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Vitellin (Vn) homeostasis is central to the fecundity of oviparous insects. Most studies have focused on the synthesis and transportation of Vn as a building block for developing eggs during vitellogenesis; however, less is known about how the utilization of this nutrient reserve affects embryonic development. Here, we show that the single ortholog of the knirps and knirps‐like nuclear receptors, KNRL, negatively regulates Vn breakdown by suppressing the expression of hydrolase genes in the brown planthopper, Nilaparvata lugens. KNRL was highly expressed in the ovary of adult females, and knockdown of KNRL by RNA interference resulted in the acceleration of Vn breakdown and the inhibition of embryonic development. Transcriptome sequencing analysis revealed that numerous hydrolase genes, including cathepsins and trypsins were up‐regulated after KNRL knockdown. At least eight of the nine significantly enriched Gene Ontology terms for the up‐regulated genes were in proteolysis‐related categories. The expression levels of five selected trypsin genes and the enzymatic activities of trypsin in the embryos were significantly increased after KNRL knockdown. Moreover, trypsin injection prolonged egg duration, delayed embryonic development, accelerated Vn breakdown and severely reduced egg hatchability, a pattern similar to that observed in KNRL‐silenced N. lugens. These observations suggest that KNRL controls Vn breakdown in embryos via the transcriptional inhibition of hydrolases. Generally, this study provides a foundation for understanding how embryo nutrient reserves are mobilized during embryogenesis and identifies several genes and pathways that may prove valuable targets for pest control. [ABSTRACT FROM AUTHOR]

Adipokinetic hormones (AKHs) are well known to mobilize lipids and carbohydrates for energy-consuming activities in insects. These neuropeptides exert their functions by interacting with AKH receptors (AKHRs) located on the plasma membrane of fat body cells, which regulates energy mobilization by stimulating lipolysis of triacylglycerols (TAG) to diacylglycerols (DAG) and conversion of glycogen into trehalose. Here, we investigated the roles of AKH/AKHR signaling system in trehalose metabolism and vitellogenesis during female reproduction in the brown planthopper, Nilaparvata lugens. Knockdown of AKHR expression by RNA interference (RNAi) resulted in a decrease of the circulating trehalose in hemolymph and significantly increased levels of two trehalases in fat bodies, indicating that the modulation of hemolymph trehalose levels by AKHR may be mediated by regulating trehalose degradation. In addition, adult females that had been injected with double-stranded RNA (dsRNA) for AKHR exhibited delayed oocyte maturation, prolonged pre-oviposition period, as well as decline in egg number and reduction in fecundity. Considering that these phenotypes resulting from AKHR silencing are similar to those of vitellogenin receptor (VgR) RNAi, we further analyzed a possible connection between AKHR and vitellogenesis. Knockdown of AKHR showed no effects on the Vg synthesis in fat bodies, whereas it significantly reduced the levels of VgR in ovaries. With RNAi-females, we observed an increase of Vg accumulation in hemolymph and a decrease of Vg deposition in ovaries. Moreover, the decrease in VgR expression and Vg incorporation by developing oocytes could be partially rescued by injection of trehalose into AKHR RNAi females. The present study has implicated trehalose in the AKH/AKHR signaling-mediated control of reproduction and provided new insight into mechanisms of AKH/AKHR regulation of trehalose metabolism in insect vitellogenesis, oocyte maturation and fecundity. [ABSTRACT FROM AUTHOR]

Lipid storage must be efficiently mobilized to sustain the energy demands during processes of exercise or starvation. In insects, adipokinetic hormone (AKH) and brummer lipase are well-known regulators of lipid mobilization. We recently demonstrated that brummer-dependent lipolysis regulates starvation resistance in the brown planthopper, Nilaparvata lugens , one of the most destructive rice pests. The present work investigated the roles of the AKH signaling system in lipid mobilization during the starvation process in N. lugens. Nl AKHR is a typical G protein-coupled receptor (GPCR) and possesses high structure and sequence similarity to other insect AKHRs. Spatial and developmental expression profiles suggested that Nl AKH is released from the corpora cardiaca to activate Nl AKHR mainly expressed in the fat body. Starvation significantly induced the expression of NlAKH and NlAKHR , indicating a potential role of the AKH signaling system in starvation resistance. To reveal the functions of the AKH signaling system, a double-stranded RNA (dsRNA)-mediated knockdown of NlAKHR and Nl AKH peptide injection was performed. The results show NlAKHR silencing decreased the levels of 1,2-diacylglycerol (DAG) in the hemolymph and increased triacylglycerol (TAG) levels in the fat body, whereas Nl AKH injection led to a critical accumulation of DAG in the hemolymph and a severe reduction of TAG content in the fat body. Knockdown of NlAKHR resulted in prolonged lifespan and high levels of whole-body TAG, indicating an inability to mobilize TAG reserves during starvation. Conversely, the Nl AKH injection reduced the survival and accelerated TAG mobilization during starvation, which further confirms the role of Nl AKH in lipolysis. Moreover, NlAKHR silencing caused obesity in N. lugens , whereas Nl AKH injection depleted organismal TAG reserves in vivo and produced a slim phenotype. These results indicate that lipid mobilization is regulated by the AKH signaling system, which is essential for adjusting body lipid homeostasis and ensuring energy supplement during starvation in N. lugens. [ABSTRACT FROM AUTHOR]

