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.     

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.     

Discontinuous gas exchange, water loss, and metabolism in Protaetia cretica (Cetoniinae, Scarabaeidae)

Insects are at high risk of desiccation because of their small size, high surface-area-to-volume ratio, and air-filled tracheal system that ramifies throughout their bodies to transport O(2) and CO(2) to and from respiring cells. Although the tracheal system offers a high-conductance pathway for the movement of respiratory gases, it has the unintended consequence of allowing respiratory transpiration to the atmosphere. When resting, many species exchange respiratory gases discontinuously, and an early hypothesis for the origin of these discontinuous gas exchange cycles (DGCs) is that they serve to reduce respiratory water loss. In this study, we test this "hygric" hypothesis by comparing rates of CO(2) exchange and water loss among flower beetles Protaetia cretica (Cetoniinae, Scarabaeidae) breathing either continuously or discontinuously. We show that, consistent with the expectations of the hygric hypothesis, rates of total water loss are higher during continuous gas exchange than during discontinuous gas exchange and that the ratio of respiratory water loss to CO(2) exchange is lower during discontinuous gas exchange. This conclusion is in agreement with other studies of beetles and cockroaches that also support the hygric hypothesis. However, this result does not exclude other adaptive hypotheses supported by work on ants and moth pupae. This ambiguity may arise because there are multiple independent evolutionary origins of DGCs and no single adaptive function underlying their genesis. Alternatively, the observed reduction in water loss during DGCs may be a side effect of a nonadaptive gas exchange pattern that is elicited during periods of inactivity.     

Oxygen-induced plasticity in tracheal morphology and discontinuous gas exchange cycles in cockroaches Nauphoeta cinerea

The function and mechanism underlying discontinuous gas exchange in terrestrial arthropods continues to be debated. Three adaptive hypotheses have been proposed to explain the evolutionary origin or maintenance of discontinuous gas exchange cycles (DGCs), which may have evolved to reduce respiratory water loss, facilitate gas exchange in high CO₂ and low O₂ micro-environments, or to ameliorate potential damage as a result of oversupply of O₂. None of these hypotheses have unequivocal support, and several non-adaptive hypotheses have also been proposed. In the present study, we reared cockroaches Nauphoeta cinerea in selected levels of O₂ throughout development, and examined how this affected growth rate, tracheal morphology and patterns of gas exchange. O₂ level in the rearing environment caused significant changes in tracheal morphology and the exhibition of DGCs, but the direction of these effects was inconsistent with all three adaptive hypotheses: water loss was not associated with DGC length, cockroaches grew fastest in hyperoxia, and DGCs exhibited by cockroaches reared in normoxia were shorter than those exhibited by cockroaches reared in hypoxia or hyperoxia.     

Monitoring of gas exchange cycles and ventilatory movements in the pine weevil Hylobius abietis: respiratory failures evoked by a botanical insecticide

The large pine weevil, Hylobius abietis (L.) (Coleoptera: Curculionidae), is the most important insect pest of young coniferous plants. The implementation of new control methods requires not only a profound knowledge of the ecology and behaviour of the pest, but particularly of its physiology. Standard metabolic rate (SMR) and discontinuous gas exchange cycles (DGCs) were recorded in parallel with abdominal ventilation movements in adults of H. abietis using a differential electrolytic respirometer‐actograph. Quiescent weevils displayed DGCs of the constriction, flutter, and ventilation phases of the CFV type, while bursts of carbon dioxide were always accompanied by abdominal pumping movements, i.e., muscular ventilation in the closed subelytral cavity (SEC). In some beetles the C phase was absent and thus (C)FV cycles were recorded. In addition, at the beginning and often at the end of a burst, the SEC was rhythmically opened and closed by movements of the last abdominal segments. Continuous pumping movements and an absence of DGCs were signs of stress imposed by handling or by a new environment, even if the beetle was not moving. All individuals showed clear DGCs after recovering from handling and apparatus stress lasting 2–3 h. The results show that in the monitoring of DGCs, it is essential to determine whether they are of the constriction, flutter, and open phases (CFO), or the CFV subtype of the constriction, flutter, and burst (CFB) cycles. Use of our simple closed‐system respirometer enables non‐invasive simultaneous recording of SMR, oxygen uptake, DGCs, and active ventilation in H. abietis and other beetles. The topical application of adult H. abietis with sublethal doses of a botanical insecticide, NeemAzal T/S, caused essential respiratory failures: cyclic gas exchange was lost and irregular pumping movements appeared. In the treated beetles normal DGCs did not resume.     

