Select Small Core Structure Carbamates Exhibit High Contact Toxicity to ��Carbamate-Resistant�� Strain Malaria Mosquitoes, Anopheles gambiae (Akron)

Acetylcholinesterase (AChE) is a proven target for control of the malaria mosquito (Anopheles gambiae). Unfortunately, a single amino acid mutation (G119S) in An. gambiae AChE-1 (AgAChE) confers resistance to the AChE inhibitors currently approved by the World Health Organization for indoor residual spraying. In this report, we describe several carbamate inhibitors that potently inhibit G119S AgAChE and that are contact-toxic to carbamate-resistant An. gambiae. PCR-RFLP analysis was used to confirm that carbamate-susceptible G3 and carbamate-resistant Akron strains of An. gambiae carry wild-type (WT) and G119S AChE, respectively. G119S AgAChE was expressed and purified for the first time, and was shown to have only 3% of the turnover number (k _cat) of the WT enzyme. Twelve carbamates were then assayed for inhibition of these enzymes. High resistance ratios (>2,500-fold) were observed for carbamates bearing a benzene ring core, consistent with the carbamate-resistant phenotype of the G119S enzyme. Interestingly, resistance ratios for two oxime methylcarbamates, and for five pyrazol-4-yl methylcarbamates were found to be much lower (4- to 65-fold). The toxicities of these carbamates to live G3 and Akron strain An. gambiae were determined. As expected from the enzyme resistance ratios, carbamates bearing a benzene ring core showed low toxicity to Akron strain An. gambiae (LC₅₀>5,000 μg/mL). However, one oxime methylcarbamate (aldicarb) and five pyrazol-4-yl methylcarbamates (4a–e) showed good to excellent toxicity to the Akron strain (LC₅₀ = 32–650 μg/mL). These results suggest that appropriately functionalized “small-core” carbamates could function as a resistance-breaking anticholinesterase insecticides against the malaria mosquito.     

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).     

STUDIES ON THE KINETICS OF ACETYLCHOLINESTERASE IN THE RESISIANT AND SUSCEPTIBLE STRAINS OF HOUSEFLY (MUSCA DOMESTICA)

Abstract Acetylcholinesterase (AChE) in the susceptible (S) and the resistant (R) strains of housefly (Musca domestica) was investigated using kinetic analysis. The V_max values of AChE for hydrolyzing acetylthiocholine (ATCh) and butyrylthiocholine (BTCh) were 4578.50 and 1716.08nmol/min/mg* protein in the R strain, and were 1884.75 and 864.72 nmol/min/mg. protein in the Sstrain, respectively. The V_max ratios of R to S enzyme were 2.43 for ATCh and 1.98 for BTCh. The K_m values of AChE for ATCh and BTCh were 0.069 and 0.034 mmol/L in the S strain, and 0.156, 0.059 mmol/L in the R strain, respectively. The K_m ratios of R to S enzyme were 2.26 for ATCh and 1.74 for BTCh. The k_i ratios of S to R enzyme for three insecticides propoxur, methomyl and paraoxon were 46.04, 4.17 and 2. 86, respectively. In addition, k_cat and k_cat/K_m for measuring turnover and catalytic efficiency of AChE were determined using eserine as titrant. The k_cat values of AChE from the R strain for both ATCh and BTCh were higher than those values from the S strain. But the values of k_cat/K_m were in contrary to the k_cat values with R enzyme compared to S enzyme. The AChE catalytic properties and sensitivity to the inhibition by three insecticides in the R and S strains of housefly were discussed based on contribution of V_max, K_m, k_i, k_cat and k_cat/K_m. All these data implied that AChE from the R strain might be qualitatively altered. We also observed an intriguing phenomenon that inhibitors could enhance the activity of AChE from the resistant strain. This “flight reaction” of the powerful enzyme might be correlated with the developing resistance of housefly to organophosphate or carbamate insecticides.     

