ABSTRACT When exposed to anoxia, insects rapidly go into a hypometabolic coma from which they can recover when exposed to normoxia again. However, prolonged anoxic bouts eventually lead to death in most insects, although some species are surprisingly tolerant. Anoxia challenges ATP, ion, pH and water homeostasis, but it is not clear how fast and to what degree each of these parameters is disrupted during anoxia, nor how quickly they recover. Further, it has not been investigated which disruptions are the primary source of the tissue damage that ultimately causes death. Here, we show, in the migratory locust ( Locusta migratoria ), that prolonged anoxic exposures are associated with increased recovery time, decreased survival, rapidly disrupted ATP and pH homeostasis and a slower disruption of ion ([K + ] and [Na + ]) and water balance. Locusts could not fully recover after 4 h of anoxia at 30°C, and at this point hemolymph [K + ] was elevated 5-fold and [Na + ] was decreased 2-fold, muscle [ATP] was decreased to ≤3% of normoxic values, hemolymph pH had dropped 0.8 units from 7.3 to 6.5, and hemolymph water content was halved. These physiological changes are associated with marked tissue damage in vivo and we show that the isolated and combined effects of hyperkalemia, acidosis and anoxia can all cause muscle tissue damage in vitro to equally large degrees. When locusts were returned to normoxia after a moderate (2 h) exposure of anoxia, ATP recovered rapidly (15 min) and this was quickly followed by recovery of ion balance (30 min), while pH recovery took 2–24 h. Recovery of [K + ] and [Na + ] coincided with the animals exiting the comatose state, but recovery to an upright position took ∼90 min and was not related to any of the physiological parameters examined.

ABSTRACT Oxygen limitation plays a key role in many pathologies; yet, we still lack a fundamental understanding of the mechanisms responsible for variation in anoxia tolerance. Most vertebrate studies suggest that anoxia tolerance involves the ability to maintain cellular ATP despite the loss of aerobic metabolism. However, insects such as adult Drosophila melanogaster are able to survive long periods of anoxia (LT 50 : ∼8 h) in a hypo-energetic state characterized by low [ATP]. In this study, we tested for possible mechanisms that allow D. melanogaster adults to survive long periods of anoxia. Adults are paralyzed within 30 s, and after 2 h of anoxia, ATP was 3% of normal, extracellular potassium concentration ([K + ] o ) increased threefold, pH dropped 1 unit, yet survival was 100%. With 0.5–6 h of anoxia, adults maintained low but constant ATP levels while [K + ] o and pH o continued to change. When returned to normoxia, adults restored [K + ] o and activity. With longer durations of anoxia, ATP levels decreased and [K + ] o rose further, and both correlated tightly with decreased survival. This response contrasts with the anoxia-sensitive larval stage (LT 50 : ∼1 h). During anoxia, larvae attempted escape for up to 30 min and after 2 h of anoxia, ATP was <1% of resting, [K + ] o increased by 50%, hemolymph pH fell by 1 unit, and survival was zero. The superior anoxia tolerance of adult D. melanogaster appears to be due to the capacity to maintain a paralytic hypometabolic state with low but non-zero ATP levels, and to be able to tolerate extreme extracellular ionic variability.

ABSTRACT Environmental changes during development have long-term effects on adult phenotypes in diverse organisms. Some of the effects play important roles in helping organisms adapt to different environments, such as insect polymorphism. Others, especially those resulting from an adverse developmental environment, have a negative effect on adult health and fitness. However, recent studies have shown that those phenotypes influenced by early environmental adversity have adaptive value under certain (anticipatory) conditions that are similar to the developmental environment, though evidence is mostly from morphological and behavioral observations and it is still rare at physiological and molecular levels. In the companion study, we applied a short-term starvation treatment to fifth instar honey bee larvae and measured changes in adult morphology, starvation resistance, hormonal and metabolic physiology and gene expression. Our results suggest that honey bees can adaptively respond to the predicted nutritional stress. In the present study, we further hypothesized that developmental starvation specifically improves the metabolic response of adult bees to starvation instead of globally affecting metabolism under well-fed conditions. Here, we produced adult honey bees that had experienced a short-term larval starvation, then we starved them for 12 h and monitored metabolic rate, blood sugar concentrations and metabolic reserves. We found that the bees that experienced larval starvation were able to shift to other fuels faster and better maintain stable blood sugar levels during starvation. However, developmental nutritional stress did not change metabolic rates or blood sugar levels in adult bees under normal conditions. Overall, our study provides further evidence that early larval starvation specifically improves the metabolic responses to adult starvation in honey bees.

