Activation of the NR2E nuclear receptor HR83 leads to metabolic detoxification-mediated chlorpyrifos resistance in Nilaparvata lugens.

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.

Effect of hepatocyte nuclear factor 4 on the fecundity of Nilaparvata lugens: Insights from RNA interference combined with transcriptomic analysis.

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.

Copper exposure enhances Spodoptera litura larval tolerance to ß-cypermethrin.

Environmental xenobiotics can influence the tolerance of insects to chemical insecticides. Heavy metals are widespread distributed, can be easily bio-accumulated in plants and subsequently within phytophagous insects via the food chains. However, less attention has been paid to the effect of heavy metal exposure on their insecticide tolerance. In this study, pre-exposure of copper (Cu, 25-100 mg kg-1) significantly enhanced the subsequent tolerance of Spodoptera litura to ß-cypermethrin, a widely used pyrethroid insecticide in crop field. Cytochrome P450 monooxygenases (CYPs) activities were cross-induced in larvae exposed to Cu and ß-cypermethrin, while the activities of glutathione S-transferase (GST) and carboxylesterase (CarE) were not affected. Application of piperonyl butoxide (PBO), a P450 synergist, effectively impaired the tolerance to ß-cypermethrin in Cu-exposed S. litura larvae with a synergistic ratio of 1.72, indicating that P450s contribute to larval tolerance to ß-cypermethrin induced by Cu exposure. Among the four CYP6AB family genes examined, only larval midgut-specific CYP6AB12 was found to be cross-induced by Cu and ß-cypermethrin. RNA interference (RNAi)-mediated silencing of CYP6AB12 effectively decreased the mRNA levels of the target gene, and significantly reduced the larval tolerance to ß-cypermethrin following exposure to Cu. These results showed that pre-exposure of heavy metal Cu enhanced larval tolerance to ß-cypermethrin in S. litura, possibly through the cross-induction of P450s. Our findings provide new insights on the relationship between heavy metals and chemical insecticides that may benefit both the risk evaluation of heavy metal contamination and development of pest management strategies.

Copper-induced H2O2 accumulation confers larval tolerance to xanthotoxin by modulating CYP6B50 expression in Spodoptera litura.

In the plant-insect arms race, plants synthesize toxic compounds to defend against herbivorous insects, whereas insects employ cytochrome P450 monooxygenases (P450s) to detoxify these phytotoxins. As ubiquitous environmental contaminants, heavy metals can be easily absorbed by plants and further accumulated in herbivorous insects through the food chains, resulting in tangible consequences for plant-insect interactions. However, whether heavy metals can influence P450 activities and thereby cause further effects on larval tolerance to phytotoxins remains unknown. In this study, we shown that prior exposure to copper (Cu) enhanced larval tolerance to xanthotoxin in Spodoptera litura, a major polyphagous pest of agriculture. P450 activities were induced in larvae exposed to Cu or xanthotoxin, and a midgut specific expressed P450 gene, CYP6B50 was cross-induced after exposure to these two toxic xenobiotics. Knocking down CYP6B50 by RNA interference (RNAi) rendered the larvae more sensitive to xanthotoxin. As defense against oxidative stress following metal exposure has been demonstrated to affect insecticide resistance, the reactive oxygen species (ROS) generation and antioxidant enzyme activities were assessed. Cu exposure caused the accumulation of hydrogen peroxide (H2O2) and enhanced the activities of superoxide dismutase (SOD) and peroxidase (POD) in larval midgut. In addition, two antioxidant response elements (AREs) were identified from the CYP6B50 promoter, indicating that Cu-induced CYP6B50 expression may be related to the ROS burst. Application of ROS scavenger N-acetylcysteine (NAC) effectively suppressed CYP6B50 expression, inhibited P450 activities and impaired larval tolerance to xanthotoxin that had been induced by Cu. These results indicate that the increase in CYP6B50 expression regulated by Cu-induced H2O2 generation contributed to the enhancement of larval tolerance to xanthotoxin in S. litura. Ingestion of heavy metals from their host plants can inadvertently boost the counter-defense system of herbivorous insects to protect themselves against plant defensive toxins.

