Supercooling in a freeze-tolerant cranefly larva, Tipula sp

The freezing curves of individuals of Tipula sp. revealed two markedly separate freezing events during winter. The initial freezing event occurred at about −3°C, the second at about −5°C. Larvae survived freezing to −10°C and contained about 30% unfreezable water. Larvae ligated close to the anus supercooled to −5°C, yet did not survive freezing. The drop in relative water content from about 76–81% gave rise to a somewhat smaller depression of haemolymph melting point than what should be expected from the water loss. Thus, the data indicated that osmolytes must have been removed rather than added to the body fluid of hibernating larvae. Ice-nucleating agents present in excised alimentary tracts and in haemolymph samples promoted freezing in the temperature range from −5 to −7°C. In larvae ligated close to the anus to prevent a leakage of rectal water, freezing occurred at temperatures equivalent to those of their excised alimentary tracts which contained the most powerful ice nucleators in the organism. Therefore, rectal fluid which moistens the body surface surrounding the anus, appeared to be the prime nucleating site. Thus, freezing is initiated when rectal fluid freezes outside the animal and ice propagates throughout the tissue. The remarkably high supercooling point of intact larvae moreover suggests that they rely upon freezing by inoculation rather than by extracellular ice nucleators.