Skip to main content
Download PDF

Two independent regulatory mechanisms synergistically contribute to P450-mediated insecticide resistance in a lepidopteran pest, Spodoptera exigua

BMC Biology volume 23, Article number: 122 (2025) Cite this article

Abstract

Background

Cytochrome P450 enzymes play a pivotal role in the detoxification of plant allelochemicals and insecticides. Overexpression of P450 genes has been proven to be involved in insecticide resistance in insects. However, the molecular mechanisms underlying the regulation of P450 genes in insects are poorly understood.

Results

Here, we determine that upregulation of CYP321B1 confers resistance to organophosphate (chlorpyrifos) and pyrethroid (cypermethrin and deltamethrin) insecticides in the resistant Spodoptera exigua strain. Enhanced expression of transcription factors CncC/Maf contributes to the increase in the expression of CYP321B1 in the resistant strain. Reporter gene assays and site-directed mutagenesis analyses confirm that a specific binding site is crucial for binding CncC/Maf to activate the expression of CYP321B1. In addition, creation of a new binding site resulting from the cis-mutations in the promoter region of CYP321B1 in the resistant strain facilitates the binding of the POU/homeodomain transcription factor Nubbin, and further enhances the expression of this P450 gene. Furthermore, we authenticate that changes in both trans- and cis-regulatory elements in the promoter region of CYP321B1 act in combination to modulate the promoter activity in a synergistic manner.

Conclusions

Collectively, these results demonstrate that two distinct but synergistic mechanisms coordinately result in the overexpression of CYP321B1 involved in insecticide resistance in an agriculturally important insect pest, S. exigua. The information on mechanisms of metabolic resistance could help to understand the development of resistance to insecticides by other pests and contribute to designing effective integrated pest management strategies for the pest control.

Background

Cytochrome P450 enzymes play an important role in the adaptation to host plants and the detoxification of xenobiotics in insects [1,2,3]. Constitutive upregulation of P450 genes has shown to be associated with insecticide resistance in insects [4, 5]. Many genes belonging to the CYP4, CYP6, CYP9 and CYP12 families have been documented to be overexpressed and lead to insecticide resistance by enhancing the metabolic detoxification of them [2, 6]. For example, transcriptional upregulation of four P450 genes CYP9M10, CYP9J34, CYP9J40 and CYP6AA7 has been shown to confer resistance to permethrin in Culex quinquefasciatus [7, 8]. Similarly, overexpression of CYP6FU1 gene leads to deltamethrin resistance in Laodelphax striatellus [9] and the enhanced expression of CYP6BQ9 gene results in the majority of deltamethrin resistance in Tribolium castaneum [10]. However, the link between the genes belonging to the CYP321 subfamily and insecticide resistance remains largely unknown in insects.

The nuclear factor erythroid 2 related factor 2 (Nrf2) acts as a trans-regulatory factor to coordinate an evolutionarily conserved transcriptional activation pathway that mediates antioxidant and detoxification responses in laboratory mice [11,12,13]. Under basal (unstressed) conditions, Nrf2 is sequestered in the cytoplasm by Keap1, a Cul3-dependent ubiquitin ligase adaptor protein that targets Nrf2 for ubiquitination and proteasomal degradation in mammalian cells [14]. Oxidative stress and electrophiles disrupt the Nrf2-Keap1 interaction allowing Nrf2 to translocate to the nucleus where it dimerizes with the small Maf proteins; the heterodimers then bind to the antioxidant response elements (AREs) of detoxifying enzyme genes, including P450s, NAD(P)H: quinone oxidoreductase-1 (NQO1), GSTs, UDP-glycosyltransferase (UGT), and heme oxygenase 1 (HO-1) to initiate their transcription [15,16,17,18]. CncC (Cap ‘n’ Collar isoform-C), the insect ortholog of mammalian transcription factor Nrf2, as a heterodimer with Maf-S (muscle aponeurosis fibromatosis) is a central regulator of xenobiotic detoxification responses and contributes to the widespread overexpression of detoxification genes in insecticide-resistant strains of Drosophila melanogaster [17,18,19]. Transcription factors CncC and Maf also have been demonstrated as the key regulators for the expression of multiple P450 genes responsible for deltamethrin resistance in T. castaneum [20] and fenpropathrin resistance in Tetranychus cinnabarinus [21].

A few studies have shown that cis-acting mutations in the promoter region of P450 genes regulate the expression of these P450 involved in insecticide resistance in insects. For example, the mutations alternating TF binding site in the 5’-promoter core region lead to the constitutive overexpression of CYP6A2 gene and a TE insertion in the promoter region of CYP6G1 upregulate the expression of this P450 gene in D. melanogaster [19, 22, 23]. Similarly, the expansion of a dinucleotide microsatellite in the promoter region of CYP6CY3 enhances the transcriptional level of this P450 gene and results in the resistance to neonicotinoid insecticides in Myzus persicae [24]. However, whether cis- and trans-acting factors act in combination to regulate detoxification genes and if so, how these mechanisms interact remains poorly understood.

The beet armyworm, Spodoptera exigua, is a widely distributed polyphagous pest that seriously damages many cultivated crops including cabbage, soybean, cotton, and others [25]. The use of chemical insecticides remains currently the major method for the control of this pest. It has developed resistance to various groups of chemical insecticide classes, including chlorinated hydrocarbons, organophosphates, carbamates, pyrethroids, IGRs and others [26], understanding the molecular mechanisms of the regulation of resistance genes is very important. Our recent analyses have shown that enhanced CYP450 enzyme activity is involved in resistance to chlorpyrifos and pyrethroids insecticides in S. exigua, and many CYP450 genes are upregulated in the resistant strain. One of these, CYP321B1 (GenBank Accession No. KX443444.1) has 41-fold higher expression in the resistant strain and might confer resistance to chlorpyrifos and pyrethroid insecticides [27]. Here we report that constitutive overexpression of the CYP321B1 gene indeed results in resistance to chlorpyrifos and pyrethroid insecticides. Upregulation of CncC/ Maf and a cis-acting mutation in the promoter region jointly control the constitutive overexpression of CYP321B1 gene in a synergistic manner. Finding the molecular mechanisms underlying the regulation of detoxification genes will help us identify new target genes for pest control.

Results

Insecticide metabolism in vitro by recombinant CYP321B1

To investigate the functional role of CYP321B1 in insecticide metabolism in vitro, the recombinant protein was expressed in the Sf9 cells. Reduced CO-difference spectrum analysis revealed that recombinant CYP321B1 protein was successfully expressed as a good-quality functional enzyme (Fig. 1A). Furthermore, catalytic activity test with the ECOD substrate indicated that the recombinant protein efficiently catalyzed 7-ethoxy coumarin with a specific activity of 0.19 pmol min−1 pmol−1 protein (Fig. 1B). High-performance liquid chromatography (HPLC) was utilized to evaluate the capacity of recombinant CYP321B1 for degrading insecticides including chlorpyrifos, cypermethrin and deltamethrin. After incubating insecticides with the microsome for 1.5 h, 30.7 ± 3.7% and 43.6 ± 4.1% of chlorpyrifos, 13.1 ± 1.0% and 30.9 ± 2.2% of cypermethrin, and 19.1 ± 2.3% and 39.7 ± 3.1% of deltamethrin were degraded by 100 and 200 μg recombinant CYP321B1 in the presence of NADPH, respectively (Fig. 1C, D and E). No depletion in insecticide was found when recombinant CYP321B1 was incubated with these insecticides without NADPH. Only trace levels of degradation of these insecticides when the proteins of non-transfected Sf9 cells were used. These results suggest that overexpression of CYP321B1 is associated with resistance to chlorpyrifos, cypermethrin and deltamethrin in S. exigua.

