Functional diversification of three delta-class glutathione S-transferases involved in development and detoxification in Tribolium castaneum

Glutathione S-transferases (GSTs) are members of a multifunctional enzyme superfamily. Forty-one GSTs have been identified in Tribolium castaneum; however, none of the 41 GSTs has been functionally characterized. Here, three delta-class GSTs, TcGSTd1, TcGSTd2 and TcGSTd3, of T. castaneum were successfully cloned and expressed in Escherichia coli. All of the studied GSTs catalysed the conjugation of reduced glutathione with 1-chloro-2,4-dinitrobenzene. Insecticide treatment showed that the expression levels of TcGSTd3 and TcGSTd2 were significantly increased after exposure to phoxim and lambda-cyhalothrin, whereas TcGSTd1 was slightly upregulated only in response to phoxim. A disc diffusion assay showed that overexpression of TcGSTD3, but not TcGSTD1 or TcGSTD2, in E. coli increased resistance to paraquat-induced oxidative stress. RNA interference knockdown of TcGSTd1 caused metamorphosis deficiencies and reduced fecundity by regulating insulin/target-of-rapamycin signalling pathway-mediated ecdysteroid biosynthesis, and knockdown of TcGSTd3 led to reduced fertility and a decreased hatch rate of the offspring, probably caused by the reduced antioxidative activity in the reproductive organs. These results indicate that TcGSTd3 and TcGSTd2 may play vital roles in cellular detoxification, whereas TcGSTd1 may play essential roles in normal development of T. castaneum. These delta-class GSTs in T. castaneum have obtained different functions during the evolution.