Synergism of resistance to phosalone and comparison of kinetic properties of acetylcholinesterase from four field populations and a susceptible strain of Colorado potato beetle

The susceptibility to phosalone and biochemical characteristics of acetylcholinesterase (AChE) were compared between susceptible (SS) strain and four field populations of Colorado potato beetle (CPB) collected from commercial potato fields of Hamedan Province in west of Iran. Bioassays involving topical application of phosalone to fourth instars revealed up to 252 fold resistance in field populations compared with the SS strain. Synergism studies showed that although esterase and/or glutathione S-transferase metabolic pathways were present and active against phosalone, they were not selected for and did not have a major role in resistance. It is likely that piperonyl butoxide (PBO) reduced phosalone toxicity by inhibiting bio-activation of phosalone. The affinity (K_m) and hydrolyzing efficiency (V_max) of AChE to selected substrates, namely, acetylthiocholine iodide (ATC), propionylthiocholine iodide (PTC), and butyrylthiocholine iodide (BTC) were examined. AChE inhibition by higher substrate concentration was evident only in the SS strain. In resistant field populations, Aliabad (Aa), Bahar (B) and Dehpiaz (Dp), substrate inhibition at higher concentrations was not seen. There was no definite optimal concentration found for any of the substrates examined. When ATC, PTC, and BTC were used as substrate, the reaction rates of AChE from Yengijeh (Yg) population increased as the concentration of all three substrates were increased, but were almost constant at concentration of ATC ? 3.98, PTC ? 2.8, and BTC ? 5 mM. The susceptible form of AChE had the most efficient ATC hydrolysis but very low BTC hydrolysis activity. In contrast, AChEs from field populations elicited relatively reduced ATC hydrolysis, but relatively increased BTC hydrolysis. The in vitro inhibition potency of some organophosphates (OPs), on AChEs of the field populations and SS strain was determined. The rank order from the most potent inhibitor to the least as determined by their bimolecular reaction constants (K_i) was ethyl paraoxon > diazoxon > methyl paraoxon for AChE from Aa, B, Dp, and Yg populations, respectively, whereas the rank order for the susceptible strain was methyl paraoxon > ethyl paraoxon > diazoxon.