RNA interference of glycerol biosynthesis suppresses rapid cold hardening of the beet armyworm, Spodoptera exigua

The beet armyworm, Spodoptera exigua, is a freeze-susceptible species that overwinters in temperate zones without diapause. A rapid cold hardening (RCH) and supercooling capacity usually play crucial roles in survival during the overwintering period. This study identified a cryoprotectant as a RCH factor of S. exigua. Pre-exposure of S. exigua larvae to 4 degrees C significantly increased survival at -10 degrees C in all developmental stages from egg to adult. RCH was dependent on the duration of the pre-exposure period. RCH also significantly enhanced the supercooling capacity. Cryoprotectant analysis using HPLC showed that the pre-exposure treatment allowed the larvae to accumulate glycerol in the hemolymph. Two genes, glycerol-3-phosphate dehydrogenase (GPDH) and glycerol kinase (GK), were identified as being associated with glycerol biosynthesis, and were cloned from S. exigua larvae. Both GPDH and GK were expressed in all developmental stages of S. exigua. RNA interference (RNAi) of either GPDH or GK significantly inhibited glycerol accumulation in the hemolymph of S. exigua. Larvae treated with RNAi for GPDH or GK exhibited a significant decrease in RCH capacity. The glycerol accumulation in response to 4 degrees C appeared to be under the control of a humoral signal, because a ligation experiment prevented glycerol accumulation in the other half of the body. This study indicates that glycerol is a RCH factor of S. exigua and its synthesis is in response to low temperature via humoral mediation.