Pharmcological Characteristics Of Neonicotinoid Insecticides On The Nicotinic Acetylcholine Receptors Of Target And Non-target Insects

Aphis gossypii Glover (Hemiptera:Aphididae) is one of the important agricultural pests in cotton fields. Nowadays, the research on cotton aphid only focused on the bioassays as well as the molecular cloning of the relevant targets genes because of the limitations of the experimental conditions and technical. So far there is no systematic, detailed research reports about the characteristics and toxicology of the nAChRs subunits of the cotton aphid. 1.In these study, we cloned all nAChRs subunits according to the genome sequence of Aphis gossypii, eight nAChRs subunits have been acquired, included seven a subunits, and one β1 subunits. Among these seven a subunits, α4 and α7 subunit have two alternative splicing variant. And the deduced amino acids were aligned with other nAChRs subunits, all nAChRs subunits of the two insects have six loops(LpA-F) which involved in ligand binding, four transmembrane motifs (TM1-4) forming the ion channel are indicated. Meanwhile two Cys residues in α-type subunits are also included. A phylogenetic tree was constructed based on the amino acid sequences of selected model insect nAChRs. This work contributes to defining the characteristics and the molecular mechanism of the cotton aphid nAChRs subunits, It is important for us to understand the chemical control as well as the prevention of the resistance generation of cotton aphid. Simultaneously, It is important to learn the structure optimization of existing pesticides and the directed molecular design of the new insecticides.2. Neonicotinoid insecticides are selective agonists of nAChRs with nitro (-NO2) or cyano (-CN) group, which have been postulated to contribute directly to their selectivity. Since imidacloprid, the first member of the neonicotinoid class of insecticides to be commercialized, up to now, Sulfoxaflor has been considered by many to be a fourth-generation neonicotinoid, four generation neonicotinoid insecticide was widly used in plant protection in nearly past 30 years. As observed in the representative response, the maximal inward currents (Imax) for imidacloprid, thiamethoxam and sulfoxaflor were only 9.35%,48.9% and 81.3% of that for Acetylcholine. In addition, the calculated EC50 values for thiamethoxam and sulfoxaflor were similar to the values of Acetylcholine, and the values for imidacloprid is 1.76-fold of that for Acetylcholine. The trend EC50values for these three neonicotinoid insecticides is in close agreement with the trend of LC50 values for these three neonicotinoid insecticides, in this study we found that there is significant correlation between EC50 and LC50 among these three neonicotinoid insecticides:imidacloprid, thiamethoxam and sulfoxaflor. However, when we test the inward currents evoked by dinotefuran, at the concentration 0.1 mM, the trace is almost undetectable, and increase the concentration tol mM, the change of the current evoked by dinotefuran is very small, the binding sites for dinotefuran may be not in the subunits Agal.These results showed the group of nitro (-NO2) or cyano (-CN) (only in sulfoxaflor among these four neonicotinoid insecticides)in neonicotinoid insecticides are postulated to contribute directly to their selectivity.3. In the commercial market about 40% registered pesticides are chiral pesticides, in this study, we show the pharmacological response of the dinotefuran and its two enantiomers S-(+)-dinotefuran and R-(-)-dinotefuran. According to the report genome sequence of the Apis mellifera, the nucleotide full-length sequences α1-9 and β1 subunits were obtained. All the a subunits were subcloned into the expression vector, we try to test the pharmacological response of the dinotefuran and its two enantiomers to the nAChRs of Apis mellifera in Xenopus laevis oocytes using two-electrode voltage clamping. In voltage-clamp electrophysiological studies, in all tested a subunits, only AmeIa1/Ratbeta2, Amela3/Ratbeta2, AmeIαa8/Ratbeta2 evoked inward currents were detected in response to applications of acetylcholine, whereas other nAChRs subunits have been not found the inward currents. Both rac-dinetofuran and its two enantiomers didn’t cause significant changes in AmeIα1-β2 and AmeIα3-β2, however, the significant right shift in EC50 to R-(-)-dinotefuran were observed inAmeIα8-2β, this is consistent with the toxicity to adult worker honeybees, In conclusion, we have shown that the enantiomer R-(-)-dinotefuran is lower toxicity to non-target insects than the enantiomer S-(+)-dinotefuran, On the basis of biological activity, ecotoxicity, and environmental behavior, it is likely that the use of pure enantiomer R-(-)-dinotefuran instead of the commercial stereoisomer mix may help to increase the bioactivity and reduce environmental pollution.