About This Project

Aquatic ectotherms experience disjoint between oxygen availability and respiratory needs at high temperatures, because O2 solubility in water decreases, while metabolic demands increase with temperature. We hypothesize, based on O2 consumption and lipid composition data, that this limit is set by inability of mitochondria to maintain proton potential and propose to directly measure this in vivo using fluorescent microscopy.

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What is the context of this research?

High temperature is a common stressor and a common selective pressure in nature. In a model organism Daphnia, a freshwater zooplankton crustacean this manifests in latitudinal clines of heat tolerance [1], indicating local adaptation. Organisms, including Daphnia can also show plastic response to heat, adjusting their physiology and biochemistry to allow higher heat tolerance [2, 3]. There is evidence that such acclimation is related to respiration [1, 3] and lipid composition [4], but no in-vivo experiments confirming this have yet been conducted. Rhodamine 123 is a commonly used fluorescent dye that targets mitochondria; accumulating up to 4000x in intact mitochondria it exhibits quenching and red shift, resulting in lower green fluorescence. It has not yet been used in-vivo.

What is the significance of this project?

Daphnia is both a classic model organism in ecological physiology and an emerging model for ecological genomics. One of the key goals of ecological genomics today is to understand selective and plastic responses to changing environments, such as elevated temperatures. A protocol for in-vivo quantification of mitochondrial function in Daphnia will therefore be useful for a range of environmental, physiological and genomics studies. Testing the central hypothesis of the project - that higher heat tolerance in heat-acclimated Daphnia is achieved by their greater ability to sustain respiration at elevated temperatures - will be a milestone in the research aiming to understand ecological physiology of thermal acclimation and adaptation in a model crustacean.

What are the goals of the project?

Goal 1 of the project is to develop an in-vivo fluorescent microscopy protocol to quantify mitochondrial membrane integrity in Daphnia, using rhodamine 123 fluorophore that is known to target mitochondria and detect mitochondrial dysfunction [5].

Goal 2 is to use this protocol to test the hypothesis that higher heat tolerance in heat-acclimated Daphnia is caused by the ability to sustain mitochondrial functions longer. The experiment will include untreated high- and low temperature-acclimated controls, heat-treated individuals and individuals treated with a sublethal concentration of mito-toxin as positive controls.