Discontinuous Gas Exchange Research Articles

COCKROACHES THAT EXCHANGE RESPIRATORY GASES DISCONTINUOUSLY SURVIVE FOOD AND WATER RESTRICTION

Metabolic rate and respiratory gas exchange patterns vary significantly both between and within species, even after a number of biotic and abiotic factors are taken into account. This suggests that such variation is of evolutionary importance, but the life history implications of this variation remain relatively poorly characterized. In the present study, we examine the effect of metabolic variation on starvation and desiccation resistance in the speckled cockroach Nauphoeta cinerea. We also compare the starvation and desiccation resistance of individuals that exchange respiratory gases continuously with those that breathe discontinuously. We show that metabolic rate has no effect on survival during food and water restriction, but cockroaches exhibiting discontinuous gas exchange cycles (DGCs) live longer than those that do not and those provisioned with water lived longer than those that were not. This finding represents the first demonstration that DGCs confer a fitness benefit, and supports the oldest hypothesis for the evolution of DGCs (which suggests that DGCs arose or are maintained to reduce respiratory water loss) as we also reveal reduced water loss (both respiratory and total) in cockroaches exhibiting discontinuous gas exchange.

Reactive oxygen species production and discontinuous gas exchange in insects

While biochemical mechanisms are typically used by animals to reduce oxidative damage, insects are suspected to employ a higher organizational level, discontinuous gas exchange mechanism to do so. Using a combination of real-time, flow-through respirometry and live-cell fluorescence microscopy, we show that spiracular control associated with the discontinuous gas exchange cycle (DGC) in Samia cynthia pupae is related to reactive oxygen species (ROS). Hyperoxia fails to increase mean ROS production, although minima are elevated above normoxic levels. Furthermore, a negative relationship between mean and mean ROS production indicates that higher ROS production is generally associated with lower . Our results, therefore, suggest a possible signalling role for ROS in DGC, rather than supporting the idea that DGC acts to reduce oxidative damage by regulating ROS production.

Regulation of gas exchange and haemolymph pH in the cockroach Nauphoeta cinerea

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

Oxygen convective uptakes in gas exchange cycles in early diapause pupae of Pieris brassicae

Oxygen convective uptakes in gas exchange cycles were directly recorded in early diapause pupae of Pieris brassicae L. (Lepidoptera; Pieridae) by means of O2 coulometric respirometry. This method was combined with flow-through CO2 respirometry, the two systems being switchable one to the other. During recording with both systems, measurements were also taken with infrared actography. The pupae displayed short discontinuous gas exchange cycles lasting 40-70 min. No true C phase was found by flow-through measurements; instead, flutter opening of the spiracles with discrete convective O2 uptakes began shortly after the O phase whereas CO2 release was suppressed by the inward directed passive suction ventilation. The F phase was characterized by a series of small CO2 bursts (flutter events). Between these bursts, novel sub-phase `miniflutter' was observed, which consisted of six to 10 miniature inspirations without any CO2 emission. During the flow-through measurements, oxygen convective uptakes were indirectly recorded by the infrared actograph as sudden extensions (lengthening) of the abdominal segments at each spiracular microopening.

Discontinuous gas exchange: new perspectives on evolutionary origins and ecological implications

Summary1. When at rest, many insects and some other arthropods exchange gasses discontinuously. Whether or not discontinuous gas exchange (DGE) is adaptive has long been controversial. Several competing evolutionary hypotheses exist, none of which enjoy unequivocal support. This Gordian knot seems now to have been cut.2. Discontinuous gas exchange appears to arise as a consequence of energy‐saving, metabolic downregulation. Moreover, many ecological circumstances, such as quiescence during daily inactivity and during seasonal resource shortages, are likely to have favoured such downregulation.3. Once the brain relinquishes control of gas exchange to the segmental ganglia, an interaction between peripheral CO2 sensing and central O2 sensing sets in, leading to the discontinuous pattern.4. The evolution of DGE may thus be readily explained, with the primary evolutionary hypothesis incorporating a non‐adaptive mechanistic component and an adaptive energy savings one.5. Paradoxically, though, DGE results in a comparatively high water loss for a given metabolic rate in insects compared with other organisms. Thus, the pattern itself may not have evolved to limit water loss under xeric conditions. Rather, variation in the components of the DGE cycle, which has formed the foundation for much debate concerning the ecological and evolutionary advantages of this gas exchange pattern, may have evolved to do so, accounting for associations between these components and environmental conditions.6. The relatively high respiratory water loss of insects may spell ecological trouble given increasing metabolic rates of ectotherms associated with rising global temperatures and global change–type drought in many regions.

