Expression of two self-enhancing antifreeze proteins from the beetle Dendroides canadensis in Drosophila melanogaster

Antifreeze proteins (AFPs) lower the freezing point of water without affecting the melting point. This difference between melting point and freezing point has been termed thermal hysteresis. Antifreeze protein genes, dafp-1 and/or dafp-4, from the freeze-avoiding insect, Dendroides canadensis, were transferred to Drosophila melanogaster via P-element-mediated transformation. The Northern and Western blots showed expression of DAFP(s) at both transcript and protein levels. The highest thermal hysteresis activity of 6.78+/-0.12 degrees C was detected in 5-day adult flies containing two copies of each of the dafp-1 and dafp-4 genes, while flies with two copies of either dafp-1 or dafp-4 had less activity, 5.52 and 3.24 degrees C, respectively (measured by nanoliter osmometer). This suggests synergistic enhancement of thermal hysteresis activity between DAFP-1 and DAFP-4 in transgenic D. melanogaster containing both DAFPs. Supercooling points without ice in contact with the insects were lowered in all 5 transgenic lines compared with controls, however, when ice was in contact with the flies, supercooling points were lowered only in the heterozygous + transgenic line. Also, transgenic D. melanogaster exhibited higher survivorship compared with controls when placed at low non-freezing temperatures (0 and 4 degrees C), however, DAFP-1 and DAFP-4 did not display any synergistic enhancement in these non-freezing survival experiments.