Functional Analysis of Insecticide Inhibition and Metabolism of Six Glutathione S-Transferases in the Rice Stem Borer, Chilo suppressalis.

The glutathione S-transferases (GSTs) are important detoxifying enzymes in insects. Our previous studies found that the susceptibility of Chilo suppressalis to abamectin was significantly increased when the CsGST activity was inhibited by glutathione (GSH) depletory. In this study, the potential detoxification mechanisms of CsGSTs to abamectin were explored. Six CsGSTs of C. suppressalis were expressed in vitro. Enzymatic kinetic parameters including Km and Vmax of recombinant CsGSTs were determined, and results showed that all of the six CsGSTs were catalytically active and displaying glutathione transferase activity. Insecticide inhibitions revealed that a low concentration of abamectin could effectively inhibit the activities of CsGSTs including CsGSTd1, CsGSTe4, CsGSTo2, CsGSTs3, and CsGSTu1. However, the in vitro metabolism assay found that the six CsGSTs could not metabolize abamectin directly. Additionally, the glutathione transferase activity of CsGSTs in C. suppressalis was significantly increased post-treatment with abamectin. Comprehensive analysis of the results in present and our previous studies demonstrated that CsGSTs play an important role in detoxification of abamectin by catalyzing the conjugation of GSH to abamectin in C. suppressalis, and the high binding affinities of CsGSTd1, CsGSTe4, CsGSTo2, CsGSTs3, and CsGSTu1 with abamectin might also suggest the involvement of CsGSTs in detoxification of abamectin via the noncatalytic passive binding and sequestration instead of direct metabolism. These studies are helpful to better understand the detoxification mechanisms of GSTs in insects.

Characterization of Glutamate-Gated Chloride Channel in Tribolium castaneum.

The glutamate-gated chloride channels (GluCls) play essential roles in signal transduction by regulating fast inhibitory synaptic transmission in the nervous system of invertebrates. While there is only one GluCl subunit in the insect, the diversity of insect GluCls is broadened by alternative splicing. In the present study, three TcGluCl variant genes were cloned from the red flour beetle Tribolium castaneum. Analysis of the characteristics of TcGluCls including sequence features, genomic structures, and alternative splicing revealed that TcGluCls had the typical structural features of GluCls and showed high homologies with the GluCls from other insect orders. The TcGluCl-encoding gene consists of nine exons and three variants (TcGluCl-3a, TcGluCl-3b, and TcGluCl-3c) were generated by the alternative splicing of exon 3, which was a highly conserved alternative splicing site in insect GluCls. Homology modeling of TcGluCl-3a showed that the exon 3 coding protein located at the N-terminal extracellular domain, and there were no steric clashes encountered between the exon 3 coding region and ivermectin/glutamate binding pocket, which indicated that the alternative splicing of exon 3 might have no impact on the binding of GluCls to glutamate and insecticide. In addition to the head tissue, TcGluCl-3a and TcGluCl-3c also had high expressions in the ovary and testis of T. castaneum, whereas TcGluCl-3b showed high expression in the midgut, suggesting the diverse physiological functions of TcGluCl variants in T. castaneum. The total TcGluCl and three variants showed the highest expression levels in the early stage larvae. The expressions of TcGluCl, TcGluCl-3b, and TcGluCl-3c were significantly increased from the late-stage larvae to the early stage pupae and indicated that the TcGluCl might be involved in the growth and development of T. castaneum. These results are helpful to further understand the molecular characteristics of insect GluCls and provide foundations for studying the specific function of the GluCl variant.

The insecticidal activity and action mode of an imidacloprid analogue, 1-(3-pyridylmethyl)-2-nitroimino-imidazolidine.

Neonicotinoids, such as imidacloprid, are key insecticides extensively used for control of Nilaparvata lugens. However, imidacloprid resistance has been reported in many Asian countries in recent years. To understand the roles of the chlorine atom of pyridyl group on insecticidal activity and resistance, the atom was removed to generate an imidacloprid analogue DC-Imi (DesChlorine Imidacloprid). DC-Imi showed significantly higher toxicity than imidacloprid in the susceptible strain of N. lugens, but had medium level cross-resistance in an imidacloprid-resistant strain. In Xenopus oocyte expressed nicotinic acetylcholine receptors (nAChRs) Nlα1/rß2, the inward currents evoked by DC-Imi were detected and could be blocked by typical nAChRs antagonist dihydro-ß-erythroidine (DHßE), which demonstrated that DC-Imi acted as an agonist on insect nAChRs. The efficacy of DC-Imi on Nlα1/rß2 was 1.8-fold higher than that of imidacloprid. In addition, the influence of an imidacloprid resistance associated mutation (Y151S) on agonist potencies was evaluated. Compared with the wild-type receptor, the mutation reduced maximal inward current of DC-Imi to 55.6% and increased half maximal effective concentration (EC50 ) to 3.53-fold. Compared with imidacloprid (increasing EC50 to 2.38-fold of wild-type receptor), Y151S mutation decreased DC-Imi potency more significantly. The results indicated that the selective and possibly high toxicities could be achieved through the modification of 6-chloro-3-pyridyl group in imidacloprid and other neonicotinoids.