Phytochemical Flavone Confers Broad-Spectrum Tolerance to Insecticides in Spodoptera litura by Activating ROS/CncC-Mediated Xenobiotic Detoxification Pathways.

Tolerance to chemical insecticides can be driven by the necessity of herbivorous insects to defend against host plant-produced phytochemicals. However, how the phytochemicals are sensed and further transduced into a defense response associated with insecticide tolerance is poorly understood. Herein, we show that pre-exposure to flavone, a flavonoid phytochemical, effectively enhanced larval tolerance to multiple synthetic insecticides and elevated detoxification enzyme activities in Spodoptera litura. RNA-Seq analysis revealed that flavone induced a spectrum of genes spanning phase I and II detoxification enzyme families, as well as two transcription factors Cap "n" collar isoform C (CncC) and its partner small muscle aponeurosis fibromatosis (MafK). Knocking down of CncC by RNA interference suppressed flavone-induced detoxification gene expression and rendered the larvae more sensitive to the insecticides. Flavone exposure elicited a reactive oxygen species (ROS) burst, while scavenging of ROS inhibited CncC-mediated detoxification gene expression and suppressed flavone-induced detoxification enzyme activation. Metabolome analysis showed that the ingested flavone was mainly converted into three flavonoid metabolites, and only 3-hydroxyflavone was found to affect the ROS/CncC pathway-mediated metabolic detoxification. These results indicate that the ROS/CncC pathway is an important route driving detoxification gene expression responsible for insecticide tolerance after exposure to the phytochemical flavone.

Inhibition of hepatocyte nuclear factor 4 confers imidacloprid resistance in Nilaparvata lugens via the activation of cytochrome P450 and UDP-glycosyltransferase genes.

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.

The KNRL nuclear receptor controls hydrolase-mediated vitellin breakdown during embryogenesis in the brown planthopper, Nilaparvata lugens.

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.

Activation of the ROS/CncC and 20-Hydroxyecdysone Signaling Pathways Is Associated with Xanthotoxin-Induced Tolerance to λ-Cyhalothrin in Spodoptera litura.

Adaptation to phytochemicals in herbivorous insects can influence tolerance to insecticides. However, it is unclear how insects use phytochemicals as cues to activate their metabolic detoxification systems. In this study, we found that dietary exposure to xanthotoxin enhanced tolerance of Spodoptera litura larvae to λ-cyhalothrin. Xanthotoxin ingestion significantly elevated the mRNA levels of 35 detoxification genes as well as the transcription factors Cap 'n' collar isoform-C (CncC) and its binding factor small muscle aponeurosis fibromatosis isoform-K (MafK). Additionally, xanthotoxin exposure increased the levels of reactive oxygen species (ROS), while ROS inhibitor N-acetylcysteine (NAC) treatment blocked xanthotoxin-induced expression of CncC, MafK, and detoxification genes and also prevented xanthotoxin-enhanced larval tolerance to λ-cyhalothrin. The 20-hydroxyecdysone (20E) signaling pathway was effectively activated by xanthotoxin, while blocking of 20E signaling transduction prevented xanthotoxin-enhanced larval tolerance to λ-cyhalothrin. Application of 20E induced the expression of multiple xanthotoxin-induced detoxification genes and enhanced λ-cyhalothrin tolerance in S. litura. NAC treatment blocked xanthotoxin-induced 20E synthesis, while the CncC agonist curcumin activated the 20E signaling pathway. These results indicate that the ROS/CncC pathway controls the induction of metabolic detoxification upon exposure to xanthotoxin, at least in part, through its regulation of the 20E signaling pathway.

Activation of CncC pathway by ROS burst regulates cytochrome P450 CYP6AB12 responsible for λ-cyhalothrin tolerance in Spodoptera litura.

