Insects that are exposed to subzero temperatures adapt by becoming either freeze tolerant (they survive if frozen) or they must become freeze avoiding and prevent freezing. The freeze avoiding beetle Cucujus clavipes has a broad latitudinal range, from North Carolina to the Brooks Range in northern Alaska above the Arctic Circle. In the interior of Alaska, this species of beetle is exposed to some of the lowest temperatures in North America and as a result has become freeze avoiding to the extreme. Overwintering Alaska populations of C. clavipes generally supercool (cool below their freezing point without freezing) to a mean of approximately -40oC, with some individuals supercooling to 58oC, before freezing. However, sometimes, larvae of this beetle species deep supercool and cannot be frozen even when cooled to 150oC. Although during deep supercooling beetle larvae do not freeze, they do turn glassy or vitrify at ~ -75oC.. During vitrification their body water turns solid, but does not crystallize to form ice.. The primary goal of this study is to identify and characterize the physiological adaptations that permit deep supercooling and vitrification. Specifically the PIs will investigate the roles of antifreeze proteins, glycerol, removal of ice nucleators that limit supercooling, diapause (reduced metabolism), and cryoprotective dehydration and concentration of antifreeze proteins.. In addition, a proteome study will identify additional proteins that may aid in deep supercooling. The broader impacts of this work includes the potential applications for: (1) cryopreservation of biological materials in a non-frozen state, (2) development of more cold tolerant plants for agriculture, and (3) the frozen food industry. Further, the work will include training for a talented high school student and several graduate students. Furthering the understanding of the how adaptations permit survival at such extreme low temperatures has the potential to create enthusiasm and attract more students to careers in biology.
