Introduction

Application of pesticides promotes agricultural development to meet global food demand. However, their overuse has caused severe resistance1 of insects to almost all classes of commercial insecticides in these insect pests2,3,4. Insects develop resistance to insecticides through two major mechanisms: (i) target-site insensitivity resulting from the mutations in the insecticide target genes5, and (ii) metabolic resistance, which results from the enhanced expression or activity of enzymes that metabolize to less toxic and/or more soluble compounds that could be excreted faster6. New insecticides are often needed to deal with target resistance; however, the creation of new insecticides faces significant challenges in design, synthesis, testing, and analysis7. Therefore, insecticide resistance management requires the development of new strategies for preventing metabolic resistance. The complex metabolic enzymes mainly include cytochrome P450 monooxygenases (P450s), carboxylesterases (CarEs) and glutathione S-transferases (GSTs)8. Overexpression of these detoxification genes has been proven to be involved in insecticide resistance in insects. For example, upregulation of CYP6BQ9 results in the majority of deltamethrin resistance in Tribolium castaneum9. Similarly, a CarE gene plays an important role in the metabolism of chlorpyrifos and leads to resistance to this compound in Nilaparvata lugens10. Finally, GSTe2 confers resistance to DDT and pyrethroid in mosquitoes11.

In arthropods, constitutive overexpression of transcription factors contributes to increasing the expression of detoxification genes responsible for metabolic resistance12,13. Whether inhibition of transcriptional regulation of detoxification genes is used as a new strategy to suppress insecticide resistance remains unknown. Among these regulators, transcription factor Nrf2 is the primary factor responsible for regulating the expression of detoxifying and antioxidant genes in mammals14. Under normal conditions, Keap1 acts as an E3 ligase adapter to retain Nrf2 in the cytoplasm8. The Nrf2-Keap1 interaction is disrupted under oxidative stress conditions and Nrf2 translocates to the nucleus, where it heterodimerizes with Maf. Subsequently, the Nrf2-Maf complex binds to the antioxidant response element (ARE) in the promoter region of genes coding for detoxifying and antioxidant enzymes, including P450s, GSTs, UGTs, NAD(P)H:quinone oxidoreductase-1 (NQO1) and heme oxygenase 1 (HO-1), which synergistically increase the efficiency of cellular defense systems15. Nrf2 ortholog in invertebrates is known as cap ‘n’ collar C (CncC) that plays a critical role in regulating the expression of detoxification genes involved in insecticide resistance16. CncC and Maf enhance the expression of CYP392A28, CYP391B1 and CYP391A1, responsible for fenpropathrin resistance in the spider mite Tetranychus cinnabarinus17. Similarly, these transcription factors also control the overexpression of P450 genes associated with deltamethrin resistance in T. castaneum and imidacloprid resistance in Leptinotarsa decemlineata18,19. Therefore, these transcription factors may serve as new target genes to screen for inhibitors of detoxification genes for pest control.

Constitutive activation of Nrf2 contributes to resistance to chemotherapy drugs in mammalian cancer cells20,21. Inhibitors of Nrf2 are often used to improve the susceptibility of cancer cells to drugs. For example, brusatol reduces the expression of Nrf2 and downstream genes, and increases the susceptibility of lung cancer cells to cisplatin22. Similarly, DMC (2’,4’-Dihydroxy-6’-methoxy-3’,5’-dimethylchalcone) reduces the expression of GST genes by inhibiting the expression of Nrf2 and preventing Nrf2 nuclear translocation, and reverses drug resistance in hepatocellular carcinoma cells23. Finally, Retinoic acid decreases the expression of Nrf2-dependent genes by blocking the binding of Nrf2 to the ARE enhancer in breast cancer cells24. However, the screening of inhibitors of CncC/Maf has not be conducted, and whether the potential inhibitors can be used for insecticide resistance management remain poorly known in insect pests. Sofalcone, a synthetic analog of sophoradin, is a type of natural phenol derived from the traditional medicinal herb Sophora subprostrata, with potent anti-inflammatory activity in the human colonic epithelial cells25.

Spodoptera exigua is a widespread and polyphagous lepidopteran pest that seriously damages many cultivated crops and causes considerable economic agricultural losses26,27,28. Currently, chemical insecticides are the major method for the control of this pest, and excessive and frequent insecticide applications have led this pest to develop resistance to many insecticides29. Therefore, finding synergists acting on novel target genes for resistance management becomes very important. Here, a high-throughput cell screening platform for identification of the inhibitors of CncC/Maf was established. We confirmed that sofalcone served as an inhibitor of CncC/Maf in vivo and in vitro. Furthermore, we determined that sofalcone significantly increased larval sensitivity to insecticides and the underlying molecular mechanisms.

