Limitations and requirements for measuring metabolic rates: a mini review.

Metabolic measurement of humans and model animals is an important aspect of biomedicine. Particularly, in the case of model animals, the limitations of currently widely used metabolic measurement methods are not widely understood. In this mini-review, I explain the theoretical underpinnings of flow-through respirometry as a linear time-invariant system, and the (usually serious) distortions of metabolic data caused by the interaction of chamber volume and flow rate. These can be ameliorated by increasing the flow rate through the chamber, though this is at the expense of the magnitude of the O2 depletion and CO2 enhancement signals from which metabolic rates are calculated. If achieved, however, the improvement in temporal response that follows higher flow rates can be marked, and allows confident and accurate measurement of resting and active energy expenditure. Applications of this approach in multiplexing gas signals from multiple cages, and in human room calorimetry, are also discussed.

Flow-through respirometry applied to chamber systems: pros and cons, hints and tips.

Flow-through respirometry is a powerful, accurate methodology for metabolic measurement that is applicable to organisms spanning a body mass range of many orders of magnitude. Concentrating on flow-through respirometry that utilizes a chamber to contain the experimental animals, we describe the most common flow measurement and control methodologies (push, pull and stop-flow) and their associated advantages and disadvantages. Objective methods for calculating air flow rates through the chamber, based on the body mass and taxon of the experimental organism, are presented. Techniques for removing the effect of water vapor dilution, including the direct measurement of water vapor pressure and mathematical compensation for its presence, are described and evaluated, as are issues surrounding the analysis of one or both of the respiratory gases (oxygen and carbon dioxide), and issues related to the mathematical correction of wash-out phenomena (response correction). Two important biomedical applications of flow-through respirometry (metabolic phenotyping and room calorimetry) are discussed in detail, and we conclude with a list of suggestions aimed primarily at investigators starting out in applying flow-through respirometry.

Low metabolic rate in scorpions: implications for population biomass and cannibalism.

Scorpions are abundant in arid areas, where their population biomass may exceed that of vertebrates. Since scorpions are predators of small arthropods and feed infrequently across multi-year lifespans, a parsimonious explanation for their observed, anomalously high biomass may be a depressed metabolic rate (MR). We tested the hypothesis that scorpion MR is significantly depressed compared with that of other arthropods, and we also measured the temperature-dependence of the MR of scorpions to quantify the interaction between large seasonal variations in desert temperatures and MR and, thus, long-term metabolic expenditure. Scorpion MR increased markedly with temperature (mean Q(10)=2.97) with considerable inter-individual variation. At 25 degrees C, the MRs of scorpions from two genera were less than 24 % of those of typical terrestrial arthropods (spiders, mites, solpugids and insects) of the same mass. It is likely, therefore, that the low MR of scorpions contributes to their high biomass in arid areas. The combination of high biomass and high production efficiency associated with low MR may also favor a density-dependent "transgenerational energy storage" strategy, whereby juveniles are harvested by cannibalistic adults that may be closely related to their juvenile prey.

Mitochondrial function in flying honeybees (Apis mellifera): respiratory chain enzymes and electron flow from complex III to oxygen.

The biochemical bases for the high mass-specific metabolic rates of flying insects remain poorly understood. To gain insights into mitochondrial function during flight, metabolic rates of individual flying honeybees were measured using respirometry, and their thoracic muscles were fixed for electron microscopy. Mitochondrial volume densities and cristae surface densities, combined with biochemical data concerning cytochrome content per unit mass, were used to estimate respiratory chain enzyme densities per unit cristae surface area. Despite the high content of respiratory enzymes per unit muscle mass, these are accommodated by abundant mitochondria and high cristae surface densities such that enzyme densities per unit cristae surface area are similar to those found in mammalian muscle and liver. These results support the idea that a unit area of mitochondrial inner membrane constitutes an invariant structural unit. Rates of O(2) consumption per unit cristae surface area are much higher than those estimated in mammals as a consequence of higher enzyme turnover rates (electron transfer rates per enzyme molecule) during flight. Cytochrome c oxidase, in particular, operates close to its maximum catalytic capacity (k(cat)). Thus, high flux rates are achieved via (i) high respiratory enzyme content per unit muscle mass and (ii) the operation of these enzymes at high fractional velocities.

