Effects of Freezing on Insulin Regulation of Intracellular Solute Concentrations in the Freeze-Tolerant Spring Peeper (genus Pseudacris)

Man’s exploration of harsh environments has often been informed by phenomena observed in animals. A prime example is the cryobiology (the study of life in cold, harsh conditions) of certain North American frog species. In this case, scientists have observed members of the frog genera Dryophytes, Pseudacris, and Rana, among others, which survive harsh North American winters. In the winter, these frogs undergo a frozen state, during which their body tissues and fluids freeze. Once spring arrives with warmer temperatures, these frogs are then able to re-emerge apparently unharmed by the process. Various studies have been conducted to explain the molecular pathways that lead to this amazing ability. This study seeks to analyze the presence of the insulin signaling pathway in species of genus Pseudacris. To accomplish this, specimens of freeze-tolerant Pseudacris species were incubated, euthanized at different stages of entering into their frozen state, and preserved at -80 C so that their proteins could later be extracted and analyzed. Specimens from non-freeze tolerant species were also prepared in similar fashion, so that their proteins could be compared to the freeze-tolerant species. The proteins extracted from these specimens were then analyzed using ELISA and western blot testing to characterize the proteins and compare insulin content between the extracts from these frogs.