昆虫对拟除虫菊酯类杀虫剂的代谢抗性机制研究进展

随着拟除虫菊酯类杀虫剂在卫生和农业害虫防治中的广泛应用,昆虫对此类杀虫剂产生抗性的报道越来越多。目前已明确昆虫对拟除虫菊酯类杀虫剂的抗性机制包括表皮穿透率下降、靶标抗性以及代谢抗性,其中代谢抗性机制较为普遍,而且其与昆虫对多种杀虫剂的交互抗性关系密切。目前,随着基因组、转录组以及蛋白质组学等新技术的发展及应用,昆虫对拟除虫菊酯类杀虫剂的代谢抗性机制研究也取得了很多新进展。昆虫体内细胞色素P450酶（P450s）、羧酸酯酶（CarE）及谷胱甘肽S-转移酶（GSTs）等重要解毒酶系的改变均与昆虫对拟除虫菊酯类杀虫剂的代谢抗性有关,其中这3类解毒酶的活性及相关基因表达量的变化是昆虫对此类杀虫剂产生代谢抗性的主要原因。明确昆虫对拟除虫菊酯类杀虫剂的代谢抗性机制,对合理使用此类杀虫剂及延缓抗药性的产生均具有重要意义。本文在总结拟除虫菊酯类杀虫剂代谢路径及相关生物酶研究概况的基础上,综述了近年来有关昆虫对此类杀虫剂代谢抗性机制研究的主要进展。     

主要农业害虫对茚虫威的抗性现状及其治理策略

茚虫威属于噁二嗪类杀虫剂，与大多数杀虫剂不同的是其进入害虫体内需要经活化代谢转变成N-去甲氧羰基代谢物（decarbomethoxylated metabolite，DCJW）后不可逆地阻断钠通道，进而发挥杀虫活性。茚虫威由于其作用机制不同于常见的使钠离子通道延迟关闭的菊酯类药剂而被广泛用于鳞翅目和一些同翅目、鞘翅目害虫的防治。抗药性是任何杀虫药剂使用后面临的问题，茚虫威也不例外，许多害虫对其产生了不同程度的抗性。昆虫对茚虫威产生抗性的机制包括酯酶活性、谷胱甘肽S-转移酶（glutathione S-transferase，GST）和P450活性的增加以及分子靶标F1845Y、V1848I、L1014P的突变，这些对茚虫威抗性机制的研究基本都是基于抗性种群和敏感种群开展的，需要进一步验证其对抗性研究的贡献度。针对我国田间害虫种群对茚虫威的抗性现状，及时实施对茚虫威有效的抗性治理是迫切的。对于茚虫威的抗性治理除了传统的杀虫药剂轮用、混用外，需要利用其作用机制特点开展抗性治理策略研究。一是充分利用其活化代谢的特点，开展组合药剂的研究应用；二是菊酯类药剂和茚虫威的作用机制均与钠离子通道有关，但是前者是使钠离子通道关闭延迟，而后者是阻断钠离子通道，开展相关基础研究，使菊酯类药剂与茚虫威合理地用于抗性治理中。本文综述了茚虫威的抗性现状、抗性机制与交互抗性、茚虫威的抗性风险评价，针对茚虫威的抗性特点提出了抗性治理策略。     

Lack of cross-resistance to indoxacarb in insecticide-resistant Spodoptera frugiperda (Lepidoptera: Noctuidae) and Plutella xylostella (Lepidoptera: Yponomeutidae)

Two field strains of the fall armyworm, Spodoptera frugiperda (JE Smith), collected from corn in north Florida showed high resistance to carbaryl (626- and 1159-fold) and moderate resistance to parathion-methyl (30- and 39-fold) as compared with a laboratory susceptible strain. A field strain of the diamondback moth, Plutella xylostella (L.) collected from cabbage in north Florida and selected for 20 generations with permethrin showed high resistance to permethrin (987-fold) as compared with a susceptible strain. However, in all instances, no cross-resistance to indoxacarb, a novel oxidiazine insecticide, was observed in these two species. Biochemical studies revealed that, in S. frugiperda, activities of detoxification enzymes (microsomal oxidase, glutathione S-transferase and general esterase) were significantly higher in the field strains than in the susceptible strain, indicating that these detoxification enzymes were not actively involved in the resistance to indoxacarb. The lack of cross-resistance between indoxacarb and permethrin in P. xylostella further supports the notion that the mode of action of these insecticides on the insect sodium channel is different.     

