During extracellular freezing, solutes in the haemolymph are concentrated, resulting in osmotic dehydration of the cells, which must be reversed upon thawing. Here, we used freeze tolerant Pyrrharctia isabella (Lepidoptera: Arctiidae) larvae to examine the processes of ion redistribution after thawing. To investigate the effect of the intensity of cold exposure on ion redistribution after thawing, we exposed caterpillars to -14°C, -20°C or -30°C for 35h. To investigate the effect of duration of cold exposure on ion redistribution after thawing, we exposed the caterpillars to -14°C for up to 6 weeks while sampling several time points. The concentrations of Na(+), K(+), Mg(2+) and Ca(2+) were measured after thawing in the haemolymph, fat body, muscle, midgut tissue and hindgut tissue. Being frozen for long durations (>3 weeks) or at low temperatures (-30°C) both result in 100% mortality, although different ions and tissues appear to be affected by each treatment. Both water distribution and ion content changes were detected after thawing, with the largest effects seen in the fat body and midgut tissue. Magnesium homeostasis appears to be vital for post-freeze survival in these larvae. The movement of ions during thawing lagged behind the movement of water, and ion homeostasis was not restored within the same time frame as water homeostasis. Failure to regain ion homeostasis after thawing is therefore implicated in mortality of freeze tolerant insects.
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