| Summary: | 碩士 === 國立臺灣大學 === 昆蟲學研究所 === 99 === Oriental fruit flies (Bactrocera dorsalis) are notorious agricultural pests, methomyl has been used to manage B. dorsalis in Taiwan since 1995. Unfortunately, several times of methomyl resistance has been observed in B. dorsalis from field investigation in 2002. In this study, the synergism bioassay, metabolic enzyme activity, acetylcholinesterase (AChE) activity and the ace gene (which can encode AChE protein) molecular assays were used to explore the possible methomyl resistant mechanisms in the methomyl-resistant strain (Meth-R, 140-fold resistance ratio) of B. dorsalis. A well-documented fenitrothion-resistant strain (Fenit-R, 538-fold resistance ratio) was used to compare with the Meth-R and susceptible strains on AChE assays. Synergism tests indicated synergistic ratios toward methomyl at 4.04-fold with piperonyl butoxide (PBO) and 4.82-fold with triphenyl phosphate (TPP) on Meth-R, and there was a 1.7-fold diethyl maleate (DEM) synergistic ratio on Meth-R as compared to the results of the susceptible strain. Enzyme activity results supported the theory that Meth-R had greater activity, close to 1.5-fold in ESTs (by α- or β-naphthyl acetate as the substrates), 1.5- to 2-fold in GSTs (by CDNB or DCNB as the substrates), and 4-fold in MFOs (by 7-ethoxycoumarin as the substrates) over the susceptible strain, which suggests that mixed function oxidases (MFOs), esterases (ESTs), and glutathione S-transferases (GSTs) are involved in methomyl resistance. No significant difference was found on the AChE activity, AChE inhibition (by using methomyl and paraoxon as inhibitors), and quantitative PCR (qPCR) of ace gene between the Meth-R and susceptible strains. The AChE of the Fenit-R strain was found to be insensitive to the paraoxon inhibitor. Although a point mutation (T659A) located at the post-translational modification region was discovered on the ace gene of the Meth-R; T659A also occurred in the susceptible strain, but with lower gene frequency (susceptible strain: homozygous genotype of T = 0.27, heterozygous = 0.55, homozygous genotype of A = 0.18; Meth-R strain: TA = 0.16; AA = 0.84). T659A might slightly influence the hydrophobicity of AChE precursor protein due to an increase of the hydrophobic results on the C-terminal region. However, there was no correlation between the Meth-R and susceptible strains or among different genotypes of T659A on the results of AChE activity or AChE inhibition assays. As a result, our study indicates that the possible methomyl resistance is the metabolic mechanism in B. dorsalis. The mechanism in methomyl resistance is different from the well-known altered target-site mechanism in organophosphate resistance in Bactrocera spp.
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