fold higher, respectively, in the field strain than in the susceptible strain. In addition, acetylcholinesterase from the field strain was 2- to 85-fold less sensitive than that from the susceptible strain to inhibition by carbamates (carbaryl, propoxur, carbofuran, bendiocarb, thiodicarb) and organophosphates (methyl paraoxon, paraoxon, dichlorvos), insensitivity being highest toward carbaryl. Kinetics studies showed that the apparent K_m value for acetylcholinesterase from the field strain was 56% of that from the susceptible strain. The results indicated that the insecticide resistance observed in the field strain was due to multiple resistance mechanisms, including increased detoxification of these insecticides by microsomal oxidases, glutathione S-transferases, hydrolases and reductases, and target site insensitivity such as insensitive acetylcholinesterase. Resistance appeared to be correlated better with detoxification enzyme activities in larval fat bodies than in larval midguts, suggesting that the larval fat body is an ideal tissue source for comparing detoxification capability between insecticide-susceptible and -resistant insects.     

Metabolic mechanisms of insecticide resistance in the western flower thrips, Frankliniella occidentalis (Pergande)

The interactions between six insecticides (methiocarb, formetanate, acrinathrin, deltamethrin, methamidophos and endosulfan) and three potential synergists (piperonyl butoxide (PBO), S,S,S-tributyl phosphorotrithioate (DEF) and diethyl maleate (DEM)) were studied by topical exposure in strains selected for resistance to each insecticide, and in a susceptible strain of Frankliniella occidentalis (Pergande). In the susceptible strain PBO produced appreciable synergism only of formetanate, methiocarb and methamidophos. Except for endosulfan, PBO synergized all the insecticides to varying degrees in the resistant strains. A very high level of synergism by PBO was found with acrinathrin, which reduced the resistance level from 3344- to 36-fold. PBO slightly synergized the carbamates formetanate (4.6-fold) and methiocarb (3.3-fold). PBO also produced a high synergism of deltamethrin (12.5-fold) and methamidophos (14.3-fold) and completely restored susceptibility to both insecticides. DEF did not produce synergism with any insecticide in the resistant strains and DEM was slightly synergistic to endosulfan (3-fold). These studies indicate that an enhanced detoxification, mediated by cytochrome P-450 monooxygenases, is the major mechanism imparting resistance to different insecticides in F occidentalis. Implications of different mechanisms in insecticide resistance in F occidentalis are discussed.     

Mechanisms responsible for high levels of permethrin resistance in the house fly

The mechanisms responsible for > 6000-fold permethrin resistance in a pyrethroid-selected strain of house fly, Learn-PyR, were investigated. Through electrophysiological, in vitro metabolism, in vivo penetration and synergism studies it was demonstrated that the resistance mechanisms consisted of enhanced metabolic detoxification via the mixed-function oxidase (MFO) system, target-site insensitivity and decreased cuticular penetration. The major resistance mechanism was the MFO-mediated detoxification. The elevated MFO activity was correlated with higher levels of cytochrome P-450, cytochrome b₅ and NADPH-cytochrome c reductase activity. The kinetics of the latter showed similar K_m but greater V_max values in the Learn-PyR than in the susceptible strain, suggesting that the elevated activity was due to an altered amount, but not an altered form, of the enzyme. The Learn-PyR strain showed widely varying levels of resistance to the pyrethroids tested. Comparison of the pyrethroid structures with the resistance ratios revealed that resistance was highest in the presence of an unsubstituted phenoxybenzyl alcohol moiety. Substitution or certain modifications of the alcohol moiety reduced the level of resistance. Structure of the acid moiety or the presence or absence of an a-CN group did not affect the resistance level. These results are discussed with reference to the resistance mechanisms present.