Tracheal hyperallometry and spatial constraints in a large beetle.

Insects exchange respiratory gases with their environment through their gas-filled tracheal system, a branched tracheal tree extending from segmental openings and terminating at fine tissue penetrating tracheoles. It was shown that the tracheal volume increases hyperallometrically with insect body size (Mb), both interspecifically and across developmental stages. In this study, we used the sixfold Mb variation in adult Batocera rufomaculata(Cerambicidae; Coleoptera) examining the allometry of adult tracheal volume (Vtr). We further explored the effect of sex and sexual maturity on tracheal gas conductance, testing the hypotheses that (i) larger body size and (ii) egg volume in gravid females would result in lower safety margins for tracheal oxygen transport due to structural restriction. We report a hyperallometric tracheal growth in both sexes of adult B. rufomaculata(mean mass exponent of 1.42 ± 0.09), similar in magnitude to previously reported values. Tracheal gas conductance was independent of Mb and reproductive state, but was significantly higher in females compared with males. We suggest that females may have pre-adapted a higher tracheal conductance required for the higher flight power output while gravid. Lack of compliant air sacs and rigid trachea may explain how gravid females retain their Vtr. However, we show that Vtr outgrows thoracic dimensions with increased B. rufomaculatasize. Hyperallometric growth of the giant cerambycid thoracic trachea could explain the previously reported hypometric scaling of flight muscles in B. rufomaculata, and the compromised long-distance flight performance of larger compared with smaller conspecifics.

Intraspecific scaling and early life history determine the cost of free-flight in a large beetle (Batocera rufomaculata).

The scaling of the energetic cost of locomotion with body mass is well documented at the interspecific level. However, methodological restrictions limit our understanding of the scaling of flight metabolic rate (MR) in free-flying insects. This is particularly true at the intraspecific level, where variation in body mass and flight energetics may have direct consequences for the fitness of an individual. We applied a 13C stable isotope method to investigate the scaling of MR with body mass during free-flight in the beetle Batocera rufomaculata. This species exhibits large intraspecific variation in adult body mass as a consequence of the environmental conditions during larval growth. We show that the flight-MR scales with body mass to the power of 0.57, with smaller conspecifics possessing up to 2.3 fold higher mass-specific flight MR than larger ones. Whereas the scaling exponent of free-flight MR was found to be like that determined for tethered-flight, the energy expenditure during free-flight was more than 2.7 fold higher than for tethered-flight. The metabolic cost of flight should therefore be studied under free-flight conditions, a requirement now enabled by the 13C technique described herein for insect flight.

Microbiome-related aspects of locust density-dependent phase transition.

Locust plagues are a notorious, ancient phenomenon. These swarming pests tend to aggregate and perform long migrations, decimating cultivated fields along their path. When population density is low, however, the locusts will express a cryptic, solitary, non-aggregating phenotype that is not considered a pest. Although the transition from the solitary to the gregarious phase has been well studied, associated shifts in the locust's microbiome have yet to be addressed. Here, using 16S rRNA amplicon sequencing, we compared the bacterial composition of solitary desert locusts before and after a phase transition. Our findings revealed that the microbiome is altered during the phase transition, and that a major aspect of this change is the acquisition of Weissella (Firmicutes). Our findings led us to hypothesize that the locust microbiome plays a role in inducing aggregation behaviour, contributing to the formation and maintenance of a swarm. Employing a mathematical model, we demonstrate the potential evolutionary advantage of inducing aggregation under different conditions; specifically, when the aggregation-inducing microbe exhibits a relatively high horizontal transmission rate. This is the first report of a previously unknown and important aspect of locust phase transition, demonstrating that the phase shift includes a shift in the gut and integument bacterial composition.

Conserved ecophysiology despite disparate microclimatic conditions in a gecko.

