氯虫苯甲酰胺诱导甜菜夜蛾细胞色素P450基因上调表达

【目的】明确氯虫苯甲酰胺对甜菜夜蛾Spodoptera exigua (Hübner)细胞色素P450基因的诱导表达作用。【方法】采用O-脱乙基香豆素法研究了低剂量氯虫苯甲酰胺处理对甜菜夜蛾幼虫中肠P450s酶活性的影响,应用Real-time PCR方法测定了其对P450基因(CYP9A9, CYP4G37,CYP4S11和CYP6B)和NADPH细胞色素P450还原酶基因(HQ852049)表达的影响。【结果】氯虫苯甲酰胺对甜菜夜蛾P450酶及相关基因的诱导作用均表现出时间效应和剂量效应,。甜菜夜蛾4龄幼虫取食0.02 mg/kg氯虫苯甲酰胺饲料至5龄, 在蜕皮后6-36 h内, 其P450s酶活性增加为对照组的1.90~2.92倍, 诱导效应高于0.01 mg/kg氯虫苯甲酰胺处理组（其P450s酶活性为对照组的1.11~1.62倍）。同时, 0.02 mg/kg氯虫苯甲酰胺处理组甜菜夜蛾中肠P450基因CYP9A9, CYP4G37和CYP6B mRNA的相对表达量分别上升为对照组的1.97~3.95, 2.46~4.29及1.53~4.48倍, NADPH细胞色素P450还原酶基因 HQ852049 的相对表达量亦增加为对照的1.85~4.08倍。【结论】结果提示,氯虫苯甲酰胺可能通过诱导3种P450基因及细胞色素P450还原酶基因 HQ852049 基因mRNA的上调表达而增强了甜菜夜蛾幼虫中肠P450s酶活性。     

三种杀虫剂亚致死剂量对草地贪夜蛾细胞色素P450基因表达的影响

【目的】为明确杀虫剂亚致死剂量对草地贪夜蛾Spodoptera frugiperda细胞色素P450基因表达的影响。【方法】本研究采用叶片浸渍法测定了3种杀虫剂[氯虫苯甲酰胺、甲氨基阿维菌素苯甲酸盐和苏云金杆菌Bacillus thuringiensis(Bt)]对草地贪夜蛾2龄幼虫的毒力,以及通过实时荧光定量PCR(real-time quantitative PCR, RT-qPCR)技术测定了这3种杀虫剂亚致死剂量(LC₁₀)处理后48 h时草地贪夜蛾2龄幼虫16个P450基因的表达量。【结果】氯虫苯甲酰胺、甲氨基阿维菌素苯甲酸盐和Bt对草地贪夜蛾2龄幼虫的LC₁₀值分别为0.931, 0.283和1 089.688 mg/L。2龄幼虫受LC₁₀氯虫苯甲酰胺胁迫后,13个P450基因(CYP4G75,CYP6AB12,CYP6B50,CYP321A7,CY321A8,CYP321A9,CYP321A10,CYP321B1,CYP337B5,CYP9A59,CYP9A58,CYP6AE44及CYP6AE43)表达上调...     

拟除虫菊酯微胶囊剂的应用研究

研究结果表明,应用二氯苯醚菊酯、氯氰菊酯和氰戊菊酯的3种微胶囊剂对赤拟谷盗、豆蚜、小菜粉蝶和粘虫室内测定及大田试验药效均为显著。处理后6天,死亡率90％左右,各天药效相当或略高于乳油。其残效期对赤拟谷盗24天,对其他3种试虫15天左右,相当于该类药剂乳油残效期的2倍。3种微胶囊剂的药效高低顺序依次为氯氰菊酯>二氯苯醚菊酯>氰戊菊酯,和其乳油效果高低的顺序基本相同。     

番茄、芦苇、水杉、皂荚粗提液与辛硫磷、氯氰菊酯混用对赤拟谷盗杀灭作用的初步试验

赤拟谷盗是一种重要的世界性储粮害虫,防治困难.本文采用浸渍法测定了番茄茎叶、芦苇全株、水杉叶、皂荚果实等4种植物材料水提液与两种杀虫剂混用对赤拟谷盗的毒力效应.结果 表明,各提取液与40%辛硫磷乳油1000倍液、5%氯氰菊酯乳油500倍液以不同比例混配时,均表现出不同程度的增效作用.药液与植物提取液以4:1、1:1体积...     

