Natural Ice-Nucleating Bacteria Increase the Freezing Tolerance of the Intertidal Bivalve Geukensia demissa

Instead of avoiding freezing, freeze tolerant invertebrates actively initiate controlled ice nucleation at relatively high sub-zero temperatures in extracellular compartments. Most produce proteinaceous ice-nucleators in their hemolymph, however the intertidal bivalve mollusc Geukensia demissa lacks this ability. Instead it utilizes at least one strain of ice-nucleation active (INA) bacteria, Pseudomonas fulva, present in seawater, to induce crystallization in the pallial fluid that fills its mantle cavity. In this study, two additional INA bacteria strains were isolated from the palial fluid of Geukensia demissa: Psychrobacter sp. and Shewanella sp. The icenucleation activity of both strains was characterized and Psychrobacter was found to consistently induce nucleation at temperatures 1-3°C higher than Shewanella. Based on 16S rRNA sequencing, neither of these bacteria have yet been identified. The effects of Psychrobacter on the freeze tolerance of summer-acclimatized Geukensia were assessed and compared to the freeze tolerance of winter-acclimatized specimens. This assessment was accomplished through whole-organism death experiments involving 12-hour periods of exposure to sub-zero temperatures and cell viability tests using a LIVE/DEAD sperm viability kit (Molecular Probes, Inc, Eugene, OR). Adding INA bacteria to summer-acclimatized Geukensia reduced their LT50 from -12.5°C to -15.0°C. The LT50 of winter-acclimatized specimens was determined to be 16.5°C. This result may be explained by the presence of cryoprotectants and multiple strains of bacteria in the winter-acclimatized specimens. Gill cell viability tests resulted in an average of 12% greater damage in summer-acclimatized Geukensia without added bacteria at -13.5°C, but no significant differences at -10°C and -15°C. This study is, to our knowledge, the first time that a bacterium has been shown to increase the survival of a freeze tolerant animal.