Isometric spiracular scaling in scarab beetles-implications for diffusive and advective oxygen transport.

The scaling of respiratory structures has been hypothesized to be a major driving factor in the evolution of many aspects of animal physiology. Here, we provide the first assessment of the scaling of the spiracles in insects using 10 scarab beetle species differing 180× in mass, including some of the most massive extant insect species. Using X-ray microtomography, we measured the cross-sectional area and depth of all eight spiracles, enabling the calculation of their diffusive and advective capacities. Each of these metrics scaled with geometric isometry. Because diffusive capacities scale with lower slopes than metabolic rates, the largest beetles measured require 10-fold higher PO2 gradients across the spiracles to sustain metabolism by diffusion compared to the smallest species. Large beetles can exchange sufficient oxygen for resting metabolism by diffusion across the spiracles, but not during flight. In contrast, spiracular advective capacities scale similarly or more steeply than metabolic rates, so spiracular advective capacities should match or exceed respiratory demands in the largest beetles. These data illustrate a general principle of gas exchange: scaling of respiratory transport structures with geometric isometry diminishes the potential for diffusive gas exchange but enhances advective capacities; combining such structural scaling with muscle-driven ventilation allows larger animals to achieve high metabolic rates when active.

Size-dependent Scaling of Stingless Bee Flight Metabolism Reveals an Energetic Benefit to Small Body Size.

Understanding the effect of body size on flight costs is critical for development of models of aerodynamics and animal energetics. Prior scaling studies that have shown that flight costs scale hypometrically have focused primarily on larger (> 100 mg) insects and birds, but most flying species are smaller. We studied the flight physiology of thirteen stingless bee species over a large range of body sizes (1-115 mg). Metabolic rate during hovering scaled hypermetrically (scaling slope = 2.11). Larger bees had warm thoraxes while small bees were nearly ecothermic; however, even controlling for body temperature variation, flight metabolic rate scaled hypermetrically across this clade. Despite having a lower mass-specific metabolic rate during flight, smaller bees could carry the same proportional load. Wingbeat frequency did not vary with body size, in contrast to most studies that find wingbeat frequency increases as body size decreases. Smaller stingless bees have greater relative forewing surface area which may help them reduce the energy requirements needed to fly. Further, we hypothesize that the relatively larger heads of smaller species may change their body pitch in flight. Synthesizing across all flying insects, we demonstrate that the scaling of flight metabolic rate changes from hypermetric to hypometric at approximately 58 mg body mass with hypermetic scaling below (slope = 1.2) and hypometric scaling (slope = 0.67) above 58 mg in body mass. The reduced cost of flight likely provides selective advantages for the evolution of small body size in insects. The biphasic scaling of flight metabolic rates and wingbeat frequencies in insects supports the hypothesis that the scaling of metabolic rate is closely related to the power requirements of locomotion and cycle frequencies.

Physiological responses to gravity in an insect.

Gravity is one of the most ubiquitous environmental effects on living systems: Cellular and organismal responses to gravity are of central importance to understanding the physiological function of organisms, especially eukaryotes. Gravity has been demonstrated to have strong effects on the closed cardiovascular systems of terrestrial vertebrates, with rapidly responding neural reflexes ensuring proper blood flow despite changes in posture. Invertebrates possess open circulatory systems, which could provide fewer mechanisms to restrict gravity effects on blood flow, suggesting that these species also experience effects of gravity on blood pressure and distribution. However, whether gravity affects the open circulatory systems of invertebrates is unknown, partly due to technical measurement issues associated with small body size. Here we used X-ray imaging, radio-tracing of hemolymph, and micropressure measurements in the American grasshopper, Schistocerca americana, to assess responses to body orientation. Our results show that during changes in body orientation, gravity causes large changes in blood and air distribution, and that body position affects ventilation rate. Remarkably, we also found that insects show similar heart rate responses to body position as vertebrates, and contrasting with the classic understanding of open circulatory systems, have flexible valving systems between thorax and abdomen that can separate pressures. Gravitational effects on invertebrate cardiovascular and respiratory systems are likely to be widely distributed among invertebrates and to have broad influence on morphological and physiological evolution.