新烟碱类杀虫剂抗药性研究进展

新烟碱类杀虫剂是一类新开发的杀虫剂。研究表明，害虫野外种群对其敏感性差弄较大，现已有多种害虫对吡虫啉和啶虫脒产生了抗性。初步研究显示，马铃薯叶甲对吡虫啉抗性以不完全隐性的常染色体遗传；抗性似不稳定，交互抗性谱随虫种而变化，抗性形成可能与多功能氧化酶和酯酶有关。合理轮用和高剂量杀死策略是治理其抗性的有效措施。     

Can selective peptides be combined efficiently with agrochemicals? A new approach to insect control

Sodium channels have been a major target for the development of insecticides such as synthetic pyrethroids. However, insecticides currently available induce resistance and present limited selectivity to insect pests. Molecular and biochemical studies, as well as binding experiments using radiolabelled neurotoxins, have shown that sodium channels expressed in various insect orders must be structurally and pharmacologically different. At least three groups of peptide neurotoxins derived from scorpion venom are highly active on insects and very weakly or practically inactive on mammals. It is proposed that various insecticides are examined for possible cooperative interactions with the peptide toxins highly active on insects, and pairs of ligands are identified that will increase the selectivity not only between mammals and insects but also between different pest and non-pest insects. This is feasible on the basis of the differential allosteric modulations observed between LqhαIT, an α-toxin highly active on insects, and brevetoxin on locust versus cockroach and rat brain sodium channels. Moreover, combination of LqhαIT with the pyrethroid deltamethrin increased the binding of [¹²⁵I] LqhαIT by more than 1.8-fold, and the combined presence of brevetoxin further increased the binding. Such allosteric modulation may provide a new approach to increase the selective activity of pesticides on target organisms by simultaneous application of allosterically interacting drugs, designed on the basis of the selective peptide toxins.     

Molecular studies of knockdown resistance to pyrethroids: cloning of domain II sodium channel gene sequences from insects

Knockdown resistance (kdr) is a target-site resistance mechanism that confers nerve insensitivity to DDT and pyrethroid insecticides. In the housefly, Musca domestica, molecular cloning of the para-type sodium channel gene has revealed two amino acid mutations that are associated with kdr and super-kdr resistance phenotypes. Both mutations are located in the domain II region of the channel; Leu1014 to Phe in the hydrophobic segment IIS6 and Met918 to Thr in the IIS4-IIS5 linker. To investigate whether these mutations also occur in other insects, we have designed degenerate primers based on conserved sequences in the domain II region of the sodium channel and used these to PCR amplify this region from insecticide-susceptible strains of eight diverse insect species representing four different insect Orders: Helicoverpa armigera, Plutella xylostella, Spodoptera littoralis (Lepidoptera), Blattella germanica (Dictyoptera), Tribolium castaneum (Coleoptera), Myzus persicae, Aphis gossypii and Phorodon humuli (Hemiptera). The primers amplified closely related para-type sodium channel sequences from each insect with a minimum of 85% amino acid identity between species. All of the sequences contained ‘susceptible’ Leu and Met residues at the positions associated with kdr and super-kdr resistance in the housefly. Recent results detailing the presence of a kdr-type Leu to Phe mutation in pyrethroid-resistant strains of two important agricultural pests, P. xylostella and M. persicae, are discussed. ©1997 SCI     

Insect resistance to insecticides

The past 40 years have seen insect resistance to insecticides develop from a scientific curiosity to an immense practical problem that threatens man's ability to control not only the insect pests of agriculture but also the insect vectors that transmit major human and animal diseases. The spread of genes for cross and multiple resistance among insect pests has rendered most of our present insecticides obsolescent and very few novel insecticides are under development as substitutes. The most feasible strategy to maintain adequate control of insect pests is integrated pest management or I P M, in which insecticide management is a useful component. However, much of our present planning for the future of insect control is carried out in ignorance of past failures. We must learn from the past if we are to retain the use of chemical insecticides as a viable component of IPM.     

