Enthralling genetic regulatory mechanisms meddling insecticide resistance development in insects: role of transcriptional and post-transcriptional events

Abstract

Insecticide resistance in insects severely threatens both human health and agriculture, making insecticides less compelling and valuable, leading to frequent pest management failures, rising input costs, lowering crop yields, and disastrous public health. Insecticide resistance results from multiple factors, mainly indiscriminate insecticide usage and mounted selection pressure on insect populations. Insects respond to insecticide stress at the cellular level by modest yet significant genetic propagations. Transcriptional, co-transcriptional, and post-transcriptional regulatory signals of cells in organisms regulate the intricate processes in gene expressions churning the genetic information in transcriptional units into proteins and non-coding transcripts. Upregulation of detoxification enzymes, notably cytochrome P450s (CYPs), glutathione S-transferases (GSTs), esterases [carboxyl choline esterase (CCE), carboxyl esterase (CarE)] and ATP Binding Cassettes (ABC) at the transcriptional level, modification of target sites, decreased penetration, or higher excretion of insecticides are the noted insect physiological responses. The transcriptional regulatory pathways such as AhR/ARNT, Nuclear receptors, CncC/Keap1, MAPK/CREB, and GPCR/cAMP/PKA were found to regulate the detoxification genes at the transcriptional level. Post-transcriptional changes of non-coding RNAs (ncRNAs) such as microRNAs (miRNA), long non-coding RNAs (lncRNA), and epitranscriptomics, including RNA methylation, are reported in resistant insects. Additionally, genetic modifications such as mutations in the target sites and copy number variations (CNV) are also influencing insecticide resistance. Therefore, these cellular intricacies may decrease insecticide sensitivity, altering the concentrations or activities of proteins involved in insecticide interactions or detoxification. The cellular episodes at the transcriptional and post-transcriptional levels pertinent to insecticide resistance responses in insects are extensively covered in this review. An overview of molecular mechanisms underlying these biological rhythms allows for developing alternative pest control methods to focus on insect vulnerabilities, employing reverse genetics approaches like RNA interference (RNAi) technology to silence particular resistance-related genes for sustained insect management.

Keywords: RNA methylation; detoxification enzymes; insecticide resistance; insects; ncRNAs; pathways.

FIGURE 1

The target site of miRNA. MiRNAs can silence the expression of target genes by binding to 5′or 3′untranslated regions (UTRs) or coding sequence (CDS) of mRNA. MiRNA can bind with different complementarities, such as incomplete pairing in UTR regions and complete pairing in the CDS region. Created with BioRender.com.

FIGURE 2

Mechanism of insecticide resistance. Due to the repeated application of insecticides, insects develop resistance. (A) Target site insensitivity: Mutations in the target receptors of insecticides. Nicotinic acetylcholine receptors (nAChRs), Gamma-aminobutyric acid (GABA), Voltage-gated sodium channel (VGSC), and Acetylcholine esterase (Ach) receptors may face resistance due to alterations in the structure or expression levels of these receptors. In permethrin susceptible insects, miR-33 upregulates the transcript level of VGSC, leading to increased binding of permethrin to VGSC. In the case of permethrin-resistant insects, the binding of permethrin to VGSC is less due to the downregulation of VGSC by miR-33. (B) Metabolic resistance: Insects possess a wide range of metabolic enzymes, such as cytochrome P450 monooxygenases (P450s), esterases, and glutathione S-transferases (GSTs), which can detoxify insecticides. miRNAs can bind to target detoxification gene mRNA molecules, leading to mRNA translational repression or degradation. The upregulation of miR-311-3p degrades the mRNA of the CYP6AB gene resulting in lower expression of CYP6AB in susceptible insects. In contrast, the resistant insects had overexpression of CYP6AB due to downregulation of miR-311-3p. (C) Reduced cuticular penetration: Insects possess a cuticle that acts as a barrier to the penetration of insecticides. Resistance can occur through the thickening or modification of the cuticle, reducing insecticide uptake. The CPR5 gene has been highly expressed in susceptible insects due to the downregulation of miR-932. However, the CPR5 gene is repressed in resistant insects due to the upregulation of miR-932. (D) Behavioral resistance: Some insects can develop behavioral changes that reduce their insecticide exposure. They may avoid treated areas, alter feeding or breeding behaviors, or exhibit reduced contact with insecticides. The involvement of microRNA in behavioral resistance has not been reported. Created with BioRender.com.

