The A302S mutation in Rdl that confers resistance to cyclodienes and limited cross-resistance to fipronil is undetectable in field populations of house flies from the USA

Fipronil is a relatively new insecticide with great potential for insect control, however widespread use of cyclodiene insecticides has selected for an A302S mutation in the Rdl (GABA gated chloride channel) allele. This mutation gives resistance to cyclodienes and limited cross-resistance to fipronil. Given the concern over the possible reduction in efficacy and/or lifetime that fipronil might be used for pest control (given the extensive use of cyclodienes in the past), it is important to know the frequency of the A302S Rdl mutation in field populations. To ascertain the relative frequency of the A302S Rdl mutation in house fly populations we used three experimental approaches. First, we attempted to select for fipronil resistance by initially treating 33,100 field collected flies and selecting 14 additional generations. We were unable to produce a highly resistant strain. Second, we directly sequenced field collected flies. Third, we tested field collected house flies with a diagnostic dose of dieldrin and then genotyped the survivors. Out of the 4750 flies tested, there were no Rdl resistance alleles detected. We conclude that the resistant Rdl allele is rare in house flies in the US due to decades without cyclodiene use and a fitness disadvantage (in the absence of cyclodienes) of the 302S Rdl allele. The limited cross-resistance provided by the cyclodiene resistant Rdl allele, combined with the very low frequency of this allele in field populations, suggests that fipronil could be a promising insecticide for house fly control.     

Application of computational methods in the analysis of pesticide target-site and resistance mechanisms

Meta-diamide insecticides including broflanilide have a high insecticidal activity by acting on RDL GABA receptors. Both membrane potential assays and docking studies suggest that the target site of meta-diamides is different from that of conventional noncompetitive inhibitors, such as fipronil. In fact, meta-diamides are effective against cyclodiene- and fipronil-resistant pests that carry target-site mutations. Dinotefuran uniquely possesses a tetrahydrofuran ring, whereas other neonicotinoids possess aromatic rings. Moreover, dinotefuran has been reported to be effective against imidacloprid-resistant strains. A docking study predicted the weak binding of dinotefuran to cytochrome P450s which are associated with imidacloprid resistance. Metabolic assays revealed that dinotefuran was not metabolized by these cytochrome P450s. These findings suggest that the lack of metabolic activity of P450s against dinotefuran causes a low level of cross-resistance.     

Cloning, developmental and tissue-specific expression of γ-aminobutyric acid (GABA) receptor alpha2 subunit gene in Spodoptera exigua (Hübner)

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     

The role of GABA and glutamate receptors in susceptibility and resistance to chloride channel blocker insecticides

Despite a point mutation in the pore-forming segment of the Rdl GABA receptor subunit that is widespread and persistent in insect populations and confers high levels of resistance to dieldrin and other polychlorocycloalkane (PCCA) insecticides, the phenylpyrazole insecticide fipronil, which binds at same site, has proven to be effective in controlling many insects, including dieldrin-resistant populations. Fipronil and its major sulfone metabolite are unique among chloride channel blocking insecticides in that they also potently block GluCls. We present here a patch clamp study of the action of fipronil sulfone on native GABA receptors and GluCl receptors from susceptible and dieldrin-resistant German cockroaches, to provide a better understanding of the effect of the Rdl mutation on the function and insecticide sensitivity of these two targets, and its role in resistance. Dieldrin blocked GABA currents with an IC₅₀ of 3 nM in wild-type cockroaches, and 383 nM in resistant insects, yielding a resistance ratio of 128. Fipronil sulfone blocked GABA currents with an IC₅₀ of 0.8 nM in susceptible insects and 12.1 nM, or 15-fold higher, in resistant insects. While both GluClD (desensitizing) and GluClN (non-desensitizing) receptors were found in German cockroach neurons, GluClN receptors were rare and could not be included in this study. GluClD receptors from resistant insects had reduced sensitivity to glutamate and a lower rate of desensitization than those from susceptible insects, but their sensitivity to block by fipronil sulfone was not significantly changed, with an IC₅₀ of 38.5 ± 2.4 nM (n = 8) in the susceptible strain and 40.3 ± 1.0 nM (n = 7) in the resistant strain. Fipronil sulfone also slowed the decay time course of GluClD currents. These results suggest that GluClD receptors contain the Rdl subunit, but their sensitivity to fipronil sulfone is not altered in resistant insects.     

