Freeze tolerance in hermit flower beetle (Osmoderma eremicola) larvae

Larvae of Osmoderma eremicola (Knoch)(Coleoptera: Scarabaeidae) were found to be freeze tolerant. Early (1-2.5 cm) and late (3-5 cm) larval stages survived freezing to -8.3°C for 96 h with 64% of body water as ice. Glycogen phosphorylase activity in the fat body was increased 6-7-fold during freezing, and glycogen stores were depleted in fat body (from 237 ± 1 to 57 ± 7 μmol/g fresh weight) and body wall (89 ± 4 to 40 ± 1 μmol/g fresh weight). However, no glycogen derived cryoprotectants (glycerol, sorbitol, glucose, fructose) were found to accumulate in tissues or haemolymph during freezing or acclimation at 9,0 and -5°C. Osmolatity (∼450 mOsmol) of haemolymph was constant during freezing and acclimation from 9.to -5°C, also suggesting the absence of low molecular weight cryoprotectants. Haemolymph trehalose levels never rose above 2.7 mM as detected by NMR. Total protein levels in fat body and body wall remained unchanged during acclimation. HPLC analysis showed increases in amino acid levels by 10-15 μmol/g fresh weight in the body wall of both early and late instar larvae during freezing to -8.3°C for 96 h, with consistent increases in glycine (7.4-8.7 μmol/g fresh weight), alanine (4.2-5.8 μmol/g fresh weight), glutamate (2.4 μmol/g fresh weight), and valine (0.8-1.6 μmol/g fresh weight). Smaller increases in glutamic acid (by 1 μmol/g fresh weight) and alanine (by 3.5 μmol/g fresh weight) were also seen in late instar fat body, whereas glycine levels remained unchanged and valine decreased slightly (by 1.4 μmol/g fresh weight) in this tissue. Proline levels, detected by NMR, ranged from 10 to 18 μmol/g fresh weight in fat body and 40-70 μmol/g fresh weight in haemolymph, making it the most abundant amino acid in fat body