FIGURE 3

miRNAs regulating detoxification genes. miRNA genes are transcribed by RNA polymerase II to primary miRNA (pri-miRNA) with one or more stem-loops. Drosha further processes the stem loop into precursor miRNA. Pre-miRNA is carried into the cytoplasm by Exportin-5, and the terminal loop of pre-miRNA is removed by ribonuclease enzyme Dicer-1 (Dcr-1), resulting in a miRNA:miRNA* duplex. The duplex is integrated into the RNA Inducing Silencing Complex (RISC), mainly made up of the Argonaute-1 (Ago-1) protein. The miRNA strand (guide strand) subsequently directs the RISC complex to the target mRNA, and the miRNA* strand (passenger strand) will be degraded. miRNAs primarily bind to the mRNA of target molecules, leading to mRNA translational repression or degradation. They are involved in the post-transcriptional regulation of detoxification gene expression involved in insecticide metabolism, and the expression is negatively correlated between microRNAs and detoxification genes. In the case of susceptible insects, the microRNA miR-2b-3p is upregulated, where these microRNAs bind to the mRNA of detoxification gene CYP9F2 and degrade them. The CYP9F2 genes produced are fewer in number, which is not sufficient for insecticide detoxification, making the insect susceptible. In contrast to susceptible insects, miR-2b-3p is downregulated, which upregulates CYP9F2 genes, and the insect creates resistance. Created with BioRender.com.

FIGURE 4

Interaction between miRNA and detoxification gene. The binding of a miRNA to its target site typically occurs in the 3′untranslated region (UTR) of the mRNA. In chlorantraniliprole exposed to P. xylostella, miR-8534-5p was downregulated, and its corresponding target CYP6B6 was upregulated. (A) 3′ UTR of CYP6B6. (B) miR-5834-5p. (C) Interaction of CYP6B6 and miR-5834-5p. Created with BioRender.com.

FIGURE 5

The regulation of insecticide resistance mechanisms involves epi-transcriptome signals, detoxification signaling pathways, and long non-coding RNAs (lncRNAs). (A) lncRNAs influence miRNA-mediated insecticide resistance regulation. Regulation mechanisms include lncRNA-miRNA-mRNA interaction and lncRNA-mediated regulation of miRNA expression. GSTu1, a detoxifying gene, is involved in the chlorantraniliprole resistance of P. xylostella. A long non-coding RNA, lnc-GSTu1-AS, interacted with GSTu1 by forming an RNA duplex, masking the binding site of microRNA, miR-8525-5p, at the GSTu1-3′ UTR. lnc-GSTu1-AS maintained the mRNA stability of GSTu1 by preventing its degradation, which could have been induced by miR-8525-5p and thus resulting in increased production of detoxification gene (GSTu1), increased the resistance of P. xylostella to chlorantraniliprole. (B) Detoxification enzyme induction pathway. This pathway involves upregulating detoxification enzymes, such as cytochrome P450 monooxygenases, esterases, and glutathione S-transferases. They are the CncC/Keap1, NR, PKA, MAPK/CREB, and AhR/ARNT pathways. The activated molecules of these pathways, such as Cncc/maf, TF, CREB, Ahr/AHRNT, and HR96/HRE, respectively, in the cytoplasm, interact with their corresponding response element in the nucleus to regulate the expression of detoxification genes through transcription. The arrows indicate the cascade of effectors in the signaling pathway. (C) N6-methyladenosine (m⁶A) is a modified form of adenosine widely involved in gene expression regulation. Mutation (T to A at position-206 bp) was observed in the 5′UTR of CYP4C64 that was observed at a much greater frequency in the thiamethoxam-resistant strains compared with the susceptible strain. The T at 206 bp helps bind m⁶A, and the overexpression of the enzyme METTL (methyltransferase) led to the development of thiamethoxam-resistant insects. CncC, Cap‘n’ Collar isoform C; Maf, Musculoaponeurotic fibrosarcoma; ARE, Antioxidant responsive element; Gas, G protein alpha unit which stimulates adenyl cyclase; AC, adenyl cyclase; cAMP, cyclic adenosine monophosphate; PKA, protein kinase A; TF, Transcription factor; XRE, xenobiotic response element; MAPK, mitogen-activated protein kinase; ERK, extracellular regulated protein kinase; P38, P38 mitogen-activated protein kinase; CREB, c-AMP response element binding protein; -P, phosphorylation; CRE, cAMP response element; NR, nuclear receptor; AhR, aryl hydrocarbon receptor; Hsp90, heat shock protein 90; ARNT, aryl hydrocarbon receptor nuclear translocator; XRE-NR, xenobiotic response element-nuclear receptor; XRE-AhR, xenobiotic response element-aryl hydrocarbon receptor. Created with BioRender.com.