Insect lipophorin receptor (LpR) belongs to the low-density lipoprotein receptor (LDLR) superfamily and plays an essential role in fecundity by mediating the incorporation of lipophorin into developing oocytes. Here we report the identification and characterization of a full-length cDNA encoding a putative LpR from the brown planthopper, Nilaparvata lugens . The deduced amino acid sequence of Nl LpR possesses the conserved structural motifs of LDLR family members, and displays a high degree of similarity with sequences from other insect LpRs. NlLpR is transcribed throughout oogenesis with its maximum level on day 7 after adult female emergence. NlLpR is highly expressed in the fat body and ovary, with relative low levels in the head, epidermis and midgut. Knockdown of NlLpR using double-stranded RNA (dsRNA) led to decreased triacylglycerol (TAG) content, retarded development of ovaries and decreased fecundity. Further functional analyses revealed that NlLpR works through nutritional signaling pathway-dependent activation of S6 kinase to regulate vitellogenin (Vg) biosynthesis during vitellogenesis and oocyte development. Disrupting of ecdysone receptor (EcR) expression and 20-hydroxyecdysone (20E) topical application demonstrated that NlLpR is regulated by ecdysone at transcript level. These results suggest that LpR is essential for Vg synthesis in the fat body and lipid uptake by developing oocytes, thus playing a critical role in insect reproduction. [ABSTRACT FROM AUTHOR]

Insulin-like peptides (ILPs) sense and transduce nutritional information and are linked to female reproduction in many insect species. Our previous studies have shown that “Target of rapamycin” (TOR) pathway functions through juvenile hormone (JH) to regulate amino acids-mediated vitellogenesis in the brown planthopper, Nilaparvata lugens , one of the most destructive rice pests in Asia. Recent reports have demonstrated that DNA methyltransferases (Dnmts) are also involved in female reproduction of N . lugens . However, the roles of ILPs and Dnmts in the nutritional regulation of female reproduction have not been fully elucidated. ILPs and Dnmts are highly expressed in the adult females after a supplement of amino acids, indicating nutrition-stimulated expression patterns of these genes. RNA interference-mediated depletion of NlILP2 or NlILP4 dramatically decreased the expression levels of NlDnmt1 and NlDnmt2 (tRNA methyltransferase), and resulted in severely impaired ovary growth as well as the substantial reduction of fecundity. Notably, NlILP2 or NlILP4 knockdown led to reduced mRNA accumulation of S6 kinase (S6K), a downstream target of the nutritional TOR pathway, and decreased vitellogenin content in the fat body. Silencing NlDnmt1 or NlDnmt2 effectively suppressed ovary development and decreased female fecundity. However, NlDnmt1 or NlDnmt2 knockdown did not influence the expression of NlILP2 and NlILP4 . We infer that amino acids act on ILPs and Dnmts to regulate vitellogenesis and oocyte maturation in N . lugens . [ABSTRACT FROM AUTHOR]