不同地区转Bt基因抗虫水稻稻谷对赤拟谷盗生长发育的影响

为了明确来自湖北武穴、孝感、随州3个不同地区的转cry1Ab/cry1Ac、cry2A、cry1C基因明恢63(分别命名为TT51、T2A-1和T1C-19)对赤拟谷盗Tribolium castaneumHerbst生长发育的影响,在室内以不同的转Bt基因稻谷继代饲养赤拟谷盗6代,结果表明:各处理赤拟谷盗的卵期3～4d,卵孵化率85%～100%,幼虫期22～27d,化蛹率85%～97%,蛹期6～7d,百蛹重0.25～0.31g,性比0.8～1.4,羽化率82%～97%,产卵前期5～7d。不同抗虫转基因水稻对赤拟谷盗各发育历期及生命表参数没有显著的影响,同一转基因事件,没有因为种植地区的不同造成对赤拟谷盗生长发育的差异。3种Bt蛋白在继代饲养的赤拟谷盗幼虫体内检测均呈阳性反应,但积累量很小,继代饲养后,没有发现在赤拟谷盗体内明显的累积。     

敲减Wingless / Wnt1基因表达对赤拟谷盗发育中的影响

Wnt信号通路是进化中高度保守的一条信号转导途径,在调控动物的胚胎轴向正常发育、胚胎分化、决定细胞极性、维持成体动态平衡等方面发挥重要作用. 该信号通路的异常激活还与肿瘤的发生密切相关. 本实验将体外人工合成的Wingless(Wg)/Wnt1基因dsRNA显微注射入赤拟谷盗晚期幼虫体内,研究Wingless/Wnt1蛋白在赤拟谷盗发育过程中发挥的作用. 实验结果显示,注射 Wingless(wg)/Wnt1基因dsRNA后,赤拟谷盗发育形成的蛹,翅膀宽度减小,翅间距明显增大,且羽化过程也受到严重影响. 此外,qPCR结果表明,赤拟谷盗Wingless(Wg)/Wnt1基因被沉默后,Cadherin-like 和 Smoothened (Smo)基因的表达显著上调,Armadillo-2基因略上调. 这些结果揭示,Wnt-1 信号通路和赤拟谷盗翅膀发育以及成虫羽化过程密切相关. 蛹翅宽减小,翅间距增大,可能是由于调控细胞粘连及细胞形态的Cadherin-like 和Armadillo-2基因的上调所引起.更重要的是,Smo基因的上调,表明了Wnt信号通路和Hedgehog信号通路在赤拟谷盗发育过程中有交互作用.     

家蝇（Musca domestica L.）成虫和幼虫对杀虫剂敏感性差异研究

报导了家蝇成虫和幼虫对杀虫剂敏感性的差异及其机理,家蝇的幼虫对杀虫剂的耐药性比成虫高,敏感性家蝇幼虫对三氟氯氰菊酯的耐药性是成虫的205．5倍,机理研究表明幼虫高GST活性与其较高的耐药性有关,三氟氯氰菊酯和二硫氰基甲烷混剂（4：1）对菊酯抗性家蝇的共毒系数达188。     

甜菜夜蛾泰安郊区田间种群对杀虫剂的抗药性水平监测

测定了山东泰安市郊区甜菜夜蛾田间种群对11种杀虫剂的抗药性。用点滴法监测结果表明：该种群3龄幼虫对氯氟氰菊酯抗性最高(2445．5倍),其次为高效氯氰菊酯(586．5倍),均属极高水平抗性;对百树菊酯(123．8倍)属高水平抗性;对毒死蜱(39．1倍)、马拉硫磷(27．4倍)及灭多威(25．1倍)属中等水平抗性;对辛硫磷(8．1倍)抗性最低,属低水平抗性。用浸叶法监测结果表明：3龄幼虫对氯氟氰菊酯(856．5倍)和高效氯氰菊酯(250．7倍)也均属极高水平抗性;对百树菊酯(134．4倍)属高水平抗性;对毒死蜱(14．3倍)属中等水平抗性;对辛硫磷(8．1倍)属低水平抗性;对抑太保(4．2倍)、拉威因(4．2倍)、抑食肼(3．4倍)及虫酰肼(3．1倍)属敏感性降低阶段。两种监测方法的监测结果基本一致。     

