Document Type

Thesis

Date of Degree Completion

Spring 2014

Degree Name

Master of Science (MS)

Department

Biology

Committee Chair

Jason Irwin

Second Committee Member

Daniel Beck

Third Committee Member

David Darda

Abstract

Pacific chorus frogs, Pseudacris regilla, have the ability to survive extensive tissue freezing during the winter months, but there has been only one study of this species' physiological responses to freezing. There is nothing known about how freeze tolerance might vary across this species' range. The goal of this study was to examine geographic variation in freeze tolerance, specifically the storage and synthesis of cryoprotectants. Survival of freezing requires that the frogs produce massive amounts of cryoprotective glucose from stored liver glycogen, leading me to hypothesize that where winters are longer and/or colder, frogs will produce more cryoprotectants and, therefore, store more liver glycogen. Frogs were collected from regions of Washington State that have very different climates: near sea-level with mild winters (Mill Creek), high ­elevation areas with heavy snowfall (Snoqualmie Pass and Table Mountain, both ~ 1300m), and a cold, dry inland site (Ellensburg: 470m). After being housed outdoors on the Central Washington University campus to naturally develop their cold tolerance, the frogs were moved in early November into artificial hibernation at 2°C. In January the frogs were frozen in a cooling bath to -2.5°C and then dissected to remove liver and thigh. I used colorimetric assays to measure glucose and glycogen concentrations in homogenized tissues. As expected, liver glycogen consistently decreased upon freezing in all populations as it was converted into glucose. Mill Creek and Ellensburg populations had the highest liver glycogen concentrations both in control and frozen frogs. Liver glucose increased with freezing in all populations, but the Ellensburg population produced 40% more glucose than the other populations. Dehydration in the tissues occurs with freezing, and this was similar in all of the populations with an approximately 10% decrease of water content in both liver and thigh samples. Overall, the pacific chorus frogs had similar trends in glycogen storage and glucose production to what is found in other freeze-tolerant frog species. Frogs that endured longer, colder winters did not produce more cryoprotectants than frogs that endured warmer winters. In fact, the Mill Creek population stored more liver glycogen than the higher elevation populations, possibly to sustain them through a warmer winter.

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