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).     

Did the first insects live in water or in air?

We evaluate the arguments and evidence for a terrestrial vs an aquatic origin for the Insecta. The evidence falls into three categories: (1) evidence that does not support one view more than the other; (2) speculative evidence, which should carry little weight; and (3) evidence that does support one view more than the other. Category 1 includes evidence from locomotory and osmoregulatory systems; plausible functions have been proposed for 'protowings' in both aquatic and terrestrial environments, while locomotory and osmoregulatory mechanisms of insects shed little light on their origins. Fossils, phylogenetic speculation, gill structure and life histories fall into category 2, in which, although speculative, the evidence favours a terrestrial origin. The earliest fossil hexapods were apparently terrestrial and unequivocally aquatic hexapods do not appear until 60–70 million years later, while sister-group relationships point to a terrestrial life style from at least the Hexapoda-Myriapoda stem group. The great variation in gill structure, even within orders, suggests convergence, and the more or less completely aquatic life histories are better interpreted as steps towards independence from land, rather than signs of an aquatic origin. Category 3 includes evidence from the tracheal system. In order to have evolved in water, a tracheal system must have first invaginated, then connected with the body wall for gas exchange with the water, and thirdly connected with the internal organs. It is difficult to envisage functions for the first two stages; on the other hand, the system could have readily evolved on land by invagination of respiratory surfaces, and then have been modified to effect gas exchange in water via gills.     

Effects of Neem EC on gas exchange, tracheal ventilation, and water loss in diapausing pupae of Pieris brassicae

Effects of Neem EC (The Indian Neem Tree Company^TM, 1% azadirachtin) on gas exchange cycles, tracheal ventilation, and water loss in diapausing pupae of the large white butterfly, Pieris brassicae L. (Lepidoptera: Pieridae), were studied using a constant volume respirometer combined with an infrared probe actograph. The non‐treated pupae displayed discontinuous gas exchange cycles (DGC) with a trend coinciding with the bursts of carbon dioxide (CO₂) release, active tracheal ventilation, and the heartbeat periods. Two independent forms of tracheal ventilation were observed, relatively vigorous abdominal shaking movements and weak abdominal pulsations. The ability to respond to mechanical excitation with abdominal movements was entirely lost on the 2nd day after treatments with Neem EC, and also a reduced tendency to use a DGC was observed. During 2–3 days after treatments, the DGCs and gas exchange microcycles were entirely lost, as was active ventilation. Before treatments, body mass loss, that is, water loss, was 0.6–0.9 mg g^?1 day^?1. After the treatments, water loss increased to 3–5 mg g^?1 day^?1. The pupae remained alive for 10–15 days after the treatments and died after having lost about 50% of their initial body mass. The absence of heartbeats measured during at least 4–5 h was the main criterion for ascertaining death of pupae. The results suggested that respiratory failures, that is, the loss of cyclic gas exchange, evoked by Neem EC were the primary cause of lethal desiccation. Thus, the hypothesis that the cyclic gas exchange is an adaptation for restricting water losses in insects was supported.     

Allosteric control of cyclic di-GMP signaling

Cyclic di-guanosine monophosphate is a bacterial second messenger that has been implicated in biofilm formation, antibiotic resistance, and persistence of pathogenic bacteria in their animal host. Although the enzymes responsible for the regulation of cellular levels of c-di-GMP, diguanylate cyclases (DGC) and phosphodiesterases, have been identified recently, little information is available on the molecular mechanisms involved in controlling the activity of these key enzymes or on the specific interactions of c-di-GMP with effector proteins. By using a combination of genetic, biochemical, and modeling techniques we demonstrate that an allosteric binding site for c-di-GMP (I-site) is responsible for non-competitive product inhibition of DGCs. The I-site was mapped in both multi- and single domain DGC proteins and is fully contained within the GGDEF domain itself. In vivo selection experiments and kinetic analysis of the evolved I-site mutants led to the definition of an RXXD motif as the core c-di-GMP binding site. Based on these results and based on the observation that the I-site is conserved in a majority of known and potential DGC proteins, we propose that product inhibition of DGCs is of fundamental importance for c-di-GMP signaling and cellular homeostasis. The definition of the I-site binding pocket provides an entry point into unraveling the molecular mechanisms of ligand-protein interactions involved in c-di-GMP signaling and makes DGCs a valuable target for drug design to develop new strategies against biofilm-related diseases.     

