The opening–closing rhythms of the subelytral cavity and associated gas exchange patterns were monitored in diapausing Leptinotarsa decemlineata beetles. Measurements were made by means of a flow-through CO2 analyser and a coulometric respirometer. Under the elytra of these beetles there is a more or less tightly enclosed space, the subelytral cavity (SEC). When the cavity was tightly closed, air pressure inside was sub-atmospheric, as a result of oxygen uptake into the tracheae by the beetle. In about half of the beetles, regular opening–closing rhythms of the SEC were observed visually and also recorded; these beetles displayed a discontinuous gas exchange pattern. The SEC opened at the start of the CO2 burst and was immediately closed. On opening, a rapid passive suction inflow of atmospheric air into the SEC occurred, recorded coulometrically as a sharp upward peak. As the CO2 burst lasted beyond the closure of the SEC, we suggest that most of the CO2 was expelled through the mesothoracic spiracles. In the remaining beetles, the SEC was continually semi-open, and cyclic gas exchange was exhibited. The locking mechanisms and structures between the elytra and between the elytra and the body were examined under a stereomicroscope and by means of microphotography. We conclude that at least some of the L. decemlineata diapausing beetles were able to close their subelytral cavity tightly, and that the cavity then served as a water-saving device.
The subelytral cavity (SEC) is an air-filled space under the elytra and above the abdomen characteristic in some beetles. The abdominal and metathoracic spiracles open into this cavity. In flightless and wingless beetles, the cavity is opened by lifting the elytra (Duncan, 2002; Duncan and Byrne, 2002; Schilman et al., 2007; Duncan et al., 2010). In winged beetles, e.g. the Colorado potato beetle, Leptinotarsa decemlineata Say 1824, the SEC is opened by lowering the last abdominal segments and closed by pressing these segments against the elytra without lifting them (Vanatoa et al., 2006).
The SEC is considered to aid in diminishing the level of respiratory and cuticular transpiration as it reduces the diffusion gradient between the tracheal system and peripheral air (Dizer, 1955; Cloudsley-Thompson, 1964). This water-conserving hypothesis for the SEC was later supported by data showing that the air in the cavity has a high water content (Ahearn, 1970; Zachariassen, 1991; Hadley, 1994; Cloudsley-Thompson, 2001). Ahearn (1970) suggested that, in desert tenebrionid beetles, a unidirectional airflow passes backwards with inspiration via the mesothoracic spiracles and expiration via the subelytral spiracles. Nicolson et al. (1984) suggested that after air has been expelled via the spiracles, CO2 accumulated in the SEC is eliminated to the atmosphere by lifting of the elytra. However, when CO2 release was measured separately and simultaneously from the mesothoracic spiracles and from the SEC in the flightless dung beetles Circellium bacchus (Duncan and Byrne, 2002, 2005; Byrne and Duncan, 2003; Duncan, 2003), there was a predominantly anterograde or tidal airflow in the tracheal system. According to Byrne and Duncan (2003), the air within the SEC in these beetles was high in water vapour and in CO2 but the concentration of O2 was below atmospheric. Dizer (1955) demonstrated that removal of the elytra resulted in a substantial increase in water loss in wingless beetles with fused elytra, but little increase in winged species capable of flight, suggesting that in the latter the cavity was not hermetically sealed. Byrne and Duncan (2003) showed that if the SEC in a flightless dung beetle was open, there would be a 74% increase of water loss rate.
Further data indicate that in beetles the SEC is needed to avoid death by desiccation. The elimination of water-conserving properties of the SEC may be used as a physiological method of controlling some insects. Treatment of last instar larvae of L. decemlineata with non-lethal doses of neem preparation (Kuusik et al., 2001a) or with a synthetic analogue of juvenile hormone (Kuusik and Kogerman, 1978) resulted in morphological failure of the elytra and high mortality rates of beetles during winter diapause. The exposure of L. decemlineata pupae in dry conditions produced elytral deformations in adults and lethal water loss during hibernation (Pelletier, 1995). The topical application of neem preparation directly on Hylobius abietis beetles caused respiratory failure: the SEC was left open and a transition from normal discontinuous gas exchange (DGE) to irregular rhythms of continuous respiration occurred (Sibul et al., 2004a). Many toxic substances are able to evoke respiratory failure in insects (reviewed in Kuusik et al., 2001a; Jõgar et al., 2005; Karise and Mänd, 2015).
