Identification of Central Regulatory Hubs in Pupal Diapause of Helicoverpa armigera Using Weighted Gene Co-Expression Network Analysis and Multiscale Embedded Network Analysis

Diapause is a vital overwintering strategy for many insects, yet its comprehensive molecular architecture remains elusive. In the polyphagous pest Helicoverpa armigera, facultative pupal diapause is key to its ecological success. To elucidate the complex diapause regulatory network, we conducted a transcriptomic analysis of diapause (DP) versus non-diapause (NP) pupal brains across early pupal development (days 2, 5, and 10). Integrated analyses, including differential expression, persistent gene identification, weighted gene co-expression network analysis (WGCNA), and multiscale embedded network analysis (MEGENA), were employed to define core regulatory modules and hubs. The number of differentially expressed genes (DEGs) increased over time, with 1781 genes persistently regulated across all time points, enriched in mitochondrial metabolism, hormone signaling, and chromatin remodeling. WGCNA revealed a diapause-associated module (red) linked to RNA processing/transcription and a development-associated module (blue) enriched for translation and mitochondrial metabolism. Network analyses pinpointed three central hub genes: DDX5 and PLK4 (downregulated in diapause, upregulated upon 20E treatment) and TAF5L (upregulated in diapause, downregulated after 20E). This study provides a systems-level view of the transcriptional landscape governing pupal diapause in H. armigera and identifies novel candidate regulators for future functional studies.