Results

Identification of sofalcone as CncC/Maf inhibitor using a CncC-luciferase reporter gene assay in the Sf9 cells

To rapidly identify the effective natural product candidates to enhance the toxicity of insecticides to insect pests, we constructed the pGL3-CncC-Core promoter plasmid containing the CncC/Maf binding sequence (Fig. 1A). These constructs were transfected into Sf9 cells, chlorpyrifos, lambda-cyhalothrin and an equal volume of DMSO (control) were added after 16 h post-transfection. The lambda-cyhalothrin- and chlorpyrifos-induced luciferase activities of the pGL3-CncC-Core promoter were 3.1-fold and 4.7-fold greater than that of the control, respectively, whereas both two insecticides did not affect the promoter activity of the control pGL3-Core (Fig. 1A), suggesting that the pGL3-CncC-Core promoter vector were successfully constructed and can be utilized to perform large-scale screening of inhibitors for blocking the promoter activity of CncC/Maf. As shown in Supplementary Fig. 1, sofalcone did not change the luciferase activity of pGL3-CncC-Core promoter construct, whereas the other twelve natural products significantly enhanced the CncC-Core promoter activities compared with the control. Interestingly, pretreatment with sofalcone dramatically inhibited the insecticide-induced promoter activities of the CncC-Core sequence by 63.8% and 71.2%, respectively (Fig. 1B). Meanwhile, MTT assay results showed that sofalcone exposure (2.5 μM) did not affect cell viability (Supplementary Fig. 2). These results indicate that sofalcone do not affect the basal promoter activities of target genes of CncC/Maf, but significantly suppresses the insecticide-induced promoter activities of these genes.

Fig. 1: The activity of insecticide-induced CncC promoter was blocked by sofalcone.
figure 1

A Determination of the luciferase (Luc) activities of core promoter and core promoter plus the CncC/Maf binding site in response to insecticides. Data are the mean ± SD of six independent assays. The data were analyzed using Tukey’s HSD. Letters a, b, c, and d denote significant differences at p < 0.05. n = 6. B Promoter activities of PGL3-core-CncC/Maf after exposure to insecticides with or without sofalcone. Error bars represent the mean ± SD values (n = 6). Different letters on the right of error bars indicate significant differences based on ANOVA with Tukey’s HSD multiple comparison test (p < 0.05).

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Sofalcone inhibited the expression of detoxification genes after exposure to insecticides in the S. exigua larvae

To investigate whether sofalcone affects the expression levels of detoxification genes after exposure to insecticides in vivo, the relative expression levels of these genes were analyzed in the S. exigua larvae exposed to DMSO, sofalcone, insecticide (chlorpyrifos or lambda-cyhalothrin) or in combination. The results showed that the transcription levels of six CYP genes (CYP321A8, CYP321A9, CYP321A16, CYP321B1, CYP9A11 and CYP9A27) were significantly overexpressed by 2.1- to 31.3-fold after exposure to each of the two insecticides, whereas the induced-expression levels of these CYP genes are significantly reduced by 31.6% - 100.0% in the larvae exposed to a mixture of insecticide and sofalcone (Fig. 2). Similarly, four GST genes (GSTe2, GSTe6, GSTe14 and GSTo2) were significantly upregulated by 1.6- to 15.7-fold after exposure to each of the two insecticides, and pretreatment with sofalcone also inhibited the insecticide-induced expression levels of these GST genes by 40.7–100.0% (Fig. 3). Meantime, pretreatment with sofalcone also reduced the insecticide-induced expression levels of two CarE genes (CarE1 and CarE5) and two ABC genes (ABCB2 and ABCC2) by 43.8–100.0%, whereas the other two CarE genes (CarE2 and CarE4) and ABCB1 were not significantly affected in response to insecticides (Fig. 4 and Supplementary Fig. 3). ABCC1 and ABCC3 were clearly upregulated by 2.4- to 3.5-fold in the presence of insecticides, and pretreatment with sofalcone did not change the expression levels of the two genes compared to treatment with insecticides alone (Supplementary Fig. 3). These results suggest that insecticide-induced increases in the expression levels of detoxification-related genes were significantly downregulated by combined treatment with insecticides and sofalcone.

Fig. 2: Sofalcone inhibited the expression of P450 genes after exposure to insecticides.
figure 2

The expression difference of CYP321A8 (A), CYP321A9 (B), CYP321A16 (C), CYP321B1 (D), CYP9A11 (E) and CYP9A27 (F) in response to insecticides with or without sofalcone was determined by RT-qPCR. Data are the mean ± SD of five independent assays. The data were analyzed using Tukey’s HSD. Different letters on the right of error bars indicate significant differences (p < 0.05). n = 5.

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Fig. 3: The transcriptional expression of insecticide-induced GST genes was reduced by sofalcone.
figure 3

RT-qPCR was performed to determine the difference in the expression of GSTe2 (A), GSTe6 (B), GSTe14 (C) and GSTO2 (D) in response to insecticides with or without sofalcone. Five biological replicates were conducted, and the 2−ΔΔCT method was utilized to calculate the relative expression levels. GAPDH and β-Actin were used to normalize expression levels. Letters on the standard error bars indicate significant differences compared with the control (ANOVA with post-hoc Tukey HSD, p < 0.05). n = 5.

