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

抗冻蛋白（Antifreeze Proteins，AFP）通过一种不寻常的非依数机制降低水的冰点，已在海洋鱼类、昆虫和其他陆生节肢动物、植物、真菌和细菌中被鉴定。在这些生物群体之间和内部，AFP的结构存在巨大的差异。已知最活跃的AFP存在于昆虫中，其中它们通常通过抑制由外部冰穿过角质层引发的接种冻结和通过抑制潜在的内部冰成核剂促进过冷来帮助易冻昆虫避免冻结。一些产生AFP的昆虫是耐冷冻的，能够在冷冻中存活，并且在这些昆虫中，AFP显然起到冷冻保护剂的作用以保护免受冷冻损伤，然而其机制尚不清楚。 已经表征了两种类型的昆虫AFP。一种是在甲虫Dendroides和Tenebrio中发现的。另一种是云杉芽虫毛虫。有趣的是，尽管AFP已在许多昆虫中发现，但它们以前从未在阿拉斯加或北极昆虫中描述过，直到最近在阿拉斯加费尔班克斯附近筛选的12种昆虫中的7种中发现了AFP。这为研究暴露于极冷冬季温度的昆虫中的AFP提供了机会。 拟议的研究有四个方面。其中之一将集中在AFPs在甲虫Cucujus clavipes越冬幼虫的耐寒性中的作用。以前对来自印第安纳州的该物种种群的研究表明，在这些幼虫中存在AFP，其具有-20至-25 ℃的较低致死温度。在20世纪70年代后期，这种来自阿拉斯加费尔班克斯附近的物种被证明具有"-55 ℃或更低"的较低致死温度。 印第安纳州和阿拉斯加种群都产生Dendroides/Tenebrio型AFP。 在这项研究中，比较了AFP在印第安纳州和阿拉斯加（来自北极圈以南的费尔班克斯附近，以及来自北极圈以上的怀斯曼附近，靠近树线的界限）人群中的作用。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的内部，这是北美最寒冷的气候之一。 该研究的第二个组成部分旨在纯化和表征一种新型AFP。 Elasmostethus interstinctus是一种在阿拉斯加内陆常见的产生AFP的昆虫。 分子探针已经证明，该错误的AFP是不同于Dendroides/Tenebrio型或云杉芽虫型AEPs。这种昆虫的丰富性、易于收集、大尺寸和暴露于非常冷的温度使其成为鉴定新昆虫AFP的极好系统。 本研究的第三个方面是筛选昆虫血淋巴中AFP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，特别是从阿拉斯加大学北极生物学研究所附近的一个真正的苔原地点，费尔班克斯，图利克野外站。这些地点提供了多种多样的昆虫。 AFP以前没有在这些地区的昆虫中进行过研究，以前也没有研究过图利克昆虫的耐寒性。这个屏幕应确定新的物种的AFP生产昆虫从非常寒冷的环境中，其中一些可能提供良好的模型系统，为未来的研究。 此外，还将确定微生境温度和选定昆虫物种的越冬存活率。这些对于协作各种生理参数的实验室测定是重要的。因此，这项研究将整合经典的生理生态学与生物化学和分子生物学研究的抗冻蛋白在这些严重的北极和亚北极环境中的昆虫。 自从在南极鱼类中首次发现AFP以来，对动物和植物中AFP的研究已经蓬勃发展，今天有数十个实验室研究这些蛋白质。 特别是昆虫AFP具有巨大的潜力，因为它们是已知的最活跃的AFP。 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、食品保存和农业中。 因此，从这些高度冷适应昆虫的AFP的研究可能会产生很大的价值的信息。

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.