The Opening–Closing Rhythms of the Subelytral Cavity Associated With

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RESEARCH ARTICLE The opening–closing rhythms of the subelytral cavity associated with gas exchange patterns in diapausing Colorado potato beetle, Leptinotarsa decemlineata Aare Kuusik1, Katrin Jõgar1,*, Luule Metspalu1, Angela Ploomi1, Enno Merivee1, Anne Must1, Ingrid H. Williams1, Külli Hiiesaar1, Ivar Sibul2 and Marika Mänd1

ABSTRACT The SEC is considered to aid in diminishing the level of The opening–closing rhythms of the subelytral cavity and associated respiratory and cuticular transpiration as it reduces the diffusion gas exchange patterns were monitored in diapausing Leptinotarsa gradient between the tracheal system and peripheral air (Dizer, 1955; decemlineata beetles. Measurements were made by means of a flow- Cloudsley-Thompson, 1964). This water-conserving hypothesis for the SEC was later supported by data showing that the air in the through CO2 analyser and a coulometric respirometer. Under the elytra of these beetles there is a more or less tightly enclosed space, the cavity has a high water content (Ahearn, 1970; Zachariassen, 1991; subelytral cavity (SEC). When the cavity was tightly closed, air Hadley, 1994; Cloudsley-Thompson, 2001). Ahearn (1970) pressure inside was sub-atmospheric, as a result of oxygen uptake suggested that, in desert tenebrionid beetles, a unidirectional into the tracheae by the beetle. In about half of the beetles, regular airflow passes backwards with inspiration via the mesothoracic opening–closing rhythms of the SEC were observed visually and also spiracles and expiration via the subelytral spiracles. Nicolson et al. recorded; these beetles displayed a discontinuous gas exchange (1984) suggested that after air has been expelled via the spiracles, CO2 accumulated in the SEC is eliminated to the atmosphere by pattern. The SEC opened at the start of the CO2 burst and was immediately closed. On opening, a rapid passive suction inflow of lifting of the elytra. However, when CO2 release was measured atmospheric air into the SEC occurred, recorded coulometrically as separately and simultaneously from the mesothoracic spiracles and from the SEC in the flightless dung beetles Circellium bacchus a sharp upward peak. As the CO2 burst lasted beyond the closure of (Duncan and Byrne, 2002, 2005; Byrne and Duncan, 2003; Duncan, the SEC, we suggest that most of the CO2 was expelled through the mesothoracic spiracles. In the remaining beetles, the SEC was 2003), there was a predominantly anterograde or tidal airflow in the continually semi-open, and cyclic gas exchange was exhibited. The tracheal system. According to Byrne and Duncan (2003), the air locking mechanisms and structures between the elytra and between within the SEC in these beetles was high in water vapour and in CO2 the elytra and the body were examined under a stereomicroscope and but the concentration of O2 was below atmospheric. Dizer (1955) by means of microphotography. We conclude that at least some of the demonstrated that removal of the elytra resulted in a substantial L. decemlineata diapausing beetles were able to close their subelytral increase in water loss in wingless beetles with fused elytra, but little cavity tightly, and that the cavity then served as a water-saving device. increase in winged species capable of flight, suggesting that in the latter the cavity was not hermetically sealed. Byrne and Duncan KEY WORDS: Discontinous gas exchange, Cyclic gas exchange, (2003) showed that if the SEC in a flightless dung beetle was open, Respiration, Water loss rate there would be a 74% increase of water loss rate. Further data indicate that in beetles the SEC is needed to avoid INTRODUCTION death by desiccation. The elimination of water-conserving The subelytral cavity (SEC) is an air-filled space under the elytra properties of the SEC may be used as a physiological method of and above the abdomen characteristic in some beetles. The controlling some insects. Treatment of last instar larvae of L. abdominal and metathoracic spiracles open into this cavity. In decemlineata with non-lethal doses of neem preparation (Kuusik flightless and wingless beetles, the cavity is opened by lifting the et al., 2001a) or with a synthetic analogue of juvenile hormone elytra (Duncan, 2002; Duncan and Byrne, 2002; Schilman et al., (Kuusik and Kogerman, 1978) resulted in morphological failure of 2007; Duncan et al., 2010). In winged beetles, e.g. the Colorado the elytra and high mortality rates of beetles during winter diapause. potato beetle, Leptinotarsa decemlineata Say 1824, the SEC is The exposure of L. decemlineata pupae in dry conditions produced opened by lowering the last abdominal segments and closed by elytral deformations in adults and lethal water loss during pressing these segments against the elytra without lifting them hibernation (Pelletier, 1995). The topical application of neem (Vanatoa et al., 2006). 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 1Department of Plant Protection, Institute of Agricultural and Environmental continuous respiration occurred (Sibul et al., 2004a). Many toxic Sciences, Estonian University of Life Sciences, Kreutzwaldi 1, Tartu 51014, Estonia. substances are able to evoke respiratory failure in insects (reviewed 2Department of Silviculture, Institute of Forestry and Rural Engineering, Estonian University of Life Sciences, Kreutzwaldi 5, Tartu 51014, Estonia. in Kuusik et al., 2001a; Jõgar et al., 2005; Karise and Mänd, 2015). DGE, in which CO2 is released periodically as bursts, is a *Author for correspondence ([email protected]) common respiratory pattern in many insects (reviewed in Lighton, K.J., 0000-0002-2330-1995 1994, 1996; Chown and Nicolson, 2004; Quinlan and Gibbs, 2006). The DGE cycle has three consecutive phases, the open or O-phase,

