SUMMARY: The widely accepted idea that bees fuel flight through the oxidation of carbohydrate is based on studies of only a few species. We tested this hypothesis as part of our research program to investigate the size-dependence of flight energetics in Panamanian orchid bees. We succeeded in measuring rates of O2 consumption and CO2 production in vivo during hovering flight, as well as maximal activities(Vmax values) in vitro of key enzymes in flight muscle energy metabolism in nine species belonging to four genera. Respiratory quotients (ratios of rates of CO2 production to O2consumption) in all nine species are close to 1.0. This indicates that carbohydrate is the main fuel used for flight. Trehalase, glycogen phosphorylase and hexokinase activities are sufficient to account for the glycolytic flux rates estimated from rates of CO2 production. High activities of other glycolytic enzymes, as well as high activities of mitochondrial oxidative enzymes, are consistent with the estimated rates of carbohydrate-fueled oxidative metabolism. In contrast, hydroxyacylCoA dehydrogenase, an enzyme involved in fatty acid oxidation, was not detectable in any species. Thoracic homogenates displayed ADP-stimulated oxidition of pyruvate + proline, but did not oxidize palmitoyl l-carnitine +proline as substrates. A metabolic map, based on data reported herein and information from the literature, is presented. The evidence available supports the hypothesis that carbohydrate serves as the main fuel for flight in bees.

Flying insects achieve the highest known mass-specific rates of O(2) consumption in the animal kingdom. Because the flight muscles account for >90% of the organismal O(2) uptake, accurate estimates of metabolic flux rates (J) in the muscles can be made. In steady state, these are equal to the net forward flux rates (v) at individual steps and can be compared with flux capacities (V(max)) measured in vitro. In flying honeybees, hexokinase and phosphofructokinase, both nonequilibrium reactions in glycolysis, operate at large fractions of their maximum capacities (i.e., they operate at high v/V(max)). Phosphoglucoisomerase is a reversible reaction that operates near equilibrium. Despite V(max) values more than 20-fold greater than the net forward flux rates during flight, a close match is found between the V(max) required in vivo (estimated using the Haldane relationship) to maintain near equilibrium and this net forward flux rate and the V(max) measured in vitro under simulated physiological conditions. Rates of organismal O(2) consumption and difference spectroscopy were used to estimate electron transfer rates per molecule of respiratory chain enzyme during flight. These are much higher than those estimated in mammalian muscles. Current evidence indicates that metabolic enzymes in honeybees do not display higher catalytic efficiencies than the homologous enzymes in mammals, and the high electron transfer rates do not appear to be the result of higher enzyme densities per unit cristae surface area. A number of possible mechanistic explanations for the higher rates of electron transfer are proposed.

Metabolic control analysis has revealed that flux through pathways is the consequence of system properties, i.e. shared control by multiple steps, as well as the kinetic effects of various pathways and processes over each other. This implies that the allometric scaling of flux rates must be understood in terms of properties that pertain to the regulation of flux rates. In contrast, proponents of models considering the scaling of branching or fractal-like systems suggest that supply rates determine metabolic rates. Therefore, the allometric scaling of supply alone provides a sufficient explanation for the allometric scaling of metabolism. Examination of empirical data from the literature of comparative physiology reveals that basal metabolic rates (BMR) are driven by rates of energy expenditure within internal organs and that the allometric scaling of BMR can be understood in terms of the scaling of the masses and metabolic rates of internal organs. Organ metabolic rates represent the sum of tissue metabolic rates while, within tissues, cellular metabolic rates are the outcome of shared regulation by multiple processes. Maximal metabolic rates (MMR, measured as maximum rates of O₂ consumption, V̇O_2max) during exercise also scale allometrically, are also subject to control by multiple processes, but are due mainly to O₂ consumption by locomotory muscles. Thus, analyses of the scaling of MMR must consider the scaling of both muscle mass and muscle energy expenditure. Consistent with the principle of symmorphosis, allometry in capacities for supply (the outcome of physical design constraints) is observed to be roughly matched by allometry in capacities for demand (i.e. for energy expenditure). However, physiological rates most often fall far below maximum capacities and are subject to multi-step regulation. Thus, mechanistic explanations for the scaling of BMR and MMR must consider the manner in which capacities are matched and how rates are regulated at multiple levels of biological organization.

Summary: Much research in comparative physiology is now performed using ‘omics’ tools and many results are interpreted in terms of the effects of changes in gene expression on energy metabolism. However, ‘metabolism’ is a complex phenomenon that spans multiple levels of biological organization. In addition rates and directions of flux change dynamically under various physiological circumstances. Within cells, message level cannot be equated with protein level because multiple mechanisms are at play in the ‘regulatory hierarchy’ from gene to mRNA to enzyme protein. This results in many documented instances wherein change in mRNA levels and change in enzyme levels are unrelated. It is also known from metabolic control analysis that the influence of single steps in pathways on flux is often small. Flux is a system property and its control tends to be distributed among multiple steps. Consequently, change in enzyme levels cannot be equated with change in flux. Approaches developed by Hans Westerhoff and colleagues, called ‘hierarchical regulation analysis’, allow quantitative determination of the extent to which ‘hierarchical regulation’, involving change in enzyme level, and ‘metabolic regulation’, involving the modulation of the activity of preexisting enzyme, regulate flux. We outline these approaches and provide examples to show their applicability to problems of interest to comparative physiologists.

Most hummingbirds and some species of nectar bats hover while feeding on floral nectar. While doing so, they achieve some of the highest mass-specific values among vertebrates. This is made possible by enhanced functional capacities of various elements of the ‘O₂ transport cascade’, the pathway of O₂ from the external environment to muscle mitochondria. Fasted hummingbirds and nectar bats fly with respiratory quotients (RQs; ) of ∼0.7, indicating that fat fuels flight in the fasted state. During repeated hover-feeding on dietary sugar, RQ values progressively climb to ∼1.0, indicating a shift from fat to carbohydrate oxidation. Stable carbon isotope experiments reveal that recently ingested sugar directly fuels ∼80 and 95% of energy metabolism in hover-feeding nectar bats and hummingbirds, respectively. We name the pathway of carbon flux from flowers, through digestive and cardiovascular systems, muscle membranes and into mitochondria the ‘sugar oxidation cascade’. O₂ and sugar oxidation cascades operate in parallel and converge in muscle mitochondria. Foraging behavior that favours the oxidation of dietary sugar avoids the inefficiency of synthesizing fat from sugar and breaking down fat to fuel foraging. Sugar oxidation yields a higher P/O ratio (ATP made per O atom consumed) than fat oxidation, thus requiring lower hovering per unit mass. We propose that dietary sugar is a premium fuel for flight in nectarivorous, flying animals.