P450 gene CYP6a13 is responsible for cross-resistance of insecticides in field populations of Spodoptera frugiperda.

Continuous and long-term use of traditional and new pesticides can result in cross-resistance among pest populations in different fields. Study on the mechanism of cross-resistance and related genes will help resistance management and field pest control. In this study, the pesticide-resistance mechanism in Spodoptera frugiperda (FAW) was studied with field populations in 3 locations of South China. Field FAW populations were highly resistant to traditional insecticides, chlorpyrifos (organophosphate) and deltamethrin (pyrethroid), and had higher levels of cytochrome P450 activity than a non-resistant laboratory strain. Inhibition of P450 activity by piperonyl butoxide significantly increased the sensitivity of resistant FAW in 3 locations to chlorpyrifos, deltamethrin and chlorantraniliprole (amide), a new type of insecticide, suggesting that P450 detoxification is a critical factor for insecticide resistance in field FAW populations. Transcriptomic analysis indicated that 18 P450 genes were upregulated in the field FAW populations collected in 3 regions and in 2 consecutive years, with CYP6a13, the most significantly upregulated one. Knockdown of CYP6a13 messenger RNA by RNA interference resulted in an increased sensitivity to the 3 tested insecticides in the field FAW. Enzyme activity and molecular docking analyses indicated that CYP6a13 enzyme was able to metabolize the 3 tested insecticides and interact with 8 other types of insecticides, confirming that CYP6a13 is a key cross-resistance gene with a wide range of substrates in the field FAW populations across the different regions and can be used as a biomarker and target for management of FAW insecticide resistance in fields.

The TIAR-mediated Nrf2 response to oxidative stress is mediated through the Nrf2 noncoding 3'untranslated region in Spodoptera litura.

Nrf2 is a key regulator in the maintenance of cellular redox balance by regulating the expression of genes related to antioxidative responses and detoxification. Nrf2 protein levels are increased in response to oxidative stress. However, the regulation of the Nrf2 3'UTR on Nrf2 translation is unclear. Here, we report that the translational activity of the 3'UTR is required for Spodoptera litura Nrf2 protein expression. Experiments showed that the 3'UTR translation activity of S. litura Nrf2 was much higher than that of the 5'UTR. RNA interference (RNAi) of the expression of T cell internal antigen-related protein (TIAR), an RNA-binding protein that interacts with the 3'UTR of S. litura Nrf2, resulted in Nrf2 mRNA movement out of translationally active polysomes and a decrease in cellular Nrf2 protein levels. TIAR interacted with poly(A)-binding protein (PABP) and translation initiation factors eIF2-2 and eIF2-3 to enhance Nrf2 translation, indicating that the 3'UTR regulates Nrf2 translation. Diethyl maleate (DEM) treatment increased reactive oxygen species (ROS) in cells and enhanced Nrf2 levels, which had been reduced by cycloheximide (CHX), an inhibitor of de novo protein synthesis; Tiar RNAi increased ROS levels in DEM-treated cells, suggesting TIAR-mediated 3'UTR involvement in Nrf2 translation in response to DEM treatment. Thus, we reveal a posttranscriptional regulation mechanism of Nrf2, in which TIAR binds with the Nrf2 mRNA 3'UTR to enhance Nrf2 translation, facilitating the increase in Nrf2 protein levels in response to oxidative stress.

Knockdown of AMP-activated protein kinase increases the insecticidal efficiency of pymetrozine to Nilaparvata lugens.

