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The present study describes and validates a novel yet simple system for simultaneous in vivo measurements of rates of aquatic CO 2 production ( Ṁ CO 2 ) and oxygen consumption ( Ṁ O 2 ), thus allowing the calculation of respiratory exchange ratios (RER). Diffusion of CO 2 from the aquatic phase into a gas phase, across a hollow fibre membrane, enabled aquatic Ṁ CO 2  measurements with a high-precision infrared gas CO 2 analyser. Ṁ O 2  was measured with a P O 2  optode using a stop-flow approach. Injections of known amounts of CO 2 into the apparatus yielded accurate and highly reproducible measurements of CO 2 content ( R 2 =0.997, P <0.001). The viability of in vivo measurements was demonstrated on aquatic dragonfly nymphs (Aeshnidae; wet mass 2.17 mg-1.46 g, n =15) and the apparatus produced precise Ṁ CO 2  ( R 2 =0.967, P <0.001) and Ṁ O 2  ( R 2 =0.957, P <0.001) measurements; average RER was 0.73±0.06. The described system is scalable, offering great potential for the study of a wide range of aquatic species, including fish., (© 2017. Published by The Company of Biologists Ltd.)

Many insects at rest breathe discontinuously, alternating between brief bouts of gas exchange and extended periods of breath-holding. The association between discontinuous gas exchange cycles (DGCs) and inactivity has long been recognised, leading to speculation that DGCs lie at one end of a continuum of gas exchange patterns, from continuous to discontinuous, linked to metabolic rate (MR). However, the neural hypothesis posits that it is the downregulation of brain activity and a change in the neural control of gas exchange, rather than low MR per se, which is responsible for the emergence of DGCs during inactivity. To test this, Nauphoeta cinerea cockroaches had their brains inactivated by applying a Peltier-chilled cold probe to the head. Once brain temperature fell to 8°C, cockroaches switched from a continuous to a discontinuous breathing pattern. Re-warming the brain abolished the DGC and re-established a continuous breathing pattern. Chilling the brain did not significantly reduce the cockroaches' MR and there was no association between the gas exchange pattern displayed by the insect and its MR. This demonstrates that DGCs can arise due to a decrease in brain activity and a change in the underlying regulation of gas exchange, and are not necessarily a simple consequence of low respiratory demand.

Insects and spiders rely on gas-filled airways for respiration in air. However, some diving species take a tiny air-store bubble from the surface that acts as a primary O(2) source and also as a physical gill to obtain dissolved O(2) from the water. After a long history of modelling, recent work with O(2)-sensitive optodes has tested the models and extended our understanding of physical gill function. Models predict that compressible gas gills can extend dives up to more than eightfold, but this is never reached, because the animals surface long before the bubble is exhausted. Incompressible gas gills are theoretically permanent. However, neither compressible nor incompressible gas gills can support even resting metabolic rate unless the animal is very small, has a low metabolic rate or ventilates the bubble's surface, because the volume of gas required to produce an adequate surface area is too large to permit diving. Diving-bell spiders appear to be the only large aquatic arthropods that can have gas gill surface areas large enough to supply resting metabolic demands in stagnant, oxygenated water, because they suspend a large bubble in a submerged web.

