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The developmental regulation of body size is a fundamental life-history characteristic that in most animals is tied to the transition from juvenile to adult form. In holometabolous insects, this transition is ostensibly initiated at the attainment of a critical weight in the final larval instar. It has been hypothesized that the size-sensing mechanism used to determine attainment of critical weight exploits oxygen limitation as a larvae grows beyond the oxygen-delivery capacity of its fixed tracheal system; that is, developmentally induced cellular hypoxia initiates the synthesis of the molting hormone ecdysone by the prothoracic gland. We tested this hypothesis in Drosophila by assaying cellular hypoxia throughout the third larval instar at 21 and 10 kPa O2, using the activity of the HIF (hypoxia inducible factor)-signaling pathway as a measure of hypoxia. While HIF signaling was elevated at low levels of environmental O2, it did not markedly increase during development at either oxygen level, and was only suppressed by hyperoxia after feeding had ceased. Further, changes in HIF signaling in the prothoracic gland alone did not alter body size or developmental time in a way that would be expected if cellular hypoxia in the prothoracic gland was part of the critical weight mechanism. Our data do show, however, that reduced HIF signaling in the prothoracic gland decreases survival and retards development at 10 kPa O2, suggesting that prothoracic HIF signaling is a necessary part of the beneficial plasticity mechanism that controls growth and development in response to low oxygen level., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Growth rate, development time, and response to environmental stressors vary tremendously across organisms, suggesting trade-offs that are affected by evolutionary or ecological factors, but such trade-offs are poorly understood. Prior studies using artificially selected lines of Manduca sexta suggest that insects with high growth rates, long development time, and large body size are more sensitive to hypoxic or hyperoxic stresses, such as reactive oxygen species (ROS) production, but the mechanisms and specific life-history associations remain unclear. Here, we manipulated the social environment to differentiate the effects of size, growth rate, and development time on oxygen sensitivity of the giant mealworm, Zophobas morio . Crowding reduced growth rates but yielded larger adults as a result of supernumerary molts and longer development times. The juvenile performance (growth rate, development time, adult mass) of crowd-reared mealworms was less sensitive to variation in atmospheric oxygen than it was for individually reared animals, consistent with the hypothesis that high growth rates are associated with increased sensitivity to ROS. Life span in normoxia was extended by crowd rearing, perhaps due to the larger size and/or increased resources of the larger adults. Life spans of crowd-reared animals were more negatively affected by hypoxia or hyperoxia than life spans of individually reared animals, possibly due to the longer total stress exposure of crowd-reared animals. These data suggest that animals with high growth rates experience a negative trade-off of performance with greater sensitivity to stress during the juvenile phase, while animals with long development times or life spans experience a negative trade-off of greater susceptibility of life span to environmental stress.

As the genetic bases to variation in anoxia tolerance are poorly understood, we used the Drosophila Genetics Reference Panel (DGRP) to conduct a genome-wide association study (GWAS) of anoxia tolerance in adult and larval Drosophila melanogaster Survival ranged from 0-100% in adults exposed to 6 h of anoxia and from 20-98% for larvae exposed to 1 h of anoxia. Anoxia tolerance had a broad-sense heritability of 0.552 in adults and 0.433 in larvae. Larval and adult phenotypes were weakly correlated but the anoxia tolerance of adult males and females were strongly correlated. The GWA identified 180 SNPs in adults and 32 SNPs in larvae associated with anoxia tolerance. Gene ontology enrichment analysis indicated that many of the 119 polymorphic genes associated with adult anoxia-tolerance were associated with ionic transport or immune function. In contrast, the 22 polymorphic genes associated with larval anoxia-tolerance were mostly associated with regulation of transcription and DNA replication. RNAi of mapped genes generally supported the hypothesis that disruption of these genes reduces anoxia tolerance. For two ion transport genes, we tested predicted directional and sex-specific effects of SNP alleles on adult anoxia tolerance and found strong support in one case but not the other. Correlating our phenotype to prior DGRP studies suggests that genes affecting anoxia tolerance also influence stress-resistance, immune function and ionic balance. Overall, our results provide evidence for multiple new potential genetic influences on anoxia tolerance and provide additional support for important roles of ion balance and immune processes in determining variation in anoxia tolerance., (Copyright © 2019 Campbell et al.)