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Fig. 4: Sofalcone diminished the mRNA levels of CarE genes when exposed to insecticides.
figure 4

The expression changes of CarE1 (A), CarE2 (B), CarE4 (C) and CarE5 (D) under the stress of insecticides in larvae of S. exigua were determined by RT-qPCR. The results are presented as the mean ± SD (n = 5). Different letters on the error bar represent the significant difference based on ANOVA with Tukey’s HSD multiple comparison test (p < 0.05).

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The insecticide-induced activities of detoxification enzymes were repressed by sofalcone

To determine the effect of sofalcone on detoxification enzyme activities, the assays of detoxification enzyme activities were performed in the larvae in the presence of insecticide with or without sofalcone. The results revealed that both chlorpyrifos and lambda-cyhalothrin increased the P450 enzyme activities by 2.0- and 1.7-fold, respectively, while cotreatment of insecticide with sofalcone dramatically reduced the insecticide-induced P450 activities by 62.5% and 59.1%, respectively (Fig. 5A). Similarly, treatment with chlorpyrifos and lambda-cyhalothrin resulted in a 2.0- and 2.2-fold increase in the GST activities, respectively, and the activities were significantly reduced by 60.3% and 72.2% after sofalcone and insecticide coapplied treatment compared with treatment with insecticide alone (Fig. 5B). In addition, treatment with the sofalcone markedly decreased the lambda-cyhalothrin- or chlorpyrifos-induced esterase activities of S. exigua larvae by 69.6% and 52.1%, respectively (Fig. 5C). These results demonstrate that sofalcone effectively represses the increase in detoxification enzyme activities after exposure to insecticides in the S. exigua larvae.

Fig. 5: Sofalcone decreased the activities of detoxification enzymes when exposed to insecticides.
figure 5

A P450 monooxygenase activity was evaluated by measuring ethoxycoumarin-O-deethylase (ECOD) activity. Significant differences (p < 0.05) in enzymatic activities are denoted using letters above bars (ANOVA with post-hoc Tukey’s HSD). n = 5. B Glutathione-S-transferase (GST) activity was measured using 1-chloro-2, 4-dinitrobenzene (CDNB) as substrate. Different letters above the bars indicate significant differences based on ANOVA followed by post-hoc Tukey’s HSD (p < 0.05). n = 5. C Esterase (EST) activity was determined by measuring a-naphthyl acetate activity. Different letters above the bars denote significant differences at p < 0.05 (ANOVA with post-hoc Tukey HSD). n = 5.

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Sofalcone suppressed the expression of detoxification genes by CncC /Maf in the larvae exposed to insecticides

To further explore the molecular mechanisms of the regulation of detoxification genes,

we cloned the upstream sequences of six CYPs, four GSTs, four CarEs and five ABCs by genomic walking. Transcription factor (TF) binding sites were predicted and analyzed using online tools JASPAR and ALGGEN (Fig. 6A, Supplementary Figs. 4–7 and Supplementary Table 5). According to the prediction of TF binding sites, four CYP450s (CYP321B1, CYP321A16, CYP9A11 and CYP9A27), three GSTs (GSTe2,GSTe6 and GSTo2), three CarEs (CarE2, CarE4 and CarE5) and three ABCs (ABCB1, ABCC1 and ABCC2) contained the AhR/Arnt binding site. The upstream sequences of three CYPs (CYP321A8, CYP321A9 and CYP9A27), two GSTs (GSTe2 and GSTe6), four CarEs (CarE1, CarE2, CarE4 and CarE5) and four ABCs (ABCB1, ABCB2, ABCC1 and ABCC3) possessed the binding sequence of ECR. PXR binding site existed in the promoter sequences of CYP321A9, CYP321B1, GSTe6, GSTe14, CarE1, CarE5, ABCB1, ABCB2 and ABCC3. The binding sites of CncC/Maf are observed in the promoter sequences of detoxification-related genes including six CYP450s (CYP321A8, CYP321A9, CYP321A16, CYP321B1, CYP9A11 and CYP9A27), four GSTs (GSTe2, GSTe6, GSTe14 and GSTo2), two CarEs (CarE1 and CarE5) and two ABCs (ABCB2 and ABCC2). These results suggest that the transcription of these detoxification genes, which were significantly increased by insecticides, may be under the control of the same cis-regulatory element and transcription factors: CncC and Maf.

