Oxygen extraction efficiency of the tidally-ventilated rectal gills of dragonfly nymphs.

Dragonfly nymphs breathe water using tidal ventilation, a highly unusual strategy in water-breathing animals owing to the high viscosity, density and low oxygen (O2) concentration of water. This study examines how well these insects extract O2 from the surrounding water during progressive hypoxia. Nymphs were attached to a custom-designed respiro-spirometer to simultaneously measure tidal volume, ventilation frequency and metabolic rate. Oxygen extraction efficiencies (OEE) were calculated across four partial pressure of oxygen (pO2) treatments, from normoxia to severe hypoxia. While there was no significant change in tidal volume, ventilation frequency increased significantly from 9.4 ± 1.2 breaths per minute (BPM) at 21.3 kPa to 35.6 ± 2.9 BPM at 5.3 kPa. Metabolic rate increased significantly from 1.4 ± 0.3 µl O2 min-1 at 21.3 kPa to 2.1 ± 0.4 µl O2 min-1 at 16.0 kPa, but then returned to normoxic levels as O2 levels declined further. OEE of nymphs was 40.1 ± 6.1% at 21.3 kPa, and did not change significantly during hypoxia. Comparison to literature shows that nymphs maintain their OEE during hypoxia unlike other aquatic tidal-breathers and some unidirectional breathers. This result, and numerical models simulating experimental conditions, indicate that nymphs maintain these extraction efficiencies by increasing gill conductance and/or lowering internal pO2 to maintain a sufficient diffusion gradient across their respiratory surface.

Air sacs are a key adaptive trait of the insect respiratory system.

Air sacs are a well-known aspect of insect tracheal systems, but have received little research attention. In this Commentary, we suggest that the study of the distribution and function of air sacs in tracheate arthropods can provide insights of broad significance. We provide preliminary phylogenetic evidence that the developmental pathways for creation of air sacs are broadly conserved throughout the arthropods, and that possession of air sacs is strongly associated with a few traits, including the capacity for powerful flight, large body or appendage size and buoyancy control. We also discuss how tracheal compression can serve as an additional mechanism for achieving advection in tracheal systems. Together, these patterns suggest that the possession of air sacs has both benefits and costs that remain poorly understood. New technologies for visualization and functional analysis of tracheal systems provide exciting approaches for investigations that will be of broad significance for understanding invertebrate evolution.

Discontinuous gas exchange in Madagascar hissing cockroaches is not a consequence of hysteresis around a fixed PCO2 threshold.

It has been hypothesised that insects display discontinuous gas-exchange cycles (DGCs) as a result of hysteresis in their ventilatory control, where CO2-sensitive respiratory chemoreceptors respond to changes in haemolymph PCO2 only after some delay. If correct, DGCs would be a manifestation of an unstable feedback loop between chemoreceptors and ventilation, causing PCO2 to oscillate around some fixed threshold value: PCO2 above this ventilatory threshold would stimulate excessive hyperventilation, driving PCO2 below the threshold and causing a subsequent apnoea. This hypothesis was tested by implanting micro-optodes into the haemocoel of Madagascar hissing cockroaches and measuring haemolymph PO2 and PCO2 simultaneously during continuous and discontinuous gas exchange. The mean haemolymph PCO2 of 1.9 kPa measured during continuous gas exchange was assumed to represent the threshold level stimulating ventilation, and this was compared with PCO2 levels recorded during DGCs elicited by decapitation. Cockroaches were also exposed to hypoxic (PO2 10 kPa) and hypercapnic (PCO2 2 kPa) gas mixtures to manipulate haemolymph PO2 and PCO2. Decapitated cockroaches maintained DGCs even when their haemolymph PCO2 was forced above or below the putative ∼2 kPa ventilation threshold, demonstrating that the characteristic oscillation between apnoea and gas exchange is not driven by a lag between changing haemolymph PCO2 and a PCO2 chemoreceptor with a fixed ventilatory threshold. However, it was observed that the gas exchange periods within the DGC were altered to enhance O2 uptake and CO2 release during hypoxia and hypercapnia exposure. This indicates that while respiratory chemoreceptors do modulate ventilatory activity in response to haemolymph gas levels, their role in initiating or terminating the gas exchange periods within the DGC remains unclear.

