Biochemical mechanisms of imidacloprid resistance in Nilaparvata lugens: over-expression of cytochrome P450 CYP6AY1.

Imidacloprid is a key insecticide extensively used for control of Nilaparvata lugens, and its resistance had been reported both in the laboratory selected strains and field populations. A target site mutation Y151S in two nicotinic acetylcholine receptor subunits and enhanced oxidative detoxification have been identified in the laboratory resistant strain, contributing importantly to imidacloprid resistance in N. lugens. To date, however, imidacloprid resistance in field population is primarily attributable to enhanced oxidative detoxification by over-expressed P450 monooxygenases. A resistant strain (Res), originally collected from a field population and continuously selected in laboratory with imidacloprid for more than 40 generations, had 180.8-fold resistance to imidacloprid, compared to a susceptible strain (Sus). Expression of different putative P450 genes at mRNA levels was detected and compared between Res and Sus strains, and six genes were found expressed significantly higher in Res strain than in Sus strain. CYP6AY1 was found to be the most different expressed P450 gene and its mRNA level in Res strain was 17.9 times of that in Sus strain. By expressing in E. coli cells, CYP6AY1 was found to metabolize imidacloprid efficiently with initial velocity calculated of 0.851 ± 0.073 pmol/min/pmol P450. When CYP6AY1 mRNA levels in Res strain was reduced by RNA interference, imidacloprid susceptibility was recovered. In four field populations with different resistance levels, high levels of CYP6AY1 transcript were also found. In vitro and in vivo studies provided evidences that the over-expression of CYP6AY1 was one of the key factors contributing to imidacloprid resistance in the laboratory selected strain Res, which might also be the important mechanism for imidacloprid resistance in field populations, when the target site mutation was not prevalent at present.

Gene knockdown by intro-thoracic injection of double-stranded RNA in the brown planthopper, Nilaparvata lugens.

RNA interference (RNAi) is a powerful strategy for gene function study in insects. Here, we described the development of a RNAi technique by microinjection of double-stranded RNA (dsRNA) in the brown planthopper Nilaparvata lugens. Based on the mortality and RNAi efficiency criteria, the conjunctive between prothorax and mesothorax was selected as the injection site and 50 nl as injection volume. Three genes with different expression patterns were selected to evaluate the RNAi efficiency. A comparable 40% decrease of gene expression was observed at the 4th day after injection for the ubiquitously expressed calreticulin and the gut specific cathepsin-B genes, but only 25% decrease at the 5th day for the central nervous system specific Nlbeta2 gene. Double injection could increase the RNAi efficiency, such as from 25% to 53% for Nlbeta2 gene. The gene knockdown technique developed in this study will be an essential post-genomic tool for further investigations in N. lugens.

Native subunit composition of two insect nicotinic receptor subtypes with differing affinities for the insecticide imidacloprid.

Neonicotinoid insecticides, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) and are used extensively to control a variety of insect pest species. The brown planthopper (Nilaparvata lugens), an insect pest of rice crops throughout Asia, is an important target species for control with neonicotinoid insecticides such as imidacloprid. Studies with nAChRs purified from N. lugens have identified two [(3)H]imidacloprid binding sites with different affinities (K(d) = 3.5 +/- 0.6 pM and 1.5 +/- 0.2 nM). Co-immunoprecipitation studies with native preparations of N. lugens nAChRs, using subunit-selective antisera, have demonstrated the co-assembly of Nlalpha1, Nlalpha2 and Nlbeta1 subunits into one receptor complex and of Nlalpha3, Nlalpha8 and Nlbeta1 into another. Immunodepletion of Nlalpha1 or Nlalpha2 subunits resulted in the selective loss of the lower affinity imidacloprid binding site, whereas immunodepletion of Nlalpha3 or Nlalpha8 caused the selective loss of the high-affinity site. Immunodepletion of Nlbeta1 resulted in a complete absence of specific imidacloprid binding. In contrast, immunodepletion with antibodies selective for other N. lugens nAChR subunits (Nlalpha4, Nlalpha6, Nlalpha7 and Nlbeta2) had no significant effect on imidacloprid binding. Taken together, these data suggest that nAChRs containing Nlalpha1, Nlalpha2 and Nlbeta1 constitute the lower affinity binding site, whereas nAChRs containing Nlalpha3, Nlalpha8 and Nlbeta1 constitute the higher affinity binding site for imidacloprid in N. lugens.

Expression and purification of functional human anthrax toxin receptor (ATR/TEM8) binding domain from Escherichia coli.

Anthrax is caused by the gram-positive, spore-forming bacterium, Bacillus anthracis. Anthrax receptors play a crucial role in the pathogenesis of the anthrax disease. Anthrax toxin receptor ATR/TEM8 VWA domain is responsible for the binding of protective antigen (PA) of B. anthracis, and thus an attractive target for structure-based drug therapies. However, the production of soluble and functional ATR/TEM8 VWA domain currently requires the use of mammalian expression systems. In this work, we expressed the ATR/TEM8 VWA domain as a fusion protein in Escherichia coli. Recombinant ATR/TEM8 VWA domain has been purified to homogeneity, and its identity has been verified by both N-terminal protein microsequencing and mass spectrometry. The purified ATR/TEM8 VWA domain exhibits very high affinity to PA based on BIAcore assay. Moreover, like the domain expressed in mammalian system, the bacterially expressed ATR/TEM8 VWA domain can block cytotoxicity induced by anthrax toxins, suggesting that the bacterially expressed ATR/TEM8 VWA domain is properly folded and fully functional.