Fig. 1
figure 1

Upregulation of CYP321B1 gene involved in resistance to chlorpyrifos, cypermethrin and deltamethrin. A Carbon monoxide difference spectra were used to assess the integrity of the recombinant CYP321B1 protein. B Enzyme activity of recombinant P450 protein was evaluated by using a model fluorescent substrate (ECOD). Error bars display SD. Chlorpyrifos (C), cypermethrin (D) and deltamethrin (E) metabolism by recombinant CYP321B1 protein. Error bars represent the mean ± SD values (n = 3). Different letters on the right of error bars indicate significant differences based on ANOVA with Tukey’s HSD multiple comparison test (p < 0.05)

Full size image

Overexpression of CYP321B1 enhanced the tolerance of D. melanogaster to insecticides

To identify whether the overexpression of CYP321B1 was involved in insecticide resistance in vivo, the GAL4/UAS system was utilized to generate the transgenic D. melanogaster flies expressing CYP321B1 (actin5C-CYP321B1). The expression level of CYP321B1 in F1 progeny was confirmed by RT-PCR (Fig. 2A and Additional file 1: Fig. S2) and RT-qPCR (Fig. 2B). When the transgenic flies expressing CYP321B1 (actin5C > CYP321B1) were exposed to 0.05 mg/L chlorpyrifos for 3 days, the mortality was 60.00% compared to 98.50% mortality in the control flies (Fig. 2C). The mortalities of flies expressing CYP321B1 exposed to 0.6 mg/L cypermethrin were 53.33% compared to the control flies which exhibited mortality of 96.67% (Fig. 2D). A significant difference was also observed after exposure to 0.4 mg/L deltamethrin, with flies expressing CYP321B1 displaying 50.00% mortality compared with 86.67% for controls (Fig. 2E). These results demonstrate that overexpression of CYP321B1 confers resistance to the three insecticides in S. exigua.

Fig. 2
figure 2

Susceptibility of transgenic flies expressing CYP321B1 to chlorpyrifos, cypermethrin and deltamethrin. A PCR was used to measure the mRNA level of CYP321B1. B The transcriptional level of CYP321B1 was analyzed by qRT-PCR. The 2.−∆∆CT method was used for quantification of the gene expression level. Student’s t-test was used for statistical analysis. Asterisks on the standard error bars indicate significant differences compared with the control. A value of P < 0.05 was considered statistically significant. Susceptibility of transgenic flies expressing CYP321B1 to chlorpyrifos (C), cypermethrin (D) and deltamethrin (E) was investigate. The progeny from the cross between the w1118 and Act5C-GAL4 lines was used as a control (actin-5C). Three independent biological replicates were carried out and the mean ± SD was displayed. A significant difference in survival between transgenic flies expressing CYP321B1 and controls is marked with asterisks on the right of error bars (Student’s t-test, p < 0.05)

Full size image

Homology modeling and docking

Homology modeling and molecular docking was utilized to determine the possible interactions of CYP321B1 protein with insecticides. These results revealed that this P450 protein were tightly bound to chlorpyrifos, cypermethrin and deltamethrin. The free binding energies of CYP321B1-chlorpyrifos, CYP321B1-cypermethrin and CYP321B1-deltamethrin complexes were −6.33, −9.70 and −9.07 kcal/mol, respectively (Fig. 3A, B and C). Chlorpyrifos and cypermethrin were docked within 14.2 Å and 14.3 Å from heme iron by hydrophobic interactions and hydrogen bonds (Fig. 3A, B and Additional file 1: Table S2). Deltamethrin was docked within a distance of 14.0 Å from heme iron by hydrophobic interactions (Fig. 3C and Additional file 1: Table S2). There was only one hydrogen bonding interaction between CYP321B1 residue ARG-104 and chlorpyrifos with a distance of 2.5 Å (Fig. 3A). One hydrogen bonding interaction existed between residue THR-373 (2.2 Å) of CYP321B1 and cypermethrin (Fig. 3B). No hydrogen bonding interaction was found between CYP321B1 and deltamethrin (Fig. 3C). These results indicated that these bonds are discernible.

Fig. 3
figure 3

Homology modeling and insecticides docking. Binding modes of chlorpyrifos (A), cypermethrin (B) and deltamethrin (C) to CYP321B1 protein. The residues of P450 protein interacting with insecticides were represented in blue sticks

Full size image

Overexpression of CncC/Maf regulates the expression of CYP321B1 gene

To explore the regulation mechanism of overexpression of CYP321B1 in the resistant strain, a 1380 bp upstream promoter sequence was identified by genomic walking approach. Predictive analysis of transcription factor binding sites showed that the promoter region of CYP321B1 gene contains the binding sites for EcR, P53, CncC/Maf, Hb, BR–C, Kr, GATA and Dfd (Fig. 4A). Our previous study has shown that the mRNA levels of CncC and Maf were dramatically upregulated in the resistant strain used in this study, whereas no significant differences in the expression levels of P53, EcR, USP, Dfd, BR–C, and Kr between the resistant and susceptible strains were observed [27]. These results imply that the expression of CYP321B1 may be mediated by transcriptional factors CncC and Maf. To further verify this hypothesis, the CYP321B1 promoter region was ligated into the reporter gene vector and co-transfected into Sf9 cells with the expression vectors containing the open reading frames of CncC and Maf (Fig. 4B). Compared to the control, the promoter activities of CYP321B1 were increased by 1.47- and 2.80-fold in the presence of CncC and Maf, respectively. Co-expression of CncC and Maf increased CYP321B1 promoter activity even more, 4.64-fold. These results demonstrate that upregulated CncC and Maf enhance the expression of CYP321B1 gene in the resistant strain.

Fig. 4
figure 4

Overexpression of CncC and Maf increase the promoter activity of CYP321B1. A Prediction of transcription factor binding sites in the promoter region of CYP321B1. The nucleotides are numbered relative to the translation start site (ATG) indicated by + 1, with sequence upstream of it preceded by “- “. The transcription start sites (Inr) and putative transcription factor binding sites are underlined. B Promoter activities of CYP321B1 in the presence of CncC and/or Maf. The data are presented as the mean ± SD. Different letters above the bars indicate significant differences based on ANOVA followed by Tukey’s HSD multiple comparison test (p < 0.05)

Full size image

Identification of CncC/Maf binding sites in the CYP321B1 promoter region

To determine the relevant CncC/Maf binding sites, the promoter region of the CYP321B1 gene was divided into different size fragments to construct promoter truncation, and the fragments were ligated into reporter gene vector (Fig. 5A). Each construct containing truncation of the CYP321B1 promoter was co-transfected with CncC and Maf constructs. The promoter activities of full-length promoter (Full) and truncation −258 to −1 bp (T3) increased by 4.4- and 4.9-fold respectively in the presence of CncC and Maf (Fig. 5B). In contrast, the promoter activities of truncation −1463 to-1234 bp (T1), truncation −1253 to-238 bp (T2) and truncation core promoter (T4) were not significantly changed by the CncC and Maf. These results confirm that the effective CncC/Maf binding site was present in the region between −258 to −1 bp, which is consistent with the prediction of CncC/Maf binding site (AATGACAACGCAAAA) located in the region.