A pH-powered mechanochemical engine regulates the buoyancy of Chaoborus midge larvae.

The freshwater aquatic larvae of the Chaoborus midge are the world's only truly planktonic insects, regulating their buoyancy using two pairs of internal air-filled sacs, one in the thorax and the other in the seventh abdominal segment. In 1911, August Krogh demonstrated the larvae's ability to control their buoyancy by exposing them to an increase in hydrostatic pressure.1 However, how these insects control the volume of their air-sacs has remained a mystery. Gas is not secreted into the air-sacs, as the luminal gas composition is always the same as that dissolved in the surrounding water.1,2 Instead, the air-sac wall was thought to play some role.3-6 Here we reveal that bands of resilin in the air-sac's wall are responsible for the changes in volume. These bands expand and contract in response to changes in pH generated by an endothelium that envelops the air-sac. Vacuolar type H+ V-ATPase (VHA) in the endothelium acidifies and shrinks the air-sac, while alkalinization and expansion are regulated by the cyclic adenosine monophosphate signal transduction pathway. Thus, Chaoborus air-sacs function as mechanochemical engines, transforming pH changes into mechanical work against hydrostatic pressure. As the resilin bands interlaminate with bands of cuticle, changes in resilin volume are constrained to a single direction along the air-sac's longitudinal axis. This makes the air-sac functionally equivalent to a cross-striated pH muscle and demonstrates a unique biological role for resilin as an active structural element.

The cibarial pump of the xylem-feeding froghopper Philaenus spumarius produces negative pressures exceeding 1 MPa.

The xylem sap of vascular plants is an unlikely source of nutrition, being both nutrient poor and held under tensions (negative pressures) that can exceed 1 MPa. But some insects feed on xylem sap exclusively, extracting copious quantities using a muscular cibarial pump. However, neither the strength of the insect's suction, nor the direct energetic cost of xylem ingestion, have ever been quantified. Philaenus spumarius froghoppers were used to address these gaps in our knowledge. Micro-CT scans of its cibarium and measurements of cibarial muscle sarcomere length revealed that P. spumarius can generate a maximum tension of 1.3 ± 0.2 MPa within its cibarium. The energetic cost of xylem extraction was quantified using respirometry to measure the metabolic rate (MR) of P. spumarius while they fed on hydroponically grown legumes, while xylem sap excretion rate and cibarial pumping frequency were simultaneously recorded. Increasing the plants' xylem tensions up to 1.1 MPa by exposing their roots to polyethylene glycol did not reduce the insects' rate of xylem excretion, but significantly increased both MR and pumping frequency. We conclude that P. spumarius can gain energy feeding on xylem sap containing previously reported energy densities and at xylem tensions up to their maximum suction capacity.

How insects transition from water to air: Respiratory insights from dragonflies.

The transition of animal life from water onto land is associated with well-documented changes in respiratory physiology and blood chemistry, including a dramatic increase in blood pCO2 and bicarbonate, and changes in ventilatory control. However, these changes have primarily been documented among ancestrally aquatic animal lineages that have evolved to breathe air. In contrast, the physiological consequences of air-breathing animals secondarily adopting aquatic gas exchange are not well explored. Insects are arguably the most successful air-breathing animals, but they have also re-evolved the ability to breathe water multiple times. The juvenile life stages of many insect lineages possess tracheal gills for aquatic gas exchange, but all shift back to breathing air in their adult form. This makes these amphibiotic insects an instructive contrast to most other animal groups, being not only an ancestrally air-breathing group of animals that have re-adapted to life in water, but also a group that undergoes an ontogenetic shift from water back to air across their life cycle. This graphical review summarizes the current knowledge on how blood acid-base balance and ventilatory control change in the dragonfly during its water-to-air transition, and highlights some of the remaining gaps to be filled.

