Resistance to malathion in field populations of Ceratitis capitata

The Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), is considered one of the most economically damaging pests of citrus orchards in Spain. Insecticide treatments for the control of this pest are mainly based on aerial and ground treatments with malathion bait sprays. However, the frequency of insecticide treatments has been increased in some areas of the Comunidad Valenciana in the last years, because of problems with the control of C. capitata. We have found that field populations from citrus and other fruit crops from different geographical areas in Spain showed lower susceptibility to malathion (6- to 201-fold) compared with laboratory populations. More importantly, differences in susceptibility could be related to the frequency of the field treatments. A resistant strain (W), derived from a field population, and a susceptible laboratory strain (C) were maintained in the laboratory. The W strain showed cross-resistance to the organophosphate fenthion (10-fold) but not to spinosad. Enzymatic assays showed that acethylcholinesterase activity was less inhibited in vivo by malathion and in vitro by malaoxon (active form of malathion) in adult flies from the W-resistant strain. Experiments to evaluate the effects of synergists revealed that the esterase inhibitor S,S,S-tributyl phosphorotrithioate (DEF) partially suppressed malathion resistance. Thus, target site insensitivity and metabolic resistance mediated by esterases might be involved in the loss of susceptibility to malathion in C. capitata. Nonetheless, additional biochemical and molecular studies will be required to confirm this hypothesis.     

The genetic basis of malathion resistance in housefly (Musca domestica L.) strains from Turkey

Organophosphate insecticide (parathion/diazinon) resistance in housefly (Musca domestica L.) is associated with the change in carboxylesterase activity. The product of MdalphaE7 gene is probably playing a role in detoxification of xenebiotic esters. In our research, we have isolated, cloned and sequenced the MdalphaE7 gene from 5 different Turkish housefly strains. High doses of malathion (600 microg/fly) were applied in a laboratory environment for one year to Ceyhan1, Ceyhan2, Adana and Ankara strains while no insecticide treatment was performed in the laboratory to Kirazli strain. Trp251 --> Ser substitution was found in the product of MdalphaE7 gene in all malathion resistant and Kirazli stocks. In addition, we checked the malathion carboxylesterase (MCE), percent remaining activities in acetylcholinesterase (AChE), glutathion-S-transferase (GST), and general esterase activities in all 5 strains used in this study. In comparing with universal standard sensitive control WHO, a high level of MCE and GST activities were observed while lower level of general esterase activities was detected in the tested strains. In addition, a higher percent remaining activities in AChE than WHO susceptible strain were observed in all malathion resistant strains.     

Investigation of resistance mechanisms to fipronil in diamondback moth (Lepidoptera: Plutellidae)

The SZ-F strain of Plutella xylostella (L.) (Lepidoptera: Plutellidae) was derived from a field-collected strain (SZ) by 20 generations of continuous selection with fipronil. The selection resulted in 300-fold increase in resistance to fipronil, and 3.5- and 6.5-fold cross-resistance to dieldrin and endosulfan, respectively, in the SZ-F strain compared with the unselected SZ strain. Analysis of detoxification enzyme activities and bioassay with synergists indicated that metabolic mechanisms are not important to fipronil resistance of the SZ-F strain and that the fipronil resistance is most likely attributed to target site insensitivity. The genomic DNA fragments flanking the second membrane-spanning region of Rdl gamma-aminobutyric acid (GABA) receptor gene from P. xylostella, PxRdl, were cloned and sequenced. A single allele of the PxRdl gene (encoding A302 or allele PxRdl-Ala) was found in both the Roth (susceptible) and SZ strains. In addition to the wild-type allele PxRdl-Ala, the resistant SZ-F strain carried a mutant PxRdl allele with the conserved amino acid replacement A302(GGC)--> S302(TCC) (allele PxRdl-Ser). The mutant PxRdl-Ser allele frequency in the SZ-F strain was 30%. After treatment of 20 mg/l fipronil on the SZ-F strain, the PxRdl-Ser allele frequency in the survivors increased to 57%. High frequency of the PxRdl-Ala allele remaining in the resistant SZ-F strain suggested that the A302S mutation in the PxRdl gene is partially associated with fipronil resistance and that other mutation(s) in the PxRdl gene or other Rdl-like subunit(s) may contribute to fipronil resistance.     

