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1.
BACKGROUND: The rynodine receptors (RyRs) are the main targets of diamide insecticides such as chlorantraniliprole. To provide the basis for a good understanding of the molecular mechanisms of diamide insecticide resistance, an RyR gene from Plutella xylostella was cloned and characterised in the present paper. RESULTS: A full‐length cDNA sequence of RyR was cloned from P. xylostella through RT‐PCR and rapid amplification of cDNA ends (RACE). The gene (named PxRyR1) is 15 753 bp long, with an open reading frame of 15 354 bp, encoding a predicted RyR of 5117 amino acids. An alternative splicing of the PxRyR1 was also cloned and named PxRyR2. The PxRyR1 shares 77–93% identity with other insect RyRs. Quantitative real‐time PCR analysis showed that the PxRyR was expressed at a high level in second‐instar larvae and adults, at a low level in prepupae and pupae and abundantly in the body wall muscle and head (respectively 6.00 and 3.12 times the expression in the gut). Western blot analysis with anti‐RyR antibodies showed that the RyR was mainly present in the body wall muscle and head, but barely present in the haemocyte and gut. CONCLUSIONS: There are at least two alternative splices of PxRyR expressed in all developmental stages and tissues in P. xylostella at various levels. The results provided the basis for further understanding of the mechanisms of resistance to diamide insecticides in P. xylostella. Copyright © 2012 Society of Chemical Industry  相似文献   

2.
Development of insecticides with unique modes of action is necessary to combat widespread insecticide resistance. A new class of insecticides has been discovered, the anthranilic diamides, that provides exceptional control through action on a novel target, the ryanodine receptor. Anthranilic diamides potently activate this receptor, releasing stored calcium from the sarcoendoplasmic reticulum causing impaired regulation of muscle contraction. Expression of a recombinant Drosophila ryanodine receptor in a lepidopteran cell line confers sensitivity to anthranilic diamides similar to that observed with native receptors. Ligand-binding studies with radiolabeled ryanodine and radiolabeled anthranilic diamide in Periplaneta americana reveal a single, saturable binding site for this chemistry distinct from that of ryanodine. Further, calcium mobilization studies using mammalian cell lines indicate anthranilic diamides exhibit >500-fold differential selectivity toward insect, over mammalian, receptors. Consequently, anthranilic diamides offer a novel pharmacological tool for calcium signaling research in addition to a unique alternative to existing pest-management strategies.  相似文献   

3.
The full-length cDNA sequence of γ-aminobutyric acid (GABA) receptor alpha2 subunit gene was cloned from Spodoptera exigua, an economically important pest species in China, through rapid amplification of cDNA ends (RACE) and polymerase chain reaction using primers based on the 5′ and 3′-ends of the mRNA. The gene (named as SeGABARα2) was 1620 bp long, with an open reading frame of 1500 bp encoding a predicted GABA receptor protein of 499 amino acids with a molecular weight of 55 kDa. The predicted GABA receptor protein shared 96.19%, 82.16%, 74.30%, 73.95%, 73.42%, 71.40%, 67.11% and 65.13% identity with other GABA receptors proteins isolated from Heliothis virescens, Plutella xylostella, Aedes aegypti, Laodelphax striatellus, Lucilia cuprina, Musca domestica, Drosophila simulans and Drosophila melanogaster, at amino acid level, respectively. The developmental changes and tissue-specificity of the relative mRNA expression levels of SeGABARα2 gene were investigated in S. exigua. The highest expression level was observed in adult and lowest was in the first instar, there was a stable expression level during the developmental period from the second instar to the adult. Expression levels were 1.41-, 1.57-, 1.45-, 1.80-, 1.82- and 1.93-fold higher in the second instar, third instar, fourth instar, fifth instar, pupa and adult than in the first instar larva, respectively. The relative mRNA expression levels of SeGABARα2 gene were 3.51-fold higher in head and 1.77-fold higher in thorax than in abdomen  相似文献   