The bimodal gas exchange strategies of dragonfly nymphs across development.

Dragonfly nymphs are aquatic and breathe water using a rectal gill. However, it has long been known that the nymphs of many species appear to possess the ability to breathe air, either during their final instar when they leave the water prior to metamorphosis, or during periods of aquatic hypoxia. The aerial gas exchange associated with these activities has not been quantified. This study used flow-through respirometry to measure the rate of aerial CO2 release (V̇CO2) from dragonfly nymphs as a proxy for their aerial gas exchange, both across development and in response to progressive aquatic hypoxia. It examined a total of four species from two families (Libellulidae and Aeshnidae). In both families, the late-final instar nymphs developed functional mesothoracic spiracles, allowing them to breathe air by positioning their head and thorax above the water's surface. While breathing air in this position, the nymphs could also ventilate their submerged rectal gill. Thus, during bimodal gas exchange in normoxic water, it was calculated that aeshnid nymphs expelled 39% of their respiratory CO2 into the air through their spiracles, while libellulid nymphs expelled 56% into the air. Decreasing the aquatic PO2 to 2.5 kPa and then below 1 kPa increased the proportion of respiratory CO2 expelled into the air from 69% to 100%, respectively. Thus, bimodally breathing late-final nymphs can vary how they partition gas exchange between their spiracles and their gill depending on aquatic PO2. Aeshnid nymphs of all developmental stages were also found to use their rectal gill as an air-breathing organ; pre-final nymphs performing 'surface skimming' while late final nymphs aspirated air bubbles directly into their gill's branchial basket. Mass-specific rates of aerial V̇CO2 also increased as the nymphs approached metamorphosis. These findings indicate that aeshnid nymphs are capable of accessing aerial O2 across development using their rectal gill as an air breathing organ, while the aquatic nymphs of both aeshnid and libellulid dragonflies undergo a progressive shift towards using the atmosphere for respiration as they approach metamorphosis.

Quantifying the acid-base status of dragonflies across their transition from breathing water to breathing air.

Amphibiotic dragonflies show a significant increase in hemolymph total CO2 (TCO2) as they transition from breathing water to breathing air. This study examined the hemolymph acid-base status of dragonflies from two families (Aeshnidae and Libellulidae) as they transition from water to air. CO2 solubility (αCO2 ) and the apparent carbonic acid dissociation constant (pKapp) were determined in vitro, and pH/bicarbonate concentration ([HCO3-]) plots were produced by equilibrating hemolymph samples with PCO2  between 0.5 and 5 kPa in custom-built rotating microtonometers. Hemolymph αCO2  varied little between families and across development (mean 0.355±0.005 mmol l-1 kPa-1) while pKapp was between 6.23 and 6.27, similar to values determined for grasshopper hemolymph. However, the non-HCO3- buffer capacity for dragonfly hemolymph was uniformly low relative to that of other insects (3.6-5.4 mmol l-1 pH-1). While aeshnid dragonflies maintained this level as bimodally breathing late-final instars and air-breathing adults, the buffer capacity of bimodally breathing late-final instar Libellula nymphs increased substantially to 9.9 mmol l-1 pH-1 Using the pH/[HCO3-] plots and in vivo measurements of TCO2 and PCO2  from early-final instar nymphs, it was calculated that the in vivo hemolymph pH was 7.8 for an aeshnid nymph and 7.9 for a libellulid nymph. The pH/[HCO3-] plots show that the changes in acid-base status experienced by dragonflies across their development are more moderate than those seen in vertebrate amphibians. Whether these differences are due to dragonflies being secondarily aquatic, or arise from intrinsic differences between insect and vertebrate gas exchange and acid-base regulatory mechanisms, remains an open question.

Studies on gas exchange in the meadow spittlebug, Philaenus spumarius: the metabolic cost of feeding on, and living in, xylem sap.

