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.

Methods
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-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+†