取食不同寄主植物对棉蚜后代抗药性的影响

测定了5种药剂对棉蚜Aphis gossypii抗氰戊菊酯、吡虫啉品系和敏感品系取食棉花、黄瓜和石榴的后代的毒力,并对它们的后代体内乙酰胆碱酯酶和羧酸酯酶的比活力做了初步探索。结果表明,氰戊菊酯抗性品系取食棉花比取食黄瓜的后代对氰戊菊酯的抗性大76.4倍,对灭多威、氧乐果、硫丹和吡虫啉的抗性也大0.5～4.6倍;取食石榴的后代对5种药剂的抗性介于取食棉花和黄瓜的之间。吡虫啉抗性品系的测定结果与氰戊菊酯抗性品系基本一致。敏感品系取食黄瓜比取食棉花的后代对5种药剂的敏感性更高。3个品系取食不同植物的后代相比,其体内乙酰胆碱酯酶的比活力,取食棉花的为取食黄瓜的2.4～2.8倍;羧酸酯酶的比活力,取食棉花的为取食黄瓜的1.8～2.4倍。证明棉蚜的抗性和敏感品系取食的寄主植物不同,可引起对药剂敏感性的变化。乙酰胆碱酯酶和羧酸酯酶活力的变化均是引起这种变化的重要因素。     

Mutations in acetylcholinesterase genes of Rhopalosiphum padi resistant to organophosphate and carbamate insecticides.

Apple grain aphid, Rhopalosiphum padi (Linnaeus), is an important wheat pest. In China, it has been reported that R. padi has developed high resistance to carbamate and organophosphate insecticides. Previous work cloned from this aphid 2 different genes encoding acetylcholinesterase (AChE), which is the target enzyme for carbamate and organophosphate insecticides, and its insensitive alteration has been proven to be an important mechanism for insecticide resistance in other insects. In this study, both resistant and susceptible strains of R, padi were developed, and their AChEs were compared to determine whether resistance resulted from this mechanism and whether these 2 genes both play a role in resistance. Bioassays showed that the resistant strain used was highly or moderately resistant to pirimicarb, omethoate, and monocrotophos (resistance ratio, 263.8, 53.8, and 17.5, respectively), and showed little resistance to deltamethrin or thiodicarb (resistance ratio, 5.2 and 3.4, respectively). Correspondingly, biochemistry analysis found that AChE from resistant aphids was very insensitive to the first 3 insecticides (I50 increased 43.0-, 15.2-, and 8.8-fold, respectively), but not to thiodicarb (I50 increased 1.1-fold). Enzyme kinetics tests showed that resistant and susceptible strains had different AChEs. Sequence analysis of the 2 AChE genes cloned from resistant and susceptible aphids revealed that 2 mutations in Ace2 and 1 in Ace1 were consistently associated with resistance. Mutation F368(290)L in Ace2 localized at the same position as a previously proven resistance mutation site in other insects. The other 2 mutations, S329(228)P in Ace1 and V435(356)A in Ace2, were also found to affect the enzyme structure. These findings indicate that resistance in this aphid is mainly the result of insensistive AChE alteration, that the 3 mutations found might contribute to resistance, and that the AChEs encoded by both genes could serve as targets of insecticides.     

A COMPARISON OF SENSITIVITY TO INHIBITOR AMONG ACETYLCHOLINESTERASE (AChE) MOLECULAR FORMS OF RESISTANT AND SUSCEPTIBLE STRAINS IN HELICOVERPA ARMIGERA*

Abstract The sensitivity of 2.8s and 8.7s acetylcholinesterase (AChE) to eserine sulfate is significantly lower in resistant (R) strain than in susceptible (S) strain in five AChE forms isolated by sucrose gradient centrifugation from cotton bollworm, Helicoverpa armigera. There are 186 and 85 times of difference in heads of adults and 10¹⁰ and 10⁵ times of difference in heads of larvae based on a comparison of I₅₀ values for 2.8s and 8.7s forms respectively. The sensitivity of 5.3s form of AChE to eserine sulfate shows 123 times of difference between R and S strains in larvae, however no difference in adults. The above results indicate that insensitive 2.8s, 8.7s and 5.3s forms of AChE may play an important role in the resistance of cotton boll‐worm to organophosphate and carbamate insecticides.     