Relationships between enzymatic flux capacities and metabolic flux rates: nonequilibrium reactions in muscle glycolysis.

The rules that govern the relationships between enzymatic flux capacities (Vmax) and maximum physiological flux rates (v) at enzyme-catalyzed steps in pathways are poorly understood. We relate in vitro Vmax values with in vivo flux rates for glycogen phosphorylase, hexokinase, and phosphofructokinase, enzymes catalyzing nonequilibrium reactions, from a variety of muscle types in fishes, insects, birds, and mammals. Flux capacities are in large excess over physiological flux rates in low-flux muscles, resulting in low fractional velocities (%Vmax = v/Vmax x 100) in vivo. In high-flux muscles, close matches between flux capacities and flux rates (resulting in fractional velocities approaching 100% in vivo) are observed. These empirical observations are reconciled with current concepts concerning enzyme function and regulation. We suggest that in high-flux muscles, close matches between enzymatic flux capacities and metabolic flux rates (i.e., the lack of excess capacities) may result from space constraints in the sarcoplasm.

Effects of ambient oxygen tension on flight performance, metabolism, and water loss of the honeybee.

Although the metabolic rate of resting insects is relatively insensitive to atmospheric O2 tensions, metabolic rates during flight increase by 20- to 100-fold above resting levels. In this study we test whether O2 delivery limits metabolic rate during unladen hovering flight of the honeybee, Apis mellifera. Below 10 kPa PO2, wing-stroke frequency decreased, and at 5 kPa, bees could not fly. However, for PO2's ranging from 39 to 10 kPa, metabolic rate and wing-stroke frequency were unaffected by PO2. Evaporative water loss rates increased by 40% at the lowest O2 tensions, which suggests that flying honeybees compensated for decreasing ambient PO2 by modulating convective ventilatory flow. Under normal sea-level conditions, O2 delivery does not limit flight metabolic rate in unladen, hovering honeybees and does not limit maximal metabolic rate. At altitudes above 3,000 m, the convective component of O2 delivery may, however, limit flight metabolic rate and flight capacity in honeybees.

Energy metabolism, enzymatic flux capacities, and metabolic flux rates in flying honeybees.

Honeybees rely primarily on the oxidation of hexose sugars to provide the energy required for flight. Measurement of VCO2 (equal to VO2, because VCO2/VO2 = 1.0 during carbohydrate oxidation) during flight allowed estimation of steady-state flux rates through pathways of flight muscle energy metabolism. Comparison of Vmax values for flight muscle hexokinase, phosphofructokinase, citrate synthase, and cytochrome c oxidase with rates of carbon and O2 flux during flight reveal that these enzymes operate closer to Vmax in the flight muscles of flying honeybees than in other muscles previously studied. Possible mechanistic and evolutionary implications of these findings are discussed.

Discontinuous gas exchange in insects.

Many insects exchange respiratory gases cyclically and discontinuously. A typical discontinuous gas exchange cycle (DGC) starts with a closed-spiracle (C) phase, during which little external gas exchange takes place, followed by a fluttering-spiracle (F) phase, which is usually dominated by diffusive oxygen uptake. The DGC is terminated by an open-spiracle (O) phase, during which accumulated CO2 escapes. This review critically examines the applicability of the DGC to insect gas exchange in general, discusses the primary mechanisms of gas exchange in the F and O phases, evaluates the widespread hypothesis that the DGC lowers respiratory water loss rates adaptively, and proposes new hypotheses concerning the evolutionary genesis of the DGC in insects and other tracheate arthropods.

Muscle efficiency and elastic storage in the flight motor of Drosophila.

