Abstract
ABSTRACTPhysiological investigations of fish gills have traditionally centred on the two principal functions of the gills: gas exchange and ion regulation. Mitochondrion-rich cells (MRCs) are primarily found within the gill filaments of fish, and are thought to proliferate in order to increase the ionoregulatory capacity of the gill in response to environmentally induced osmotic challenges. However, surprisingly little attention has been paid to the metabolic function of mitochondria within fish gills. Here, we describe and validate a simple protocol for the permeabilization of fish gills and subsequent measurement of mitochondrial respiration rates in vitro. Our protocol requires only small tissue samples (8 mg), exploits the natural structure of fish gills, does not require mechanical separation of the gill tissue (so is relatively quick to perform), and yields accurate and highly reproducible measurements of respiration rates. It offers great potential for the study of mitochondrial function in gills over a wide range of fish sizes and species.
Highlights
It has been known for a long time that fish gills are highly metabolically active – Keys and Willmer (1932) reported the presence of mitochondrion-rich cells (MRCs) from fish gill epithelial tissue in their seminal studies of chloride excretory cells
Enzyme activity was measured for citrate synthase (CS) and cytochrome c oxidase (COX)
We believe that measuring mitochondrial function in fish gills complements well-established techniques for exploring other aspects of gill function as it provides insight into the energetic cost of these other functions
Summary
It has been known for a long time that fish gills are highly metabolically active – Keys and Willmer (1932) reported the presence of mitochondrion-rich cells (MRCs) from fish gill epithelial tissue in their seminal studies of chloride excretory cells. Permeabilization of tissues by detergents has been suggested as a more physiologically relevant approach as it leaves the physical structure of mitochondria and other organelles intact, and eliminates any centrifugation bias. A study performed on oyster gills used traditional methods requiring the mechanical shredding of gill tissues followed by permeabilization with detergents during the run (Cahill et al, 2013). Including the permeabilization step during the measurement of mitochondrial function leaves the possibility of over-permeabilization during prolonged runs, and the mechanical separation of tissue fibres required to facilitate permeabilization can be time consuming and challenging to sustain sample-to-sample consistency (Larsen et al, 2014)
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