The widespread and extensive application of insecticides have promoted the development of resistance in the brown planthopper Nilaparvata lugens (Stål), one of the most important rice pests in Asia. To better understand the underlying molecular mechanisms of metabolic resistance to insecticides, a chlorpyrifos-resistant (CR) strain of N. lugens was selected and its possible resistance mechanism was investigated. Synergistic tests using carboxylesterases (CarEs) inhibitor triphenyl phosphate (TPP) decreased the resistance of N. lugens to chlorpyrifos, and CarE activities could be induced by low concentrations of chlorpyrifos. Subsequently, a gene putatively encoding CarE, namely NlCarE , predominant in the midgut and ovary was isolated and characterized. The expression levels of NlCarE were detected and compared between the CR and a susceptible (SS) strain of N. lugens . Consistent with the increased CarE activity, this gene was overexpressed in the CR strain compared to the SS strain. The transcript levels of NlCarE were up-regulated by chlorpyrifos exposure, showing dose- and time-dependent expression patterns. Furthermore, RNA interference (RNAi)-mediated knockdown of NlCarE followed by insecticide application significantly increased the susceptibility of N. lugens to chlorpyrifos. These results demonstrate that NlCarE plays an important role in chlorpyrifos detoxification and its overexpression may be involved in chlorpyrifos resistance in N. lugens . [ABSTRACT FROM AUTHOR]

The brown planthopper, Nilaparvata lugens , possesses a strong adaptability to extreme temperature and insecticide stresses. Heat shock proteins (Hsps) are highly conserved molecular chaperones and play a pivotal role in response to various environmental stresses in insects. However, little is known about the response of Hsps to stresses in N. lugens . In the present study, an inducible Hsp70 ( Nl Hsp70) was isolated from this insect and transcriptional expression patterns of NlHsp70 under temperature and insecticide stresses were analyzed. The full-length of NlHsp70 was 2805 bp with an open reading frame (ORF) of 1896 bp, showing high homology to its counterparts in other species. Expression of NlHsp70 was not altered by heat shock for 1 h, nor following recovery from thermal stress. Conversely, decreased expression of NlHsp70 was observed in response to cold shock. In addition, the expression of NlHsp70 increased after imidacloprid exposure. RNA interference experiment combined with insecticide injury assay also demonstrated that NlHsp70 was essential for resistance against insecticide exposure. These observations indicated that NlHsp70 was an important gene involved in the resistance or tolerance to environmental stresses in N. lugens . Interestingly, weak changes in mRNA expression levels of two thermal-inducible Hsp genes, NlHsp90 and NlHsc70 were observed in imidacloprid-exposed N. lugens adults, suggesting that different Hsps may respond differential to the extreme temperature and insecticide stresses. [ABSTRACT FROM AUTHOR]

Previous studies have demonstrated differences in thermotolerance between two wing morphs of Nilaparvata lugens , the most serious pest of rice across the Asia. To reveal the molecular regulatory mechanisms underlying the differential thermal resistance abilities between two wing morphs, a full-length of transcript encoding heat shock cognate protein 70 (Hsc70) was cloned, and its expression patterns across temperature gradients were analyzed. The results showed that the expression levels of NlHsc70 in macropters increased dramatically after heat shock from 32 to 38 °C, while NlHsc70 transcripts in brachypters remained constant under different temperature stress conditions. In addition, NlHsc70 expression in the macropters was significantly higher than that in brachypters at 1 and 2 h recovery from 40 °C heat shock. There was no significant difference in NlHsc70 mRNA expression between brachypters and macropters under cold shock conditions. Therefore, NlHsc70 was indeed a constitutively expressed member of the Hsp70 family in brachypters of N. lugens , while it was heat-inducible in macropters. Furthermore, the survival rates of both morphs injected with NlHsc70 dsRNA were significantly decreased following heat shock at 40 °C or cold shock at 0 °C for 1 h. These results suggested that the up-regulation of NlHsc70 is possibly related to the thermal resistance, and the more effective inducement expression of NlHsc70 in macropters promotes a greater thermal tolerance under temperature stress conditions. [ABSTRACT FROM AUTHOR]

The brown planthopper, Nilaparvata lugens , is one of the most destructive pests damaging rice in Asia and exhibits wing dimorphism, with brachypters possessing severely reduced wings and macropters bearing fully developed wings. Previous studies have shown that macropters are more heat resistant than brachypters. To understand the molecular mechanism underlying the differential thermotolerance abilities of these two morphs, a full-length Hsp gene, NlHsp90 was cloned from N. lugen . Our results showed that the relative expression levels of NlHsp90 in N. lugens females increased with the rise of temperature. Interestingly, NlHsp90 in macropters females could be induced at lower temperature (32 °C) than that in brachypters (34 °C), and the NlHsp90 mRNA levels in macropters were significantly higher than those in brachypters from 34 to 40 °C. In addition, the maximum expression levels of NlHsp90 were achieved much earlier in macropters, and NlHsp90 mRNA levels in macropters were significantly higher than those in brachypters from 1 to 6 h of recovery after temperature stress. Furthermore, knockdown of NlHsp90 by dsRNA injection reduced survival in both morphs with a greater reduction in the macropters relative to that of the brachyters. These results indicated that NlHsp90 plays an important role for thermotolerance in N. lugens , and there is difference on induction between two morphs. [ABSTRACT FROM AUTHOR]

