ROS‐dependent phosphorylation of insulin receptor and insulin receptor substrate during pupal diapause development in the cotton bollworm, Helicoverpa armigera

Insect diapause is a critical adaptive strategy characterized by developmental arrest and enhanced stress tolerance, orchestrated by intricate molecular networks. The insulin signaling pathway, which governs growth, metabolism, and stress responses, has been implicated in diapause regulation. However, the precise roles of insulin receptor (InR) and insulin receptor substrate (IRS) in modulating pupal diapause remain incompletely understood, particularly in the economically important pest Helicoverpa armigera. Here, we show that InR and IRS are associated with pupal diapause development through redox-sensitive phosphorylation cascades. Temporal profiling revealed a marked upregulation of phosphorylated InR (p-InR) and IRS (p-IRS) in diapause-destined pupal brains, concomitant with elevated phosphorylation of Akt (p-Akt) and ERK (p-ERK). Functional validation in Helicoverpa zea (HzAm1) cells, via overexpression and RNA interference (RNAi), showed that IRS positively regulates Akt and ERK signaling. Further mechanistic studies indicated that reactive oxygen species (ROS) act upstream of the phosphorylation of InR, IRS, Akt, and ERK in vitro and in vivo; these effects were reversible upon ROS scavenging. Further investigations revealed that increased p-ERK was associated with global protein ubiquitination, suggesting this kinase as a key regulator of proteasomal degradation in diapause development. Our findings delineate a ROS-InR-IRS-Akt/ERK signaling framework associated with diapause development and proteostatic regulation in H. armigera, thereby advancing our understanding of insect diapause biology and providing candidate molecular targets for future pest-management interventions.