Abstract
The bay mussel, Mytilus trossulus, is one of the few animals that can survive internal ice formation. Freeze tolerant intertidal animals, like M. trossulus, may freeze and thaw many times during the winter, depending on air and ocean temperatures. Freezing can cause protein denaturation, leading to an induction of the heat shock response with expression of proteins like HSP70, and an increase in ubiquitin conjugated proteins. There has been little work on the mechanisms of freeze tolerance in intertidal species, limiting our understanding of this survival strategy. Additionally, this limited research has focused solely on the effects of single freezing events, but the act of repeatedly crossing the freezing threshold may present novel physiological or biochemical stressors that have yet to be discovered. We predicted that repeated freeze exposures would increase mortality, upregulate HSP70 expression, and increase ubiquitin conjugates in mussels, relative to single, prolonged freeze exposures. Mytilus trossulus from Vancouver, Canada were repeatedly frozen for a combination of 1 × 8 hours, 4 × 2 hours, or 2 × 4 hours. We then compared mortality, HSP70 expression, and ubiquitin quantity across experimental groups. We found a single 8-hour freeze caused significantly more mortality than repeated freeze-thaw cycles. We also found that HSP70 and ubiquitin expression was upregulated exclusively after freeze-thaw cycles, suggesting that freeze-thaw cycles offer a period of damage repair between freezes. This indicates that freeze-thaw cycles, which happen naturally in the intertidal, are crucial for M. trossulus survival in sub-zero temperatures.