Functional and evolutionary analysis of key enzymes triacylglycerol lipase, glycogen hydrolases in the glycerol and glucose biosynthesis pathway and cellular chaperones for freeze-tolerance of the Rice stem borer, Chilo suppressalis.

Freeze-tolerance is an important physiological trait for terrestrial environmental adaptation and intraspecific geographic-lineage diversification in ectothermic animals, yet there remains a lack of systematic studies on its underlying genetic mechanisms and evolution. To address this problem, we employed the widely distributed rice pest, the Chilo suppressalis, as a model to explore the genetic mechanisms and evolutionary history of freeze-tolerance. First, we systematically characterized its antifreeze mechanisms by performing functional validation of potential key genes in laboratory-reared lines. This revealed the functional roles of glycerol biosynthesis in freeze-tolerance, including the triacylglycerol-originated pathway via triacylglycerol lipase (Tgl) hydrolysis and the glycogen-originated pathway via α-amylase (Aa) and maltase (Ma) hydrolysis, as well as the roles of the cellular chaperones Hsc70 and Hsf1. Then, we investigated the evolution of freeze-tolerance by collecting representative geographical samples and performing population genetic analyses, which suggested differentiated strategies of cold adaptation in different geographic populations. Taken together, our findings demonstrate the functional basis of cold resistance in Chilo suppressalis and reveal the evolutionary history of freeze-tolerance in natural populations, providing insights into organismal freeze-tolerance and clues for pest control.