Identification of the Rdl mutation in laboratory and field strains of the cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae)

In many insect species, resistance to cyclodiene insecticides is caused by amino acid substitutions at a single residue (A302) within the M2 transmembrane region of the gamma-aminobutyric acid (GABA) receptor sub-unit termed Rdl (resistance to dieldrin). These mutations (A302S and A302G) have also been shown to confer varying levels of cross-resistance to fipronil, a phenylpyrazole insecticide with a similar mode of action to cyclodienes. To investigate the possible occurrence of these mutations in the cat flea, Ctenocephalides felis (Bouché), a 176-bp fragment of the cat flea Rdl gene, encompassing the mutation site, was PCR amplified and sequenced from nine laboratory flea strains. The A302S mutation was found in eight of the nine strains analysed, although the relative frequency of the mutant allele varied between strains. Only one strain (R6) was found to be homozygous for the S302 allele in all the individuals tested, and this correlated with previous reports of low-level fipronil resistance in this strain. A PCR-based diagnostic assay, capable of screening individual fleas for this mutation, was developed and used to survey a range of fleas collected at random from veterinary clinics in the UK and USA. The A302S mutation was present at a high frequency in these domestic pet populations.     

Detection of insecticide resistance-associated mutations in cat flea Rdl by TaqMan-allele specific amplification

We are interested in correlating the performance of fipronil in populations of cat fleas (Ctenocephalides felis) with potential cross-resistance conferred by point mutations in the Resistance to dieldrin gene, Rdl. Here we report the sequencing of exon 7 of the cat flea Rdl gene and the documentation of a putative resistance-associated mutation predicting the replacement of alanine302 with a serine. We describe two polymerase chain reaction (PCR) based diagnostics for the detection of this mutation. First, PCR mediated detection of a restriction endonuclease polymorphism (PCR REN) using a BsmAI site created by the resistance-associated mutation. Second, a TaqMan assay using allele specific fluorogenic probes and the TaqMan 5^′ specific nuclease. We describe how such diagnostics can be used in the diagnosis of resistance in the field and laboratory.     

Mode of action of the plant-derived silphinenes on insect and mammalian GABAA receptor/chloride channel complex

The silphinenes are tricyclic sesquiterpenes that have antifeedant and toxic effects in insects and structural similarity to the known GABA antagonist, picrotoxinin. In murine synaptoneurosomes, silphinenes block GABA-stimulated influx of ³⁶Cl⁻ with EC₅₀s in the range of 10-30 μM. In insects, silphinenes were tested in neurophysiological recordings of central neurons from third instar Drosophila melanogaster larvae. Silphinenes reversed the blockage of neuronal firing induced by GABA, but had little effect below 100 μM. The structure-activity profile observed in the murine chloride flux assay was also observed in the larval neurophysiological assay, indicating little selectivity for the silphinenes. A reference silphinene was equally active on nerve preparations from the rdl strain of D. melanogaster, which is resistant to channel-blocking antagonists via an altered GABA receptor. This latter finding suggests that silphinenes interact with the insect GABA receptor in a manner somewhat different from PTX, and that rdl resistance in the field may have little effect on silphinene efficacy.     

Acetylcholinesterase point mutations putatively associated with monocrotophos resistance in the two-spotted spider mite

Molecular mechanisms of monocrotophos resistance in the two-spotted spider mite (TSSM), Tetranychus urticae Koch, were investigated. A monocrotophos-resistant strain (AD) showed ca. 3568- and 47.6-fold resistance compared to a susceptible strain (UD) and a moderately resistant strain (PyriF), respectively. No significant differences in detoxification enzyme activities, except for the cytochrome P450 monooxygenase activity, were found among the three strains. The sensitivity of acetylcholinesterase (AChE) to monocrotophos, however, was 90.6- and 41.9-fold less in AD strain compared to the UD and PyriF strains, respectively, indicating that AChE insensitivity mechanism plays a major role in monocrotophos resistance. When AChE gene (Tuace) sequences were compared, three point mutations (G228S, A391T and F439W) were identified in Tuace from the AD strain that likely contribute to the AChE insensitivity as predicted by structure analysis. Frequencies of the three mutations in field populations were predicted by quantitative sequencing (QS). Correlation analysis between the mutation frequency and actual resistance levels (LC₅₀) of nine field populations suggested that the G228S mutation plays a more crucial role in resistance (r² = 0.712) compared to the F439W mutation (r² = 0.419). When correlated together, however, the correlation coefficient was substantially enhanced (r² = 0.865), indicating that both the F439W and G228S mutations may work synergistically. The A391T mutation was homogeneously present in all field populations examined, suggesting that it may confer a basal level of resistance.     