Animals vary tremendously in their capacities to survive anoxia, and the mechanisms responsible are poorly understood. Adult Drosophila melanogaster are rapidly paralyzed and survive up to 12 h of anoxia, whereas larvae vigorously attempt escape but then die if anoxia exceeds 2 h. Here we use nuclear magnetic resonance methods to compare the metabolome of larvae and adult D. melanogaster under normoxic conditions and after various anoxic durations up to 1 h. Glucose increased during anoxia in both larvae and adults, so anoxic death by carbohydrate limitation is unlikely for either stage. Lactate and alanine were the primary anaerobic end products in both adults and larvae. During the first 30 min of anoxia, larvae accumulated anaerobic end products (predominately lactate) at a higher rate, suggesting that larvae may experience greater initial acid-base disruption during anoxic exposures. Adult Drosophila did not possess higher levels of putative protective metabolites; however, these increased during anoxia in adults and decreased in larvae. Metabolites that decreased during anoxia in larvae included mannitol, xylitol, glycerol, betaine, serine, and tyrosine, perhaps due to use as fuels, antioxidants, or binding to denatured proteins. Adults showed significant increases in glycine, taurine, and the polyols glycerol, mannitol, and xylitol, suggesting that adults upregulate protective metabolites to prevent damage. Our results suggest that lower initial metabolic demand due to paralytic hypometabolism and capacities to upregulate protective metabolites may assist the better anoxia tolerance of adult Drosophila .

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Insects can experience functional hypoxia, a situation in which O 2 supply is inadequate to meet oxygen demand. Assessing when functional hypoxia occurs is complex, because responses are graded, age and tissue dependent, and compensatory. Here, we compare information gained from metabolomics and transcriptional approaches and by manipulation of the partial pressure of oxygen. Functional hypoxia produces graded damage, including damaged macromolecules and inflammation. Insects respond by compensatory physiological and morphological changes in the tracheal system, metabolic reorganization, and suppression of activity, feeding, and growth. There is evidence for functional hypoxia in eggs, near the end of juvenile instars, and during molting. Functional hypoxia is more likely in species with lower O 2 availability or transport capacities and when O 2 need is great. Functional hypoxia occurs normally during insect development and is a factor in mediating life-history trade-offs.

Hypometric scaling of aerobic metabolism in animals has been widely attributed to constraints on oxygen (O 2 ) supply in larger animals, but recent findings demonstrate that O 2 supply balances with need regardless of size. Larger animals also do not exhibit evidence of compensation for O 2 supply limitation. Because declining metabolic rates (MRs) are tightly linked to fitness, this provides significant evidence against the hypothesis that constraints on supply drive hypometric scaling. As an alternative, ATP demand might decline in larger animals because of performance-safety tradeoffs. Larger animals, which typically reproduce later, exhibit risk-reducing strategies that lower MR. Conversely, smaller animals are more strongly selected for growth and costly neurolocomotory performance, elevating metabolism., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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 Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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.

Holometabolous insects undergo dramatic morphological and physiological changes during ontogeny. In particular, the larvae of many holometabolous insects are specialized to feed in soil, water or dung, inside plant structures, or inside other organisms as parasites where they may commonly experience hypoxia or anoxia. In contrast, holometabolous adults usually are winged and live with access to air. Here, we show that larval Drosophila melanogaster experience severe hypoxia in their normal laboratory environments; third instar larvae feed by tunneling into a medium without usable oxygen. Larvae move strongly in anoxia for many minutes, while adults (like most other adult insects) are quickly paralyzed. Adults survive anoxia nearly an order of magnitude longer than larvae (LT50: 8.3 versus 1 h). Plausibly, the paralysis of adults is a programmed response to reduce ATP need and enhance survival. In support of that hypothesis, larvae produce lactate at 3× greater rates than adults in anoxia. However, when immobile in anoxia, larvae and adults are similarly able to decrease their metabolic rate, to about 3% of normoxic conditions. These data suggest that Drosophila larvae and adults have been differentially selected for behavioral and metabolic responses to anoxia, with larvae exhibiting vigorous escape behavior likely enabling release from viscous anoxic media to predictably normoxic air, while the paralysis behavior of adults maximizes their chances of surviving flooding events of unpredictable duration. Developmental remodeling of behavioral and metabolic strategies to hypoxia/anoxia is a previously unrecognized major attribute of holometabolism., (© 2015. Published by The Company of Biologists Ltd.)