The discontinuous gas exchange cycle (DGC) is a three-phase breathing pattern displayed by many insects at rest. The pattern consists of an extended breath-hold period (closed phase), followed by a sequence of rapid gas exchange pulses (flutter phase), and then a period in which respiratory gases move freely between insect and environment (open phase). This study measured CO(2) emission in resting locusts Locusta migratoria throughout ontogeny, in normoxia (21 kPa P(O2)), hypoxia (7 kPa P(O2)) and hyperoxia (40 kPa P(O2)), to determine whether body mass and ambient O(2) affect DGC phase duration. In normoxia, mean CO(2) production rate scales with body mass (M(b); g) according to the allometric power equation , closed phase duration (C; min) scales with body mass according to the equation C=8.0M(b)(0.38±0.29), closed+flutter period (C+F; min) scales with body mass according to the equation C+F=26.6M (0.20±0.25)(b) and open phase duration (O; min) scales with body mass according to the equation O=13.3M(b) (0.23±0.18). Hypoxia results in a shorter C phase and longer O phase across all life stages, whereas hyperoxia elicits shorter C, C+F and O phases across all life stages. The tendency for larger locusts to exhibit longer C and C+F phases might arise if the positive allometric scaling of locust tracheal volume prolongs the time taken to reach the minimum O(2) and maximum CO(2) set-points that determine the duration of these respective periods, whereas an increasingly protracted O phase could reflect the additional time required for larger locusts to expel CO(2) through a relatively longer tracheal pathway. Observed changes in phase duration under hypoxia possibly serve to maximise O(2) uptake from the environment, whereas the response of the DGC to hyperoxia is difficult to explain, but could be affected by elevated levels of reactive oxygen species.

Flying insects achieve the highest mass-specific aerobic metabolic rates of all animals. However, few studies attempt to maximise the metabolic cost of flight and so many estimates could be sub-maximal, especially where insects have been tethered. To address this issue, oxygen consumption was measured during tethered flight in adult locusts Locusta migratoria, some of which had a weight attached to each wing (totalling 30-45% of body mass). Mass-specific metabolic rate increased from 28±2 μmol O(2) g(-1) h(-1) at rest to 896±101 μmol O(2)g(-1) h(-1) during flight in weighted locusts, and to 1032±69 μmol O(2) g(-1) h(-1) in unweighted locusts. Maximum metabolic rate of locusts during tethered flight (m(O(2)); μmol O(2) h(-1)) increased with body mass (M(b); g) according to the allometric equation m(O(2))=994M(b)(0.75±0.19), whereas published metabolic rates of moths and orchid bees during hovering free flight (h(O(2))) are approximately 2.8-fold higher, h(O(2))=2767M(b)(0.72±0.08). The modest flight metabolic rate of locusts is unlikely to be an artefact of individuals failing to exert themselves, because mean maximum lift was not significantly different from that required to support body mass (95±8%), mean wingbeat frequency was 23.7±0.6 Hz, and mean stroke amplitude was 105±5 deg in the forewing and 96±5 deg in the hindwing - all of which are close to free-flight values. Instead, the low cost of flight could reflect the relatively small size and relatively modest anatomical power density of the locust flight motor, which is a likely evolutionary trade-off between flight muscle maintenance costs and aerial performance.

Weibel and Taylor's theory of symmorphosis predicts that the structural components of the respiratory system are quantitatively adjusted to satisfy, but not exceed, an animal's maximum requirement for oxygen. We tested this in the respiratory system of the adult migratory locust Locusta migratoria by comparing the aerobic capacity of hopping and flight muscle with the morphology of the oxygen cascade. Maximum oxygen uptake by flight muscle during tethered flight is 967±76 μmol h(-1) g(-1) (body mass specific, ±95% confidence interval CI), whereas the hopping muscles consume a maximum of 158±8 μmol h(-1) g(-1) during jumping. The 6.1-fold difference in aerobic capacity between the two muscles is matched by a 6.4-fold difference in tracheole lumen volume, which is 3.5×10(8)±1.2×10(8) μm(3) g(-1) in flight muscle and 5.5×10(7)±1.8×10(7) μm(3) g(-1) in the hopping muscles, a 6.4-fold difference in tracheole inner cuticle surface area, which is 3.2×10(9)±1.1×10(9) μm(2) g(-1) in flight muscle and 5.0×10(8)±1.7×10(8) μm(2) g(-1) in the hopping muscles, and a 6.8-fold difference in tracheole radial diffusing capacity, which is 113±47 μmol kPa(-1) h(-1) g(-1) in flight muscle and 16.7±6.5 μmol kPa(-1) h(-1) g(-1) in the hopping muscles. However, there is little congruence between the 6.1-fold difference in aerobic capacity and the 19.8-fold difference in mitochondrial volume, which is 3.2×10(10)±3.9×10(9) μm(3) g(-1) in flight muscle and only 1.6×10(9)±1.4×10(8) μm(3) g(-1) in the hopping muscles. Therefore, symmorphosis is upheld in the design of the tracheal system, but not in relation to the amount of mitochondria, which might be due to other factors operating at the molecular level.

