The remarkable adaptability of insects is clearly demonstrated in examples of overwintering responses. Some insects have even been known to inhabit areas where temperatures reach -75 degrees C. Although researchers have been able to identify many physiological and biochemical parameters that may contribute to survival at such low temperatures, little is known about the underlying cellular mechanisms for these responses and how they are regulated. One major obstacle has been the lack of an in vitro model, i.e. where tissues or cells from these insects are maintained in culture and subjected to experimental manipulation. Recent research in my laboratory has now established a system for the long-term culturing of fat body cells derived from cold-hardy beetles shown to produce antifreeze proteins during the winter. The proposed study uses this system to more directly investigate the physiological and cellular mechanisms of antifreeze protein biosynthesis and regulation. First, it will characterize the cell types that predominate in vitro, how they relate to intact tissues, their degree of developmental maturation, and their ability to produce antifreeze proteins, using very sensitive immunological and fluorescent labeling techniques. We will then examine the cellular means by which these proteins may be stored and then secreted, through ultrastructural immunolocalization with electron microscopy, and by charting transport of newly synthesized antifreezes from cells to culture media. Concurrently, we will examine how hormones and low temperature may directly influence antifreeze protein production and secretion by these cells. Thus, this study introduces a powerful and state-of-the-art tool to research on insect fat body, and low temperature adaptations, and provides the first cellular analyses of environmental and hormonal control of antifreeze protein production and secretion.