Developmental plasticity and stability in the tracheal networks supplying Drosophila flight muscle in response to rearing oxygen level.

While it is clear that the insect tracheal system can respond in a compensatory manner to both hypoxia and hyperoxia, there is substantial variation in how different parts of the system respond. However, the response of tracheal structures, from the tracheoles to the largest tracheal trunks, have not been studied within one species. In this study, we examined the effect of larval/pupal rearing in hypoxia, normoxia, and hyperoxia (10, 21 or 40kPa oxygen) on body size and the tracheal supply to the flight muscles of Drosophila melanogaster, using synchrotron radiation micro-computed tomography (SR-µCT) to assess flight muscle volumes and the major tracheal trunks, and confocal microscopy to assess the tracheoles. Hypoxic rearing decreased thorax length whereas hyperoxic-rearing decreased flight muscle volumes, suggestive of negative effects of both extremes. Tomography at the broad organismal scale revealed no evidence for enlargement of the major tracheae in response to lower rearing oxygen levels, although tracheal size scaled with muscle volume. However, using confocal imaging, we found a strong inverse relationship between tracheole density within the flight muscles and rearing oxygen level, and shorter tracheolar branch lengths in hypoxic-reared animals. Although prior studies of larger tracheae in other insects indicate that axial diffusing capacity should be constant with sequential generations of branching, this pattern was not found in the fine tracheolar networks, perhaps due to the increasing importance of radial diffusion in this regime. Overall, D. melanogaster responded to rearing oxygen level with compensatory morphological changes in the small tracheae and tracheoles, but retained stability in most of the other structural components of the tracheal supply to the flight muscles.

Multigenerational Effects of Rearing Atmospheric Oxygen Level on the Tracheal Dimensions and Diffusing Capacities of Pupal and Adult Drosophila melanogaster.

Insects are small relative to vertebrates, and were larger in the Paleozoic when atmospheric oxygen levels were higher. The safety margin for oxygen delivery does not increase in larger insects, due to an increased mass-specific investment in the tracheal system and a greater use of convection in larger insects. Prior studies have shown that the dimensions and number of tracheal system branches varies inversely with rearing oxygen in embryonic and larval insects. Here we tested whether rearing in 10, 21, or 40 kPa atmospheric oxygen atmospheres for 5-7 generations affected the tracheal dimensions and diffusing capacities of pupal and adult Drosophila. Abdominal tracheae and pupal snorkel tracheae showed weak responses to oxygen, while leg tracheae showed strong, but imperfect compensatory changes. The diffusing capacity of leg tracheae appears closely matched to predicted oxygen transport needs by diffusion, perhaps explaining the consistent and significant responses of these tracheae to rearing oxygen. The reduced investment in tracheal structure in insects reared in higher oxygen levels may be important for conserving tissue PO2 and may provide an important mechanism for insects to invest only the space and materials necessary into respiratory structure.

Temperature and the Ventilatory Response to Hypoxia in Gromphadorhina portentosa (Blattodea: Blaberidae).

In general, insects respond to hypoxia by increasing ventilation frequency, as seen in most other animals. Higher body temperatures usually also increase ventilation rates, likely due to increases in metabolic rates. In ectothermic air-breathing vertebrates, body temperatures and hypoxia tend to interact significantly, with an increasing responsiveness of ventilation to hypoxia at higher temperatures. Here, we tested whether the same is true in insects, using the Madagascar hissing cockroach, Gromphadorhina portentosa (Schaum) (Blattodea: Blaberidae). We equilibrated individuals to a temperature (beginning at 20 °C), and animals were exposed to step-wise decreases in PO2 (21, 15, 10, and 5 kPa, in that order), and we measured ventilation frequencies from videotapes of abdominal pumping after 15 min of exposure to the test oxygen level. We then raised the temperature by 5 °C, and the protocol was repeated, with tests run at 20, 25, 30, and 35 °C. The 20 °C animals had high initial ventilation rates, possibly due to handling stress, so these animals were excluded from subsequent analyses. Across all temperatures, ventilation increased in hypoxia, but only significantly at 5 kPa PO2 Surprisingly, there was no significant interaction between temperature and oxygen, and no significant effect of temperature on ventilation frequency from 25 to 35 °C. Plausibly, the rise in metabolic rates at higher temperatures in insects is made possible by increasing other aspects of gas exchange, such as decreasing internal PO2, or increases in tidal volume, spiracular opening (duration or amount), or removal of fluid from the tracheoles.

