Summary
Mitochondrial content, central to aerobic metabolism, is thought to be controlled by a few transcriptional master regulators, including nuclear respiratory factor 1 (NRF-1), NRF-2 and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). Though well studied in mammals, the mechanisms by which these factors control mitochondrial content have been less studied in lower vertebrates. We evaluated the role of these transcriptional regulators in seasonal changes in white muscle cytochrome c oxidase (COX) activity in eight local fish species representing five families: Centrarchidae, Umbridae, Esocidae, Gasterosteidae and Cyprinidae. Amongst centrarchids, COX activity was significantly higher in winter for pumpkinseed (2-fold) and black crappie (1.3-fold) but not bluegill or largemouth bass. In esociforms, winter COX activity was significantly higher in central mudminnow (3.5-fold) but not northern pike. COX activity was significantly higher in winter-acclimatized brook stickleback (2-fold) and northern redbelly dace (3-fold). Though mudminnow COX activity increased in winter, lab acclimation to winter temperatures did not alter COX activity, suggesting a role for non-thermal cues. When mRNA was measured for putative master regulators of mitochondria, there was little evidence for a uniform relationship between COX activity and any of NRF-1, NRF-2α or PGC-1α mRNA levels Collectively, these studies argue against a simple temperature-dependent mitochondrial response ubiquitous in fish, and suggest that pathways which control mitochondrial content in fish may differ in important ways from those of the better studied mammals.
Methodology
Fish
Eight local fish species were caught in Lake Opinicon and its nearby marshlands, about 50 km north of Kingston, ON, Canada. Lake Opinicon is a large (787 ha, or 7.87 km2), but relatively shallow (∼50% is <5 m; maximum depth <11 m) and wind-exposed lake (Keast and Fox, 1992). Because of these physical conditions, it is classified as a polymictic lake (Agbeti and Smol, 1995), with water mixing in summer as well as spring and autumn, thereby reducing thermal variation regionally (Crowder et al., 1977; Keast and Fox, 1992). The four sunfish (Centrarchidae) representatives, pumpkinseed (Lepomis gibbosus, Linnaeus), bluegill (Lepomis macrochirus, Rafinesque), black crappie (Pomoxis nigromaculatus, Lesueur) and largemouth bass (Micropterus salmoides, Lacépède), as well as northern pike (Esox lucius, Linnaeus) were caught by hook and line. Brook stickleback (Culaea inconstans, Kirtland), northern redbelly dace (Chrosomus eos, Cope) and central mudminnow (Umbra limi, Kirtland) were caught in minnow traps in the marsh around the lake. No longer than 1 h after being caught, animals were anaesthetized in buffered (0.8 g l–1 NaHCO3) 0.4 g l–1 tricaine methanesulphonate and killed by cutting the spinal cord. White muscle samples were taken from the epaxial muscle below the dorsal fin but above the lateral line and immediately frozen in liquid nitrogen. Fish were caught in summer 2008 and 2009 and in winter/spring 2009/2010 (Fig. 1). Morphometric data including mass, length and condition factor of the fish are summarized in Table 1. As these fish were sampled from their natural environment, experiencing the whole range of seasonally changing stimuli, it is not surprising that some of them showed significant differences in their condition factors between summer and winter.
Acclimation experiment
Mudminnows, caught in the marsh area around Lake Opinicon during summer 2008, were held in a 723 l tank attached to a flow-through system in the Animal Care facilities of Queen's University, Kingston. Water was supplied by the city system taken from Lake Ontario and filtered through a carbon filter prior to serving the fish tank. Fish were acclimated to the ambient water temperature of Lake Ontario for 4 weeks before the first sampling event. During the beginning of the experiment, water temperatures of Lake Ontario were the same as those in the marshlands around Lake Opinicon, but decreased at a slightly lower rate than field water temperatures throughout the experiment. Samples were taken every 27–42 days (Fig. 2A). Acclimation temperatures were defined as the mean temperature over the 3 weeks prior to each sampling event (Fig. 2A).
Cytochrome c oxidase activity
For enzyme extraction, white muscle samples were powdered under liquid nitrogen. About 50 mg was homogenized in 20 volumes of cold extraction buffer (25 mmol l–1 K2HPO4, 1 mmol l–1 EDTA, 0.6 mmol l–1 lauryl maltoside, pH 7.4) using a 7 ml Tenbroeck tissue grinder (Wheaton Industries, Millville, NJ, USA). Homogenates were not centrifuged and were mixed prior to enzyme measurements. Enzyme activity was determined spectrophotometrically (Molecular Devices, Sunnyvale, CA, USA) at 550 nm and 25°C in 96-well plates (Corning, Corning, NY, USA) using assay buffer (25 mmol l–1 K2HPO4, 0.6 mmol l–1 lauryl maltoside, pH 7.4) and reduced cytochrome c (0.05 mmol l–1) as the substrate. Cytochrome c was reduced by ascorbic acid, then dialysed exhaustively in 25 mmol l–1 K2HPO4 (pH 7.4) and frozen in aliquots. All samples were measured in triplicate.