Fig. 5
figure 5

CncC and Maf regulate the expression of CYP321B1 by binding to a specific site present in the region between −258 to −1 bp. A Schematic representation of the CYP321B1 promoter truncations. B Analysis of CYP321B1 promoter truncations. The cells were harvested at 48 h after transfection and the luciferase activity was quantified. Significant differences in the luciferase activity were identified using Student’s t-test and are indicated using an asterisk. A value of p < 0.05 was considered statistically significant

Full size image

Furthermore, we introduced mutations into the CncC/maf binding site located in the CYP321B1 region (Fig. 6A). In the construct M-1, the binding site AATGACAACGCAAAA was mutated to AAgactAAtttAAAA based on the wild-type WT-1 construct. Similarly, the construct WT-2 contained the binding site AATGACAACGCAAAA which was replaced with AAgactAAtttAAAA in the construct M-2. A significant decrease in the promoter activity of the construct M1 was observed when compared with that of the wild-type WT-1 (Fig. 6B). Similarly, compared to the wild-type WT-2, the promoter activity of the construct M2 was markedly reduced by 8.6-fold. These results demonstrate that the specific CncC/Maf binding site is responsible for the activation of CYP321B1 promoter.

Fig. 6
figure 6

Mutations in CncC/Maf binding sites block the ability of these transcription factors to regulate the expression of CYP321B1. A Schematic representation of mutated CYP321B1 promoter constructs. B Analysis of CYP321B1 promoter with the mutation of CncC/Maf binding sequence. The CYP321B1 promoter constructs with or without mutations were transfected with CncC/Maf constructs into sf9 cells. The cells were harvested at 48 h after transfection, and the luciferase activities were measured. The data were shown as the mean ± SD of three independent transfections. The results were analyzed using Student’s t-test and significant differences (p < 0.05) are indicated with an asterisk

Full size image

Cis-acting mutations in the promoter region enhanced the expression of CYP321B1 gene

To investigate the genetic variation degrees of the promoter region of CYP321B1 derived from the resistant and susceptible strains, the upstream promoter sequences of CYP321B1 derived from six resistant and six susceptible individuals were cloned and sequenced. Phylogenetic analysis revealed that the twelve promoter sequences were clustered into three different groups (Fig. 7A). Among them, all six sequences derived from resistant insects are distributed in Type 1, and most of the sequences cloned from susceptible insects are grouped into Type 3. Subsequently, alignment of promoter sequences derived from the resistant and susceptible strains identified several mutations that differentiate the strains (Additional file 1: Fig. S1). To confirm whether these mutations contribute to the increase in the promoter activity of CYP321B1 in the resistant strain, the promoter sequences from the resistant and susceptible strains were constructed into the promoter vector. Compared to the susceptible strain, a 2.1-fold increase in the promoter activity of CYP321B1 from the resistant strain was observed (Fig. 7B), suggesting that higher promoter activity is also one of the reasons for the overexpression of CYP321B1 in the resistant strains.

Fig. 7
figure 7

Cis-acting mutations in the promoter region enhanced the expression of CYP321B1 gene. A Phylogenetic relationship of CYP321B1 promoter sequences from the susceptible and resistant strains. The phylogenetic tree was inferred using maximum likelihood using MEGA5. Nodes with distance bootstrap values (1000 replicates) are shown. B Analysis of the activity of the CYP321B1 resistant promoter (DNA5’-R) and susceptible promoter (DNA5’-S). Error bars display SD. Letters to the right of bars denote significant differences at p < 0.05 (ANOVA with post-hoc Tukey HSD). C The activity of progressive 5’ deletion constructs of CYP321B1 promoter from the susceptible and resistant strains. Error bars display SD. Letters to the right of bars denote significant differences at p < 0.05 (ANOVA with post-hoc Tukey HSD). D The luciferase activity of progressive 5’-deletion constructs from −353 to −167 of CYP321B1 promoter. The average relative luciferase activity and standard errors of three independent experiments are presented. Different letters on the right of error bars indicate significant differences based on ANOVA with Tukey’s HSD multiple comparison test (p < 0.05)

Full size image

To further identify the region of the promoter that is responsible for the differences in promoter activity between the resistant and susceptible strains, a series of truncations (−1463 ~ −1131, −1130 ~ −689, −688 ~ −354, −353 ~ −168 and −167 ~ −1) were constructed. As shown in Fig. 7C, the transcriptional activities of P (−1463/−1), P (−1130/−1), P (−688/−1) and P (−353/−1) showed significant differences between the resistant and susceptible strains. In contrast, no difference in the promoter activity of P (−167/−1) was observed. These results indicate that the mutations contributing to the difference in the promoter activity were located in the fragment located between – 353 to – 167 bp. To further identify this region, two additional truncations (−258/−1 and −210/−1) were constructed and assayed in Sf9 cells. The luciferase assays showed that these two truncations remain the obvious difference in the promoter activity in the resistant and susceptible strains, suggesting that the mutations responsible for the difference in the activity of CYP321B1 promoter from the resistant and susceptible strains were located in the fragment, −258 ~ −210 (Fig. 7D). Subsequently, analysis of resistant and susceptible promoter sequences revealed that five mutation sites were found in the fragment from −258 to −210 (Fig. 8A). To verify if these mutations can affect promoter activity, P (−258/−1)-R was selected as standard and two mutation constructs P (−258/−1)-R-M1 and P (−258/−1)-R-M2 were generated to recover these mutations. This result showed that the reporter activity of the mutant construct (M2) was significantly decreased compared with the control construct P (−258/−1)-R, indicating that the cis-acting element resulting from mutation at the M2 site can significantly upregulate the expression of CYP321B1 gene (Fig. 8B). However, the mutant construct M1 has on significant effect on the promoter activity. Surprisingly, the M2 mutation is located in a predicted binding site of the POU/homeodomain transcription factor, Nubbin, indicating that this transcription factor may regulate the overexpression of CYP321B1 in the resistant strain.

Fig. 8
figure 8

Creation of a new binding site for the POU/homeodomain transcriptional regulator, Nubbin, resulting from the mutations in the CYP321B1 promoter region from the resistant strain, enhances the transcriptional level of this gene. A Alignment of CYP321B1 promoter sequences from the resistant and susceptible strains. Two mutation regions (M1 and M2) are marked under the corresponding nucleotides. B The luciferase activity of recovery mutation constructs in the region from −258 to −210. Data are the mean ± SD of three independent assays. Different letters on the right of error bars indicate significant differences based on ANOVA with Tukey’s HSD multiple comparison test (p < 0.05). C Transcription factor CncC/Maf and cis-acting mutation coordinately enhance the promoter activity of CYP321B1 gene. The promoter constructs (DNA5’-R and DNA5’-S) were transfected with or without CncC/Maf constructs into sf9 cells. Data are the mean ± SD of three independent assays. The data were analyzed using Tukey’s HSD. Letters a, b, c and d denote significant differences at p < 0.05

Full size image

Trans-acting factors CncC/Maf and cis-acting mutations synergistically contribute to the increase in the expression of CYP321B1 in the resistant strain

To authenticate whether the changes in both cis-acting elements and trans-acting factors coordinately control the expression of CYP321B1 gene, the CncC and Maf constructs, and the promoter constructs with (DNA 5'-R) or without the Nubbin binding site (DNA5'-S) were co-transfected and assayed in sf9 cells. The promoter constructs DNA5'-S co-transfected with empty vector pIB served as the control. Compared to the control, the DNA5'-R construct showed a 1.9-fold increase in the promoter activity in the absence of CncC and Maf, and then overexpression of CncC and Maf enhanced the promoter activity of DNA5'-S and DNA 5'-R by 4.3- and 8.4-fold, respectively (Fig. 8C). Amazingly, the increase in the promoter activity mediated by both cis-acting mutations and CncC/Maf overexpression (8.4-fold) were much greater than the sum of the individual effect (1.9-fold + 4.3-fold). These results demonstrate that overexpression of trans-acting factors CncC/Maf and cis-acting mutations in the promoter region synergistically regulate the overexpression of CYP321B1 gene in the resistant strain.

