Gene_locus Report for: bemta-ACHE1

Two acetylcholinesterases (AChEs) are present in Bemisia tabaci (BtAChE1 and BtAChE2). A conserved AChE mutation conferring organophosphate (OP) resistance (F392W in BtAChE1) is saturated in field populations despite its potential fitness cost, and a highly conserved amino acid residue forming the backdoor of AChE is substituted with a unique amino acid (Y390N in BtAChE1) in B. tabaci. Thus, the roles and relationships of the two amino acid substitutions in the evolutionary adaptation of B. tabaci remain to be elucidated, and little information is available on the catalytic and molecular properties of BtAChE1 and BtAChE2. To determine which AChE is a more relevant target of OPs and carbamates, the molecular and kinetic properties of BtAChE1 and BtAChE2 were investigated. Both BtAChE1 and BtAChE2 were exclusively expressed in head and thorax but not in abdomen, bound to the membrane via GPI anchoring, and present as dimeric forms. Soluble monomeric form was detected only in BtAChE2. The catalytic activity of baculovirus-expressed BtAChE1 was 19.5-fold higher than that of BtAChE2. The inhibition assay revealed that the F392W mutation in BtAChE1 enhanced resistance to OPs. The artificial substitution of N390 (wild form) to Y (putative ancient form) led to reduced catalytic efficiency and increased inhibition by glycoalkaloids, suggesting that the Y390N substitution in BtAChE1 may have been required for Solanaceae host adaptation. BtAChE1 was proven to function as a main catalytic enzyme for ACh hydrolysis, thus being the main target of OPs and carbamates.

Pyrethroid and organophosphate resistance-associated mutations have been recently reported in the whitefly Bemisia tabaci (Gennadius), a major pest of protected and outdoor crops worldwide. Here, we developed simple PCR-agarose gel visualization based assays for reliably monitoring the L925I and T929V pyrethroid resistance mutations in the B. tabaci para-type voltage gated sodium channel and the iAChE F331W organophosphate resistance mutation in the acetylcholinesterase enzyme ace1. PCR-RFLP assays were developed for detecting the L925I and the F331W resistance mutations. A highly specific PASA was developed for detecting the T929V mutation. The molecular diagnostic tools were used to monitor the frequency of the resistance mutations in a large number of field caught Q biotype B. tabaci from Crete (Greece), where both organophosphates and pyrethroids are extensively used. The F331W mutation was fixed in all field individuals examined. The pyrethroid resistance mutations were detected in high frequencies: 0.38 and 0.54 for L925I and T929V, respectively. The simple diagnostics are accurate and robust, to be used alongside classical bioassays to prevent ineffective insecticide applications, and for early identification of the spreading of resistant Q biotype populations into new regions around the globe.

Organophosphate (OP) insecticides are inhibitors of the enzyme acetylcholinesterase (AChE), which terminates nerve impulses by catalyzing the hydrolysis of the neurotransmitter acetylcholine. Previous biochemical studies in Bemisia tabaci (Hemiptera: Aleyrodidae) proposed the existence of two molecular mechanisms for OPs' resistance: carboxylesterase- (COE) mediated hydrolysis or sequestration and decreased sensitivity of AChE. Here, two acetylcholinesterase genes, ace1 and ace2, have been fully cloned and sequenced from an OP-resistant strain and an OP-susceptible strain of B. tabaci. Comparison of nucleic acid and deduced amino acid sequences revealed only silent nucleotide polymorphisms in ace2, and one mutation, Phe392Trp (Phe331 in Torpedo californica), in ace1 of the resistant strain. The Phe392Trp mutation is located in the acyl pocket of the active site gorge and was recently shown to confer OP insensitivity in Culex tritaeniorhynchus. In addition, we also report on the isolation of two carboxylesterase genes (coe1 and coe2) from B. tabaci, the first carboxylesterases to be reported from this species. We show that one of the genes, coe1, is overexpressed ( approximately 4-fold) in the OP-resistant strain, and determine, by quantitative PCR, that the elevated expression is not related to gene amplification but probably to modified transcriptional control. Lastly, we bring new biochemical evidence that support the involvement of both AChE insensitivity and COE metabolism in resistance to OP insecticides in the resistant strain.