4.
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.  相似文献   

5.
Insecticidal tests using diazinon showed that the mortality of Plutella xylostella larvae parasitized by Cotesia plutellae was reduced by 4.6-fold compared to that of the nonparasitized hosts. The use of chemicals with synergistic effect to insecticides in toxicity assay helps to elucidate the kind of enzyme involved in lowering insect mortality. Synergism of diethyl maleate and piperonyl butoxide with diazinon resulted to 2.4- and 1.9-fold increase, respectively, in susceptibility of parasitized larvae compared to those of nonparasitized larvae. These results indicated the possibility that the decrease in susceptibility to diazinon was due to the elevated activities of glutathione-S-transferase (GST) and cytochrome P450 monooxygenase (CYP), respectively. The GST activities in parasitized larvae were significantly higher than those of nonparasitized ones starting from three days post-parasitization until emergence of parasitoid larva. High GST activities during late parasitism could be attributed to both enzyme activities toward diazinon of parasitized P. xylostella larva itself and C. plutellae larva inside larval host. High GST activity one day after parasitization, although statistical significance was not detected, was caused by polydnavirus (PDV) and the venom of C. plutellae not by parasitoid larvae. Artificial injection of PDV plus venom demonstrated that the resulting increase in GST activity is similar to the increase brought by parasitization. High CYP activity after 3 days post-parasitization in parasitized larva was attributed mainly to the activity of parasitoid larva. Carboxylesterase activity in the parasitized host remained at a high level, while that in the nonparasitized host decreased slightly as pupation approaches. On the other hand, acetylcholinesterase activity also remained constant after parasitization until larval emergence, while that of the nonparasitized hosts decreased gradually as the host larvae approach pupation. These results were supported by inhibition tests using diazoxon in vitro.  相似文献   

6.
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7.
昆虫鱼尼丁受体及以其为靶标的杀虫剂的研究进展   总被引：9，自引：3，他引：6  
植物保护领域以昆虫鱼尼丁受体(ryanodine receptor,RyR)为靶标的杀虫剂的研发取得了突破性进展。对近年来RyR在分子结构、功能调节,以及对以RyR为靶标的杀虫剂的作用机制方面的研究进展进行了综述。昆虫RyR与哺乳动物RyR仅有约47%的同源性,因而是一个有效的杀虫剂靶标。昆虫RyR克隆与表达技术的成熟为新型杀虫剂的开发和作用机制的研究提供了有力的工具。昆虫RyR单通道、配体结合和免疫学特性的研究补充了RyR的电生理学数据。近年来开发的新型RyR杀虫剂通过激活害虫鱼尼丁敏感的细胞内钙离子释放通道来达到杀虫的效果。  相似文献   

8.
The insecticidal activity of four forms of Hong Jing (HJ) allylisothiocyanate (AITC), AITC + cypermethrin (HJA, HJB, and HJC) with ratio of (1:1, 4:1, and 2:1), pure AITC (HJD), and two forms of Hong Du (HD) AITC, AITC + chlorpyrifos (HDA and HDB) with ratio of (2:1 and 2:1), respectively, were studied on the major cruciferous insect larvae Plutella xylostella (L.) and Pieris rapae (L.) by combining both spraying and dipping methods. The P. rapae was more susceptible than P. xylostella larvae. The LC50 values 72 h after treatment of AITC forms (HJB, HJA, HJC, HJD, HDB, and HDA) on the P. rapae were; 0.07, 0.08, 0.16, 0.83, 0.26, 1.08 gL−1, and 0.69, 0.26, 5.45, 0.93, 3.01, 5.98 gL−1 on the P. xylostella, respectively. The toxicity of some of the AITC forms was very close to or better than that of the commercial contact insecticides such as chlorpyrifos (LC50 = 0.03 and 0.04 gL−1 on P. rapae and P. xylostella, respectively), and cypermethrin (0.65 and 0.78 gL−1, respectively, against P. rapae and P. xylostella). The ultrastructural studies on the integument of the third larval instar of P. xylostella treated by sub-lethal concentration (LC20) of HJB, HJD, and HDB were carried out by using transmission electron microscope. The more pronounced alterations in the hypodermis and mitochondria cells. They exhibited changes in all treated samples. The hypodermis was almost completely destroyed, and the mitochondria exhibited morphological alterations, represented by enlargement, matrix rarefaction and vacuolization of the mitochondria matrix, quantity of cristae reduced, and density electron matrix lessened. These AITC forms have potential as contact insecticides, and the ultra structural observations confirm the insecticidal efficiency of different AITC forms on P. rapae and P. xylostella.  相似文献   