Discussion

Cytochrome P450s are the primary enzyme family that is associated with resistance to insecticides in insect species [28,29,30]. Although the importance of CYP450s in conferring resistance has been well known, the underlying transcriptional regulatory mechanisms of these P450 genes remain largely unknown. In the present study, we determine that upregulation of CYP321B1 results in resistance to chlorpyrifos, permethrin and deltamethrin in the resistant strain. Two transcription factors CncC and Maf were overexpressed in the resistant strain and we demonstrate that these regulate the expression of CYP321B1 by binding to one specific site in the promoter region of this P450 gene. In addition, we identify that cis-acting mutations in the promoter region of CYP321B1 create a cis-regulatory element that facilitates binding of a POU/homeodomain transcription factor Nubbin, enhancing the expression of this P450 gene. Altogether, these data demonstrate that the combined action of two genetically independent mechanisms synergistically results in the overexpression of CYP321B1 in the resistant strain of S. exigua (Fig. 9).

Fig. 9
figure 9

Schematic representation of trans-acting factors and cis-acting mutation involved in the overexpression of CYP321B1 gene conferring resistance to insecticides in S. exigua. In the resistant strain, constitutive overexpression of trans-acting factors CncC and Maf, and cis-acting mutations in the resistant promoter creating the binding site for the POU/homeodomain transcription factor Nubbin synergistically enhance the expression of CYP321B1 gene associated with insecticide resistance; In the susceptible strain, CncC and Maf only regulate the basic expression of CYP321B1 gene, and there is no binding site for Nubbin. Therefore, the expression level of CYP321B1 gene is low which makes the larvae susceptible to insecticides

Full size image

Insect P450s are known to play an important role in detoxifying insecticides and plant toxins [1, 31,32,33,34]. Constitutive overexpression of cytochrome P450 genes has been found in insecticide-resistant strains and has been proven to be involved in insecticide resistance [35,36,37]. For example, upregulation of CYP6G1 gene in D. melanogaster confers resistance to DDT and imidacloprid [38]. Similarly, the CYP6M2, overexpressed in a resistant population of A. gambiae, results in the resistance to DTT and pyrethroids [39]. Finally, overexpression of CYP6CM1 leads to the cross-resistance between imidacloprid and pymetrozine in Bemisia tabaci. A previous study has documented that CYP321B1 may play an important role in chlorpyrifos and β-cypermethrin detoxification in Spodoptera litura, however, the relevant metabolic analysis is not conducted [40]. In this study, transgenic strains of D. melanogaster expressing CYP321B1 showed higher levels of tolerance to chlorpyrifos, cypermethrin and deltamethrin than the control flies with the same genetic background. Furthermore, recombinant P450 protein metabolized these insecticides. Our results determine that the increased expression of CYP321B1 is associated with resistance to chlorpyrifos, cypermethrin and deltamethrin in vivo and in vitro. These results suggest that the functional roles of CYP321B1 in conferring resistance to insecticides are highly conserved in lepidopteran pests and will contribute to investigating the functional role of CYP321B genes in insecticide resistance in other pests. Simultaneously, these studies indicate that insect P450s exhibit significant flexibility in metabolizing structurally diverse insecticides belonging to different mode of action classes. The beet armyworm has developed high resistance and cross-resistance to insecticides [41], which may not only be caused by a certain CYP450. Although our previous study has reported that CYP321A8 confers cross-resistance to insecticides [27], a new P450 gene, CYP321B1, which not only metabolize pyrethroid insecticides but also degrade organophosphate chlorpyrifos is found in this study. These results suggest that CYP321B1 may be one of the reasons for the cross-resistance between organophosphate and pyrethroid insecticides, and can be used as a biomarker and target for management of S. exigua insecticide resistance in fields.

In mammals, Nrf2 is identified as an important regulation factor responsible for the activation of P450 genes involved in the detoxification of xenobiotic compounds [15]. Similarly, Nrf2 homolog, CncC and its heterodimer partner Maf regulate the expression of multiple P450 genes coding for enzymes that are associated with metabolism and detoxification of insecticides in insects. For example, CncC and Maf are constitutively overexpressed in a fenpropathrin-resistant T. cinnabarinus strain, and increase the expression of CYP391A1, CYP391B1 and CYP392A28, which result in the resistance to this insecticide [21]. Similarly, CncC and Maf enhance the expression of four cytochromes P450 genes (CYP6BJa/b, CYP6BJ1v1, CYP9Z25, and CYP9Z29), which are involved in imidacloprid resistance in Leptinotarsa decemlineata [42]. Our previous study has shown that the transcriptional levels of CncC and Maf are upregulated in the resistant strain used in this paper [27]. Here, we determine that CncC and Maf regulate the expression of CYP321B1 by binding to one specific regulatory element in the promoter region of this P450. In addition, we have previously shown that the increased expression levels of three p450 genes (CYP321A8, CYP321A16 and CYP332A1), which confer resistance to insecticides in S. exigua, are regulated by these two key regulatory factors, CncC and Maf [27, 43]. These data suggest that the functional roles of CncC/Maf in mediating the expression of a series of detoxification genes conferring resistance to insecticides were conserved in insects. Therefore, these transcription factors can serve as new target genes to screen for inhibitors of detoxification genes for pest control. In this study, the CncC/Maf binding site was determined by reporter gene assays and site-directed mutagenesis analyses. This result is very important for improving the accuracy of in silico prediction of the binding sites of these transcription factors in insects.

In this study, we also identify that cis-acting mutations in the promoter region of CYP321B1 from the resistant strain enhance the transcriptional level of this P450 gene. A few studies have documented that insertions/deletions or mutations in the promoter region contribute to insecticide resistance by increasing the expression of P450 genes. For example, a single nucleotide substitution in the promoter region of CYP9M10 enhances the expression of this gene in Culex quinquefasciatus [44]. Similarly, the changes of cis-regulatory elements result in the upregulation of CYP6P9a and CYP6P9b involved in resistance to pyrethroid insecticides in the major African malaria vector Anopheles funestus [45]. Finally, multiple cis-acting mutations in the promoter region lead to the overexpression of CYP6FU1, which is responsible for deltamethrin resistance in L. striatellus [46]. Together with the results of our study, these findings illustrate that changes in cis-acting mutations in the promoter region often confer resistance to insecticides by controlling the overexpression of the P450 genes associated with the metabolism of insecticides in insects. Meantime, these mutations can act as markers for identification of the frequency and distribution of resistance, and further design effective integrated pest management programmes. In this study, we indicate that creation of a new binding site for the POU/homeodomain transcriptional regulator, Nubbin, resulting from the mutation in the CYP321B1 promoter region from the resistant strain, enhances the transcriptional level of this gene compared with the susceptible strain. In D. melanogaster, Nubbin regulates the expression of genes that are involved in immune, differentiation, stress responses and metabolism [47]. Here, we showed that Nubbin may act as a regulator of P450 genes in S. exigua.

