Gene_locus Report for: aphgo-cxest

BACKGROUND: RNA interference (RNAi) is an effective tool to examine the function of individual genes. Carboxylesterases (CarE, EC 3.1.1.1) are known to play significant roles in the metabolism of xenobiotic compounds in many insect species. Previous studies in our laboratory found that CarE expression was up-regulated in Aphis gossypii (Glover) (Hemiptera: Aphididae) adults of both omethoate and malathion resistant strains, indicating the potential involvement of CarE in organophosphorus (OP) insecticide resistance. Functional analysis (RNAi) is therefore warranted to investigate the role of CarE in A. gossypii to OPs resistance. RESULT: CarE expression in omethoate resistant individuals of Aphis gossypii was dramatically suppressed following ingestion of dsRNA-CarE. The highest knockdown efficiency (33%) was observed at 72 h after feeding when dsRNA-CarE concentration was 100 ng/microL. The CarE activities from the CarE knockdown aphids were consistent with the correspondingly significant reduction in CarE expression. The CarE activity in the individuals of control aphids was concentrated in the range of 650-900 mOD/per/min, while in the individuals of dsRNA-CarE-fed aphids, the CarE activity was concentrated in the range of 500-800 mOD/per/min. In vitro inhibition experiments also demonstrated that total CarE activity in the CarE knockdown aphids decreased significantly as compared to control aphids. Bioassay results of aphids fed dsRNA-CarE indicated that suppression of CarE expression increased susceptibility to omethoate in individuals of the resistant aphid strains. CONCLUSION: The results of this study not only suggest that ingestion of dsRNA through artificial diet could be exploited for functional genomic studies in cotton aphids, but also indicate that CarE can be considered as a major target of organophosphorus insecticide (OPs) resistance in A. gossypii. Further, our results suggest that the CarE would be a propitious target for OPs resistant aphid control, and insect-resistant transgenic plants may be obtained through plant RNAi-mediated silencing of insect CarE expression.

Carboxylesterases provide key mechanisms of resistance to insecticides, particularly organophosphates (OPs), in insects. One resistance mechanism is a qualitative change in the properties of a carboxylesterase. Two mutant forms, G151D and W271L, have been observed, mostly in dipteran species, to affect substrate specificity of enzymes. But whether these two single mutations can commonly change character of insect carboxylesterases is unknown. In our study carboxylesterase genes from seven insects distributed among four orders were cloned, mutated at position 151 or 271 and expressed in Escherichia coli. The kinetics of the purified recombinant proteins was examined towards an artificial carboxylester and two OP insecticides. The G/A151D and W271L mutation significantly reduced carboxylesterase activity in 87.5% and 100% cases, respectively, and at the same time conferred OP hydrolase activities in 62.5% and 87.5% cases, respectively. Thus, the change at position 271 is more effective to influence substrate specificity than that at position 151. These results may suggest that these two mutations have the potential to cause insecticide resistance broadly in insects.

Levels of insecticide resistance, carboxylesterase activity, carboxylesterase expression, and the cDNA sequence of carboxylesterase gene were investigated in malathion resistant and susceptible strains of cotton aphids, Aphis gossypii (Glover). The resistant strain (MRR) exhibited 80.6-fold resistance to malathion compared to the susceptible strain (MSS) in cotton aphids. Five substrates, alpha-naphthyl acetate (alpha-NA), beta-naphthyl acetate (beta-NA), alpha-naphthyl propionate (alpha-NPr), alpha-naphthyl butyrate (alpha-NB), alpha-naphthyl caprylate (alpha-NC) and S-methyl thiobutyrate (S-MTB) were used to determine carboxylesterase activity in MRR and MSS strains of cotton aphids. Carboxylesterase activity was significantly higher in MRR strain than in MSS strain, 3.7-fold for alpha-NA, 3.0-fold for beta-NA, 2.0-fold for alpha-NPr, 2.9-fold for alpha-NB and 1.6-fold for alpha-NC, While for S-MTB, there was nearly no difference between the two strains. Two site mutations (K14Q and N354D) with high frequency were also found by sequence analysis in the MRR strain, compared with the MSS strain. The levels of gene expression for carboxylesterase of both MRR and MSS strains were determined by real-time quantitative PCRs. Compared with the MSS strain, the relative transcription levels and gene copy numbers of the carboxylesterase were 1.99- and 4.42-fold in the MRR strain, respectively. These results indicated that the increased expression of the carboxylesterase resulted from the increased transcription levels of carboxylesterase mRNA and gene copy numbers and combined with the site mutants might play role in cotton aphid resistance to malathion.

