| As a worldwide distribute pest, Cotton aphid, Aphis gossypii Glover, attracts lots of attentions. The rapid development of resistance to insecticides has brought the difficulty in the field control of this important pest. However, little has been understood about its neuro-targets of insecticides, such as acetylcholinesterase (AChE, EC3.1.1.7), nicotinic acetylcholine receptor (nAChR) and sodium channel. In this paper, systematic studies on the neuro-targets of conventional insecticides have been perfonned including protein purification, toxicological study, gene clone, molecular phylogenetic analysis and molecular monitoring of pesticide resistance.1 Caging method for rearing cotton aphidTo obtain better results in experiments and reduce the labor consumption, a new method for rearing cotton aphid was developed. Transparent film of A4 size and gauze were used to make cages, with which cotton aphid could be reared on the cotton plants cultivated separately in the lab. Compared with agar-leaves and Blackman box method, the new cotton seedling caging method had some advantages: less time and labor consuming, no limitation of seasons, and better living conditions for aphids on living cotton plants.To see if an alternative could be used, the influence of host plants, cotton, hibiscus and pumpkin, on cotton aphids was studied. The results showed that the acetylcholinesterase and carboxylesterase varied with the host plants of the aphids, which also affected the insecticide tolerance level to some extent. Reared on cotton, the aphids had the AChE with highest affinity to ATChl. On hibiscus, the aphids had the lowest esterase activity and the lowest tolerance to organophosphate insecticides.2 Selection of strains with different resistance levelWith the cotton seedling caging method, laboratory strains of cotton aphids with different resistance level were set up. A susceptible strain 171B was introduced from Rothamsted Experiment Station, UK. Five resistant strains were selected from the aphids collected from different districts in China, Nanjing, Taishan, Laiyan, Anyan and Beijing.Leaf-dip bioassay revealed that Anyan strain was the highest while Taishan strain the lowest resistant to conventional organophosphorus (OP) and carbamate pesticides, Methamidophos, Monocrotophos, Pirimicarb and Thiodicarb. Generally, the resistant levels of five resistant strains were arranged as: Anyan>Beijing>Laiyan>Nanjing and Taishan. Compared with that from 171B, the sensitivity of acetylcholinesterase (AChE) from Laiyan, Anyan and Beijing strain apparently decreased. The AChE of Nanjing and Taishan strain were still relatively sensitive. The apparent decrease of the Km value indicated that AChE from Anyan and Beijing strain had alternated. Esterase (total esterase and carboxylesterase) activity increased dramatically in different resistant strains. The Km value of carboxylesterase from Anyan strain reached 2460.4umol L-1 and that from Beijing strain decreased to 84.4umol L-1. It could be deduced that pesticide resistance in Nanjing and Taishan strain mainly attributed to increased pesticide sequestration or detoxification, while the pesticides resistance in Laiyan, Anyan and Beijing strain attributed to both increased pesticides sequestration or detoxification and reduced sensitivity of AChE. 3 Studies on acetylcholinesterase from cotton aphid 3.1 Protein purificationA simple and effective method was set up to purify acetylcholinesterase from the cotton aphid, Aphis gossypii Glover. The procedure involved filtration on a sephadex G-25 column, separation with sephadex G-200 and procainamide affinity column. AChE from both susceptible and resistant strains were purified to a single band as resolved on denaturing polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity increased by 35,100 and 33,680-fold with a yield of 30.3% and 29.8%, respectively. The molecular mass of the purified AChE was about 63,500 Dalton as determined by SDS-PAGE. However, three bands resolved on PAGE gel electrophoresis, leadi... |
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