Two acetylcholinesterases (AChEs) are present in Bemisia tabaci (BtAChE1 and BtAChE2). A conserved AChE mutation conferring organophosphate (OP) resistance (F392W in BtAChE1) is saturated in field populations despite its potential fitness cost, and a highly conserved amino acid residue forming the backdoor of AChE is substituted with a unique amino acid (Y390N in BtAChE1) in B. tabaci. Thus, the roles and relationships of the two amino acid substitutions in the evolutionary adaptation of B. tabaci remain to be elucidated, and little information is available on the catalytic and molecular properties of BtAChE1 and BtAChE2. To determine which AChE is a more relevant target of OPs and carbamates, the molecular and kinetic properties of BtAChE1 and BtAChE2 were investigated. Both BtAChE1 and BtAChE2 were exclusively expressed in head and thorax but not in abdomen, bound to the membrane via GPI anchoring, and present as dimeric forms. Soluble monomeric form was detected only in BtAChE2. The catalytic activity of baculovirus-expressed BtAChE1 was 19.5-fold higher than that of BtAChE2. The inhibition assay revealed that the F392W mutation in BtAChE1 enhanced resistance to OPs. The artificial substitution of N390 (wild form) to Y (putative ancient form) led to reduced catalytic efficiency and increased inhibition by glycoalkaloids, suggesting that the Y390N substitution in BtAChE1 may have been required for Solanaceae host adaptation. BtAChE1 was proven to function as a main catalytic enzyme for ACh hydrolysis, thus being the main target of OPs and carbamates.

BACKGROUND: Previously, we had reported that the majority of the Bemisia tabaci Mediterranean (MED) populations converged from two dominant genetic clusters (cluster 1 and 2) to one (cluster 2) during one year in greenhouse tomatoes in Korea. To find possible mechanisms for this phenomenon, we investigated the concurrent changes in resistance traits of the two clusters for three insecticide classes (organophosphate, pyrethroid, and neonicotinoid). RESULTS: Since the resistance mutation frequencies in regional samples were either high (i.e., the voltage-sensitive sodium channel L925I/T929V mutations and the F392 acetylcholinesterase 1 mutation) or zero (the nicotinic acetylcholine receptor R81T mutation), no meaningful correlation between the resistance allele frequency and genetic cluster was deduced. However, the actual resistance levels to all three insecticide classes were significantly higher in cluster 2 than those in cluster 1, suggesting that cluster 2 has a higher resistance potential. Furthermore, thiamethoxam treatment to the mixed population of clusters 1 and 2 over three generations exhibited a strong tendency of population change from cluster 1 to cluster 2. CONCLUSION: Our results demonstrated that the insecticide resistance trait is one of the driving forces for rapid genetic cluster change in the B. tabaci MED populations.

The whitefly Bemisia tabaci (Genn.) is a pest and vector of plant viruses to crop and ornamental plants worldwide. Using RNA interference (RNAi) to down regulate whitefly genes by expressing their homologous double stranded RNAs in plants has great potential for management of whiteflies to reduce plant virus disease spread. Using a Tobacco rattle virus-derived plasmid for in planta transient expression of double stranded RNA (dsRNA) homologous to the acetylcholinesterase (AChE) and ecdysone receptor (EcR) genes of B. tabaci, resulted in significant adult whitefly mortality. Nicotiana tabacum L. plants expressing dsRNA homologous to B. tabaci AChE and EcR were constructed by fusing sequences derived from both genes. Mortality of adult whiteflies exposed to dsRNA by feeding on N. tabacum plants, compared to non-dsRNA expressing plants, recorded at 24-hr intervals post-ingestion for three days, was >90% and 10%, respectively. Analysis of gene expression by real time quantitative PCR indicated that whitefly mortality was attributable to the down-regulation of both target genes by RNAi. Results indicated that knock down of whitefly genes involved in neuronal transmission and transcriptional activation of developmental genes, has potential as a bio-pesticide to reduce whitefly population size and thereby decrease virus spread.