9.
Acetylcholinesterase (AChE) is the primary target of organophosphate (OP) and carbamate (CB) insecticides. Many insect species have been shown to have two different AChE genes. The amino acid identity between the two lepidopteran AChEs is lower than 40%, and potential differences in enzymatic function have not been characterized. In this study, the cDNAs encoding two AChEs (Boma-AChE1 and Boma-AChE2) from Bombyx mandarina were sequenced, and the corresponding proteins were heterologously expressed to compare their enzymatic properties and interactions with insecticides in vitro. Both of these enzymes had high specific activities for acetylthiocholine iodide. Studies on substrate and inhibitor specificities confirmed that both enzymes belong to AChE. Insecticide inhibition assays indicated that Boma-AChE1 was more sensitive than Boma-AChE2 to eight of the nine insecticides tested. However, Boma-AChE2 was more sensitive than Boma-AChE1 to one of the OP insecticides, heptenophos. The results suggested that two AChEs from a lepidopteran insect have distinct catalytic properties and responses to different inhibitors.  相似文献   

10.
Decreased insecticides cuticular penetration, as one of resistant mechanisms in insect, has been extensively documented. Laccases, are enzymes with p-diphenol oxidase activity, was related to the cuticular tanning in insect. In this study, one laccase 2 gene (CpLac2) was cloned from Culex pipiens pallens. The CpLac2 contains an open reading frame (ORF) of 2289 bp and encodes a putative 762 amino acid protein. The deduced protein of CpLac2 was more similar to laccase 2 than other insect laccases, and shared the highest identity with laccases from the same family mosquito, Aedes aegypti and Anopheles gambiae. The developmental expression model of CpLac2 in C. pipiens pallens was measured by RT-PCR. The result showed the CpLaC2 was abundantly expressed in egg, the 4th instar larva and pupa, which suggested the role of CpLac2 for egg chorion tanning and cuticular sclerotization. Meanwhile, the expression of CpLac2 in fenvalerate-susceptible and -resistant strains of C. pipiens pallens was measured by real-time PCR. The result revealed the CpLac2 was significant higher expressed in resistant strain than in susceptible strain. The overexpression of CpLac2 in resistant strain suggested that resistance could derive from reinforcement of the cuticle, which decreased the penetration of insecticide in cuticle.  相似文献   

11.
氯虫苯甲酰胺对小菜蛾鱼尼丁受体基因mRNA表达量的影响   总被引：3，自引：1，他引：2  
采用实时荧光定量PCR研究比较了氯虫苯甲酰胺、甲氨基阿维菌素苯甲酸盐和溴虫腈对小菜蛾鱼尼丁受体基因mRNA表达量的影响,研究分析了低剂量氯虫苯甲酰胺连续处理和间断处理对小菜蛾鱼尼丁受体基因表达量的影响,以及对氯虫苯甲酰胺产生抗性的小菜蛾与敏感小菜蛾鱼尼丁受体基因表达量的差异。结果表明:氯虫苯甲酰胺可提高小菜蛾鱼尼丁受体基因表达量,而甲氨基阿维菌素苯甲酸盐和溴虫腈对其表达量均无影响;0.05和0.01 mg/L的氯虫苯甲酰胺均可使小菜蛾鱼尼丁受体基因表达量增加;对氯虫苯甲酰胺产生抗性的小菜蛾鱼尼丁受体基因表达量是敏感小菜蛾的5.79倍。研究结果有助于阐明氯虫苯甲酰胺的作用机理,并可能为筛选作用于鱼尼丁受体的新化合物提供参考。  相似文献   

12.
BACKGROUND: Phthalic acid diamide derivatives are among the most important classes of synthetic insecticides. In this study, a 3,3‐dichloro‐2‐propenyloxy group, the essential active group of pyridalyl derivatives, was incorporated into phthalic acid diamide derivatives with the aim of combining the active groups to generate more potent insecticides. RESULTS: Thirty‐one new phthalic acid diamides were obtained, and these were characterised by 1H and 13C NMR. The structure of N2‐[1,1‐dimethyl‐2‐(methoxy)ethyl]‐3‐iodo‐N1‐[4‐(3,3‐dichloro‐2‐propenyloxy)‐3‐(trifluoromethyl)phenyl]‐1,2‐benzenedicarboxamide was determined by X‐ray diffraction crystallography. The insecticidal activities of the compounds against Plutella xylostella were evaluated. The title compounds exhibited excellent larvicidal activities against P. xylostella. Structure‐activity relationships revealed that varying the combination of aliphatic amide and aromatic amide moieties, or the nature and position of substituent Y on the aniline ring, could aid the design of structures with superior performance. CONCLUSION: A series of novel phthalic acid diamides containing a 3,3‐dichloro‐2‐propenyloxy group at the 4‐position of the aniline ring were designed and synthesised. Structure‐activity relationships with the parent structure provided information that could direct further investigation on structure modification. Copyright © 2012 Society of Chemical Industry  相似文献   