The effect of ambient humidity and metabolic rate on the gas-exchange pattern of the semi-aquatic insectAquarius remigis

We have examined the effects of temperature on metabolic rate and respiratory pattern in the water strider Aquarius remigis. As temperature was increased from 10 to 30°C, the metabolic rate of the insects increased and the respiratory pattern transitioned from discontinuous, to cyclic, to continuous. The discontinuous gas-exchange cycle (DGC) was observed even in insects standing on water when the respirometry chamber was being perfused with humid (>95% relative humidity) air. Comparisons of insects at 20°C in humid and dry air showed no statistically significant differences in metabolic rate or respiratory pattern (P>0.05). The proportion of time that the spiracles were closed was greater at 10°C than at 20°C (P<0.01), and greater at 20°C than at 30°C (P<0.05). These results are compatible with the hypothesis that the respiratory patterns of insects are determined by the relationship between oxygen supply and oxygen demand. There was no evidence in this insect that humidity had any effect on the respiratory pattern. The results are discussed in the context of the ongoing discussion in the literature of the origin, maintenance and adaptive significance of the DGC in insects.

Gas exchange patterns of Pterostichus niger (Carabidae) in dry and moist air

Gas exchange patterns of adult male Pterostichus niger Schaller after hydration (i.e. given access to food and water) are compared in dry air [5–7% relative humidity (RH)] and moist air (90–97% RH) by means of flow-through CO2 respirometry combined with infrared probe actography. Of thirty beetles examined, slightly more than 50% showed continuous gas exchange and are not considered further. Of the remaining beetles, the majority (approximately 71%) display a pattern of cyclic gas exchange in both dry and moist air (i.e. CO2 gas is released in bursts, with a low level of CO2 release during the interburst periods). A minority of the beetles (four out of 30) are found to exhibit discontinuous gas exchange in both dry and moist air; this is characterized by three clearly separated states of the spiracles: closed (C), flutter (F) and open (O) phases. The pattern of cyclic gas exchange is associated with weak abdominal pulsations. After switching from moist to dry air, a small modulation of the discontinuous gas exchange cycles (maximum mean CO2 production rate) occurs, providing no clear support for the hygric theory of discontinuous gas exchange in this species (i.e. that it serves to restrict respiratory water loss).

Discontinuous Gas Exchange in Insects: Is It All in Their Heads?

Some insects display an intermittent pattern of gas exchange while at rest, often going hours between breaths. These discontinuous gas exchange cycles (DGCs) are known to have evolved independently within five insect orders, but their possible adaptive benefit and evolutionary origin remain an enigma. Current research is primarily concerned with testing three adaptive hypotheses: that DGCs originally evolved or are currently maintained to (1) limit respiratory water loss, (2) enhance gas exchange in subterranean environments, or (3) limit oxidative damage. These adaptive explanations fail to unite a range of apparently contradictory observations regarding the insects that display DGCs and the conditions under which they occur. Here we argue that DGCs are explained by circadian, developmental, or artificially induced reductions in brain activity. We conclude that this pattern results from the thoracic and abdominal ganglia regulating ventilation in the absence of control from higher neural centers, and it is indicative of a sleeplike state.

A new look at discontinuous respiration in pupae of Hyalophora cecropia (Lepidoptera: Saturniidae): Haemocoelic pressure, extracardiac pulsations and O&lt;sub&gt;2 &lt;/sub&gt;consumption