Cross-resistance to insecticides in a malathion-resistant strain of Ceratitis capitata (Diptera: Tephritidae)

Resistance to malathion has been reported in field populations of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), in areas of Spain where an intensive use of this insecticide was maintained for several years. The main goal of this study was to determine whether resistance to malathion confers cross-resistance to different types of insecticides. Susceptibility bioassays showed that the malathion-resistant W-4Km strain (176-fold more resistant to malathion than the susceptible C strain) has moderate levels of cross-resistance (three- to 16-fold) to other organophosphates (trichlorphon, diazinon, phosmet and methyl-chlorpyrifos), the carbamate carbaryl, the pyrethroid lambda-cyhalothrin, and the benzoylphenylurea derivative lufenuron, whereas cross-resistance to spinosad was below two-fold. The W-4Km strain was selected with lambda-cyhalothrin to establish the lambda-cyhalothrin-resistant W-1Klamda strain (35-fold resistant to lambda-cyhalothrin). The synergistic activity of the esterase inhibitor DEF with lambda-cyhalothrin and the increase in esterase activity in the W-1Klamda strain suggests that esterases may be involved in the development of resistance to this insecticide. Our results showed that resistance to malathion may confer some degree of cross-resistance to insecticides currently approved for the control of Mediterranean fruit fly in citrus crops (lambda-cyhalothrin, lufenuron, and methyl-chlorpyrifos). Especially relevant is the case of lambda-cyhalothrin, because we have shown that resistance to this insecticide can rapidly evolve to levels that may compromise its effectiveness in the field.     

有机磷和拟除虫菊酯抗性棉铃虫靶标抗性的分子机理(英文)

对有机磷和拟除虫菊酯抗性 (R)棉铃虫靶标抗性的分子机理 ,即乙酰胆碱酯酶 (AChE)和钠通道敏感度降低进行了研究。根据AChE的动力学常数表明 ,R品系AChE的活性和Vmax值分别是S品系的 1 0 9和 1 2 3倍 ,但R品系的AChE的Km 值仅是S品系的 0 6 7倍。R品系AChE对DDVP和马拉硫磷的Ki值分别是S品系的 0 4 4和 0 55。这表明AChE发生了质的变化。还应用PCR技术对抗性棉铃虫的击倒抗性 (kdr)进行了鉴定 ,克隆了钠通道的IIS6序列、IIS5和IIS6连接片段以及II和III连接片段 ,测序后比较了R和S品系以及其它昆虫的同源性 ,结果在氨基酸水平未发现有任何差异 ,这表明该抗性棉铃虫品系不涉及kdr。     

Mechanisms of monocrotophos resistance in cotton bollworm,Helicoverpa armigera (Hübner)

Insensitive acetylcholinesterase was identified as a resistance mechanism by comparing biochemical analysis with a laboratory selected monocrotophos resistant cotton bollworm (RR: 200) and the susceptible strain. The cDNA encoding AChE was cloned by the method of RACE (rapid amplification of cDNA ends). The complete AChE gene deduced from the cDNA consisted of a putative signal peptide of 32 amino acid residues, a mature protein of 615 residues, 5' untranslated regions (UTR) of 315 bp and 3' UTR of 324 bp. The coding sequence had a high degree of homology to the AChE from other insect species reported in the GenBank. After comparing analysis of the entire AChE gene sequence from 5 resistant and 6 susceptible cotton bollworm individuals, nine mutations were identified. One of them, the Ala/Thr mutation, is likely to be responsible for the AChE insensitivity to monocrotophos.     

