Mortality caused by extracellular freezing is associated with fragmentation of nuclear DNA in larval haemocytes of two drosophilid flies.

The great complexity of extracellular freezing stress, involving mechanical, osmotic, dehydration and chemical perturbations of the cellular milieu, hampers progress in understanding the nature of freezing injury and the mechanisms to cope with it in naturally freeze-tolerant insects. Here, we show that nuclear DNA fragmentation begins to occur in larval haemocytes of two fly species, Chymomyza costata and Drosophila melanogaster, before or at the same time as the sub-zero temperature is reached that causes irreparable freezing injury and mortality in freeze-sensitive larval phenotypes. However, when larvae of the freeze-tolerant phenotype (diapausing-cold acclimated-hyperprolinemic) of C. costata were subjected to severe freezing stress in liquid nitrogen, no DNA damage was observed. Artificially increasing the proline concentration in freeze-sensitive larvae of both species by feeding them a proline-enriched diet resulted in a decrease in the proportion of nuclei with fragmented DNA during freezing stress. Our results suggest that proline accumulated in diapausing C. costata larvae during cold acclimation may contribute to the protection of nuclear DNA against fragmentation associated with freezing stress.