Dual roles of glucose in the freeze-tolerant earthwormDendrobaena octaedra: cryoprotection and fuel for metabolism

Abstract

SUMMARYEctothermic animals inhabiting the subarctic and temperate regions have evolved strategies to deal with periods of continuous frost during winter. The earthworm Dendrobaena octaedra is freeze tolerant and accumulates large concentrations of glucose upon freezing. The present study investigates the roles of glucose accumulation for long-term freeze tolerance in worms kept frozen at -2°C for 47 days. During this period, worms were sampled periodically for determination of survival and for measurements of glucose, glycogen, lactate, alanine and succinate. In addition we performed calorimetric measurements to assess metabolic rate of frozen and unfrozen worms. Long-term freezing was associated with a gradual depletion of glucose and worms that succumbed during this period were always characterised by low glucose and glycogen levels. The anaerobic waste products lactate and alanine increased slightly whereas succinate levels remained constant. However, it is argued that other waste products (particularly propionate) could be the primary end product of a continued anaerobic metabolism. Calorimetric measures of the metabolic rate of frozen worms were in accord with values calculated from the reduction in glucose assuming that most (~90%) glucose was metabolised anaerobically. Both estimates of metabolic rate demonstrated a 10-fold metabolic depression associated with freezing. Thus, in addition to the suspected role of glucose as cryoprotectant, the present study demonstrates that glucose accumulation is vital to ensure substrate for long-term anaerobic metabolism in frozen worms. On the basis of the estimated metabolite levels, we calculate that the combined effect of metabolic depression and large glucose stores enables a projected 3 months survival of freezing at -2°C of the 'average' D. octaedra. Such conditions are very likely to occur in the northern distribution ranges of this stress-tolerant earthworm.

Effect of repeated freeze-thaw cycles on geographically different populations of the freeze tolerant worm Enchytraeus albidus (Oligochaeta)

“…Unfortunately, we do not have good records of microclimatic thermal conditions from the origin of the populations, and are therefore unable to assess whether freeze-thaw events are more pronounced in the more temperate or the more arctic climates (Table 1). However, for the present study, it is our working hypothesis that freeze-thaw events are at least as frequent in the more mild winter temperate conditions as in the colder arctic areas, where temperatures are often found to remain below 0°C for extended periods (Calderon et al, 2009). Table S2 for detailed statistics).…”

“…Coulson et al, 1995;Calderon et al, 2009;Coulson, 2013;Sinclair et al, 2013). Freeze-thaw events may entail energetic costs as the re-establishment of osmotic and ionic gradients after freezing could be costly.…”

“…These changes include loss of mobility, loss of internal convective transport, synthesis of cryoprotectants and antifreeze proteins to protect cells and inhibit recrystallization, and establishment of metabolic depression that may be important to reduce the winter energy consumption (Ramløv, 2000;Bale, 2002;Calderon et al, 2009;Marshall and Sinclair, 2012b). Oligochaetes, including E. albidus, are no exception to these generalized responses.…”

“…Oligochaetes, including E. albidus, are no exception to these generalized responses. Thus, freeze-tolerant oligochaetes are characterised by a large build-up of glycogen reserves prior to winter, a large accumulation of cryoprotectants following freezing and a (moderate) metabolic depression during the frozen period (Berman and Leirikh, 1985;Holmstrup et al, 1999;Rasmussen and Holmstrup, 2002;Overgaard et al, 2007;Slotsbo et al, 2008;Calderon et al, 2009;Overgaard et al, 2009;Silva et al, 2013;Fisker et al, 2014). The relationship between winter survival and accumulation of glycogen and glucose is, however, more complex than first assumed, as accumulated glucose can serve both as a cryoprotectant and as a fermentable fuel (Calderon et al, 2009;Fisker et al, 2014).…”

Temperature dependence and ontogenetic changes of metabolic rate of an endemic earthworm Dendrobaena mrazeki

“…• C using calorimetry (Calderon et al 2009). The decrease of Q 10 in some intervals of temperature was interpreted several times as metabolic adaptation based on metabolic rate "regulation" inside limits of "optimal" temperature (Uvarov 1998;Uvarov & Scheu 2004b).…”