Animals develop in atmospheric hypoxia in a wide range of habitats, and tissues may experience O2 limitation of ATP production during postembryonic development if O2 supply structures do not keep pace with growing O2 demand during ontogeny. Most animal species are stunted by postembryonic development in hypoxia, showing reduced growth rates and size in moderate hypoxia (5-15 kPa Po2). In mammals, the critical Po2 that limits resting metabolic rate also falls in this same moderate hypoxic range, so stunted growth may simply be due to hypoxic limits on ATP production. However, in most invertebrates and at least some lower vertebrates, hypoxic stunting occurs at Po2 values well above those that limit resting metabolism. Studies with diverse model organisms have identified multiple homologous O2-sensing signaling pathways that can inhibit feeding and growth during moderate hypoxia. Together, these comparative and model organism-based studies suggest that hypoxic stunting of growth and size can occur as programmed inhibition of growth, often by inhibition of insulin stimulation of growth processes. Furthermore, there is increasing evidence that these same O2 signaling pathways can be utilized during normal animal development to ensure matching of O2 supply and demand structures and in mediation of variation in animal performance.

One hypothesis for the small size of insects relative to vertebrates, and the existence of giant fossil insects, is that atmospheric oxygen levels constrain insect body sizes because oxygen delivery is more challenging in larger insects. This study tested this hypothesis in dragonflies by measuring the oxygen sensitivity of flight metabolic rates and behavior during hovering for 11 species of dragonflies that ranged in mass by an order of magnitude. We measured flight times and flight metabolic rates in seven oxygen concentrations ranging from 30% to 2.5% to assess the sensitivity of their flight to atmospheric oxygen. We also assessed the oxygen sensitivity of flight in low-density air (nitrogen replaced with helium) in order to increase the metabolic demands of hovering flight. Lowered atmospheric densities did induce higher flight metabolic rates. Flight behavior was more sensitive to decreasing oxygen levels than flight metabolic rate. The oxygen sensitivity of flight metabolic rates and behaviors were not correlated with body size, indicating that larger insects are able to maintain an oxygen supply-to-demand balance even during flight., (© 2014. Published by The Company of Biologists Ltd.)