Discontinuous respiration in diapausing pupae of Cecropia silkworms was monitored by means of several electronic methods, including recording changes in haemocoelic pressure, monitoring respiratory movements by strain-gauge sensors and nanorespirographic recording of O2 consumption and CO2 output. It appears that, in contrast to previous concepts of stereotypic discontinuous respiration cycles (DGC) driven by accumulation of gaseous CO2 in the body, the new results indicate that CO2 remains dissolved in liquid carbonate buffers during interburst periods. In other words, there is no accumulation of gaseous CO2 within the air filled tracheal space between the bursts. The bursts of CO2 are caused by homeostatically regulated enzymatic hydration by carbonic anhydrase of metabolically produced carbonic acid. The chemically produced gaseous CO2 was exhaled mainly by a bulk outflow through selectively opened or pulsating spiracles. The output of CO2 was enhanced by actively regulated, unidirectional ventilation. The deep depressions in haemocoelic pressure, caused by permanent closure of all spiracular valves for long periods, appeared to be a specific feature of diapausing saturniid pupae. Physiologically, it has circulatory, not respiratory functions. The original definition of spiracular fluttering resulted from a misinterpretation of previously unknown extracardiac pulsations in haemocoelic pressure. The coordinated pulsation of the spiracular valves with extracardiac pulsations produce a very efficient, unidirectional ventilation of the whole tracheal system. According to the new results, the discontinuous respiration cycles of diapausing Cecropia pupae can be briefly described as follows: (1) Spiracular valves are kept permanently closed during the periods of deep depressions, they remain closed for some 99% of the time with occasional snap opening (passive inspirations) during prolonged interburst periods and more than 50% closed during the bursts; (2) During the long interburst periods, CO2 is retained in liquid carbonate buffers, while the relatively high (after the burst) or low (toward the next burst) rate of O2 consumption creates an internal vacuum, which is homeostatically compensated for by the snap-opening of one or just a few spiracular valves (passive suction inspirations); (3) The CO2 gas, produced enzymatically by carbonic anhydrase, enters the air filled tracheal system and leaves the body by diffusion, a bulk outflow, or actively regulated unidirectional ventilation (fluttering spiracles). The selective advantage of this actively regulated respiratory system for water retention in pupae is discussed.

Phenotypic plasticity of gas exchange pattern and water loss in Scarabaeus spretus (Coleoptera: Scarabaeidae): deconstructing the basis for metabolic rate variation

Investigation of gas exchange patterns and modulation of metabolism provide insight into metabolic control systems and evolution in diverse terrestrial environments. Variation in metabolic rate in response to environmental conditions has been explained largely in the context of two contrasting hypotheses, namely metabolic depression in response to stressful or resource-(e.g. water) limited conditions, or elevation of metabolism at low temperatures to sustain life in extreme conditions. To deconstruct the basis for metabolic rate changes in response to temperature variation, here we undertake a full factorial study investigating the longer- and short-term effects of temperature exposure on gas exchange patterns. We examined responses of traits of gas exchange [standard metabolic rate (SMR); discontinuous gas exchange (DGE) cycle frequency; cuticular, respiratory and total water loss rate (WLR)] to elucidate the magnitude and form of plastic responses in the dung beetle, Scarabaeus spretus. Results showed that short- and longer-term temperature variation generally have significant effects on SMR and WLR. Overall, acclimation to increased temperature led to a decline in SMR (from 0.071+/-0.004 ml CO(2) h(-1) in 15 degrees C-acclimated beetles to 0.039+/-0.004 ml CO(2) h(-1) in 25 degrees C-acclimated beetles measured at 20 degrees C) modulated by reduced DGE frequency (15 degrees C acclimation: 0.554+/-0.027 mHz, 20 degrees C acclimation: 0.257+/-0.030 mHz, 25 degrees C acclimation: 0.208+/-0.027 mHz recorded at 20 degrees C), reduced cuticular WLRs (from 1.058+/-0.537 mg h(-1) in 15 degrees C-acclimated beetles to 0.900+/-0.400 mg h(-1) in 25 degrees C-acclimated beetles measured at 20 degrees C) and reduced total WLR (from 4.2+/-0.5 mg h(-1) in 15 degrees C-acclimated beetles to 3.1+/-0.5 mg h(-1) in 25 degrees C-acclimated beetles measured at 25 degrees C). Respiratory WLR was reduced from 2.25+/-0.40 mg h(-1) in 15 degrees C-acclimated beetles to 1.60+/-0.40 mg h(-1) in 25 degrees C-acclimated beetles measured at 25 degrees C, suggesting conservation of water during DGE bursts. Overall, this suggests water conservation is a priority for S. spretus exposed to longer-term temperature variation, rather than elevation of SMR in response to low temperature acclimation, as might be expected from a beetle living in a relatively warm, low rainfall summer region. These results are significant for understanding the evolution of gas exchange patterns and trade-offs between metabolic rate and water balance in insects and other terrestrial arthropods.

