Modulation of cyclic CO2 release in response to endogenous changes of metabolism during pupal development of Zophobas rugipes (Coleoptera: Tenebrionidae)

Understanding the mechanisms of gas exchange regulation in insects currently is a hot topic of insect physiology. Endogenous variation of metabolism during pupal development offers a great opportunity to study the regulation of respiratory patterns in insects. Here we show that metabolic rates during pupal development of the tenebrionid beetle Zophobas rugipes reveal a typical U-shaped curve and that, with the exception of 9-day-old pupae, the time between two bursts of CO₂ (interburst phase) was the only parameter of cyclic CO₂ gas exchange patterns that was adjusted to changing metabolic rates. The volume of CO₂ released in a burst was kept constant, suggesting a regulation for accumulation and release of a fixed amount of CO₂ throughout pupal development. We detected a variety of discontinuous and cyclic gas exchange patterns, which were not correlated with any periods of pupal development, suggesting a high among individual variability. An occasional occurrence of continuous CO₂ release patterns at low metabolic rates was very likely caused by single defective non-occluding spiracles.     

Rhythms of passive and active ventilation, and circulation recorded in diapausing pupae of Mamestra brassicae using constant volume respirometry

Abstract.  The periodically occurring convective inflow of air into the tracheal system, or passive suction ventilation, together with the cyclic bursts of release of CO₂ and active ventilation, is recorded in diapausing pupae of Mamestra brassicae . A constant volume respirometer combined with an opto-cardiograph-actograph is used. In all pupae with a metabolic rate of 0.025–0.054 mL g⁻¹ h⁻¹, the bouts of almost imperceptible abdominal contractions are recorded during the bursts of carbon dioxide release and this mode of active ventilation is qualified as extracardiac haemocoelic pulsations. The pupae whose metabolic rate is 0.052–0.075 mL⁻¹ g⁻¹ h⁻¹ show more vigorous abdominal contractions. The results demonstrate that, in diapausing pupae, characterized with low metabolic rates, both passive suction ventilation, referred to also as passive suction inspiration, and active ventilation occurs. In approximately 50% of the pupae, each gas exchange microcycle during the interburst periods begins with a miniature PSI followed by a microburst of CO₂ release; in approximately 30% of the individuals, passive suction inspirations occur separately from CO₂ microbursts; in the remaining pupae, miniature ones without microbursts of CO₂ are recorded. A typical event is heartbeat reversion: in longer periods, the heart peristalses are directed forward (anterograde of heartbeat) and, in shorter periods, the heart peristalses are directed backward (retrograde of heartbeat). At 0 °C, the cyclic release of CO₂ and miniature passive suction inspirations during the interburst periods are preserved at lower frequencies but active ventilation is lost.     

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 (18.7 nmol g⁻¹ min⁻¹ vs. 7.9 nmol g⁻¹ min⁻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.     

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.     

The role of the spiracles in gas exchange during development of Samia cynthia (Lepidoptera, Saturniidae)

Spiracles and the tracheal system of insects allow effective delivery of respiratory gases. During development, holometabolous insects encounter large changes in the functional morphology of gas exchange structures. To investigate changes in respiratory patterns during development, CO₂-release was measured in larvae, pre-pupae and pupae of Samia cynthia (Lepidoptera, Saturniidae). Gas exchange patterns showed great variability. Caterpillars had high metabolic rates and released carbon dioxide continuously. Pre-pupae and pupae showed typical discontinuous gas exchange cycles (DGC) at reduced metabolic rates. Changes in gas exchange patterns can partly be explained with low metabolic rates during pupation. Sequential blocking of spiracles in pre-pupae and pupae reduced spiracle conductance with tracheal conductance remaining unaffected. Analysis of gas exchange patterns indicates that caterpillars and pre-pupae use more than 14 spiracles simultaneously while pupae only use 8 to 10 spiracles. Total conductance is not a simple multiple of single spiracles, but may be gradually adaptable to gas exchange demands. Surprisingly, moth pupae showed a DGC if all except one spiracle were blocked. The huge conductance of single spiracles is discussed as a pre-adaptation to high metabolic demands at the beginning and the end of the pupal as well as in the adult stage.     