亚致死浓度氰氟虫腙对小菜蛾三种解毒酶及P450 mRNA表达量的影响

【目的】为探讨亚致死浓度氰氟虫腙对小菜蛾Plutella xylostella(L.)3种解毒酶和细胞色素P450基因表达量的影响。【方法】采用叶片浸渍法,测定用氰氟虫腙LC10和LC25处理72 h后小菜蛾羧酸酯酶、谷胱甘肽S-转移酶和多功能氧化酶3种解毒酶的活性;应用实时荧光定量PCR(Real-time PCR)方法测定了其在24、48和72 h时对P450基因(Px CYP4、Px CYP6和Px CYP9家族)表达的影响。【结果】结果表明,氰氟虫腙LC10和LC25处理对羧酸酯酶活性影响不显著;对谷胱甘肽S-转移酶活性存在显著抑制作用(P<0.05);LC25处理后多功能氧化酶活性显著增加(P<0.05),为对照的1.39倍。同时,LC25氰氟虫腙处理组小菜蛾P450基因Px CYP4M19、Px CYP6BF1V2、Px CYP6CN1和Px CYP6CV2 m RNA的相对表达量分别为对照组的2.33~8.5倍、1.32~3.48倍、1.18~2.53倍、2.02~7.22倍。【结论】结果显示,氰氟虫腙可能通过诱导这4种P450基因m RNA的上调表达而增强了小菜蛾多功能氧化酶酶活性。     

3种拟除虫菊酯类农药对草地贪夜蛾的毒力测定及田间应用效果评价

评价使用氟氯氰菊酯、溴氰菊酯、高效氯氟氰菊酯喷雾和喇叭口点施处理对草地贪夜蛾的活性与防效,为草地贪夜蛾综合防控提供技术支持。采用喷雾法测定了3种农药原药对草地贪夜蛾3龄幼虫毒力;采用喷雾和喇叭口点施2种方式田间施用5.7%氟氯氰菊酯乳油、25 g/L溴氰菊酯乳油、5.0%高效氯氟氰菊酯乳油,药后第1天、第3天、第7天调查挂牌标记玉米上草地贪夜蛾活虫数,计算防治效果。3种拟除虫菊酯农药对草地贪夜蛾3龄幼虫LC 50值大小顺序依次为氟氯氰菊酯(29.80 mg/L)<高效氯氟氰菊酯(42.39 mg/L)<溴氰菊酯(49.88 mg/L);3种拟除虫菊酯类农药在54.00 g a.i./ha剂量下均能明显降低草地贪夜蛾田间虫口数量,同等剂量防效氟氯氰菊酯乳油>高效氯氟氰菊酯乳油>溴氰菊酯微乳剂。由于草地贪夜蛾低龄幼虫和高龄幼虫取食部位有差异,喷雾法防治低龄幼虫的效果好,喇叭口点施防治高龄幼虫的效果好。拟除虫菊酯类农药对草地贪夜蛾具有较好的防治效果,喇叭口点施方式节省药剂、提高对高龄幼虫的防效,在防治草地贪夜蛾等害虫方面具有广阔的应用前景。     

Molecular analysis of CYP321A1, a novel cytochrome P450 involved in metabolism of plant allelochemicals (furanocoumarins) and insecticides (cypermethrin) in Helicoverpa zea

Cytochrome P450 monooxygenases play a significant role in the detoxification of hostplant allelochemicals and synthetic insecticides in Lepidoptera. In the corn earworm Helicoverpa zea, a noctuid of considerable economic importance, metabolisms of xanthotoxin, a toxic furanocoumarin, and alpha-cypermethrin, an insecticide, are mediated by at least one P450 with a catalytic site capable of accepting both substrates. To further the characterization of P450s in this species, we have cloned three full-length cDNAs encoding two CYP4M subfamily members and a novel CYP321A subfamily member. RNA analyses have demonstrated that the CYP321A1 gene is highly induced (51-fold) in larval midguts in response to xanthotoxin but not cypermethrin. Both CYP4M genes are expressed at negligible levels that are not increased by xanthotoxin or cypermethrin. Baculovirus-mediated expression of the full-length CYP321A1 cDNA has demonstrated that the CYP321A1 protein metabolizes xanthotoxin and angelicin, like the CYP6B1 protein in the furanocoumarin specialist Papilio polyxenes, and alpha-cypermethrin, like the CYP6B8 protein previously characterized in H. zea. In contrast, the CYP4M7 protein does not metabolize xanthotoxin at any detectable level. We conclude that at least two xanthotoxin-inducible P450s from highly divergent subfamilies (CYP6B and CYP321A) contribute to the resistance of H. zea larvae to toxic furanocoumarins and insecticides. Genomic PCR analysis indicates that the CYP321A1 gene has evolved independently from the CYP6B genes known to be present in this insect.     