Although few studies have reported that cis- and/or trans-acting factors regulate the expression of P450 genes that are associated with insecticide resistance, how these regulatory mechanisms interact, is unclear. For example, a 15 bp insert in the promoter region of CYP6D1 enhances the transcriptional level of this P450 gene involved in resistance to pyrethroid insecticides in housefly [48]. Further studies showed that an unknown trans-acting factor located in chromosome 2 also results in the increased expression of CYP6D1, however, whether this unknown trans-acting factor and the 15 bp insert in the promoter region act in combination to regulate the expression of CYP6D1 has not been identified [49]. In this study, our results showed that the combined effect of cis- and trans-acting factors was much greater than the sum of their individual effects, suggesting that cis- and trans-acting factors regulate the expression of CYP321B1 in a synergistic manner. Altogether, these studies suggest that insect pests have evolved two independent regulatory mechanisms that work in concert to resist insecticides. The beet armyworm has developed high resistance to various groups of chemical insecticide classes, and even cross-resistance was found in the filed populations [41]. Although our previous study has indicated that CncC/Maf and mutations in the promoter region control the expression of CYP321A8 [27], new transcription factor and regulatory mechanism contributing to insecticide resistance is reported in this study. These results suggest that insect pests have evolved complex and diverse regulatory mechanisms that contribute to the adaptation of resistant populations to insecticides and there may be other regulatory mechanisms that need to be further explored.

Conclusions

In summary, the current study identified upregulation of CYP321B1 confers resistance to chlorpyrifos, cypermethrin and deltamethrin in the resistant strain. Furthermore, constitutive overexpression of transcription factors CncC /Maf is associated with the enhanced expression of CYP321B1 by binding to a special binding site in the promoter region of this P450 gene. In addition, cis-acting mutations in the promoter region of CYP321B1 creating a cis-regulatory element that recruits the binding of the transcription factor Nubbin, results in the upregulated expression of this P450 gene. Finally, these results present the evidence that cis- and trans-acting factors synergistically upregulate the expression of P450 gene conferring resistance to insecticides in an agriculturally important insect pest and provide us with more insights into the design of effective integrated pest management strategies for the pest control.

Methods

Insect strains

The Wuhan susceptible strain of S. exigua used in this study was obtained from Wuhan Kernel Bio-pesticide Company, Hubei, China and the chlorpyrifos resistant strain was collected from Allium fistulosum in Huizhou, Guangdong province, China [27]. According to our previous method [26], 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% [25].

DNA/RNA extraction and cDNA synthesis

Total RNAs were extracted from larvae using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. First-strand cDNAs were synthesized using the Supermo III RT Kit (BioTeKe, Beijing, China) according to the operation manual and stored at −25 °C. An Insect DNA Kit (Omega Bio-Tek Inc., Norcross, Georgia, USA) was utilized to isolate the S. exigua Genomic DNA.

Functional expression of CYP321B1 in Sf9 cells

CYP321B1 was expressed in Sf9 cells using a baculovirus expression system according to our previous approaches [27]. All the primers are shown in Additional file 1: Table S1. Empty pFastBacHTA vector served as a negative control. Bacmids were transfected into sf9 cells using FUGEGE transfection reagent (Promega, Madison, WI, USA), and virus titer was tested by plaque assay and qPCR [50]. Various multiplicity of infection ratios was used to determine the optimal conditions. After 48 h, the infected cells were collected and washed with 0.1 M potassium phosphate buffer (PH 7.4, with 20% glycerin), and the microsome of the membrane fraction was prepared according to standard procedures [51] and stored at − 80 °C. The concentration of total protein was quantified by the Bradford method using bovine serum albumin as the standard [52]. P450 content was measured in reduced samples using CO-difference spectra [53].

Enzyme activities to model substrate

The fluorescent P450 model probe substrate7-ethoxy coumarin (ECOD) was used to identify the O-dealkylation of recombinant P450s as described previously [54]. The assay was carried out at 30 °C in 0.1 M potassium phosphate buffer (pH 7.4) with 0.1 mg microsomes, 100 mM ECOD and 500 μM cumene hydroperoxide (CuOOH) for a final volume of 200 μL. Microsomes and substrate ECOD were incubated at 30 °C for 5 min before adding CuOOH. The fluorescence was measured in a TECAN microplate reader (Infinite™ M200) at 380 nm excitation, 460 nm emission and 30 °C for 15 min. P450 activity was calculated based on the 7-hydroxycoumarin standard curve and was expressed as mean pmoles of 7-OH per pmole of microsomal protein/min ± SD.

HPLC analysis of insecticide metabolism

For chlorpyrifos, cypermethrin and deltamethrin metabolism, in vitro reactions were carried out according to our previous methods [27]. The non-insertion microsomes served as the control. The reactions were performed at 30 °C with 700 rpm/min oscillation for 1.5 h, and stopped by adding 500 μL acetonitrile and incubated for further 20 min. Finally, the quenched reactions were centrifuged at 16,000 g for 10 min and 200 μL supernatant was transferred to HPLC vials and analyzed immediately by reverse phase HPLC using a C18 column (Ameritech Technology, USA, 4.6 × 250 mm in length, 5 μm particle size) with 80% methanol, 90% methanol and 82% acetonitrile as the mobile phase for chlorpyrifos, cypermethrin and deltamethrin, respectively, with a flow rate of 1 ml min−1. The quantity of chlorpyrifos, cypermethrin and deltamethrin remaining in the samples was determined at a monitoring absorbance wavelength of 289, 230 and 240 nm, respectively. The insecticide was quantified by peak integration and calculated based on the standard curves.

Construction of transgenic D. melanogaster and bioassays

The UAS-CYP321B1 strain was constructed according to the previous methods [27]. UAS-CYP321B1 flies were generated and crossed with Act5C-GAL4 lines and the offspring were used in the insecticide bioassays. The mRNA level of CYP321B1 was analyzed by RT-PCR and qRT-PCR. All primers are shown in in Additional file 1: Table S1. For insecticide bioassays, each assay needed ten adult flies (2–5 days old) and the progeny from the w1118 and Act5C-GAL4 strains served as controls. The flies (2–5-day-old) were added to each vial with 10 ml corn meal medium containing 0.05 mg/L chlorpyrifos, 0.4 mg/L deltamethrin and 0.6 mg/L permethrin. At least six replicates were used for each experiment.

Homology modeling and docking

Three-dimensional structures of insecticides (chlorpyrifos, cypermethrin and deltamethrin) were obtained from the PubChem servers. The P450 protein model was generated by using SWISS-MODEL. AutoDock v4.2 software was used to perform the molecular docking between P450 protein and insecticides [55]. The modeling and docking results were visualized in PyMOL.

Cloning and sequencing the 5’-flanking regions

Four different restriction enzymes including DraI, EcoRV, PvuII, and StuI, were used to digest the genomic DNAs according to the operation manual of the Universal Genome Walker Kit (Clontech, Palo Alto, CA, USA). Genome Walker adaptors were ligated into the DNA fragments using T4 DNA ligase. The target sequences were amplified using LATaq polymerase (Takara, Japan), and ligated into the PMD-19 T vectors and sequenced. The primers are shown in Additional file 1: Table S3. All the sequences have been deposited in GenBank (Accession nos: MK327549 and MK327550). The putative TF binding sites in 5’-flanking regions were predicted and analyzed by the online software JASPAR and ALLGEN [46, 56].

