Collaborative research: Deep Supercooling to -100C or Lower in Alaska Populations of the Beetle Cucujus clavipes

暴露在零下温度下的昆虫要么变得耐寒(如果结冰它们就会存活)，要么必须变得避免结冰和防止结冰。这种避冻甲虫的纬度范围很广，从北卡罗来纳州到阿拉斯加北部北极圈上方的布鲁克斯山脉。在阿拉斯加的内陆地区，这种甲虫暴露在北美最低的温度下，因此变得极端地避免结冰。在阿拉斯加越冬的棒状隐翅虫种群通常过冷(低于冰点而不结冰)，平均温度约为-40摄氏度，有些个体在结冰前过冷到58摄氏度。然而，有时，这种甲虫的幼虫会深度过冷，即使冷却到150摄氏度也不能冷冻。尽管在深度过冷期间，甲虫幼虫不会冻结，但在-75摄氏度的温度下，它们会变成玻璃状或玻璃化。在玻璃化过程中，它们体内的水会变成固体，但不会结晶成冰。这项研究的主要目标是识别和描述允许深度过冷和玻璃化的生理适应。具体地说，PI将调查抗冻蛋白、甘油、限制过冷的冰核的去除、滞育(新陈代谢减少)、低温保护脱水和抗冻蛋白浓度的作用。此外，一项蛋白质组研究将确定可能有助于深度过冷的其他蛋白质。这项工作的更广泛影响包括潜在的应用：(1)非冷冻状态下生物材料的超低温保存，(2)开发更耐寒的农业植物，以及(3)冷冻食品工业。此外，这项工作将包括培训一名有才华的高中生和几名研究生。进一步了解适应是如何在如此极端的低温下生存的，有可能激发热情，吸引更多的学生投身生物学职业。

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.