Intra-individual variation allows an explicit test of the hygric hypothesis for discontinuous gas exchange in insects

The hygric hypothesis postulates that insect discontinuous gas exchange cycles (DGCs) are an adaptation that reduces respiratory water loss (RWL), but evidence is lacking for reduction of water loss by insects expressing DGCs under normal ecological conditions. Larvae of Erynnis propertius (Lepidoptera: Hesperiidae) naturally switch between DGCs and continuous gas exchange (CGE), allowing flow-through respirometry comparisons of water loss between the two modes. Water loss was lower during DGCs than CGE, both between individuals using different patterns and within individuals using both patterns. The hygric cost of gas exchange (water loss associated with carbon dioxide release) and the contribution of respiratory to total water loss were lower during DGCs. Metabolic rate did not differ between DGCs and CGE. Thus, DGCs reduce RWL in E. propertius, which is consistent with the suggestion that water loss reduction could account for the evolutionary origin and/or maintenance of DGCs in insects.

Tradeoffs between metabolic rate and spiracular conductance in discontinuous gas exchange of Samia cynthia (Lepidoptera, Saturniidae)

The insect tracheal system is a unique respiratory system, designed for maximum oxygen delivery at high metabolic demands, e.g. during activity and at high ambient temperatures. Therefore, large safety margins are required for tracheal and spiracular conductance. Spiracles are the entry to the tracheal system and play an important role in controlling discontinuous gas exchange (DGC) between tracheal system and atmosphere in moth pupae. We investigated the effect of modulated metabolic rate (by changing ambient temperature) and modulated spiracular conductance (by blocking all except one spiracles) on gas exchange patterns in Samia pupae. Both, spiracle blocking and metabolic rates, affected respiratory behavior in Samia cynthia pupae. While animals showed discontinuous gas exchange cycles at lower temperatures with unblocked spiracles, the respiratory patterns were cyclic at higher temperatures, with partly blocked spiracles or a combination of these two factors. The threshold for the transition from a discontinuous (DGC) to a cyclic gas exchange (cycGE) was significantly higher in animals with unblocked spiracles (18.7nmolg−1min−1 vs. 7.9nmolg−1min−1). These findings indicate an important influence of spiracle conductance on the DGC, which may occur mostly in insects showing high spiracular conductances and low metabolic rates.

Cockroaches breathe discontinuously to reduce respiratory water loss

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

COCKROACHES HOLD BREATH TO CONSERVE WATER

![Figure][1] When some insects stop rushing around and settle down, they switch to a discontinuous respiration mode where they cycle between closing, `fluttering' (rapidly opening and closing) and opening the spiracles that they breathe through. Why inactive insects switch to this form of

Water loss and gas exchange by eggs of Manduca sexta: Trading off costs and benefits

Like all terrestrial organisms, insect eggs face a tradeoff between exchanging metabolic gases (O2 and CO2) and conserving water. Here I summarize the physiology underlying this tradeoff and the ecological contexts in which it may be important. The ideas are illustrated primarily by work from my laboratory on eggs of the sphingid moth Manduca sexta. In particular, I discuss: (1) dynamic changes in metabolic demand and water loss during development; and (2) how the eggshell layers and embryonic tracheal system control the traffic of gases between the embryo and its environment. Subsequently, I identify three areas with interesting but unresolved issues: (1) what eggs actually experience in their microclimates, focusing particularly on the leaf microclimates relevant to eggs of M. sexta; (2) how egg experience influences whether or not hatchling larvae succeed in establishing feeding sites on host plants; and (3) whether Hetz and Bradley's [Hetz, S.K., Bradley, T.J., 2005. Insects breathe discontinuously to avoid oxygen toxicity. Nature 433, 516−519] oxygen toxicity hypothesis for discontinuous gas-exchange cycles applies to insect eggs.

Gas exchange patterns of bumble bee foragers before and after exposing to lowered temperature

The gas exchange patterns are known to vary between insect species, individuals and even intra-individually. Using volumetric-manometric and flow-through respirometry combined with IR-actography we studied how periods of low temperature affect the respiratory patterns of bumble bee Bombus terrestris foragers. We have shown, in this study, that there is a change in the respiratory patterns of individual B. terrestris foragers after exposing to low temperatures. The bumble bees seemed to become more inactive. The different respiratory patterns appeared in succession and the transition from one pattern to another was associated with the change from an active to a resting state. Typical patterns after exposition to low temperature were discontinuous gas exchange cycles (DGCs).