Experimental evolution of evolutionary potential in fluctuating environments

Variation is the raw material for evolution. Evolutionary potential is determined by the amount of genetic variation, but evolution can also alter the visibility of genetic variation to natural selection. Fluctuating environments are suggested to maintain genetic variation but they can also affect environmental variance, and thus, the visibility of genetic variation to natural selection. However, experimental studies testing these ideas are relatively scarce. In order to determine differences in evolutionary potential we quantified variance attributable to population, genotype and environment for populations of the bacterium Serratia marcescens. These populations had been experimentally evolved in constant and two fluctuating environments. We found that strains that evolved in fluctuating environments exhibited larger environmental variation suggesting that adaptation to fluctuations has decreased the visibility of genetic variation to selection.     

Selection on a eumelanic ornament is stronger in the tropics than in temperate zones in the worldwide‐distributed barn owl

Spatial variation in the pattern of natural selection can promote local adaptation and genetic differentiation between populations. Because heritable melanin‐based ornaments can signal resistance to environmentally mediated elevation in glucocorticoids, to oxidative stress and parasites, populations may vary in the mean degree of melanic coloration if selection on these phenotypic aspects varies geographically. Within a population of Swiss barn owls (Tyto alba), the size of eumelanic spots is positively associated with survival, immunity and resistance to stress, but it is yet unknown whether Tyto species that face stressful environments evolved towards a darker eumelanic plumage. Because selection regimes vary along environmental gradients, we examined whether melanin‐based traits vary clinally and are expressed to a larger extent in the tropics where parasites are more abundant than in temperate zones. To this end, we considered 39 barn owl species distributed worldwide. Barn owl species living in the tropics displayed larger eumelanic spots than those found in temperate zones. This was, however, verified in the northern hemisphere only. Parasites being particularly abundant in the tropics, they may promote the evolution of darker eumelanic ornaments.     

Experimental evolution of infectious behaviour in a facultative ectoparasite

Parasitic lifestyles have evolved many times in animals, but how such life‐history strategies evolved from free‐living ancestors remains a great puzzle. Transitional symbiotic strategies, such as facultative parasitism, are hypothesized evolutionary stepping stones towards obligate parasitism. However, to consider this hypothesis, heritable genetic variation in infectious behaviour of transitional symbiotic strategies must exist. In this study, we experimentally evolved infectivity and estimated the additive genetic variation in a facultative parasite. We performed artificial selection experiments in which we selected for either increased or decreased propensity to infect in a facultatively parasitic mite (Macrocheles muscaedomesticae). Here, infectiousness was expressed in terms of mite attachment to a host (Drosophila hydei) and modelled as a threshold trait. Mites responded positively to selection for increased infectivity; realized heritability of infectious behaviour was significantly different from zero and estimated to be 16.6% (±4.4% SE). Further, infection prevalence was monitored for 20 generations post‐selection. Selected lines continued to display relatively high levels of infection, demonstrating a degree of genetic stability in infectiousness. Our study is the first to provide an estimate of heritability and additive genetic variation for infectious behaviour in a facultative parasite, which suggests natural selection can act upon facultative strategies with important implications for the evolution of parasitism.     

A test of the oxidative damage hypothesis for discontinuous gas exchange in the locust Locusta migratoria

The discontinuous gas exchange cycle (DGC) is a breathing pattern displayed by many insects, characterized by periodic breath-holding and intermittently low tracheal O(2) levels. It has been hypothesized that the adaptive value of DGCs is to reduce oxidative damage, with low tracheal O(2) partial pressures (PO(2) ≈ 2-5 kPa) occurring to reduce the production of oxygen free radicals. If this is so, insects displaying DGCs should continue to actively defend a low tracheal PO(2) even when breathing higher than atmospheric levels of oxygen (hyperoxia). This behaviour has been observed in moth pupae exposed to ambient PO(2) up to 50 kPa. To test this observation in adult insects, we implanted fibre-optic oxygen optodes within the tracheal systems of adult migratory locusts Locusta migratoria exposed to normoxia, hypoxia and hyperoxia. In normoxic and hypoxic atmospheres, the minimum tracheal PO(2) that occurred during DGCs varied between 3.4 and 1.2 kPa. In hyperoxia up to 40.5 kPa, the minimum tracheal PO(2) achieved during a DGC exceeded 30 kPa, increasing with ambient levels. These results are consistent with a respiratory control mechanism that functions to satisfy O(2) requirements by maintaining PO(2) above a critical level, not defend against high levels of O(2).     

