Epigenetic remodeling of DNA methylation is associated with pupal diapause regulation in Helicoverpa armigera

Diapause is a key seasonal adaptation that enables insects to survive predictable environmental adversity, yet the molecular mechanisms linking environmental cues to stable developmental decisions remain incompletely understood. We hypothesized that DNA methylation contributes to the epigenetic regulation of pupal diapause in the cotton bollworm Helicoverpa armigera. To test this hypothesis, we combined gene expression analyses, pharmacological manipulation, whole-genome bisulfite sequencing (WGBS), transcriptome profiling, and functional validation of candidate genes. Diapause-destined pupae exhibited reduced expression of DNA methylation-associated factors, accompanied by lower genomic 5-methylcytosine levels. Pharmacological inhibition of DNA methylation delayed pupal development and induced a reversible diapause-like arrest in a subset of nondiapause pupae. Integrated methylome and transcriptome analyses revealed extensive stage-dependent remodeling of DNA methylation and gene expression, identifying pathways associated with developmental regulation, energy metabolism, and amino acid metabolism. Among the methylation-associated candidates, BCAT1 was consistently upregulated during diapause, and pharmacological inhibition of BCAT1 promoted diapause termination, supporting its involvement in diapause regulation. Taken together, our findings identify DNA methylation as an important epigenetic component associated with diapause programming and provide a framework for understanding how seasonal environmental signals shape developmental plasticity in insects.