Lin Hui Yao, M.Sc. Chemistry, 2007

Profile of FoxO proteins and MnSOD in two cold-hardy insect species exposed to low temperature.



Expression of FoxO genes and proteins in two insects with different overwintering strategies were studied with Western blotting and RT-PCR. Epiblema scudderiana, a freeze-avoiding gall moth, and Eurosta solidaginis, a freeze-tolerant gall fly were used as model organisms to assess the role of FoxO transcription factors and one of their downstream proteins, manganese superoxide dismutase (MnSOD) in insect cold hardiness. FoxO3a gene and protein levels, as well as MnSOD protein, were analyzed inEpiblema scudderiana over time courses of exposure to 5°C or ‑15°C. Significant changes in total Foxo3a protein, the amount of phosphorylated inactive FoxO3a, FoxO3a mRNA levels, and MnSOD content were identified under different conditions. For example, reduced phospho-FoxO3a (Ser 253) content in larvae exposed to -15°C suggested enhanced transcriptional activation of genes under FoxO3a control at subzero temperatures whereas MnSOD content increased in larvae exposed to 5°C. Sequencing of FoxO3a mRNA from Epiblema scudderiana showed that the transcription factor was highly conserved as compared with mammals. FoxO3a, FoxO1 and MnSOD protein levels were also measured in the freeze tolerant insect Eurosta solidaginis subjected to 5 and ‑15°C exposures as well as a time course of thawing after freezing at -15°C for 1 week. The results implied that FoxO3a and FoxO1 functioned oppositely during cold, freezing and thawing of the larvae. Expression of MnSOD increased with 5°C exposure, remained elevated when larvae were frozen at -15°C, and decreased after thawing. Differential expression of FoxOs and MnSOD in both species suggest that MnSOD may not be transcriptionallycontrolled by FoxO3a or FoxO1 in cold hardy insects.