Moth sex-pheromone biosynthesis is inhibited by the herbicide diclofop

Pheromones of nocturnal moths are derived from fatty acids produced as a result of the activity of acetyl-CoA carboxylase. This timely production is initiated in nocturnal moths by a tropic peptide, pheromone biosynthesis activating neuropeptide released into the hemolymph. In monocotyledonous plants, specific plastid acetyl-CoA carboxylase is inhibited by herbicides that target the eukaryotic form of the enzyme. We report evidence that these herbicides can also target pheromone biosynthesis by a moth, thereby implicating the acetyl-CoA carboxylase as a key regulatory enzyme in the pheromone biosynthetic pathway. These findings, whilst indicating the possible action of such herbicides on non-target organisms, also suggest a novel alternative method of insect pest management, which precludes sex-pheromone production and mating success, thereby reducing insect population growth.     

Physiological basis for antagonism of clethodim by imazapic on goosegrass (Eleusine indica (L.) Gaertn.)

Greenhouse and laboratory experiments were conducted to determine the effect of imazapic on the herbicidal activity of clethodim on goosegrass. Imazapic did not affect absorption of [¹⁴C]clethodim by goosegrass. Averaged across the two treatments of clethodim alone and clethodim plus imazapic, absorption was 36 and 89% of applied [¹⁴C]clethodim at 0.5 and 96 h, respectively. The majority of [¹⁴C]clethodim (79% of applied) was absorbed by 24 h. Translocation of ¹⁴C was not affected by imazapic, and 3.6% of applied ¹⁴C had translocated into the portion of the shoot below the treated leaf at 96 h after treatment. Metabolism of clethodim was not affected by the presence of imazapic. Three major metabolites of clethodim were detected in treated tissue at all harvest intervals. The majority (58%) of [¹⁴C]clethodim was converted to a relative polar metabolite form 96 h after treatment, whether clethodim was applied alone or in the presence of imazapic. One day after treatment, the photosynthetic rate in plants treated with imazapic decreased below the rate in the non-treated check, and was less for 8 days, the duration of the study. These data suggest that the antagonism of clethodim by imazapic may be caused by imazapic reducing the photosynthetic rate of goosegrass and therefore the sensitivity of ACCase to clethodim.     

聚乙二醇8000对棉铃虫微粒体蛋白沉淀作用的研究

为建立棉铃虫Helicoverpa armigera微粒体P450的分离纯化方案,比较了不同浓度下聚乙二醇8000(PEG8000)对棉铃虫幼虫中肠和脂肪体微粒体蛋白的沉淀作用。结果表明,终浓度为8.0×104 mg/L的PEG8000可以使中肠和脂肪体微粒体蛋白的78%沉淀下来,其中包含的细胞色素P450分别占中肠和脂肪体P450总量的28%和34%。SDS-PAGE显示,中肠微粒体经8.0×104 mg/L的PEG8000沉淀后,被沉淀蛋白的分子质量主要集中在14.1 ~ 40 kD和66 ~97 kD范围内。     

棉铃虫对甲氧虫酰肼的抗性遗传力

(δp=0.5),抗性现实遗传力为实际筛选估计值的一半,当杀死率为80%和90%时,棉铃虫对甲氧虫酰肼的抗性增加10倍,分别需要34.48代和27.40代.田间条件下,由于等位基因频率变化、环境变异等因素的影响,抗性增加10倍则需要更长的时间.     

拟除虫菊酯类杀虫剂对棉铃虫雌蛾化学通讯系统的影响

用低致死剂量（LD30）氰菊酯和氰戊菊酯胸部点滴处理棉铃虫3龄幼虫后，研究拟除虫菊酯类杀虫剂对存活雌蛾化学通讯系统的影响。结果表明，杀虫剂对雌蛾的求偶行为不产生明显影响，对照组和杀虫剂处理组雌蛾的求偶高峰均为第3个暗期的8-8．5h，求偶率均可达73％-82％。氰戊菊酯处理组中雌蛾性信息素各组分滴度和总量与对照无显著性差异，但溴氰菊酯处理组中雌蛾性信息素的主组分Z11-16：Ald和次要组分Z9-16：Ald、Z7-16：Ald的滴度及信息素总量均显著高于对照组，并且两个活性组分Z11-16：Ald和Z9-16：Ald的比例与对照组雌蛾有显著差异。     