The temperature size rule in arthropods: independent of macro-environmental variables but size dependent.

Temperature is a key factor that affects the rates of growth and development in animals, which ultimately determine body size. Although not universal, a widely documented and poorly understood pattern is the inverse relationship between the temperature at which an ectothermic animal is reared and its body size (temperature size rule [TSR]). The proximate and ultimate mechanisms for the TSR remain unclear. To explore possible explanations for the TSR, we tested for correlations between the magnitude/direction of the TSR and latitude, temperature, elevation, habitat, availability of oxygen, capacity for flight, and taxonomic grouping in 98 species/populations of arthropods. The magnitude and direction of the TSR was not correlated with any of the macro-environmental variables we examined, supporting the generality of the TSR. However, body size affected the magnitude and direction of the TSR, with smaller arthropods more likely to demonstrate a classic TSR. Considerable variation among species exists in the TSR, suggesting either strong interactions with nutrition, or selection based on microclimatic or seasonal variation not captured in classic macro-environmental variables.

How locusts breathe.

Insect tracheal-respiratory systems achieve high fluxes and great dynamic range with low energy requirements and could be important models for bioengineers interested in developing microfluidic systems. Recent advances suggest that insect cardiorespiratory systems have functional valves that permit compartmentalization with segment-specific pressures and flows and that system anatomy allows regional flows. Convection dominates over diffusion as a transport mechanism in the major tracheae, but Reynolds numbers suggest viscous effects remain important.

The effect of developmental stage on the sensitivity of cell and body size to hypoxia in Drosophila melanogaster.

Animals reared in hypoxic environments frequently exhibit smaller body sizes than when reared under normal atmospheric oxygen concentrations. The mechanisms responsible for this widely documented pattern of body size plasticity are poorly known. We studied the ontogeny of responses of Drosophila melanogaster adult body size to hypoxic exposure. We hypothesized that there may be critical oxygen-sensitive periods during D. melanogaster development that are primarily responsive to body size regulation. Instead, our results showed that exposure to hypoxia (an atmospheric partial pressure of oxygen of 10 kPa) during any developmental stage (embryo, larvae and pupae) leads to smaller adult size. However, short hypoxic exposures during the late larval and early pupal stages had the greatest effects on adult size. We then investigated whether the observed reductions in size induced by hypoxia at various developmental stages were the result of a decrease in cell size or cell number. Abdominal epithelial cells of flies reared continuously in hypoxia were smaller in mean diameter and were size-limited compared with cells of flies reared in normoxia. Flies reared in hypoxia during the embryonic, larval or pupal stage, or during their entire development, had smaller wing areas than flies reared in normoxia. Flies reared during the pupal stage, or throughout development in hypoxia had smaller wing cells, even after controlling for the effect of wing size. These results suggest that hypoxia effects on the body size of D. melanogaster probably occur by multiple mechanisms operating at various developmental stages.

Critical oxygen partial pressures and maximal tracheal conductances for Drosophila melanogaster reared for multiple generations in hypoxia or hyperoxia.

In Drosophila melanogaster and other insects, increases in atmospheric oxygen partial pressure (aPO(2)) tend to increase adult body size and decrease tracheal diameters and tracheolar proliferation. If changes in tracheal morphology allow for functional compensation for aPO(2), we would predict that higher aPO(2) would be associated with higher critical PO(2) values (CritPO(2)) and lower maximal tracheal conductances (G(max)). We measured CritPO(2) and G(max) for adult and larval vinegar flies reared for 7-9 generations in 10, 21 or 40 kPa O(2). The CritPO(2), CO(2) emission rates and G(max) values were generally independent of the rearing PO(2) these flies had experienced, suggesting that minimal functional changes in tracheal capacities occurred in response to rearing PO(2). Larvae were able to continue activity during 20 min of anoxia. The lack of multigenerational rearing PO(2) effects on tracheal function suggests that the functional compensation at the whole-body level due to tracheal morphological changes in response to aPO(2) may be minimal; alternatively the benefits of such compensation may occur in specific tissues or during processes not assessed by these methods. In larvae, the CritPO(2) and the capacity for movement in anoxia suggest adaptations for life in hypoxic organic matter.