Multiple Sites of Gas Exchange

SYNOPSIS. For many years the general body surface has been recognizedas the primitive site of respiratory gas exchange in the animalkingdom. Even in simple animals such as the sea anemones, however,some specialization has occurred. In epifaunal species the tentacularcrown appears to be the major site of gas exchange while inone infaunal species dermal papillae are more important. Inboth, the columnar body wall remains an additional site of O₂uptake. In the polychaete annelids an evolutionary trend can be seenfrom the predominance of the general body surface over simple,metameric gills to the predominance of highly specialized andbranched anterior gills over the general body surface. n theoligochaetes reversion to the general body surface was followedin at least one species to specialization of the tail regionto form a "lung." Multiple sites clearly exist in the molluscs, but their relativeimportance is poorly known. Several sites exist in the echinoderms,the best known being the approximately equal allocation of O₂uptake between modified tube feet and respiratory trees in theholothurians. Multiple sites of gas exchange persist even inthe crustaceans, in temporal as well as spatial dimensions.Water breathers revert to cutaneous O₂ uptake following a molt,and air breathers have evolved an array of gas exchangers adaptedto their new medium. This array is so diverse that it does notprovide a single selection pressure for hemocyanin O₂ bindingproperties which, when compared broadly, remain indistinguishablefrom those of water breathers found in similar thermal regimes.     

Ventilation of the branchial chambers in the amphibious West African freshwater crab,Sudanonautes (Convexonautes) aubryi monodi (Balss, 1929) (Brachyura,Potamonautidae)

The anatomy of the respiratory system of the savanna-zone African freshwater crab, Sudanonautes (Convexonautes) aubryi monodi [Balss, 1929], has been examined and has been found to be adapted for both aerial and aquatic gas exchange. The activities of the scaphognathites and the directions of flow of the ventilatory stream have been recorded in stressed, active and resting specimens during their exposure to a wide range of conditions from deep water to dry land.Ventilation of the branchial chambers during aquatic gas exchange in Sudanonautes kept in deep water is shown to consist of a rapid, predominantly forward water flow similar to that of fully-aquatic species. Ventilation of the branchial chambers during aerial gas exchange in Sudanonautes on land is shown to consist of a relatively slow forward air flow. This flow is continuous in post-operative crabs, pulsatile in active crabs and completely immobile in resting crabs.A second method of ventilation of the branchial chambers during aerial gas exchange is shown to consist of a pulsatile reversed air flow. This occurs (1) when Sudanonautes is kept in very shallow water and active or stressed; (2) when it has recently moved on to land; and (3) when it is completely immersed and exhibiting aerial gas exchange under water. The unusual phenomenon of aerial gas exchange under water is reported here for the first time in any species of crab.Bimodal ventilation of the branchial chambers occurs in stressed or active crabs partly immersed in shallow water. This consists of an alternation between forward water flow and reversed air flow.The morphology of the branchial chambers in Sudanonautes, and observational data on the patterns of ventilation of the branchial chambers, are discussed in relation to those described for other air-breathing decapod crustaceans.     

Discontinuous gas exchange in dung beetles: patterns and ecological implications

This study correlates a distinctive pattern of external gas exchange, referred to as the discontinuous gas exchange cycle (DGC), observed in the laboratory, with habitat associations of five species of telecoprid dung beetles. The beetles were chosen from a variety of habitats that would be expected to present different amounts of water stress. All five species exhibited DGC. Sisyphus fasciculatus has been recorded only in woodland areas, and does not have strict spiracular control during its DGC. Anachalcos convexus and Scarabaeus rusticus are associated with open mesic habitats. Both species exhibit a distinct DGC, previously found in some other insect species, but intermediate within this study group. Sc. flavicornis and Circellium bacchus are typically found in arid regions, and have the most unusual form of DGC, with spiracular fluttering during the burst phase. These results support the hypothesis that spiracular fluttering reduces respiratory water loss. From this study we conclude that the DGC is an ancestral adaptation, most probably as a result of anoxic environments in underground burrows, but that spiracular control is enhanced to reduce respiratory water loss in beetle species that live in arid habitats. Received 4 August 1999 / Accepted: 7 October 1999     

Selection on the Drosophila seminal fluid protein Acp62F

Sperm competition and sexual conflict are thought to underlie the rapid evolution of reproductive proteins in many taxa. While comparative data are generally consistent with these hypotheses, few manipulative tests have been conducted and those that have provided contradictory results in some cases. Here, we use both comparative and experimental techniques to investigate the evolution of the Drosophila melanogaster seminal fluid protein Acp62F, a protease inhibitor for which extensive functional tests have yielded ambiguous results. Using between‐species sequence comparisons, we show that Acp62F has been subject to recurrent positive selection. In addition, we experimentally evolved populations polymorphic for an Acp62F null allele over eight generations, manipulating the opportunities for natural and sexual selection. We found that the Acp62F null allele increased in frequency in the presence of natural selection, with no effect of sexual selection.     