嗜线虫致病杆菌对棉铃虫的生物活性研究

嗜线虫致病杆菌HB310(Xenorhabdus nematophila HB310)菌液中主要的杀虫活性物质是一种高分子量的复合蛋白—毒素Ⅱ。以该菌液和毒素Ⅱ分别饲喂棉铃虫Helicoverpa armigera幼虫,检测其对棉铃虫生长发育的影响,同时通过生化分析研究了该毒素对幼虫中肠内几种蛋白酶活力的影响。结果表明:菌液和毒素Ⅱ对棉铃虫幼虫的取食量和生长发育均有显著的影响,取食拌有菌液和毒素Ⅱ的人工饲料的棉铃虫食量明显减少,发育速度延缓,发育历期比对照明显推迟;尽管一直取食混合原菌液(6.5×108 cells/mL)人工饲料的棉铃虫2龄幼虫前期死亡率很低,但是其生长发育几乎完全被抑制,该处理组所有幼虫均不能化蛹;原菌液对4龄幼虫的食量、发育历期、蛹重及化蛹率均有显著影响。菌液对棉铃虫幼虫的影响与菌液的浓度和幼虫的龄期成反比,稀释50倍的菌液对2龄和4龄幼虫的生长发育仍有一定影响;毒素Ⅱ(51.9 μg/mL)对4龄棉铃虫的生长发育也有明显的抑制作用。短时间(2 d)饲喂原菌液后更换正常饲料,仅延缓了棉铃虫幼虫的发育历期,而对其化蛹率、蛹重及羽化率均无明显影响。饲喂毒素Ⅱ的棉铃虫幼虫中肠主要蛋白酶的活性受到明显的抑制。     

Functional expression of Helicoverpa armigera CYP9A12 and CYP9A14 in Saccharomyces cerevisiae

Elevated oxidative detoxification is a major mechanism responsible for pyrethroid resistance in Helicoverpa armigera from Asia. Constitutive overexpression of CYP9A12 and CYP9A14 was associated with pyrethroid resistance in the YGF strain of H. armigera. CYP9A12 and CYP9A14 were functionally expressed in the W(R) strain of yeast (Saccharomyces cerevisiae) transformed with a plasmid shuttle vector pYES2. The cell lysates prepared from yeast transformed with CYP9A12 and CYP9A14, respectively, exhibited considerable O-demethylation activities against two model substrates p-nitroanisole (0.59 and 0.42 nmol p-nitrophenol min⁻¹ mg protein⁻¹) and methoxyresorufin (2.98 and 5.41 pmol resorufin min⁻¹ mg protein⁻¹), and clearance activity against the pyrethroid esfenvalerate (8.18 and 4.29 pmol esfenvalerate min⁻¹ mg protein⁻¹). These results provide important evidence on the role of CYP9A12 and CYP9A14 in conferring pyrethroid resistance in H. armigera, and also demonstrate that the yeast expression system can provide necessary redox environment for insect P450s to metabolize xenobiotics.     

棉铃虫抗辛硫磷品系的代谢抗性机理

通过重复回交和药剂选择,将棉铃虫Phoxim-R抗性品系对辛硫磷的抗性导入到BK77敏感品系中,得到棉铃虫BK77-R抗性品系,BK77-R和BK77为一对近等基因系。BK77-R抗性品系对辛硫磷的抗性达155倍,对溴氰菊酯有高水平交互抗性(抗性倍数248倍),对灭多威和硫丹有中等水平交互抗性, 分别为31倍和11倍,对丙溴磷有低水平交互抗性(4倍)。在BK77-R抗性品系中,脱叶磷(DEF,酯酶抑制剂)对辛硫磷、灭多威和硫丹具有增效作用,增效倍数分别为7倍、2倍 和1.9倍;增效醚(PBO,氧化酶抑制剂)对溴氰菊酯、灭多威和辛硫磷的增效倍数分别为21倍、2.2倍和1.7倍。与BK77敏感品系相比,BK77-R抗性品系的酯酶和多功能氧化酶活性均显著提高,而谷胱甘肽S-转移酶活性没有明显变化。上述结果表明,酯酶解毒代谢在棉铃虫BK77-R品系对辛硫磷的抗性中起重要作用,酯酶和多功能氧化酶解毒作用增强是该抗性品系对不同类型药剂产生交互抗性的重要原因。     