All insects studied to date show reduced growth rates in hypoxia. Drosophila melanogaster reared in moderate hypoxia (10 kPa PO2) grow more slowly and form smaller adults, but the mechanisms responsible are unclear, as metabolic rates are not oxygen-limited. It has been shown that individual fruit flies do not grow larger in hyperoxia (40 kPa PO2), but populations of flies evolve larger size. Here we studied the effect of acute and chronic variation in atmospheric PO2 (10, 21, 40 kPa) on feeding behavior of third instar larvae of D.melanogaster to assess whether oxygen effects on growth rate can be explained by effects on feeding behavior. Hypoxic-reared larvae grew and developed more slowly, and hyperoxic-rearing did not affect growth rate, maximal larval mass or developmental time. The effect of acute exposure to varying PO2 on larval bite rates matched the pattern observed for growth rates, with a 22% reduction in 10 kPa PO2 and no effect of 40 kPa PO2. Chronic rearing in hypoxia had few effects on the responses of feeding rates to oxygen, but chronic rearing in hyperoxia caused feeding rates to be strongly oxygen-dependent. Hypoxia produced similar reductions in bite rate and in the volume of tunnels excavated by larvae, supporting bite rate as an index of feeding behavior. Overall, our data show that reductions in feeding rate can explain reduced growth rates in moderate hypoxia for Drosophila, contributing to reduced body size, and that larvae cannot successfully compensate for this level of hypoxia with developmental plasticity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Rearing oxygen level is known to affect final body size in a variety of insects, but the physiological mechanisms by which oxygen affects size are incompletely understood. In Manduca sexta and Drosophila melanogaster, the larval size at which metamorphosis is initiated largely determines adult size, and metamorphosis is initiated when larvae attain a critical mass. We hypothesized that oxygen effects on final size might be mediated by oxygen effects on the critical weight and the ecdysone titers, which regulate growth rate and the timing of developmental transitions. Our results showed that oxygen affected critical weight, the basal ecdysone titers and the timing of the ecdysone peak, providing clear evidence that oxygen affected growth rate and developmental rate. Hypoxic third instar larvae (10% oxygen) exhibited a reduced critical weight, slower growth rate, delayed pupariation, elevated baseline ecdysone levels and a delayed ecdysone peak that occurred at a lower larval mass. Hyperoxic larvae exhibited increased basal ecdysone levels, but no change in critical weight compared with normoxic larvae and no significant change in timing of pupariation. Previous studies have shown that nutrition is crucial for regulating growth rate and the timing of developmental transitions. Here we show that oxygen level is one of multiple cues that together regulate adult size and the timing and dynamics of growth, developmental rate and ecdysone signaling.

Constraints on oxygen delivery potentially limit animal body size. Because diffusion rates are highly distance dependent, and because tracheal length increases with size, gas exchange was traditionally thought to be more difficult for larger insects. As yet the effect of body size on critical oxygen partial pressure (P(crit)) has not been measured for any clade of insect species for which there are interspecific data on tracheal scaling. We addressed this deficiency by measuring P(crit) over a 4150-fold mass range (ratio of largest to smallest species mean) of two families of Coleoptera (Tenebrionidae and Scarabaeidae). We exposed adult beetles to progressively lower oxygen levels and measured their ability to maintain CO(2) release rates. Absolute metabolic rates increased hypometrically with beetle body mass (M) at both normoxic (M(0.748)) and hypoxic (M(0.846)) conditions. P(crit), however, was independent of body size. Maximum overall conductances for oxygen from air to mitochondria (G(O(2),max)) matched metabolic rates as insects became larger, likely enabling the similar P(crit) values observed in large and small beetles. These data suggest that current atmospheric oxygen levels do not limit body size of insects because of limitations on gas exchange. However, increasing relative investment in the tracheal system in larger insects may produce trade-offs or meet spatial limits that constrain insect size.

Insects in general, and Drosophila in particular, are much more capable of surviving anoxia than vertebrates, and the mechanisms involved are of considerable biomedical and ecological interest. Temperature is likely to strongly affect both the rates of damage occurring in anoxia and the recovery processes in normoxia, but as yet there is no information on the effect of this crucial variable on recovery rates from anoxia in any animal. We studied the effects of temperature, and thus indirectly of metabolic flux rates, on survival and recovery times of individual male Drosophila melanogaster following anoxia and O(2) reperfusion. Individual flies were reared at 25°C and exposed to an anoxic period of 7.5, 25, 42.5 or 60 min at 20, 25 or 30°C. Before, during and after anoxic exposure the flies' metabolic rates (MRs), rates of water loss and activity indices were recorded. Temperature strongly affected the MR of the flies, with a Q(10) of 2.21. Temperature did not affect the slope of the relationship between time to recovery and duration of anoxic exposure, suggesting that thermal effects on damage and repair rates were similar. However, the intercept of that relationship was significantly lower (i.e. recovery was most rapid) at 25°C, which was the rearing temperature. When temperatures during exposure to anoxia and during recovery were switched, recovery times matched those predicted from a model in which the accumulation and clearance of metabolic end-products share a similar dependence on temperature.

