Cryoprotective and osmotic responses to cold acclimation and freezing in freeze-tolerant and freeze-intolerant earthworms

In this paper we present the results of physiological responses to winter acclimation and tissue freezing in a freeze-tolerant Siberian earthworm, Eisenia nordenskioeldi, and two freeze-intolerant, temperate earthworm species, Lumbricus rubellus and Aporrectodea caliginosa. By analysing the physiological responses to freezing of both types we sought to identify some key factors promoting freeze tolerance in earthworms. Winter acclimation was followed by a significant increase in osmolality of body fluids in E. nordenskioeldi, from 197 mosmol kg-1 in 10 degrees C-acclimated animals to 365 mosmol kg-1 in animals acclimated to 0 degrees C. Cold acclimation did not cause any change in body fluid osmolality in the two freeze-intolerant species. As a response to ice formation in the body, the freeze-intolerant species produced copious amounts of slime and expulsion of coelomic fluids, and thereby lost 10-30% of their total water content. Contrary to this, the freeze-tolerant species did not lose water upon freezing. At temperatures down to -6.5 degrees C, the ice content in the freeze-tolerant E. nordenskioeldi was significantly lower than in L. rubellus. At lower temperatures there were no differences in ice content between the two species. Cold acclimated, but unfrozen, specimens of all three species had low levels of ammonia, urea, lactate, glycerol and glucose. As a response to ice formation, glucose levels significantly increased within the first 24 h of freezing. This was most pronounced in E. nordenskioeldi where a 153-fold increase of glucose was seen (94 mmol.l-1). In L. rubellus and A. caliginosa a 19-fold and 17-fold increase in glucose was seen. This is the first study on physiological mechanisms promoting freeze tolerance in E. nordenskioeldi, or any other oligochaete. Our results suggest that the cryoprotective system of this species more closely resembles that of freeze-tolerant anurans, which synthesize cryoprotectants only after tissues begin to freeze, than that of cold-hardy invertebrates which exhibit a preparatory accumulation of cryoprotectants during seasonal exposure to low temperature.