Collaborative Research: Studies of Antifreeze Proteins in Arctic and Nearctic Insects

抗冻蛋白（AFPs）通过一种不寻常的非聚合机制降低水的冰点，已经在海洋鱼类、昆虫和其他陆生节肢动物、植物、真菌和细菌中被发现。在这些生物群体之间和内部，afp的结构都有巨大的差异。已知最活跃的afp是在昆虫中发现的，它们通常通过抑制外部冰穿过角质层引起的接种性冻结，以及通过抑制潜在的内部冰核促进过冷，来帮助对冷冻敏感的昆虫避免冻结。少数产afp的昆虫具有抗冻性，能够在冷冻中存活，在这些昆虫中，afp显然是一种冷冻保护剂，可以防止冻害，但其机制尚不清楚。昆虫afp有两种类型。一种是在石竹甲和黄粉甲中发现的。另一种来自云杉毛虫。有趣的是，尽管在许多昆虫中发现了afp，但直到最近在阿拉斯加费尔班克斯附近筛选的12种昆虫中有7种发现了afp，才在阿拉斯加或北极昆虫中发现了它们。这为研究暴露在极冷冬季温度下的昆虫体内的afp提供了一个机会。建议的研究有四个方面。其中一项研究将集中于afp在Cucujus clavipes甲虫越冬幼虫的耐寒性中的作用。先前对来自印第安纳州的这一物种种群的研究表明，这些幼虫中存在afp，其致死温度较低，为-20至-25摄氏度。在20世纪70年代末，阿拉斯加费尔班克斯附近的这一物种被证明具有“-55摄氏度或更低”的较低致死温度。印第安纳州和阿拉斯加的种群都产生树突虫/黄粉虫型afp。在这项研究中，比较了afp在印第安纳州和阿拉斯加州（位于北极圈以南的费尔班克斯附近，以及位于北极圈以上、接近树线极限的怀斯曼附近）种群中的作用。因此，人们可以在一个巨大的纬度范围内研究这个物种，从温带气候到阿拉斯加内陆，这是北美最冷的气候之一。该研究的第二个组成部分旨在纯化和表征一种新型AFP。臭虫是一种产afp的昆虫，常见于阿拉斯加内陆。分子探针表明，该昆虫的AFP不同于树endroides / tenbrio型或云杉芽虫型aep。这种昆虫的丰富、易于收集、体型大、暴露在极冷的温度下，使其成为鉴定新的昆虫AFP的良好系统。本研究的第三个方面是筛选昆虫血淋巴中抗冻蛋白的热滞活性特征。这将确定哪些物种有afp。昆虫将从阿拉斯加内陆针叶林的两个地点收集，靠近费尔班克斯和怀斯曼附近，特别是在阿拉斯加大学费尔班克斯北极生物学研究所附近的一个真正的苔原地点，图利克野外站。这些地点提供了种类繁多的昆虫。以前没有在这些地区的昆虫中调查过afp，也没有研究过来自Toolik的昆虫的耐寒性。这一筛选可以在极寒环境中发现产生afp的昆虫新品种，其中一些可能为未来的研究提供很好的模型系统。此外，还将确定微生境温度和选定昆虫物种的越冬存活率。这些是重要的协作实验室确定各种生理参数。因此，本研究将经典生理生态学与生物化学和分子研究结合起来，研究这些严酷的北极和亚北极环境中昆虫的抗冻蛋白。自从在南极鱼类中首次发现afp以来，对动物和植物中afp的研究迅速发展，今天有几十个实验室在研究这些蛋白质。昆虫是目前已知的活性最高的蛋白质，在基础和应用方面都具有巨大的潜力。afp存在许多可能的用途，特别是在生物医学材料的冷冻保存，食品保存和农业。因此，研究这些高度适应寒冷的昆虫的afp可能会产生非常有价值的信息。

Antifreeze Proteins (AFPs), which lower the freezing point of water by an unusual non-colligative mechanism, have been identified in marine fish, insects and other terrestrial arthropods, plants, fungi and bacteria. There is tremendous variation in the structures of AFPs both between and within these groups of organisms. The most active known AFPs are found in insects, where they typically assist freeze-susceptible insects to avoid freezing by inhibiting inoculative freezing initiated by external ice across the cuticle, and by promoting supercooling by inhibiting potential internal ice nucleators. A few AFP-producing insects are freeze-tolerant, able to survive freezing, and in these the AFPs apparently function as a cryoprotectant to protect from freeze damage, however the mechanism is unknown. Two types of insect AFPs have been characterized. One is found in the beetles Dendroides and Tenebrio. The other is from the spruce budworm caterpillar. Interestingly, although AFPs have been found in many insects, they have not previously been described in Alaskan or Arctic insects until recently when AFPs were found in 7 of 12 insects screened from near Fairbanks, Alaska. This presents an opportunity to study AFPs in insects that are exposed to extremely cold winter temperatures. There are four aspects to the proposed study. One of these will concentrate on the role of AFPs in the cold tolerance of overwintering larvae of the beetle Cucujus clavipes. Previous studies on populations of this species from Indiana showed the presence of AFPs in these larvae which have a lower lethal temperature of -20 to -25 C. In the late 1970's this species from near Fairbanks, Alaska was shown to have a lower lethal temperature of "-55 C or less". Both the Indiana and Alaskan populations produce the Dendroides/Tenebrio-type AFPs. In this study, comparisons of the role of AFPs in populations from Indiana and Alaska (from near Fairbanks, just south of the Arctic Circle, and from near Wiseman, above the Arctic Circle and near the limit of treeline). Thus, one can study this species over a tremendous latitudinal range, from a temperate climate to the interior of Alaska, one of the coldest climates in North America. A second component of the study aims to purify and characterize a new type of AFP. The stinkbug Elasmostethus interstinctus is an AFP-producing insect common in interior Alaska. Molecular probes have demonstrated that the bug's AFP is different from either the Dendroides/Tenebrio-type or the spruce budworm-type AEPs. The abundance, ease of collection, large size and exposure to very cold temperatures in this insect make it an excellent system in which to identify a new insect AFP. The third aspect of this study is to screen the hemolymph of insects for thermal hysteresis activity characteristic of AFPs. This will identify which species have AFPs. Insects will be collected from two sites in the taiga of interior Alaska, near Fairbanks and near Wiseman, and especially from a true tundra site in the vicinity of the Institute of Arctic Biology, University of Alaska, Fairbanks, Toolik Field Station. These sites provide a wide diversity of insects. AFPs have not been previously investigated in insects from these regions, and no aspect of the cold tolerance of insects from Toolik has previously been studied. This screen should identify new species of AFP-producing insects from very cold environments, some of which may provide excellent model systems for future study. In addition, microhabitat temperatures and overwintering survival of selected insect species will be determined. These are important to collaborate laboratory determinations of various physiological parameters. Thus this study will integrate classical physiological ecology with biochemistry and molecular studies of antifreeze proteins in insects from these severe arctic and subarctic environments. Since their initial discovery in Antarctic fish the study of AFPs in both animals and plants has burgeoned, and today scores of laboratories study these proteins. In particular insect AFPs have tremendous potential for both basic and applied work since they are the most active AFPs known. Numerous possible uses of AFPs exist, especially in cryopreservation of biomedical materials, food preservation and agriculture. Therefore, the study of AFPs from such highly cold adapted insects may yield information of great value.