Drosophila melanogaster is a model genetic organism with an exceptional hypoxia tolerance relative to mammals. Forward genetic, microarray, and P-element manipulations and selection experiments have revealed multiple mechanisms of severe hypoxia tolerance, including RNA editing, downregulation of metabolism, and prevention of protein unfolding. Drosophila live in microbe-rich, semiliquid food in which hypoxia likely indicates deteriorating environments. Hypoxia reduces growth and size by multiple mechanisms, influencing larval feeding rates, protein synthesis, imaginal cell size, and control of molting. In moderate hypoxia, these effects appear to occur without ATP limitation and are instead mediated by signaling systems, including hypoxia-inducible factor and atypical guanyl cyclase sensing of oxygen, with downstream actions on behavior, anabolism, and the cell cycle. In hypoxia, flies develop smaller sizes, but size does not evolve, whereas in hyperoxia, flies evolve larger sizes without exhibiting developmental size plasticity, suggesting differential evolutionary responses to natural versus novel directions of oxygen change.

Oxygen provides the substrate for most ATP production, but also serves as a source of reactive oxygen species (ROS), which can induce cumulative macromolecular oxidative damage and cause aging. Pure oxygen atmospheres (100 kPa) are known to strongly reduce invertebrate lifespan and induce aging-related physiological changes. However, the nature of the relationship between atmospheric oxygen, oxidative stress, and lifespan across a range of oxygen levels is poorly known. Developmental responses are likely to play a strong role, as prior research has shown strong effects of rearing oxygen level on growth, size and respiratory system morphology. In this study, we examined (1) the effect of oxygen on adult longevity and (2) the effect of the oxygen concentration experienced by larvae on adult lifespan by rearing Drosophila melanogaster in three oxygen atmospheres throughout larval development (10, 21 and 40 kPa), then measuring the lifespan of adults in five oxygen tensions (2, 10, 21, 40, 100 kPa). We also assessed the rate of protein carbonyl production for flies kept at 2, 10, 21, 40 and 100 kPa as adults (all larvae reared in normoxia). The rearing of juveniles in varying oxygen treatments affected lifespan in a complex manner, and the effect of different oxygen tensions on adult lifespan was non-linear, with reduced longevity and heightened oxidative stress at extreme high and low atmospheric oxygen levels. Moderate hypoxia (10 kPa) extended maximum, but not mean lifespan.

Insects are small relative to vertebrates, possibly owing to limitations or costs associated with their blind-ended tracheal respiratory system. The giant insects of the late Palaeozoic occurred when atmospheric PO(2) (aPO(2)) was hyperoxic, supporting a role for oxygen in the evolution of insect body size. The paucity of the insect fossil record and the complex interactions between atmospheric oxygen level, organisms and their communities makes it impossible to definitively accept or reject the historical oxygen-size link, and multiple alternative hypotheses exist. However, a variety of recent empirical findings support a link between oxygen and insect size, including: (i) most insects develop smaller body sizes in hypoxia, and some develop and evolve larger sizes in hyperoxia; (ii) insects developmentally and evolutionarily reduce their proportional investment in the tracheal system when living in higher aPO(2), suggesting that there are significant costs associated with tracheal system structure and function; and (iii) larger insects invest more of their body in the tracheal system, potentially leading to greater effects of aPO(2) on larger insects. Together, these provide a wealth of plausible mechanisms by which tracheal oxygen delivery may be centrally involved in setting the relatively small size of insects and for hyperoxia-enabled Palaeozoic gigantism.

Greater oxygen availability has been hypothesized to be important in allowing the evolution of larger invertebrates during the Earth's history, and across aquatic environments. We tested for evolutionary and developmental responses of adult body size of Drosophila melanogaster to hypoxia and hyperoxia. Individually reared flies were smaller in hypoxia, but hyperoxia had no effect. In each of three oxygen treatments (hypoxia, normoxia or hyperoxia) we reared three replicate lines of flies for seven generations, followed by four generations in normoxia. In hypoxia, responses were due primarily to developmental plasticity, as average body size fell in one generation and returned to control values after one to two generations of normoxia. In hyperoxia, flies evolved larger body sizes. Maximal fly mass was reached during the first generation of return from hyperoxia to normoxia. Our results suggest that higher oxygen levels could cause invertebrate species to evolve larger average sizes, rather than simply permitting evolution of giant species.

