Detoxification of insecticides, allechemicals and heavy metals by glutathione S-transferase SIGSTE1 in the gut of Spodoptera litura

Insect glutathione S-transferases （GSTs） play important roles in detoxifying toxic compounds and eliminating oxidative stress caused by these compounds. In this study, detoxification activity of the epsilon GST S1GSTE1 in Spodoptera litura was analyzed for several insecticides and heavy metals. S1GSTE1 was significantly up-regulated by chlor- pyrifos and xanthotoxin in the midgut of S. litura. The recombinant S1GSTE1 had Vmax （reaction rate of the enzyme saturated with the substrate） and Km （michaelis constant and equals to the substrate concentration at half of the maximum reaction rate of the enzyme） values of 27.95 ± 0.88μmol/min/mg and 0.87 ± 0.028 mmol/L for glutathione, respec- tively, and Vmax and Km values of 22.96 ± 0,78μmol/min/mg and 0.83 ± 0.106 mmol/L for 1-chloro-2,4-dinitrobenzene, respectively. In vitro enzyme indirect activity assay showed that the recombinant S1GSTE 1 possessed high binding activities to the insecticides chlor- pyrifos, deltamethrin, malathion, phoxim and dichloro-diphenyl-trichloroethane （DDT）. S1GSTE1 showed higher binding activity to toxic heavy metals cadmium, chromium and lead than copper and zinc that are required for insect normal growth. Western blot analysis showed that S1GSTE1 was induced in the gut of larvae fed with chlorpyrifos or cadmium. S1GSTE1 also showed high peroxidase activity. All the results together indicate that S1G- STE1 may play an important role in the gut orS. litura to protect the insect from the toxic effects of these compounds and heavy metals.

Expression, subcellular localization and protein-protein interaction of four isoforms of EcR/USP in the common cutworm

Ecdysone receptor （EcR） and ultraspiracle （USP） form heterodimers to mediate ecdysteroid signaling during molting and metamorphosis. Various EcR/USP heterodimers have been reported. However, it is unclear what kind of EcR/USP combination is adopted by lepidopteran insects during the larval-pupal metamorphosis and whether the EcR/USP heterodimer varies among different tissues. To address these questions, two isoforms of each EcR and USP were cloned from the common cutworm, their messenger RNA expression patterns were examined by real-time quantitative polymerase chain reaction in different tissues during the larval-pupal metamorphosis and in the midgut in response to hormonal induction. Furthermore, their subcellular localization and protein-protein interaction were explored by transient expression and far-western blotting, respectively. All the four genes were significantly up-regulated in prepuae and/or pupae. The expression profiles of EcRB1 and USP1 were nearly identical to each other in the epidermis, fat body and midgut, and a similar situation also applied to EcRA and USP2. The three genes responded to 20-hydroxyecdysone （20E） induction except for USP2, and USP1 could be up-regulated by both 20E and juvenile hormone. The four proteins mainly localized in the nucleus and the nuclear localization was promoted by 20E. The protein-protein interaction between each EcR and USP was found in vitro. These results suggest that two types of EcR/USP heterodimer （EeRA/USP2 and EcRB 1/USP1） may exist simultaneously in the common cutworm, and the latter should play more important roles during the larval-pupal metamorphosis. In addition, the types of EcR/USP heterodimer do not vary in the tissues which undergo histolysis and regeneration during metamorphosis.