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).     

Abdominal movements, heartbeats and gas exchange in pupae of the Colorado potato beetle, Leptinotarsa decemlineata

The rhythms of abdominal movements, heartbeats and gas exchange in the pupae of Leptiontarsa decemlineata (Say) were recorded simultaneously using an electrolytic respirometer and infrared gas analyser, both combined with contact thermography. Abdominal pulsations and heartbeat occurred periodically with little variance among individuals. The abdominal pulsations and heartbeat pauses varied individually within large limits, with the frequency of abdominal pulsations being six to seven times lower than that of the heart pulses. A proportion of the pupae (20%) showed discontinuous gas exchange with large, actively ventilated CO₂ bursts, whereas others (≈ 25%) exhibited continuous regular microcycles (flutter) with abrupt intake of air into the tracheae before discrete microbursts of carbon dioxide. The abdominal pulsations exerted only a minor influence on ventilation during the microcycles. More than 90% of the bursts of abdominal movement coincided with a series of forward directed heartbeats, but interspersed between the bouts of abdominal movement commonly two to three heartbeat pulses were observed that were not associated with abdominal movements. A period of abdominal movement associated with a heartbeat pulse was commonly initiated by one or two vigorous strokes of abdominal rotation.     

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 1-1.5 kPa 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.     

Supradian and infradian cycles in oxygen uptake of diapausing pupae of Pieris brassicae

The general characteristics of diapause respiration in P. brassicae are described, together with an examination of short-term (supradian) and long-term (infradian) variation in oxygen uptake. Supradian cycles occur approximately every 3 hr at 10°C and are shown by closed box analyses to be initiated by carbon dioxide bursts. Maximal rates of oxygen uptake occur shortly after the burst in carbon dioxide release, not at the start of the burst as recorded in other diapausing species. The frequency of supradian cycles is directly related to temperature and metabolism in accordance with the characteristics of discontinuous carbon dioxide release.Infradian cycles of between 3 and 7 days duration are recorded for both oxygen uptake and net exchange rates. Peaks in oxygen demand occur on average every 4 days at 10°C, and are related in frequency to the level of metabolism of individual pupae. Just before post-diapause development, oxygen demands fall to about half their normal levels; these changes are associated with appropriate changes in the frequency of supradian and infradian cycles.     

Gas exchange pattern in the two‐spot ladybird beetle,Adalia bipunctata,differs in dry vs. moist air

Gas exchange patterns in the ladybird beetle, Adalia bipunctata (L.) (Coleoptera: Coccinellidae), were investigated using an infrared gaseous analyser (IRGA) and a coulometric O₂ respirometer (manometric–volumetric system). Before testing, the beetles were kept either in dry (dehydrated) or moist (hydrated) conditions for 1 day. Their subsequent gas exchange patterns did not depend on their state of humidity but rather were controlled by the humidity of the insect chamber during gas exchange measurement. If this chamber contained dry air, the beetles exhibited CO₂ release by burst, which we interpreted as cyclic gas exchange (CGE) with inter‐burst periods, but if the chamber was switched to contain moist air, then cyclic CO₂ release was soon abandoned and a pattern of continuous gas exchange appeared. Measurements with the coulometric respirometer in moist air showed that continuous gas exchange was often associated with weak abdominal pulsations, which we interpreted as active ventilation. Their metabolic rate was lower during gas exchange cycles than during continuous gas exchange. We revealed that in the ladybird beetle metabolic rate increased in moist air when the gas exchange pattern transitioned from cyclic to continuous.     

