Temperature-metabolism relationships above and below the body fluid freezing point in the freeze-tolerant white worm, Enchytraeus albidus

Enchytraeus albidus is a freeze-tolerant oligochaete with potential for long-term cryopreservation storage as live feed in aquaculture. In this study, we measured the metabolic rate (MR) of E. albidus across a range of temperatures (+20 to -14 °C) in both unfrozen and frozen states using intermittent flow-through CO₂ respirometry. Our results demonstrate an exponential decrease in MR with declining temperature, and a markedly steeper decline below the freezing point of body fluids. Frozen worms exhibited a Q₁₀ of 24.1, in contrast to 2.2 in unfrozen worms, indicating significant metabolic depression associated with freezing. We argue that this depression is strongly correlated with intracellular osmolality, which increases due to freeze-induced dehydration. Theoretical calculations based on MR data suggest that glycogen stores could support frozen aerobic metabolism for up to 3-4 months at -14 °C, aligning with overwinter survival observed in Arctic populations. These findings indicate that E. albidus primarily relies on aerobic metabolism when frozen and highlight its suitability for long-term storage as live feed under moderate freezing conditions. Our results provide valuable physiological knowledge for optimizing industrial cryopreservation strategies to balance survival and shelf-life in freeze-tolerant invertebrates.