中华卵索线虫寄生对棉铃虫保幼激素和蜕皮激素的影响

中华卵索线虫Ovomermis sinensis Chen广泛寄生于粮、棉、油等多种作物害虫体内,生防潜力极大,但其体外培养尚未获得成功[1],阻碍了该线虫生物杀虫剂的开发与应用,因此,对线虫寄生期生长机制的研究具有重要意义。目前,对中华卵索线虫寄生后宿主血淋巴的相关研究主要集中在营养成分、生化物质等方面。王茂先等[2]对该线虫感染棉铃虫     

Tolerance to acetolactate synthase and acetyl-coenzyme A carboxylase inhibiting herbicides in Vulpia bromoides is conferred by two co-existing resistance mechanisms

Vulpia bromoides is a grass species naturally tolerant to acetolactate synthase (ALS) and acetyl-coenzyme A carboxylase (ACCase) inhibiting herbicides. The mechanism of tolerance to ALS herbicides was determined as cytochrome P450-monooxygenase mediated metabolic detoxification. The ALS enzyme extract partially purified from V. bromoides shoot tissue was found to be as sensitive as that of herbicide susceptible Lolium rigidum to ALS-inhibiting sulfonylurea (SU), triazolopyrimidine (TP), and imidazolinone (IM) herbicides. Furthermore, phytotoxicity of the wheat-selective SU herbicide chlorsulfuron was significantly enhanced in vivo in the presence of the known P450 inhibitor malathion. In contract, the biochemical basis of tolerance to ACCase inhibiting herbicides was established as an insensitive ACCase. In vitro ACCase inhibition assays showed that, compared to a herbicide susceptible L. rigidum, the V. bromoides ACCase was moderately (4.5- to 9.5-fold) insensitive to the aryloxyphenoxypropionate (APP) herbicides diclofop, fluazifop, and haloxyfop and highly insensitive (20- to >71-fold) to the cyclohexanedione (CHD) herbicides sethoxydim and tralkoxydim. No differential absorption or de-esterification of fluazifop-P-butyl was observed between the two species at 48 h after herbicide application, and furthermore V. bromoides did not detoxify fluazifop acid as rapidly as susceptible L. rigidum. It is concluded that two co-existing resistance mechanisms, i.e., an enhanced metabolism of ALS herbicides and an insensitive target ACCase, endow natural tolerance to ALS and ACCase inhibiting herbicides in V. bromoides.     

嗜线虫致病杆菌北京变种CB6菌株杀虫蛋白对棉铃虫幼虫的生物活性

嗜线虫致病杆菌北京变种Xenorhabdus var.pekingensis CB6菌株是本研究室自主分离的新菌株,其代谢物对棉铃虫具有很强的拒食和抑制生长活性.为了进一步明确代谢物中杀虫蛋白的生物活性,作者用饲料染毒法和叶碟法测定了杀虫蛋白对棉铃虫不同龄期幼虫取食和生长发育的影响.结果表明,杀虫蛋白对棉铃虫幼虫有很强的抑制生长作用,用64μg/g含杀虫蛋白饲料饲喂初孵、1、2和3龄幼虫5天的生长抑制率分别达95.37%、92.73%、87.15%和88.64%,并明显延长幼虫发育历期,影响幼虫的化蛹及蛹的羽化.杀虫蛋白对5龄棉铃虫幼虫拒食效果明显,幼虫饲喂经1.6mg/mL杀虫蛋白处理的叶片24h,选择性拒食率和非选择性拒食率分别为76.22%和85.42%.当蛋白浓度为0.32mg/mL时,24h选择性拒食和非选择性拒食率分别为68.39%和74.75%.     

新疆地区棉铃虫自然种群对Bt棉的抗性频率监测

为监测新疆棉区棉铃虫Helicoverpa armigera(Hübner)田间种群对Bt棉的抗性频率,在2010年和2011年分别采集石河子和喀什地区莎车的棉铃虫单雌系,以Cry1Ac毒蛋白作为人工饲料,用单雌系F1/F2代法进行棉铃虫种群抗性个体检测。2010年筛选了123个石河子的棉铃虫单雌系,2 011年筛选了152个莎车的棉铃虫单雌系。两地的棉铃虫种群均没有筛选到相对平均发育级别≥0.8的抗Bt棉个体,估算出石河子和莎车的棉铃虫种群的抗性频率低于10-3。莎车F2单雌系与其对应的F1单雌系相对平均发育级别有明显差异。研究表明新疆石河子地区田间棉铃虫种群仍保持敏感状态,喀什地区田间棉铃虫种群对Bt棉的耐受性增高。     

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.     

