Oxygen convective uptakes in gas exchange cycles were directly recorded in early diapause pupae of Pieris brassicae L. (Lepidoptera; Pieridae) by means of O2 coulometric respirometry. This method was combined with flow-through CO2 respirometry, the two systems being switchable one to the other. During recording with both systems, measurements were also taken with infrared actography. The pupae displayed short discontinuous gas exchange cycles lasting 40–70 min. No true C phase was found by flow-through measurements; instead, flutter opening of the spiracles with discrete convective O2 uptakes began shortly after the O phase whereas CO2 release was suppressed by the inward directed passive suction ventilation. The F phase was characterized by a series of small CO2 bursts (flutter events). Between these bursts, novel sub-phase `miniflutter' was observed, which consisted of six to 10 miniature inspirations without any CO2 emission. During the flow-through measurements, oxygen convective uptakes were indirectly recorded by the infrared actograph as sudden extensions (lengthening) of the abdominal segments at each spiracular microopening.
Breathing in many insects is characterised by discontinuous gas exchange cycles (DGCs), during which carbon dioxide is released periodically. To date, the focus of studies of DGCs has been on CO2 release as measured by flow-through CO2 respirometry, whereas O2 consumption during the DGC has been little studied.
Classically, the DGC consists of three phases (Schneiderman, 1960; Lighton, 1996; Chown and Nicolson, 2004). During the open (O) phase, the spiracles are open and CO2 is released in a burst. The O phase is followed by the facultative closed (C) phase, when the spiracles are closed and little or no gas exchange occurs. Within the C phase, sub-atmospheric pressure is created in the tracheae because of O2 consumption by the tissues and CO2 buffering by means of bicarbonates in the tissue and haemolymph (Wobschall and Hetz, 2004). After the C phase, the flutter (F) phase occurs, during which the spiracles open and close rapidly in succession (fluttering). When open, air is sucked through the spiracles into the tracheae by convection along the negative pressure gradient. This is known as passive suction ventilation (PSV); thus PSV occurs in the absence of muscular movement (see Miller, 1974; Miller, 1981). PSV during the F phase has been described as a mechanism for restricting water loss in insects (see Kestler, 1978; Kestler, 1982; Kestler, 1991). DGC is regarded as CFO cycles when CO2 bursts are not actively ventilated by abdominal pumping, but as CFV (V, ventilation) cycles when the CO2 bursts are associated with pumping movements (Kestler, 1985; Kestler, 2003).
Discontinuous O2 uptakes and CO2 release during the O phase has been demonstrated in some Carabidae beetles and in pupae of the Cecropia moth, Hyalophora cecropia, using heat conductivity detectors or diaferometers (Punt et al., 1957). Simultaneous measurements of O2 uptake and CO2 release by flow-through respirometry in the tok-tok beetle, Psammodes striatus, showed a peak of O2 consumption at the beginning of the O phase, together with a burst of CO2 release (Lighton, 1988). Oxygen uptake also peaked during the release of CO2 in the giant burrowing cockroach, Macropanesthia rhinoceros (Woodman et al., 2007).
The F phase cannot be detected by flow-through O2 respirometry without a significant diffusive component, because the inward bulk flow of air into the tracheal system is functionally equivalent to a minute and probably undetectable reduction in the flow rate of air through the respirometer chamber (Lighton, 1988; Lighton, 1994). Thus, single microopenings of the spiracles and air convective uptakes into the tracheae cannot be detected by flow-through respirometry.
Special techniques are required to record PSV during microopening of the spiracles in the F phase. Schneiderman used cannulated spiracles to measure partial pressure and thus described the rhythms of passive air uptake in silkworm pupae (H. cecropia) (Schneiderman, 1960). Sláma recorded a sawtooth pattern of abdominal retractions with contact transducers in lepidopteran pupae (including the large cabbage white Pieris brassicae) (Sláma, 1984; Sláma, 1988). This pattern was caused by the microopening of the spiracles and passive inspirations. A similar pattern of passive inspirations was recorded by Sláma and Neven in young pupae of the codling moth, Cydia pomonella (Sláma and Neven, 2001). Hetz et al. used miniaturized amperometric sensors to make direct O2 measurements within the tracheal system of lepidopteran pupae (Hetz et al., 1994). Wobschall and Hetz recorded O2 uptake directly in diapausing Atlas moth (Attacus atlas) pupae by simultaneous measurements of tracheal pressure and volume changes (plethysmometry) in the tracheal system, while combining CO2 measurements by flow-through respirometry (Wobschall and Hetz, 2004). Coulometric (volumetric-manometric) respirometry has been used to directly record O2 convective uptakes in diapausing 2–5 month old pupae of the cabbage moth, Mamestra brassicae (Jõgar et al., 2007), and P. brassicae (Jõgar et al., 2004; Jõgar et al., 2005; Jõgar et al., 2008). However, there is a lack of information about the gas exchange patterns, including O2 convective uptake during the initiation phase of diapause (early diapause) (see Kostál, 2006; Belozerov, 2009).
