Kenneth C. Welch

Energy metabolism in orchid bee flight muscles : carbohydrate fuels all

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 O2 consumption) 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.

Dietary sugar as a direct fuel for flight in the nectarivorous bat Glossophaga soricina

SUMMARY It is thought that the capacity of mammals to directly supply the energetic needs of exercising muscles using recently ingested fuels is limited. Humans, for example, can only fuel about 30%, at most, of exercise metabolism with dietary sugar. Using indirect calorimetry, i.e. measurement of rates of O2 consumption and CO2 production, in combination with carbon stable isotope techniques, we found that nectarivorous bats Glossophaga soricina use recently ingested sugars to provide ∼78% of the fuel required for oxidative metabolism during their energetically expensive hovering flight. Among vertebrate animals, only hummingbirds exceed the capacity of these nectarivorous bats to fuel exercise with dietary sucrose. Similar experiments performed on Annas (Calypte anna) and rufous (Selasphorus rufus) hummingbirds show that they use recently ingested sugars to support ∼95% of hovering metabolism. These results support the suggestion that convergent evolution of physiological and biochemical traits has occurred among hovering nectarivorous animals, rendering them capable of a process analogous to aerial refueling in aircraft.

Allometric scaling of flight energetics in Panamanian orchid bees: a comparative phylogenetic approach

SUMMARY The relationship between body size and flight energetics was studied in the clade of tropical orchid bees, in order to investigate energy metabolism and evolution. Body mass, which varied from 47 to 1065 mg, was found to strongly affect hovering flight mass-specific metabolic rates, which ranged from 114 ml CO2 h-1 g-1 in small species to 37 ml CO2 h-1 g-1 in large species. Similar variation of wingbeat frequency in hovering flight occurred among small to large species, and ranged from 250 to 86 Hz. The direct relationship between such traits was studied by the comparative method of phylogenetically independent contrasts (PIC), using a new molecular phylogeny generated from the cytochrome b gene partial sequences. We found wingbeat frequency variation is satisfactorily explained by variation in wing loading, after corrections for body mass and phylogeny. The correlated evolution of mass-specific metabolic rate, wingbeat frequency and wing loading was also revealed after correcting for phylogeny and body mass. Further, the effect of body size on flight energetics can be understood in terms of a relationship between wing form and kinematics, which directly influence and explain the scaling of metabolic rate in this group of bees.

Fiber type homogeneity of the flight musculature in small birds.

Studies of medium- and large-bodied avian species have suggested that variation in flight muscle composition is related to differences in flight behavior. For example, slow-twitch or tonic fibers are generally found only in the flight muscles of non-volant or soaring/gliding birds. However, we know comparatively little about fiber composition of the muscles of the smallest birds. Here we describe the fiber composition of muscles from the wings, shoulders, and legs of two small avian species, which also display very high wingbeat frequencies: Annas hummingbirds (Calypte anna) and zebra finches (Taeniopygia guttata). All flight muscles examined in both species contained exclusively fast oxidative glycolytic (FOG) fibers. These unique results suggest that fast oxidative fibers are both necessary and sufficient for the full range of flight behaviors in these small-bodied birds. Like all other studied birds, the zebra finch gastrocnemius, a tarsometatarsal extensor, contained a mixture of FOG (27.1%), slow oxidative (SO, 12.7%), and fast glycolytic (FG, 60.2%) fibers. By contrast, the hummingbird gastrocnemius lacked FG fibers (85.5% FOG, 14.5% SO), which may reflect the reduced role of the hindlimb during take-off. We further hypothesize that thermogenic requirements constrain fiber type heterogeneity in these small endothermic vertebrates.

Oxidation rate and turnover of ingested sugar in hovering Anna's(Calypte anna) and rufous (Selasphorus rufus)hummingbirds

SUMMARY Hummingbirds obtain most of their dietary calories from floral nectar ingested during hovering flight. Despite the importance of dietary sugar to hummingbird metabolism, the turnover of newly ingested carbon in the pool of actively metabolized substrates has not been adequately characterized in hovering hummingbirds. By combining respirometry with stable carbon isotope analysis of respired breath, we show that in rufous (Selasphorus rufus) and Annas (Calypte anna) hummingbirds at high foraging frequencies, utilization of newly ingested sugars increased over a period of 30–45 min until it accounted for virtually 100% of the fuel oxidized. This newly ingested sugar disappears from the actively metabolized pool of substrates over a similar time course. These results demonstrate that turnover of carbon in the pool of actively metabolized substrates is rapid; carbon from ingested sucrose is available for oxidation for 30–45 min before being cleared. By monitoring expired CO2 for the appearance and disappearance of the signature characteristic of newly ingested sugar and then calculating energy budgets using video recordings of hummingbird activity, we estimated the proportion of recently ingested sugar used to fuel ongoing metabolism as well as the proportion devoted to energy storage. Consistent differences between species in the percentage of ingested cane sugar oxidized during the 2 h experimental periods suggest that individuals of each species adopted energy intake patterns appropriate to their needs. This approach provides a means by which to examine the partitioning of dietary carbon intake between energy expenditure and storage using non-invasive, field-compatible techniques.