13.
测定了玫烟色棒束孢原始亲本菌株PFCF-O、退化亲本菌株PFCF-D和单孢分离子菌株PF-CF-D58、PFCF-D20、PFCF-D50的菌落生长速率、产孢量、孢子萌发率和胞外蛋白酶水平等生物学指标以及各菌株对小菜蛾2龄幼虫的校正死亡率。结果表明,菌株PFCF-O的生长速率呈先快后慢的趋势,产孢量为2.7×107个/mL,孢子萌发率为62.59%,胞外蛋白酶水平为1.16,对小菜蛾2龄幼虫校正死亡率达97.74%。而菌株PFCF-D、PFCF-D20、PFCF-D50和PFCF-D58间平均生长速率和孢子萌发率无显著差异,产孢量变化范围为1.05×107~2.34×107个/mL,胞外蛋白酶水平变化范围为1.02~1.07,各生物学指标及其对小菜蛾2龄幼虫的校正死亡率(19.86%~51.44%)都显著低于菌株PFCF-O。相关性分析结果显示,玫烟色棒束孢对小菜蛾的致病力与菌株生长初期的生长速率、产孢量、孢子萌发率和胞外蛋白酶水平呈极显著的正相关关系,与菌株生长后期的生长速率呈极显著负相关关系。  相似文献   

14.
Since insecticide insensitivity of acetylcholinesterase (AChE) was, found about 40 years ago, a cause of the resistance to organophosphates in the spider mite, more than 30 insect and Acarus species have added to the instance. Based on the 3-dimensional analysis of Torpedo AChE structure and sequencing of Drosophila AChE gene (Ace), amino acid substitutions conferring the insensitivity have been found in Drosophila melanogaster. However, no amino acid substitution responsible for the AChE insensitivity had been found in insects and Acari except Brachicera flies until the second type of AChE paralogous to Ace was discovered in Schizaphis graminus and Anopheles gambiae. Sequencing of Ace-paralogous AChE cDNAs has been followed in insect species of various orders. Now, various amino acid substitutions are found and correspond to different biochemical properties of insensitive AChEs in relation to the function of substituted amino acids in the 3-dimensional structure. Existence of two AChE genes raises questions about differentiation of the two genes, site of gene expression, and function of each enzyme.  相似文献   

15.
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.  相似文献   

16.
Acetylcholinesterase (AChE), which is encoded by the ace gene, catalyzes the hydrolysis of the neurotransmitter acetylcholine to terminate nerve impulses at the postsynaptic membrane. AChE is a primary target of many insecticides including organophosphates (OP) and carbamates (CB). In this study, full-length cDNA sequences of two ace genes (Nlace1 and Nlace2) were sequenced from the brown planthopper (BPH) Nilaparvata lugens, the most destructive insect pest of rice crops. Nlace1 cDNA is 2842 nucleotides long and contains an ORF potentially encoding a 790 amino acid peptide. Nlace2 cDNA is 2852 bp in length and contains an ORF that potentially encodes a 672 amino acid peptide. NlAChE1 has an identity of 40% with NlAChE2 at the amino acid sequence level. Phylogenetic analysis of 59 AChEs from 32 animal species showed that NlAChE1 is most closely related to AChE1s from Blattella germanica and Nephotettix cincticeps, while NlAChE2 is most closely related to AChE2 from N. cincticeps. Quantitative RT-PCR analysis showed that Nlace1 is expressed at a much higher level than Nlace2 in all developmental stages and tissues, demonstrating that NlAChE1 may be the dominant AChE form of the two enzymes. This result will help reveal the resistance mechanism of N. lugens to organophosphorous and carbamate insecticides and promote development of more selective insecticides targeting the main NlAChE1.  相似文献   

17.
八种杀虫剂对黑粪蚊的防治效果及残留分析   总被引：2，自引：1，他引：1  
选择8种杀虫剂,分别在实验室和菇房条件下研究其对黑粪蚊的防治效果和对平菇菌丝生长的影响,并分析施药后不同时间平菇中的农药残留.结果表明,40%辛硫磷EC、40%氧乐果EC、48%毒死蜱EC对黑粪蚊具有很好的防治效果,但对平菇菌丝的抑制率明显高于其它药剂;10%吡虫啉WP、4.5%高效氯氰菊酯EC不仅对黑粪蚊成、幼虫具有良好的防治效果,而且使用后对平菇菌丝生长的影响较小;2.5%高效氯氟氰菊酯EW、4.5%高效氯氰菊酯EC、1.8%阿维菌素EC等使用5天后均未检测到农药残留,5%氟虫腈SC和10%吡虫啉WP药后15天仍能检测到残留.  相似文献   

