Significance: Most studies of pesticide resistance have focused on the identification and functional analysis of resistance genes, but few studies have considered the signaling pathways involved in their regulation. In this work, we discovered that overexpression of a P450 that confers resistance to neonicotinoid insecticides in whitefly is trans -regulated by the transcription factor CREB . Further studies demonstrated that exposure to neonicotinoid insecticides activates a key pathway involved in the cellular response to extracellular signals, the MAPK signaling pathway, that activates CREB by phosphorylation. CREB then binds to a specific site on the promoter of CYP6CM1 resulting in its increased expression. These findings reveal mechanisms underlying the regulation of P450-mediated pesticide resistance and also provide a potential target for pest control.

N⁶-methyladenosine (m⁶A) is the most prevalent messenger RNA modification in eukaryotes and an important posttranscriptional regulator of gene expression. However, the biological roles of m⁶A in most insects remain largely unknown. Here, we show that m⁶A regulates a cytochrome P450 gene (CYP4C64) in the global whitefly pest, Bemisia tabaci, leading to insecticide resistance. Investigation of the regulation of CYP4C64, which confers resistance to the insecticide thiamethoxam, revealed a mutation in the 5′ untranslated region of this gene in resistant B. tabaci strains that introduces a predicted m⁶A site. We provide several lines of evidence that mRNA methylation of the adenine at this position, in combination with modified expression of m⁶A writers, acts to increase expression of CYP4C64 and resistance. Collectively, these results provide an example of the epitranscriptomic regulation of the xenobiotic response in insects and implicate the m⁶A regulatory axis in the development of insecticide resistance.

Trade-offs between evolutionary gain and loss are prevalent in nature, yet their genetic basis is not well resolved. The evolution of insect resistance to insecticide is often associated with strong fitness costs; however, how the fitness trade-offs operates remains poorly understood. Here, we show that the mitogen-activated protein kinase (MAPK) pathway and its upstream and downstream actors underlie the fitness trade-offs associated with insecticide resistance in the whitefly Bemisia tabaci . Specifically, we find a key cytochrome P450 gene CYP6CM1 , that confers neonicotinoids resistance to in B. tabaci , is regulated by the MAPKs p38 and ERK through their activation of the transcription factor cAMP-response element binding protein. However, phosphorylation of p38 and ERK also leads to the activation of the transcription repressor Cap “n” collar isoform C (CncC) that negatively regulates exuperantia (Ex), vasa (Va), and benign gonial cell neoplasm (Bg ), key genes involved in oogenesis, leading to abnormal ovary growth and a reduction in female fecundity. We further demonstrate that the transmembrane G protein-coupled receptor (GPCR) neuropeptide FF receptor 2 (NPFF2) triggers the p38 and ERK pathways via phosphorylation. Additionally, a positive feedback loop between p38 and NPFF2 leads to the continuous activation of the MAPK pathways, thereby constitutively promoting neonicotinoids resistance but with a significant reproductive cost. Collectively, these findings provide fundamental insights into the role of cis-trans regulatory networks incurred by GPCR–MAPK signaling pathways in evolutionary trade-offs and applied knowledge that can inform the development of strategies for the sustainable pest control.

Ongoing host–pathogen interactions can trigger a coevolutionary arms race, while genetic diversity within the host can facilitate its adaptation to pathogens. Here, we used the diamondback moth ( Plutella xylostella ) and its pathogen Bacillus thuringiensis (Bt) as a model for exploring an adaptive evolutionary mechanism. We found that insect host adaptation to the primary Bt virulence factors was tightly associated with a short interspersed nuclear element (SINE - named SE2) insertion into the promoter of the transcriptionally activated MAP4 K4 gene. This retrotransposon insertion coopts and potentiates the effect of the transcription factor forkhead box O (FOXO) in inducing a hormone-modulated Mitogen-activated protein kinase (MAPK) signaling cascade, leading to an enhancement of a host defense mechanism against the pathogen. This work demonstrates that reconstructing a cis – trans interaction can escalate a host response mechanism into a more stringent resistance phenotype to resist pathogen infection, providing a new insight into the coevolutionary mechanism of host organisms and their microbial pathogens.