A novel acetylcholinesterase gene in mosquitoes codes for the insecticide target and is non-homologous to the ace gene in Drosophila

Acetylcholinesterase (AChE) is the target of two major insecticide families, organophosphates (OPs) and carbamates. AChE insensitivity is a frequent resistance mechanism in insects and responsible mutations in the ace gene were identified in two Diptera, Drosophila melanogaster and Musca domestica. However, for other insects, the ace gene cloned by homology with Drosophila does not code for the insensitive AChE in resistant individuals, indicating the existence of a second ace locus. We identified two AChE loci in the genome of Anopheles gambiae, one (ace-1) being a new locus and the other (ace-2) being homologous to the gene previously described in Drosophila. The gene ace-1 has no obvious homologue in the Drosophila genome and was found in 15 mosquito species investigated. In An. gambiae, ace-1 and ace-2 display 53% similarity at the amino acid level and an overall phylogeny indicates that they probably diverged before the differentiation of insects. Thus, both genes are likely to be present in the majority of insects and the absence of ace-1 in Drosophila is probably due to a secondary loss. In one mosquito (Culex pipiens), ace-1 was found to be tightly linked with insecticide resistance and probably encodes the AChE OP target. These results have important implications for the design of new insecticides, as the target AChE is thus encoded by distinct genes in different insect groups, even within the Diptera: ace-2 in at least the Drosophilidae and Muscidae and ace-1 in at least the Culicidae. Evolutionary scenarios leading to such a peculiar situation are discussed.     

MOLECULAR MECHANISMS OF TARGET RESISTANCE IN THE ORGANOPHOSPHATE- AND PYRETHROID-RE-SISTANT HELICOVERPA ARMIGERA (HUBNER)

Abstract  The molecular mechanisms of target resistance, i. e. acetyicholinesterase (AChE) and sodium channel insensitivity, in the organophosphate(0P)- and pyrethroid(Py)-resistant (R) Helicoverpa armigera were investigated. The activity and V_max of AChE from R strain were 1.09– and 1.23-fold of the susceptible(S) strain respectively, but the K_M value of AChE in R strain was only 0.67-fold of S stain. The K_i values of AChE from the R strain were 0.44 for DDVP and 0.50 for malathion respectively, as compared with those of the S strain. These data showed that the AChE from R strain might be qualitatively altered. The knockdown resistance ( kdr ) of the resistant H. armigera was also identified by PCR technique. The fragments of IIS6, linker of 11–111 and a part of IIS5-IIs6 in the sodium channel were cloned and sequenced, and then compared to amino acid sequences from the R and S strains. and other insect species. There was no difference to be found at amino acid level in the above fragments cloned. It was suggested that the Rdr was not involved in the resistance of R strain.     

Determination of malathion and diazinon resistance by sequencing the Md alpha E7 gene from Guatemala, Colombia, Manhattan, and Thailand housefly (Musca domestica L.) strains

Organophosphate (OP) insecticides (parathion/diazinon) resistance in housefly (Musca domestica L.) is associated with the change in carboxylesterase activity. The product of alpha E7 gene, which is a member of alpha-esterase gene cluster, is probably playing a role in detoxification of the xenobiotic esters. In parathion/diazinon resistant M. domestica species Gly137 to Asp substitution was found in the active center of the product of alpha E7 gene. In malathion (an OP) resistant M. domestica strains Trp251 to Ser substitution was identified in the active center of the Md alpha E7. In our research, to understand the allelic diversity of the Md alpha E7, the gene was partially sequenced from four different housefly strains from different localities (Guatemala, Manhattan (USA), Colombia (USA) and Thailand). It was found out that; in Thailand strain one allele has Cys residue at the position of 251, the other allele contains a Trp for the same site. In Colombia strain, one allele has Asp137, the other allele contains a Gly residue at this point. The Manhattan and Guatemala strains have Asp137 and Trp251 residues on their both alleles at these two different positions.     