Spittlebugs (superfamily Cercopoidea) live within a mass of frothy, spittle-like foam that is produced as a by-product of their xylem-feeding habits. The wet spittle represents a unique respiratory environment for an insect, potentially acting either as a reserve of trapped oxygen (O2) or as a significant barrier to O2 diffusion from the surrounding atmosphere. Feeding on xylem sap under tension is also assumed to be energetically expensive, potentially placing further constraints on their gas exchange. To understand the respiratory strategies used by spittlebugs, this study measured the PO2  within the spittle of the meadow spittlebug, Philaenus spumarius, as well as the non-feeding metabolic rate (RMR) and respiratory quotient (RQ) of both nymphs and adults. The metabolic rate of nymphs feeding on xylem was also measured. In separate experiments, the ability of a nymph to obtain O2 from bubbles while submerged in foam was determined using a glass microscope slide coated in an O2-sensitive fluorophore. We determined that P. spumarius breathes atmospheric O2 by extending the tip of its abdomen outside of its spittle, rather than respiring the O2 trapped in air bubbles within the foam. However, spittlebugs can temporarily use these air bubbles to breathe when forcibly submerged. V̇O2  and V̇CO2  did not differ statistically within life stages, giving a RQ of 0.92 for nymphs and 0.95 for adults. Feeding on xylem was found to increase the nymphs' V̇CO2  by only 20% above their RMR. From this cost of feeding, cibarial pump pressures were estimated to be between -0.05 and -0.26 MPa.

Metabolic fuel use after feeding in the zebrafish (Danio rerio): a respirometric analysis.

We used respirometric theory and a new respirometry apparatus to assess, for the first time, the sequential oxidation of the major metabolic fuels during the post-prandial period (10 h) in adult zebrafish fed with commercial pellets (51% protein, 2.12% ration). Compared with a fasted group, fed fish presented peak increases of oxygen consumption (78%), and carbon dioxide (80%) and nitrogen excretion rates (338%) at 7-8 h, and rates remained elevated at 10 h. The respiratory quotient increased slightly (0.89 to 0.97) whereas the nitrogen quotient increased greatly (0.072 to 0.140), representing peak amino acid/protein usage (52%) at this time. After 48-h fasting, endogenous carbohydrate and lipid were the major fuels, but in the first few hours after feeding, carbohydrate oxidation increased greatly, fueling the first part of the post-prandial specific dynamic action, whereas increased protein/amino acid usage predominated from 6 h onwards. Excess dietary protein/amino acids were preferentially metabolized for energy production.

Changes in hemolymph total CO2 content during the water-to-air respiratory transition of amphibiotic dragonflies.

Dragonflies (Odonata, Anisoptera) are amphibiotic; the nymph is aquatic and breathes water using a rectal gill before metamorphosing into the winged adult, which breathes air through spiracles. While the evolutionary and developmental transition from water breathing to air breathing is known to be associated with a dramatic rise in internal CO2 levels, the changes in blood-gas composition experienced by amphibiotic insects, which represent an ancestral air-to-water transition, are unknown. This study measured total CO2 (TCO2) in hemolymph collected from aquatic nymphs and air-breathing adults of Anax junius, Aeshna multicolor (Aeshnidae), Libellula quadrimaculata and Libellulaforensis (Libellulidae). Hemolymph PCO2  was also measured in vivo in both aeshnid nymphs and marbled crayfish (Procambarus fallax. f. virginalis) using a novel fiber-optic CO2 sensor. The hemolymph TCO2 of the pre- and early-final instar nymphs was found to be significantly lower than that of the air-breathing adults. However, the TCO2 of the late-final instar aeshnid nymphs was not significantly different from that of the air-breathing adults, despite the late-final nymphs still breathing water. TCO2 and PCO2  were also significantly higher in the hemolymph of early-final aeshnid nymphs compared with values for the water-breathing crayfish. Thus, while dragonfly nymphs show an increase in internal CO2 as they transition from water to air, from an evolutionary standpoint, the nymph's ability to breathe water is associated with a comparatively minor decrease in hemolymph TCO2 relative to that of the air-breathing adult.

The transition from water to air in aeshnid dragonflies is associated with a change in ventilatory responses to hypoxia and hypercapnia.

