Comp aratrve Ultrastructure of Fat Body Cells of Freeze-susceptible and Freeze-tolerant Eurosta solidaginis Larvae After Chemical Fixation and High Pressure Freezing

In response to low environmental temperatures third instar larvae of the goldenrod gall fly, Eurosta solidaginis, undergo the process of cold-hardening in which they acquire freeze-tolerance. Cold-hard-ening includes the elevation of the supercooling point, the temperature at which body water spontaneously freezes, and the accumulation of the low-molecular-mass cryoprotectants, glycerol, sorbitol and trehalose. Although it is generally believed that freezing survival is only possible if the ice lattice is restricted to the extracellular space, the larval fat body cells survive intracellular ice formation. Fat body cells offreeze-susceptible (September-collected) and freeze-tolerant (January-col-lected) third instar larvae were prepared for transmission electron microscopic analysis using both conventional chemical fixation and high pressure freezing with freeze substitution. High pressure cryofixation procedures were superior to conventional fixation in both freeze-susceptible and freeze-tolerant cells. Endogenous cryoprotectants in freeze-tolerant larvae contributed to the superior ultrastructure of cytoplasmic organelles and nucleoplasm in high-pressure frozen fat body cells, while freeze-susceptible larvae, which lack high levels of cryoprotectants, contained fat body cells which sustained ice crystal damage following cryofixation. Mitochondria in fat body cells prepared using high pressure freezing/freeze substitution were characterized by smooth outer membranes and uniforml