Microscale differences in the habitats organisms occupy can influence selection regimes and promote intraspecific variation of traits. Temperature-dependent traits can be locally adapted to climatic conditions or be highly conserved and insensitive to directional selection under all but the most extreme regimes, and thus be similar across populations. The opposing slopes of Nahal Oren canyon in the Carmel Mountains, Israel, are strikingly different: the south-facing slope receives intensive solar radiation, is hot and supports mostly annual vegetation, whereas the north-facing slope is ~10°C cooler, more humid, and supports Mediterranean woodland. We examined whether these differences manifest in the thermal physiology of a common gecko species Ptyodactylus guttatus in controlled laboratory conditions. We predicted that geckos from the hotter south-facing slope would prefer higher temperatures, have faster gut passage times, lower metabolic and evaporative water loss rates, and start diel activity earlier compared with north-facing slope conspecifics. Contrary to these predictions, there were no differences between any of the ecophysiological traits in geckos from the opposing slopes. Nevertheless, our data showed that individuals from the north-facing slope were generally more active in earlier hours of the afternoon compared with south-facing individuals. We suggest that P. guttatus individuals disperse between the slopes and either gene-flow or behavioral plasticity deter local adaptation, resulting in similar physiological traits. Perhaps a stronger contrast in climatic conditions and a stronger barrier are needed to result in interpopulation divergence in temperature-dependent traits.

From chemoreception to regulation: filling the gaps in understanding how insects control gas exchange.

Insects coordinate the opening and closing of spiracles with convective ventilatory movements to produce considerable intraspecific and interspecific variation in gas exchange patterns. But fundamental questions remain regarding how these movements are coordinated and modulated by central and peripheral respiratory chemoreceptors, and where these chemoreceptors are located and how they function. Recent findings have revealed regions of the CNS that generate coordinated respiratory motor activity, while peripheral neurons sensitive to respiratory gases have been identified in Drosophila. Importantly, plasticity in structure and function of neural elements of respiratory control indicate the need for caution when generalizing the mechanistic basis for breathing in insects, and an adaptive explanation for breathing pattern variability.

Dated phylogeny and ancestral range estimation of sand scorpions (Buthidae: Buthacus) reveal Early Miocene divergence across land bridges connecting Africa and Asia.

Sand scorpions of the genus Buthacus Birula, 1908 (Buthidae C.L. Koch, 1837) are widespread in the sandy deserts of the Palearctic region, occurring from the Atlantic coast of West Africa across the Sahara, and throughout the Middle East to Central Asia. The limits of Buthacus, its two species groups, and many of its species remain unclear, and in need of revision using modern systematic methods. The study presented here set out to investigate the phylogeny and biogeography of the Buthacus species occurring in the Levant, last studied in 1980. A phylogenetic analysis was performed on 104 terminals, including six species collected from more than thirty localities in Israel and other countries in the region. Three mitochondrial and two nuclear gene loci were sequenced for a total of 2218 aligned base-pairs. Morphological datasets comprising 22 qualitative and 48 quantitative morphological characters were compiled. Molecular and morphological datasets were analyzed separately and simultaneously with Bayesian Inference, Maximum Likelihood, and parsimony. Divergence time and ancestral range estimation analyses were performed, to understand dispersal and diversification. The results support a revised classification of Levantine Buthacus, and invalidate the traditional species groups of Buthacus, instead recovering two geographically-delimited clades, an African clade and an Asian clade, approximately separated by the Jordan Valley (the Jordan Rift Valley or Syro-African Depression), the northernmost part of the Great Rift Valley. The divergence between these clades occurred in the Early Miocene (ca. 19 Ma) in the Levant, coinciding temporally with the existence of two land bridges, which allowed faunal exchange between Africa and Asia.

The maternal foam plug constitutes a reservoir for the desert locust's bacterial symbionts.

A hallmark of the desert locust's ancient and deserved reputation as a devastating agricultural pest is that of the long-distance, multi-generational migration of locust swarms to new habitats. The bacterial symbionts that reside within the locust gut comprise a key aspect of its biology, augmenting its immunity and having also been reported to be involved in the swarming phenomenon through the emission of attractant volatiles. However, it is still unclear whether and how these beneficial symbionts are transmitted vertically from parent to offspring. Using comparative 16S rRNA amplicon sequencing and direct experiments with engineered bacteria, we provide evidence for vertical transmission of locust gut bacteria. The females may perform this activity by way of inoculation of the egg-pod's foam plug, through which the larvae pass upon hatching. Furthermore, analysis of the composition of the foam revealed chitin to be its major component, along with immunity-related proteins such as lysozyme, which could be responsible for the inhibition of some bacteria in the foam while allowing other, more beneficial, strains to proliferate. Our findings reveal a potential vector for the transgenerational transmission of symbionts in locusts, which contributes to the locust swarm's ability to invade and survive in new territories.

Post-feeding thermophily in a scorpion is associated with rapid digestion and recovery of maximal nocturnal activity.