Toxicity and neurophysiological effects of selected insecticides on the mole cricket, Scapteriscus vicinus (Orthoptera: Gryllotalpidae)

Mole crickets (Scapteriscus spp.) are severe subterranean pests of turfgrasses, commonly targeted with neurotoxic insecticides. Ideally insecticides used against mole crickets should induce quick knockdown or mortality to minimize damage caused by their tunneling. However, neurophysiological effects of insecticides on mole crickets are mostly unknown. The aims of this study were to investigate neurophysiological and toxic effects of several insecticides on tawny mole cricket (Scapteriscus vicinus Scudder) adults and nymphs, and potential synergy between pyrethroid and neonicotinoid insecticides. Bifenthrin, fipronil, and the combination of bifenthrin + imidacloprid provided the fastest median mortality when injected. The combination of bifenthrin + imidacloprid elicited faster toxicity than either active ingredient alone. Imidacloprid, bifenthrin, and bifenthrin + imidacloprid caused immediate knockdown, whereas fipronil immobilized mole crickets within 1-2 h. Acephate, bifenthrin, fipronil, imidacloprid, and bifenthrin + imidacloprid caused significant neuroexcitation. Bifenthrin + imidacloprid resulted in greater increases of spontaneous neural activity than the additive effects of imidacloprid and bifenthrin alone. Excitatory compounds acting at sodium and chloride channels (bifenthrin and fipronil) were the most toxic against S. vicinus. Combining a sodium channel toxin (bifenthrin) and a synaptic toxin (imidacloprid) led to greater than additive neurophysiological and toxic effects, which to our knowledge provides the first documented evidence of synergistic neurological “potentiation”.     

Sequence variation in the two-component histidine kinase gene of Botrytis cinerea associated with resistance to dicarboximide fungicides

The complete two-component histidine kinase gene (Bos1) was sequenced from eight dicarboximide-resistant (D^R) and six-sensitive (D^S) field isolates of Botrytis cinerea. Comparisons in the predicted amino acid sequences of Bos1 showed that each two D^R isolates had a single point mutation at amino acid position 365 from an isoleucine to serine (I365S) or to an asparagine (I365N). Three D^R isolates were characterized with a change from glutamine to proline at position 369 (Q369P) as well as an asparagine to serine at the position 373 (N373S). These mutations located within the 90-amino-acid repeats of Bos1 have been reported previously. One new mutation, however, was found in the D^R isolate 65-E8. In this isolate, a null mutation at the amino acid position 1040 in the Bos1 was detected. Inoculation tests showed that this isolate was nearly nonpathogenic to cucumber. After the Bos1 gene from the sensitive isolate 38B1 was transferred into the resistant isolate 65-E8, all transformants tested were sensitive to iprodione and capable of infecting cucumber. DNA fingerprint generated by micro-satellite primed-PCR showed that isolates were not clustered based on their sensitivity to iprodione. Results from this study indicate that mutations in the Bos1 gene associated with dicarboximide resistance are diverse in B. cinerea, and the Bos1 gene is associated with virulence of B. cinerea.     

Cloning of the acetylcholinesterase 1 gene and identification of point mutations putatively associated with carbofuran resistance in Nilaparvata lugens

Molecular mechanisms of carbofuran resistance in the brown planthopper, Nilaparvata lugens Stål, were investigated. A carbofuran-resistant strain (CAS) showed approximately 45.5- and 15.1-fold resistance compared with a susceptible strain (SUS) and a non-selected field strain (FM), respectively. Activities of the esterase and mixed-function oxidase were approximately 2.8- and 1.6-fold higher, respectively, in the CAS strain than in the SUS strain, suggesting that these enzymes play a minor role in carbofuran resistance. Interestingly, the insensitivity of acetylcholinesterase (AChE) to carbofuran was approximately 5.5- and 3.7-fold higher in the CAS strain compared to the SUS and FM strains, respectively, indicating that AChE insensitivity is associated with carbofuran resistance. Western blot analysis identified two kinds of AChEs, of which the type-1 AChE (encoded from Nlace1, which is paralogous to the Drosophila AChE gene) was determined to be the major catalytic AChE in N. lugens. The open reading frame of Nlace1 is composed of 1989 bp (approximately 74 kD) and revealed 52.5% and 24.3% amino acid sequence identities to those of Nephotettix cincticeps and Drosophila melanogaster, respectively. Screening of point mutations identified four amino acid substitutions (G119A, F/Y330S, F331H and H332L) in the CAS strain that likely contribute to AChE insensitivity. The frequencies of these mutations were well correlated with resistance levels, confirming that they are associated with reduced sensitivity to carbofuran in N. lugens. These point mutations can be useful as genetic markers for monitoring resistance levels in field populations of N. lugens.     

