Evolution of freezing susceptibility and freezing tolerance in terrestrial arthropods

Arthropods have evolved various adaptations to survive adverse seasons and it has long been discussed why some arthropods are freezing-susceptible and some are freezing-tolerant. However, which mode of frost resistance came first during the course of evolution? A commonly held opinion is that no choice of strategy has been offered in evolution, because each species of arthropod may have its own evolutionary and natural history, leading to cold-hardiness. Freezing tolerance is more frequent in holometabolous insect orders and partially used by certain vertebrates, like some terrestrially hibernating amphibians and reptiles. Supported by phylogenetic, ontogenetic and ecological arguments, we suggest here that freezing tolerance is more recent than freezing susceptibility in the course of arthropods evolution. In addition, we observe that three basic modes of freezing resistance in insect species exist in the field: (i) permanent or year-round freezing-susceptible species, (ii) alternative or seasonal freezing-susceptible/freezing-tolerant species, (iii) permanent or year-round freezing tolerant species.     

Field ecology of freezing: Linking microhabitat use with freezing tolerance in Litoria ewingii

A small number of vertebrate species, including some frogs, are freezing tolerant and survive ice forming in their bodies under ecologically relevant conditions. Habitat use information is critical for interpreting laboratory studies of freezing tolerance, but there is often little known about the winter habitat and behaviours of the species under study. This work describes microhabitats used by the freezing‐tolerant frog Litoria ewingii Duméril and Bibron 1841 and their temperature characteristics. In winter, L. ewingii used microhabitats with wood, located further away from water than in summer. Microhabitat temperature records showed that frog microhabitats regularly fell below the temperature at which frog body fluids freeze (?1°C), and cooled substantially more slowly than did the air temperature. Temperatures were highly variable between microhabitats, seasons and years, with a minimum of ?2.4°C and a maximum cooling rate of 0.77°C h^?1. Frozen frogs were observed to recover in the field, demonstrating freezing tolerance. Both the characteristics of microhabitats and their selection are important in ensuring freezing survival.     

Flash freezing of erythrocyte suspensions

Freezing whole blood in bulk usually results in severe cellular destruction through the action of ice crystals and osmotic effects in the freezing liquid. The potential of flash freezing blood aerosols onto a liquid nitrogen surface as a means of inhibiting cellular damage was studied in this work. Three commercial spraying devices were employed to spray-freeze either whole blood or concentrated erythrocyte suspensions, using hydroxyethyl starch (HES) as a cryoprotectant. The integrity and viability of the processed cells were assessed by measuring gross rheological properties and the extent of hemolysis. Cells were found to be susceptible to the very high shear stresses imposed by some of the spraying devices. Bulk freezing of blood, even in the presence of the cryoprotectant, resulted in complete cellular destruction. Whereas flash freezing was capable of substantially reducing the level of hemolysis to 12.6% and preserving the cellular deformability.     

Peripheral tissue freezing in cryosurgery

The recently formulated bioheat equation of Weinbaum and Jiji which accounts for the vascular ultrastructure and blood perfusion was applied to the freezing of peripheral tissue. Using quasi-steady approximation the temperature distribution in the two-phase tissue and the motion of the frozen front were determined. Results are in good agreement with Pennes' bioheat equation.     

Deep freezing of horse embryos

Fourteen horse embryos recovered non-surgically on Days 6-8 after ovulation (Day 0) were cooled slowly to - 35 degrees C (7 embryos) or - 40 degrees C (7 embryos) and stored in liquid nitrogen (- 196 degrees C) for 4-98 days. Surgical transfer of the thawed embryos to unmated recipient mares that had ovulated - 2 to + 1 days with respect to the embryo donors resulted initially in the establishment of 4 conceptuses. However, only one mare maintained her pregnancy to term.