Discontinuous gas exchange in the Pseudoscorpion Garypus californicus is regulated by hypoxia,not hypercapnia

The discontinuous gas exchange cycle (DGC) of the pseudoscorpion Garypus californicus is characterized by periodic bursts of CO(2) emission and by high rates of interburst CO(2) emission. We investigated the mechanism that triggers the burst phase by manipulating ambient oxygen partial pressures (Po(2)). The ventilatory trigger in most land animals is hypercapnia; in insects, for example, the burst phase is triggered when endotracheal Pco(2) reaches about 4 kPa. In insects with a DGC, hypoxia induces prolonged interburst phases because spiracular conductance is elevated to supply oxygen to the tissues, thus delaying the onset of the hypercapnia-triggered burst phase because CO(2) accumulates more slowly. In G. californicus, hypoxia induced a decrease in interburst phase length, while hyperoxia increased its duration relative to normoxia. This is opposite to the condition in insects. In addition, CO(2) emission fell during the interburst phase as ambient Po(2) rose, also opposite to the condition in insects. Thus, the burst phase is triggered in G. californicus (and presumably in other pseudoscorpions) not by hypercapnia but by hypoxia, a situation that is seldom encountered in terrestrial animals.     

Tracheal compression in pupae of the beetle Zophobas morio

Insects that are small or exhibit low metabolic rates are considered to not require active ventilation to augment diffusive gas exchange. Some pupae with low metabolic rates exhibit abdominal pumping, a behaviour that is known to drive tracheal ventilation in the adults of many species. However, previous work on pupae suggests that abdominal pumping may serve a non-respiratory role. To study the role of abdominal pumping in pupa of the beetle Zophobas morio, we visualized tracheal dynamics with X-rays while simultaneously measuring haemolymph pressure, abdominal movement, and CO₂ emission. Pupae exhibited frequent tracheal compressions that were coincident with both abdominal pumping and pulsation of pressure in the haemolymph. However, more than 63% of abdominal pumping events occurred without any tracheal collapse and hence ventilation, suggesting that the major function of the abdominal pump is not respiratory. In addition, this study shows that the kinematics of abdominal pumping can be used to infer the status of the spiracles and internal behaviour of the tracheal system.     

The role of the spiracles in gas exchange during development of Samia cynthia (Lepidoptera, Saturniidae).

Spiracles and the tracheal system of insects allow effective delivery of respiratory gases. During development, holometabolous insects encounter large changes in the functional morphology of gas exchange structures. To investigate changes in respiratory patterns during development, CO2-release was measured in larvae, pre-pupae and pupae of Samia cynthia (Lepidoptera, Saturniidae). Gas exchange patterns showed great variability. Caterpillars had high metabolic rates and released carbon dioxide continuously. Pre-pupae and pupae showed typical discontinuous gas exchange cycles (DGC) at reduced metabolic rates. Changes in gas exchange patterns can partly be explained with low metabolic rates during pupation. Sequential blocking of spiracles in pre-pupae and pupae reduced spiracle conductance with tracheal conductance remaining unaffected. Analysis of gas exchange patterns indicates that caterpillars and pre-pupae use more than 14 spiracles simultaneously while pupae only use 8 to 10 spiracles. Total conductance is not a simple multiple of single spiracles, but may be gradually adaptable to gas exchange demands. Surprisingly, moth pupae showed a DGC if all except one spiracle were blocked. The huge conductance of single spiracles is discussed as a pre-adaptation to high metabolic demands at the beginning and the end of the pupal as well as in the adult stage.     

Roles of aorta,ostia and tracheae in heartbeat and respiratory gas exchange in pupae of Troides rhadamantus Staudinger 1888 and Ornithoptera priamus L. 1758 (Lepidoptera,Papilionidae)