Received 4 May 2016; Accepted 20 August 2016 the facultative closure or C-phase and the flutter or F-phase. During Journal of Experimental Biology

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Wobschall and Hetz (2004) registered O2 uptake in diapausing List of abbreviations atlas moth, Attacus atlas, pupae by simultaneously measuring the C-phase closed phase tracheal pressure and volume changes in the tracheal system CGE cyclic gas exchange (plethysmometry). Sláma (1984, 1988) used the micro- DGE discontinuous gas exchange anemometric method and also measured changes in haemocoelic F-phase flutter phase pressure using electronic strain-gauge transducers. Constant- IR infrared IRA IR actograph volume coulometric respirometry has been used to record the IRGA infrared gas analyser (flow-through analyser) peaks due to PSIs in diapausing pupae of Pieris brassicae (Jõgar O-phase open phase et al., 2004, 2008, 2011) and Mamestra brassicae (Jõgar et al., PSI passive suction inspiration 2007, 2014). This method has also been used to record PSI peaks at PSV passive suction ventilation the openings of the SEC in adult H. abietis (Sibul et al., 2004a,b) RH relative humidity and in L. decemlineata beetles (Vanatoa et al., 2006). SEC subelytral cavity – WLR water loss rate 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 O-phase, CO2 is expelled by burst. During the C-phase, the the SEC were also investigated. spiracles are closed and presumably no gas exchange occurs. In the tracheae, the pressure level is reduced because the tissues consume MATERIALS AND METHODS O2 and the tissues as well as haemolymph buffer CO2 by means of Insects bicarbonates (Miller, 1974; Sláma, 2010). Throughout the F-phase, Adult L. decemlineata were collected in autumn from potato fields the spiracles open and close rapidly when the negative pressure near Tartu, Estonia (58°21′N, 26°39′E). They were reared in the gradient allows intake of air through them into the tracheae. This laboratory on fresh potato leaves under short day conditions phenomenon is described as passive suction ventilation (PSV) or [10 h:14 h light:dark, 18–20°C, 60–70% relative humidity (RH)]. passive suction inspiration (PSI) (see Sláma, 1988), and occurs Diapausing beetles were placed in 1 l glass jars half-filled with without muscular contractions (Miller, 1974, 1981). The PSI during lightly moistened peat, into which they burrowed; these jars were this phase may be a mechanism to restrict water loss in insects, as the held in a refrigerator at 5–6°C, 40–50% RH, in darkness. air inflow impedes the release of CO2 gas and water (see Kestler, Experiments were started when the beetles had entered deep 1978, 1980, 1982, 1991). diapause. To ensure that beetles reached a stable diapause state, they In contrast to the DGE, in cyclic gas exchange (CGE), CO2 is were kept in the peat for 1 month. Before measurements were taken, released periodically by bursts with a little also being released beetles were removed from the refrigerator and adapted to room during the interburst periods (Gibbs and Johnson, 2004). A third conditions, at 21±2°C and 35±10% RH for 1 day. Then they were common gas exchange pattern is continuous respiration, in which placed individually into Petri dishes (diameter 9 cm). They were CO2 release is more or less steady (Hadley, 1994). weighed using an analytical balance (Explorer Balances, Ohaus Several hypotheses have been proposed to explain the adaptive Corporation, Pine Brook, NJ, USA) to within 0.1 mg. Preliminary benefit of DGE cycles or their mechanistic origin (reviewed in measurements by means of flow-through respirometry were