Atmospheric hypoxia limits selection for large body size in insects.

BACKGROUND: The correlations between Phanerozoic atmospheric oxygen fluctuations and insect body size suggest that higher oxygen levels facilitate the evolution of larger size in insects. METHODS AND PRINCIPAL FINDINGS: Testing this hypothesis we selected Drosophila melanogaster for large size in three oxygen atmospheric partial pressures (aPO(2)). Fly body sizes increased by 15% during 11 generations of size selection in 21 and 40 kPa aPO(2). However, in 10 kPa aPO(2), sizes were strongly reduced. Beginning at the 12(th) generation, flies were returned to normoxia. All flies had similar, enlarged sizes relative to the starting populations, demonstrating that selection for large size had functionally equivalent genetic effects on size that were independent of aPO(2). SIGNIFICANCE: Hypoxia provided a physical constraint on body size even in a tiny insect strongly selected for larger mass, supporting the hypothesis that Triassic hypoxia may have contributed to a reduction in insect size.

Increase in tracheal investment with beetle size supports hypothesis of oxygen limitation on insect gigantism.

Recent studies have suggested that Paleozoic hyperoxia enabled animal gigantism, and the subsequent hypoxia drove a reduction in animal size. This evolutionary hypothesis depends on the argument that gas exchange in many invertebrates and skin-breathing vertebrates becomes compromised at large sizes because of distance effects on diffusion. In contrast to vertebrates, which use respiratory and circulatory systems in series, gas exchange in insects is almost exclusively determined by the tracheal system, providing a particularly suitable model to investigate possible limitations of oxygen delivery on size. In this study, we used synchrotron x-ray phase-contrast imaging to visualize the tracheal system and quantify its dimensions in four species of darkling beetles varying in mass by 3 orders of magnitude. We document that, in striking contrast to the pattern observed in vertebrates, larger insects devote a greater fraction of their body to the respiratory system, as tracheal volume scaled with mass1.29. The trend is greatest in the legs; the cross-sectional area of the trachea penetrating the leg orifice scaled with mass1.02, whereas the cross-sectional area of the leg orifice scaled with mass0.77. These trends suggest the space available for tracheae within the leg may ultimately limit the maximum size of extant beetles. Because the size of the tracheal system can be reduced when oxygen supply is increased, hyperoxia, as occurred during late Carboniferous and early Permian, may have facilitated the evolution of giant insects by allowing limbs to reach larger sizes before the tracheal system became limited by spatial constraints.

Cuticular lipid mass and desiccation rates in Glossina pallidipes: interpopulation variation.

Tsetse flies, Glossina pallidipes (Diptera: Glossinidae) are said to have strong dispersal tendencies. Gene flow among these populations is estimated to be the theoretical equivalent of no more than one or two reproducing flies per generation, thereby raising the hypothesis of local regimes of natural selection. Flies were sampled from four environmentally diverse locations in Kenya to determine whether populations are homogeneous in desiccation tolerance and cuticular lipids. Cuticular hydrocarbon fractions known to act as sex pheromones do not differ among populations, thereby eliminating sexual selection as an isolating mechanism. Cuticular lipid quantities vary among populations and are not correlated with prevailing temperatures, humidities, and normalized density vegetation indices. Females demonstrate a stronger correlation than males between cuticular lipid mass and body weight. Desiccation rates also vary among populations, but are not correlated with the amounts of cuticular lipid. Chemical analysis of cuticular hydrocarbons by gas chromatography-mass spectroscopy shows that one of the four populations has more 11,15- and 11,21-dimethyl-31 hydrocarbon on females. These results are discussed in the context of population differences and estimates of gene flow.

Phenotypic plasticity and geographic variation in thermal tolerance and water loss of the tsetse Glossina pallidipes (Diptera: Glossinidae): implications for distribution modelling.