A postprandial increase in metabolic rate is typical in all studied animal groups. The phenomenon, termed specific dynamic action (SDA), is understudied in terrestrial arthropods, and arachnids in particular. To the best of our knowledge, this is the first report of SDA properties in scorpions, which are temperature-dependent as in other poikilotherms. Metabolic rates of scorpions are low compared with similarly-sized arthropods, and as they often feed on relatively large prey the cost of digestion is expected to be notable. This prompted us to study the extent of SDA and its characteristics in scorpions at two different ecologically-relevant temperatures. We also hypothesized that post-feeding behavioral thermoregulation would reflect benefits to the scorpion energy balance. On average, fed adult Hottentotta judaicus (Buthidae) expressed a 3 °C increase in preferred surface temperature, although we did not find evidence for lower costs of digestion at higher temperatures. However, SDA duration was significantly shorter at 30 compared with 25 °C. Fast processing of their meal at 30 °C was correlated with recovery of elevated nocturnal metabolic rates, which are not digestion-related. This suggests that post-feeding choice of higher temperatures accelerates digestion and recovery of other elevated metabolic states such as locomotion and lower sensory threshold, which may enhance foraging success.

Locust Bacterial Symbionts: An Update.

As one of the world's most infamous agricultural pests, locusts have been subjected to many in-depth studies. Their ability at one end of their behavioral spectrum to live as solitary individuals under specific conditions, and at the other end of the spectrum to form swarms of biblical scale, has placed them at the focus of vast research efforts. One important aspect of locust ecology is that of their interactions with the bacteria that reside in and on them. Although this aspect of locust ecology has been little studied relative to the mainstream locust research, these bacteria have been shown both to affect locust immunity and to participate in maintaining swarm integrity through the secretion of attractant volatiles. The interaction between locusts and their bacteria seems, however, to be bi-directional, with the bacteria themselves, as recently shown, being influenced by their host's swarming tendencies. This seems to be a consequence of the bacterial composition in the locust's gut, reproductive organs, and integument undergoing change with the change in their host's behavior. In this review we describe the current state of knowledge of the locust-bacteria interactions (data exists mainly for the desert and the migratory locusts), as well as highlighting some newly-gained understanding; and offer perspectives for future research.

Dynamics of bacterial composition in the locust reproductive tract are affected by the density-dependent phase.

The important role that locust gut bacteria play in their host biology is well accepted. Among other roles, gut bacteria are suggested to be involved in the locust swarming phenomenon. In addition, in many insect orders, the reproductive system is reported to serve as a vector for trans-generation bacterial inoculation. Knowledge of the bacterial composition of the locust reproductive tract is, however, practically absent. Here we characterized the reproductive system bacterial composition of gregarious and solitary females. We investigated its temporal dynamics and how it interacts with the locust phase, by comparative sampling and 16S rRNA amplicon sequencing. We revealed that the bacterial composition of the locust female reproductive tract is mostly constructed of three core genera: Micrococcus, Acinetobacter and Staphylococcus. While solitary females maintained a consistent bacterial composition, in the gregarious phase this consortium demonstrated large temporal shifts, mostly manifested by Brevibacterium blooms. These data are in accord with our previous report on the dynamics of locust hindgut bacterial microbiota, further indicating that locust endosymbionts are affected by their host population density. These newly understood dynamics may have implications beyond their contribution to our knowledge of locust ecology, as aggregation and mass migration are prevalent phenomena across many migrating animals.

Respiratory gas levels interact to control ventilatory motor patterns in isolated locust ganglia.

Large insects actively ventilate their tracheal system even at rest, using abdominal pumping movements, which are controlled by a central pattern generator (CPG) in the thoracic ganglia. We studied the effects of respiratory gases on the ventilatory rhythm by isolating the thoracic ganglia and perfusing its main tracheae with various respiratory gas mixtures. Fictive ventilation activity was recorded from motor nerves controlling spiracular and abdominal ventilatory muscles. Both hypoxia and hypercapnia increased the ventilation rate, with the latter being much more potent. Sub-threshold hypoxic and hypercapnic levels were still able to modulate the rhythm as a result of interactions between the effects of the two respiratory gases. Additionally, changing the oxygen levels in the bathing saline affected ventilation rate, suggesting a modulatory role for haemolymph oxygen. Central sensing of both respiratory gases as well as interactions of their effects on the motor output of the ventilatory CPG reported here indicate convergent evolution of respiratory control among terrestrial animals of distant taxa.