Fig. 6: CncC/Maf regulates the expression of CYP321A9 gene.
figure 6

A Prediction of transcription factor binding sites in a ~1.2 kb region of the promoter of CYP321A9 gene. Nucleotides are numbered relative to the translation start site (ATG) indicated by +1. The predicted binding sites for transcription factors sites are underlined. B The activity of each promoter construct in reporter gene assays in the presence or absence of CncC/Maf is shown. Error bars display SD. Different letters above the bars indicate significant differences based on ANOVA followed by post-hoc Tukey’s HSD (p < 0.05). n = 4.

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To determine whether the transcription factors CncC and Maf regulate the expression of detoxification genes, the fragment of CYP321A9 putative promoter was ligated into the reporter gene vector and co-transfected with the CncC and Maf ligated into the expression plasmid. These results showed that the overexpression of Maf slightly enhanced the transcription activity of CYP321A9 promoter, and CncC significantly increased the promoter activity of CYP321A9 by 3.8-fold. Co-transfection with CncC and Maf resulted in a 6.5-fold increase in the promoter activity of CYP321A9 (Fig. 6B). These results determine that the transcription factors CncC and Maf control the transcription levels of detoxification genes in S. exigua.

To examine whether the expression of CncC and Maf was inhibited by sofalcone after exposure to insecticides in larvae, the mRNA levels of the two genes were analyzed by RT- qPCR. The results showed that the mRNA levels of CncC induced by chlorpyrifos or lambda-cyhalothrin were about 5.3- and 5.0-fold higher than that of control, respectively. Compared to the presence of insecticides alone, cotreatment with sofalcone and insecticides decreased the expression of CncC by 73.5% and 67.7%, respectively (Fig. 7A). Similarly, lambda-cyhalothrin- or chlorpyrifos-induced Maf expression was 3.1- and 2.9-fold, respectively. Also, sofalcone significantly prevented the lambda-cyhalothrin- or chlorpyrifos-induced upregulation of Maf (Fig. 7B). These results demonstrate that sofalcone concomitant treatment inhibits insecticide-induced expression of detoxification genes by transcription factors CncC /Maf.

Fig. 7: Sofalcone suppressed the expression of CncC and Maf after exposure to insecticides.
figure 7

Relative expression of transcription factors CncC (A) and Maf (B) when exposed to insecticides in the presence or absence of sofalcone was determined by RT-qPCR. The 2−ΔΔCT method was used for the quantification of the gene expression level. The cycle threshold (Ct) values for the genes were normalized to the Ct values of GAPDH and β-Actin. Data are the mean ± SD of five independent assays. The data were analyzed using Tukey’s HSD. Letters a, b and c denote significant differences at p < 0.05. n = 5.

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Sofalcone decreased the ROS content when exposed to insecticides

Our previous studies proved that the expression of detoxification genes was associated with oxidative stress induced by insecticides30, so we measured the ROS content, including GSH-Px activity, hydrogen peroxide level and MDA content at 0, 6, 24, 48, and 72 h after exposure to insecticides with or without sofalcone. These results revealed that both chlorpyrifos and lambda-cyhalothrin significantly enhanced the GSH-Px activities at 6, 24, 48, and 72 h posttreatment, and the activity kept growing during the exposure period. Sofalcone prevented the increase in the GSH-Px activities in response to insecticides compared to independent treatment with insecticides (Fig. 8A, B). A significant increase in the production of hydrogen peroxide was observed in the larvae treated with chlorpyrifos for 48 or 72 h. A similar increase in the H2O2 levels was found after exposure to lambda-cyhalothrin. When challenged with sofalcone and insecticides, the insecticide-induced increase in the production of hydrogen peroxide was dramatically inhibited in the S. exigua larvae (Fig. 8C, D). Like the GSH-Px activity and H2O2 content, MDA also accumulated under chlorpyrifos stress for 24, 48, or 72 h. Compared to the control, lambda-cyhalothrin treatment elevated the MDA content at 6, 24, 48 or 72 h. Similarly, co-treatments with sofalcone and lambda-cyhalothrin or chlorpyrifos dramatically decreased the MDA content (Fig. 8E, F). These results confirm that sofalcone blocked the insecticide-induced expression of detoxification genes through the ROS-dependent CncC/Maf in S. exigua.

Fig. 8: Sofalcone decreased the ROS content when exposed to insecticides.
figure 8

The activity of GSH-Px under the stress of chlorpyrifos (A) and lambda-cyhalothrin (B) with or without sofalcone was assessed by using a GSH-Px assay kit. n = 5. A Hydrogen peroxide assay kit was used to determine the activity of Hydrogen peroxide after exposure to chlorpyrifos (C) and lambda-cyhalothrin (D) with or without sofalcone. n = 5. The content of MDA exposed to chlorpyrifos (E) and lambda-cyhalothrin (F) with or without sofalcone was analyzed by using an MDA assay kit. The results are presented as the mean ± SD (n = 5). Blue asterisks on the standard error bars indicate significant differences between the treatment with insecticide only and control. Red asterisks represent significant differences between the combined treatment of insecticides and sofalcone and the treatment of insecticides alone. The significant difference was analyzed using a Student’s t-test (p < 0.05).