Using the tsetse, Glossina pallidipes, we show that physiologic plasticity (resulting from temperature acclimation) accounts for among-population variation in thermal tolerance and water loss rates. Critical thermal minimum (CT(Min)) was highly variable among populations, seasons, and acclimation treatments, and the full range of variation was 9.3 degrees C (maximum value = 3.1 x minimum). Water loss rate showed similar variation (max = 3.7 x min). In contrast, critical thermal maxima (CT(Max)) varied least among populations, seasons, and acclimation treatments, and the full range of variation was only approximately 1 degree C. Most of the variation among the four field populations could be accounted for by phenotypic plasticity, which in the case of CT(Min), develops within 5 days of temperature exposure and is lost rapidly on return to the original conditions. Limited variation in CT(Max) supports bioclimatic models that suggest tsetse are likely to show range contraction with warming from climate change.

Discontinuous gas exchange in insects: a clarification of hypotheses and approaches.

Many adult and diapausing pupal insects exchange respiratory gases discontinuously in a three-phase discontinuous gas exchange cycle (DGC). We summarize the known biophysical characteristics of the DGC and describe current research on the role of convection and diffusion in the DGC, emphasizing control of respiratory water loss. We summarize the main theories for the evolutionary genesis (or, alternatively, nonadaptive genesis) of the DGC: reduction in respiratory water loss (the hygric hypothesis), optimizing gas exchange in hypoxic and hypercapnic environments (the chthonic hypothesis), the hybrid of these two (the chthonic-hygric hypothesis), reducing the toxic properties of oxygen (the oxidative damage hypothesis), the outcome of interactions between O(2) and CO(2) control set points (the emergent property hypothesis), and protection against parasitic invaders (the strolling arthropods hypothesis). We describe specific techniques that are being employed to measure respiratory water loss in the presence or absence of the DGC in an attempt to test the hygric hypothesis, such as the hyperoxic switch and H(2)O/CO(2) regression, and summarize specific areas of the field that are likely to be profitable directions for future research.

Insect gas exchange patterns: a phylogenetic perspective.

Most investigations of insect gas exchange patterns and the hypotheses proposed to account for their evolution have been based either on small-scale, manipulative experiments, or comparisons of a few closely related species. Despite their potential utility, no explicit, phylogeny-based, broad-scale comparative studies of the evolution of gas exchange in insects have been undertaken. This may be due partly to the preponderance of information for the endopterygotes, and its scarcity for the apterygotes and exopterygotes. Here we undertake such a broad-scale study. Information on gas exchange patterns for the large majority of insects examined to date (eight orders, 99 species) is compiled, and new information on 19 exemplar species from a further ten orders, not previously represented in the literature (Archaeognatha, Zygentoma, Ephemeroptera, Odonata, Mantodea, Mantophasmatodea, Phasmatodea, Dermaptera, Neuroptera, Trichoptera), is provided. These data are then used in a formal, phylogeny-based parsimony analysis of the evolution of gas exchange patterns at the order level. Cyclic gas exchange is likely to be the ancestral gas exchange pattern at rest (recognizing that active individuals typically show continuous gas exchange), and discontinuous gas exchange probably originated independently a minimum of five times in the Insecta.

Metabolic rate in the whip-spider, Damon annulatipes (Arachnida: Amblypygi).

Metabolic rate estimates as well as a measure of their repeatability and response to laboratory acclimation are provided for the amblypygid Damon annulatipes (Wood). This species (mean +/- S.E. mass: 640+/-66 mg) shows continuous gas exchange, as might be expected from its possession of book lungs, and at 21 degrees C has a metabolic rate of 30.22+/-2.87 microl CO2 h(-1) (approximately 229.6+/-21.8 microW, R.Q. = 0.72). The intraclass correlation coefficient (r=0.74-0.89) indicated substantial repeatability in metabolic rate which did not change with laboratory acclimation over a period of 2 weeks. By contrast, absolute metabolic rate declined by c. 16-33%, although this was not a consequence of changes in mass (which were non-significant over the same period). Rather, it appears that a reduction in overall stress or activity in the laboratory might have been responsible for the decline in mass-independent metabolic rate. At the intraspecific level, metabolic rate scaled as microW = 342 M(0.857), where mass is in grams. Metabolic rates of this species are in keeping with its sedentary behaviour such that for a given body size they are lower than those of most arthropods (spiders and insects), higher than the very sedentary ticks, and equivalent to scorpions. These findings have implications for the understanding of the evolution of metabolic rates in arthropods.