Reporter and expression constructs

Promoter region sequences (DNA5’-R and DNA5’-S) were amplified and inserted into the firefly luciferase reporter vector PGL3-Basic (Promega, Madison, WI, USA) between the XhoI and KpnI restriction sites. Various promoter truncations were amplified using the full promoter region DNA as a template. A Mut Express II Fast Mutagenesis Kit was utilized to generate the mutated promoter truncations following the manufacturer’s instructions [57]. The open reading frames from CncC and Maf were ligated into the expression vector, pIB/V5-His (Invitrogen, San Diego, CA, USA). The primers used in this study are shown in Additional file 1: Table S4.

Luciferase reporter assays

The luciferase reporter assays were performed according to the previous description [58]. The sf9 cells were seeded in 24-well cell culture plates with a density of 4 × 105 cells/well. 0.5 μg of the promoter construct, 0.5 μg expression plasmid of CncC or/and Maf and 0.02 μg of pRL-CMV (Promega, Madison, WI, USA) were transfected using 2 μL of FUGENE transfection reagent (Promega, Madison, WI, USA) following the operation manual. 2 μL of FUGENE transfection reagent was used to transfect a mixture of 1 μg promoter constructs (DNA5’-R, DNA5’-S, promoter truncations and mutated promoter truncations) and 0.02 μg pRL-CMV. After 48 h post-transfection, the cells were harvested and the luciferase activities were measured.

Statistical analysis

These data were analyzed using SPSS 16.0 software (SPSS Inc., Chicago, IL, USA) and were presented as the mean ± standard deviation (SD). Student’s t-test was carried out for the comparison of two samples. One-way ANOVA with Tukey’s HSD tests was used to determine the statistical significance of differences among more than two groups. A value of P < 0.05 was considered statistically significant.

Data availability

All data generated or analyzed during this study are included in the manuscript and Supplementary material. Supplementary material contains all primers used to amplify gene sequences and construct expression vectors. S. exigua genomic sequences have been deposited in GenBank (Accession nos: MK327549 and MK327550) [59, 60].

Abbreviations

Nrf2:

Nuclear factor erythroid 2 related factor 2

AREs:

Antioxidant response elements

NQO1:

Quinone oxidoreductase-1

UGT:

UDP-glycosyltransferase

HO-1:

Heme oxygenase 1

CncC:

Cap ‘n’ Collar isoform-C

Maf-S:

Muscle aponeurosis fibromatosis

HPLC:

High-performance liquid chromatography

ECOD:

Substrate7-ethoxy coumarin

CuOOH:

Cumene hydroperoxide

SD:

Standard deviation

References

  1. Feyereisen R. 8 – Insect CYP Genes and P450 Enzymes. Insect Biochem Mol Biol. 2012;8:236–316.

    Article  Google Scholar 

  2. Li X, Schuler M, Berenbaum M. Molecular mechanisms of metabolic resistance to synthetic and natural xenobiotics. Annu Rev Entomol. 2007;52(1):231–53.

    Article  PubMed  Google Scholar 

  3. Mitchell SN, Stevenson BJ, Muller P, Wilding CS, Egyir-Yawson A, Field SG, Hemingway J, Paine MJ, Ranson H, Donnelly MJ. Identification and validation of a gene causing cross-resistance between insecticide classes in Anopheles gambiae from Ghana. Proc Natl Acad Sci USA. 2012;109(16):6147–52.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Joußen N, Agnolet S, Lorenz S, Schöne SE, Ellinger R, Schneider B, Heckel DG. Resistance of Australian Helicoverpa armigera to fenvalerate is due to the chimeric P450 enzyme CYP337B3. Proc Natl Acad Sci USA. 2012;109(38):15206–11.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Riveron JM, Irving H, Ndula M, Barnes KG, Ibrahim SS, Paine MJ, Wondji CS. Directionally selected cytochrome P450 alleles are driving the spread of pyrethroid resistance in the major malaria vector Anopheles funestus. Proc Natl Acad Sci USA. 2013;110(1):252–7.

    Article  CAS  PubMed  Google Scholar 

  6. Rewitz KF, O’Connor MB, Gilbert LI. Molecular evolution of the insect Halloween family of cytochrome P450s: phylogeny, gene organization and functional conservation. Insect Biochem Mol Biol. 2007;37(8):741–53.

    Article  CAS  PubMed  Google Scholar 

  7. Li T, Cao C, Yang T, Zhang L, He L, Xi Z, Bian G, Liu N. A G-protein-coupled receptor regulation pathway in cytochrome P450-mediated permethrin-resistance in mosquitoes, Culex quinquefasciatus. Sci Rep. 2015;5:17772.

    Article  CAS  PubMed  Google Scholar 

  8. Gong Y, Li T, Feng Y, Liu N. The function of two P450s, CYP9M10 and CYP6AA7, in the permethrin resistance of Culex quinquefasciatus. Sci Rep. 2017;7(1):587.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Elzaki MEA, Miah MA, Peng Y, Zhang H, Jiang L, Wu M, Han Z. Deltamethrin is metabolized by CYP6FU1, a cytochrome P450 associated with pyrethroid resistance, Laodelphax striatellus. Pest Manag Sci. 2017;74(6):1265–71.

    Google Scholar 

  10. Zhu F, Parthasarathy R, Bai H, Woithe K, Kaussmann M, Nauen R, Harrison DA, Palli SR. A brain-specific cytochrome P450 responsible for the majority of deltamethrin resistance in the QTC279 strain of Tribolium castaneum. Proc Natl Acad Sci USA. 2010;107(19):8557–62.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Kensler TW, Wakabayashi N, Biswal S. Cell Survival Responses to Environmental Stresses Via the Keap1-Nrf2-ARE Pathway. Annu Rev Pharmacol Toxicol. 2007;47(1):89–116.

    Article  CAS  PubMed  Google Scholar 

  12. Slocum SL, Kensler TW. Nrf2: control of sensitivity to carcinogens. Arch Toxicol. 2011;85(4):273–84.

    Article  CAS  PubMed  Google Scholar 

  13. Niture SK, Kaspar JW, Shen J, Jaiswal AK. Nrf2 signaling and cell survival. Toxicol Appl Pharmacol. 2010;244(1):37–42.

    Article  CAS  PubMed  Google Scholar 

  14. Kobayashi A, Kang MI, Okawa H, Ohtsuji M, Zenke Y, Chiba T, Igarashi K, Yamamoto M. Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2. Mol Cell Biol. 2004;24(16):7130–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Guoxiang S, Ah-Ng K. Nrf2 plays an important role in coordinated regulation of Phase II drug metabolism enzymes and Phase III drug transporters. Biopharm Drug Dispos. 2010;30(7):345–55.

    Google Scholar 

  16. Jaiswal AK. Nrf2 signaling in coordinated activation of antioxidant gene expression. Free Radic Biol Med. 2004;36(10):1199–207.

    Article  CAS  PubMed  Google Scholar 

  17. Misra JR, Horner MA, Geanette L, Thummel CS. Transcriptional regulation of xenobiotic detoxification in Drosophila. Gene Dev. 2011;25(17):1796.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Misra JR, Lam G, Thummel CS. Constitutive activation of the Nrf2/Keap1 pathway in insecticide-resistant strains of Drosophila. Insect Biochem Mol Biol. 2013;43(12):1116–24.