Spatiotemporal dynamics and genome-wide association genome-wide association analysis of desiccation tolerance in Drosophila melanogaster.

Water availability is a major environmental challenge to a variety of terrestrial organisms. In insects, desiccation tolerance varies predictably over spatial and temporal scales and is an important physiological determinant of fitness in natural populations. Here, we examine the dynamics of desiccation tolerance in North American populations of Drosophila melanogaster using: (a) natural populations sampled across latitudes and seasons; (b) experimental evolution in field mesocosms over seasonal time; (c) genome-wide associations to identify SNPs/genes associated with variation for desiccation tolerance; and (d) subsequent analysis of patterns of clinal/seasonal enrichment in existing pooled sequencing data of populations sampled in both North America and Australia. A cline in desiccation tolerance was observed, for which tolerance exhibited a positive association with latitude; tolerance also varied predictably with culture temperature, demonstrating a significant degree of thermal plasticity. Desiccation tolerance evolved rapidly in field mesocosms, although only males showed differences in desiccation tolerance between spring and autumn collections from natural populations. Water loss rates did not vary significantly among latitudinal or seasonal populations; however, changes in metabolic rates during prolonged exposure to dry conditions are consistent with increased tolerance in higher latitude populations. Genome-wide associations in a panel of inbred lines identified twenty-five SNPs in twenty-one loci associated with sex-averaged desiccation tolerance, but there is no robust signal of spatially varying selection on genes associated with desiccation tolerance. Together, our results suggest that desiccation tolerance is a complex and important fitness component that evolves rapidly and predictably in natural populations.

Intricate but tight coupling of spiracular activity and abdominal ventilation during locust discontinuous gas exchange cycles.

Discontinuous gas exchange (DGE) is the best studied among insect gas exchange patterns. DGE cycles comprise three phases, which are defined by their spiracular state: closed, flutter and open. However, spiracle status has rarely been monitored directly; rather, it is often assumed based on CO2 emission traces. In this study, we directly recorded electromyogram (EMG) signals from the closer muscle of the second thoracic spiracle and from abdominal ventilation muscles in a fully intact locust during DGE. Muscular activity was monitored simultaneously with CO2 emission, under normoxia and under various experimental oxic conditions. Our findings indicate that locust DGE does not correspond well with the commonly described three-phase cycle. We describe unique DGE-related ventilation motor patterns, coupled to spiracular activity. During the open phase, when CO2 emission rate is highest, the thoracic spiracles do not remain open; rather, they open and close rapidly. This fast spiracle activity coincides with in-phase abdominal ventilation, while alternating with the abdominal spiracle and thus facilitating a unidirectional air flow along the main trachea. A change in the frequency of rhythmic ventilation during the open phase suggests modulation by intra-tracheal CO2 levels. A second, slow ventilatory movement pattern probably serves to facilitate gas diffusion during spiracle closure. Two flutter-like patterns are described in association with the different types of ventilatory activity. We offer a modified mechanistic model for DGE in actively ventilating insects, incorporating ventilatory behavior and changes in spiracle state.

Real-life effectiveness and safety of salbutamol Steri-Neb™ vs. Ventolin Nebules® for exacerbations in patients with COPD: Historical cohort study.

INTRODUCTION: Ventolin Nebules® (reference product; GlaxoSmithKline) was the first licensed nebulizer solution containing the rapid-onset, short-acting ß2-agonist salbutamol. Salbutamol Steri-Neb™ (comparator; Teva Pharmaceuticals, Inc.) has the same chemical composition as the reference product. This study evaluated whether the effectiveness of the comparator is non-inferior to the reference product alongside concomitant medications during real-life clinical management of COPD exacerbations. Safety in terms of adverse events (AEs) was also examined. METHODS: This matched (1:1) historical cohort study evaluated data from 2 UK primary care databases on patients prescribed the salbutamol comparator or reference. The study included a 1-year baseline period, starting 1 year before the index prescription date, and 1-year outcome period. Cohorts were matched for baseline COPD respiratory medications. The primary outcome was analysis of non-inferiority for the comparator versus reference product for the rate of moderate and severe COPD exacerbations. Non-inferiority was satisfied if the 95% confidence interval (CI) upper limit for mean differences in proportions between treatments was <15%. Secondary outcomes were examined through rate ratios (RR) of severe exacerbations and AEs. RESULTS: After matching, 1191 patients were included in each cohort. Adjusted upper 95% CI for the difference in proportion of patients experiencing moderate or severe exacerbations between comparator and reference groups was 0.032 (3.2%), demonstrating non-inferiority. No significant differences were observed in rates of moderate and severe exacerbations (RR: 1.00; 95% CI: 0.91, 1.10), severe exacerbations (RR: 0.76; 95% CI: 0.49, 1.17), or AEs (RR: 0.98; 95% CI: 0.78, 1.22) after adjusting for baseline confounders. No significant differences across cohorts were observed for rates of any AE or death. CONCLUSION: This matched cohort study of real-life management of COPD patients supports the salbutamol comparator as non-inferior to the reference product, providing an effective treatment alternative for COPD exacerbations. Comparator and reference safety profiles were similar.