Coulometric respirometry was combined with flow-through CO2 respirometry. We suppose that the flutter events observed by coulometric O2 measurements can usefully be directly compared with flow-through CO2 respirometry.
The main aim of the present investigation was to describe the pattern of O2 convective uptakes and associated body movements in young pupae of P. brassicae. To achieve this, coulometric O2 respirometry was combined with flow-through CO2 respirometry, and discrete O2 discrete uptake was simultaneously recorded indirectly using an infrared (IR) actograph.
MATERIALS AND METHODS
For laboratory experiments, eggs of Pieris brassicae (Linnaeus 1758) (second generation) were collected from cabbage fields near Tartu, Estonia (58°23′N, 26°41′E), during July and August 2009. They were reared in a laboratory under short-day conditions (12 h:12 h light:dark) at 21±1°C and ambient air humidity (55–65% relative humidity). The larvae that hatched from the eggs were fed on leaves cut from cabbage plants, which were replaced with a fresh supply daily. After pupation, each pupa was placed in an Eppendorff tube and kept in laboratory conditions.
For the experiments, twenty-five 2 week old (14±2 days) pupae were used. Each pupa was weighed to 0.1 mg with an analytical balance before experimentation (Explorer Balances, max. 62 g; Ohaus Corporation, Nänikon, Switzerland). Pupal body mass ranged from 0.383 to 0.411 g (Table 1). During respiratory measurements, temperature and humidity conditions were recorded using a digital HygroClip probe (HygroPalm, Rotronic Company, Basserdorf, Switzerland). All measurements were made at 21±1°C and ambient air humidity (50–55% relative humidity). The Eppendorff tube with pupa was used as the insect chamber in the respiratory systems; this avoided handling stress. The respiratory measurements of the first 30 min were discarded; recordings in both systems lasted at least 3 h. Tests with each individual were made twice. We confirmed, with preliminary experiments, that the switch from still air to flowing air (120 ml min–1) and vice versa did not significantly change the frequency of the DGC. Body movements were visually observed under a stereomicroscope (SZ-ZTW, Olympus, Japan).
Coulometric respirometers usually work in an interrupted regime (on–off) of electrolysis (e.g. Heusner et al., 1982). By contrast, our coulometric respirometry (a volumetric manometric system) was characterised by a continuously (uninterrupted) O2-compensating system (Kuusik, 1977; Kuusik et al., 1996; Tartes et al., 1999; Tartes et al., 2002). This setup has also been described by Lighton (Lighton, 2008). This respirometer ensures continuous and adequate replacement of consumed O2 with electrolytically produced O2. The insect itself plays an active role in this self-regulating system. The rates of O2 production and O2 consumption by the insect are indicated on graphs as VO2 (ml h–1). The system also records transient changes in the rate of release of CO2. In our respirometer, we did not use the switching electrodes of electrolysis; instead, the electrolysis current was directly connected with a photoelement. High sensitivity of the respirometer to pressure changes in the respiration chamber was achieved by replacing the standard photodiode with the photosensitive element of a transistor (KT302A, Semitronics, Freeport, NY, USA), which has a very small photosensitive area (approximately 0.5 mm2). In this way, the smallest movement in the meniscus of ethanol inside the U-shaped capillary was reflected as a signal on the recording trace (Fig. 1). The electrolysis current depended on the intensity of the light falling on the phototransistor. The ethanol meniscus in the glass capillary served as a shutter to screen the photosensitive area from light. The electrochemical equivalent of O2 generation has been reported as 209.5 μl O2 mA–1 h–1 (Taylor, 1977). This value was used to convert the readings of the event recorder to O2 consumption values (μl O2 h–1 or μmol O2 h–1).
The coulometric respirometer allowed simultaneous recording of O2 consumption, sudden O2 (air) uptake (known as PSV) by convection into the tracheae at microopenings of the spiracles, discrete CO2 releases by bursts, abdominal pumping movements and heartbeat patterns (see Jõgar et al., 2004; Jõgar et al., 2007). Rapid changes in pressure (lasting seconds) in the insect chamber, caused by active body movements of the insect or other rapid events, were not compensated and led to corresponding rapid changes in the electrolysis current, reflected as spikes on recordings. Thus, our coulometric respirometer also served as an activity detector.
