Although seasonal changes in the freeze-tolerance of third-instar larvae of Eurosta solidaginis have been well documented for the whole organism, the nature of this cold-hardiness at the cellular level has not been examined. Seasonal changes in the survival of fat body cells from E. solidaginis larvae were assessed using fluorescent vital dyes after freezing at -10, -25 or -80 °C for 24 h both in vivo and in vitro. Cells frozen in vitro were frozen with glycerol, with sorbitol (both of which enhanced cell survival) or without cryoprotectants. Both cellular and organismal survival were low in August when larvae were not freeze-tolerant, then increased dramatically during September and October before leveling off from November to January. This observation for cells frozen without cryoprotectants indicates that the cells themselves have adapted. The single most important factor influencing cell survival, as determined by logistic regression modeling, was the time of larval collection, which reflects the level of cold-hardiness achieved by field acclimation. Cells frozen in vivo exhibited greater survival than did those frozen in vitro, even with the addition of cryoprotectants. Since no differences were observed between cells frozen with glycerol or sorbitol, the role of the multi-component cryoprotectant system present in E. solidaginis should be investigated.

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