Upper thermal tolerance and oxygen limitation in terrestrial arthropods.

The hypothesis of oxygen limitation of thermal tolerance proposes that critical temperatures are set by a transition to anaerobic metabolism, and that upper and lower tolerances are therefore coupled. Moreover, this hypothesis has been dubbed a unifying general principle and extended from marine to terrestrial ectotherms. By contrast, in insects the upper and lower limits are decoupled, suggesting that the oxygen limitation hypothesis might not be as general as proposed. However, no direct tests of this hypothesis or its predictions have been undertaken in terrestrial species. We use a terrestrial isopod (Armadillidium vulgare) and a tenebrionid beetle (Gonocephalum simplex) to test the prediction that thermal tolerance should vary with oxygen partial pressure. Whilst in the isopod critical thermal maximum declined with declining oxygen concentration, this was not the case in the beetle. Efficient oxygen delivery via a tracheal system makes oxygen limitation of thermal tolerance, at a whole organism level, unlikely in insects. By contrast, oxygen limitation of thermal tolerances is expected to apply to species, like the isopod, in which the circulatory system contributes significantly to oxygen delivery. Because insects dominate terrestrial systems, oxygen limitation of thermal tolerance cannot be considered pervasive in this habitat, although it is a characteristic of marine species.

Metabolism of the sub-Antarctic caterpillar Pringleophaga marioni during cooling, freezing and thawing.

Although general models of the processes involved in insect survival of freezing exist, there have been few studies directly investigating physiological processes during cooling, freezing and thawing, without which these models remain hypothetical. Here, we use open-flow respirometry to investigate the metabolism of the freeze-tolerant sub-Antarctic caterpillar Pringleophaga marioni Viette (Lepidoptera: Tineidae) during cooling, freezing and thawing and to compare animals exposed to non-lethal (-5.8 degrees C) and lethal (-6.0 degrees C, after which caterpillars are moribund for several days, and -18 degrees C, after which caterpillars are completely unresponsive) freezing stress. We found a large decrease in metabolic rate (that is not associated with freezing) at -0.6+/-0.1 degrees C and calculated a Q10 of 2.14 x 10(3) at this breakpoint. This breakpoint is coincident with the critical thermal minimum (CTmin) and is hypothesised to be a metabolic manifestation of the latter, possibly a failure of the Na+/K(+)-ATPase pump. This provides a plausible link between processes at the cellular level and observations of the action of the CTmin at tissue and whole-organism levels. Caterpillars froze at -4.6+/-0.1 degrees C and had detectable metabolism when frozen. Post-thaw, metabolic rates were lower than pre-freezing measurements. Post-thaw metabolic rates did not differ between temperatures that did and did not kill the caterpillars, which suggests that mortality may be a result of a breakdown in processes at the organismal, rather than cellular, level of organisation.

Water-balance characteristics respond to changes in body size in subantarctic weevils.

Several environmental factors leading to size-dependent mortality influence insect body size. Few investigations have been concerned with the ways in which the mechanisms underlying variation in water-balance characteristics evolve in response to changes in body size that occur independently of water-balance requirements. Using an explicitly phylogenetic analysis, we show how body size has changed over time in the Ectemnorhinus group of weevils and how water-balance characteristics have evolved in response to this change and changes in habitat use. The basal species in the group are all large bodied and from moist environments. In response to a change in resource availability, there was a marked decline in size within the group. Despite the reduction in water content and dehydration tolerance that this meant, evolution of low whole-animal water-loss rates and high tolerance of dehydration resulted in conservation of desiccation resistance. The return to moist habitats in the group resulted in a reduction in dehydration tolerance and an increase in water-loss rate. Thus, dehydration tolerance and water-loss rate respond rapidly both when there is selection for water conservation and when this requirement is relaxed. Future laboratory selection experiments might usefully explore both directions of water-balance evolution.