Objective This study aimed to explore the genes associated with diapause in Rhagoletis batava obseuriosa Kol. and to elucidate the molecular mechanisms underlying its diapause regulation.

Methods  Transcriptomic analysis was performed using RNA-Seq technology on two types of pupae: emerging pupae (EP, capable of eclosion within the year) and dispause pupae (DP, incapable of eclosion within the year). Differential expression of the RboHSP70 gene family during diapause was further investigated.

Results Comparative transcriptomic analysis between EP and DP identified 25,538 differentially expressed genes (DEGs) of which 18,437 genes were down-regulated and 7,101 genes were up-regulated in DP compared to EP. GO functional annotation and KEGG pathway analysis revealed significant enrichment of DEGs in stress response, protein folding, and metabolic pathways. Heat shock proteins (HSP70s) were highlighted as key diapause regulators. Further characterization of the HSP70 family identified 20 diapause-associated members (RboHSP70-1 to RboHSP70-20). Physicochemical characterization showed an average molecular weight of 71.82 kDa and an isoelectric point (pI) of 5.49. Phylogenetic analysis classified these members into five groups (Group I–V). while motif analysis indicated that most sequences contained 17 conserved motifs, Transcriptional profiling demonstrated that 16 RboHSP70 genes were significantly up-regulated in diapause pupae, with RboHSP70-13 showing the most pronounced up-regulation, suggesting its pivotal role in diapause regulation.

Conclusion This study provides molecular and theoretical foundations for identifying and functionally characterizing key genes involved in diapause regulation in Rhagoletis batava obseuriosa.