    Article  CAS  PubMed  Google Scholar 

  19. Wan H, Liu Y, Li M, Zhu S, Li X, Pittendrigh BR, Qiu X. Nrf2/Maf-binding-site-containing functional Cyp6a2 allele is associated with DDT resistance in Drosophila melanogaster. Pest Manag Sci. 2014;70(7):1048–58.

    Article  CAS  PubMed  Google Scholar 

  20. Kalsi M, Palli SR. Transcription factors, CncC and Maf, regulate expression of CYP6BQ genes responsible for deltamethrin resistance in Tribolium castaneum. Insect Biochem Mol Biol. 2015;65:47–56.

    Article  CAS  PubMed  Google Scholar 

  21. Shi L, Wang M, Zhang Y, Shen G, Di H, Wang Y, He L. The expression of P450 genes mediating fenpropathrin resistance is regulated by CncC and Maf in Tetranychus cinnabarinus (Boisduval). Comp Biochem Physiol C Toxicol Pharmacol. 2017;198:28–36.

    Article  CAS  PubMed  Google Scholar 

  22. Schmidt JM, Good RT, Appleton B, Sherrard J, Raymant GC, Bogwitz MR, Martin J, Daborn PJ, Goddard ME, Batterham P, Robin C. Copy number variation and transposable elements feature in recent, ongoing adaptation at the Cyp6g1 locus. PLoS Genet. 2010;6(6):e1000998.

  23. Chung H, Bogwitz MR, McCart C, Andrianopoulos A, Ffrench-Constant RH, Batterham P, Daborn PJ. Cis-regulatory elements in the Accord retrotransposon result in tissue-specific expression of the Drosophila melanogaster insecticide resistance gene Cyp6g1. Genetics. 2007;175(3):1071–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Bass C, Zimmer CT, Riveron JM, Wilding CS, Wondji CS, Kaussmann M, Field LM, Williamson MS, Nauen R. Gene amplification and microsatellite polymorphism underlie a recent insect host shift. Proc Natl Acad Sci USA. 2013;110(48):19460–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Lai T, Su J. Effects of chlorantraniliprole on development and reproduction of beet armyworm, Spodoptera exigua (Hübner). J Pest Sci. 2011;84(3):381–6.

    Article  Google Scholar 

  26. Lai T, Su J. Assessment of resistance risk in Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae) to chlorantraniliprole. Pest Manag Sci. 2011;67(11):1468–72.

    Article  CAS  PubMed  Google Scholar 

  27. Hu B, Huang H, Hu S, Ren M, Wei Q, Tian X. Esmail Abdalla Elzaki M, Bass C, Su J, Reddy Palli S: Changes in both trans- and cis-regulatory elements mediate insecticide resistance in a lepidopteron pest,Spodoptera exigua. PLoS Genet. 2021;17(3):e1009403.

  28. Feyereisen R. Insect p450 enzymes. Annu Rev Entomol. 1999;44(44):507–33.

    Article  CAS  PubMed  Google Scholar 

  29. Ibrahim SS, Riveron JM, Stott R, Irving H, Wondji CS. The cytochrome P450 CYP6P4 is responsible for the high pyrethroid resistance in knockdown resistance-free Anopheles arabiensis. Insect Biochem Mol Biol. 2016;68:23–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Joussen N, Heckel DG, Haas M, Schuphan I, Schmidt B. Metabolism of imidacloprid and DDT by P450 CYP6G1 expressed in cell cultures of Nicotiana tabacum suggests detoxification of these insecticides in Cyp6g1-overexpressing strains of Drosophila melanogaster, leading to resistance. Pest Manag Sci. 2008;64(1):65–73.

    Article  CAS  PubMed  Google Scholar 

  31. Daborn PJ, Yen JL, Bogwitz MR, Le Goff G, Feil E, Jeffers S, Tijet N, Perry T, Heckel D, Batterham P, Feyereisen R, Wilson TG, ffrench-Constant RH. A single p450 allele associated with insecticide resistance in Drosophila. Science. 2002;297(5590):2253–6. https://doiorg.publicaciones.saludcastillayleon.es/10.1126/science.1074170. PMID: 12351787.

  32. Liu N, Li M, Gong Y, Liu F, Li T. Cytochrome P450s–Their expression, regulation, and role in insecticide resistance. Pestic Biochem Physiol. 2015;120:77–81.

    Article  CAS  PubMed  Google Scholar 

  33. Mao YB, Cai WJ, Wang JW, Hong GJ, Tao XY, Wang LJ, Huang YP, Chen XY. Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. Nat Biotechnol. 2007;25(11):1307–13.

    Article  CAS  PubMed  Google Scholar 

  34. Rupasinghe SG, Wen Z, Chiu TL, Schuler MA. Helicoverpa zea CYP6B8 and CYP321A1: different molecular solutions to the problem of metabolizing plant toxins and insecticides. Protein Eng Des Sel. 2007;20(12):615–24.

    Article  CAS  PubMed  Google Scholar 

  35. Chiu TL, Wen Z, Rupasinghe SG, Schuler MA. Comparative molecular modeling of Anopheles gambiae CYP6Z1, a mosquito P450 capable of metabolizing DDT. Proc Natl Acad Sci USA. 2008;105(26):8855–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Daborn PJ. Characterization of Drosophila melanogaster cytochrome P450 genes. Proc Natl Acad Sci U S A. 2009;106(14):5731–6.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Li X, Baudry J, Berenbaum MR, Schuler MA. Structural and functional divergence of insect CYP6B proteins: From specialist to generalist cytochrome P450. Proc Natl Acad Sci U S A. 2004;101(9):2939–44.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Daborn P, Boundy S, Yen J, Pittendrigh B, Ffrench-Constant R. DDT resistance in Drosophila correlates with Cyp6g1 over-expression and confers cross-resistance to the neonicotinoid imidacloprid. Mol Genet Genomics. 2001;266(4):556–63.

    Article  CAS  PubMed  Google Scholar 

  39. Mitchell SN, Stevenson BJ, Müller P, Wilding CS, Egyir-Yawson A, Field SG, Hemingway J, Paine MJI, Ranson H, Donnelly MJ. Identification and validation of a gene causing cross-resistance between insecticide classes in Anopheles gambiae from Ghana. Proc Natl Acad Sci U S A. 2012;109(16):6147–52.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Wang R-L, Zhu-Salzman K, Baerson SR, Xin X-W, Li J, Su Y-J, Zeng R-S. Identification of a novel cytochrome P450 CYP321B1 gene from tobacco cutworm (Spodoptera litura) and RNA interference to evaluate its role in commonly used insecticides. Insect Sci. 2017;24(2):235–47.

    Article  CAS  PubMed  Google Scholar 

  41. Su J, Sun XX. High level of metaflumizone resistance and multiple insecticide resistance in field populations of Spodoptera exigua (Lepidoptera: Noctuidae) in Guangdong Province. China Crop Prot. 2014;61(3):58–63.

    Article  CAS  Google Scholar 

  42. Kalsi M, Palli SR. Transcription factor cap n collar C regulates multiple cytochrome P450 genes conferring adaptation to potato plant allelochemicals and resistance to imidacloprid in Leptinotarsa decemlineata (Say). Insect Biochem Mol Biol. 2017;83:1–12.

    Article  CAS  PubMed  Google Scholar 

  43. Bo H, Miaomiao R, Jianfeng F, Sufang H, Xia W, Elzaki MEA, Chris B, Palli SR, Jianya S. Xenobiotic transcription factors CncC and maf regulate expression of CYP321A16 and CYP332A1 that mediate chlorpyrifos resistance in Spodoptera exigua. J Hazard Mater. 2020;398:122971.