The Effect of Density-Dependent Phase on the Locust Gut Bacterial Composition.

The desert locust demonstrates density-dependent phase polyphenism: For extended periods it appears in a non-aggregating, non-migrating phenotype, known as the solitary phase. When circumstances change, solitary individuals may aggregate and transform to the gregarious phenotype, which have a strong propensity for generating large swarms. Previous reports have suggested a role for gut-bacteria derived volatiles in the swarming phenomenon, and suggested that locusts are capable of manipulating their gut microbiome according to their density-dependent phases. Here, we directly tested this hypothesis for the first time. Using locusts of both phases from well-controlled laboratory cultures as well as gregarious field-collected individuals; and high-throughput sequencing. We characterized the hindgut bacterial community composition in the two phases of the desert locust. Our findings demonstrate that laboratory-reared gregarious and solitary locusts maintain a stable core of Enterobacter. However, while different generations of gregarious locust experience shifts in their Enterobacter's relative abundance; the solitary locusts maintain a stable gut microbiome, highly similar to that of the field-collected locusts. Tentative phase differences in wild populations' microbiome may thus be an indirect effect of environmental or other factors that push the swarming individuals to homogenous gut bacteria. We therefore conclude that there are phase-related differences in the population dynamics of the locust hindgut bacterial composition, but there is no intrinsic density-dependent mechanism directly affecting the gut microbiome.

Real-life effectiveness and safety of the inhalation suspension budesonide comparator vs the originator product for the treatment of patients with asthma: a historical cohort study using a US health claims database.

OBJECTIVE: The objective of this study was to determine whether the effectiveness of budesonide comparator is non-inferior to budesonide reference in the prevention of asthma exacerbations. Asthma-related hospitalizations and safety were also examined. METHODS: This study used a matched, historic cohort design. Data were drawn from the Clinformatics™ Data Mart US claims database and included a 1-year baseline, starting 1 year before the index prescription date, and a 1-year outcome period. Patients received budesonide comparator or reference treatment. The primary outcome was the rate of asthma exacerbations. Non-inferiority for budesonide comparator vs budesonide reference was established if the 95% confidence interval (CI) upper limit of mean difference in proportions between treatments was <15%. Secondary outcomes examined rate of asthma-related hospitalizations and adverse events (AEs). RESULTS: The budesonide comparator and reference-matched cohorts each included 3109 patients. The adjusted upper 95% CI for the difference in proportions of patients experiencing asthma exacerbations was 0.035 (3.5%), demonstrating non-inferiority. Cohorts did not significantly differ in the rate of asthma exacerbations (adjusted rate ratio [RR]=1.04, 95% CI: 0.95-1.14) or rate of asthma-related hospitalizations (adjusted RR=1.10, 95% CI: 0.99-1.24) after adjusting for baseline confounders. No asthma exacerbations occurred during the outcome period in 72.9% of budesonide comparator patients and 71.8% of budesonide reference patients. No asthma-related hospitalizations occurred in 77.9% of patients in the budesonide comparator cohort and 79.0% of patients in the budesonide reference cohort. The most frequent AEs were throat irritation (≤0.4% of patients) and hoarseness/dysphonia (0.02% of patients). AEs did not significantly differ between treatment cohorts. CONCLUSION: In this real-life study, non-inferiority of the budesonide comparator vs reference was met for the primary end point of asthma exacerbation rates. Asthma-related hospitalization and AE rates did not differ between the two treatment cohorts. The budesonide comparator is an effective and safe treatment alternative for asthma exacerbations.

