Glutathione S-transferase genes in the epsilon group were reported to function in insecticide resistance. SlGSTE12 was validated to be overexpressed in pyrethroid-and organophosphate-resistant populations of Spodoptera litura compared to a susceptible population. A functional study of heterologously expressed SlGSTE12 showed that K m and V max for 1-chloro-2,4dinitrobenzene (CDNB) conjugating activity were 0.70 ± 0.18 mmol L −1 and 90.6 ± 9.4 nmol mg −1 min −1 , respectively. β-Cypermethrin and cyhalothrin showed much weaker inhibition of SlGSTE12 activity to CDNB conjugation than fenvalerate, chlorpyrifos, and phoxim. Ultrahigh-performance liquid chromatography analysis showed that SlGSTE12 had significant metabolism activity to fenvalerate and phoxim both in vitro and in Escherichia coli, especially to chlorpyrifos, and slight metabolism activity toward cyhalothrin only in vitro. Silencing of SlGSTE12 by RNAi increased the mortality to fenvalerate, cyhalothrin, and chlorpyrifos significantly. SlGSTE12 also had a significant antioxidant ability against cumene hydroperoxide. Our study suggested that SlGSTE12 could metabolize phoxim, fenvalerate, cyhalothrin, and especially chlorpyrifos. SlGSTE12 might also participate in pyrethroid and organophosphate resistance by antioxidant activity.

BACKGROUND: Spodoptera litura is an important agricultural pest and has developed serious resistance to multiple insecticides. The resistance level to several insecticides is reported to be unstable, but the mechanism is less reported.RESULTS: Chlorpyrifos and phoxim resistance level in a field-collected population of S. litura declined continuously from the first to the tenth generation and remained stable at the 11th and 12th generations without insecticide exposure. Synergist experiment showed that diethyl maleate and piperonyl butoxide significantly increased mortality to chlorpyrifos and phoxim in the first and sixth generations, but not in the 12th generation. The expression of 31 identified glutathione S-transferase (GST) genes in the third-instar larvae of S. litura in the first, sixth and 12th generations was determined, and eight genes were seen to decrease significantly in the sixth and 12th generations compared with the first generation. SlGSTe9 was selected for further functional study as it had higher abundance and significantly higher expression in the chlorpyrifos-resistant population than in the susceptible population. The recombinant protein of SlGSTE9 showed metabolism activity to chlorpyrifos in vitro and in Escherichia coli, but not to phoxim. Silencing of SlGSTe9 increased the cumulative mortality to chlorpyrifos significantly. SlGSTE9 also showed antioxidant activity to cumene hydroperoxide. CONCLUSION: Our results suggest that SlGSTe9 is directly involved in chlorpyrifos resistance stability, but not in phoxim. SlGSTE9 may also participate in insecticides resistance by relieving the oxidase stress induced by insecticides.

Glutathione S‐transferases (GSTs) were reported to participate in insecticide resistance by metabolic and antioxidant activities. In our previous study, an ε class gene of GSTs, SlGSTe8 in Spodoptera litura, was screened out to be upregulated in a population resistant to pyrethroids and organophosphates. SlGSTe8 was highly expressed in the larvae stage, and the digestive tissue, foregut, midgut and hindgut, while the relative expression level was low in the pupae stage and other tissues. To further explore its role in the resistance to pyrethroids and organophosphates, the metabolic activity to insecticides by its recombinant protein was determined by Ultra Performance Liquid Chromatography, and its antioxidant enzyme activity was evaluated by disc diffusion assay. The recombinant protein showed significant metabolic activity to phoxim and chlorpyrifos, but not to fenvalerate, cyhalothrin or β‐cypermethrin. After incubation, the depletion rate of chlorpyrifos is 85.3%, higher than that of phoxim (17.5%). Also, the inhibition zone around filter discs decreased significantly after exposure to cumene hydroperoxide in recombinant plasmid than vector only, suggesting significant antioxidant activity of SlGSTE8. Further modelling and docking analysis indicated that the 3D structure of SlGSTE8 was well shaped for phoxim and chlorpyrifos, with the binding energy −5.58 and −5.15 kcal/mol, respectively. Our work provides evidence that SlGSTe8 in S. litura plays important roles in phoxim and chlorpyrifos resistance.

Ribavirin is an antiviral drug showing high and delayed toxicity to the destructive agricultural pest Spodoptera litura. Larvae fed with artificial diets containing ribavirin could not molt successfully and showed abnormal phenotypes, including cuticle melanization and heavy wrinkle of the newly formed procuticle. RNA-Seq analysis suggested that ribavirin has great negative influence on cuticle. Quantitative real-time-polymerase chain reaction results indicated that ribavirin treatment decreased the expression of key genes in juvenile hormone (JH) biosynthesis (CYP15C1 and JH acid methyltransferase) and most cuticle protein genes, whereas the genes in melanin biosynthesis and bursicon genes were upregulated by ribavirin treatment. These results coincided with the decreased titer of JH I, JH II, and JH III determined by enzyme-linked immunosorbent assay, the much thinner procuticle layer exhibited by histopathological examination, and the cuticle melanization after ribavirin treatment. These results provided a valuable theoretical basis for the creation of green insecticides targeting JH and the development of new insecticide derivatives from 1,2,4-triazole.