Transcriptional Regulation Networks of Cold Hardiness in Galeruca daurica (Coleoptera: Chrysomelidae) and HSF1-Mediated HSP26 and HSP68 Expression Validation

Background Galeruca daurica (Joannis), a notorious pest in Northern China, seriously threatens Inner Mongolia grassland ecosystems and economy, due to its strong cold hardiness and huge outbreak in a short time. Despite previous studies emphasized molecular mechanisms in response to low-temperature stress, especially based on studies of key gene functions on cold hardiness, there is a lack of in-depth research on transcriptional regulation mechanisms. Results The G . daurica eggs, collected from the grassland of Xianghuang Banner of Xilin Gol League in Inner Mongolia, were reared to the 1st and 2nd instar larvae at different temperatures. RNA-Seq analyzed differentially expressed genes (DEGs) in larvae reared in different states. Weighted gene co-expression network analysis (WGCNA) identified cold hardiness-related gene modules, and regulatory network analyses screened key transcription factors (TFs). The Gene Transcription Regulation Database (GTRD) predicted HSF1 target genes. The genes of HSF1 , HSP26 , and HSP68 were knocked down to evaluate the impacts on expression and cold hardiness. The blue module showed the highest cold hardiness correlation, with HSF1 as a key regulator. Silencing HSF1 decreased HSP26 and HSP68 expression, and knocking down these HSPs reduced the expression of HSF1 . Silencing HSF1 , HSP28 , and HSP86 can increase super-cooling points (SCPs) and freezing points (FPs) significantly. HSF1 regulates HSP26 and HSP68 expression to enhance the cold hardiness of G. daurica . These results elucidate the molecular mechanisms underlying cold hardiness and provide new insights into the adaptability of insects to extreme environments. Conclusions The RNA-Seq analysis of G. daurica has revealed a potential regulatory relationship among HSF1 , HSP26 , and HSP68 . The increased SCPs and FPs after RNAi support their roles in low-temperature tolerance. These findings provide preliminary insights into the molecular basis of cold hardiness in G. daurica.