Dragonflies are amphibiotic, spending most of their lives as aquatic nymphs before metamorphosing into terrestrial, winged imagoes. Both the nymph and the adult use rhythmic abdominal pumping movements to ventilate their gas exchange systems: the nymph tidally ventilates its rectal gill with water, while the imago pumps air into its tracheal system through its abdominal spiracles. The transition from water to air is known to be associated with changes in both respiratory chemosensitivity and ventilatory control in vertebrates and crustaceans, but the changes experienced by amphibiotic insects have been poorly explored. In this study, dragonfly nymphs (Anax junius) and imagoes (Anax junius and Aeshna multicolor) were exposed to hypoxia and hypercapnia while their abdominal ventilation frequency and amplitude was recorded. Water-breathing nymphs showed a significant increase in abdominal pumping frequency when breathing hypoxic water (<10 kPa O2), but no strong response to CO2, even in severe hypercapnia (up to 10 kPa CO2). In contrast, both species of air-breathing imago increased their abdominal pumping amplitude when exposed to either hypoxia or hypercapnia, but did not show any significant increase in frequency. These results demonstrate that aquatic dragonfly nymphs possess a respiratory sensitivity that is more like other water breathing animals, being sensitive to hypoxia but not hypercapnia, while their air-breathing adult form responds to both respiratory challenges, like other terrestrial insects. Shifting from ventilating a rectal gill with water to ventilating a tracheal system with air is also associated with a change in how abdominal ventilation is controlled; nymphs regulate gas exchange by varying frequency while imagoes respond by varying amplitude.

The mechanisms underlying the production of discontinuous gas exchange cycles in insects.

This review examines the control of gas exchange in insects, specifically examining what mechanisms could explain the emergence of discontinuous gas exchange cycles (DGCs). DGCs are gas exchange patterns consisting of alternating breath-hold periods and bouts of gas exchange. While all insects are capable of displaying a continuous pattern of gas exchange, this episodic pattern is known to occur within only some groups of insects and then only sporadically or during certain phases of their life cycle. Investigations into DGCs have tended to emphasise the role of chemosensory thresholds in triggering spiracle opening as critical for producing these gas exchange patterns. However, a chemosensory basis for episodic breathing also requires an as-of-yet unidentified hysteresis between internal respiratory stimuli, chemoreceptors, and the spiracles. What has been less appreciated is the role that the insect's central nervous system (CNS) might play in generating episodic patterns of ventilation. The active ventilation displayed by many insects during DGCs suggests that this pattern could be the product of directed control by the CNS rather than arising passively as a result of self-sustaining oscillations in internal oxygen and carbon dioxide levels. This paper attempts to summarise what is currently known about insect gas exchange regulation, examining the location and control of ventilatory pattern generators in the CNS, the influence of chemoreceptor feedback in the form of O2 and CO2/pH fluctuations in the haemolymph, and the role of state-dependent changes in CNS activity on ventilatory control. This information is placed in the context of what is currently known regarding the production of discontinuous gas exchange patterns.

A novel technique for the precise measurement of CO2 production rate in small aquatic organisms as validated on aeshnid dragonfly nymphs.

The present study describes and validates a novel yet simple system for simultaneous in vivo measurements of rates of aquatic CO2 production (MCO2 ) and oxygen consumption (MO2 ), thus allowing the calculation of respiratory exchange ratios (RER). Diffusion of CO2 from the aquatic phase into a gas phase, across a hollow fibre membrane, enabled aquatic MCO2  measurements with a high-precision infrared gas CO2 analyser. MO2  was measured with a PO2  optode using a stop-flow approach. Injections of known amounts of CO2 into the apparatus yielded accurate and highly reproducible measurements of CO2 content (R2=0.997, P<0.001). The viability of in vivo measurements was demonstrated on aquatic dragonfly nymphs (Aeshnidae; wet mass 2.17 mg-1.46 g, n=15) and the apparatus produced precise MCO2  (R2=0.967, P<0.001) and MO2  (R2=0.957, P<0.001) measurements; average RER was 0.73±0.06. The described system is scalable, offering great potential for the study of a wide range of aquatic species, including fish.