  44. Itokawa K, Komagata O, Kasai S, Tomita T. A single nucleotide change in a core promoter is involved in the progressive overexpression of the duplicated CYP9M10 haplotype lineage in Culex quinquefasciatus. Insect Biochem Mol Biol. 2015;66:96–102.

    Article  CAS  PubMed  Google Scholar 

  45. Mugenzi LMJ, Menze BD, Tchouakui M, Wondji MJ, Irving H, Tchoupo M, Hearn J, Weedall GD, Riveron JM, Wondji CS. Cis-regulatory CYP6P9b P450 variants associated with loss of insecticide-treated bed net efficacy against Anopheles funestus. Nat Commun. 2019;10(1):4652.

    Article  PubMed  PubMed Central  Google Scholar 

  46. Pu J, Sun H, Wang J, Wu M, Wang K, Denholm I, Han Z. Multiple cis-acting elements involved in up-regulation of a cytochrome P450 gene conferring resistance to deltamethrin in smal brown planthopper, Laodelphax striatellus (Fallen). Insect Biochem Mol Biol. 2016;78:20–8.

    Article  CAS  PubMed  Google Scholar 

  47. Dantoft W, Davis MM, Lindvall JM, Tang X, Uvell H, Junell A, Beskow A, Engström Y. The Oct1 homolog Nubbin is a repressor of NF-κB-dependent immune gene expression that increases the tolerance to gut microbiota. BMC Biol. 2013;11:99.

    Article  PubMed  PubMed Central  Google Scholar 

  48. Gao J, Scott JG. Role of the transcriptional repressor mdGfi-1 in CYP6D1v1-mediated insecticide resistance in the house fly. Musca domestica Insect Biochem Mol Biol. 2006;36(5):387–95.

    Article  CAS  PubMed  Google Scholar 

  49. Lin GG, Scott JG. Investigations of the constitutive overexpression of CYP6D1 in the permethrin resistant LPR strain of house fly (Musca domestica). Pestic Biochem Physiol. 2011;100(2):130–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Lo H-R, Chao Y-C. Rapid Titer Determination of Baculovirus by Quantitative Real-Time Polymerase Chain Reaction. Biotechnol Progr. 2004;20(1):354–60.

    Article  CAS  Google Scholar 

  51. Shi Y, Wang H, Liu Z, Wu S, Yang Y, Feyereisen R, Heckel DG, Wu Y. Phylogenetic and functional characterization of ten P450 genes from the CYP6AE subfamily of Helicoverpa armigera involved in xenobiotic metabolism. Insect Biochem Mol Biol. 2018;93:79–91.

    Article  CAS  PubMed  Google Scholar 

  52. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72(1):248–54.

    Article  CAS  PubMed  Google Scholar 

  53. Omura T, Sato R. The carbon monoxide-binding pigment of liver microsomes i evidence for its hemoprotein nature. J Biol Chem. 1964;239(239):2370–8.

    Article  CAS  PubMed  Google Scholar 

  54. De SG, Cuany A, Brun A, Amichot M, Rahmani R, Bergé JB. A microfluorometric method for measuring ethoxycoumarin-O-deethylase activity on individual Drosophila melanogaster abdomens: interest for screening resistance in insect populations. Anal Biochem. 1995;229(1):86.

    Article  Google Scholar 

  55. Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem. 2010;31(2):455–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Mathelier A, Fornes O, Arenillas DJ, Chen CY, Denay G, Lee J, Shi W, Shyr C, Tan G, Worsley-Hunt R, et al. JASPAR 2016: a major expansion and update of the open-access database of transcription factor binding profiles. Nucleic Acids Res. 2016;44(D1):D110–115.

    Article  CAS  PubMed  Google Scholar 

  57. Hu B, Hu S, Huang H, Wei Q, Ren M, Huang S, Tian X, Su J. Insecticides induce the co-expression of glutathione S-transferases through ROS/CncC pathway in Spodoptera exigua. Pestic Biochem Physiol. 2019;155:58–71.

    Article  CAS  PubMed  Google Scholar 

  58. Hu B, Huang H, Wei Q, Ren M, Mburu DK, Tian X, Su J. Transcription factors CncC/Maf and AhR/ARNT coordinately regulate the expression of multiple GSTs conferring resistance to chlorpyrifos and cypermethrin in Spodoptera exigua. Pest Manag Sci. 2019;75(7):2009–19.

    Article  CAS  PubMed  Google Scholar 

  59. Su J, Hu B, Ren M: Spodoptera exigua cytochrome P450 CYP321b1 (CYP321b1) gene, promoter region and partial cds. 2020. NCBI. Accession: MK327549. https://www.ncbi.nlm.nih.gov/search/all/?term=MK327549.

  60. Su J, Hu B, Ren M: Spodoptera exigua cytochrome P450 CYP321b1 (CYP321b1) gene, promoter region and partial cds. 2020. NCBI. Accession: MK327550. https://www.ncbi.nlm.nih.gov/nuccore/MK327550.

Download references

Acknowledgements

Not applicable.

Funding

The work was supported by the National Natural Science Foundation of China (No. 32000333,32072452 and 31925007) and Natural Science Foundation of Jiangsu Province (BK20221292).

Author information

Authors and Affiliations

  1. Jiangsu Key Laboratory of Sericultural and Animal Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, 212100, China

    Bo Hu, Yuting Zhang, Zhiping Xing & Anjiang Tan

  2. Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture and Rural Affairs, Sericultural Scientific Research Center, Chinese Academy of Agricultural Sciences, Zhenjiang, 212100, China

    Bo Hu, Yuting Zhang, Zhiping Xing & Anjiang Tan

  3. School of Medicine, Linyi University, Linyi, 276000, China

    Xiangzhu Chen

  4. Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education), College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China

    Cong Rao, Kuitun Liu & Jianya Su

Authors
  1. Bo Hu
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Yuting Zhang
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Zhiping Xing
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Xiangzhu Chen
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Cong Rao
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Kuitun Liu
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Anjiang Tan
    View author publications

    You can also search for this author inPubMed Google Scholar

  8. Jianya Su
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

Conceptualization, J.Y.S.; Methodology, B.H., Y.T.Z., X.Z.C, A.J.T and J.Y.S.; Investigation, B.H. and Z.P.X.; Visualization, B.H., Y.T.Z. and C.R.; Writing – Original Draft, B.H. and J.Y.S.; Writing – Review & Editing, B.H., J.Y.S., A.J.T and K.T.L.; Funding Acquisition, B.H., A.J.T and J.Y.S. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Anjiang Tan or Jianya Su.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

12915_2025_2228_MOESM1_ESM.docx

Additional file 1: Table S1. Primers for the eukaryotic expression of P450 genes from S. exigua. Table S2. The primers used for cloning 5’-flanking regions. Table S3. The primers used for reporter and promoter constructs. Figure S1. Alignment of upstream sequences of CYP321B1 gene from susceptible and resistant strains of S. exigua.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hu, B., Zhang, Y., Xing, Z. et al. Two independent regulatory mechanisms synergistically contribute to P450-mediated insecticide resistance in a lepidopteran pest, Spodoptera exigua. BMC Biol 23, 122 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12915-025-02228-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12915-025-02228-5

Keywords

BMC Biology

ISSN: 1741-7007

Contact us