A General Signal Pathway to Regulate Multiple Detoxification Genes Drives the Evolution of Helicoverpa armigera Adaptation to Xenobiotics

Abstract

The study of insect adaptation to the defensive metabolites of host plants and various kinds of insecticides in order to acquire resistance is a hot topic in the pest-control field, but the mechanism is still unclear. In our study, we found that a general signal pathway exists in H. armigera which can regulate multiple P450s, GSTs and UGTs genes to help insects decrease their susceptibility to xenobiotics. Knockdown of HaNrf2 and HaAhR expression could significantly increase the toxicity of xenobiotics to H. armigera, and simultaneously decrease the gene expression of P450s, GSTs and UGTs which are related to the xenobiotic metabolism and synthesis of insect hormone pathways. Then, we used EMSA and dual luciferase assay to verify that a crosstalk exists between AhR and Nrf2 to regulate multiple P450s, GSTs and UGTs genes to mediate H. armigera susceptibility to plant allelochemicals and insecticides. The detoxification genes' expression network which can be regulated by Nrf2 and AhR is still unknown, and there were also no reports about the crosstalk between AhR and Nrf2 that exist in insects and can regulate multiple detoxification genes' expression. Our results provide a new general signaling pathway to reveal the adaptive mechanism of insects to xenobiotics and provides further insight into designing effective pest-management strategies to avoid the overuse of insecticides.

Keywords: detoxification genes; regulation; transcription factor; xenobiotics.

Figure 1

The effect on the susceptibility of xenobiotics by RNAi HaNrf2 and HaAhR in H. armigera. (A) RT-qPCR analysis of HaNrf2 gene expression after RNAi knockdown; (B) RT-qPCR analysis of HaAhR gene expression after RNAi knockdown; (C) mortality of H. armigera fed on cotton leaves coated with dsNrf2 or dsAhR; (D) mortality after HaNrf2 and HaAhR knockdown in H. armigera fed an artificial diet containing 0.02 mg/g gossypol; (E) mortality after HaNrf2 and HaAhR knockdown in H. armigera fed an artificial diet containing 0.05 mg/g 2-tridecanone; (F) mortality after HaNrf2 and HaAhR knockdown in H. armigera fed an artificial diet containing 0.05 μg/g chlorantraniliprole or 0.1 μg/g chlorfenapyr; (G) resistance of dsNrf2 and dsAhR coated cotton leaves against H. armigera. Means and SEs from three biological replicates are shown. Different letters on error bars show significant differences (p < 0.05).

Figure 2

Down-regulation of P450 (A,B,E), GST (C,F) and UGT (D,G) detoxification genes putatively regulated by HaNrf2 and HaAhR. Means and SEs from three biological replicates are shown. Asterisks on error bars show significant differences (p < 0.05).

Figure 3

Transcription factor binding sites prediction (A–C) and RT-qPCR validation (D–I) of down-regulated P450, GST and UGT genes associated with HaNrf2 and HaAhR knockdown and xenobiotics (gossypol, 2-tridecanone and chlorantraniliprole) treatment. Analysis of the P450, GST and UGT genes’ promoter regions were performed to identify possible regulatory elements. Antioxidant response element (ARE) for Nrf2 (ARE/Nrf2: TMANNRTGAYNNNGCRWWW; DMATGCTKWGT; WCAGHAW), and xenobiotic response element (XRE) from the aryl hydrocarbon receptor (XRE/AhR: TNGCGTG; NCACGCNNN; GAKKTGCGTGASAAGAG; MRGSYTCTTCTCACGCAACTCC; KYKKCTCACGCWRYW; DCACGCAASKCHSAAW; CACGTGCAA). Element position “+1” is defined relative to the start codon ATG. Asterisks on error bars show significant differences (p < 0.05).