The involvement of carboxylesterases (CarEs) in resistance to chlorpyrifos has been confirmed by the synergism analysis in Nilaparvata lugens. However, the function of specific CarE gene in chlorpyrifos resistance and the transcriptional regulatory mechanism are obscure. Herein, the expression patterns of 29 CarE genes in the susceptible and chlorpyrifos-resistant strains were analyzed. Among them, CarE3 , CarE17 and CarE19 were overexpressed in the resistant strain, and knockdown of either CarE gene by RNA interference significantly increased the susceptibility to chlorpyrifos. Remarkably, knockdown of CarE17 reduced the enzymatic activity of CarE by 88.63 % and showed a much greater effect on increasing chlorpyrifos toxicity than silencing other two CarE genes. Overexpression of CarE17 in Drosophila melanogaster decreased the toxicity of chlorpyrifos to transgenic fruit flies. Furthermore, the region between − 205 to + 256 of CarE17 promoter sequence showed the highest promoter activity, and 16 transcription factors (TFs) were predicted from this region. Among these TFs, Lim1β and C15 were overexpressed in the resistant strain. Knockdown of either TF resulted in reduced CarE17 expression and a decrease in resistance of N. lugens to chlorpyrifos. These results indicate that the constitutive overexpression of Lim1β and C15 induces CarE17 expression thus conferring chlorpyrifos resistance in N. lugens. • Three CarE genes were involved in chlorpyrifos resistance of N. lugens. • Metabolic resistance of N. lugens to chlorpyrifos is extremely attributed to CarE17. • Potential binding sites for homeobox TFs were predicted from CarE17 promoter. • Lim1β and C15 induces CarE17 expression thus increasing chlorpyrifos resistance. [ABSTRACT FROM AUTHOR]

Vitellogenin receptors (VgRs), members of the low-density lipoprotein receptor (LDLR) superfamily, are responsible for taking vitellogenin (Vg) into developing oocytes. Here the first full-length VgR cDNA from a hemipteran insect, the brown planthopper ( Nilaparvata lugens ), was cloned and sequenced. The complete mRNA sequence was 6174 bp in length with an open reading frame (ORF) of 5796 bp encoding 1931 amino acid residues. N. lugens VgR ( Nl VgR) contained two ligand-binding domains with five LDLR Class A cysteine-rich repeats in the first domain and eight in the second domain, which was similar to other insect VgRs. Nl VgR was specifically expressed in the ovary, and the mRNA level started to increase after adult female emergence, with a peak on day 7 in the adult stage, and then declined. Western blot analysis of Nl VgR protein revealed an ovary-specific expression pattern, which was consistent with NlVgR transcript detection. Injection with NlVgR double-stranded RNA (dsRNA) significantly disturbed NlVgR , which led to a decrease in Nl Vg protein content in the ovaries, an accumulation of Nl Vg protein in the hemolymph, the arrested development of ovaries, and the failure of insects to reproduce. Besides, Nl VgR expression was significantly upregulated after the topical application of juvenile hormone (JH) III. These results suggest that VgR is critical for Vg uptaking of oocytes and it plays an important role in insect fecundity. [ABSTRACT FROM AUTHOR]

Nilaparvata lugens (Stål) (Hemiptera: Geometroidea), a serious rice pest in many countries of Asia, causes a great loss in rice production every year. RNA interference (RNAi) is a powerful technology for gene function study in insects and a potential tool for pest control. As a core component of RNAi pathway, Dicer-2 (Dcr-2) protein determines the production of small interfering RNA (siRNA) and is crucial for the efficiency of RNAi. In this study, the full-length complementary DNA (cDNA) of N. lugens Dcr-2 ( NlDcr-2) was first cloned and analyzed, and then the RNAi experiment was conducted to explore the function of NlDcr-2 gene. The complete Dcr-2 cDNA of N. lugens was 4 971 bp in length with an open reading frame (ORF) of 1,656 amino acids. Phylogenetic and protein domain analysis showed that the predicted NlDcr-2 protein was similar to Tribolium castaneum. In the RNAi experiment, the messenger RNA level of NlDcr-2 was significantly reduced by NlDcr-2 double-stranded RNA (dsRNA) (dsDcr-2). Fifty-five per cent decrease of NlDcr-2 was found after 4 days of unremitting feeding. No significant effect was observed on the development of N. lugens after dsRNA ingestion. [ABSTRACT FROM AUTHOR]

