Ectothermic animals in the temperate and arctic zones of the world are sometimes exposed to severe cold during winter, yet they survive the potentially fatal conditions. Some of these cold tolerant ectotherms deal with very low temperatures by physiological mechanisms that promote supercooling. Others, like the larvae of the rice stem borer (Chilo suppressalis)are able to survive ice formation in their extracellular fluids and are able to withstand freezing at temperatures as low as -25°C. When water freezes in the extracellular fluid, an osmotic gradient is generated across the cell membrane so that water must move out of the cells and/or osmolytes must move in to maintain the osmotic balance. This is true for the rice stem borer where the cells loose water and simultaneously accumulate glycerol during freezing. A recent study by Yohei Izumi and colleagues investigated the importance of water and glycerol transport across the cell membrane for freeze tolerance in rice stem borer larvae.
The studies were performed on larvae that were overwintering and in diapause (arrested development) or in larvae in a non-diapause state, as only the overwintering larvae are freeze tolerant. The team extracted fat bodies from the larvae and suspended these in a medium. Using these fat bodies they assessed freeze tolerance by monitoring the appearance of freeze damaged or dead cells that could be coloured using trypan blue. The study showed that only the fat bodies from overwintering larvae were able to tolerate freezing but that they could only do so when the medium was supplied with high concentrations of glycerol (0.25 mol l-1). In contrast, fat bodies from non-diapausing larvae became severely damaged after freezing. Even when the medium's glycerol concentration was increased to 0.75 mol l-1the freeze tolerance of non-diapausing larvae's did not approach that of the overvintering larvae. This shows that the presence of glycerol is essential for freeze tolerance, but that high concentrations of cryoprotectants alone cannot assure freeze tolerance and other mechanisms must also be present.
The authors hypothesised that aquaporins, membrane bound water channel proteins, could play a role in freeze tolerance as these may be capable of rapid transport of water and small neutral solutes such as glycerol across the cell membrane. To investigate this, the group added mercuric chloride, an inhibitor of aquaporins, to the medium containing glycerol and found that this reduced glycerol and water transport in and out of the cells. Moreover this inhibition obliterated freeze tolerance from the fat bodies of the overwintering larvae. Thus, freeze tolerance in overwintering larvae seems reliant on the ability to rapidly transport water and glycerol during freezing. This was further supported by the finding that non-diapausing larvae, that could not endure freezing, were unable to attain the same rates of glycerol transport as the overwintering larvae.
The study by Izumi and colleagues clearly shows that in addition to the presence of cryoprotectants the ability to rapidly move water and cryoprotectants across membranes is essential to ensure freeze tolerance in the rice stem borer. This finding may provide further insight into the synergistic modifications that occur during winter acclimation and/or diapause in many freeze or desiccation resistant ectothermic animals.