Respiration rhythms and heartbeats of diapausing Colorado potato beetles, Leptinotarsa decemlineata, at low temperatures

Discontinuous gas exchange cycles (DGCs), active muscular ventilation, microcycles of repetitive openings, and heartbeats of diapausing adult Colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), were studied at low temperatures (0, 5, and 10 °C) using an electrolytic respirometer combined with an infrared actograph. The DGC of the adult constriction-flutter-open type was the main respiration mode in fully quiescent beetles at temperatures from 5 to 10 °C. The CO₂ bursts were actively ventilated at temperatures above 5 °C. During the flutter period, a series of microcycles appeared, but no muscular contractions associated with the microcycles were detected. We identified this respiration mode as discontinuous suction ventilation.
The hydration condition of the beetles did not influence the frequency of the gas exchange cycles, but dehydrated beetles showed significantly longer flutter periods and shorter ventilation periods than hydrated beetles. The heartbeat frequencies were influenced by both temperature and hydration status.
We conclude from the results that DGCs are used at rest in adult L. decemlineata under various environmental conditions and also at low temperatures. Our results showed that DGCs are the main respiration mode of resting adult Colorado potato beetle irrespective of its hydration state and temperature. Our method resolves O₂ uptake and subsequent CO₂ release in flutter and ventilation periods and shows that diffusion is replaced by convection to reduce water loss in adult beetles.     

The adaptive significance of drought escape in Avena barbata, an annual grass

Drought strongly influences plant productivity, suggesting that water limitation has shaped the evolution of many plant physiological traits. One functional strategy that plants employ to cope with decreasing water availability is drought escape. For drought-escaping species, high metabolic activity (gas exchange) and rapid growth are hypothesized to confer a fitness advantage, because this enables a plant to complete its life cycle before the most intense period of drought. By growing an annual grass species (Avena barbata) under well-watered or water-limited conditions in a greenhouse, we directly tested whether high photosynthesis, increased stomatal opening, and early flowering are adaptive under drought. We measured phenotypic selection on instantaneous gas exchange and flowering time as well as the underlying biochemical traits that regulate photosynthesis. We found strong selection for earlier flowering in the dry environment, but no evidence that increased photosynthesis was adaptive under drought. Photosynthetic rate (A) and stomatal conductance (gs) were both adaptively neutral in the dry environment. Increased photosynthetic capacity (Amax) was maladaptive in the dry environment, perhaps because of the respiratory cost associated with maintaining excess enzyme and substrate capacity. There was no correlational selection on the combination of physiology and flowering time in the dry environment, suggesting that accelerated development and high gas exchange may not need to be tightly linked to promote drought escape. In contrast, there was selection for both high photosynthetic function (Amax and A) and early flowering in the well-watered environment. These combinations of traits may have been favored because they maximize both energy and time available for reproduction. Our results suggest that the benefit of increased photosynthesis for plant fitness may be strongest in the absence of drought stress.     

It takes two: Heritable male effects on reproductive timing but not clutch size in a wild bird population*

Within-population variation in the traits underpinning reproductive output has long been of central interest to biologists. Since they are strongly linked to lifetime reproductive success, these traits are expected to be subject to strong selection and, if heritable, to evolve. Despite the formation of durable pair bonds in many animal taxa, reproductive traits are often regarded as female-specific, and estimates of quantitative genetic variation seldom consider a potential role for heritable male effects. Yet reliable estimates of such social genetic effects are important since they influence the amount of heritable variation available to selection. Based on a 52-year study of a nestbox-breeding great tit (Parus major) population, we apply “extended” bivariate animal models in which the heritable effects of both sexes are modeled to assess the extent to which males contribute to heritable variation in seasonal reproductive timing (egg laying date) and clutch size, while accommodating the covariance between the two traits. Our analyses show that reproductive timing is a jointly expressed trait in this species, with (positively covarying) heritable variation for laydate being expressed in both members of a breeding pair, such that the total heritable variance is 50% larger than estimated by traditional models. This result was robust to explicit consideration of a potential male-biased environmental confound arising through sexually dimorphic dispersal. In contrast to laydate, males’ contribution to heritable variation in clutch size was limited. Our study thus highlights the contrasting extent of social determination for two major components of annual reproductive success, and emphasizes the need to consider the social context of what are often considered individual-level traits.