Increased production of detoxification enzymes appears to be the primary route for insecticide resistance in many crop pests. However, the mechanisms employed by resistant insects for overexpression of detoxification genes involved in insecticide resistance remain obscure. We report here that the NR2E nuclear receptor HR83 plays a critical role in chlorpyrifos resistance by regulating the expression of detoxification genes in the brown planthopper (BPH), Nilaparvata lugens. HR83 was highly expressed in the fat body and ovary of adult females in chlorpyrifos-resistant BPHs. Knockdown of HR83 by RNA interference showed no effect on female fecundity, whereas caused a decrease of resistance to chlorpyrifos. This treatment also led to a dramatic reduction in the expression of multiple detoxification genes, including four UDP-glycosyltransferases (UGTs), three cytochrome P450 monooxygenases (P450s) and four carboxylesterases (CarEs). Among these HR83 -regulated genes, UGT-1-3 , UGT-2B10 , CYP6CW1 , CYP4CE1 , CarE and Esterase E4-1 were over-expressed both in the fat body and ovary of the resistant BPHs. Functional analyses revealed that UGT-2B10 , CYP4CE1 , CarE and Esterase E4-1 are essential for the resistance of BPH to chlorpyrifos. Generally, this study implicates HR83 in the metabolic detoxification–mediated chlorpyrifos resistance and suggests that the regulation of detoxification genes may be an ancestral function of the NR2E nuclear receptor subfamily. [Display omitted] • HR83 was highly expressed in chlorpyrifos-resistant BPHs. • Knockdown of HR83 decreased the resistance of BPH to chlorpyrifos. • HR83 was involved in the regulation of the expression of multiple detoxification genes. • HR83 -affected UGT-2B10 and CYP4CE1 were associated with chlorpyrifos resistance. [ABSTRACT FROM AUTHOR]

As a multigene superfamily of Phase II detoxification enzymes, uridine diphosphate (UDP)-glycosyltransferases (UGTs) play important roles in the metabolism of xenobiotics including insecticides. In this study, 5-nitrouracil, an inhibitor of UGT enzyme activity, effectively increased the toxicity of chlorpyrifos to the chlorpyrifos-resistant strain of Nilaparvata lugens , one of the most resistant rice pests. The enzyme content of UGT in the resistant strain was significantly higher than that in the susceptible strain. Among 20 identified UGT genes, UGT386H2 , UGT386J2 , UGT386N2 and UGT386P1 were found significantly overexpressed in the resistant strain and can be effectively induced by chlorpyrifos. These four UGT genes were most highly expressed in the midgut and/or fat body, two main insect detoxification tissues. Amino acid sequence alignments revealed that these four UGTs contained a variable N-terminal substrate-binding domain and a conserved C-terminal sugar donor-binding domain. Furthermore, homology modeling and molecular docking analyses showed that these UGTs could stably bind to chlorpyrifos and chlorpyrifos oxon, with the binding free energies from −19.4 to −110.62 kcal mol−1. Knockdown of UGT386H2 or UGT386P1 by RNA interference dramatically increased the susceptibility of the resistant strain to chlorpyrifos. These findings suggest that overexpression of these two UGT genes contributes to chlorpyrifos resistance in N. lugens. [Display omitted] • Four UGTs were found to be overexpressed in chlorpyrifos-resistant N. lugens. • The overexpressed UGT genes were primarily expressed in midgut and/or fat body. • Chlorpyrifos could stably bind to these UGTs in molecular docking. • Overexpression of UGT386H2 and UGT386P1 was responsible for chlorpyrifos resistance. [ABSTRACT FROM AUTHOR]