A butterfly effect: highly insecticidal resistance caused by only a conservative residue mutated of drosophila melanogaster acetylcholinesterase

Acetylcholinesterase (AChE) and its mutation recently emerged as a significant research area, due to its resistance against organophosphate and carbamate insecticides. Residue G265, which is always a conservative residue, mutated to A265 is the most frequent mutant of AChE in Drosophila populations. However, only this mutation caused a ‘butterfly effect’ that gives high insecticidal resistance. Herein, the models of sensitive strain (Dm-S) and the resistance strain (Dm-R) were constructed, to give a total of 2000 ps molecular dynamics simulation and to reveal the insecticidal resistance mechanism, with implied, the active gorge of Dm-R was much less flexible than that of Dm-S. The “back door” channel was widened to accelerate the detoxication against insecticides by the conformation changing of W83 and I161. All the distances (S238-H480, S238-G150, S238-G151, Y71-M153) in Dm-R became smaller than those in Dm-S, which may deeply influence the binding between the insecticides and DmAChE.     

Partial purification and characterization of trypsin-like proteinases from insecticide-resistant and -susceptible strains of the maize weevil,Sitophilus zeamais

Serine proteinases from three strains of Sitophilus zeamais (Coleoptera: Curculionidae), one susceptible and two resistant to insecticides — one exhibiting fitness cost (resistant cost strain) and the other lacking it (resistant no-cost strain), were partially purified using an aprotinin–agarose affinity column providing purification factors ranging from 36.5 to 51.2%, with yields between 10 and 15% and activity between 529 and 875 µM/min/mg protein with the substrate N-α-benzoyl-l-Arg-p-nitroanilide (L-BApNA). SDS-PAGE of the purified fraction revealed a 56,000 Da molecular mass band in all strains and a 70,000 Da band more visible in the resistant no-cost strain. The purified proteinases from all strains were inhibited by phenylmethyl sulphonyl fluoride (PMSF), N-α-tosyl-l-lysine chloromethyl ketone (TLCK), aprotinin, benzamidine and soybean trypsin inhibitor (SBTI) characterizing them as trypsin-like serine proteinases. Trypsin-like proteinases from the resistant strains exhibited higher affinity for L-BApNA. The resistant no-cost strain exhibited V_max-values 1.5- and 1.7-fold higher than the susceptible and resistance cost strains, respectively. A similar trend was also observed when using N-α-p-tosyl-L-Arg methyl ester (L-TAME) as substrate. These results provide support to the hypothesis that the enhanced serine proteinase activity may be playing a role in mitigating physiological costs associated with the maintenance of insecticide resistance mechanisms in some maize weevil strains.     

A tryptophan in the bottleneck of the catalytic gorge of an invertebrate acetylcholinesterase confers relative resistance to carbamate and organophosphate inhibitors

Amphioxus, an invertebrate chordate, has two acetylcholinesterases (AChEs): cholinesterase 1 (ChE1) and cholinesterase 2 (ChE2). ChE1 is up to 329-fold more resistant to a variety of carbamate and organophosphate inhibitors, including a number of insecticides, when compared with ChE2. One difference between the two enzymes is at the position homologous to Phe331 in Torpedo AChE. In Torpedo AChE, this residue is a component of the hydrophobic subsite and defines one side of the bottleneck in the catalytic gorge of the enzyme. In ChE1, the homologous residue is Trp353; in ChE2, it is Phe353. We used site-directed mutagenesis to investigate the proposal that the resistance of ChE1 to inhibition by carbamates and organophosphates was due to this difference, creating a ChE1 W353F mutant to widen the bottleneck. The mutation virtually abolishes the difference in sensitivity to the inhibitors. The ChE1 W353F mutant is only 2- to 3-fold more resistant than ChE2 to carbamates and is actually 2.5- to 10-fold more sensitive to inhibition by organophosphates. The differences in resistance are due to different affinities of the enzymes for the inhibitors, not different reactivities. Molecular modeling supports the proposal that the difference in inhibition is due to the width of the bottleneck of the gorge. Our results have implications for insecticide resistance in insects, in particular mosquitoes and aphids.     

