Studies On The Kinetics Of Acetylcholinesterase And Ace Gene In The Dichlorvos-resistant Strain Of The Housefly (Musca Domestica)

Acetylcholinesterase (AChE, EC 3.1.1.7) keeps the normal transmission of nerve impulses at cholinergic synapses by hydrolyzing acetycholine (ACh) released from the presynaptic membrane. AChE is the target of action by the OP and CB insecticides. The inhibition of AChE by organophosphate (OP) and carbamate (CB) insecticides accumulates ACh in the synaptic gap and causes desensitization of the ACh receptor, leading to a blockage of the signal transmission. Now insecticide resistance is very common in pest insects due to the frequent and intensive use of insecticides to control pests. Insecticide resistance is a micro-evolution phenomenon, which provides a very useful tool to study the adaptation of insects to selection pressure. In this study, we selected a dichlorvos-resistant (DRR) strain of housefly (Musca domestica) with dichlorvos in our Lab and measured the resistant spectrum by topical application. The properties and kinetic parameters of AChEs from the DRR and susceptible strains (SS) were determined by Ellman's method. The cDNA of the ace gene encoding AChE from DRR strains had been cloned using RT-PCR and sequenced. The major results obtained are summarized as following:1. The DRR strain exhibited high resistance to carbofuran (602.06), paraoxon (460.82) and monocrotophos (120.05); middle resistance to omethoate (93.12),dichlorvos (86.13), methamidophos (38.13), methomyl (36.04), propoxur (33), dimethoate (29.30) and parathion (14.15); and low resistance todiazinon(4.95),triazophos (2.826) and chlorpyrifos (2.35), compared with the SS strain.2. Biochemical properties of AChEs from the DRR strain suggested that the temperature optimum of the DRR AChE was found 36℃. The optimum pH was 8.14. The optimum substrate for AChEs from DRR was ATCh and the optimum concentration was 2mmol/L. These results were all different from the AChE from SS and CRR strains.3. The kinetic parameters for AChEs from the DRR and SS strains showed that the Michaelis constant (K_m) and maximum reaction velocity (Vmax) ratios of the DRR to SS strains were 2.76 and 2.84, respectively, suggesting that the capability of ATCh hydrolyzed by the DRR AChE was higher than the SS AChE. The bimolecular rate constant (k_i) of AChE from DRR was much smaller than that of AChE from SS. The differences in k_i may be mainly attributed to the decreased affinity of these compounds for the DRR AChE, which indicated that the DRR AChE might be qualitatively altered. 4. The cDNA of ace gene encoding AChE from the DRR strains of housefly was cloned using RT-PCR and sequenced. 1971bp fragment was amplified from the DRR cDNA by RT-PCR. The cDNA of the ace gene contained 1821 bp ORF (open reading frame), which encoded a putative protein of 607 amino acid residues. Comparison with the sequence of the SS ace allowed evidencing eight mutations in the DRR ace.Based on the above results, we could conclude that the decreased affinity of the OPs and CBs for AChE from the DRR strain was the main reason conferred the insensitivity to these inhibitors, which was associated with modified catalytic efficiency of the altered AChE; that eight single mutations in the DRR ace could provide specific insecticide resistance; that the higher level and wide resistant spectrum might be achieved by combining several mutations in the same AChE protein.