Neuromuscular control of wingbeat kinematics in Anna's hummingbirds (Calypte anna)

SUMMARY Hummingbirds can maintain the highest wingbeat frequencies of any flying vertebrate – a feat accomplished by the large pectoral muscles that power the wing strokes. An unusual feature of these muscles is that they are activated by one or a few spikes per cycle as revealed by electromyogram recordings (EMGs). The relatively simple nature of this activation pattern provides an opportunity to understand how motor units are recruited to modulate limb kinematics. Hummingbirds made to fly in low-density air responded by moderately increasing wingbeat frequency and substantially increasing the wing stroke amplitude as compared with flight in normal air. There was little change in the number of spikes per EMG burst in the pectoralis major muscle between flight in normal and low-density heliox (mean=1.4 spikes cycle−1). However the spike amplitude, which we take to be an indication of the number of active motor units, increased in concert with the wing stroke amplitude, 1.7 times the value in air. We also challenged the hummingbirds using transient load lifting to elicit maximum burst performance. During maximum load lifting, both wing stroke amplitude and wingbeat frequency increased substantially above those values during hovering flight. The number of spikes per EMG burst increased to a mean of 3.3 per cycle, and the maximum spike amplitude increased to approximately 1.6 times those values during flight in heliox. These results suggest that hummingbirds recruit additional motor units (spatial recruitment) to regulate wing stroke amplitude but that temporal recruitment is also required to maintain maximum stroke amplitude at the highest wingbeat frequencies.

Flight muscle enzymes and metabolic flux rates during hovering flight of the nectar bat, Glossophaga soricina: Further evidence of convergence with hummingbirds

Given their high metabolic rates, nectarivorous diet, and ability to directly fuel their energetically-expensive flight using recently-ingested sugar, we tested the hypothesis that Pallas long tongued nectar bats (Glossophaga soricina) possess flight muscles similar to those of hummingbirds with respect to enzymatic flux capacities in bioenergetic pathways. In addition, we compared these biochemical capacities with flux rates achieved in vivo during hovering flight. Rates of oxygen consumption (V(O(2))) were measured during hover-feeding and used to estimate rates of ATP turnover, glucose and long-chain fatty acid oxidation per unit mass of flight muscle. Enzyme V(max) values at key steps in glucose and fatty acid oxidation obtained in vitro from pectoralis muscle samples exceed those found in the locomotory muscles of other species of small mammals and resemble data obtained from hummingbird flight muscles. The ability of nectar bats and hummingbirds to hover in fed and fasted states, fueled almost exclusively by carbohydrate or fat, respectively, allowed the estimation of fractional velocities (v/V(max)) at both the hexokinase and carnitine palmitoyltransferase-2 steps in glucose and fatty acid oxidation, respectively. The results further support the hypothesis of convergent evolution in biochemical and physiological traits in nectar bats and hummingbirds.

Identification of a new murine eosinophil major basic protein (mMBP) gene: Cloning and characterization of mMBP-2

We have identified a new eosinophil major basic protein gene family member in the mouse and have given it the designation murine major basic protein‐2 (mMBP‐2). The gene was initially characterized as a unique expressed sequence tag (EST) clone having significant identity to the previously recognized member of this gene family, mMBP‐1. The EST was used to screen and isolate mMBP‐2 from a bone marrow cDNA library. In addition, a genomic clone of mMBP‐2 was isolated and this gene was shown to be physically linked to within 100 kb of mMBP‐1 on the central region of mouse chromosome 2. Progressive similarity alignment of the deduced mMBP‐2 open reading frame demonstrates the apparent conservation of the “pre‐pro‐mature” protein structure found in the other known mammalian MBPs. Mature mMBP‐2 maintains the cationic nature associated with these proteins with a predicted pI of 9.95. However, unlike the human MBPs, which display a three orders of magnitude charge difference [hMBP‐1 (pI11.4) vs. hMBP‐2 (pI8.7)], mMBP‐2 is only slightly less cationic than mMBP‐1 (pI 10.5). Expression studies demonstrate that transcription of the mMBP‐2 gene parallels mMBP‐1 and is confined to hematopoietic compartments engaged in eosinophilopoiesis. Moreover, using mMBP‐1 knockout mice and immunohistochemistry with an antisera that recognizes both mMBP‐1 and ‐2, we demonstrate that mMBP‐2 protein expression is restricted to eosinophil lineage‐committed cells. J. Leukoc. Biol. 67: 567–576; 2000.

The sugar oxidation cascade: aerial refueling in hummingbirds and nectar bats.

Summary 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.

Carbon stable-isotope tracking in breath for comparative studies of fuel use

Almost half a century ago, researchers demonstrated that the ratio of stable carbon isotopes in exhaled breath of rats and humans could reveal the oxidation of labeled substrates in vivo, opening a new chapter in the study of fuel use, the fate of ingested substrates, and aerobic metabolism. Until recently, the combined use of respirometry and stable‐isotope tracer techniques had not been broadly employed to study fuel use in other animal groups. In this review, we summarize the history of this approach in human and animal research and define best practices that maximize its utility. We also summarize several case studies that use stable‐isotope measurements of breath to explore the limits of aerobic metabolism and substrate turnover among several species and various physiological states. We highlight the importance of a comparative approach in revealing the profound effects that phylogeny, ecology, and behavior can have in shaping aerobic metabolism and energetics as well as the fundamental biological principles that underlie fuel use and metabolic function across taxa. New analytical equipment and refinement of methodology make the combined use of respirometry and stable‐isotope tracer techniques simpler to perform, less costly, and more field ready than ever before.