Fenitroxon insensitive acetylcholinesterases of the housefly, Musca domestica associated with point mutations

The cDNA of AChE in the housefly, Musca domestica, was sequenced and individual flies were genotyped by this gene in an inhibition assay of AChE activity with an organophaspate, fenitroxon. Mutations at Gly(342) and Tyr(407), which are reportedly conserved in resistant strains of Drosophila, were associated with the insensitivity to fenitroxon. Two other mutations, Ile(162) and Val(260), did not have an apparent effect on insensitivity. However, the four mutations are located in the active site of the enzyme, and therefore the non-neutral mutations in this gene are considered to cause the insensitivity of AChE in the development of insecticide resistance of the housefly.     

杀虫药剂抗性家蝇品系乙酰胆碱酯酶基因的特征分析

乙酰胆碱酯酶(AChE)是有机磷和氨基甲酸酯类杀虫药剂的作用靶标,这两大类杀虫药剂的广泛应用导致了昆虫对抗性的选择。靶标的修饰是某些昆虫产生抗性的分于机理,这种抗性是和AChE的变更型相关的,这些变更型的酶显示出对杀虫药剂的不被感性。利用RT-PCR和Streptavidin偶联磁珠技术从两种抗性家蝇(Musca domestica)品系D3和Kash中分别分离了AChE基因并测定了其按苷酸颅序。eDNA的可读框长2082bp．由此推导出了AChE的氨基酸顺序,通过与敏感家蝇品系Cooper的比较,发现了一些核苷酸顺序差异和4个氨基酸点突变,其中3个替代可能与杀虫药剂不敏感性有关。这一结果表明D3和Kash均属于CH₂抗性类型。     

Mutation in ace1 associated with an insecticide resistant population of Plutella xylostella

Insensitive acetylcholinesterase (AChE) was determined to be involved in an EPN-resistant (ER) strain and a contaminated susceptible (CS) strain of diamondback moth (DBM, Plutella xylostella L.), as estimated by AChE inhibition assay using DDVP as a inhibitor in a nondenaturing electrophoresis gel. The ER strain exhibited very high AChE insensitivity, high resistance ratio, and two point mutations (G324A, A298S) in ace1-type AChE gene (Pxace1). The CS strain showed low AChE insensitivity, low resistance ratio, and it has only one point mutation (G324A). These findings suggest that the A298S mutation, along with reported G324A mutation (Baek et al, 2005), can be important in the development of organophosphate resistance. These results also suggest that the A298S mutation could be a good candidate for a molecular diagnosis marker for resistance monitoring. Three molecular diagnosis methods (Quantitative Sequencing; QS, PCR amplification of specific alleles; PASA and restriction fragment length polymorphism; RFLP) were developed which successfully detected specific resistance associated point mutations. Seven local population DBMs were surveyed and showed high insecticide resistance levels and a A298S mutation in Pxace1. These methods can be used to monitor the resistance allele in field population of DBMs and resistance management strategy.     

A single nucleotide polymorphism in the acetylcholinesterase gene of the predatory mite Kampimodromus aberrans (Acari: Phytoseiidae) is associated with chlorpyrifos resistance

The predatory mite Kampimodromus aberrans (Oudemans) (Acari: Phytoseiidae) is one of the most important biocontrol agents of herbivorous mites in European perennial crops. The use of pesticides, such as organophosphate insecticides (OPs), is a major threat to the success of biocontrol strategies based on predatory mites in these cropping systems. However, resistance to OPs in K. aberrans has recently been reported. The present study investigated the target site resistance mechanisms that are potentially involved in OP insensitivity. In the herbivorous mite Tetranychus urticae Koch (Acari: Tetranychidae), resistance to OPs is due to a modified and insensitive acetylcholinesterase (AChE; EC: 3.1.1.7) that bears amino acid substitution F331W (AChE Torpedo numbering). To determine whether the predators and prey have evolved analogous molecular mechanisms to withstand the same selective pressure, the AChE cDNA from a putative orthologous gene was cloned and sequenced from susceptible and resistant strains of K. aberrans. No synonymous mutation coding for a G119S substitution was determined to be strongly associated with the resistant phenotype instead of the alternative F331W. Because the same mutation in T. urticae AChE was not associated with comparable levels of chlorpyrifos resistance, the role of the G119S substitution in defining insensitive AChE in K. aberrans remains unclear. G119S AChE genotyping can be useful in ecological studies that trace the fate of resistant strains after field release or in marker-assisted selection of improved populations of K. aberrans to achieve multiple resistance phenotypes through gene pyramiding. The latent complexity of the target site resistance in K. aberrans vs. that of T. urticae is also discussed in the context of data from the genome project of the predatory mite Metaseiulus occidentalis (Nesbitt) (Acari: Phytoseiidae).     

