Physiological Mechanisms of Hypoxia Tolerance

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This project will investigate adaptations to severe oxygen lack in the freshwater turtle, Chrysemys picta bellii, generally regarded as the air-breathing vertebrate most tolerant to oxygen lack. Specific objectives are to understand the role of the turtle's shell in lactic acid buffering, to clarify the fate of lactic acid and its rate of removal during recovery from a period of anoxia, to explore further the response of the turtle's heart and heart muscle to oxygen lack, and to define the mechanisms and limitations of oxygen uptake directly from the water. A broad objective is to test the hypothesis that this animal possesses no extraordinary resistance to anoxia per se, but that its effective shell buffering and its metabolic depression slow the secondary effects of hypoxia and thereby greatly extend its tolerance duration. To achieve these specific objectives, the following approaches will be used. 1) Shell function: Shell blood supply will be established using corrosion casts. Shell blood flow will be measured using proton-activated microspheres that trap in the capillaries, and the importance of shell blood flow to exchange processes evaluated by tying off selected blood vessels. The kinetics and limits of shell/blood Ca2+ and lactate exchange will be tested by incubating shell samples in acid media, and the mechanical consequences of demineralization of shell following anoxia determined. 2) Lactic acid metabolism: Lactic acid removal from the system during recovery from anoxic submergence will be compared in turtles at rest and while engaged in moderate aerobic activity swimming in a flume. Lactate metabolism by skeletal muscle will be tested using radioactively-labeled lactate, and by measuring lactate uptake by isolated muscle strips. Excretion of lactate during recovery will be tested at different plasma lactate concentrations using labeled lactate. 3) Heart function: Cardiac responses to graded hypoxia will be observed in isolated ventricular strips, in perfused hearts in vitro, and in intact surgically cannulated turtles with ECG electrodes. 4) Oxygen uptake from water: Oxygen consumption of turtles submerged in aerated water at 3 and 10oC will be measured, and the effect of blocking possible avenues of oxygen uptake will be assessed. The importance of hemoglobin to aquatic O2 uptake at low temperature will be evaluated by comparing turtles with normal and experimentally-reduced hemoglobin levels in their blood. To achieve the general objective, the comparative approach will be utilized. Shell and cardiac muscle characteristics of selected species of turtles, both aquatic and terrestrial, representing various lineages will be studied, and compared to the painted turtle. If possible, these will be animals whose anoxic tolerance has already been studied. Shell size, shell mechanical properties, mineral concentrations, and in vitro ion exchange capacities will be measured for each species. In addition, resistance to oxygen lack by ventricular muscle strips will be studied on each animal. For the latter protocol, two outgroup reptiles, an aquatic snake and a terrestrial lizard will also be studied. Hypoxia is a fundamental environmental stress experienced by many organisms. Understanding the particular adaptations that permit a specialized animal such as the freshwater turtle to resist this stress has considerable general significance. This project therefore can contribute to knowledge on a particular animal specialist, and can also reveal principles that may apply to other less-adapted species. Because the project consists of a variety of individual projects, it also lends itself well to student involvement and will provide opportunities for research experience and training to both undergraduate and graduate students.