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Synergistic effects of sofalcone with insecticides against S. exigua

To identify the synergistic effects of sofalcone on the S. exigua response to insecticides, sofalcone was coapplied with chlorpyrifos or lambda-cyhalothrin to third-instar larvae of the susceptible and resistant strains of S. exigua. The results showed that sofalcone significantly reduced the resistance of S. exigua to chlorpyrifos and lambda-cyhalothrin by 3.36- and 3.52-fold, respectively, whereas the mortalities of susceptible strain were not markedly increased by the cotreatment with sofalcone and insecticide compared to the treatment with insecticide alone (Table 1). Treatment with sofalcone alone did not affect the survival rate of larvae (Supplementary Fig. 8). These results demonstrate that sofalcone can be used as a synergist to increase the insecticidal effect of insecticides for agricultural and household use.

Table 1 Synergism of sofalcone on the toxicity of insecticides against susceptible and resistant strains of S. exigua
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Discussion

To cope with the stress of various toxic xenobiotics from the environment, insects have evolved a complex detoxification system, including cytochrome P450s, glutathione S-transferases and esterases. Increased expression of CYP genes has been proven to be implicated in resistance to insecticides in insects31. For example, the CYP6BQ genes are constitutively overexpressed in a deltamethrin-resistant strain of T. castaneum and result in resistance to this compound18. Similarly, transcriptional upregulation of four P450 genes CYP9M10, CYP9J34, CYP9J40 and CYP6AA7 leads to permethrin resistance in Culex quinquefasciatus32,33. In this study, all these CYP genes (CYP321A8, CYP321A9, CYP321A16, CYP321B1, CYP9A11 and CYP9A27) were upregulated in the larvae of S. exigua upon exposure to lambda-cyhalothrin and chlorpyrifos. Our previous study has shown that the six CYP genes are constitutively overexpressed in the resistant strain used in this paper, and three of the six genes, CYP321A8, CYP321A16 and CYP332A1 have been proven to be associated with chlorpyrifos resistance13,34. Together with the results of our study, these findings illustrate that overexpression of multiple CYP genes is involved in resistance to insecticides in insects. Insect GSTs are a super-family of multifunctional enzymes that are involved in insecticide resistance11,35,36. In N. lugens, four GST genes, GSTs1, GSTs2, GSTe1, and GSTm1, are overexpressed in the imidacloprid-resistant strain, and the RNAi test indicates that the four GST genes contribute to the development of imidacloprid resistance37. Similarly, GSTe2 and GSTd1 play an important role in detoxification of thiamethoxam and fenpropathrin in Asian citrus psyllid Diaphorina citri38. Here we showed that GSTe2, GSTe6, GSTe14 and GSTo2 were significantly upregulated when exposed to insecticides. Our previous work has shown that GSTe6 and GSTo2 are significantly upregulated in the resistant strain, and confer resistance to chlorpyrifos and cypermethrin39. These data suggest that enhanced expression of GST genes is associated with the development of insecticide resistance in insects. Carboxylesterases (CarEs) are important detoxification enzymes, which play a crucial role in the development of insecticide resistance in agricultural pests40. For example, upregulation of CarE genes results in indoxacarb resistance in Spodoptera litura41 and overexpression of CarE8 gene confers resistance to beta-cypermethrin and phoxim in Plutellaxylostella42. In this paper, we revealed that both lambda-cyhalothrin and chlorpyrifos treatments significantly increased the expression levels of two CarE genes, CarE1 and CarE5. These results suggested that the two CarE genes might play important roles in the detoxification of insecticides and the development of insecticide resistance in S. exigua. These studies, together with our findings, clearly demonstrate that upregulated expression of multiple detoxification genes confers resistance to insecticides in insects.

Although the importance of detoxification enzymes in conferring resistance to insecticides, the molecular mechanisms underlying the regulation of detoxification genes in insects are poorly understood43. In this study, our results showed that the binding site of CncC/Maf was present in the promoter region of detoxification genes, which were upregulated after exposure to insecticides. Furthermore, we determine that transcription factors CncC/Maf regulate the expression of CYP321A9 gene. Our previous studies have proven that CncC/Maf are constitutively overexpressed in the resistant strain used in this study, and regulate the expression of multiple CYP and GST genes involved in insecticide resistance in S. exigua13,30,34,39. Here, the increase in the expression of CncC and Maf was also observed after exposure to insecticides. These results authenticate that transcription factors CncC/Maf mediate the expression of a series of detoxification genes associated with insecticide resistance in S. exigua. Similarly, CncC/Maf modulate the overexpression of CYP6BQ genes, which are responsible for deltamethrin resistance in T. castaneum44, and increase the expression of CYP392A28, CYP391B1 and CYP391A1, and result in fenpropathrin resistance in T. cinnabarinus17. Together these findings determine that CncC/Maf are the key regulators of various detoxification genes involved in insecticide resistance in insects. In mammals, the Nrf2 is activated during the redox changes and regulates the expression of antioxidant and detoxifying enzymes to maintain homeostasis45. Our previous work has shown that ROS activates the CncC/Maf pathway and enhances the expression of GST genes after exposure to insecticides30. In this study, both lambda-cyhalothrin and chlorpyrifos increased the ROS content, indicating that ROS mediates the expression of detoxification genes by the CncC/Maf pathway in S.exigua. Similarly, in S. litura, ROS elevates the transcriptional levels of CncC and GSTe1 when challenged with chlorpyrifos46. These findings determine that ROS-activated CncC/Maf regulating the expression of detoxication genes in response to insecticides is conserved in insects47,48. Therefore, these transcription factors can serve as new target genes to screen inhibitors for pest control.

