Mechanistic understanding about the nature of cellular cryoinjury and mechanisms by which some animals survive freezing while others do not is currently lacking. Here, we exploited the broadly manipulable freeze tolerance of larval malt flies (Chymomyza costata) to uncover cell and tissue morphological changes associated with freeze mortality. Diapause induction, cold acclimation and dietary proline supplementation generate malt fly variants ranging from weakly to extremely freeze tolerant. Using confocal microscopy and immunostaining of the fat body, Malpighian tubules and anterior midgut, we described tissue and cytoskeletal (F-actin and α-tubulin) morphologies among these variants after exposure to various cold stresses (from chilling at -5°C to extreme freezing at -196°C), and upon recovery from cold exposure. 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. Larvae that recovered from a freezing challenge repaired F-actin aggregation but not lipid droplet coalescence or α-tubulin structure. Our observations indicate that lipid coalescence and damage to α-tubulin are non-lethal forms of freeze injury, and suggest that repair or removal (rather than protection) of actin proteins is a potential mechanism of acquired freeze tolerance.

Faculty of Science University of South Bohemia Branišovská 31 370 05 České Budějovice Czech Republic

Institute of Entomology Biology Centre of the Academy of Sciences of the Czech Republic 370 05 České Budějovice Czech Republic

References provided by Crossref.org

Extracellular freezing induces a permeability transition in the inner membrane of muscle mitochondria of freeze-sensitive but not freeze-tolerant Chymomyza costata larvae

Insect mitochondria as targets of freezing-induced injury

Transcriptional analysis of insect extreme freeze tolerance

Evidence for non-colligative function of small cryoprotectants in a freeze-tolerant insect