Abstract
The bay mussel, Mytilus trossulus, is an animal that can survive extracellular ice formation. Depending on air and ocean temperatures, freeze tolerant intertidal organisms, like M. trossulus, may freeze and thaw many times during the winter. Freezing can cause protein denaturation, leading to an induction of the heat shock response with expression of chaperone proteins like the 70 kDa heat shock protein (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. Mytilus are important ecosystem engineers and provide habitat for other intertidal species, thus understanding their physiology under thermal extremes is important for preserving shoreline health. 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 h, 2 × 4 h, or 4 × 2 h. We then compared mortality, HSP70 expression, and the quantity of ubiquitin-conjugated proteins across experimental groups. We found a single 8-h freeze caused significantly more mortality than repeated freeze–thaw cycles. We also found that HSP70 and ubiquitinated protein 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.
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All data is deposited on Github at https://github.com/laurentjgill/mussel-freeze-thaw.
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Acknowledgements
K.E.M. is supported by an NSERC Discovery grant, J.R.K. is supported by a NSERC CGS-M, and L.T.G. was supported by a NSERC USRA during this work. The authors thank Dr. Christopher Harley, Dr. Patricia Schulte, and three anonymous reviewers for their assistance and ideas which improved this manuscript greatly.
Funding
This study was funded by Natural Sciences and Engineering Research Council of Canada, RGPIN-2019-04239, Katie Elizabeth Marshall.
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Gill, L.T., Kennedy, J.R. & Marshall, K.E. Proteostasis in ice: the role of heat shock proteins and ubiquitin in the freeze tolerance of the intertidal mussel, Mytilus trossulus. J Comp Physiol B 193, 155–169 (2023). https://doi.org/10.1007/s00360-022-01473-2
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DOI: https://doi.org/10.1007/s00360-022-01473-2