Insects could minimize the high energetic costs of flight in two ways: by employing high-efficiency muscles and by using elastic elements within the thorax to recover energy expended accelerating the wings. However, because muscle efficiency and elastic storage have proven difficult variables to measure, it is not known which of these strategies is actually used. By comparison of mechanical power measurements based on gas exchange with simultaneously measured flight kinematics in Drosophila, a method was developed for determining both the mechanical efficiency and the minimum degree of elastic storage within the flight motor. Muscle efficiency values of 10 percent suggest that insects may minimize energy use in flight by employing an elastic flight motor rather than by using extraordinarily efficient muscles. Further, because of the trade-off between inertial and aerodynamic power throughout the wing stroke, an elastic storage capacity as low as 10 percent may be enough to minimize the energetic costs of flight.

Bioenergetic and kinematic consequences of limblessness in larval Diptera.

We report the cost of transport and kinematics of terrestrial locomotion by larval blowflies (Protophormia terraenovae, Diptera: Calliphoridae). We contrast inter- and intra-individual methods for estimating minimum cost of transport (MCOT) and the relationship between speed, contraction frequency and distance traveled per contraction. The minimum cost of transport calculated from intra-individual data is 2297 +/- 317 J kg-1 m-1 (S.E.M.) and the MCOT calculated from inter-individual comparisons is statistically indistinguishable at 1910 +/- 327 J kg-1 m-1. These values are almost ten times higher than the predicted value for a similar-sized limbed arthropod. Fly larvae travel by repeated peristaltic contractions and individual contractions cost about the same amount as individual strides in limbed insects. Both contraction frequency and distance traveled per contraction increase linearly with speed. Doubling the contraction frequency or the distance traveled per contraction approximately doubles speed. The cost of transport in fly larvae is among the highest recorded for terrestrial locomotion, confirming the suggestion that biomechanical and kinematic properties of limbless organisms with hydraulic skeletons lead to very high costs of transport.

Ventilation in the adults of Amblyomma hebraeum and A. marmoreum (Acarina, Ixodidae), vectors of heartwater in southern Africa.

The objective of this study was to establish the major features of respiratory gas exchange in unfed adults of the ticks Amblyomma hebraeum and A. marmoreum, both vectors of heartwater in Southern Africa. Carbon dioxide emission of ticks was measured at 25 degrees C using flow-through respirometry in order to determine standard metabolic rate (SMR) and the temporal pattern of gaseous emission. For both species, SMR was extremely low and approximately 100 fold less than that predicted for an insect of equivalent body mass. Ventilation in inactive ticks was discontinuous and characterized by periodic bursts of CO2 emissions during spiracular opening. The main selective advantage of this type of ventilation is believed to lie in a reduction of respiratory water loss. The periodicity of CO2 bursts was less frequent in A. marmoreum (every 2.5 h) compared to A. hebraeum (every 1.5 h) suggesting that A. marmoreum is more efficient at conserving respiratory water loss. It is suggested that future research into water balance physiology of ticks should address the role of ventilatory patterns in determining off-host survival and habitat associations.

Mitochondrial respiration in hummingbird flight muscles.

Respiration rates of muscle mitochondria in flying hummingbirds range from 7 to 10 ml of O2 per cm3 of mitochondria per min, which is about 2 times higher than the range obtained in the locomotory muscles of mammals running at their maximum aerobic capacities (VO2max). Capillary volume density is higher in hummingbird flight muscles than in mammalian skeletal muscles. Mitochondria occupy approximately 35% of fiber volume in hummingbird flight muscles and cluster beneath the sarcolemmal membrane adjacent to capillaries to a greater extent than in mammalian muscles. Measurements of protein content, citrate synthase activity, and respiratory rates in vitro per unit mitochondrial volume reveal no significant differences between hummingbird and mammalian skeletal muscle mitochondria. However, inner membrane surface areas per unit mitochondrial volume [Sv(im,m)] are higher than those in mammalian muscle. We propose that both mitochondrial volume densities and Sv(im,m) are near their maximum theoretical limits in hummingbirds and that higher rates of mitochondrial respiration than those observed in mammals are achieved in vivo as a result of higher capacities for O2 delivery and substrate catabolism.

Photosynthesis by inflated pods of a desert shrub, Isomeris arborea.