Taylor and Weibel's theory of symmorphosis predicts that structures of the respiratory system are matched to maximum functional requirements with minimal excess capacity. We tested this hypothesis in the respiratory system of the migratory locust, Locusta migratoria, by comparing the aerobic capacity of the jumping muscles with the morphology of the oxygen cascade in the hopping legs using an intraspecific allometric analysis of different body mass (M(b)) at selected juvenile life stages. The maximum oxygen consumption rate of the hopping muscle during jumping exercise scales as M(b)(1.02±0.02), which parallels the scaling of mitochondrial volume in the hopping muscle, M(b)(1.02±0.08), and the total surface area of inner mitochondrial membrane, M(b)(0.99±0.10). Likewise, at the oxygen supply end of the insect respiratory system, there is congruence between the aerobic capacity of the hopping muscle and the total volume of tracheoles in the hopping muscle, M(b)(0.99±0.16), the total inner surface area of the tracheoles, M(b)(0.99±0.16), and the anatomical radial diffusing capacity of the tracheoles, M(b)(0.99±0.18). Therefore, the principles of symmorphosis are upheld at each step of the oxygen cascade in the respiratory system of the migratory locust.

The hemimetabolous migratory locust Locusta migratoria progresses through five instars to the adult, increasing in size from 0.02 to 0.95 g, a 45-fold change. Hopping locomotion occurs at all life stages and is supported by aerobic metabolism and provision of oxygen through the tracheal system. This allometric study investigates the effect of body mass (Mb) on oxygen consumption rate (MO2, μmol h(-1)) to establish resting metabolic rate (MRO2), maximum metabolic rate during hopping (MMO2) and maximum metabolic rate of the hopping muscles (MMO2,hop) in first instar, third instar, fifth instar and adult locusts. Oxygen consumption rates increased throughout development according to the allometric equations MRO2=30.1Mb(0.83±0.02), MMO2=155Mb(1.01±0.02), MMO2,hop=120Mb(1.07±0.02) and, if adults are excluded, MMO2,juv=136Mb(0.97±0.02) and MMO2,juv,hop=103Mb(1.02±0.02). Increasing body mass by 20-45% with attached weights did not increase mass-specific MMO2 significantly at any life stage, although mean mass-specific hopping MO2 was slightly higher (ca. 8%) when juvenile data were pooled. The allometric exponents for all measures of metabolic rate are much greater than 0.75, and therefore do not support West, Brown and Enquist’s optimised fractal network model, which predicts that metabolism scales with a 3⁄4-power exponent owing to limitations in the rate at which resources can be transported within the body.

Ventilatory control of internal CO(2) plays an important role in regulating extracellular acid-base balance in terrestrial animals. While this phenomenon is well understood among vertebrates, the role that respiration plays in the acid-base balance of insects is in need of much further study. To measure changes in insect haemolymph pH, we implanted micro pH optodes into the haemocoel of cockroaches (Nauphoeta cinerea). They were then exposed to normoxic, hypoxic, hyperoxic and hypercapnic atmospheres while their haemolymph pH, VCO(2) and abdominal ventilation frequency were measured simultaneously. Intratracheal O(2) levels were also measured in separate experiments. It was found that cockroaches breathing continuously control their ventilation to defend a haemolymph pH of 7.3, except under conditions where hypoxia (<10% O(2)) induces hyperventilation, or where ambient hypercapnia is in excess of haemolymph (>1% CO(2)). In contrast, intratracheal O(2) levels fluctuated widely, but on average remained above 15% in normoxic (21% O(2)) atmospheres. Decapitation caused the cockroaches to display discontinuous gas exchange cycles (DGCs). The alternating periods of ventilation and apnoea during DGCs caused haemolymph pH to fluctuate by 0.11 units. Exposure to hypoxia caused haemolymph pH to increase and initiated brief bouts of spiracular opening prior to the active ventilation phase. The spontaneous occurrence of DGCs in decapitated cockroaches indicates that central pattern generators in the thoracic and abdominal ganglia generate the periodic gas exchange pattern in the absence of control from the cephalic ganglion. This pattern continues to maintain gas exchange, but with less precision.

