Visualizing cellular cryoinjury: why some animals survive freezing while others do not

Image description: Fat body cells (red, anti-beta tubulin antibody) and Malpighian tubules (green, anti-F-actin antibody) from an extremely freeze-tolerant malt fly larva frozen at -30°C.

Under the right conditions, larvae of the malt fly, Chymomyza costata (Diptera: Drosophilidae) can survive freezing to -196°C in a fully-hydrated state. Such a physiological feat is not only impressive but may hold the key to applications in cryopreservation. To understand why cells do or do not survive freezing, we exploited the malt fly's broadly-manipulable freeze tolerance. Using various rearing conditions (modified temperature, photoperiod, and/or proline supplementation) we generated three larval variants ranging from weakly freeze tolerant (some surviving to as low as -10°C) to extremely freeze tolerant (surviving at -196°C). The malt fly larval variants were exposed to temperatures between -5°C and −196°C, and some were allowed to recover from cold exposure. We then employed confocal microscopy and cytoskeletal (F-actin and α-tubulin) immunostaining to visualize the morphologies of the fat body, Malpighian tubules, and anterior midgut (nearly 2,700 images). Fat body tissue appeared to be the most susceptible to cryoinjury: freezing caused coalescence of lipid droplets, loss of α-tubulin structure, and apparent aggregation of F-actin. A combination of diapause and cold acclimation substantially lowered the temperature at which these morphological disruptions occurred and correlated with better survival. Larvae that recovered from a freezing challenge repaired F-actin aggregation but surprisingly did not repair lipid droplet coalescence or α-tubulin structure (in fact, this morphological disruption became worse!). Our observations suggest that lipid coalescence and damage to α-tubulin are non-lethal forms of freeze 'injury', while repair or removal (rather than protection) of actin proteins is a potentially important mechanism of freeze tolerance.

Des Marteaux, L., Štětina, T., & Košťál, V. (2018). Insect fat body cell morphology and response to cold stress is modulated by acclimation. Journal of Experimental Biology 221(21):jeb.189647. doi: 10.1242/jeb.189647