DGE, in which CO2 is released periodically as bursts, is a common respiratory pattern in many insects (reviewed in Lighton, 1994, 1996; Chown and Nicolson, 2004; Quinlan and Gibbs, 2006). The DGE cycle has three consecutive phases, the open or O-phase, the facultative closure or C-phase and the flutter or F-phase. During the O-phase, CO2 is expelled by burst. During the C-phase, the spiracles are closed and presumably no gas exchange occurs. In the tracheae, the pressure level is reduced because the tissues consume O2 and the tissues as well as haemolymph buffer CO2 by means of bicarbonates (Miller, 1974; Sláma, 2010). Throughout the F-phase, the spiracles open and close rapidly when the negative pressure gradient allows intake of air through them into the tracheae. This phenomenon is described as passive suction ventilation (PSV) or passive suction inspiration (PSI) (see Sláma, 1988), and occurs without muscular contractions (Miller, 1974, 1981). The PSI during this phase may be a mechanism to restrict water loss in insects, as the air inflow impedes the release of CO2 gas and water (see Kestler, 1978, 1980, 1982, 1991).
cyclic gas exchange
discontinuous gas exchange
infrared gas analyser (flow-through analyser)
passive suction inspiration
passive suction ventilation
water loss rate
In contrast to the DGE, in cyclic gas exchange (CGE), CO2 is released periodically by bursts with a little also being released during the interburst periods (Gibbs and Johnson, 2004). A third common gas exchange pattern is continuous respiration, in which CO2 release is more or less steady (Hadley, 1994).
Several hypotheses have been proposed to explain the adaptive benefit of DGE cycles or their mechanistic origin (reviewed in Kestler, 1985; Lighton, 1988; Chown and Nicolson, 2004; Chown et al., 2006; Contreras et al., 2014; Matthews and Terblanche, 2015). Originally, DGE was considered to be an adaptation to restrict respiratory water loss (Edney, 1977; Hadley, 1994; Lighton, 1988, 1996; Chown et al., 2002, 2006), although only a small fraction of the overall water loss is thought to be lost through respiration (reviewed in, for example, Quinlan and Lighton, 1999; Quinlan and Hadley, 1993; Chown, 2002).
Currently, the main method for studying gas exchange patterns and metabolic rates is by flow-through CO2 respirometry. Oxygen uptake generally tracks CO2 release, but O2 consumption is much more difficult to measure and these measurements are often omitted (Quinlan and Gibbs, 2006) or rarely performed. For example, simultaneous CO2 and O2 flow-through measurements have been performed in the tok-tok beetle Psammodes striatus (Lighton, 1988) and in the dung beetle C. bacchus (Duncan and Byrne, 2002) to study the role of the SEC.
However, flow-through respirometry cannot enable recording of the regular passive suction uptake of air (oxygen) into the tracheae or from the atmosphere into the SEC. Moreover, it is unable to detect the real F-phase with PSI in the absence of a recordable diffusive component. This is because of the inward bulk flow of air into the tracheal system, which is practically equal to a small and presumably undetectable decrease in the flow rate of air through the respirometer chamber (Lighton, 1988, 1994, 1996). Thus, other methods should be used.
Wobschall and Hetz (2004) registered O2 uptake in diapausing atlas moth, Attacus atlas, pupae by simultaneously measuring the tracheal pressure and volume changes in the tracheal system (plethysmometry). Sláma (1984, 1988) used the micro-anemometric method and also measured changes in haemocoelic pressure using electronic strain-gauge transducers. Constant-volume coulometric respirometry has been used to record the peaks due to PSIs in diapausing pupae of Pieris brassicae (Jõgar et al., 2004, 2008, 2011) and Mamestra brassicae (Jõgar et al., 2007, 2014). This method has also been used to record PSI peaks at the openings of the SEC in adult H. abietis (Sibul et al., 2004a,b) and in L. decemlineata beetles (Vanatoa et al., 2006).
The aim of the present study was to record the opening–closing rhythms of the SEC in diapausing L. decemlineata beetles, and also to examine how the state of this cavity influences the gas exchange pattern. The mechanisms and structures involved in the closure of the SEC were also investigated.
MATERIALS AND METHODS
Adult L. decemlineata were collected in autumn from potato fields near Tartu, Estonia (58°21′N, 26°39′E). They were reared in the laboratory on fresh potato leaves under short day conditions [10 h:14 h light:dark, 18–20°C, 60–70% relative humidity (RH)]. Diapausing beetles were placed in 1 l glass jars half-filled with lightly moistened peat, into which they burrowed; these jars were held in a refrigerator at 5–6°C, 40–50% RH, in darkness. Experiments were started when the beetles had entered deep diapause. To ensure that beetles reached a stable diapause state, they were kept in the peat for 1 month. Before measurements were taken, beetles were removed from the refrigerator and adapted to room conditions, at 21±2°C and 35±10% RH for 1 day. Then they were placed individually into Petri dishes (diameter 9 cm). They were weighed using an analytical balance (Explorer Balances, Ohaus Corporation, Pine Brook, NJ, USA) to within 0.1 mg. Preliminary measurements by means of flow-through respirometry were conducted on 12 beetles that displayed DGE cycles with clear opening–closing rhythms, and six individuals with the SEC continually open and that displayed CGE. To supress the beetles’ activity, we used the knocking method described by Kestler (1991) and Metspalu et al. (2002). Tonic immobility was achieved by knocking on the insect vessel. The neuromuscular basis of tonic immobility is summarized by Roeder (1953).
