Abstract
Freezing is an extreme stress to living cells, and so freeze-tolerant animals often accumulate protective molecules (termed cryoprotectants) to prevent the cellular damage caused by freezing. The bay mussel, Mytilus trossulus, is an ecologically important intertidal invertebrate that can survive freezing. Although much is known about the biochemical correlates of freeze tolerance in insects and vertebrates, the cryoprotectants that are used by intertidal invertebrates are not well characterized. Previous work has proposed two possible groups of low-molecular weight cryoprotectants in intertidal invertebrates: osmolytes and anaerobic byproducts. In our study, we examined which group of candidate cryoprotectants correlate with plasticity in freeze tolerance in mussels using 1H NMR metabolomics. We found that the freeze tolerance of M. trossulus varies on a seasonal basis, along an intertidal shore-level gradient, and with changing salinity. Acclimation to increased salinity (30 ppt compared with 15 ppt) increased freeze tolerance, and mussels were significantly more freeze tolerant during the winter. Mussel freeze tolerance also increased with increasing shore level. There was limited evidence that anaerobic byproduct accumulation was associated with increased freeze tolerance. However, osmolyte accumulation was correlated with increased freeze tolerance after high salinity acclimation and in the winter. The concentration of most low molecular weight metabolites did not vary with shore level, indicating that another mechanism is likely responsible for this pattern of variation in freeze tolerance. By identifying osmolytes as a group of molecules that assist in freezing tolerance, we have expanded the known biochemical repertoire of the mechanisms of freeze tolerance.