A rapid O2 convective uptake resulted in adequate air volume decrease in the insect chamber and the ethanolic meniscus shifted down by a fraction of a millimetre. As a result, more light fell on the sensitive area of the transistor and an upward signal was recorded (Fig. 2). Downward signals indicated CO2 release by bursts (Tartes et al., 1999). The volume of air uptake was estimated by extracting air from the insect chamber with a microsyringe (1 μl volume, Agilent Technologies, Espoo, Finland). The calibration of convective air uptake is shown in Fig. 2.
Flow-through CO2 respirometry
The infrared gas analyser or flow-through CO2 respirometer (Infralyt-4, Saxon Junkalor GmbH, Dessau, Germany) was used to confirm that the presumed CO2 signals, i.e. the downward spikes on the recording trace of the electrolytic respirometer, were actually due to CO2 bursts, and to measure them quantitatively. The respirometer was calibrated at different flow rates by means of calibration gases (Trägergase, Saxon Junkalor GmbH) and with gas injection. An air flow rate of 120 ml min–1 was used. The insect chamber could be switched either to the flow-through CO2 respirometer or to the coulometric respirometer without disturbing the insect (Fig. 1). During the measurements with coulometric respirometry, the empty respiration chamber served to determine the baseline of the measurements.
Both the coulometric (electrolytic) respirometer and the flow-through respirometer were combined with an IR insect cardiograph (opto-cardiography); we refer to this as the IR actograph, because it records not only heartbeats but also all other abdominal contractions, including muscular ventilation. An IR-emitting diode was placed on one side of the respirometer chamber near the ventral side of the abdomen, while an IR-sensitive diode (TSA6203, Mikrotechna, Prague, Czech Republic) was placed on the opposite side of the chamber (see Metspalu et al., 2001; Metspalu et al., 2002). The light from the IR diode (BP104, Mikrotechna) was modulated by contractions of the heart and skeletal muscles. The level of output voltage reflected the vigour of the muscular contractions of the insect (Hetz et al., 1999). Sudden extensions (lengthening) of abdominal segments (PSV) are recorded as relatively long upward spikes synchronous with microopenings of the spiracles (Figs 3, 4). Weak regular muscular contractions of the abdomen resulted in two-phased relatively short spikes on the recording traces we refer to as abdominal pulsations (Fig. 5). Regular, high-frequency, low-amplitude signals were interpreted as heartbeats (Fig. 3).
Data acquisition and statistics
Computerised data acquisition and analysis were performed using DAS 1401 A/D hardware and TestPoint software (Keithley, Metrabyte, Cleveland, OH, USA) with a sampling rate of 10 Hz. Four bipolar channels allowed simultaneous recording of four events. Mean (±s.d.) standard metabolic rate was calculated automatically using STATISTICA (version 8, StatSoft, Tulsa, OK, USA). Statistical comparisons were made with one-way ANOVA (analysis of variance). Significant ANOVAs were followed with the Fisher's least significant difference (LSD) test. The significance level was set at P<0.05.
At the initiation phase of diapause (14±2 days old), P. brassicae pupae displayed DGCs lasting 40–70 min (Table 1), whereas the duration of CO2 release by burst was 2–6 min (3.1±0.1). Recordings by flow-through respirometry showed a typical pattern. After the O-phase CO2 emission had ceased, the C phase began, which was followed by the F phase with small bursts of CO2 release (Fig. 3). No true C phase was found by flow-through respirometry. Shortly after the end of the O phase, coulometry revealed convective O2 uptake. During this time, CO2 release was suppressed by the inward-directed PSV. This convective O2 uptake indicates an earlier beginning of the F phase than detectable with the flow-through system. Thus the F phase lengthened on account of the C phase. At each microopening of the spiracles and passive convective oxygen uptake event, signals of abdominal lengthening were recorded (Figs 3, 4). Recordings of coulometric respirometry showed clear, gradually shortened signals due to convective oxygen uptake (Fig. 4). Each of the two to three first microopenings and O2 uptakes lasted less than 0.5 s. Oxygen convective uptakes during the interburst period increased metabolic rate by 5–6% compared with the metabolic rate when these uptakes were absent. Flow-through measurements also indicated abdominal lengthening concurrent with the small bursts of CO2 during flutter (Fig. 5).