Impact of sequential exposure of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and imidacloprid against susceptible and resistant strains of <Emphasis Type="Italic">Musca domestica</Emphasis>

Insecticide resistance in the housefly Musca domestica is hampering pest management. However, entomopathogens, possibly in combination with insecticides, may have control potential against resistant houseflies. This study investigates the combination of the entomopathogenic fungus Beauveria bassiana and the neonicotinoid insecticide, imidacloprid against a susceptible and a resistant housefly strain, respectively under laboratory conditions. The fungus and insecticide were tested alone and in combinations at LC₃₀. Significant and synergistic interactions between B. bassiana and imidacloprid were observed with increased mortality rates of the combined treatment as compared to individual treatment in housefly strains 772a (susceptible) and 766b (resistant). Significant differences in the GST and P450 activities for both strains were found. Female 766b flies caused 15- to 237-fold increases in gene expression of xenobiotic response genes for B. bassiana and 23- to 120-fold changes for imidacloprid. The combination of B. bassiana and imidacloprid caused significant synergistic interaction when applied against two housefly strains irrespective of order of application. The effect was highest when the insecticide was applied first. The resistant housefly strain had elevated detoxification enzymes and higher expression of detoxification genes, but showed the same level of susceptibility to the combined fungus/insecticide treatment as the susceptible strain.     

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.     

Sequence variation and regulatory variation in acetylcholinesterase genes contribute to insecticide resistance in different populations of Leptinotarsa decemlineata

Although insect herbivores are known to evolve resistance to insecticides through multiple genetic mechanisms, resistance in individual species has been assumed to follow the same mechanism. While both mutations in the target site insensitivity and increased amplification are known to contribute to insecticide resistance, little is known about the degree to which geographic populations of the same species differ at the target site in a response to insecticides. We tested structural (e.g., mutation profiles) and regulatory (e.g., the gene expression of Ldace1 and Ldace2, AChE activity) differences between two populations (Vermont, USA and Belchow, Poland) of the Colorado potato beetle, Leptinotarsa decemlineata in their resistance to two commonly used groups of insecticides, organophosphates, and carbamates. We established that Vermont beetles were more resistant to azinphos‐methyl and carbaryl insecticides than Belchow beetles, despite a similar frequency of resistance‐associated alleles (i.e., S291G) in the Ldace2 gene. However, the Vermont population had two additional amino acid replacements (G192S and F402Y) in the Ldace1 gene, which were absent in the Belchow population. Moreover, the Vermont population showed higher expression of Ldace1 and was less sensitive to AChE inhibition by azinphos‐methyl oxon than the Belchow population. Therefore, the two populations have evolved different genetic mechanisms to adapt to organophosphate and carbamate insecticides.     