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.     

Fipronil resistance mechanisms in the rice stem borer, Chilo suppressalis Walker

Fipronil resistance mechanisms were studied between the laboratory susceptible strain and the selective field population of rice stem borer, Chilo suppressalis Walker in the laboratory. The borer population was collected from Wenzhou county, Zhejiang province. After five generations of selection, fipronil resistance ratio was 45.3-fold compared to the susceptible strain. Synergism experiments showed that the synergistic ratios of PBO, TPP and DEF on fipronil in susceptible and resistant strains of C. suppressalis were 7.55-, 1.93- and 2.91-fold, respectively, and DEM showed no obvious synergistic action on fipronil. Activities of carboxylesterase and microsomal-O-demethylase in the resistant strain were 1.89- and 1.36-fold higher that in susceptible strain, and no significant difference of glutathione-S-transferase activity was found between the resistant and susceptible strains. The K_m and V_max experiments also demonstrated that fipronil resistance of C. suppressalis was closely relative to the enhanced activities of carboxylesterase and microsomal-O-demethylase. Moreover, cross-resistance between fipronil and other conventional insecticides and the multiple resistant properties of the original Wenzhou’s population were also discussed.     

Glutamate-activated chloride channels: Unique fipronil targets present in insects but not in mammals

Selectivity to insects over mammals is one of the important characteristics for a chemical to become a useful insecticide. Fipronil was found to block cockroach GABA receptors more potently than rat GABA_A receptors. Furthermore, glutamate-activated chloride channels (GluCls), which are present in cockroaches but not in mammals, were very sensitive to the blocking action of fipronil. The IC₅₀s of fipronil block were 30 nM in cockroach GABA receptors and 1600 nM in rat GABA_A receptors. Moreover, GluCls of cockroach neurons had low IC₅₀s for fipronil. Two types of glutamate-induced chloride current were observed: desensitizing and non-desensitizing, with fipronil IC₅₀s of 800 and 10 nM, respectively. We have developed methods to separately record these two types of GluCls. The non-desensitizing and desensitizing currents were selectively inhibited by trypsin and polyvinylpyrrolidone, respectively. In conclusion, in addition to GABA receptors, GluCls play a crucial role in selectivity of fipronil to insects over mammals. GluCls form the basis for development of selective and safe insecticides.     

Selection of Anopheles stephensi with DDT and dieldrin and cross-resistance spectrum to pyrethroids and fipronil

The field strain of Anopheles stephensi, the main malaria vector in south of Iran, was colonized in laboratory and selected with DDT and dieldrin in two separate lines for 3 generations to a level of 19.5- and 14-fold for DDT and dieldrin resistance, respectively. Synergist tests with chlorofenethol (DMC) and piperonyl butoxide (PBO) on the selected strains indicated that dehydrochlorination and oxidative detoxification might be the underlying mechanisms involved in the resistance to dieldrin and DDT in selected strains. DDT selection decreased susceptibility to DDT and pyrethroids including lambdacyhalothrin, permethrin deltamethrin and cyfluthrin. The result also showed that selection with dieldrin caused negative and positive cross-resistance to pyrethroid and fipronil, respectively. Based on these results, it can be concluded that besides metabolic resistance mechanisms, other factors such as mutation in γ aminobutyric acid (GABA) and voltage-gated sodium channels (Kdr) might be involved.     

三唑磷与氟虫腈复配对粘虫的增效机理研究

以粘虫Mythimna separate 4龄幼虫为试虫,从表皮穿透、生物转化、作用靶标3个方面研究了三唑磷·氟虫腈复配剂的增效机理。结果表明,三唑磷与氟虫腈复配后,相互促进了对方对粘虫表皮的穿透作用;三唑磷对酸性磷酸酯酶(ACPase)的竞争性抑制与氟虫腈对ACPase的非竞争性抑制与反竞争性抑制的混合型抑制相结合,使复配剂对ACPase的抑制能力大幅度提高;对谷胱甘肽S-转移酶(GSTs)的联合抑制系数(JIC)达到425.87,表现出显著的增效作用;三唑磷和氟虫腈对乙酰胆碱酯酶(AChE)只有微弱的交互作用。表皮穿透率的提高及其对ACPase和GSTs抑制能力的增强可能是该复配剂对粘虫增效的重要机理。     