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.

Stomata actively regulate internal aeration of the sacred lotus Nelumbo nucifera.

The sacred lotus Nelumbo nucifera (Gaertn.) possesses a complex system of gas canals that channel pressurized air from its leaves, down through its petioles and rhizomes, before venting this air back to the atmosphere through large stomata found in the centre of every lotus leaf. These central plate stomata (CPS) lie over a gas canal junction that connects with two-thirds of the gas canals within the leaf blade and with the larger of two discrete pairs of gas canals within the petiole that join with those in the rhizome. It is hypothesized that the lotus actively regulates the pressure, direction and rate of airflow within its gas canals by opening and closing these stomata. Impression casting the CPS reveal that they are open in the morning, close at midday and reopen in the afternoon. The periodic closure of the CPS during the day coincides with a temporary reversal in airflow direction within the petiolar gas canals. Experiments show that the conductance of the CPS decreases in response to increasing light level. This behaviour ventilates the rhizome and possibly directs benthic CO2 towards photosynthesis in the leaves. These results demonstrate a novel function for stomata: the active regulation of convective airflow.

Reversible brain inactivation induces discontinuous gas exchange in cockroaches.

Many insects at rest breathe discontinuously, alternating between brief bouts of gas exchange and extended periods of breath-holding. The association between discontinuous gas exchange cycles (DGCs) and inactivity has long been recognised, leading to speculation that DGCs lie at one end of a continuum of gas exchange patterns, from continuous to discontinuous, linked to metabolic rate (MR). However, the neural hypothesis posits that it is the downregulation of brain activity and a change in the neural control of gas exchange, rather than low MR per se, which is responsible for the emergence of DGCs during inactivity. To test this, Nauphoeta cinerea cockroaches had their brains inactivated by applying a Peltier-chilled cold probe to the head. Once brain temperature fell to 8°C, cockroaches switched from a continuous to a discontinuous breathing pattern. Re-warming the brain abolished the DGC and re-established a continuous breathing pattern. Chilling the brain did not significantly reduce the cockroaches' MR and there was no association between the gas exchange pattern displayed by the insect and its MR. This demonstrates that DGCs can arise due to a decrease in brain activity and a change in the underlying regulation of gas exchange, and are not necessarily a simple consequence of low respiratory demand.

Performance correlates of resting metabolic rate in garden skinks Lampropholis delicata.

Resting metabolic rates can vary greatly between individuals of the same species. These differences are generally repeatable and show moderate-to-high heritability, suggesting that they could be a target for natural selection. The present study therefore aimed to determine if inter-individual differences in resting metabolic rates (RMR) in garden skinks Lampropholis delicata were associated with inter-individual differences in a suite of physiological and behavioural variables: aerobic capacity, burst sprinting speed and thermal preference. Whole-animal measures of aerobic capacity and RMR were significantly positively correlated, but mass-independent measures were not. Burst sprinting speed and thermal preference were also not correlated with RMR.

Physical gills in diving insects and spiders: theory and experiment.

Insects and spiders rely on gas-filled airways for respiration in air. However, some diving species take a tiny air-store bubble from the surface that acts as a primary O(2) source and also as a physical gill to obtain dissolved O(2) from the water. After a long history of modelling, recent work with O(2)-sensitive optodes has tested the models and extended our understanding of physical gill function. Models predict that compressible gas gills can extend dives up to more than eightfold, but this is never reached, because the animals surface long before the bubble is exhausted. Incompressible gas gills are theoretically permanent. However, neither compressible nor incompressible gas gills can support even resting metabolic rate unless the animal is very small, has a low metabolic rate or ventilates the bubble's surface, because the volume of gas required to produce an adequate surface area is too large to permit diving. Diving-bell spiders appear to be the only large aquatic arthropods that can have gas gill surface areas large enough to supply resting metabolic demands in stagnant, oxygenated water, because they suspend a large bubble in a submerged web.