18.
Pollen beetle, Meligethes aeneus F. (Coleoptera: Nitidulidae) is a major univoltine pest of oilseed rape in many European countries. Winter oilseed rape is cultivated on several million hectares in Europe and the continuous use of pyrethroid insecticides to control pollen beetle populations has resulted in high selection pressure and subsequent development of resistance. Resistance to pyrethroid insecticides in this pest is now widespread and the levels of resistance are often sufficient to result in field control failures at recommended application rates. Recently, metabolic resistance mediated by cytochrome P450 monooxygenases was implicated in the resistance of several pollen beetle populations from different European regions. Here, we have also investigated the possible occurrence of a target-site mechanism caused by modification of the pollen beetle para-type voltage-gated sodium channel gene. We detected a single nucleotide change that results in an amino acid substitution (L1014F) within the domain IIS6 region of the channel protein. The L1014F mutation, often termed kdr, has been found in several other insect pests and is known to confer moderate levels of resistance to pyrethroids. We developed a pyrosequencing-based diagnostic assay that can detect the L1014F mutation in individual beetles and tested more than 350 populations collected between 2006 and 2010 in 13 European countries. In the majority of populations tested the mutation was absent, and only samples from two countries, Denmark and Sweden, contained pollen beetles heterozygous or homozygous for the L1014F mutation. The mutation was first detected in a sample from Denmark collected in 2007 after reports of field failure using tau-fluvalinate, and has since been detected in 7 out of 11 samples from Denmark and 25 of 33 samples from Sweden. No super-kdr mutations (e.g. M918T) known to cause resistance to pyrethroids were detected. The implications of these results for resistance management strategies of pollen beetle populations in oilseed rape crops are discussed.  相似文献   

19.
Previous studies performed in our laboratory have measured the effect of atrazine exposure on cytochrome P450-dependent monooxygenase activity and have found increased activity in midge larvae (Chironomus tentans) as a result of atrazine exposure (1-10 ppm). Here we report the cloning and expression of a specific C. tentans CYP4 gene that is responsive to atrazine induction with an open reading frame of 1678 bp which encodes a putative protein of 559 amino acid residues. Alignments of deduced amino acid sequences with other insect P450 genes and phylogenetic analysis indicated a high degree of similarity to other insect CYP4 genes. Northern blotting analysis employing a fragment of 1200 bp from the CYP4 gene as a probe indicated that the CYP4 gene was expressed in all developmental stages, but was expressed at highest levels in late instar larvae. Additionally, over-expression of CYP4 in C. tentans exposed to atrazine (10 mg/l) confirms the ability of atrazine to induce specific P450 genes and provides insight into potential consequences of atrazine exposure in aquatic organisms.  相似文献   

20.
The residual toxicity of two selective insecticides, teflubenzuron (acylurea) and Bacillus thuringiensis Berliner ssp. aizawai (microbial), to laboratory and field strains of Plutella xylostella L. was shown in the laboratory to be significantly affected by leaf nutritional status, other host-plant resistance factors, cultivation method and plant age. With plants offering some degree of host-plant resistance, the toxicity of the insecticides was either increased or decreased compared with highly susceptible plants, depending on the specific nature of the plant-herbivore interaction. Differences in residual toxicity of the insecticides varied up to nine-fold on different host plants (=host-plant- + insecticide-induced mortality) despite less than four-fold differences in P. xylostella mortality in controls (=host-plant-induced mortality alone). Host-plant nutritional status also had a substantial effect on the damage potential of larvae. Different response times by P. xylostella to the two insecticides tested on host plants of varying nutritional status were related to the contrasting modes of action of the respective active ingredients. The present studies suggest that insecticides applied to Brassica oleracea L. var. capitata with partial plant resistance may contribute to improved control of P. xylostella. A conceptual model is used to describe likely mortality responses by macrophagous larvae to insecticides applied to plants of varying resistance status. The implications of the findings in relation to the integrated management of P. xylostella are considered.  相似文献   

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