The cellular mechanisms of insect physiological plasticity and freeze tolerance

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The long-term objective of my research program is to understand how insects tolerate low temperatures, facilitating their ability to overwinter in temperate climates. Researchers have known that some insects are freeze-tolerant for almost 300 years, yet we have limited understanding of the how internal freezing causes damage and the mechanisms that freeze-tolerant insects use to combat that damage. My current focus is on the cell biology of freeze tolerance, using the spring field cricket Gryllus veletis as a model system. These crickets can substantially modify their cold tolerance, transitioning from freeze-intolerant to freeze-tolerant when exposed (acclimated) to six weeks of fall-like conditions (low temperatures, short days). My students and I will pursue three linked objectives to determine how acclimation induces cellular mechanisms that facilitate insect freeze tolerance. For objective 1, we will characterize freeze-induced damage at the cellular level. We will use a combination of cell biology and biochemical assays to determine whether cricket cells and their macromolecules (proteins, membranes, DNA) are damaged during freezing, and whether that damage can be repaired post-freeze. This will increase our understanding of the challenges associated with freezing, and facilitate additional investigations that test whether specific molecules or genes protect against particular types of damage. For objective 2, we will determine how acclimation alters cell structure and function. The changes in gene expression during fall acclimation suggest that freeze-tolerant crickets adjust their cell biology in several ways. We will test these hypotheses using immunohistochemistry, membrane lipidomics, and cryoprotectant quantitation assays. These experiments will determine whether fall acclimation induces changes to cell structure and function and will deepen our knowledge of potential mechanisms that can mitigate damage caused by freezing. For objective 3, we will test which molecules and cellular processes help protect cells during freezing. We will first use RNA interference (RNAi) to knock down expression of genes involved in the cellular changes from objective 2. We will then test how preventing the function of target genes affects whole animal and cellular freeze tolerance using methods similar to objective 1. This will provide much-needed information on the genes and associated cellular processes that are important for the mechanisms underlying freeze tolerance. My proposed research program applies techniques (cell biology, RNAi) that have been lacking in the field of insect freeze tolerance. These experiments have potential to substantially advance our understanding of how insects survive internal ice formation. More broadly, this work has potential applications to insect pest management, cryopreservation protocols in the biomedical and food industries, and determining how overwintering insects may respond to climate change.