In non-diapausing pupae of the two birdwing butterfly species Troides rhadamantus and Ornithoptera priamus (Lepidoptera, Papilionidae) heart activity and CO₂ release rates were measured simultaneously within the initial half of pupal development. Heartbeat patterns in these pupae consist of three different types of activity: Continuous forward-pulse periods of different duration with a frequency range of about 0.25–0.52 s⁻¹, continuous backward-pulse periods with lower frequencies (0.15–0.29 s⁻¹) and intermittent backward-pulse periods when short series of three to 10 single heartbeats at frequencies of 0.12–0.35 s⁻¹ alternated with heart pauses of 2–10 min. CO₂ release was discontinuous (CFO-type) from about four to 12 days after pupation in Troides rhadamantus and from about four to 18 days in Ornithoptera priamus. Mean CO₂ release rates were very low in both species (10–30 nmol g⁻¹ min⁻¹). After this period, heart pauses occurred more frequently, probably indicating the onset of metamorphosis and the beginning partial histolysis of the heart. Infrared-optical and thermometrical measurements of heartbeat indicated that haemolymph transport within the dorsal vessel in forward direction is more effective than in backward direction. This is deduced from the higher heartbeat frequency and heartbeat amplitude of the forward pulsations. Results from ultrasonic doppler velocimetry suggest that haemolymph flow velocity is highest during the relatively long diastasis of 2–3 s (30–40 mm s⁻¹), while minimum particle speed (about 20 mm s⁻¹) is at the end of systole and the beginning of diastole. This would mean that haemolymph velocity is highest between two consecutive peristaltic waves. In contrast to the haemolymph velocity, the speed of the peristaltic wave measured with the infrared transmission technique was lower (about 8.4–22 mm s⁻¹ in Troides, 10–23 mm s⁻¹ in Ornithoptera) and remained constant during forward pulse periods. During backward beating the speed was lower (8–20 mm s⁻¹ in Troides, 9–17 mm s⁻¹ in Ornithoptera) and decreased during backward pulse periods. During day two to seven in Troides and day three to nine in Ornithoptera, spiracular opening periods coincided with changes in heartbeat direction from backward to forward pulsations. A possible influence is the more efficient convective haemolymph mixing in the haemocoel during forward heartbeat. The mixing allows to bring the haemolymph in close contact with the tracheal system where the discharge of CO₂ takes place. Heartbeat may therefore serve for shortening the diffusion pathways for a rapid transition into the tracheal system during the open period of the spiracles.     

Pump out the volume—The effect of tracheal and subelytral pressure pulses on convective gas exchange in a dung beetle, Circellium bacchus (Fabricus)

Many flightless beetles like the large apterous dung beetle Circellium bacchus, possess a subelytral cavity (SEC) providing an extra air space below the elytra which connects to the tracheal system (TS) via metathoracic and abdominal spiracles. By measuring subelytral and intratracheal pressure as well as body movements and gas exchange simultaneously in a flow-through setup, we investigated the contribution of convection on Circellium respiratory gas exchange.No constriction phase was observed. TS and SEC pressures were always around atmospheric values. During interburst phase open abdominal spiracles and a leaky SEC led to small CO₂-peaks on a continuous CO₂ baseline, driven by intermittent positive tracheal pressure peaks in anti-phase with small negative subelytral pressure peaks caused by dorso-ventral tergite action.Spiracle opening was accompanied by two types of body movements. Higher frequency telescoping body movements at the beginning of opening resulted in high amplitude SEC and TS pressure peaks. High frequency tergite movements caused subelytral pressure peaks and led to a saw tooth like CO₂ release pattern in a burst. We propose that during the burst open mesothoracic spiracles increase the compliance of the subelytral cavity allowing big volumes of tracheal air being pulled out by convection.     