小菜蛾对几种常用药剂的敏感性测定

采用叶片浸溃法,在2001年10月和2002年10月分别测定了福州上街蔬菜基地的小菜蛾,对10种杀虫剂的敏感性。结果表明,上街菜区小菜蛾对灭多威、杀虫双、辛硫磷、毒死蜱、杀灭菊酯和功夫菊酯等农药极不敏感,已不能有效控制小菜蛾的危害。而小菜蛾对氟啶脲、阿维菌素、氟虫腈和虫螨腈比较敏感。通过两次毒力测定比较(2002年10月的LC50;2001年10月的LC50),结果表明,大多数杀虫剂敏感性变化较小。但小菜蛾对阿维菌素敏感性明显下降,毒力倍数达6．65倍,对氟虫腈毒力倍数相差1．48倍。此外,2002年10月小菜蛾对功夫菊酯的LC50为2001年10月的2．02倍。用浸叶法和浸溃法测定多杀菌素、氟虫腈与虫螨腈推荐浓度对小菜蛾幼虫的毒杀作用。结果表明3种药剂浸叶法处理效果均较理想。     

The expression of Spodoptera exigua P450 and UGT genes: tissue specificity and response to insecticides

Cytochrome P450 and UDP-glucosyltransferase (UGT) as phase I and phase II metabolism enzymes, respectively, play vital roles in the breakdown of endobiotics and xenobiotics. Insects can in crease the expression of detoxificatio n enzymes to cope with the stress from xenobiotics including insecticides. However, the molecular mechanisms for insecticide detoxification in Spodoptera exigua remain elusive, and the genes conferring insecticide metabolisms in this species are less well reported. In this study, 68 P450 and 32 UGT genes were identified. Phylogenetic analysis showed gene expansions in CYP3 and CYP4 clans of P450 genes and UGT33 family of this pest. P450 and UGT genes exhibited specific tissue expression patterns. Insecticide treatments in fat body cells of S. exigua revealed that the expression levels of P450 and UGT genes were significantly influenced by challenges of abamectin, lambda-cyhalothrin, chlorantraniliprole, metaflumizone and indoxacarb. Multiple genes for detoxification were affected in expression levels after insecticide exposures. The results demonstrated that lambda-cyhalothrin, chlorantraniliprole, metaflumizone and indoxacarb induced similar responses in the expression of P450 and UGT genes in fat body cells;eight P450 genes and four UGT genes were co-up-regulated significantly, and no or only a few CYP/UGT genes were down-regulated significantly by these four insecticides. However, abamectin triggered a distinct response for P450 and UGT gene expression;more P450 and UGT genes were down-regulated by abamectin than by the other four compounds. In con elusion, P450 and UGT genes from S. exigua were identified, and different responses to abamectin suggest a different mechanism for insecticide detoxification.     

An independent occurrence of the chimeric P450 enzyme CYP337B3 of Helicoverpa armigera confers cypermethrin resistance in Pakistan

The increasing resistance level of insect pest species is a major concern to agriculture worldwide. The cotton bollworm, Helicoverpa armigera, is one of the most important pest species due to being highly polyphagous, geographically widespread, and resistant towards many chemical classes of insecticides. We previously described the mechanism of fenvalerate resistance in Australian populations conferred by the chimeric cytochrome P450 monooxygenase CYP337B3, which arose by unequal crossing-over between CYP337B1 and CYP337B2. Here, we show that this mechanism is also present in the cypermethrin-resistant FSD strain from Pakistan. The Pakistani and the Australian CYP337B3 alleles differ by 18 synonymous and three nonsynonymous SNPs and additionally in the length and sequence of the intron. Nevertheless, the activity of both CYP337B3 proteins is comparable. We demonstrate that CYP337B3 is capable of metabolizing cypermethrin (trans- and especially cis-isomers) to the main metabolite 4'-hydroxycypermethrin, which exhibits no intrinsic toxicity towards susceptible larvae. In a bioassay, CYP337B3 confers a 7-fold resistance towards cypermethrin in FSD larvae compared to susceptible larvae from the Australian TWB strain lacking CYP337B3. Linkage analysis shows that presence of CYP337B3 accounts for most of the cypermethrin resistance in the FSD strain; up-regulation of other P450s in FSD plays no detectable role in resistance. The presence or absence of CYP337B3 can be easily detected by a simple PCR screen, providing a powerful tool to rapidly distinguish resistant from susceptible individuals in the field and to determine the geographical distribution of this resistance gene. Our results suggest that CYP337B3 evolved twice independently by unequal crossing-over between CYP337B2 and two different CYP337B1 alleles.     

