The Mode Of Action Of A Nitroconjugated Neonicotinoid And The Study Of Its Functional Target

With the development and application of imidacloprid, which developed by German Bayer and Japanese special pesticide companies, neurotoxicants acting on insect nAChRs has become to the focus of the development and research in insecticides. Neonicotinoid insecticides, such as imidacloprid, are selective agonists of insect nAChRs and are used extensively in areas of crop protection and animal health care to control a variety of insect pest species. Many researchers transform the functional groups and study structure-activity relationship of these compounds. Accordingly, they have developed a series of highly efficient neonicotinoids, as acetamiprid, nitenpyram, and thiamethoxam, and so on.The-NO2or-CN group in all commercial neonicotinoids is in trans-configuration, just like imidacloprid and clothianidin. Recently, as a molecular design strategy to keep nitro or cyno in cis-position, some potential neonicotinoids with fused heterocycles or bulky groups fixed cis-configuration have been synthesized and show insecticidal activity against a wide range of insects (Tian et al.,2007; Shao et al.,2008,2009). In this paper, a typical nitroconjugated neonicotinoid, cycloxaprid, was selected to study its pharmacological properties and action mode.1. The mode of action of a nitroconjugated neonicotinoid IPPA152201To understand the action mode of these nitroconjugated neonicotinoids, a representative member IPPA152201was chosen to perform toxicity and pharmacology studies. IPPA152201showed a comparable toxicity with imidacloprid against Nilaparvata lugens in a susceptible strain and had no significant cross-resistance in an imidacloprid resistant strain. IPPA152201showed good efficacies on the isolated cockroach neurons and the evoked responses by IPPA152201could be blocked by the typical nAChRs antagonists methyllycaconitine citrate (MLA) and dihydro-b-erythroidine (DHβE). The efficacy of IPPA152201on hybrid receptors Nlα1/β2in Xenopus oocytes and response inhibition by ML A and DHβE were also observed. These data demonstrate that IPPA152201acts on insect nAChRs as an agonist.In addition, the influence of a N1α1mutation (Y151S), which has been linked to the lab-generated neonicotinoid resistance in N. lugens, has been examined. Compared to the wildtype Nlα1/(32, this mutation reduced/max for IPPA152201to63.2%and caused a1.5-fold increase in EC50, which is much smaller than the effects on imidacloprid. The high insecticidal activity and little influence by Y151S mutation make IPPA152201to be a potential insecticide to manage N. lugens.2..Molecular cloning of nicotinic acetylcholine receptor from Americana periplanetaUsing RT-PCR with general Primers designed on insect nAChRs subunits and RACE techniques, we identified two nAChRs subunits in A. periplaneta. These subunits show high identities to other insects nAChRs subunits, all two subunits display the characteristics of nAChRs a subunits properties. These proteins include all typical properties of nAChRs subunits, such as the extracellular N-terminal region with conserver residues within loops A,B and C which are involved in ligand binding, the cys-loop consisting of two disulphide bond-forming cysteines separated by13amino acid residues, four well-conserved transmembrane regions(TM1-4) and a variable region between TM3and TM4. Based on the identities to insect nAChRs subunits, these proteins are denominated as Paa6and Paa7.3. The putative target subunit of Cycloxaprid on insect nAChRsElectrophysiology studies showed that, cycloxaprid was acting on subunit al of nAChRs, as the same of imidacloprid and the other neonicotinoid. But it is not reported that whether the compound was in the roles of subunit a6(potential type of homo-oligomers). One main aim of this paper is to establish a method to determine the putative target of new compounds with RNA interference and bioassay. With interference of a6subunit in Americana periplaneta, we do bioassay and compare the result with that of the untreatment control. In these experiments, compounds included cycloxaprid, imidacloprid and spinosad. The results showed the relationship between compounds and the treated gene.During the bioassay in Americana periplaneta, we used a novel feeding method, compared to the topical application method, which was found more suitable for preparations those are soluble in water easily. In RNAi experiments, the injection and feeding method were tested and Real-time quantitative PCR was used to analyze the expression level of target genes after RNAi. The results showed that two methods both obtained the best efficiencies in the4th day after siRNA treatment and the highest suppressing efficiencies were about70%.Three days after siRNA treatment, bioassay was performed. To spinosad, mortality of the interference group is only about a third of the normal group, which determined that a6subunit is really the functional target of spinosad, as reported. To cycloxaprid, the result shows that mortality of the interference group is lower than the normal group, so we conclude that cycloxaprid is also acting on a6subunit of nAChRs. Otherwise, the lower expression of subunit a6has little effect on imidacloprid roles.4. Phenomenon of Cycloxaprid act on Americana periplanetaDuring the bioassay, we treat cockroaches with different concentrations of cycloxaprid, which make their abdomens expand. After dissecting, swollen foregut was observed. Because of this, cockroaches could not feed and digest, which makes them die about three days after cycloxaprid treatment. The phenomenon was the first reported here. It is suggested that cycloxaprid not only acts on insect nervous system, but may also have other target, such as one in digestive system.