Cyclic CO(2) emissions during the high temperature pulse of fluctuating thermal regime in eye-pigmented pupae of Megachile rotundata

Megachile rotundata (Hymenoptera: Megachilidae), the primary pollinator used in alfalfa seed production, may need to be exposed to low-temperature storage to slow the insects' development to better match spring emergence with the alfalfa bloom. It has been demonstrated that using a fluctuating thermal regime (FTR) improves the tolerance of pupae to low temperatures. Carbon dioxide emission rates were compared between four different FTRs, all with a base temperature of 6 °C and a daily high-temperature pulse. Four different high-temperature pulses were examined, 15 or 25 °C for 2 h and 20 °C for 1 or 2 h. A subset of pupae at the FTR base temperature of 6 °C exhibited continuous gas exchange and, once ramped to 20 or 25 °C, shifted to cyclic gas exchange. As temperatures were ramped down from the high-temperature pulse to 6 °C, the pupae reverted to continuous gas exchange. The following conclusions about the effect of FTR on the CO₂ emissions of M. rotundata pupae exposed to low-temperature storage during the spring incubation were reached: 1) the high temperature component of the FTR was the best predictor of respiratory pattern; 2) neither pupal body mass nor days in FTR significantly affected which respiratory pattern was expressed during FTRs; 3) cyclic gas exchange was induced only in pupae exposed to temperatures greater than 15 °C during the FTR high temperature pulse; and 4) a two hour pulse at 25 °C doubled the number of CO₂ peaks observed during the FTR pulse as compared to a two hour pulse at 20 °C.     

四种蜚蠊的呼吸信号特征比较

在20℃下,利用CO₂红外分析仪采集了美洲大蠊Periplaneta americana、褐斑大蠊P.brunnea、澳洲大蠊P.australasiae和德国小蠊Blattella germanica的呼吸信号。结果表明,它们均具有典型的不连续气体交换循环（discontinuous gas exchange cycle, DGC）呼吸模式,且一个DGC可分为爆发间期和爆发期2个阶段。4种蜚蠊雄性成虫DGC的特征各异：美洲大蠊完成一个DGC周期平均约需24.55 min,明显长于褐斑大蠊(11.67 min)和澳洲大蠊(10.75 min),德国小蠊周期最短,仅为4.41 min;对于爆发间期历时在整个DGC历时所占的比例,美洲大蠊最大,平均为57％,德国小蠊次之,为48％,褐斑大蠊和澳洲大蠊比较接近,分别为37％和36％。德国小蠊单位体重的CO₂平均释放速率最大,而另外3种蜚蠊的差异不明显。4种蜚蠊爆发期CO₂释放体积均随DGC历时增加而增加,美洲大蠊和德国小蠊单位体重的CO₂平均释放率随DGC历时增加而减少,在褐斑大蠊和澳洲大蠊中它们关系不明显。美洲大蠊、褐斑大蠊和澳洲大蠊CO₂平均释放率和爆发期CO₂释放体积与体重呈正相关,在德国小蠊中关系不明显;4种蜚蠊DGC各阶段历时和DGC频率与它们体重的关系均不明显。     

Effects of flow rate and temperature on cyclic gas exchange in tsetse flies (Diptera, Glossinidae)

Air flow rates may confound the investigation and classification of insect gas exchange patterns. Here we report the effects of flow rates (50, 100, 200, 400 ml min⁻¹) on gas exchange patterns in wild-caught Glossina morsitans morsitans from Zambia. At rest, G. m. morsitans generally showed continuous or cyclic gas exchange (CGE) but no evidence of discontinuous gas exchange (DGE). Flow rates had little influence on the ability to detect CGE in tsetse, at least in the present experimental setup and under these laboratory conditions. Importantly, faster flow rates resulted in similar gas exchange patterns to those identified at lower flower rates suggesting that G. m. morsitans did not show DGE which had been incorrectly identified as CGE at lower flow rates. While CGE cycle frequency was significantly different among the four flow rates (p < 0.05), the direction of effects was inconsistent. Indeed, inter-individual variation in CGE cycle frequency exceeded flow rate treatment variation. Using a laboratory colony of closely related, similar-sized G. morsitans centralis we subsequently investigated the effects of temperature, gender and feeding status on CGE pattern variation since these factors can influence insect metabolic rates. At 100 ml min⁻¹ CGE was typical of G. m. centralis at rest, although it was significantly more common in females than in males (57% vs. 43% of 14 individuals tested per gender). In either sex, temperature (20, 24, 28 and 32 °C) had little influence on the number of individuals showing CGE. However, increases in metabolic rate with temperature were modulated largely by increases in burst volume and cycle frequency. This is unusual among insects showing CGE or DGE patterns because increases in metabolic rate are usually modulated by increases in frequency, but either no change or a decline in burst volume.     