斜纹夜蛾抗药性监测及茚虫威对其解毒代谢酶的影响

【目的】斜纹夜蛾Spodoptera litura (Fabricius)是主要的农业害虫之一。本研究旨在明确该害虫在湖南省5个主要蔬菜种植区的抗药性水平,并探讨该害虫对茚虫威的抗性与解毒代谢酶活性之间的关系,为斜纹夜蛾有效防控及抗性治理提供依据。【方法】采用浸叶法测定了2014-2016年湖南5地斜纹夜蛾田间种群对10种杀虫剂的抗性水平;将斜纹夜蛾敏感种群3龄幼虫在死亡率40%~70%的选择压下用茚虫威进行汰选,比较了斜纹夜蛾敏感种群和抗茚虫威种群的羧酸酯酶、谷胱甘肽S-转移酶和多功能氧化酶对硝基苯甲醚O-脱甲基活性。【结果】湖南5地斜纹夜蛾田间种群对有机磷类杀虫剂产生了26.9~220.2倍的抗性,对氨基甲酸酯类杀虫剂产生了68.3~890.8倍的抗性,对拟除虫菊酯类杀虫剂产生了21.0~267.2倍的抗性,对相对较新型杀虫剂（甲维盐、阿维菌素、茚虫威和溴虫腈）产生了5.2~53.4倍的抗性。经茚虫威汰选后第14代[抗性倍数(resistance ratio, RR)=26.43]斜纹夜蛾羧酸酯酶、谷胱甘肽S-转移酶和对硝基苯甲醚O-脱甲基酶活性分别上升2.86, 1.01和1.83倍。【结论】斜纹夜蛾对多种药剂产生了不同水平的抗性,斜纹夜蛾幼虫羧酸酯酶和对硝基苯甲醚O-脱甲基活性增强可能是斜纹夜蛾对茚虫威的抗性上升的重要因素。     

Characterization of 7-ethoxycoumarin-O-deethylase from malathion resistant and susceptible strains of Drosophila melanogaster

Mixed-function oxidase activity in a D. melanogaster strain carrying at least two closely linked malathion resistance genes on chromosome 3 was compared with the susceptible Canton S strain. The kinetics of O-deethylation of 7-ethoxycoumarin (7-ECD activity) with respect to pH, temperature, substrate and cofactor (NADPH) affinities and the response to metal salts of both strains were similar. The resistant strain had approx. 5-fold greater 7-ECD specific activity, and a parallel increase in total cytochrome P-450 content. Developmental stage, sex and nutritional state affected Drosophila 7-ECD activity. The intestine, fat body and Malpighian tubules contained the largest 7-ECD specific activity. Both susceptible and resistant strains had similar patterns of 7-ECD expression and differed only in total activity. In addition to more cytochrome P-450, the resistant strain had increased amounts of two microsomal, heme-staining polypeptides (M_r = 50 and 54 K after SDS-PAGE). The results suggest that the genetic change in the resistant strain involves the regulation of the Drosophila cytochrome P-450 system.     

Investigating the Molecular Mechanisms of Organophosphate and Pyrethroid Resistance in the Fall Armyworm Spodoptera frugiperda

The fall armyworm Spodoptera frugiperda is an economically important pest of small grain crops that occurs in all maize growing regions of the Americas. The intensive use of chemical pesticides for its control has led to the selection of resistant populations, however, to date, the molecular mechanisms underlying resistance have not been characterised. In this study the mechanisms involved in the resistance of two S. frugiperda strains collected in Brazil to chlorpyrifos (OP strain) or lambda-cyhalothrin (PYR strain) were investigated using molecular and genomic approaches. To examine the possible role of target-site insensitivity the genes encoding the organophosphate (acetylcholinesterase, AChE) and pyrethroid (voltage-gated sodium channel, VGSC) target-site proteins were PCR amplified. Sequencing of the S. frugiperda ace-1 gene identified several nucleotide changes in the OP strain when compared to a susceptible reference strain (SUS). These result in three amino acid substitutions, A201S, G227A and F290V, that have all been shown previously to confer organophosphate resistance in several other insect species. Sequencing of the gene encoding the VGSC in the PYR strain, identified mutations that result in three amino acid substitutions, T929I, L932F and L1014F, all of which have been shown previously to confer knockdown/super knockdown-type resistance in several arthropod species. To investigate the possible role of metabolic detoxification in the resistant phenotype of the OP and PYR stains all EST sequences available for S. frugiperda were used to design a gene-expression microarray. This was then used to compare gene expression in the resistant strains with the susceptible reference strain. Members of several gene families, previously implicated in metabolic resistance in other insects were found to be overexpressed in the resistant strains including glutathione S-transferases, cytochrome P450s and carboxylesterases. Taken together these results provide evidence that both target-site and metabolic mechanisms underlie the resistance of S. frugiperda to pyrethroids and organophosphates.     

