Comparative ultrastructure of fat body cells of freeze-susceptible and freeze-tolerant Eurosta solidaginis larvae after chemical fixation and high pressure freezing

Abstract

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-hardening 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 of freeze-susceptible (September-collected) and freeze-tolerant (January-collected) 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 uniformly electron dense mitochondrial matrix. Conventionally prepared samples exhibited crenated cytoplasmic organelles with undulating membrane profiles; smooth profiles characterized high-pressure frozen structures. Lipid droplets were abundant in all fat body cells, however glycogen, the energy source of cryoprotectants, was variable in both freeze-susceptible and freeze-tolerant cells. Coalescence of lipid droplets, induced by intracellular freezing at progressively lower temperatures, caused displacement of interior cytoplasmic organelles including the nucleus to the cell cortex. This shift in cytoplasm had no affect on survival of the cells.