DOI: 10.5061/dryad.tht76hf4j
title: Consistent changes in muscle metabolism underlie dive performance across multiple lineages of diving ducks

description: 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.
description: Frozen 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<sup>-1</sup> cm<sup>-1</sup>)
 Wavelength (nm) pH Glycolysis  
      
     
 Hexokinase 3 mM
 Mg•ATP 10 mM Glucose
 Glucose-6-Phosphate Dehydrogenase
 6.22 340
 7.2   10 mM
 MgCl<sub>2</sub>  
      
     1.5 mM NADP+†
      
     
 Pyruvate Kinase 10 mM
 MgCl<sub>2</sub> 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
 MgCl<sub>2</sub>  
 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
 MgCl<sub>2</sub>  
 Lactate Dehydrogenase (2 units)
      
  
 0.3 mM NADH+†  
      
     15 mM
 Phosphoenolpyruvate  
      
   Adenylate Kinase 10 mM
 MgCl<sub>2</sub> 2.5 mM MgADP
 Hexokinase 6.22
 340 7.2 
   10 mM
 Glucose   Glucose-6-Phosphate
 Dehydrogenase    
     1.5 mM NADP+†
      
    
description: The .csv file can be opened with Microsoft Excel, Google Sheets,
 etc.