| As a w orldwide-distributed p est, c otton a phid, Aphis g ossypii G lover, a ttracts 1 ots o f attention. The rapid development of resistance to insecticides has brought difficulty in the field control of this important pest. Acetylcholinesterase (AChE) is a target for organophosphate (OP) and carbamate insecticides. Insecticides resistance due to the insensitivity of acetylcholinesterase to the inhibition by these two groups of insecticides has been found in many insects including cotton aphid. Carboxylesterase (CarE) also plays an important role in organophosphate resistance of cotton aphid. In this paper, comparing studies on AChE and CarE from resistant and susceptible strains have been performed, including their stability, toxicology and sub-cellular distribution.1 Study of stability of crude acetylcholinesterase from cotton aphidThe crude preparation of AChE is usually used in pest resistance study. However, AChE is not stable and easy to breakdown. Studies on the factors affecting the stability of the enzyme would be helpful for getting correct results in the further researches. So, series of experiments were carried out to demonstrate if and how temperature, glycerol and bovine serum albumin (BSA) affect the stability of the enzyme.The results showed that the lower temperature when crude AChE preparation was stored at, the more stable it was. Glycerol could improve its stability effectively when AChE was stored at -20C. But BSA couldn't significantly improve the stability of the enzyme in crude preparations when stored at higher temperature. It was suggested that crude AChE preparations should be stored at -20C and with some glycerol.AChE was also found having the tendency to lose its activity gradually short after it was extracted, and the stability of the crude AChE prepared from different strains, resistant and susceptible, were apparently different. So, the enzyme preparations stored for similar time should be used for comparing study of their activity.2 Comparison of acetylcholinesterase and carboxylesterase from different aphid strainsWith improved caging method of Li fei (2001), four laboratory strains of cotton aphid were set up. A susceptible strain 171B was introduced from Rothamsted Experiment Strains, UK. Three resistant strains were bred from the aphids collected from different districts in China, Nanjing, Beijing and Anyang. With the AChE preparations from these 4 strains, theI50 and Ki of two OP and one carbamate insecticides were tested. Compared with that from 171B, the AChE from Anyang strain was found apparently insensitive to all the insecticides tested. But the sensitivity of AChE from Nanjing and Beijing strains were similar as that from 171B. However, the esterase (total esterase and carboxylesterase) activity increased dramatically in all the three strains. So, it could be deduced that pesticide resistance in Nanjing and Beijing strains only attributed to increased pesticide sequestration or detoxification by esterase, while the resistance in Anyang strain attributed to both increased esterase activity and reduced sensitivity of AChE.The inhibition curve test was also carried out with the same insecticides and cotton aphid strains. The results showed the similar insensitivity of the AChE from different strains. But no flat on the curves was observed as usually expected when different enzymes present. So, it was thought it is not reliable to identify different enzymes with the flat on inhibition curves.3 Subcellular distribution of acetylcholinesterase and carboxylesterase from different aphids strainsWith differential centrifugation, subcellular distribution of AChE and CarE from susceptible strain (171B) and resistance strain (Anyang strain) were studied. Results showed that both AChE and CarE from susceptible strain and resistance strain were differently sub-cellular distributed. AChE activities in resistant strain were mainly distributed in cell nucleus, cell debris and mitochondrion. While AChE activities in susceptible strain were mainly... |
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