Studies On Target Resistance Of Chilo Suppressalis Walker To Triazophos

Rice stem borer, Chilo suppressalis (Walker), is one of the most important insect pests in Asia. Because most of its life-cycle is spent inside rice stems, this pest is difficult to control. Only insecticides that combine good penetration or systemicity, low toxicity to fish, and affordability to farmers are appropriate for stem borer control. In the past, hexachlorocyolohexane (BHC), chlordimeform and monosultap were used extensively in China. However, the first two have been banned for ecotoxicological reasons, and monosultap has lost its effectiveness due to resistance. The organophosphate triazophos initially proved an excellent replacement for monosultap, but recently its effectiveness has declined. A previous publication confirmed resistance to triazophos in C. suppressalis from some locations in China, and suggested reduced sensitivity of acetylcholinesterase-the target site of organophosphates-to be the primary resistance mechanism. In this paper, we reported a mutation in Ch-AChEl consistently associated with resistance, and an assay based on restriction fragment length polymorphism (RFLP) analysis was developed to diagnose A314S genotypes in field populations. The results were summarized as follows:1. Cloning and sequence analysis of AChEl from C. suppressalisThe Rapid Amplification of cDNA Ends (RACE) procedure was employed to obtain full-length sequence of AChEl from C. suppressalis. The result showed that it is 2521 bp in full-length. The 5'and 3'untranslated regions (UTR) were 235 bp and 204 bp, respectively. The complete amino acid sequence deduced from the cDNA consisted of 17 residues for the putative signal peptide and 677 residues for the mature protein. The sequence of this gene has the features shared by members of AChE family. Compared with AChE in Torpedo californica, the functional motifs were highly conserved:the catalytic triads (S313, E439 and H553 in Ch-AChE1, and S266, E395 and H509 in Ch-AChE2), the characteristic'FGESAG' motif surrounding the active serine,3 intra-chain disulfide bridges (C181-C208, C367-380 and C515-C637 in Ch-AChEl, and C115-C143, C320-C335 and C471-C590 in Ch-AChE2), a anionic choline-binding site (W198 in Ch-AChE1 and W133 in Ch-AChE2), acyl pocket residues (W346, F402 and F443 in Ch-AChE1, and W299, F358 and F399 in Ch-AChE2) that accommodate the acyl moiety of the active site, and the oxyanion hole (G232, G233 and A314 in Ch-AChEl, and G179, G180 and A267 in Ch-AChE2) that helps to stabilize the tetrahedral molecule during catalysis. The C-termimal Cys residues that contributed to an intermolecular dimerization were Cys660 and Cys608, respectively. Following these Cys residues, the protein has amino acid tails enriched in hydrophobic residues as predicted using ProtScale. Thus, it is proved to be the AChE gene. Otherwise, this gene shares only 47%similarity with previously reported AChE2 in this pest, but has a high degree of homology to AChEls from other Lepidopetra species. All these results firmly established that the amplified cDNA fragment is the sequence of AChE 1 gene in rice stem borer. 2. Mutation in AChEl associated with triazophos resistance in the rice stem borerOne strain of the rice stem borer was selected in the lab by exposure to increasing concentrations of triazophos. Insecticide bioassays showed that the resistant strain (Tp-R) exhibited a 1172-fold resistance ratio to the insecticide, compared to a susceptible strain (Gy-S). Previous work suggested that insensitive AChE may also involved in triazophos resistance mechanism of rice stem borer. To verify the hypothesis, we compared the full-length AChEs cDNA sequences from five monocrotophos-resistant and five susceptible rice stem borer individuals. Sequence analysis found an amino acid mutation A314S in Ch-AChEl (corresponding to A201 in Torpedo californica AChE) that was consistently associated with the occurrence of resistance. This alanine residue is located in the characteristic'FGESAG'motif surrounding the active serine, and lies in the oxyanion hole that contributes to stabilizing the tetrahedral molecule during catatysis. The alanine to serine substitution changes the side group from-CH3 to-CH2OH, which is believed to alter the conformation of the adjacent serine of the catalytic triad and to affect the interaction between AChE and both substrates and inhibitors. The mutation A314S in Ch-AChEl is likely to be responsible for the AChE insensitivity to triazophos.3. Frequency of the mutation in field populations with different resistanceThe mutation GCG to TCG in Ch-acel removes an MspAl I restriction site from the wild type allele. An RFLP-PCR assay was therefore designed using primers able to amplify specifically a 758-bp fragment encompassing the mutation site in Ch-ace1 from genomic DNA, and by checking the digestion of the resulting fragment with MspAl I. The PCR product amplified from the wild type allele was completely cut into two pieces (534 bp and 224 bp long). That amplified from the heterozygote was partially cut and presented as three bands (758 bp,534 bp and 224 bp long), whereas that amplified from the mutated allele remained intact.With this method, individual larvae of C. suppressalis collected from different field sites were analysed, and results showed a strong correlation between mutation frequencies and levels of triazophos resistance. The RFLP-PCR assay developed to diagnose the A314S mutation in field populations provides a potentially valuable way of monitoring resistance and investigating the relative frequencies of resistance genotypes. It overcomes many of the difficulties encountered with time-consuming bioassays against C. suppressalis, and offers the prospect of a rapid and high throughput kit to assist with choosing insecticides and combating resistance in this species.4. Eukaryotic expression and transcript comparison in vivo of AChEs from rice stem borerReal-time PCR was performed to compare the transcript levels of Ch-AChEl and Ch-AChE2 from individual fourth-instar larva. The results showed that the transcript level of Ch-AChE1 was higher than Ch-AChE2's.Based on cloned AChEs from rice stem borer, the insect Bac-to-Bac expression system was selected to construct the eukaryotic expression plasmids of wild AChEl and AChE2. Meanwhile, plasmids of wild AChEl was changed into plasmids of mutant AChE1 by site-directed mutagenesis. The constructed donor plasmids of pFastBac1-AChE1,pFastBac 1-AChE2 and pFastBac 1-AChE 1 Mu were transfected into the DH10BAC, and then select the positive clone and extract the Bacmid, using Bacmid to transfect the Tn cells obtain the recombine virus, then amplificate the first generation virus and infect the Tn cells to express the target protein.All of the three recombinant proteins showed the obvious band through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. But the wild recombinant AChE1 and mutant showed no enzyme activity, the recombinant AChE2 showed enzyme activity.