The tobacco whitefly B-biotype Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) is a worldwide pest of many crops. In China, chlorpyrifos has been used to control this insect for many years and is still being used despite the fact that some resistance has been reported. To combat resistance and maintain good control efficiency of chlorpyrifos, it is essential to understand resistance mechanisms. A chlorpyrifos resistant tobacco whitefly strain (NJ-R) and a susceptible strain (NJ-S) were derived from a field-collected population in Nanjing, China, and the resistance mechanisms were investigated. More than 30-fold resistance was achieved after selected by chlorpyrifos for 13 generations in the laboratory. However, the resistance dropped significantly to about 18-fold in only 4 generations without selection pressure. Biochemical assays indicated that increased esterase activity was responsible for this resistance, while acetylcholine esterase, glutathione S-transferase, and microsomal-O-demethylase played little or no role. F392W mutations in acel were prevalent in NJ-S and NJ-R strains and 6 field-collected populations of both B and Q-biotype from locations that cover a wide geographical area of China. These findings provide important information about tobacco whitefly chlorpyrifos resistance mechanisms and guidance to combat resistance and optimize use patterns of chlorpyrifos and other organophosphate and carbamate insecticides.

Pyrethroid and organophosphate resistance-associated mutations have been recently reported in the whitefly Bemisia tabaci (Gennadius), a major pest of protected and outdoor crops worldwide. Here, we developed simple PCR-agarose gel visualization based assays for reliably monitoring the L925I and T929V pyrethroid resistance mutations in the B. tabaci para-type voltage gated sodium channel and the iAChE F331W organophosphate resistance mutation in the acetylcholinesterase enzyme ace1. PCR-RFLP assays were developed for detecting the L925I and the F331W resistance mutations. A highly specific PASA was developed for detecting the T929V mutation. The molecular diagnostic tools were used to monitor the frequency of the resistance mutations in a large number of field caught Q biotype B. tabaci from Crete (Greece), where both organophosphates and pyrethroids are extensively used. The F331W mutation was fixed in all field individuals examined. The pyrethroid resistance mutations were detected in high frequencies: 0.38 and 0.54 for L925I and T929V, respectively. The simple diagnostics are accurate and robust, to be used alongside classical bioassays to prevent ineffective insecticide applications, and for early identification of the spreading of resistant Q biotype populations into new regions around the globe.

Organophosphate (OP) insecticides are inhibitors of the enzyme acetylcholinesterase (AChE), which terminates nerve impulses by catalyzing the hydrolysis of the neurotransmitter acetylcholine. Previous biochemical studies in Bemisia tabaci (Hemiptera: Aleyrodidae) proposed the existence of two molecular mechanisms for OPs' resistance: carboxylesterase- (COE) mediated hydrolysis or sequestration and decreased sensitivity of AChE. Here, two acetylcholinesterase genes, ace1 and ace2, have been fully cloned and sequenced from an OP-resistant strain and an OP-susceptible strain of B. tabaci. Comparison of nucleic acid and deduced amino acid sequences revealed only silent nucleotide polymorphisms in ace2, and one mutation, Phe392Trp (Phe331 in Torpedo californica), in ace1 of the resistant strain. The Phe392Trp mutation is located in the acyl pocket of the active site gorge and was recently shown to confer OP insensitivity in Culex tritaeniorhynchus. In addition, we also report on the isolation of two carboxylesterase genes (coe1 and coe2) from B. tabaci, the first carboxylesterases to be reported from this species. We show that one of the genes, coe1, is overexpressed ( approximately 4-fold) in the OP-resistant strain, and determine, by quantitative PCR, that the elevated expression is not related to gene amplification but probably to modified transcriptional control. Lastly, we bring new biochemical evidence that support the involvement of both AChE insensitivity and COE metabolism in resistance to OP insecticides in the resistant strain.