Glycerol biosynthesis plays an essential role in mediating cold tolerance the red imported fire ant, Solenopsis invicta.

The red imported fire ant (RIFA), Solenopsis invicta Buren is native to South America and known as a global problematic invasive species. At low temperatures, several investigations have demonstrated an increase in glycerol as a primary rapid cold hardening (RCH) component and an increase in the supercooling point. Two genes, glycerol-3-phosphate dehydrogenase (GPDH) and glycerol kinase (GK), have been identified as being involved in the glycerol production process. In this study, one GPDH and two GK sequences were extracted from RIFA transcriptome analysis (Si-GPDH, Si-GK1, and Si-GK2). All three genes were expressed in different body parts and different tissues of S. invicta that Si-GK2 showed a higher expression level than the others. According to gene expression levels by qRT-PCR analysis, the highest expression levels of three genes were observed in fat body tissues. After 1 h of exposure to low temperatures (5°C or lower), the mRNA levels of these genes significantly increased, according to expression analyses. RNA interference (RNAi) of Si-GPDH or Si-GK1 and Si-GK2 exhibited a significant downregulation at the mRNA level. The mortality rate of treated RIFA by double-stranded RNA (dsRNA) specific to GPDH and GK2 significantly increased at low temperatures. This study indicates that GPDH and GK2 as glycerol biosynthesis genes in RIFA have a high expression level to synthesize a high level of glycerol as an RCH factor and they play crucial roles in survival during the cold period.