Binding of three Cry1A toxins in resistant and susceptible strains of cotton bollworm (Helicoverpa armigera)

Evolution of resistance by pests is the greatest threat to the continuous success of theBacillus thuringiensis (Bt) toxins used in conventional sprays or in transgenic plants. The most common mechanism of insect resistance to Bt is reduced binding of toxins to target sites in the brush border membrane of the larval mid-gut. In this paper, binding experiments were performed with three ¹²⁵I-Cry1A toxins and the brush border membrane vesicles from Cry1Ac resistant or susceptible strains of Helicoverpa armigera. The homologous competition test showed that there was no significant difference in Cry1Ac-binding affinity, but the concentration of Cry1Ac-binding sites dramatically decreased in the resistant strain (R_t decreased from 5.87 ± 1.40 to 2.23 ± 0.80). The heterologous competition test showed that there were three Cry1Ac-binding sites in the susceptible strain. Among them, site 1 bound with all three Cry1A toxins, site 2 bound with both Cry1Ab and Cry1Ac, and site 3 only bound with Cry1Ac. In the Cry1Ac resistant strain, the binding capability of site 1 with Cry1Ab decreased and site 2 did not bind with Cry1Ac. It is suggested that the absence of one binding site is responsible for H. armigera resistance to Cry1Ac. This result also showed that the resistance fitted the “mode 1” pattern of Bt resistance described previously.     

杀虫药剂混用对棉铃虫乙酰胆碱酯酶和羧酸酯酶的联合抑制作用研究

选用辛硫磷、对硫磷、敌敌畏和氧乐果4种有机磷类杀虫剂以及灭多威和甲萘威两种氨基甲酸酯类杀虫剂分别以1∶1、1∶3和3∶1的比例混用,以棉铃虫Helicoverpa armigera为试虫,分别测得单剂和混剂对其乙酰胆碱酯酶(AChE)和羧酸酯酶(CarE)的抑制中浓度(I50),以联合抑制系数作为增效作用的参考指标进行了比较。研究发现,两种药剂不同配比的混剂对同种酯酶的联合抑制作用也往往不同。对AChE和CarE同时具有抑制作用的只有其中的对硫磷＋甲萘威(1∶3)、辛硫磷+对硫磷(1∶3)和灭多威+氧乐果(1∶3),而甲萘威+敌敌畏(1∶3)、甲萘威＋氧乐果(1∶1)、辛硫磷+甲萘威(1∶1)、对硫磷+氧乐果(1∶1)、对硫磷+氧乐果(1∶3)、敌敌畏+氧乐果(1∶3)、灭多威+对硫磷(1∶1) 7种混剂对AChE和CarE都具有拮抗作用。     

Inhibition of P-glycoprotein ATPase and its transport function of Helicoverpa armigera by morin, quercetin and phloroglucinol

Flavonoids (morin, quercetin and phloroglucinol) were tested for their ability to modulate the function of P-glycoprotein ATPase of the insecticide resistant pest Helicoverpa armigera (Ha-Pgp). Flavonoids in the presence of ethylparaoxon or cypermethrin significantly reduced both larval weight as well as survival rate 40-50%. Morin and quercetin inhibited the activity of Ha-Pgp ATPase by 80-90%, whereas phloroglucinol inhibited ATPase activity by 40% at 100 μM concentration. These flavonoids inhibited the verapamil, ethylparaoxon and cypermethrin-stimulated Ha-Pgp ATPase activity. Morin, quercetin and phloroglucinol binding were quantitated by quenching of the intrinsic Trp fluorescence of purified Ha-Pgp ATPase. Drug transport was monitored in proteoliposomes containing Ha-Pgp ATPase using the high affinity fluorescent substrate tetramethylrosamine (TMR) in real time. Addition of the morin and quercetin mediated the collapse of the TMR concentration gradient generated by Ha-Pgp ATPase. The inhibition studies on Ha-Pgp ATPase activity may contribute towards understanding new strategies of the pest to overcome insecticide resistance.     