The reasons why many insects breathe discontinuously at rest are poorly understood and hotly debated. Three adaptive hypotheses attempt to explain the significance of these discontinuous gas exchange cycles (DGCs), whether it be to save water, to facilitate gas exchange in underground environments or to limit oxidative damage. Comparative studies favour the water saving hypothesis and mechanistic studies are equivocal but no study has examined the acclimation responses of adult insects chronically exposed to a range of respiratory environments. The present research is the first manipulative study of such chronic exposure to take a strong-inference approach to evaluating the competing hypotheses according to the explicit predictions stemming from them. Adult cockroaches (Nauphoeta cinerea) were chronically exposed to various treatments of different respiratory gas compositions (O(2), CO(2) and humidity) and the DGC responses were interpreted in light of the a priori predictions stemming from the competing hypotheses. Rates of mass loss during respirometry were also measured for animals acclimated to a range of humidity conditions. The results refute the hypotheses of oxidative damage and underground gas exchange, and provide evidence supporting the hypothesis that DGCs serve to reduce respiratory water loss: cockroaches exposed to low humidity conditions exchange respiratory gases for shorter durations during each DGC and showed lower rates of body mass loss during respirometry than cockroaches exposed to high humidity conditions.

Unlike all other diving insects, backswimmers of the genus Anisops can exploit the pelagic zone by temporarily achieving near-neutral buoyancy during the course of a dive. They begin a dive positively buoyant due to the large volume of air carried in their ventral air-stores, but rapidly enter a protracted period of near-neutral buoyancy before becoming negatively buoyant. This dive profile is due to haemoglobin found in large tracheated cells in the abdomen. Fibre optic oxygen probes placed in the air-stores of submerged bugs revealed that oxygen partial pressure (P(O(2))) dropped in a sigmoid curve, where a linear decline preceded a plateau between 5.1 and 2.0 kPa, before a final drop. Buoyancy measurements made by attaching backswimmers to a sensitive electronic balance showed the same three phases. Inactivating the haemoglobin by fumigating backswimmers with 15% CO eliminated both buoyancy and P(O(2)) plateaus. Oxygen unloaded from the haemoglobin stabilises the air-store during the neutrally buoyant phase after a decrease in volume of between 16% and 19%. Using measurements of air-store P(O(2)) and volume, it was calculated that during a dive the haemoglobin and air-store contribute 0.25 and 0.26 microl of oxygen, respectively.

Semi-fossorial animals (burrowing surface foragers) need to balance the competing morphological requirements of terrestrial and burrowing locomotion. These species rarely show the same degree of claw, forelimb and pectoral girdle structural development that fully fossorial forms (burrowing subterranean foragers) do, but nevertheless invest considerable amounts of energy in burrow systems. The compromise between terrestrial and burrowing locomotion was investigated by measuring net costs of burrowing and pedestrian transport in the spinifex hopping mouse, Notomys alexis, a species that forages in open areas in arid environments and is adapted for saltatorial locomotion. The net cost of transport by burrowing of hopping mice was found to be more expensive than for specialised fossorial species, and burrows were estimated to represent an energy investment equivalent to the terrestrial locomotion expected to be incurred in 17-100 days. A phylogenetically independent-contrasts approach revealed that morphological specialisation for burrowing was associated with low maximum running speeds in fossorial mammals and, for non-fossorial rodents and marsupials, maximum running speed was positively correlated with an index of habitat structure that ranged from arboreal to open desert. The high terrestrial speeds attainable by this semi-fossorial species by saltatory locomotion apparently outweigh the energetic savings that would be associated with burrowing specialisation.