Molecular diagnostic for detecting the cytochrome b G143S - QoI resistance mutation in Cercospora beticola

The mutation G143S has been associated with high-level strobilurin resistance in laboratory mutant strains of Cercospora beticola, one of the most destructive pathogens in sugar beet plants. By using allele specific primers (PASA-PCR) and agarose gel visualization, a molecular diagnostic was developed for the detection of the G143S resistance mutation. This assay is simple and applicable in low tech laboratory settings, with high reliability when a relatively large proportion of mutated mitochondrial alleles are present in the resistant strains. To achieve detection of resistant alleles at low frequencies, a more sensitive Real Time PCR based assay capable of discriminating resistant (S143) genotypes in frequencies as low as 1:10,000 resistant:sensitive alleles was developed. Both diagnostics were successfully validated in laboratory strains. Subsequently, a large number of C. beticola isolates from QoI-treated sugar beet experimental fields in Greece were screened for resistance to Qo fungicides using these diagnostics and classic bioassays. No proportion of the 143S resistant allele was detected in all field isolates tested, which was in agreement with the phenotypes revealed by the biotests confirming that the efficacy of QoIs against C. beticola has been sustained in Greece 7 years after their introduction.     

Effects of insecticides on plant-growth-promoting activities of phosphate solubilizing rhizobacterium Klebsiella sp. strain PS19

In this study, four technical grade insecticides, fipronil, pyriproxyfen, imidacloprid and thiamethoxam were applied at the recommended and the higher doses to investigate their effects on plant growth-promoting activities of phosphate-solubilizing Klebsiella sp. strain PS19, isolated from mustard rhizosphere. All tested insecticides displayed a concentration-dependent inhibition in plant growth promoting traits, like, inorganic phosphate solubilization, biosynthesis of phytohormones and siderophores, of rhizobacterial strain PS19. For example, the phosphate-solubilizing activity of Klebsiella sp. PS 19 was reduced maximally by 95%, at 3900 μg l⁻¹ pyriproxyfen over control. At the recommended rate, the magnitude of toxicity of insecticides to plant growth promoting traits was less severe compared to the higher doses. The sequence of insecticide-toxicity expressed as percent decrease, determined at highest dose rate of each insecticide, over control was: pyriproxyfen (95) = imidacloprid (95) > thiamethoxam (94) > fipronil (85), for phosphate-solubilizing activity while for salicylic acid (SA) it was: thiamethoxam > pyriproxyfen = imidacloprid > fipronil. The impact of the highest dose rate of insecticides on 2,3-dihydroxybenzoic acid (DHBA) was almost equal to those observed for SA. Thiamethoxam decreased the indole acetic acid (IAA) synthesis maximally by 86% whereas fipronil had least toxicity and reduced it by 67% relative to the control. Among the experimental insecticides, pyriproxyfen at 3900 μg l⁻¹ in general, had the greatest toxic effects for plant growth promoting activities of the test strain. The study inferred that insecticides affect the plant beneficial activities of rhizobacteria adversely. These findings are likely to add a new insight into the pest management practices.     

Regulation of cytochrome P450 expression in Drosophila: Genomic insights

Genomic tools such as the availability of the Drosophila genome sequence, the relative ease of stable transformation, and DNA microarrays have made the fruit fly a powerful model in insecticide toxicology research. We have used transgenic promoter-GFP constructs to document the detailed pattern of induced Cyp6a2 gene expression in larval and adult Drosophila tissues. We also compared various insecticides and xenobiotics for their ability to induce this cytochrome P450 gene, and show that the pattern of Cyp6a2 inducibility is comparable to that of vertebrate CYP2B genes, and different from that of vertebrate CYP1A genes, suggesting a degree of evolutionary conservation for the “phenobarbital-type” induction mechanism. Our results are compared to the increasingly diverse reports on P450 induction that can be gleaned from whole genome or from “detox” microarray experiments in Drosophila. These suggest that only a third of the genomic repertoire of CYP genes is inducible by xenobiotics, and that there are distinct subsets of inducers/induced genes, suggesting multiple xenobiotic transduction mechanisms. A relationship between induction and resistance is not supported by expression data from the literature. The relative abundance of expression data now available is in contrast to the paucity of studies on functional expression of P450 enzymes, and this remains a challenge for our understanding of the toxicokinetic aspects of insecticide action.     

Molecular characterization of two acetylcholinesterase genes from the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae)

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.