Carboxylesterases (CarEs) play an important role in detoxifying insecticides in insects. Over-expression and structural modification of CarEs have been implicated in the development of organophosphate (OP) insecticide resistance in insects. A previous study identified four nonsynonymous mutations (resulting in four amino acid residue substitutions) in the open reading frame of the carboxylesterase gene of resistant cotton aphids compared to the omethoate susceptible strain, which has possibly influenced the development of resistance to omethoate (a systemic OP insecticide). The current study further characterized the function of these mutations, both alone and in combination, in the hydrolysis of OP insecticides. The metabolism results suggest that the combination of four mutations, mainly existing in the laboratory-selected OP-resistant cotton aphid population, increased the OP hydrolase activity (approximately twofold) at the cost of detectable carboxylesterase activity. The functional studies of single or multiple mutations suggest the positive effect of H104R, A128V and T333P on the acquisition of OP hydrolase activity, especially the combination of H104R with A128V or T333P. K484R substitution decreased both the OP hydrolase activity and the CarE activity, indicating that this mutation primarily drives the negative effect on the acquisition of OP hydrolase activity amongst these four mutations in the resistant strain. The modelling and docking results are basically consistent with the metabolic results, which strongly suggest that the structural gene modification is the molecular basis for the OP resistance in this laboratory-selected cotton aphid strain.

BACKGROUND: RNA interference (RNAi) is an effective tool to examine the function of individual genes. Carboxylesterases (CarE, EC 3.1.1.1) are known to play significant roles in the metabolism of xenobiotic compounds in many insect species. Previous studies in our laboratory found that CarE expression was up-regulated in Aphis gossypii (Glover) (Hemiptera: Aphididae) adults of both omethoate and malathion resistant strains, indicating the potential involvement of CarE in organophosphorus (OP) insecticide resistance. Functional analysis (RNAi) is therefore warranted to investigate the role of CarE in A. gossypii to OPs resistance. RESULT: CarE expression in omethoate resistant individuals of Aphis gossypii was dramatically suppressed following ingestion of dsRNA-CarE. The highest knockdown efficiency (33%) was observed at 72 h after feeding when dsRNA-CarE concentration was 100 ng/microL. The CarE activities from the CarE knockdown aphids were consistent with the correspondingly significant reduction in CarE expression. The CarE activity in the individuals of control aphids was concentrated in the range of 650-900 mOD/per/min, while in the individuals of dsRNA-CarE-fed aphids, the CarE activity was concentrated in the range of 500-800 mOD/per/min. In vitro inhibition experiments also demonstrated that total CarE activity in the CarE knockdown aphids decreased significantly as compared to control aphids. Bioassay results of aphids fed dsRNA-CarE indicated that suppression of CarE expression increased susceptibility to omethoate in individuals of the resistant aphid strains. CONCLUSION: The results of this study not only suggest that ingestion of dsRNA through artificial diet could be exploited for functional genomic studies in cotton aphids, but also indicate that CarE can be considered as a major target of organophosphorus insecticide (OPs) resistance in A. gossypii. Further, our results suggest that the CarE would be a propitious target for OPs resistant aphid control, and insect-resistant transgenic plants may be obtained through plant RNAi-mediated silencing of insect CarE expression.

Carboxylesterases provide key mechanisms of resistance to insecticides, particularly organophosphates (OPs), in insects. One resistance mechanism is a qualitative change in the properties of a carboxylesterase. Two mutant forms, G151D and W271L, have been observed, mostly in dipteran species, to affect substrate specificity of enzymes. But whether these two single mutations can commonly change character of insect carboxylesterases is unknown. In our study carboxylesterase genes from seven insects distributed among four orders were cloned, mutated at position 151 or 271 and expressed in Escherichia coli. The kinetics of the purified recombinant proteins was examined towards an artificial carboxylester and two OP insecticides. The G/A151D and W271L mutation significantly reduced carboxylesterase activity in 87.5% and 100% cases, respectively, and at the same time conferred OP hydrolase activities in 62.5% and 87.5% cases, respectively. Thus, the change at position 271 is more effective to influence substrate specificity than that at position 151. These results may suggest that these two mutations have the potential to cause insecticide resistance broadly in insects.