Figure 4

RNAi HaNrf2 and HaAhR affects the biosynthesis and metabolism of insect hormones. (A) Analysis of genes’ promoter regions was performed to identify possible regulatory elements. Antioxidant response element (ARE) for Nrf2 (ARE/Nrf2: TMANNRTGAYNNNGCRWWW; DMATGCTKWGT; WCAGHAW), and xenobiotic response element (XRE) from the aryl hydrocarbon receptor (XRE/AhR: TNGCGTG; NCACGCNNN; GAKKTGCGTGASAAGAG; MRGSYTCTTCTCACGCAACTCC; KYKKCTCACGCWRYW; DCACGCAASKCHSAAW; CACGTGCAA). Element position “+1” is defined relative to the start codon ATG. (B) RNAi HaNrf2 and HaAhR effects on the genes’ expression in synthesis pathway of insect hormone. Genes both down-regulated following treatment with dsHaNrf2 or dsHaAhR are shown with solid blue lines. Genes only down-regulated following treatment with dsHaNrf2 are shown with solid green lines.

Figure 5

The promoter activity of CYP6B2 (A), CYP6B6 (B) and CYP6B7 (C) induced by 2-tridecanone and the deletion of XRE/AhR or ARE/Nrf2 elements affects 2-tridecanone-induced CYP6B2 (D), CYP6B6 (E) and CYP6B7 (F) promoter activity. Element position “+1” is defined relative to the start codon ATG. Different letters on error bars show significant differences (p < 0.05).

Figure 6

Different xenobiotics (gossypol and chlorantraniliprole) induced the promoter activity of CYP6AE14 (A), GSTD1s (B) and UGT40M1 (C) genes, which also transiently transfected with dsNrf2, dsAhR, HaAhR or HaNrf2. Different letters on error bars show significant differences (p < 0.05).

Figure 7

(A) sEMSA analysis of the expression of HaNrf2 and HaAhR in nuclear cells after being treated by 2-tridecanone. Nuclear extracts (NE) were prepared from un-induced (un-induced NE) or 2-tridecanone-induced (induced NE) (125 μM) homologous H. armigera fat body cells. Lane 1, only Nrf2 probe, no nuclear extracts; Lane 2, Nrf2 probe and Nrf2 antibody with nuclear extracts from induced cells, in competition with unrelated nonspecific sequence competitors (200-fold greater than that of the labeled probe); Lane 3, Nrf2 probe and Nrf2 antibody with nuclear extracts from induced cells, in competition with unlabeled probe (concentration 200-fold greater than that of the labeled probe); Lane 4, Nrf2 probe and Nrf2 antibody with nuclear extracts from un-induced cells; Lane 5, Nrf2 probe and Nrf2 antibody with nuclear extracts from 2-tridecanone-induced cells; Lane 6, only AhR probe, no nuclear extracts; Lane 7, AhR probe and AhR antibody with nuclear extracts from 2-tridecanone-induced cells, in competition with unrelated nonspecific sequence competitors (200-fold greater than that of the labeled probe); Lane 8, AhR probe and AhR antibody with nuclear extracts from induced cells, in competition with unlabeled probe (concentration 200-fold greater than that of the labeled probe); Lane 9, AhR probe and AhR antibody with nuclear extracts from un-induced cells; Lane 10, AhR probe and AhR antibody with nuclear extracts from induced cells. (B) 2-Tridecanone induced HaNrf2 transfer into the nucleus. DAPI: DAPI staining the nucleus region; Red: using recombinant pAC-v5-Red-Nrf2 plasmid to show the localization of Nrf2; Merger: subcellular localization of Nrf2; 2-tridecanone: the subcellular localization of Nrf2 when the fat body cells were treated with 125 μM 2-tridecanone, Ethanol: the subcellular localization of Nrf2 when the fat body cells were treated with ethanol. Scale bar in 2-tridecanone-1, Ethanol-1: 100 μm; Scale bar in Ethanol-2, Ethanol-3, 2-Tridecanone-2, 2-Tridecanone-3: 20 μm.

Figure 8

The crosstalk between HaNrf2 and HaAhR regulates the P450s, UGTs and GSTs genes in H. armigera. (A) EMSA method to verify the HaNrf2 binding sits in the promoter of HaAhR; (B) EMSA method to verify the HaAhR binding sits in the promoter of HaAhR; (C) overexpressed HaNrf2 and HaAhR effects on the promoter activity of HaAhR. Element position “+1” is defined relative to the start codon ATG. Different letters on error bars show significant differences (p < 0.05).

Figure 9

A schematic illustration of the proposed model of how the crosstalk between HaNrf2 and HaAhR regulate the P450s, GSTs and UGTs genes and mediate the susceptibility of xenobiotics in H. armigera. Xenobiotics are represented by a red dot.