Insecticide application remains currently the major method for pest control. However, insect pests have developed serious resistance to multiple insecticides based on the Arthropod Pesticide Resistance Database (APRD 2016). Therefore, new ideas and methods are needed to delay insecticide resistance and extend the life of existing insecticides. In this study, a high-throughput cell screening platform for quickly identifying the inhibitors of CncC/Maf was established. Based on the platform, we found that sofalcone inhibited the insecticide-induced promoter activities of target genes of CncC/Maf in sf9 cells. Furthermore, our results revealed that sofalcone reduced insecticide resistance via inhibition of the expression of CncC/Maf-dependent detoxification genes in S. exigua larvae (Fig. 9). Sofalcone belongs to chalcone compounds, and its chemical structure is shown in Supplementary Fig. 9 according to these databases49,50,51. These results demonstrate that inhibitors of CncC/Maf can serve as synergists for insecticide resistance management. Although a cell line expressing ARE-luciferase is utilized to screen natural inhibitors of Nrf2 pathway for controlling the chemoresistance of cancer cells22, a similar approach has not been reported in insects. Such knowledge is important as it can help us establish cell screening platforms for the inhibitors and activators of the other transcription factors. Traditional synergists are often enzyme-specific inhibitors and their specificity limits their widespread application. For example, piperonyl butoxide (PBO), S,S,S-tributyl phosphorotrithioate (DEF) and diethyl maleate (DEM) are used as the enzyme inhibitors of P450s, CarEs and GSTs, respectively2,52,53. In this study, the insecticide-induced enzyme activities of P450s, CarEs and GSTs were significantly inhibited by sofalcone, suggesting that inhibitors of CncC/Maf have a broad-spectrum synergistic effect with insecticides in insect pests, which facilitates their widespread application. In this study, our results demonstrate that inhibition of transcriptional regulation of detoxification genes contributes to insecticide resistance management. Based on the innovative strategy, other inhibitors, and nucleic acid pesticides (dsRNA) can also be used to control insect pests. This method could delay insecticide resistance, extend the life of existing insecticides, and reduce pesticide usage and economic costs.

Fig. 9: Schematic of sofalcone as a synergist for insecticide management.
figure 9

Insecticides induce ROS generation which activates the CncC/Maf pathway responsible for the expression of multiple detoxication genes associated with insecticide resistance in insects. Sofalcone can increase larval susceptibility to insecticides by inhibiting the activity of the ROS/CncC-dependent detoxifying enzyme and downregulating the expression levels of detoxification genes in insect pests.

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In conclusion, we confirm that CncC/Maf play a vital role in regulation of detoxification genes associated with insecticide resistance in a lepidopteran pest, S. exigua. A cell screening platform for identification of the inhibitors of CncC/Maf was developed. We determine that sofalcone can be used as a CncC/Maf inhibitor in vitro and in vivo. Furthermore, we present evidence that sofalcone significantly inhibits the insecticide-induced expression levels and enzyme activities of ROS/CncC-mediated detoxification genes and further can greatly improve the susceptibility of S. exigua larvae to insecticides. Therefore, CncC/Maf inhibitors can function as broad-spectrum synergists in preventing insecticide resistance in insect pests. Our study provides an innovative and universal strategy to increase insecticidal activity and decrease application doses for sustainable crop protection based on the suppression of transcriptional regulation of detoxification genes.

Methods

Insects

The susceptible strain of S. exigua used in this study was obtained from Wuhan Kernel Bio-pesticide Company, Hubei, China. The resistant strain of S. exigua was collected in 2016 from Welsh Onion, Allium fistulosum, in Huizhou, Guangdong province, China.

According to our previous method54, the resistant strain was selected for continuous generations by exposing neonate larvae to chlorpyrifos at a LC70 concentration. Larvae were reared on an artificial diet at 25 °C under a 16-h light/8-h dark photoperiod with a relative humidity of 60 ± 5%26,55.