Involvement of a sodium channel mutation in pyrethroid resistance in Cydia pomonella L, and development of a diagnostic test

Populations of the codling moth, Cydia pomonella L (Lepidoptera, Tortricidae) have developed resistance to several classes of insecticide such as benzoylureas, juvenile hormone analogues, ecdysone agonists and pyrethroids, but the corresponding resistance mechanisms have not been extensively studied. Knockdown resistance (kdr) to pyrethroid insecticides has been associated with point mutations in the para sodium channel gene in a great variety of insect pest species. We have studied two susceptible strains (S and Sv) and two resistant strains (Rt and Rv) of C pomonella that exhibited 4- and 80-fold resistance ratios to deltamethrin, respectively. The region of the voltage-dependent sodium channel gene which includes the position where kdr and super-kdr mutations have been found in Musca domestica L was amplified. The kdr mutation, a leucine-to-phenylalanine replacement at position 1014, was found only in the Rv strain. In contrast, the super-kdr mutation, a methionine-to-threonine replacement at position 918, was not detected in any C pomonella strain. These data allowed us to develop a PCR-based diagnostic test (PASA) to monitor the frequency of the kdr mutation in natural populations of C pomonella in order to define appropriate insecticide treatments in orchards.     

蔬菜蚜虫抗药性现状及抗性治理策略

蚜虫是为害蔬菜作物的一类重要害虫,如不防治会给蔬菜生产造成重大经济损失。长期以来主要依靠使用农药防治蔬菜蚜虫,但由于化学农药的不合理使用,蔬菜蚜虫对有机磷、拟除虫菊酯、氨基甲酸酯、新烟碱等多种类型的杀虫药剂均产生了不同程度的抗性。本文对常见的蔬菜蚜虫的抗药性现状、抗药性机理以及治理策略进行了论述,以期为该类害虫的可持续控制提供参考。     

Insecticidal 3-benzamido-N-phenylbenzamides specifically bind with high affinity to a novel allosteric site in housefly GABA receptors

γ-Aminobutyric acid (GABA) receptors (GABARs) are an important target for existing insecticides such as fiproles. These insecticides act as noncompetitive antagonists (channel blockers) for insect GABARs by binding to a site within the intrinsic channel of the GABAR. Recently, a novel class of insecticides, 3-benzamido-N-phenylbenzamides (BPBs), was shown to inhibit GABARs by binding to a site distinct from the site for fiproles. We examined the binding site of BPBs in the adult housefly by means of radioligand-binding and electrophysiological experiments. 3-Benzamido-N-(2,6-dimethyl-4-perfluoroisopropylphenyl)-2-fluorobenzamide (BPB 1) (the N-demethyl BPB) was a partial, but potent, inhibitor of [³H]4′-ethynyl-4-n-propylbicycloorthobenzoate (GABA channel blocker) binding to housefly head membranes, whereas the 3-(N-methyl)benzamido congener (the N-methyl BPB) had low or little activity. A total of 15 BPB analogs were tested for their abilities to inhibit [³H]BPB 1 binding to the head membranes. The N-demethyl analogs, known to be highly effective insecticides, potently inhibited the [³H]BPB 1 binding, but the N-methyl analogs did not even though they, too, are considered highly effective. [³H]BPB 1 equally bound to the head membranes from wild-type and dieldrin-resistant (rdl mutant) houseflies. GABA allosterically inhibited [³H]BPB 1 binding. By contrast, channel blocker-type antagonists enhanced [³H]BPB 1 binding to housefly head membranes by increasing the affinity of BPB 1. Antiparasitic macrolides, such as ivermectin B_1a, were potent inhibitors of [³H]BPB 1 binding. BPB 1 inhibited GABA-induced currents in housefly GABARs expressed in Xenopus oocytes, whereas it failed to inhibit l-glutamate-induced currents in inhibitory l-glutamate receptors. Overall, these findings indicate that BPBs act at a novel allosteric site that is different from the site for channel blocker-type antagonists and that is probably overlapped with the site for macrolides in insect GABARs.     

