Survival of Intracellular Freezitg, Lipid Coalescence and Osmotic Fragility in Fat Body Cells of the Frceze-tolerant

Atthough it is generally believed that under natural conditionso freeze-tolerant organisms can survive only if ice formation is restricted to the extracellular space, in 1959 R. W. Salt reported that fat body cefls of the freeze-tolerant gall fly, Eurosta solidaginis (Diptera: Tephritidae) ' survive intracellular freezing. Using cryomicroscopy, intratellular freezing was observed at-4.6*0.1oC for cells in Grace'i media. Freezing was apparently caused by inoculative freezing from outside the cell, since fat body cells in oil cooled to below- l5oc.without internal ice formation. Viability of cells was assessed using fluorescent vital dyes immediately following freezing for 24h at temperatures between-5 and- 80oC. At- 25oC ' or lower few cells survived freezing in Graceos medium alone. At- 25 and- 80oC, cells frozen in Grace's media supplemented with I M glycerol exhibited rates of survival similar to those in whole larvae. No larvae (n:2Olsurvived freezing to-80oC, but more-than 60oh of the fat body cells survived this treatment. Most faf body cells survived osmotic concentrations from 0-25 to 2 times that of the normal concentration of Grace's media (340 mOsm) for?Ah at 4"C. More than ffio/o of the cells survived 5 M glycerol in Grace's media under these conditions. An unusual response ofthese cells to freezing is the coalescence ofintracellular lipid droplets upon thawing. The magnitude of coalescence increases with decreasing temperature and increasing duration of exposure, but decreases with the addition of glycerol to the media. Coalescence in itself is not indicative of injury' since larvae frozen under conditions that cause extensive coalescence readily survived to complete their development and emerge as adults. Cold-hardiness Freeze tolerance Cryoprotection Glycero