Levels of insecticide resistance, carboxylesterase activity, carboxylesterase expression, and the cDNA sequence of carboxylesterase gene were investigated in malathion resistant and susceptible strains of cotton aphids, Aphis gossypii (Glover). The resistant strain (MRR) exhibited 80.6-fold resistance to malathion compared to the susceptible strain (MSS) in cotton aphids. Five substrates, alpha-naphthyl acetate (alpha-NA), beta-naphthyl acetate (beta-NA), alpha-naphthyl propionate (alpha-NPr), alpha-naphthyl butyrate (alpha-NB), alpha-naphthyl caprylate (alpha-NC) and S-methyl thiobutyrate (S-MTB) were used to determine carboxylesterase activity in MRR and MSS strains of cotton aphids. Carboxylesterase activity was significantly higher in MRR strain than in MSS strain, 3.7-fold for alpha-NA, 3.0-fold for beta-NA, 2.0-fold for alpha-NPr, 2.9-fold for alpha-NB and 1.6-fold for alpha-NC, While for S-MTB, there was nearly no difference between the two strains. Two site mutations (K14Q and N354D) with high frequency were also found by sequence analysis in the MRR strain, compared with the MSS strain. The levels of gene expression for carboxylesterase of both MRR and MSS strains were determined by real-time quantitative PCRs. Compared with the MSS strain, the relative transcription levels and gene copy numbers of the carboxylesterase were 1.99- and 4.42-fold in the MRR strain, respectively. These results indicated that the increased expression of the carboxylesterase resulted from the increased transcription levels of carboxylesterase mRNA and gene copy numbers and combined with the site mutants might play role in cotton aphid resistance to malathion.

A laboratory susceptible strain YSS of cotton aphid, Aphis gossypii (Glover), was selected with omethoate in successive generations in the laboratory to develop a resistant strain (YRR). After 24 generations, the resistance ratio of YRR strain was increased by 124.7-fold compared with the omethoate-susceptible strain. Carboxylesterase activity was significantly lower in the omethoate-resistant strain than in the omethoate-susceptible strain when alpha-naphthyl acetate alphay-NA was used as substrate. A carboxylesterase gene had been fully cloned and equenced from both omethoate-susceptible and resistant strains. Comparison of both nucleic acid and deduced amino acid sequences revealed four nucleotide acid differences between the omethoate-susceptible and resistant strain, which resulted in four amino acid substitutions (His104Arg, Ala128Val, Thr333Asp, Lys484Arg). Knowledge of the structure of a related enzyme acetylcholinesterase) suggests that one of these substitutions (His104Arg) lies within the active site of the enzyme. The GenBank accession numbers of carboxylesterase genes of omethoate-resistant (YRRAA) and susceptible (YSSAA) A. gossypii are AY485216 and AY485214 respectively

Resistance to omethoate was suppressible by the hydrolytic enzyme inhibitor SSS-tributyl phosphorotrithioate in a laboratory-selected resistant cotton aphid, Aphis gossypii Glover, strain, suggesting the involvement of hydrolytic enzymes in the detoxification process. The kinetic properties of carboxylesterases from both resistant and susceptible cotton aphids were characterized by four acyl ester substrates: alpha-naphthyl acetate (alpha-NA), alpha-naphthyl butyrate (alpha-NB), alpha-naphthyl phosphate (alpha-NP), and beta-naphthyl phosphate (beta-NP). No significant differences of carboxylesterase activity were found between resistant and susceptible strains by using either alpha-NP or beta-NP as substrates. In contrast, the susceptible A. gossypii exhibited significantly higher activity compared with resistant aphids with either alpha-NA or alpha-NB as substrates. To understand the molecular basis of this esterase-mediated resistance, carboxylesterase genes from both strains were cloned. Two genes share 99.4% identity at the nucleic acid level and 99.2% identity at the amino acid level. The full length of the cDNA opening reading frame is 1581 bp, encoding 526 amino acids. Four amino acid substitutions, Thr210 --> Met210, Asn294 --> Lys294, Gly408 --> Asp408, and Ser441 --> Phe441, were identified in the resistant strain. Probing of Southern blots with the 0.5 kb esterase fragment showed the same banding patterns and intensities with genomic DNA extracts from both resistant and susceptible A. gossypii. Furthermore, the MspI and HpaII fragments are the same in both strains, indicating there is no methylation of sequences detected by the probe. The combined results suggest that the structural gene substitution is likely the molecular basis of the organophosphate resistance in this laboratory-selected cotton aphid strain.