Chemicals

Lambda-cyhalothrin (95% TG) and chlorpyrifos (96.5% TG) were provided by Jiangsu Yangnong Chemical Co., Ltd, and Nanjing Red Sun Chemical Co., Ltd, respectively. Sofalcone, 4-hydroxychalcone and 2’-hydroxy-4’-methoxychalcone were purchased from Shanghai Merrill Co., Ltd. Lsoliquiritigenin, 2’-hydroxy-4,4’,6’-trimethoxychalcone, trans-chalcone, cianidanol, 4-chlorochalcone, 4-nitrochalcone, chalcone and 4-fluorochalcone were purchased from Sigma-Aldrich. Halofuginone and 4-methoxychalcone were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

Sample preparation

Preliminary experiments were performed to identify the dose of synergists that showed no detrimental effects on 3rd instar larvae. One hundred milligrams per liter of sofalcone had no effects on larval survival and this concentration was used in this study. The leaf dip method was used for sample preparation according to our previous studies2,56. The third-instar larvae of the resistant strain were transferred into plastic Petri dishes containing cabbage leaf discs supplemented with LC30 concentration of insecticide (743 mg/L chlorpyrifos or 1048 mg/L lambda-cyhalothrin) with or without sofalcone and leaf discs that were treated with 0.1% Triton X-100 were used as the control. At least 20 third-instar larvae were exposed to each treatment. Each replicate contained four larvae and five replicates were prepared for each treatment. After 48 h, the larvae were collected and stored at −80 °C for RNA isolation.

RNA and DNA extraction

Total RNAs were isolated from the third-instar larvae of S. exigua using a TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. The quality and quantity of total RNA were evaluated using a NanoDrop Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE) and agarose gel electrophoresis, respectively. The cDNAs were synthesized using the HiScriptTM Q Select RT SuperMix Kit (Vazyme, Nanjing, China) according to the standard protocol. An Insect DNA Kit (Omega, USA) was utilized to extract the genomic DNA of larvae.

Cloning of ABCs and CarEs

According to the transcriptome and genome data of S. exigua27,57, putative sequences of one ABC and two esterase genes (ABCB2, CarE1 and CarE2) were obtained, and the sequences of these genes (MK275645- MK275647) were verified by gene cloning and sequencing. All primers are shown in Supplementary Table 1.

Quantitative real-time PCR (RT-qPCR)

RT-qPCR was performed using an ABI 7500 Fast Real-Time PCR System (Applied Biosystems). The primers were designed using Primer Premier 5 software (Supplementary Table 2). A 20 μL reaction mixture consisting of 10 μL of AceQ qPCR SYBR Green Master Mix kit (Vazyme, China), 1 μL of cDNA template, 0.8 μL of 10 μM forward and reverse primers was carried out. The PCR program was: 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 40 s. Melting curves were added to determine the specificity of PCR products. β-Actin and GAPDH served as housekeeping genes58. All RT-qPCR analyses were performed in five biological replicates. The 2−∆∆Ct method was utilized to calculate the relative expression levels of genes59. All methods and data were confirmed to follow the guidelines for the minimum information for publication of RT- qPCR experiments60.

Cloning and sequencing the 5’-flanking regions

Four restriction enzymes were used to digest the genomic DNAs for generating four pools according to the operation manual of the Universal Genome Walker Kit (Clontech, Palo Alto, CA, USA). The digested DNA fragments were ligated to Genome Walker adapters using T4 DNA ligase. Specific primers were designed to amplify the targeted sequences using LATaq polymerase (Takara, Japan). All primers are shown in Supplementary Table 3. The PCR products were ligated into the PMD-19 T vectors (Takara, Japan) and sequenced. The online software JASPAR (Relative profile score threshold > 80%) and ALLGEN (Maximum matrix dissimilarity rate >15%) were used to predict the transcription factor binding sites61,62.

Luciferase reporter assays

To screen the inhibitors of transcription factors CncC/Maf, the CncC/Maf binding sequence was subcloned into a CYP321A8-core-PGL3 vector containing the CYP321A8 core promoter at the Xho Ι and Hind III restriction enzyme sites. The CYP321A8- (−142/-1) plasmid was used as the template13. To identify whether CncC and Maf regulate the expression of CYP321A9, the upstream sequence of CYP321A9 gene (about 1200 bp) was ligated into the PGL3-Basic vector and the ORF of CncC and Maf was cloned into the pIB/V5-His vector. All primers are displayed in Supplementary Table 4.

Sf9 cells were cultured in sf-900 II SFM (Life Technologies, Carlsbad, USA) medium at 27 °C and an MTT assay Kit (Jiancheng, Nanjing, China) was used to evaluate the cytotoxicity according to the manufacturer’s protocol. Briefly, the cells (2×105 cells/mL) were seeded in 96-well culture plates and treated with sofalcone (1.25-10 μM) for 36 h. Then, the MTT was added into the cells and incubated at 27 °C for 4 h. Cell viability was measured according to manufacturer’s instructions.