Evolutionary analysis of herbivorous insects in natural and agricultural environments

Herbivorous insects offer a remarkable example of the biological diversity that formed the foundation for Darwin's theory of evolution by natural selection. The ability of insects to evolve resistance rapidly to insecticides and host‐plant resistance present a continual challenge for pest management. This paper considers the manner in which genetic constraints, host‐plant availability and trade‐offs affect the evolution of herbivorous insects in natural and agricultural environments, and the extent to which lessons learned from studying natural systems may be applied to improve insect resistance management in agricultural systems. Studies on the genetic architecture of adaptation by herbivores to host plants and to insecticides are reviewed. The genetic basis of resistance is an important component of simulation models that predict the evolution of resistance. These models often assume monogenic resistance, but available data suggest that this assumption may be overly narrow and that modeling of resistance as oligogenic or polygenic may be more appropriate. As omics (e.g. genomics and proteomics) technologies become more accessible, a better understanding of the genetic basis of resistance will be possible. Trade‐offs often accompany adaptations by herbivores. Trade‐offs arise when the benefit of a trait, such as the ability to feed on a novel host plant or to survive in the presence of an insecticide, is counterbalanced by fitness costs that decrease fitness in the absence of the selective agent. For resistance to insecticides, and resistance to insecticidal transgenic crops in particular, fitness costs may act as an evolutionary constraint and delay or prevent the evolution of resistance. An important observation is that certain ecological factors such as host plants and entomopathogens can magnify fitness costs, which is termed ecological negative cross‐resistance. The application of omics technologies may allow for more efficient identification of factors that will impose ecological negative cross‐resistance, thereby bolstering insect resistance management. Copyright © 2009 Society of Chemical Industry     

Neonicotinoids-from zero to hero in insecticide chemistry

In recent years, neonicotinoids have been the fastest-growing class of insecticides in modern crop protection, with widespread use against a broad spectrum of sucking and certain chewing pests. As potent agonists, they act selectively on insect nicotinic acetylcholine receptors, their molecular target site. The discovery of neonicotinoids can be considered as a milestone in insecticide research and facilitates greatly the understanding of the functional properties of insect nicotinic acetylcholine receptors. Because of the relatively low risk for non-target organisms and environment, the high target specificity of neonicotinoid insecticides and their versatility in application methods, this important class has to be maintained globally for integrated pest management strategies and insect resistance management programmes. This review comprehensively describes particularly the origin, structure and bonding as well as associated properties of neonicotinoid insecticides.     

Modeling insect resistance to insecticides using velvetbean caterpillar (Anticarsia gemmatalis) as an example

Evolution of insect resistance to insecticides, using the velvetbean caterpillar (Anticarsia gemmatalis) as an example, was evaluated with the help of a computer simulation model. The effects of the following factors were studied: mortality rate, dominance and initial frequency of the resistant gene, migration rate, reproductive disadvantage and action level. According to results of the model, mortality rate, genetic dominance and frequency, and insect migration were the most critical factors involved with the rate of evolution of insect resistance to insecticides. Mortality rate can be directly managed, and migration indirectly through preservation of refugia, as part of a program of resistance management, which should include insecticides with different modes of action on the insect, and at minimum efficient rates. © 1998 SCI     

Quantitative structure-activity relationships of monoterpenoid binding activities to the housefly GABA receptor

BACKGROUND: Monoterpenoids are a large group of plant secondary metabolites. Many of these naturally occurring compounds have shown good insecticidal potency on pest insects. Previous studies in this laboratory have indicated that some monoterpenoids have positive modulatory effects on insect GABA receptors. In this study, the key properties of monoterpenoids involved in monoterpenoid binding activity at the housefly GABA receptor were determined by developing quantitative structure‐activity relationship (QSAR) models, and the relationship between the toxicities of these monoterpenoids and their GABA receptor binding activities was evaluated. RESULTS: Two QSAR models were determined for nine monoterpenoids showing significant effects on [³H]‐TBOB binding and for nine p‐menthane analogs with at least one oxygen atom attached to the ring. The Mulliken charges on certain carbon atoms, the log P value and the total energy showed significant relationships with binding activities to the housefly GABA receptor in these two QSAR models. CONCLUSIONS: From the QSAR models, some chemical and structural parameters, including the electronic properties, hydrophobicity and stability of monoterpenoid molecules, were suggested to be strongly involved in binding activities to the housefly GABA receptor. These findings will help to understand the mode of action of these natural insecticides, and provide guidance to predict more monoterpenoid insecticides. Copyright © 2012 Society of Chemical Industry     

Target-site resistance to pyrethroids in European populations of pollen beetle, Meligethes aeneus F.