First, V̇CO2 was measured with an infrared gas analyser (IRGA; flow-through CO2 respirometer LI-COR model 7000, Lincoln, NB, USA); then, in beetles displaying DGE cycles, V̇O2 was measured by coulometric respirometry. During flow-through measurements, the state of the SEC was visually observed through the transparent insect chamber under an Olympus SZ-CTV stereo microscope (Olympus Optical Co. Ltd, Tokyo, Japan). All respiratory measurements lasted at least 2 h.
The electrolytic respirometer and IRGA data were combined with an infrared (IR) cardiograph of the insects, which we refer to as the IR actograph (IRA), because it records not only heart pulses but also all other abdominal contractions, including muscular ventilating. An IR-emitting diode was placed on one side of the chamber near the ventral side of the abdomen, while an IR-sensitive diode was placed on the opposite side of the chamber (see Metspalu et al., 2001, 2002). The light from the IR-emitting diode was modulated by the contractions of the heart and skeletal muscles. The amplitude of output voltage fluctuations reflected the vigour of the muscular contractions (see S. K. Hetz, Untersuchungen zu Atmung, Kreislauf und Säure-Basen-Regulation an Puppen der tropischen Schmetterlingsgattungen Ornithoptera, Troides und Attacus, PhD thesis, Friedrich-Alexander-Universität, Erlangen-Nürnberg, 1994; Hetz et al., 1999). Abdominal contractions resulted in downward spikes while muscular relaxations were directed upward.
Coulometric respirometers usually work in an interrupted regime (on–off) of electrolysis (e.g. Taylor, 1977; 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, 2002). This setup has also been described by 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, at the same time, O2 consumption by the insect are indicated on graphs as V̇O2 in 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 is achieved by replacing the standard photodiode by the photosensitive element of a transistor (Silicon Phototransistor Type OP505W, Optek Technology Inc., TX, USA), having a very small photosensitive area (about 0.5 mm2). In this way, the smallest movement in the meniscus of ethanol inside the U-shaped capillary is reflected as a signal on the recording trace. The electrolysis current depends on the intensity of the light falling on the phototransistor. The ethanol meniscus in the glass capillary serves 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 h−1 (Taylor, 1977). This value was used to convert the readings of the event recorder to O2 consumption values for our experimental conditions.
The coulometric respirometer allowed simultaneous recording of O2 consumption, sudden O2 (air) uptake (known as PSI) by convection into the tracheae at micro-openings of the spiracles, discrete CO2 releases by bursts, abdominal pumping movements and heartbeat patterns (see Jõgar et al., 2004, 2007). Rapid changes in pressure (lasting seconds) in the insect chamber, caused by active body movements of the insect or other rapid events, are not compensated and lead to corresponding rapid changes in the electrolysis current, reflected as spikes on recordings. Thus, our coulometric respirometer also served as an activity detector.
Flow-through CO2 respirometry
CO2 release was measured using an IRGA. Ambient gas from outside the laboratory was scrubbed of CO2 and water by passing it through columns containing soda lime and Drierite/Ascarote. The CO2 channel was calibrated with commercially available span gas (Linde AG, Höllriegelskreuth, Germany). We used an air flow rate of 33 ml min−1, controlled by an electronic flow meter (model 5067-0223, Agilent Technologies, USA). Respirometry was carried out in dry air; the insect chamber (1.5 ml) was perfused with dry (4–5% RH), CO2-free air. The humidity and temperature of air entering the insect chamber were continuously measured using a HygroPalm 3 for digital HygroClips probes (Rotronic Instrument Corp., USA). The empty chamber served to determine the baseline for the measurements. All measurements were performed in a thermostat Sanyo incubator (Sanyo MR-254, Japan).
An Eclipse FN1 (Nikon, Japan) light microscope at a magnification of 40–200× and NIS Elements Imaging software version 4.30 (Nikon, Japan) were used for stacked photo-micrograph acquisition (automated multi-focus imaging, step size 1.4 µm with 20× lens, 14 µm with 4× lens). The number of stacked images ranged from 115 to 255.