Insecticides are the main tools used to control Nilaparvata lugens (Stål), a serious pest of rice in Asia. However, repeated application of insecticides has caused many negative effects. Reducing the amount of insecticide used, while maintaining good pest population control, would be valuable. AMP-activated protein kinase (AMPK), a sensor of cellular energy status, helps to maintain insect energy balance at the cellular and whole-body level. The role of AMPK in insect response to insecticide stimulation is unknown. We studied the functions of AMPK catalytic subunit alpha (NlAMPKα) in the development of N. lugens and in response to pymetrozine, an insecticide used to control insect pests with piercing-sucking mouthparts. A phylogenetic analysis of protein sequences from 12 species in six orders showed that insects have only the AMPKα 2 subtype. RNA interference against NlAMPKα demonstrated that blocking the AMPK pathway led to a decrease in the systemic ATP level and an increase in N. lugens mortality. NlAMPKα responded to the energy stress caused by pymetrozine treatment, which activated downstream energy metabolic pathways to compensate for the energy imbalance. However, the ATP level in pymetrozine- treated nymphs was not increased, suggesting that ATP is consumed more than synthesized. When NlAMPKα expression was reduced in pymetrozine-treated nymphs by RNAi, the ATP level was decreased and the mortality was significantly increased. At day eight post 0.5 g/3 L of pymetrozine and dsNlAMPKα treatment, nymph survival was 29.33%, which was similar to the 27.33% survival of 1 g/3 L pymetrozine-treated nymphs. Addition of dsNlAMPKα can reduce the concentration of pymetrozine used by 50% while providing comparable efficacy. These results indicate that AMPK helps maintain the energy metabolism of N. lugens in response to pymetrozine treatment. Knockdown of NlAMPKα increases the insecticidal efficiency of pymetrozine to N. lugens.

Analyses of microRNAs and transcriptomes in the midgut of Spodoptera litura feeding on Brassica juncea.

Spodoptera litura is a destructive agricultural pest in tropical and subtropical areas. Understanding the molecular mechanisms of S. litura adaptation to its preferred host plants may help identify target genes useful for pest control. We used high-throughput sequencing to characterize the expression patterns of messenger RNAs (mRNAs) and microRNAs (miRNAs) in the midgut of S. litura fed on Brassica juncea for 6 h and 48 h. A total of 108 known and 134 novel miRNAs were identified, 29 miRNAs and 237 mRNAs were differentially expressed at 6 h of B. juncea feeding, 26 miRNAs and 433 mRNAs were differentially expressed at 48 h. For the mRNAs, the up-regulated genes were mostly enriched in detoxification enzymes (cytochrome P450, esterase, glutathione S-transferase, uridine diphosphate-glucuronosyl transferase), while the down-regulated genes were mostly enriched in proteinases and immune-related genes. Furthermore, most detoxification enzymes begin to up-regulate at 6 h, while most digestion and immune-related genes begin to up- or down-regulate at 48 h. Eighteen and 37 differently expressed transcription factors were identified at 6 h and 48 h, which may regulate the functional genes. We acquired 136 and 41 miRNA versus mRNA pairs at 6 h and 48 h, respectively. Some down-regulated and up-regulated miRNAs were predicted to target detoxification enzymes and proteinases, respectively. Real-time quantitative polymerase chain reaction of nine randomly selected miRNAs and 28 genes confirmed the results of RNA-seq. This analyses of miRNA and mRNA transcriptomes provides useful information about the molecular mechanisms of S. litura response to B. juncea.

Inhibition of the glutathione biosynthetic pathway increases phytochemical toxicity to Spodoptera litura and Nilaparvata lugens.

Phytochemicals are toxic to insects, but their insecticidal efficiencies are usually low compared to synthetic insecticides. Understanding the mechanism of insect adaptation to phytochemicals will provide guidance for increasing their efficacy. Reduced glutathione (GSH) is a scavenger of reactive oxygen species (ROS) induced by phytochemicals. However, in insects, the pathway of GSH biosynthesis in response to phytochemicals is unclear. We found that exposure to 0.5% indole-3-methanol (I3C), xanthotoxin, and rotenone (ROT) significantly retarded the growth of Spodoptera litura larvae. The oxidative stress in S. litura larvae exposed to phytochemicals was increased. The up-regulation of glutamate cysteine ligase but not glutathione reductase revealed that the de novo synthesis pathway is responsible for GSH synthesis in phytochemical-treated larvae. Treatment with the inhibitor (BSO) of γ-glutamylcysteine synthetase (gclc), a subunit of glutamate cysteine ligase, resulted in decreases of GSH levels and GST activities, increases of ROS levels in I3C-treated larvae, which finally caused midgut necrosis and larval death. Treatment with BSO or I3C alone did not cause larval death. The addition of GSH could partly reduce the influence of I3C and BSO on S. litura growth. Nilaparvata lugens gclc RNAi confirmed the result of BSO treatment in S. litura. N. lugens gclc RNAi significantly increased the mortality of ROT-sprayed N. lugens, in which ROS levels were significantly increased. All data indicate that gclc is involved in insect response to phytochemical treatment. Treatment with dsgclc will increase the insecticidal efficacy of plant-derived compounds.