Spiracle activity in moth pupae—The role of oxygen and carbon dioxide revisited

After decades of intensive research, the actual mechanism behind discontinuous gas exchange in insects has not been fully understood. One open question concerns the actual way (closed, flutter, and open) of how spiracles respond to tracheal gas concentrations. As the results of a classic paper [Burkett, B.N., Schneiderman, H.A., 1974. Roles of oxygen and carbon dioxide in the control of spiracular function in cecropia pupae. Biological Bulletin 147, 274–293] allow ambiguous interpretation, we thus reexamined the behavior of the spiracles in response to fixed, controlled endotracheal gas concentrations.The tracheal system of diapausing pupae of Attacus atlas (Saturniidae, Lepidoptera) was flushed with gas mixtures varying in PO2 and PCO2 while the behavior of the spiracles was monitored using changes in the pressure signal. This novel pressure based technique proved to be superior to classic visual observation of single spiracles. A two-dimensional map of the spiracle behavior in response to endotracheal PO2 and PCO2 was established. Typically, it contained two distinct regions only, corresponding to “closed” and “open” spiracles. A separate “flutter” region was missing. Because fluttering is commonly observed in moth pupae, we suggest that the intermittent spiracle opening during a flutter phase is an effect of non-steady-state conditions within the tracheal system. For low PCO2 the minimum PO2 resulting in open spiracles was linearly dependent upon PCO2. Above a threshold of 1–1.5kPa CO2 the spiracles were open irrespective of PO2. We propose a hypothetical spiracular control model, which is simple and explains the time course of endotracheal partial pressures during all phases of discontinuous gas exchange.

Rhinoceros beetles modulate spiracular opening to regulate haemolymph pH

Insects are the most diverse and numerous terrestrial air-breathing animals, yet a comprehensive understanding of their respiratory physiology remains elusive. This is clearly seen in the debate surrounding the origins and function of discontinuous gas exchange cycles (DGCs) displayed by many insects at rest. While many hypotheses have been proposed to explain the function of DGCs, no general underlying mechanism driving patterns of insect ventilation has been universally accepted. In the present study, we simultaneously measured in vivo pH fluctuations and patterns of CO2 emission in rhinoceros beetles Xylotrupes ulysses. Beetles were restrained within a flow-through respirometry chamber with a fibre optic pH probe implanted in the haemolymph through the dorsal surface of their thorax. DGCs caused haemolymph pH to fluctuate by 0.1 units, with internal pH declining while the spiracles were closed and rapidly climbing when the spiracles opened. Exposure to hypoxia (5% oxygen in nitrogen) caused the spiracles to open widely and elicited a large burst of CO2, causing haemolymph pH to increase from 7.0 to 7.3. If the spiracles were held open by continued exposure to hypoxia, the pH remained high. Once normoxia was restored, the beetles held their spiracles shut, and only resumed normal opening behaviour once internal pH declined to pre-hypoxia levels. The protracted breath hold following hypoxic exposure indicates that the beetles preferentially retain CO2 in order to restore their haemolymph pH. We therefore propose that insects modulate gas exchange to regulate internal pH, and that DGCs represent an extreme example of this strategy.

Respiratory accommodations in Rhodnius prolixus in response to increased metabolic rates

It has been proposed that discontinuous respiration may have evolved in insects to decrease oxidative damage to tissues when at rest. However if metabolic rate (MR) increases, or the capacity of the respiratory system to deliver O2 to active tissues is altered, respiratory pattern in insects may change to meet aerobic demands. Specifically, we expect that as MR increases insects will transition from discontinuous (DGC) to cyclic and finally to continuous respiration. Preliminary studies have shown that a tenfold increase in MR (via temperature) in Rhodnius changed respiratory patterns from DGC to continuous respiration. However, Rhodnius respiratory responses due to feeding‐induced MR increase are not known. In Rhodnius prolixus metabolic rates (and body volume) may increase up to ten‐fold after a blood meal. They therefore provide an excellent model for studying the effects of specific dynamic action (SDA) on respiratory pattern. We expect that as metabolic rates increase, insects will transition to the different patterns as previously observed. However, due to the large effect of the meal on body volume and MR, differences in the overall MR required to induce the different patterns may be altered.Funding: APS Porter Physiology Development Award

PHYSIOLOGICAL PRINCIPLES MEET INFORMATION EXPLOSION

![Figure][1] J. L. Nation's 2nd edition of `Insect Physiology and Biochemistry', which builds on the 1st edition published in 2002, provides a timely, updated snapshot of modern insect physiology. The book strives to be comprehensive, and the array of topics covered is impressive – from