The sub-lethal effects of repeated freezing in the woolly bear caterpillar Pyrrharctia isabella

Repeated freeze-thaw cycles are common and are increasing in frequency with climate change in many temperate locations, yet understanding of their impact on freeze-tolerant insects is extremely limited. We investigated the effects of repeated freezing and thawing on the freeze-tolerant final instar caterpillars of the moth Pyrrharctia isabella (Lepidoptera: Arctiidae) by subjecting individuals to either a single sustained 35 h freeze or five 7 h freezes. Sub-lethal effects were quantified with changes in three broad groups of measures: (1) cold hardiness, (2) metabolic rate and energy reserves and (3) survival after challenge with fungal spores. Repeated freeze-thaw cycles increased mortality to almost 30% and increased tissue damage in Malpighian tubules and hemocytes. Repeated freezing increased caterpillar glycerol concentration by 0.82 mol l(-1). There were no changes in metabolic rate or energy reserves with repeated freezing. For the first time, we report increased survival after immune challenge in caterpillars after freezing and suggest that this may be linked to wounding during freezing. We suggest that little repair of freezing damage is possible in P. isabella caterpillars and repeated freeze-thaw cycles may present significant challenges to survival in this species.