Abstract
Spodoptera litura is a destructive agricultural pest and has evolved resistance to multiple insecticides, especially pyrethroids. At present, the resistance mechanism to pyrethroids remains unclear. Four field-collected populations, namely CZ, LF, NJ and JD, were identified to have high resistance to pyrethroids comparing to pyrethroid-susceptible population (GX), with resistant ratio ranging from 11.5- to 9123.5-fold. To characterize pyrethroid resistance mechanism, the transcriptomes between two pyrethroid-resistant (LF and NJ) and a pyrethroid-susceptible (GX) populations were compared by RNA-sequencing. Results showed that multiple differentially expressed genes were enriched in metabolism-related GO terms and KEGG pathways. 35 up-regulated metabolism-related unigenes were selected to verify by qRT-PCR and 15 unigenes, including 4 cytochrome P450s (P450s), 5 glutathione S-transferase (GSTs), 1 UDP-glycosyltransferase (UGT), 4 carboxylesterases (COEs) and 1 and ATP-binding cassette transporters (ABC), were all up-regulated in the four pyrethroid-resistant populations. The expression levels of CYP3 and GST3, which were annotated as CYP6A13 and GSTE1, respectively, showed positive correlation with their pyrethroid resistance levels among the four pyrethroid-resistant populations. While the expression levels of CYP5, CYP12, COE4 and ABC5 showed good correlation with their pyrethroid resistance levels in at least three populations. UGT5 had the highest expression level among the tested UGT genes in the four pyrethroid-resistant populations. RNAi mediated silencing of CYP6 increased the cumulative mortality treated by beta cypermethrin and cyhalothrin significantly, while silencing of GST3 increased the cumulative mortality treated by fenvalerate significantly. CYP3, CYP5, CYP6, CYP12, GST3, COE4, UGT5 and ABC5 play important roles in pyrethroid resistance among the four pyrethroid-resistant populations. Our work provides a valuable clue for further study of pyrethroid resistance mechanisms in S. litura.
Highlights
Spodoptera litura is a destructive agricultural pest and has evolved resistance to multiple insecticides, especially pyrethroids
Results showed that multiple differentially expressed genes were enriched in metabolismrelated Gene Ontology (GO) terms and KyotoEncyclopedia of Genes and Genomes (KEGG) pathways. 35 up-regulated metabolism-related genes were screened to verify by qRT-PCR
Metabolism-based resistance to pyrethroids was in a serious situation in S. litura and over-expressed P450s were the major reason for pyrethroid resistance based on the synergism experiments and enrichment analysis of differentially expressed genes (DEGs)
Summary
Insect culture and bioassayGX population of S. litura was kindly provided by Guangxi Tianyuan Biochemistry Corp., Ltd and served as susceptible population. LF, NJ, CZ and JD populations were collected from Langfang, Nanjing, Changzhou, and Shanghai, respectively. They had been reared on artificial diet in the laboratory without exposure to any insecticide for 3 years. Insecticides of pyrethroid (fenvalerate, beta cypermethrin and cyhalothrin), organophosphorus (phoxim, profenofos and chlorpyrifos), carbamates (methomyl), diamide (chlorantraniliprole and cyantraniliprole), chloronicotinyl (imidacloprid) and emamectin benzoate were used for bioassay. PBO, DEF, and DEM were dissolved in acetone and applied 1 h before insecticide treatment at 10 μg larva-1 (synergist at this dosage had no effects on larval mortalities), respectively, and pyrethroid insecticides were applied at the tenth of LD50. Mortality was checked after exposure to fenvalerate, beta cypermethrin, cyhalothrin, phoxim, profenofos, chlorpyrifos, methomyl, imidacloprid and emamectin benzoate for 48 h, and to chlorantraniliprole and cyantraniliprole for 72 h [38]
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