Sf9 cells were maintained in 24-well cell plates at a density of 4 × 105 cells. Then the 1 μg pGL3-CncC-Core promoter plasmid and 0.02 μg pRL-CMV were co-transfected to the cells using 2 μL FUGENE transfection reagent. After 16 h post-transfection, natural compounds (2.5 μM), chlorpyrifos (100 μM), Lambda-cyhalothrin (100 μM), or an equal volume of DMSO (control) was added. The cells were pretreated with 2.5 μM sofalcone for 4 h before the insecticide treatment. Similarly, 0.5 μg CYP321A9 promoter construct, 0.5 μg expression plasmid and 0.02 μg control pRL-CMV were co-transfected to the cells with the help of 2 μL FUGENE transfection reagent. Finally, the cells were collected after 48 h and luciferase activities were measured according to our previous method39. Each experiment was replicated four to six times.

Enzyme assays

Twenty 3rd-instar larvae were homogenized and microsomal fraction was obtained as described previously63. Each sample contained four larvae and five samples were prepared for each treatment. P450 monooxygenase activities were identified by measuring thoxycoumarin-O-deethylase (ECOD) activities on microsomal fractions as described previously by De Sousa et al.64. The microsomal fraction was resuspended and protein content was identified by the Bradford method63. The fluorescence was measured in a SpectraMax M5 multimode reader at 380 nm excitation, 460 nm emission and 30 °C for 15 min. P450 activities were expressed as mean picomoles of 7-OH per mg of microsomal protein/min ± SD.

GST enzyme activity was determined by measuring 1-chloro-2, 4-dinitrobenzene (CDNB) activity assay as described previously by Yang et al.65. 10 μL enzyme solution was mixed with 100 μL 1.2 mM CDNB (Sigma-Aldrich, USA) and 100 μL 6 mM glutathione (Sigma-Aldrich, USA) in each well. Enzyme activities were measured in a SpectraMax M5 multimode reader at 340 nm for 10 min (25 °C). Enzyme activities were expressed as micromole glutathione conjugate per min per mg of protein.

Esterase activity was measured using a-naphthyl acetate (a-NA) as a substrate according to the procedures developed by Yang et al.65. 10 μL enzyme solution and 200 μL substrate solution (0.1 mL 100 mM a-NA, 10 mg Coomassie Brilliant Blue G-250, and 5 mL 0.2 M pH 6.0 phosphate buffer) were added and mixed in each well. Enzyme activity was measured in a SpectraMax M5 multimode reader at 450 nm for 10 min (27 °C). EST activity is presented as nmole naphthol min-1 mg protein-1.

Hydrogen Peroxide, MDA and GSH-Px assays

The hydrogen Peroxide assay was performed with a hydrogen Peroxide assay kit (Jiancheng, Nanjing, China). Twenty 3rd-instar larvae were homogenized on ice in a homogenization buffer, and centrifugated at 10,000 × g for 10 min. Each sample contained four larvae and five samples were prepared for each treatment. One milliliter of supernatant solution was mixed with solution 1 and solution 2. The quantity of hydrogen peroxide was measured at 405 nm using a SpectraMax M5 multimode reader. The content of hydrogen Peroxide was presented as μmol/g protein.

An MDA assay kit (Jiancheng, Nanjing, China) was used to identify the MDA amount. The larvae were homogenized in PBS buffer on the ice. After the homogenization, the samples were centrifuged at 12,000 rpm at 4 °C for 15 min. According to the manufacturer’s protocol, MDA content was detected at 532 nm using a SpectraMax M5 multimode reader. Results were expressed as μmol/g protein.

Similarly, the GSH-Px activity was quantified by measuring the decrease of glutathione at 412 nm using H2O2 as substrate following the manufacturer’s guidebook (Jiancheng, Nanjing, China) by a microplate reader. The results of GSH-Px activity were expressed as μmol/g protein.

Toxicological bioassay and synergism assay

The toxicity of insecticides to S. exigua was assayed by the leaf dip method as described previously56. Leaf discs cut from the cabbage were dipped into insecticide suspensions for 30 s. At least 30, 3rd-instar larvae were needed for each treatment and three independent assays were repeated. 100 mg/L of sofalcone had no effects on larval survival and this concentration was used in synergism assays. POLO-Plus Software 2.0 (Leora Software, Petaluma, USA) was used to estimate the LC50 values and 95% fiducial limits (FLs). Synergism ratio was obtained by analyzing differences between LC50 of insecticide alone and LC50 of insecticide with the synergist66.

Statistical analysis

Statistical analysis was carried out using SPSS 16.0 software (SPSS Inc., Chicago, USA) and the data were presented as mean ± SD (standard deviation). Significant differences were calculated using a one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test or Student’s t-test. The level of significance was set at p < 0.05.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.