Between two large CO2 bursts, a series of small bursts of CO2 were recorded by coulometric respirometry (Fig. 6A). Each small burst started with a brief uptake of air into the tracheae, recorded by the IR actograph as a sudden extension of the abdomen, indirectly indicating air (O2) uptake (Fig. 5). Between two consecutive small CO2 bursts, a series of air uptakes (miniature inspirations) were recorded, which we considered as `miniflutter' (Fig. 6A,B; Table 1). These uptakes were irregular with respect to spike height and interval. During such miniflutters, no emissions of CO2 were recorded.
Simultaneous recording with the IR actograph during flow-through CO2 respirometry indicated that pupae differed in the type of body movement associated with the respiratory patterns of CO2 release. In some pupae, CO2 bursts were always concurrent with abdominal ventilating movements (CFV cycles) (always group, N=9) (Fig. 7A), whereas in others (occasionally group, N=10), only some CO2 bursts were concurrent with abdominal ventilating movements (Fig. 7B). In a few pupae (never group, N=6), CO2 bursts occurred without active ventilation (CFO cycles) (Fig. 7C). Ventilating movements (amplitude 1–2 V) associated with CO2 bursts were visible externally as twisting abdominal movements.
Active ventilation during the bursts of CO2 showed individual variation in the vigour of contractions and their number (from one to 15). In pupae with only one to five muscular (active) ventilating movements accompanying the burst, as well as in those lacking active ventilation, a relatively low level of CO2 release was observed. In contrast, pupae with vigorously ventilated bursts showed a significantly higher level of CO2 release (Fig. 7A). Each burst lasted 3–6 min in the always group of pupae, but 2–2.5 min in the occasionally and never groups. Statistical comparison of CO2 release frequency (ANOVA, F24.2=41.8, P>0.05) did not show a significant difference. The energy cost of muscular ventilation during a burst was not studied.
In some pupae showing no active ventilation during CO2 release by bursts, very regular low amplitude (0.1–0.2 V) pulsations (57–70 min–1) were recorded; these we interpreted as heartbeats (Fig. 3).
Our results showed that, in the initiation phase of diapause, P. brassicae pupae display relatively short DGCs (40–70 min), with CO2 bursts lasting 2–6 min. This contrasts with earlier studies, using P. brassicae pupae more than 2 months old, which displayed longer DGCs (8–23 h) (Harak et al., 1999; Jõgar et al., 2004; Jõgar et al., 2005) with CO2 bursts lasting 13–18 min (Harak et al., 1999; Kuusik et al., 1980; Tartes et al., 1999). The young pupae we used with their short DGCs were convenient for studying flutter events. They had a relatively high metabolic rate; in 2–3 month old pupae metabolic rate is at least two times lower (12–28 ml O2 g–1 h–1) (Kuusik 1977; Jõgar et al., 2004; Jõgar et al., 2005).
Commonly, after the O phase, a period with no CO2 release occurs (C phase); later, the CO2 level was marginally elevated (F phase) (Chown et al., 2006). The present study revealed that in young P. brassicae pupae, the DGC measured with flow-through respirometry was characterised by a C phase, at the end of which a series of O2 convective uptakes was found. Thus, the C phase in those pupae was not as closed as previously thought. Between two large CO2 bursts, almost regular small CO2 bursts were recorded. Each small burst started with sudden uptake of air into the trachea (PSV). The main finding in the present study was a series of irregular microopenings of the spiracle(s) with convective O2 uptakes (mini-flutter) found between small bursts. During the mini-flutter, no recordable CO2 emission occurred.
There are several examples where the interburst period consists of discrete small CO2 bursts. Such bursts were described by Lighton (Lighton, 1988) in the tok-tok beetle, P. striatus; in this beetle, each burst was accompanied by active abdominal movement. Discrete CO2 emissions during the F phase have also been reported by Duncan et al. in the tenebrionid beetle, Pimelia grandis (Duncan et al., 2002), and by Kovac et al. in resting honeybees Apis mellifera (Kovac et al., 2007). However, spiracle openings within the F phase were commonly observed to be irregular with respect to frequency and amplitude, if inferred from the CO2 release pattern (e.g. Wobshall and Hetz, 2004).
