Supercooling capacity along with up-regulation of glycerol content in an overwintering butterfly, Parnassius bremeri

Abstract

Eggs of an endangered butterfly, Parnassius bremeri, hatch during December and young larvae grow during winter and early spring. Cold tolerance is required for survival and growth of the young larvae. Supercooling points (SCPs) were below −20°C for the young larvae compared to −9.8°C for the last fifth instar (L5) larvae, which developed during May. To investigate the depression of SCPs in young third instar (L3) larvae, polyols and free amino acids in hemolymph were quantified using a BioLC. Compared to L5 larvae, L3 larvae had higher polyols (glycerol and mannitol). However, L5 larvae had much higher levels of trehalose than L3 larvae in hemolymph. Increase of glycerol in L3 larvae was explained by enhanced expression of genes associated with glycerol metabolism. RNA-Seq analysis indicated that expression level of glycerol-3-phosphate dehydrogenase in L3 larvae was higher than that of L5 larvae and more number of glycerol kinase contigs was detected in L3 larvae. In contrast, trehalose-6-phosphate dehydrogenase was highly expressed in L5 larvae by more than 3.2 folds compared to L3 larvae. These results suggest that deep supercooling along with glycerol and other small cryoprotectants is associated with cold tolerance of young larvae of P. bremeri.