Cryoprotective effects of thermal hysteresis protein on survivorship of frozen gut cells from the freeze‐tolerant centipede Lithobius forficatus

Abstract

AbstractThermal hysteresis proteins (THPs), which depress the freezing point of water by a non‐colligative process, are common in freeze‐avoiding polar marine fish and terrestrial arthropods where they function as antifreezes. However, some freeze‐tolerant arthropods and plants also produce THPs in winter, raising the question of whether these proteins are involved in the abilities of these organisms to survive the freezing of their extracellular fluids. The centipede Lithobius forficatus is one of these freeze‐tolerant, THP‐producing organisms.Cells isolated from the midgut of both winter‐ and summer‐collected L. forficatus were frozen at various temperatures, either with or without the addition of THPs purified from the beetle Dendroides canadensis, and the survival of the cells was evaluated. The presence of THPs (0.02 mg/ml) in the bathing medium produced a statistically significant increase in cell survival, indicating a cryoprotective function for the THPs. The LT50 (temperature of 50% survival) of cells for summer centipedes was shifted from −8.2°C to −15.0°C, and that of cells of winter centipedes was shifted from −12.1°C to −15.1°C by addition of THPs. Also, cells from summer‐collected centipedes which had been incubated in media containing THPs and then washed in buffer without THPs to remove the protein from the media prior to freezing demonstrated significantly increased survivorship after freezing (LT50 = −14.5). Immunofluorescence studies of these cells indicated that the THPs were present in the cells and/or on the cell membrane. The presence of THPs in the medium also provided some protection to cells frozen under conditions designed to promote the potentially damaging process of recrystallization. (THPs are well known for their ability to inhibit recrystallization.) Therefore, THPs do appear to function as a component in the suite of adaptations which permit freeze tolerance in L. forficatus. © 1995 Wiley‐Liss, Inc.