Our flow-through CO2 measurements showed no CO2 release for a short time after the O phase. The coulometric respirometry and IR actographic recordings showed rapid and clear uptakes of air shortly after the O phase, indicating the beginning of the F phase. An earlier study by Tartes et al. revealed that air convective uptakes began immediately after the O phase (Tartes et al., 2002). Air convective uptakes, shortly after large CO2 bursts, also occurred in old diapausing M. brassicae pupae (Jõgar et al., 2007). We suggest that, at the beginning of the flutter, air uptakes were convective but later were diffusive-convective. These results concur with the plethysmometry flow-through measurements of Wobschall and Hetz (Wobschall and Hetz, 2004), revealing that the convective uptakes of O2 dominate at the beginning of the flutter phase but that, in the later F phase, diffusion takes over from convection as the chief mechanism of O2 uptake. Wobschall and Hetz showed that, in diapausing moth pupae (A. atlas), uptake of air into the tracheal system at the beginning of the F phase along the negative hydrostatic pressure gradient may initially inhibit CO2 release from the tracheae (Wobschall and Hetz, 2004). We suppose that, at the beginning of the F phase of P. brassicae pupae, CO2 emission was also inhibited. One may suppose that water is conserved only at the beginning of the F phase when clear convective O2 uptakes (PSV) occur, but not later when diffusion is the dominating mechanism of the fluttering period.
The duration of the F phase may be underestimated, as the F phase may start before the CO2 measurements can detect it (Hetz et al., 1994; Wobschall and Hetz, 2004). In pupae of P. brassicae, the duration of the F phase may also be longer than estimated by the flow-through system, as far as CO2 release was prevented at the beginning of the F phase by convective air uptakes. Wobschall and Hetz showed that small volume and pressure decreases occurred between the microopenings in the F phase (Wobschall and Hetz, 2004). This confirmed a small but significant contribution of suction ventilation during each microopening (see also Kestler, 1985). In our measurements in P. brassicae pupae, each miniature inspiration was synchronised with rapid extension of the abdomen, confirming that a convective component was always present in O2 uptakes. We observed in P. brassicae a relatively longer flutter phase compared with that in some other insects, such as the carabid Pterostichus niger (Kivimägi et al., 2011) and the bumblebee Bombus terrestris (Karise et al., 2010).
Gas exchange patterns are known to vary between and within individuals (see Chown, 2001; Chown et al., 2002; Marais and Chown, 2003). In the present study, variation was found between individuals in the duration of CO2 release and metabolic rates, but not in DGC frequency. Individuals displayed different gas exchange cycles. Most showed DGCs with all CO2 bursts actively ventilated, a few with no bursts actively ventilated, and others with only some bursts actively ventilated.
Beside active ventilation, some other types of body movements, differing in frequency and amplitude, were observed in the present study. The regular but periodically occurring abdominal pulsations in P. brassicae pupae correspond, in our opinion, to the extracardiac hemocoelic pulsations described in lepidopteran pupae (Sláma, 1984; Sláma, 1999). These pulsations and other abdominal movements play an important role in the regulation of pupal respiration and haemolymph circulation (Sláma and Neven, 2001). In P. brassicae pupae, we interpreted high-frequency but low-amplitude signals, accompanied by CO2 release in bursts, as heartbeats. In a previous study using thermographic measurements, we demonstrated heartbeat reversal, correlated with gas exchange cycles and twisting abdominal movements in diapausing P. brassicae pupae (Jõgar et al., 2005). Heartbeat reversal correlated with gas exchange cycles has also been reported in saturnid moth pupae (Wasserthal, 1996; Hetz et al., 1999; Sláma, 2003).
Manometric O2 respirometry methods have been criticized and their readings mistrusted because these methods usually do not allow separation of active and resting metabolism (see Van Voorhies et al., 2008). Nevertheless, some volumetric manometric methods, including our coulometric respirometry, are regarded as useful (Klok and Chown, 2005). We are convinced that in gas exchange studies of insects, coulometric respirometry supplemented by the flow-through method has clear advantages. Lighton pointed out that coulometric continuously recording respirometry deserves to be more widely used (Lighton, 2008).
In summary, in this study we have shown that the pattern of gas exchange in P. brassicae pupae may be effectively investigated by the combined use of coulometric respirometry and flow-through CO2 systems by switching the same respiration chamber from one system to the other without disturbing the insect. By combining IR actography in parallel with both types of respirometry, it was possible to record rapid air uptakes. Thus, on our recording traces, the patterns of microopenings of the spiracles were clearly indicated.
The research was supported by the Estonian Science Foundation (grant nos 7130 and 6722) and Estonian target financing project no. SF170057s09.
We thank the editor and two anonymous reviewers, who provided helpful and much-appreciated comments on earlier drafts of the manuscript.