ABSTRACT Comparisons of the carbon, nitrogen and phosphorus (P) content of plants and insect herbivores suggests that P limitation and herbivore foraging to balance P intake could be common. However, the lack of synthetic diets for testing the effects of lower ranges of dietary P has been a major impediment to experimental assessment of the ecological importance of, and physiological responses to, P limitation for terrestrial herbivores. We manipulated dietary P content (%P) over its observed range in terrestrial foliage using artificial diets containing near-optimal content of other nutrients for the grasshopper Schistocerca americana . Over much of the ecologically relevant range, when consuming single diets over a lifetime, higher P stimulated growth rates and increased survival, with an optimal dietary %P of 0.25–0.50% when measured throughout development. Excessive dietary P (1%) reduced growth and survival. However, with only short-term (3 day) confinement to single diets, dietary P had no effect on food consumption or growth rates. During these short exposures, fifth (but not third) instar hoppers increased the proportion of P excreted relative to P assimilated as dietary P increased. Target experiments demonstrated that, when given a choice, grasshoppers select among foods to attain a P intake target of 0.6%. These data suggest that P limitation could be common for terrestrial insect herbivores and that they can exhibit ingestive and post-ingestive mechanisms to attain sufficient but not excessive P.

ABSTRACT 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.

SUMMARY 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.

SUMMARY 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.

SUMMARY 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.

SUMMARY 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.

SUMMARY 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.

SUMMARY Rhythmic tracheal compression is a prominent feature of internal dynamics in multiple orders of insects. During compression parts of the tracheal system collapse, effecting a large change in volume, but the ultimate physiological significance of this phenomenon in gas exchange has not been determined. Possible functions of this mechanism include to convectively transport air within or out of the body, to increase the local pressure within the tracheae,or some combination thereof. To determine whether tracheal compressions are associated with excurrent gas exchange in the ground beetle Pterostichus stygicus , we used flow-through respirometry and synchrotron x-ray phase-contrast imaging to simultaneously record CO 2 emission and observe morphological changes in the major tracheae. Each observed tracheal compression (which occurred at a mean frequency and duration of 15.6±4.2 min –1 and 2.5±0.8 s, respectively) was associated with a local peak in CO 2 emission, with the start of each compression occurring simultaneously with the start of the rise in CO 2 emission. No such pulses were observed during inter-compression periods. Most pulses occurred on top of an existing level of CO 2 release, indicating that at least one spiracle was open when compression began. This evidence demonstrates that tracheal compressions convectively pushed air out of the body with each stroke. The volume of CO 2 emitted per pulse was 14±4 nl, representing approximately 20% of the average CO 2 emission volume during x-ray irradiation, and 13% prior to it. CO 2 pulses with similar volume, duration and frequency were observed both prior to and after x-ray beam exposure, indicating that rhythmic tracheal compression was not a response to x-ray irradiation per se . This study suggests that intra-tracheal and trans-spiracular convection of air driven by active tracheal compression may be a major component of ventilation for many insects.

SUMMARY We use a factorial experimental design to test whether rearing at colder temperatures shifts the lower thermal envelope for flight of Drosophila melanogaster Meigen to colder temperatures. D. melanogaster that developed in colder temperatures (15°C) had a significant flight advantage in cold air compared to flies that developed in warmer temperatures(28°C). At 14°C, cold-reared flies failed to perform a take-off flight∼47% of the time whereas warm-reared flies failed ∼94% of the time. At 18°C, cold- and warm-reared flies performed equally well. We also compared several traits in cold- and warm-developing flies to determine if cold-developing flies had better flight performance at cold temperatures due to changes in body mass, wing length, wing loading, relative flight muscle mass or wing-beat frequency. The improved ability to fly at low temperatures was associated with a dramatic increase in wing area and an increase in wing length (after controlling for wing area). Flies that developed at 15°C had∼25% more wing area than similarly sized flies that developed at 28°C. Cold-reared flies had slower wing-beat frequencies than similarly sized flies from warmer developmental environments, whereas other traits did not vary with developmental temperature. These results demonstrate that developmental plasticity in wing dimensions contributes to the improved flight performance of D. melanogaster at cold temperatures, and ultimately, may help D. melanogaster live in a wide range of thermal environments.

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 O 2, 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×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.

SUMMARY The volume of a tracheal system influences breath-holding capacity and provides an index of an insect's investment in its respiratory system. Here,we describe a new, generally applicable method to measure tracheal volume that enables repeatable determinations on live animals. Animals are isolated in a closed chamber of a known volume and equilibrated with a helium:oxygen gas mixture. The chamber is then rapidly flushed with a nitrogen:oxygen gas mixture to eliminate the helium surrounding the animal, and sealed. After a period of time sufficient to allow equilibration of helium between tracheal system and chamber air, a gas sample is taken from the chamber, and tracheal volumes are calculated from the helium content of the sample, using a gas chromatograph. We show that relative investment in the tracheal system increases with age/size in the grasshopper; tracheal volume scales with mass to the power 1.3. This increased proportional investment in the tracheal system provides a mechanistic basis for the enhanced respiratory capacity of older grasshoppers. Tracheal volumes decrease strongly as grasshoppers grow within an instar stage, explaining reduced safety margins for oxygen delivery. Finally, tracheal volumes are smaller in gravid females than males, probably due to compression of air sacs by eggs.