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.

Why is maximal insect body size relatively small compared to that of vertebrates? Possibly insect body size is limited by the capacity of the tracheal respiratory system to delivery oxygen down longer and longer tracheae to the tissues. If so, one possible outcome would be that larger insect species would have a smaller safety margin for oxygen delivery (higher critical P(O2), P(c)). We tested this idea by exposing inactive adult grasshoppers of a range of species and body sizes (0.07-6.4 g) to progressively lower oxygen atmospheres and measuring their ventilation frequency and their ability to maintain metabolic rate (indexed by CO(2) emission rate). We analyzed effects of body size on these parameters by simple linear regressions, as well as methods to control for phylogenetic relatedness among species. We found interspecific variation in P(c), but P(c) did not significantly correlate with body mass (average P(c) across all species = 4 kPa). Maximal tracheal system conductance scaled approximately with mass(0.7), and estimated ventilation in hypoxia (ventilatory frequency x tidal volume) scaled directly with mass, suggesting that convection is the major mechanism of gas exchange in all these species. These comparative data strengthen the growing body of evidence that body size does not affect the safety margin for oxygen delivery in insects.

Resting insects are extremely tolerant of hypoxia. However, oxygen requirements increase dramatically during flight. Does the critical atmospheric P (O)(2) (P(c)) increase strongly during flight, or does increased tracheal conductance allow even flying insects to possess large safety margins for oxygen delivery? We tested the effect of P(O)(2) on resting and flying CO(2) emission, as well as on flight behavior and vertical force production in flying locusts, Schistocerca americana. The P(c) for CO(2) emission of resting animals was less than 1 kPa, similar to prior studies. The P(c) for flight bout duration was between 10 and 21 kPa, the P(c) for vertical force production was between 3 and 5 kPa, and the P(c) for CO(2) emission was between 10 and 21 kPa. Our study suggests that the P(c) for steady-state oxygen consumption is between 10 and 21 kPa (much higher than for resting animals), and that tracheal oxygen stores allowed brief flights in 5 and 10 kPa P(O)(2) atmospheres to occur. Thus, P(c) values strongly increased during flight, consistent with the hypothesis that the excess oxygen delivery capacity observed in resting insects is substantially reduced during flight.

Structural changes in the tracheal system during development have the potential to allow insects to compensate for varying oxygen availability. Despite possible compensation, oxygen level during development may also affect insect body size. We investigated how atmospheric oxygen level affects the dimensions of the main dorsal tracheae (DT) and masses of larval Drosophila melanogaster (Meigen) reared for up to six generations in 10%, 21% or 40% O2 at 25 degrees C. Wandering-stage third-instar larvae were weighed every other generation, and the dimensions of the DT were measured. Hypoxia produced significantly lighter larvae after one generation of exposure, while hyperoxia did not affect larval mass. Atmospheric oxygen content did not significantly change the diameters of the anterior portions of the main tracheae; however, the posterior diameters were strongly affected. During the first generation of exposure, tracheal diameters were inversely proportional to rearing oxygen levels, demonstrating that developmental plasticity in DT diameters can partially (8-15%) compensate for variation in atmospheric oxygen level. After multiple generations in differing atmospheres and two further generations in 21% O2, larvae had tracheal diameters inversely related to their historical oxygen exposure, suggesting that atmospheric oxygen can produce heritable changes in insect tracheal morphology.

