Summary
The influence of body size on metabolic rate, muscle enzyme activities and the underlying patterns of mRNA for these enzymes were explored in an effort to explain the genetic basis of allometric variation in metabolic enzymes. We studied two pairs of sister species of centrarchid fish: black bass (largemouth bass Micropterus salmoides and smallmouth bass Micropterus dolomieui) and sunfish (pumpkinseed Lepomis gibbosus and bluegill Lepomis macrochirus). Our goal was to assess the regulatory basis of both intraspecific and interspecific variation relative to body size, as well as to gain insights into the evolutionary constraints within lineages. Whole animal routine metabolic rate showed scaling coefficients not significantly different from 1, ranging from (+0.87 to +0.96). However, there were significant effects of body size on the specific activities of oxidative and glycolytic enzymes. Mass-specific activity of the oxidative enzyme citrate synthase (CS) scaled negatively with body size in each species, with scaling coefficients ranging from –0.15 to –0.19, whereas the glycolytic enzyme pyruvate kinase (PK) showed positive scaling, with scaling coefficients ranging from +0.08 to +0.23. The ratio of mass-specific enzyme activity in PK to CS increased with body size, whereas the ratio of mRNA transcripts of PK to CS was unaffected, suggesting the enzyme relationships were not due simply to transcriptional regulation of both genes. The mass-dependent differences in PK activities were best explained by transcriptional regulation of the muscle PK gene; PK mRNA was a good predictor of PK specific enzyme activity within species and between species. Conversely, CS mRNA did not correlate with CS specific enzyme activities, suggesting post-transcriptional mechanisms may explain the observed inter-specific and intraspecific differences in oxidative enzymes.
Methodology
Animal and tissue collection
All fish were collected using angling or seine nets. Smallmouth bass Micropterus dolomieui Lacepède 1802 were collected from Lake Ontario (44°15′, 76°31′). Largemouth bass Micropterus salmoides Lacepède 1802, pumpkinseed Lepomis gibbosus L. and bluegill Lepomis macrochirus Rafinesque 1819 were collected from Lake Opinicon, Ontario (44°35′, 76°20′). All fish were allowed to recover in flow-though tanks (420 l) for at least 12 h prior to experimental procedures.
Oxygen consumption measurements
Respiration measurements were performed in standard glass aquaria, with removable, sealable tops constructed of Plexiglass™. Fish were captured by dipnet and placed in respirometry chambers (50–100 ml g–1 fish) held at 20°C. The containers were closed with water flowing into the chamber for approximately 20 min. At this point the containers were sealed, air bubbles removed with a syringe and respiration measurements commenced. Oxygen levels were measured continuously using a fluorescent fiber optic probe (foxy R probe, Ocean Optics, Dunedin, FL, USA) until oxygen levels had declined by 10%. Oxygen consumption rates were calculated using linear regression and expressed relative to fish mass. Our goal was to measure respiration in animals freshly captured from the natural environment, and thus we chose to minimize the effects of holding time and food deprivation (Glass, 1968). Though the animals recovered overnight after capture, we cannot demonstrate that the duration of the adjustment period following transfer to the respirometry chamber (20 min) was sufficient to ensure that the fish exhibited a true routine metabolic rate. However, the respiration measurements obtained from these fish are in close agreement with other studies on these same species (see Discussion).
Enzyme assays and DNA extraction
Fish were anaesthetized in a solution of tricaine methane sulphonate (0.4 g l–1) and sodium bicarbonate (0.8 g l–1). After fish were killed, their masses were recorded and white muscle samples taken from the epaxial region near the dorsal fin. Muscle samples were rapidly frozen in liquid nitrogen and stored at –80°C. Tissues were powdered in liquid nitrogen and stored at –80°C.
Enzyme extracts were prepared by homogenizing powdered tissue in 20 volumes of homogenization buffer (20 mmol l–1 Hepes, 1 mmol l–1 EDTA, 0.1% Triton X-100, pH 7.2) using a ground-glass homogenizer. Homogenates were used directly without centrifugation. Enzyme activities were assayed using a Spectromax plate spectrometer (Molecular Devices, Sunnyvale, CA, USA) in 96-well format at 25°C.
Pyruvate kinase (PK) activity, measured within 2 h of homogenization, was assayed in 50 mmol l–1 Hepes (pH 7.4), 5 mmol l–1 ADP, 100 mmol l–1 KCl, 10 mmol l–1 MgCl2, 0.15 mmol l–1 NADH, 0.01 mmol l–1 fructose 1,6-biphosphate, 5 mmol l–1 phosphoenolpyruvate and excess lactate dehydrogenase (10 units ml–1). All substrate levels were saturating.