Ultrastructural effects of lethal freezing on brain, muscle and Malpighian tubules from freeze-tolerant larvae of the gall fly, Eurosta solidaginis

In preparation for winter low temperatures, larvae of the gall fly, Eurosta solidaginis, accumulate the cryoprotectants glycerol, sorbitol, and trehalose. The fat body cells of these freeze-tolerant larvae can survive intracellular freezing to −80°C for 48 h even though no whole larvae survive this treatment. We hypothesized that some other tissue was more susceptible to freezing and therefore may be responsible for larval death. This paper compares the ultrastructure of brain, muscle, and Malpighian tubules between non-lethally frozen and lethally frozen freeze-tolerant larvae. The nuclei of cortical brain cells from lethally frozen larvae exhibited clumped chromatin and nuclear membranes with occasional expansions or `blebs' of the intermembranous space, while the cytoplasm contained swollen spheres of endoplasmic reticulum. In contrast, non-lethally frozen brain contained nuclei with evenly dispersed chromatin, smooth nuclear membranes and a cytoplasm free of swollen endoplasmic reticulum. Muscle tissue of lethally frozen larvae contained disrupted myofilaments surrounding the Z-line in comparison to non-lethally frozen muscle which had myofilaments extending all the way to the Z-line. Alterations of Malpighian tubule cells from lethally frozen larvae included an extracted cytoplasm with swollen and rounded mitochondria. In contrast, Malpighian tubule cells from non-lethally frozen larvae had a more concentrated cytoplasm with many rod-shaped mitochondria. Results show alterations to all three tissue types due to lethal freezing. The brain tissue contained the most observable alterations and therefore may be the most susceptible to lethal freeze damage.