An experimental evolution study confirms that discontinuous gas exchange does not contribute to body water conservation in locusts.

The adaptive nature of discontinuous gas exchange (DGE) in insects is contentious. The classic 'hygric hypothesis', which posits that DGE serves to reduce respiratory water loss (RWL), is still the best supported. We thus focused on the hygric hypothesis in this first-ever experimental evolution study of any of the competing adaptive hypotheses. We compared populations of the migratory locust (Locusta migratoria) that underwent 10 consecutive generations of selection for desiccation resistance with control populations. Selected locusts survived 36% longer under desiccation stress but DGE prevalence did not differ between these and control populations (approx. 75%). Evolved changes in DGE properties in the selected locusts included longer cycle and interburst durations. However, in contrast with predictions of the hygric hypothesis, these changes were not associated with reduced RWL rates. Other responses observed in the selected locusts were higher body water content when hydrated and lower total evaporative water loss rates. Hence, our data suggest that DGE cycle properties in selected locusts are a consequence of an evolved increased ability to store water, and thus an improved capacity to buffer accumulated CO2, rather than an adaptive response to desiccation. We conclude that DGE is unlikely to be an evolutionary response to dehydration challenge in locusts.

Discontinuous gas-exchange cycle characteristics are differentially affected by hydration state and energy metabolism in gregarious and solitary desert locusts.

The termination of discontinuous gas exchange cycles (DGCs) in severely dehydrated insects casts doubt on the generality of the hygric hypothesis, which posits that DGCs evolved as a water conservation mechanism. We followed DGC characteristics in the two density-dependent phases of the desert locust Schistocerca gregaria throughout exposure to an experimental treatment of combined dehydration and starvation stress, and subsequent rehydration. We hypothesized that, under stressful conditions, the more stress-resistant gregarious locusts would maintain DGCs longer than solitary locusts. However, we found no phase-specific variations in body water content, water loss rates (total and respiratory) or timing of stress-induced abolishment of DGCs. Likewise, locusts of both phases re-employed DGCs after ingesting comparable volumes of water when rehydrated. Despite comparable water management performances, the effect of exposure to stressful experimental conditions on DGC characteristics varied significantly between gregarious and solitary locusts. Interburst duration, which is affected by the ability to buffer CO2, was significantly reduced in dehydrated solitary locusts compared with gregarious locusts. Moreover, despite similar rehydration levels, only gregarious locusts recovered their initial CO2 accumulation capacity, indicating that cycle characteristics are affected by factors other than haemolymph volume. Haemolymph protein measurements and calculated respiratory exchange ratios suggest that catabolism of haemolymph proteins may contribute to a reduced haemolymph buffering capacity, and thus a compromised ability for CO2 accumulation, in solitary locusts. Nevertheless, DGC was lost at similar hydration states in the two phases, suggesting that DGCs are terminated as a result of inadequate oxygen supply to the tissues.

The effect of discontinuous gas exchange on respiratory water loss in grasshoppers (Orthoptera: Acrididae) varies across an aridity gradient.

The significance of discontinuous gas-exchange cycles (DGC) in reducing respiratory water loss (RWL) in insects is contentious. Results from single-species studies are equivocal in their support of the classic 'hygric hypothesis' for the evolution of DGC, whereas comparative analyses generally support a link between DGC and water balance. In this study, we investigated DGC prevalence and characteristics and RWL in three grasshopper species (Acrididae, subfamily Pamphaginae) across an aridity gradient in Israel. In order to determine whether DGC contributes to a reduction in RWL, we compared the DGC characteristics and RWL associated with CO2 release (transpiration ratio, i.e. the molar ratio of RWL to CO2 emission rates) among these species. Transpiration ratios of DGC and continuous breathers were also compared intraspecifically. Our data show that DGC characteristics, DGC prevalence and the transpiration ratios correlate well with habitat aridity. The xeric-adapted Tmethis pulchripennis exhibited a significantly shorter burst period and lower transpiration ratio compared with the other two mesic species, Ocneropsis bethlemita and Ocneropsis lividipes. However, DGC resulted in significant water savings compared with continuous exchange in T. pulchripennis only. These unique DGC characteristics for T. pulchripennis were correlated with its significantly higher mass-specific tracheal volume. Our data suggest that the origin of DGC may not be adaptive, but rather that evolved modulation of cycle characteristics confers a fitness advantage under stressful conditions. This modulation may result from morphological and/or physiological modifications.