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

University Of Notre Dame, Notre Dame IN

Investigators

Abstract

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.