Molecular cloning and mRNA expression of a ryanodine receptor gene in the cotton bollworm, Helicoverpa armigera

Ryanodine receptors (RyRs) are the targets of novel diamide insecticides. The cotton bollworm, Helicoverpa armigera, is one of the most important cotton pests in the world. In this study, we report the full-length RyR cDNA sequence (named as HaRyR) of H. armigera. The 16,083-bp contiguous sequence encoded 5, 142 amino acid residues, which shares 80% and 78% overall identities with its homologues in Nilaparvata lugens (NlRyR) and Drosophila melanogaster (DmRyR), respectively. All hallmarks of RyR proteins are conserved in the HaRyR, including the GXRXGGGXGD motif conserved in the Ca²⁺ release channels and four copies of RyR domain unique to RyR channels. The previously identified seven lepidopteran-specific RyR residues were also found in HaRyR (N⁴⁹⁷⁷, N⁴⁹⁷⁹, N⁴⁹⁹⁰, L⁵⁰⁰⁵, L⁵⁰³⁶, N⁵⁰⁶⁸ and T⁵¹¹⁹). An amino acid sequence alignment showed that the N-terminal region of HaRyR (residues 188–295) shared high sequence identity with NlRyR (94%) and DmRyR (92%), and moderate sequence identity (47–50%) with three rabbit RyR isoforms, while the short segment of the C-terminal transmembrane region of HaRyR (residues 4632–4676) exhibited moderate sequence identity with NlRyR (69%) and DmRyR (67%), and low sequence identity (19–28%) with three rabbit RyR isoforms. In addition, expression analysis of HaRyR revealed that the mRNA expression level in eggs was significantly lower than in third instar larvae, pupae and adults, and anatomical regulation of HaRyR expression was also observed with the highest expression level in head compared with thorax and abdomen. Our results lay a foundation for comprehensive structural and functional characterization of HaRyR and for understanding of the molecular mechanisms of toxicity selectivity of diamide insecticides among different species.     

Methylparathion- and carbofuran-induced mitochondrial dysfunction and oxidative stress in Helicoverpa armigera (Noctuidae: Lepidoptera)

The cotton bollworm, Helicoverpa armigera is a polyphagous pest of several crops in Asia, Africa, and the Mediterranean Europe. Organophosphate and carbamate insecticides are used on a large-scale to control Helicoverpa. Therefore, we studied the effect of methylparathion and carbofuran, an organophosphate and carbamate insecticide, respectively, on oxidative phosphorylation and oxidative stress in H. armigera larvae to gain an understanding of the different target sites of these insecticides. It was observed that state III and state IV respiration, respiratory control index (RCI), and P/O ratios were inhibited in a dose-dependent manner by methylparathion and carbofuran under in vitro and in vivo conditions. Methylparathion and carbofuran inhibited complex II by ∼45% and 30%, respectively. Lipid peroxidation, H₂O₂ content, and lactate dehydrogenase (LDH) activity increased and glutathione reductase (GR) activity decreased in a time- and dose-dependent manner in insecticide-fed larvae. However, catalase activity was not affected in insecticide-fed larvae. Larval growth decreased by ∼64% and 67% in larvae fed on diets with 100 μM of methylparathion and carbofuran. The results suggested that both the insecticides impede the mitochondrial respiratory functions and induced lipid peroxidation, H₂O₂, and LDH leak, leading to oxidative stress in cells, which contribute to deleterious effects of these insecticides on the growth of H. armigera larvae, along with their neurotoxic effects.     

棉铃虫对Cry1Ac抗性的稳定性及其对适合度的影响

为明确棉铃虫Helicoverpa armigera（Hübner）对苏云金芽胞杆菌Bacillus thuringiensis（Bt）Cry1Ac毒蛋白抗性的稳定性及其适合度变化,利用生物测定的方法研究了Cry1Ac抗性品系棉铃虫转到正常饲料饲养后的抗性衰退及再次筛选后抗性的恢复情况,并比较了敏感、抗性和抗性衰退后各品系间的适合度差异。在失去选择压的情况下,高抗品系棉铃虫对Cry1Ac的抗性迅速衰退,经过4代后抗性水平由最初的3626.67倍下降到1436.67倍;到第12代时抗性水平已低于10倍,随后品系保持较稳定的低抗水平;当重新进行抗性再筛选时,其抗性水平可快速恢复,抗性倍数快速回升,5代后恢复到1123.33倍。与敏感品系相比,高抗棉铃虫品系的适合度明显降低,相对适合度仅为0.33,但转到正常饲料连续饲养14代后,棉铃虫适合度明显上升,相对适合度为0.87,主要表现为卵孵化率和幼虫存活率等显著提高。