The photosynthetic capacity and carbon metabolism of the fruits of Isomeris arborea (Capparidaceae), an evergreen shrub endemic to the desert and coastal habitats of Southern California and Baja California, are described. The inflated structure of the pods of I. arborea provides a model system for experimental studies of fruit photosynthesis in native plants since the gas concentration of the internal space can be manipulated and monitored separately from the external pod environment. CO2 released by seed respiration is partially contained in the inner gas space of the pods, resulting in an elevated CO2 environment inside the fruit (500 to 4000 µmol mol-1 depending on the stage of fruit development). A portion of this CO2 is assimilated by the inner layers of the pericarp, but a larger fraction leaks out. The photosynthetic layers of the pericarp use two different sources of CO2: the exocarp fixes exogenous CO2 while the endocarp fixes CO2 released by seed respiration into the pod cavity. Even though the total weight of the fruit increases during development, the combined rates of fixation of externally and internally supplied CO2 remained constant (10-11 µmol CO2 pod-1 h-1). After the pods attain maximum volume, the major change in gas exchange that takes place during fruit growth is a gradual increase in the amount of respiratory CO2 released by the seeds. This shifts the CO2 balance of the fruit from positive, in young fruits, to negative in mature fruits. Pericarp photosynthesis helped support not only the cost of fruit maintenance, but also the cost of fruit growth, particularly during the first stages of fruit development. During later fruiting stages insufficient carbon is fixed to fully supply either respiration or growth.

Fuel selection in rufous hummingbirds: ecological implications of metabolic biochemistry.

Hummingbirds in flight display the highest rates of aerobic metabolism known among vertebrates. Their flight muscles possess sufficient maximal activities of hexokinase and carnitine palmitoyltransferase to allow the exclusive use of either glucose or long-chain fatty acids as metabolic fuels during flight. Respiratory quotients (RQ = VCO2/VO2) indicate that fatty acid oxidation serves as the primary energy source in fasted resting birds, while subsequent foraging occurs with a rapid shift towards the use of carbohydrate as the metabolic fuel. We suggest that hummingbirds building up fat deposits in preparation for migration behave as carbohydrate maximizers (or fat minimizers) with respect to the metabolic fuels selected to power foraging flight.

Standard energy metabolism of a desert harvester ant, Pogonomyrmex rugosus: Effects of temperature, body mass, group size, and humidity.

Pogonomyrmex rugosus (Hymenoptera: Formicidae) is an important seed predator in the Mojave Desert of the southwestern United States. Its standard rate of O(2) consumption ( Vo(2)) varied significantly with temperature ( Vo(2) = 10((-1.588 + 0.0315T)), where Vo(2) is ml.g(-1).hr(-1) and T is body temperature in degrees C). The ratio of the Vo(2) values at 10 degrees C increments in body temperature, Q(10), also varied with temperature; methods of calculating Vo(2) from temperature with a shifting Q(10) are described. Vo(2) also varied with body mass ( Vo(2) = 0.0462M(0.669), where Vo(2) is ml.hr(-1) and M is body mass in g). Vo(2) was inversely related to relative humidity and was independent of group size. The rise in Vo(2) at low relative humidities was caused by increased activity and resulted in higher rates of net water loss. The primary metabolic adaptation to xeric conditions in P. rugosus appears to be a lower-than-predicted metabolic rate.

Oxygen consumption during hover-feeding in free-ranging Anna hummingbirds.

Rates of oxygen consumption during hover-feeding of wild, unrestrained, adult male Anna hummingbirds (Calypte anna) were measured with an artificial outdoor feeder converted into a respirometer mask. A computer sampled changes in O2 concentration in air drawn through the mask, automatically detecting the presence of a hummingbird from a drop in the O2 concentration, and photoelectrically timing the duration over which the feeder functioned as a mask. Birds coming to the feeder were weighed on a trapeze perch suspended from a force transducer. Feeding bouts consisted of sallies which carried the head in and out of the feeding mask about once a second. The volume of O2 consumed per feeding sally was linearly related to the length of the sally. The energy cost of hover-feeding in five hummingbirds, mean mass 4.6 g, was 41.5 +/- 6.3 ml O2 g-1 h-1.