The overexpression of acetylcholinesterase compensates for the reduced catalytic activity caused by resistance-conferring mutations in Tetranychus urticae

The mutations (G228S, A391T and F439W) and duplication of the acetylcholinesterase (AChE) gene (Tuace) are involved in monocrotophos resistance in the two-spotted spider mites, Tetranychus urticae (Kwon et al., 2010a, Kwon et al., 2010b). The overexpression of T. urticae AChE (TuAChE) as a result of Tuace duplication was confirmed in several field-collected populations by Western blotting using an AChE-specific antibody. To investigate the effects of each mutation on the insensitivity and fitness cost of AChE, eight variants of TuAChE were expressed in vitro using the baculovirus expression system. Kinetic analysis revealed that the G228S and F439W mutations confer approximately 26-fold and 99-fold increases in the insensitivity to monocrotophos, respectively, whereas the insensitivity increased over 1165-fold in the AChE with double mutations. Nevertheless, the presence of these mutations reduced the catalytic efficiency of AChE significantly. In particular, the TuAChE having both mutations together exhibited a 17.8～27.1-fold reduced catalytic efficiency, suggesting an apparent fitness cost in the monocrotophos-resistant mites. The A391T mutation did not change the kinetic properties of either the substrate or inhibitor when present alone but mitigated the negative impacts of the F439 mutation. To simulate the catalytic activity of the overexpressed TuAChE in two T. urticae strains (approximately 6 copies for AD strain vs. 2 copies for PyriF strain), appropriate TuAChE variants were combined to make up the desired AChE copies and mutation frequencies, and their enzyme kinetics were determined. The reconstituted 6-copy and 2-copy TuAChEs exhibited catalytic efficiency levels comparable to those of a single-copy wildtype TuAChE, suggesting that, if mutations are present, multiple copies of AChE are required to restore a normal level of catalytic activity in the monocrotophos-resistant mites. In summary, the present study provides clear evidence that Tuace duplication resulted in the proportional overexpression of AChE, which was necessary to compensate for the reduced catalytic activity of AChE caused by mutations.     

Development and Characterization of Diamondback Moth Resistance to Transgenic Broccoli Expressing High Levels of Cry1C

A field-collected colony of the diamondback moth, Plutella xylostella, had 31-fold resistance to Cry1C protoxin of Bacillus thuringiensis. After 24 generations of selection with Cry1C protoxin and transgenic broccoli expressing a Cry1C protein, the resistance that developed was high enough that neonates of the resistant strain could complete their entire life cycle on transgenic broccoli expressing high levels of Cry1C. After 26 generations of selection, the resistance ratios of this strain to Cry1C protoxin were 12,400- and 63,100-fold, respectively, for the neonates and second instars by a leaf dip assay. The resistance remained stable until generation 38 (G38) under continuous selection but decreased to 235-fold at G38 when selection ceased at G28. The Cry1C resistance in this strain was seen to be inherited as an autosomal and incompletely recessive factor or factors when evaluated using a leaf dip assay and recessive when evaluated using Cry1C transgenic broccoli. Saturable binding of ¹²⁵I-Cry1C was found with brush border membrane vesicles (BBMV) from both susceptible and Cry1C-resistant strains. Significant differences in Cry1C binding to BBMV from the two strains were detected. BBMV from the resistant strain had about sevenfold-lower affinity for Cry1C and threefold-higher binding site concentration than BBMV from the susceptible strain. The overall Cry1C binding affinity was just 2.5-fold higher for BBMV from the susceptible strain than it was for BBMV from the resistant strain. These results suggest that reduced binding is not the major mechanism of resistance to Cry1C.     

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.