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Cold-hardening during long-term acclimation in a freeze-tolerant woolly bear caterpillar, Pyrrharctia isabella

ABSTRACT The banded woolly bear caterpillar, Pyrrharctia isabella (Lepidoptera: Erebidae), overwinters in leaf litter and survives freezing under natural conditions. Following 18 weeks of cold acclimation at 5°C, all caterpillars could survive 1 week of continuous freezing at −20°C or seven cycles of freezing–thawing at −20°C, but none survived freezing at −80°C. Field-collected caterpillars had a temperature of crystallization of −7.7±0.5°C that decreased significantly to −9.5±0.6°C after 12 weeks of acclimation at 5°C. Hemolymph levels of free proline, total amino acids and proteins reached a peak during the first 4 weeks of acclimation; concomitantly, hemolymph osmolality increased markedly during this interval (from 364 to 1282 mosmol kg−1). In contrast, hemolymph pH decreased during the first 4 weeks of acclimation before this trend reversed and pH values gradually returned to initial values. However, pH reached its peak value following 1 week at −20°C, but decreased after longer periods of freezing. During cold acclimation, cholesterol levels decreased in the hemolymph and the membrane fraction of fat body but not in other tissues. Lethal freezing at −80°C reduced cell survival in foregut tissue and caused leakage of free proline, total amino acids and proteins from tissues into the hemolymph. The addition of glycerol to the bathing medium reduced freezing injury in fat body cells, as evidenced by reduced leakage of amino acids and proteins. Summary: After an extended period of cold acclimation, woolly bear caterpillars survived freezing at −20°C. Cold-hardening markedly increased hemolymph osmolality, as well as hemolymph levels of free proline, total amino acids and proteins.