Consistent changes in muscle metabolism underlie dive performance across multiple lineages of diving ducks

Diving animals must sustain high activity with limited O2 stores to successfully capture prey. Studies suggest that increasing body O2 stores supports breath-hold diving, but less is known about metabolic specializations that underlie underwater locomotion. We measured maximal activities of 10 key enzymes in locomotory muscles (gastrocnemius, pectoralis) to identify biochemical changes associated with diving in pathways of oxidative and substrate-level phosphorylation and compared them across three groups of ducks — the strong diving sea ducks (Mergini, 8 spp.), the mid-tier diving pochards (Aythyini, 3 spp.), and the non-diving dabblers (Anatini, 5 spp.). Relative to dabblers, both diving groups had increased activities of succinate dehydrogenase and cytochrome c oxidase, and sea ducks further showed increases in citrate synthase (CS) and hydroxyacyl-coA dehydrogenase (HOAD). Both diving groups had relative decreases in capacity for anaerobic metabolism (lower ratio of lactate dehydrogenase to CS), with sea ducks also showing a greater capacity for oxidative phosphorylation and lipid oxidation (lower ratio of pyruvate kinase to CS, higher ratio of HOAD to hexokinase). These data suggest that the locomotory muscles of diving ducks are specialized for sustaining high rates of aerobic metabolism, emphasizing the importance of body O2 stores for dive performance in these species.The .csv file can be opened with Microsoft Excel, Google Sheets, etc.Funding provided by: University of MiamiCrossref Funder Registry ID: http://dx.doi.org/10.13039/100006686Award Number: Kushlan Endowment for Waterbird Biology and ConservationFunding provided by: University of MiamiCrossref Funder Registry ID: http://dx.doi.org/10.13039/100006686Award Number: Department of BiologyFrozen tissue samples from the pectoralis and gastrocnemius of 16 species of ducks were homogenized in 20 volumes of ice-cold homogenization buffer (100 mM potassium phosphate, 1 mM EGTA, 1 mM EDTA, 0.1% Triton X-100; pH 7.2), then centrifuged for 2 minutes at 2,000 rpm. The supernatant was collected and used in the subsequent assays. Each assay was conducted at avian body temperature (41°C) with substrate concentrations previously found to be saturating (Dawson et al., 2016). The substrates and their concentrations used for each enzymatic assay are listed in the table below. All chemicals were sourced from Merck (Darmstadt, Germany) unless otherwise noted. Assays were run in triplicate for 30 minutes, and the change in absorbance was measured using a Spectramax Plus 384 spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). The section of the curve with the steepest slope was isolated, and the maximal activity for each enzyme was calculated as the difference between the reaction rate with all substrates present minus the background reaction rate (the rate in the presence of an inhibitor or without the key substrate) and is reported as units of micromole substrate per gram tissue (U/g) per minute. The calculation for activity is: ((Vmax/(extinction coefficient*path length/assay volume per well))/(homogenate volume per well/homogenate dilution*homogenate tissue)) Enzyme Assay Components Control Substrate Coupling Enzymes Extinction Coefficient (mM-1 cm-1) Wavelength (nm) pH Glycolysis Hexokinase 3 mM Mg•ATP 10 mM Glucose Glucose-6-Phosphate Dehydrogenase 6.22 340 7.2 10 mM MgCl2 1.5 mM NADP+† Pyruvate Kinase 10 mM MgCl2 10 mM Phosphoenolpyruvate Lactate Dehydrogenase 6.22 340 7.2 2.5 mM Mg•ADP 0.15 mM NADH† Lactate Dehydrogenase 0.3 mM NADH* 5 mM Pyruvate 6.22 340 7.2 Beta Oxidation 3-hydrocy-acyl-CoA Dehydrogenase 0.15 mM NADH* 0.15 mM Acetoacetyl Coenzyme-A 6.22 340 7.2 Krebs Cycle Citrate Synthase 0.15 mM Acetyl CoA 0.5 mM Oxaloacetate 14.15 412 8 0.15 mM DTNB† Electron Transport Chain Complex II 0.3 mM KCN 20 mM Succinate 21.9 600 7.5 0.05 mM DCPIP† 0.05 mM DUB Complex IV - 0.2 mM reduced Cytochrome C* 28.5 550 7 ATP Synthase 2.5 mM Mg•ADP 0.01 mM Oligomycin Hexokinase 6.22 340 7.5 10 mM MgCl2 Glucose-6-Phosphate Dehydrogenase 10 mM Glucose 1.5 mM NADP+† ATP Turnover Creatine Kinase 6 mM ATP 15 mM Creatine Pyruvate Kinase (2 units) 6.22 340 7.2 10 mM MgCl2 Lactate Dehydrogenase (2 units) 0.3 mM NADH+† 15 mM Phosphoenolpyruvate Adenylate Kinase 10 mM MgCl2 2.5 mM MgADP Hexokinase 6.22 340 7.2 10 mM Glucose Glucose-6-Phosphate Dehydrogenase 1.5 mM NADP+