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

抗冻蛋白(AFP)通过一种不寻常的非结合机制降低水的冰点，已在海鱼、昆虫和其他陆地节肢动物、植物、真菌和细菌中发现。在这些生物体之间和内部，AFP的结构都有巨大的差异。已知的最活跃的AFP存在于昆虫中，它们通常通过抑制外部冰在角质层上引发的接种性冻结来帮助易冻昆虫避免结冰，并通过抑制潜在的内部冰核来促进过冷。一些产生甲胎蛋白的昆虫是耐寒的，能够在冰冻中存活，在这些昆虫中，甲胎蛋白显然是一种低温保护剂，可以防止冰冻破坏，但其机制尚不清楚。已鉴定了两种类型的昆虫AFP。一种是在甲虫树枝和拟步甲虫中发现的。另一种来自云杉幼虫毛虫。有趣的是，尽管在许多昆虫中都发现了AFP，但之前还没有在阿拉斯加或北极的昆虫中描述过AFP，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。这为研究暴露在极端寒冷冬季温度下的昆虫的AFP提供了机会。这项拟议的研究包括四个方面。其中一项将集中在AFP在金龟子幼虫越冬耐寒中的作用。先前对印第安纳州该物种种群的研究表明，在这些幼虫中存在AFP，其致死温度较低，为-20至-25摄氏度。20世纪70年代末，S发现，来自阿拉斯加费尔班克斯附近的这一物种的致死温度较低，为“-55摄氏度或更低”。印第安纳州和阿拉斯加的种群都产生树状线虫/拟南线虫类型的AFP。在这项研究中，比较了AFP在印第安纳州和阿拉斯加(位于北极圈以南的费尔班克斯附近，以及怀斯曼附近、北极圈上方和树线界限附近)种群中的作用。因此，人们可以在巨大的纬度范围内研究这种物种，从温带气候到北美最冷的气候之一阿拉斯加的内陆。这项研究的第二部分旨在提纯和鉴定一种新型AFP。臭虫Elasmostetus interstinctus是一种产甲胎蛋白的昆虫，常见于阿拉斯加内陆。分子探针已经证明，该虫的AFP既不同于树枝类/黄粉虫类型的AEP，也不同于云杉芽孢子型AEP。这种昆虫的丰富、易于采集、体型大以及暴露在非常低的温度下，使其成为鉴定新昆虫AFP的极佳系统。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。这将确定哪些物种具有AFP。昆虫将从阿拉斯加内陆泰加的两个地点收集，分别位于费尔班克斯和怀斯曼附近，特别是从阿拉斯加大学费尔班克斯北极生物研究所、费尔班克斯和图利克野战站附近的一个真正的苔原地点收集。这些地点提供了种类繁多的昆虫。AFPS在这些地区的昆虫中还没有被研究过，也没有关于Toolik昆虫耐寒性的任何方面的研究。这一筛选将从非常寒冷的环境中鉴定出产生甲胎蛋白的新物种，其中一些可能为未来的研究提供优秀的模式系统。此外，还将确定选定昆虫物种的微生境温度和越冬存活率。这些对于协作实验室测定各种生理参数是重要的。因此，这项研究将把经典的生理生态学与生物化学和分子研究结合起来，研究来自这些严酷的北极和亚北极环境的昆虫的抗冻蛋白。自从在南极鱼类中首次发现AFP以来，对AFP在动植物中的研究迅速发展，如今已有数十家实验室研究这些蛋白质。特别是，昆虫AFP在基础工作和应用工作方面都具有巨大的潜力，因为它们是已知的最活跃的AFP。AFP有许多可能的用途，特别是在生物医学材料的超低温保存、食品保存和农业中。因此，对这种高度耐寒昆虫的AFP进行研究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.