Abstract
Galeruca daurica (Joannis) is a pest species with serious outbreaks in the Inner Mongolian grasslands in recent years, and its larvae and eggs are extremely cold-tolerant. To gain a deeper understanding of the molecular mechanism of its cold-tolerant stress response, we performed de novo transcriptome assembly of G. daurica via RNA-Seq and compared the differentially expressed genes (DEGs) of first- and second-instar larvae grown and developed indoors and outdoors, respectively. The results show that cold tolerance in G. daurica is associated with changes in gene expression mainly involved in the glycolysis/gluconeogenesis pathway, the fatty acid biosynthesis pathway and the production of heat shock proteins (HSPs). Compared with the control group (indoor), the genes associated with gluconeogenesis, fatty acid biosynthesis and HSP production were up-regulated in the larvae grown and developed outdoors. While the changes in these genes were related to the physiological metabolism and growth of insects, it was hypothesized that the proteins encoded by these genes play an important role in cold tolerance in insects. In addition, we also investigated the expression of genes related to the metabolic pathway of HSPs, and the results show that the HSP-related genes were significantly up-regulated in the larvae of G. daurica grown and developed outdoors compared with the indoor control group. Finally, we chose to induce significant expression differences in the Hsp70 gene (Hsp70A1, Hsp70-2 and Hsp70-3) via RNAi to further illustrate the role of heat stress proteins in cold tolerance on G. daurica larvae. The results show that separate and mixed injections of dsHSP70A1, dsHsp70-2 and dsHsp70-3 significantly reduced expression levels of the target genes in G. daurica larvae. The super-cooling point (SCP) and the body fluid freezing point (FP) of the test larvae were determined after RNAi using the thermocouple method, and it was found that silencing the Hsp70 genes significantly increased the SCP and FP of G. daurica larvae, which validated the role of heat shock proteins in the cold resistance of G. daurica larvae. Our findings provide an important theoretical basis for further excavating the key genes and proteins in response to extremely cold environments and analyzing the molecular mechanism of cold adaptation in insects in harsh environments.