Data acquisition and statistics
Computerised data acquisition and analysis were performed using DAS 1401 A/D (analog–digital) 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. Data were analysed using Student's t-test, with StatSoft v10 (StatSoft Inc., Tulsa, OK, USA). The significance level was set at P<0.05.
Visual observation and recording of the opening–closing rhythms of the SECs of many L. decemlineata beetles showed that the cavity was opened by lowering the last abdominal segments, and closed by pressing these segments upon the elytra, while the elytra were not lifted. Commonly, the SEC was opened and immediately closed, and one or two movements were done by the last abdominal segments. The SEC was usually opened near the start of CO2 emission, while later it was kept in a closed state until the subsequent burst.
Using flow-through CO2 respirometry, we studied how the state of the SEC was associated with gas exchange patterns. The clear DGC with open, closed and flutter phases was exhibited only in beetles with the regular opening–closing rhythms described above. The characteristics of DGE are given in Table 1. The IRA recording, made simultaneously with flow-through CO2 measurements, showed spikes due to opening–closing of the SEC (Fig. 1). The burst of CO2 started some minutes before the SEC opened, and continued after the closing of the cavity; this was why we concluded that most CO2 was released from the mesothoracic spiracles and not through the SEC.
In beetles whose SEC was continually semi-open or leaking, mainly CGE was recorded, i.e. during the interburst period noticeable volumes of CO2 were released, while no C-phase was observed. In these beetles, irregular abdominal contractions were typical and no periods of active ventilation occurred (Fig. 2).
We compared body mass loss rate, interpreted as water loss rate (WLR), between beetles with normal opening–closing rhythms of the SEC and individuals in which a section of the SEC was removed (WLR=4.26±0.68 and 6.90±0.45 µl g−1 h−1, respectively). When we removed a small fraction of the elytra, we did not observe any haemolymph. Thus, water loss increased substantially when the SEC was open. Comparison of WLRs in beetles before and after the wings were removed during DGE showed that the WLR was significantly higher in beetles with removed wings (Student's t-test WLR, t=−7.74, d.f.=10, P<0.05).
The coulometric respirometry recording shows that on opening of the SEC, a sharp upward peak occurs at the start of the CO2 burst (Fig. 3). This upward peak resulted in rapid uptake of air into the SEC from the atmosphere by the principle of PSI, indicating that there was sub-atmospheric air pressure inside the SEC. In some individuals the peak of air uptake was absent, suggesting that their SEC was not tightly closed, i.e. it was leaking.
After opening of the SEC by removal of the elytra, the large upward peaks were lost and a clear pattern of O-, C- and F-phases of gas exchange appeared in the coulometric recording, suggesting DGE cycles. The F-phase was recognised by spikes of micro-opening of the spiracles and PSIs (Fig. 4). From these spikes, we measured the duration of every micro-opening, which in the first spikes of PSIs was 0.4–0.6 s, while in later spikes it was 1.5–1.8 s.
The elytral locking mechanism
Detailed visual examination and microphotography of L. decemlineata elytra using a stereomicroscope revealed that the medial (sutural) margins of two elytra are asymmetrically designed: the narrow sutural face of one elytron consists of two ridges, a dorsal ridge and a ventral ridge with a groove between them. The ridge (key) of one elytron locks into the groove of the other. This lock-and-key construction forms the medial sutural lock of the two elytra (Fig. 5).
A similar lock-and-key structure locks the elytra to the body. Along the inner surface of the outer margin of the elytron is a soft rounded ridge. Along the body side on the pleural area there is a groove. Elytron-to-body locking occurs by pressing the soft elytron ridge into the groove on the pleural area of the body side (Fig. 6). These interlocking structures probably allow the SEC to be closed tightly.
We suggest that the elytra provide complete cover and also protection for the delicate hindwings and abdomen against external disturbances, but also reduce overall water loss in individuals that are able to close the SEC tightly (Fig. 7).
The Colorado potato beetle, L. decemlineata, uses several strategies for hibernating in conditions of different humidity and one of these is to tightly close its SEC to restrict water loss. In our experiments, the opening–closing rhythms of the SEC were visually observed and recorded by an IRA and coulometric respirometer. During the DGE cycles, the SEC was opened and immediately closed at the start of the CO2 burst emission. Therefore, we suggest from our whole-body measurements in L. decemlineata beetles, as representative of insects with flying ability, that most of the CO2 was expelled from the body via the mesothoracic spiracles, and not from the SEC. That these spiracles are the main outlet for CO2 emission was also demonstrated in flightless dung beetles by Duncan (2002, 2003) and Byrne and Duncan (2003).