Pharmacodynamics of malathion and carbaryl in susceptible and multiresistant German cockroaches (Dictyoptera: Blattellidae)

Studies with malathion and carbaryl were done to compare toxicity; absorption, metabolism, internal accumulation, and excretion; and in vivo inhibition of acetylcholinesterase (AChE) after topical applications to adult male susceptible (S, Orlando normal) or multiresistant (R, HRDC) German cockroaches, Blattella germanica (L.). Compared with the S strain, R cockroaches were highly resistant to malathion (about 33-fold) and only moderately resistant or tolerant to carbaryl (about 5-fold). Tests with topically applied 14C-labeled malathion and carbaryl indicated that both compounds penetrated rapidly and radioactive products were readily excreted. Rates of absorption or excretion in S and R strains did not differ significantly. Both insecticides were extensively metabolized; each yielded the same array and similar concentrations of metabolites in insects from either strain. In contrast, metabolic detoxification of malathion and carbaryl was significantly greater in R cockroaches when the insects were treated by injection. Strains did not differ significantly in the in vitro inhibition of brain AChE by either malaoxon or carbaryl. However, dramatic differences were observed between strains in the in vivo inhibition of AChE during a 6-h test period after topical treatment with malathion, and moderate but significant differences occurred between strains in the in vivo inhibition of AChE by carbaryl. These data suggest that the strong resistance to malathion and moderate resistance or tolerance to carbaryl in R cockroaches is probably a result of enhanced capability for metabolic detoxification.     

抑制剂存在下不同种群烟粉虱乙酰胆碱酯酶残余活性频率分布及其与抗药性的关系

为了探讨烟粉虱Bemisia tabaci不同种群个体乙酰胆碱酯酶敏感性差异及其与抗药性的关系, 我们选用室内饲养的烟粉虱SUD S敏感品系和6个田间抗性种群, 采用酶标板酶动力学法测定了各品系 (种群）乙酰胆碱酯酶对抑制剂的敏感性反应以及抑制剂存在时各抗性种群个体乙酰胆碱酯酶残余活性频率分布。结果表明: 在抑制剂浓度为300 μmol/L时, 敏感品系乙酰胆碱酯酶的活性基本上被完全抑制, 可以明显地区分敏感品系与田间抗性种群。在抑制剂浓度为2 000 μmol/L时, 各抗性种群个体乙酰胆碱酯酶残余活性频率分布差异明显, 其中ZZ-R种群和FZ-R种群的乙酰胆碱酯酶残余活性频率分布相似, 大部分个体的乙酰胆碱酯酶残余活性分布在1.00~1.80 mOD/min之间; SM-R种群和ND-R种群的乙酰胆碱酯酶残余活性频率分布也相似, 大部分个体的乙酰胆碱酯酶残余活性分布在0.40~1.00 mOD/min之间; LY-R和NP-R种群大部分个体的乙酰胆碱酯酶残余活性分别分布在1.00~1.60 mOD/min和0.80~1.20 mOD/min之间。各抗性种群乙酰胆碱酯酶高残余活性 (大于1.00 mOD/min）个体频率与对敌敌畏的抗性水平之间具有明显相关性, 相关系数为0.86 (P<0.05）。考虑到乙酰胆碱酯酶对抑制剂作用不敏感是一些昆虫对有机磷和氨基甲酸酯类杀虫剂抗性的重要机制之一, 建议可以将乙酰胆碱酯酶对敌敌畏的敏感性作为烟粉虱抗药性生化检测的一个参考指标。     