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.     

害虫对新烟碱类杀虫剂的抗药性及其治理策略

烟碱和新烟碱类杀虫剂都是作为后突触烟碱乙酰胆碱受体(nAChRs)的激动剂作用于昆虫中枢神经系统,但这两类杀虫剂存在明显不同的选择毒性:烟碱类对哺乳动物毒性较高,而杀虫活性低;新烟碱类具有高杀虫活性,而对哺乳动物低毒。由于新烟碱类杀虫剂的作用方式独特,对以前使用的如拟除虫菊酯类、氯化烃类、有机磷类和氨基甲酸酯类等杀虫剂很少或无交互抗性,该类杀虫剂为防治一些世界性重大害虫(包括对以前使用的杀虫剂具有长期抗性的害虫)作出了重要贡献。但现已发现不少害虫对新烟碱类杀虫剂产生了抗性。文章就害虫对新烟碱类杀虫剂的抗性概况、抗性机理和抗性治理策略进行了综述。     

昆虫鱼尼丁受体及以其为靶标的杀虫剂的研究进展

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。     

Culex pipiens pallens: identification of genes differentially expressed in deltamethrin-resistant and -susceptible strains

Insecticide-resistance is a major obstacle to controlling insect vectors of microorganisms that cause human diseases. Identification of genes associated with resistance to insecticides has been a valuable tool for understanding mechanisms underlying resistance to commonly used insecticides such as deltamethrin. To identify such genes, we used suppression subtractive hybridization to obtain 809 differentially expressed clones in deltamethrin resistant versus susceptible laboratory strains of Culex pipiens pallens. Using cDNA microarrays and reverse Northern blots, a subset of 16 clones was confirmed to have greater than 3-fold difference in expression levels. Within this subset, we identified 2 clones uniquely expressed in the deltamethrin-resistant strain, eight clones exhibiting higher expression in the resistant strain and six in the susceptible strain. Of these 16 clones, 13 clones have sequence homology to known genes, such as ribosomal RNA, ribosome proteins, trypsin, and chymotrypsin-like proteins. Our data suggests resistance to deltamethrin may be a polygenic phenotype.     

Scorpion neurotoxins: structure/function relationships and application in agriculture

Continued use of non‐specific chemical insecticides poses potential risks to the environment and to human health resulting from non‐target toxicity and increased insect resistance to these agents. Scorpions produce anti‐insect selective polypeptide toxins that bind to and modulate voltage‐sensitive ion channels in excitable tissues, thus offering alternative, environmentally safer means for insect pest control. Despite this potential, little is known about their structural elements dictating anti‐insect preference, which may be useful for the design of selective insecticides. We used a bacterial system for expression and genetic dissection of two pharmacologically distinct scorpion toxins: alpha and excitatory. By exploiting a multi‐disciplinary approach consisting of mutagenesis, protein chemistry, electrophysiology, binding and toxicity assays, and structural studies, we elucidated the bioactive surface of two anti‐insect toxins, LqhαIT and Bj‐xtrIT. In both polypeptides the bioactive surface is composed of residues surrounding the C‐terminal region. In addition, a direct, immediate approach in using the toxin genes was demonstrated by engineering baculoviruses with cDNAs encoding LqhIT2 (depressant toxin), and LqhIT1 (excitatory toxin) resulting in viral vectors with significantly improved insecticidal efficacy. © 2000 Society of Chemical Industry     

Biophysical aspects of nervous activity in relation to studies on the mode of action of insecticides

The spread to the nervous system of topically applied neurotoxic insecticides is discussed. During spread different insecticides may distribute themselves between fluid and solid phases within the insect's tissues in greatly differing ratios. These differences are reflected in their concentrations in the haemolymph, which is probably the medium mainly responsible for distributing insecticides within the insect, and also the main source from which insecticides enter the nervous system. Evidence for spread via more restricted pathways, such as tracheae or the nerves themselves, is conflicting. The mode of action of neurotoxic insecticides is briefly reviewed, including the role of secondarily released neuroactive stress substances, and the extent to which resistance is expressed at the site of action is also discussed. Finally, the nervous system is examined for new sites of insecticidal action which, if attacked, could give greater specificity.