Changes in the material properties of the shell during simulated aquatic hibernation in the anoxia-tolerant painted turtle.

Western painted turtles ( Chrysemys picta bellii ) tolerate anoxic submergence longer than any other tetrapod, surviving more than 170 days at 3°C. This ability is due, in part, to the shell and skeleton simultaneously releasing calcium and magnesium carbonates, and sequestering lactate and H+ to prevent lethal decreases in body fluid pH. We evaluated the effects of anoxic submergence at 3°C on various material properties of painted turtle bone after 60, 130, and 167-170 days, and compared them to normoxic turtles held at the same temperature for the same time periods. To assess changes in the mechanical properties, beams (4×25 mm) were milled from the plastron and broken in a three-point flexural test. Bone mineral density, CO2 concentration (a measure of total bone HCO3 - /CO3 2- ), and elemental composition were measured using microCT, HCO3 - /CO3 2- titration, and inductively coupled plasma mass spectrometry (ICP-MS), respectively. Tissue mineral density of the sampled bone beams were not significantly altered by 167-170 days of aquatic overwintering in anoxic or normoxic water, but bone CO2 and Mg were depleted in anoxic compared normoxic turtles. At this time point, the plastron beams from anoxic turtles yielded at stresses that were significantly smaller and strains significantly greater than the plastron beams of normoxic turtles. When data from anoxic and normoxic turtles were pooled, plastron beams had a diminished elastic modulus after 167-170 days compared to control turtles sampled on Day 1, indicating an effect of prolonged housing of the turtles in 3°C water without access to basking sites. There were no changes in the mechanical properties of the plastron beams at any of the earlier time points in either group. We conclude that anoxic hibernation can weaken the painted turtle's plastron, but likely only after durations that exceed what it might naturally experience. The duration of aquatic overwintering, regardless of oxygenation state, is likely to be an important factor determining the mechanical properties of the turtle shell during spring emergence.