Frequent insecticide use poses an environmental hazard and also selects for insecticide tolerance. Increased metabolic detoxification by cytochrome P450 monooxygenases (P450s) is the most common mechanism of insecticide tolerance. However, the underlying regulatory mechanisms remain unknown. We studied the midgut-specific P450 gene, CYP6AB12, associated with λ-cyhalothrin tolerance. Its regulatory pathway was investigated in the tobacco cutworm, Spodoptera litura (Fabricius). P450 activities and CYP6AB12 transcript levels increased after λ-cyhalothrin exposure. Inhibiting P450 activities with piperonyl butoxide and silencing CYP6AB12 by double-stranded RNA (dsRNA) injection decreased larval tolerance to λ-cyhalothrin. λ-Cyhalothrin exposure induced the expression of the cap 'n' collar isoform C (CncC) and muscle aponeurosis fibromatosis (Maf), increased hydrogen peroxide (H2O2) contents and elevated antioxidant enzyme activities. CncC knockdown by dsRNA feeding suppressed CYP6AB12 expression and decreased larval tolerance to λ-cyhalothrin. In contrast, application of the CncC agonist curcumin induced CYP6AB12 expression and enhanced insecticide tolerance. Ingestion of the reactive oxygen species (ROS) scavenger N-acetylcysteine reduced H2O2 accumulation, suppressed the expression of CncC, Maf and CYP6AB12 and led to increased larval susceptibility to λ-cyhalothrin. The results demonstrate that in S. litura, λ-cyhalothrin induces cytochrome P450 CYP6AB12 via elicitation of the ROS burst and activation of the CncC pathway.

Adipokinetic hormone regulates cytochrome P450-mediated imidacloprid resistance in the brown planthopper, Nilaparvata lugens.

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.

Adipokinetic Hormone Receptor Mediates Lipid Mobilization to Regulate Starvation Resistance in the Brown Planthopper, Nilaparvata lugens.

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. NlAKHR 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 NlAKH is released from the corpora cardiaca to activate NlAKHR 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 NlAKH 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 NlAKH 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 NlAKH injection reduced the survival and accelerated TAG mobilization during starvation, which further confirms the role of NlAKH in lipolysis. Moreover, NlAKHR silencing caused obesity in N. lugens, whereas NlAKH 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.

Deficiency of Brummer Impaires Lipid Mobilization and JH-Mediated Vitellogenesis in the Brown Planthopper, Nilaparvata lugens.

Provisioning of sufficient lipids and vitellogenin to the oocytes is an indispensable process for fecundity of oviparous insects. Acute mobilization of lipid reserves in insects is controlled by the Brummer (Bmm), an orthologous of human adipose triglyceride lipase (ATGL). To investigate the functional roles of brummer-mediated lipolysis in the fecundity of the brown planthopper, Nilaparvata lugens, RNA interference (RNAi) analyses were performed with double-stranded RNA (dsRNA) against NlBmm in adult females. Knockdown of NlBmm expression resulted in obesity and blocked lipid mobilization in the fat body. In addition, NlBmm silencing led to retarded ovarian development with immature eggs and less ovarioles, decreased number of laid eggs, prolonged preoviposition period and egg duration. Furthermore, severe reductions of vitellogenin and its receptor abundance were observed upon NlBmm knockdown. The transcript levels of NlJHE (juvenile hormone esterase) which degrades JH were up-regulated, whereas the expression levels of JH receptors NlMet (Methoprene-tolerant) and NlTai (Taiman) and their downstream transcription factors NlKr-h1 (Krüppel-homolog 1) and NlBr (Broad-Complex) were down-regulated after suppression of NlBmm. JH-deficient females exhibited impaired vitellogenin expression, whereas JH exposure stimulated vitellogenin biosynthesis. Moreover, JH topical application partially rescued the decrease in vitellogenin expression in the NlBmm-deficient females. These results demonstrate that brummer-mediated lipolytic system is essential for lipid mobilization and energy homeostasis during reproduction in N. lugens. In addition to the classical view of brummer as a direct lipase with lipolysis activity, we propose here that brummer-mediated lipolysis works through JH signaling pathway to activate vitellogenesis and oocyte maturation that in turn regulates female fecundity.

Adipokinetic Hormone Receptor Mediates Trehalose Homeostasis to Promote Vitellogenin Uptake by Oocytes in Nilaparvata lugens.

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.