Discontinuous gas exchange patterns of beet armyworm pupae, Spodoptera exigua (Lepidoptera: Noctuidae): effects of Bacillus thuringiensis Cry1C toxin, pupal age and temperature

Abstract. Changes in the discontinuous gas exchange cycle of pupal beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), exposed or not to Cry1C Bacillus thuringiensis toxin, are examined against developmental age (1–7 days) and at different temperatures (10–25 °C) using flow through respirometry. Both exposed and nonexposed pupae exhibit discontinuous gas exchange, but only at 10 °C; the frequency of cyclic release of CO₂ increases with increasing temperatures. The three phases of the discontinuous gas exchange cycle are distinct for both treatment groups. However, the duration of each phase is significantly greater for pupae exposed previously to toxin. The closed phase is 40 ± 14% longer, the flutter phase 23 ± 19% longer, and the open phase is 28 ± 12% longer when pupae were exposed to toxin. Respiratory water loss is 4.5 ± 1.3% for toxin exposed pupae and 2.1 ± 2.4% for unexposed pupae. Furthermore, the exposed pupae have significantly greater cuticular permeability (26.01 ± 1.9 µg cm⁻² h⁻¹ mmHg⁻¹) than the nonexposed pupae (9.64 ± 0.9 µg cm⁻² h⁻¹ mmHg⁻¹). However, in both strains, cuticular transpiration (>93%) far exceeds respiratory transpiration. Overall, total water loss is significantly greater in pupae whose larvae are exposed to toxin compared with pupae from nontreated larvae. Toxin exposed pupae have a mean cycle duration of 60 ± 2.5 min whereas that of nonexposed pupae is 42 ± 1.8 min.(ml g⁻¹ h⁻¹) of the open phase is greater earlier in pupal life followed by a minimum at mid-pupal stage and an increase at late-pupal development in both treatment groups. Combining all 7 days, closed, flutter and open phase (ml g⁻¹ h⁻¹), pupae exposed to toxin produce significantly more CO₂ during each phase. On average, toxin exposed pupae produce 52 ± 12, 43 ± 10 and 15 ± 37% more CO₂ than the untreated pupae during the closed, flutter and open phases, respectively. Therefore, the present study reinforces the need to use insects of similar developmental age in studies of insect respiration patterns and energy metabolism.     

Involvement of respiratory processes in the transient knockout of net CO2 uptake in Mimosa pudica upon heat stimulation

Leaf photosynthesis of the sensitive plant Mimosa pudica displays a transient knockout in response to electrical signals induced by heat stimulation. This study aims at clarifying the underlying mechanisms, in particular, the involvement of respiration. To this end, leaf gas exchange and light reactions of photosynthesis were assessed under atmospheric conditions largely eliminating photorespiration by either elevated atmospheric CO₂ or lowered O₂ concentration (i.e. 2000 μmol mol^?1 or 1%, respectively). In addition, leaf gas exchange was studied in the absence of light. Under darkness, heat stimulation caused a transient increase of respiratory CO₂ release simultaneously with stomatal opening, hence reflecting direct involvement of respiratory stimulation in the drop of the net CO₂ uptake rate. However, persistence of the transient decline in net CO₂ uptake rate under illumination and elevated CO₂ or 1% O₂ makes it unlikely that photorespiration is the metabolic origin of the respiratory CO₂ release. In conclusion, the transient knockout of net CO₂ uptake is at least partially attributed to an increased CO₂ release through mitochondrial respiration as stimulated by electrical signals. Putative CO₂ limitation of Rubisco due to decreased activity of carbonic anhydrase was ruled out as the photosynthesis effect was not prevented by elevated CO₂.