Hepatocyte nuclear factor 4 (HNF4) plays essential roles in regulating lipid metabolism and glucose homeostasis in female insects. However, little is known about the role of HNF4 in insect fecundity. Here we demonstrate that HNF4 regulates female fecundity by affecting egg hatching in the brown planthopper (BPH) Nilaparvata lugens. HNF4 was highly expressed in the ovary and fat body of female adult. RNA interference-mediated HNF4 knockdown resulted in a dramatic reduction in egg hatchability and caused a severe block in embryonic development, while showed no significant effects on ovary development and egg laying. Transcriptome sequencing analysis showed that 72 genes encoding ribosome proteins were significantly down-regulated in the HNF4 -silenced BPH and "ribosome" was the most-enriched pathway for the down-regulated genes. These results suggest that HNF4 controls the dynamics of egg structure, likely through its regulation of ribosome protein genes, which in turn affects the embryonic development and egg hatching. • HNF4 was highly expressed in the fat body and ovary of adult BPH female. • HNF4 knockdown resulted in a dramatic reduction in egg hatching rate. • Transcriptome sequencing analysis identified 3544 DEGs (1797 up-regulated and 1747 down-regulated) in the ovary of HNF4 -silenced BPH. • 72 ribosome genes were significantly down-regulated after HNF4 knockdown. • KEGG analysis showed that "ribosome" was the most-enriched pathway for the down-regulated genes. [ABSTRACT FROM AUTHOR]

Increasing evidence indicates that insect resistance to synthesized insecticides is regulated by the nuclear receptors. However, the underlying mechanisms of this regulation are not clear. Here, we demonstrate that inhibition of hepatocyte nuclear factor 4 (HNF4) confers imidacloprid resistance in the brown planthopper (BPH) Nilaparvata lugens by regulating cytochrome P450 and UDP-glycosyltransferase (UGT) genes. An imidacloprid-resistant strain (Res) exhibited a 251.69-fold resistance to imidacloprid in comparison to the susceptible counterpart (Sus) was obtained by successive selection with imidacloprid. The expression level of HNF4 in the Res strain was lower than that in Sus, and knockdown of HNF4 by RNA interference significantly enhanced the resistance of BPH to imidacloprid. Comparative transcriptomic analysis identified 1400 differentially expressed genes (DEGs) in the HNF4 -silenced BPHs compared to controls. Functional enrichment analysis showed that cytochrome P450- and UGT-mediated metabolic detoxification pathways were enriched by the up-regulated DEGs after HNF4 knockdown. Among of them, UGT-1-7 , UGT-2B10 and CYP6ER1 were found to be over-expressed in the Res strain, and knockdown of either gene significantly decreased the resistance of BPH to imidacloprid. This study increases our understanding of molecular mechanisms involved in the regulation of insecticide resistance and also provides potential targets for pest management. Image 1 • Hepatocyte nuclear factor 4 (HNF4) negatively regulates imidacloprid resistance. • Transcriptomic analysis identified seven metabolic resistance pathways after HNF4 silencing. • Inhibition of HNF4 activates P450s and UDP-glycosyltransferases (UGTs). • P450s and UGTs are involved in the imidacloprid resistance. [ABSTRACT FROM AUTHOR]

Insect resistance to chemical insecticide is a global problem that presents an ongoing threat to sustainable agriculture. Although the increased production of detoxification enzymes has been frequently implicated in resistance development, the mechanisms employed by insecticide-resistant insects for overexpression of these genes remain elusive. Here we report that neuropeptide adipokinetic hormone (AKH) negatively regulates the expression of CYP6ER1 and CYP6AY1 , two important cytochrome P450 monooxygenases (P450s) that confer resistance to neonicotinoid imidacloprid in the brown planthopper (BPH). Imidacloprid exposure suppresses AKH synthesis in the susceptible BPH, and AKH is inhibited in the imidacloprid-resistant strain. RNA interference (RNAi) and AKH peptide injection revealed that imidacloprid exposure inhibits the AKH signaling cascade and then provokes reactive oxygen species (ROS) burst. These in turn activate the transcription factors cap 'n' collar isoform-C (CncC) and muscle aponeurosis fibromatosis (MafK). RNAi and ROS scavenger assays showed that ROS induces CYP6ER1 expression by activating CncC and MafK , while ROS mediates induction of CYP6AY1 through another unidentified pathway in the resistant BPH. Collectively, these results provide new insights into the regulation of insecticide resistance and implicate both the neuropeptide AKH-mediated ROS burst and transcription factors are involved in the overexpression of P450 detoxification genes in insecticide-resistant insects. Image 1 • The inhibition of AKH upon exposure to imidacloprid provokes ROS burst. • ROS burst activates CncC and MafK, which then induces the expression of CYP6ER1. • ROS induces the expression of CYP6AY1 through a CncC/MafK-independent pathway. • CYP6AY1 and CYP6ER1 confer resistance to imidacloprid in the BPH. [ABSTRACT FROM AUTHOR]