We hypothesized that oxygen delivery becomes more difficult for insects and tracheate arthropods as they progress throughout an intermolt period. During this time, body mass can more than double, yet the major tracheae and spiracles cannot be increased in size until molting. Also, tissue growth could compress air sacs used for convective gas exchange. To test these possibilities, we investigated the effect of within-instar growth on respiratory parameters, including CO(2) emission rate, ventilation frequency, tidal volume and critical oxygen partial pressure (P(O2)) for first-, third- and fifth-instar juveniles and adults of the American locust Schistocerca americana. We found that late-stage grasshoppers tended to have 40% higher total CO(2) emission rates but 15% lower mass-specific CO(2) emission rates and 35% higher ventilation frequencies than early-stage animals. Maximal tracheal system conductance decreased by 20-33% at the end of an instar, possibly due to compression of air sacs. In addition, animals nearing the end of an instar had higher critical P(O2) values for abdominal pumping, and late-stage adults had 50% lower tidal volumes, suggesting that increases in tissue mass throughout an instar may hinder the ability of animals to breathe deeply. Late-stage adults had lower critical P(O2) values for CO(2) emission, although this pattern was not found in any juvenile instars, indicating that late-stage juveniles compensate for decreased conductance by increasing ventilation frequency or the use of diffusive gas exchange. Our data suggest that late-stage arthropods are more vulnerable to hypoxia and may have reduced aerobic capacities and lower tissue P(O2)s than early-stage arthropods.

We tested the hypothesis that oxygen delivery from the atmosphere to the tissues becomes more difficult as grasshoppers increase in body size throughout development due to increases in tracheal length. If this is true, then older, larger grasshoppers should have smaller safety margins [higher critical oxygen partial pressures (P(O2)s)] for oxygen delivery than younger, smaller grasshoppers. We exposed grasshoppers of first, third and fifth instars and adults to decreasing levels of atmospheric O2 and measured their ventilatory responses. Contrary to our prediction, we found that larger grasshoppers had critical P(O2)s eight times lower than juveniles due in part to their threefold lower mass-specific metabolic rates and their ability to quadruple convective gas exchange. Adults more than doubled abdominal pumping frequency and increased tidal volume by 25% as P(O2) decreased fourfold, whereas the youngest juveniles showed no such responses. This study indicates that juveniles may be more susceptible to hypoxia in natural situations, such as exposure to high altitude or restricted burrows. Also, larger size is not necessarily correlated with a smaller safety margin for oxygen delivery in insects.

Ventilatory efferent discharges in the isolated central nervous system (CNS) of the lubber grasshopper Taeniopoda eques were recorded and the change of ventilatory rates were measured in correlation to changing concentrations of oxygen and carbon dioxide. These in vitro responses were compared to the ventilatory rates of intact animals exposed to various gas tensions. We found highly significant effects of oxygen on the ventilatory response of both the in vitro CNS and the intact animals, with ventilatory rates inverse to the concentration of oxygen. Application of changing carbon dioxide concentrations showed significant effects on ventilatory frequencies in isolated CNS of the American desert locust (Schistocerca americana). Our results provide the first direct evidence for the existence of oxygen and carbon dioxide receptors located in the insect CNS.

Many physiological systems appear to have safety margins, with excess capacity relative to normal functional needs, but the significance of such excess capacity remains controversial. In this study, we investigate the effects of parasitic tracheal mites (Acarapis woodi) on the safety margin for oxygen delivery and flight performance of honeybees. Tracheal mites did not affect the flight metabolic rate of honeybees in normoxic (21% oxygen) or hyperoxic (40% oxygen) air, but did reduce their metabolic rate relative to uninfected bees when flying in hypoxic air (5 or 10% oxygen), demonstrating that mites reduced the safety margin for tracheal oxygen delivery. The negative effects of mites on flight metabolic rate in hypoxic atmospheres were graded with the number of mites per trachea. For example, in 10% oxygen atmospheres, flight metabolic rate was reduced by 20% by moderate mite infection and by 40% by severe mite infection. Thus, the safety margin for oxygen delivery in honeybees allows them to retain normal flight metabolic rate and behavior despite tracheal mite infection under most conditions. However, the reduction in tracheal gas-exchange capacity may constrain activities requiring the highest metabolic rates, such as flying in cool weather. In support of this hypothesis, bees that were unable to return to the hive during late-winter flights showed significantly higher levels of mite infection than bees that returned safely.

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.