Mechanisms of organophosphate resistance in a field population of oriental migratory locust, Locusta migratoria manilensis (Meyen)

The susceptibilities to three organophosphate (OP) insecticides (malathion, chlorpyrifos, and phoxim), responses to three metabolic synergists [triphenyl phosphate (TPP), piperonyl butoxide (PBO), and diethyl maleate (DEM)], activities of major detoxification enzymes [general esterases (ESTs), glutathione S-transferases (GSTs), and cytochrome P450 monooxygenases (P450s)], and sensitivity of the target enzyme acetylcholinesterase (AChE) were compared between a laboratory-susceptible strain (LS) and a field-resistant population (FR) of the oriental migratory locust, Locusta migratoria manilensis (Meyen). The FR was significantly resistant to malathion (57.5-fold), but marginally resistant to chlorpyrifos (5.4) and phoxim (2.9). The malathion resistance of the FR was significantly diminished by TPP (synergism ratio: 16.2) and DEM (3.3), but was unchanged by PBO. In contrast, none of these synergists significantly affected the toxicity of malathion in the LS. Biochemical studies indicated that EST and GST activities in the FR were 2.1- to 3.2-fold and 1.2- to 2.0-fold, respectively, higher than those in the LS, but there was no significant difference in P450 activity between the LS and FR. Furthermore, AChE from the FR showed 4.0-fold higher activity but was 3.2-, 2.2-, and 1.1-fold less sensitive to inhibition by malaoxon, chlorpyrifos-oxon, and phoxim, respectively, than that from the LS. All these results clearly indicated that the observed malathion resistance in the FR was conferred by multiple mechanisms, including increased detoxification by ESTs and GSTs, and increased activity and reduced sensitivity of AChE to OP inhibition.     

Highly-substrate active isoenzyme acetylcholinesterase-II,in rosy eye mutant of Aedes aegypti mosquito

Insecticide bioassays were carried out on larvae and adults of rosy eye mutant and wildtype strains of A. aegypti. Both the strains were equally susceptible to DDT, malathion and deltamethrin. Biochemical assays showed an increase in acetylcholinesterase enzyme (AChE) activity in all the stages of mutant strain with both the substrates i.e. acetylthiocholine iodide and S-butyrylthiocholine iodide. However, there was no difference in the percent inhibition of enzyme activity with propoxur in these two strains. Polyacrylamide gel electrophoresis performed in native conditions on the homogenates of adults of rosy eye mosquitoes showed that AChE-II allele was highly active with the substrate acetylthiocholine iodide as compared to wildtype strain. Frequency of the highly active AChE-II allele in the mutant strain was about 68%, whereas it was about 5% in the wildtype strain.     

Biochemical genetics of altered acetylcholinesterase resistance to insecticides in the house fly

Resistance to the organophosphate insecticide tetrachlorvinphos was examined in a house fly (Musca domestica L.) strain with an altered acetylcholinesterase (AChE) of decreased sensitivity to inhibition by the insecticide. Genetic tests showed that both resistance and the altered AChE were controlled by semidominant gene(s) on chromosome II. The gene for resistance was five crossover units from the mutant marker stubby wing (stw). A house fly strain was prepared in which resistance was introduced in to a susceptible stw strain by recombination. Biochemical assays revealed that the altered AChE was introduced along with resistance. Assays of the AChE of resistant and susceptible stw strains by two independent methods showed that the enzyme from resistant flies was 30 times more slowly inhibited by tetrachlorvinphos than the enzyme from susceptible flies.This work was supported in part by NIH Grant ES 00901.Technical Article 13340, Texas Agricultural Experiment Station.