SUMMARY Developing vertebrates increase both their locomotory power output and endurance due to ontogenetic improvements in anaerobic and aerobic metabolic capacities. Do similar patterns hold for insect locomotion, or do longer tracheal lengths create problems for oxygen delivery in older animals? We forced developing American locust grasshoppers ( Schistocerca americana ) to jump repeatedly and examined the effect of development on power output, endurance, lactate concentration, oxygen consumption and the oxygen sensitivity of jump performance. As previously shown, power outputs,relative leg lengths and leg cuticular content increased with age. A key finding of this study is that both lactate concentration and aerobic metabolic rate of the jumping muscle increase with age, explaining how the increased leg cuticular stiffness can result in increased power output. After two minutes of jumping, grasshoppers rely completely on aerobic ATP production. The rise in mass-specific, active aerobic metabolic rates with age indicates that problems with longer tracheae can be overcome; however, the reduced endurance, higher lactate concentrations and increased oxygen sensitivity of locomotory performance in older animals indicate that larger/older grasshoppers have smaller safety margins for oxygen delivery during hopping.

SUMMARY The respiratory system of growing caterpillars is challenged in two distinct ways as they develop from hatchlings to fifth instars preparing for pupation. First, across instars, body sizes and tracheal lengths increase substantially. Second, within each instar, animal mass can more than double while major tracheal respiratory system structures, such as spiracles and large tracheae, are fixed in size until molting. To test whether these growth processes result in a decrease in O 2 delivery capacity relative to tissue oxygen needs, we exposed feeding Manduca sexta larvae of various ages to decreasing levels of atmospheric O 2 and measured their metabolic rate and ability to feed. We found that near the beginning of all instars, M. sexta were able to maintain gas exchange and feed down to approximately 5 kPa O 2 , indicating that these insects are able to match tracheal O 2 delivery to increased metabolic rates across instars. However, gas exchange and feeding of caterpillars nearing the molt were limited at much higher O 2 levels (up to 15 kPa O 2 ), suggesting that caterpillars have limited capacities to increase tracheal O 2 delivery as O 2 consumption rates increase within instars. It seems possible that the safety margin for O 2 delivery may disappear completely in the last hours before ecdysis, providing an ultimate if not proximate explanation for the necessity of molting.

SUMMARY 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% O 2 at 25°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% O 2 , larvae had tracheal diameters inversely related to their historical oxygen exposure,suggesting that atmospheric oxygen can produce heritable changes in insect tracheal morphology.

SUMMARY We assessed the energetic and aerodynamic limits of hovering flight in the carpenter bee Xylocopa varipuncta . Using normoxic, variable-density mixtures of O 2 , N 2 and He, we were able to elicit maximal hovering performance and aerodynamic failure in the majority of bees sampled. Bees were not isometric regarding thorax mass and wing area, both of which were disproportionately lower in heavier individuals. The minimal gas density necessary for hovering ( MGD ) increased with body mass and decreased with relative thoracic muscle mass. Only the four bees in our sample with the highest body mass-specific thorax masses were able to hover in pure heliox. Wingbeat frequency and stroke amplitude during maximal hovering were significantly greater than in normodense hovering, increased significantly with body mass during normodense hovering but were mass independent during maximal hovering. Reserve capacity for wingbeat frequency and stroke amplitude decreased significantly with increasing body mass, although reserve capacity in stroke amplitude (10–30%) exceeded that of wingbeat frequency(0–8%). Stroke plane angle during normodense hovering was significantly greater than during maximal hovering, whereas body angle was significantly greater during maximal hovering than during normodense hovering. Power production during normodense hovering was significantly less than during maximal hovering. Metabolic rates were significantly greater during maximal hovering than during normodense hovering and were inversely related to body mass during maximal and normodense hovering. Metabolic reserve capacity averaged 34% and was independent of body mass. Muscle efficiencies were slightly higher during normodense hovering. The allometry of power production,power reserve capacity and muscle efficiency were dependent on the assumed coefficient of drag ( C D ), with significant allometries most often at lower values of C D . Larger bees operate near the envelope of maximal performance even in normodense hovering due to smaller body mass-specific flight muscles and limited reserve capacities for kinematics and power production.

SUMMARY 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 O 2 s)] for oxygen delivery than younger, smaller grasshoppers. We exposed grasshoppers of first, third and fifth instars and adults to decreasing levels of atmospheric O 2 and measured their ventilatory responses. Contrary to our prediction, we found that larger grasshoppers had critical P O 2 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 O 2 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.