Metabolic imidacloprid resistance in the brown planthopper,Nilaparvata lugens,relies on multiple P450 enzymes

Target insensitivity contributing to imidacloprid resistance in Nilaparvata lugens has been reported to occur either through point mutations or quantitative change in nicotinic acetylcholine receptors (nAChRs). However, the metabolic resistance, especially the enhanced detoxification by P450 enzymes, is the major mechanism in fields. From one field-originated N. lugens population, an imidacloprid resistant strain G25 and a susceptible counterpart S25 were obtained to analyze putative roles of P450s in imidacloprid resistance. Compared to S25, over-expression of twelve P450 genes was observed in G25, with ratios above 5.0-fold for CYP6AY1, CYP6ER1, CYP6CS1, CYP6CW1, CYP4CE1 and CYP425B1. RNAi against these genes in vivo and recombinant tests on the corresponding proteins in vitro revealed that four P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, played important roles in imidacloprid resistance. The importance of the four P450s was not equal at different stages of resistance development based on their over-expression levels, among which CYP6ER1 was important at all stages, and that the others might only contribute at certain stages. The results indicated that, to completely reflect roles of P450s in insecticide resistances, their over-expression in resistant individuals, expression changes at the stages of resistance development, and catalytic activities against insecticides should be considered. In this study, multiple P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, have proven to be important in imidacloprid resistance.     

Exposure to herbicides reduces larval sensitivity to insecticides in Spodoptera litura (Lepidoptera: Noctuidae)

Herbicides and insecticides are widely used in modern agriculture. It has been reported in various studies that application of insecticides can increase tolerance of herbivorous insects to insecticides. However, limited information exists on susceptibility to insecticides when insects are exposed to herbicides. This study was conducted to investigate the potential impact of the herbicides trifluralin and 2-methyl-4-chlorophenoxyacetic acid sodium salt (MCPA-Na) on the susceptibility of the nocturnal moth Spodoptera litura to the insecticides X-cyhalothrin, phoxim and bifenthrin. We found that larvae exposed to trifluralin or MCPA-Na became significantly less susceptible to both insecticides than nonexposed control larvae. Herbicide-treated larvae did not show altered growth under the used test conditions. However, heads of herbicide-treated larvae showed increased expression of the acetylcholinesterase genes SI Ace I and SI Ace 2. Moreover, the fat body and midgut of herbicide-treated larvae displayed elevated expression of detoxification genes (the carboxylesterase gene SI CarE;the glutathione S-transferase genes SlGSTe2 and SlGSTe3\ the cytochrome P450 monooxygenase genes CYP6B48, CYP9A40 and CYP321B1). The CYP6B48 gene exhibited highest inducibility. In conclusion, the data of this study suggest that exposure of S. litura larvae to herbicides may stimulate detoxification mechanisms that compromise the efficacy of insecticides.     

防治新入侵害虫蜂巢小甲虫杀虫剂的室内筛选

蜂巢小甲虫Aethina tumida是一种新入侵为害蜜蜂蜂巢的危险性害虫,筛选出具有较高毒力水平的化学药剂防治该虫迫在眉睫。本文采用浸渍法测定了10种杀虫剂对蜂巢小甲虫幼虫的毒力。试验结果表明,10 mg/L高效氯氰菊酯、啶虫脒和功夫菊酯在24 h内对蜂巢小甲虫幼虫的致死率达到36.67%、33.33%和29.39%,而10 mg/L高效氯氰菊酯和功夫菊酯在48 h对蜂巢小甲虫幼虫的致死率均达到100%。啶虫脒、高效氯氰菊酯和功夫菊酯在24 h的LC_(50)分别为10.47 mg/L、16.94 mg/L和19.1 mg/L。其它受试的杀虫剂如多杀菌素、氟虫腈、氟啶脲、虫酰肼、敌百虫、阿维菌素和甲维盐的触杀致死率相对较低。筛选结果显示,啶虫脒、高效氯氰菊酯和功夫菊酯可作为目前防治蜂巢小甲虫的的重要参考防治药剂。