| Terrestrial insects have evolved molecular and biochemical adaptations that have allowed them to exploit virtually all climates on earth including adaptations that allow survival at subzero temperatures during the winter months by insects living in temperate and polar climates. Two strategies of insect cold-tolerance have emerged: freeze-tolerance and freeze-avoidance. Freeze-tolerant insects can endure extracellular ice formation, whereas freeze-avoiding species strongly depress the supercooling point of their body fluids to remain liquid over the winter. The research reported in this thesis analyzed molecular strategies of cold tolerance, hypometabolism and hypoxia tolerance in both kinds of insects using as models the freeze-avoiding gall moth, Epiblema scudderiana, and the freeze-tolerant gall fly, Eurosta solidaginis . Activities of ion motive ATPases [Na+K+ATPase, sarco(endo)plasmic Ca2+ATPase] were strongly reduced over the winter months, contributing to energy-saving hypometabolism, and in vitro studies indicated that the control mechanism involved was reversible protein phosphorylation. Differential gene expression in response to cold, subzero or hypoxia exposures was evaluated using three different methods (cDNA library construction and screening, nylon macroarrays, DNA microarrays) to identify key protein products that could contribute to cold and hypoxic tolerance. These studies identified a wide variety of genes as cold responsive that have never previously been associated with cold tolerance. Enzyme activities, transcript levels, and DNA content of mitochondrial genes were evaluated under cold and hypoxia stresses and indicated a role for mitochondria in cold and hypoxia tolerance that is more dynamic that previously understood and that differs significantly between freeze avoiding and freeze tolerant species. Other studies indicated an important role for signal transduction via the mitogen-activated protein kinase (MAPK) superfamily in cold and hypoxia tolerance, particularly the role of the p38 MAPKs, which showed clear differences between the freeze-tolerant and freeze-avoiding species. Finally, examination of the nuclear factor (NF)-kappaB signaling pathway indicated that the suppression of the inflammatory response appears to be significant in the winter survival of both species and may be a crucial difference between hypoxia-tolerance and hypoxia-intolerance. |