Our observations in L. decemlineata demonstrated characteristic convective uptake or passive suction inflow, i.e. inspiration (PSI) of air into the cavity when the pressure inside was sub-atmospheric. A similar large PSI peak at the opening of the SEC and its immediate closure at the start of the CO2 burst has also been recorded in H. abietis beetles (Sibul et al., 2004a,b). The decrease in air pressure in the SEC indicated oxygen depletion into the tracheae. Similar oxygen uptake from the SEC into the tracheae has been described in dung beetles, but here the leaky elytra would enable air to enter the SEC by passive mass flow, without opening of the SEC by lifting the elytra (Duncan et al., 2010).
The flightless beetles with fused elytra are able to hermetically seal the SEC and to open it again by lifting the elytra (Gorb, 1998; Frantsevich et al., 2005). Our visual examination of L. decemlineata elytra under the stereomicroscope suggests that these beetles are able to close their SEC, if not hermetically then at least tightly, by means of special structures: the sutural (medial) ridges of two elytra forming a lock. A tight seal between the elytra and the body is possible because of the close fit of the ridge on the edge of the elytron and the groove along the body side at the pleural area. A similar elytron-to-elytron locking system has been described in some other winged beetles, but elytra-to-body locking in flying and flightless beetles is performed by microtrichia (Gorb, 1998; Frantsevich et al., 2005).
Water loss increased by 62% in L. decemlineata beetles when the SEC was opened by removing a section of an elytron. Therefore, we hypothesise that L. decemlineata beetles with regular opening–closing rhythms of the SEC are able to close this cavity tightly or even hermetically, and that it serves as a water-saving device. A similar water-conserving role of the SEC has been described in flightless dung beetles, in which water loss increased by 74% when the SEC was open (Duncan, 2003).
After removing the elytra and wings from L. decemlineata, all three DGE phases appeared, including the F-phase when monitored by the coulometric respirometer, which recorded PSIs into the tracheae and air uptake from the atmosphere into the SEC. This pattern of flutter looked very similar to the flutter described in M. brassicae pupae (see Jõgar et al., 2014). The coulometric measurements in L. decemlineata revealed that only for a short time at the beginning of the F-phase was the gas exchange purely or predominantly convective, presuming no efflux of CO2 and water vapour, but that later gas exchange was instead diffusive. Our results are consistent with those of Wobschall and Hetz (2004) that in a diapausing lepidopteran pupa during the initial period of F-phase only the brief spiracular openings allow O2 uptake mainly by convection or mass flow, which conserves water; later O2 uptake occurs mainly by diffusion, not conserving water.
Our measurements showed that the release of CO2 was always accompanied by active ventilation or pumping in L. decemlineata beetles. Kestler (,1980, 1985, 2003; P. Kestler, Die diskontinuierliche Ventilation bei Periplaneta americana L. und anderen Insekten, PhD thesis, Julius-Maximilians-University, Würzburg, 1971) demonstrated that suction ventilation and active muscular ventilation are strategies for water retention in some insects.
Passive suction uptake or inspiration of air from the surrounding atmosphere into the SEC and into the tracheae plays an important role in L. decemlineata respiration. This has previously been demonstrated in several other insects (Kestler, 1980, 1985; Sláma, 1988; Wobschall and Hetz, 2004; Jõgar et al., 2011, 2014). The SEC also plays an important role in insect respiration and as a water-conserving device (Byrne and Duncan, 2003; Schilman et al., 2007; Duncan et al., 2010; Chown, 2011).
Our experimental data reveal that about half of the L. decemlineata beetles were able to close their SEC tightly and that it then served as a water-saving device. In about half of the beetles, the SEC was continuously open and, from our earlier data (Kuusik et al., 2001a,b), these beetles are not able to survive the winter period. We demonstrated that the opening rhythms of the SEC could be studied in more detail when traditional flow-through CO2 respirometry was supplemented with still-air coulometric respirometry, which also works as an activity indicator.
A.K. conceived the project and wrote the manuscript. K.J. analysed the data and wrote the manuscript. L.M. revised the manuscript and assisted with experimental planning. A.P. revised the manuscript and presented data. E.M. and A.M. took microphotographs. I.H.W. and M.M. revised the manuscript. K.H. and I.S. collected data from the experiments. All authors approved the manuscript.
The research was supported by the Estonian Science Foundation (grant nos 9449 and 9450), institutional research funding IUT36-2 of the Estonian Ministry of Education and Research, and research funding 3-2_8/4304-1/2015 of the Environmental Investment Centre.
The authors declare no competing or financial interests.