Kestler, 1985; Lighton, 1988; Chown and Nicolson, 2004; Chown conducted on 12 beetles that displayed DGE cycles with clear et al., 2006; Contreras et al., 2014; Matthews and Terblanche, 2015). opening–closing rhythms, and six individuals with the SEC Originally, DGE was considered to be an adaptation to restrict continually open and that displayed CGE. To supress the beetles’ respiratory water loss (Edney, 1977; Hadley, 1994; Lighton, 1988, activity, we used the knocking method described by Kestler (1991) 1996; Chown et al., 2002, 2006), although only a small fraction of and Metspalu et al. (2002). Tonic immobility was achieved by the overall water loss is thought to be lost through respiration knocking on the insect vessel. The neuromuscular basis of tonic (reviewed in, for example, Quinlan and Lighton, 1999; Quinlan and immobility is summarized by Roeder (1953). V̇ Hadley, 1993; Chown, 2002). First, CO2 was measured with an infrared gas analyser (IRGA; Currently, the main method for studying gas exchange patterns flow-through CO2 respirometer LI-COR model 7000, Lincoln, NB, V̇ and metabolic rates is by flow-through CO2 respirometry. Oxygen USA); then, in beetles displaying DGE cycles, O2 was measured by uptake generally tracks CO2 release, but O2 consumption is much coulometric respirometry. During flow-through measurements, the more difficult to measure and these measurements are often omitted state of the SEC was visually observed through the transparent (Quinlan and Gibbs, 2006) or rarely performed. For example, insect chamber under an Olympus SZ-CTV stereo microscope simultaneous CO2 and O2 flow-through measurements have been (Olympus Optical Co. Ltd, Tokyo, Japan). All respiratory performed in the tok-tok beetle Psammodes striatus (Lighton, 1988) measurements lasted at least 2 h. and in the dung beetle C. bacchus (Duncan and Byrne, 2002) to study the role of the SEC. Infrared actography However, flow-through respirometry cannot enable recording of The electrolytic respirometer and IRGA data were combined with an the regular passive suction uptake of air (oxygen) into the tracheae infrared (IR) cardiograph of the insects, which we refer to as the IR or from the atmosphere into the SEC. Moreover, it is unable to actograph (IRA), because it records not only heart pulses but also all detect the real F-phase with PSI in the absence of a recordable other abdominal contractions, including muscular ventilating. An diffusive component. This is because of the inward bulk flow of air IR-emitting diode was placed on one side of the chamber near the into the tracheal system, which is practically equal to a small and ventral side of the abdomen, while an IR-sensitive diode was placed presumably undetectable decrease in the flow rate of air through the on the opposite side of the chamber (see Metspalu et al., 2001, respirometer chamber (Lighton, 1988, 1994, 1996). Thus, other 2002). The light from the IR-emitting diode was modulated by the methods should be used. contractions of the heart and skeletal muscles. The amplitude of Journal of Experimental Biology

3413 RESEARCH ARTICLE Journal of Experimental Biology (2016) 219, 3412-3419 doi:10.1242/jeb.142489 output voltage fluctuations reflected the vigour of the muscular 0.16 contractions (see S. K. Hetz, Untersuchungen zu Atmung, A 0.14 Kreislauf und Säure-Basen-Regulation an Puppen der tropischen Schmetterlingsgattungen Ornithoptera, Troides und Attacus, PhD 0.12 thesis, Friedrich-Alexander-Universität, Erlangen-Nürnberg, 1994; Hetz et al., 1999). Abdominal contractions resulted in downward 0.1 spikes while muscular relaxations were directed upward. 0.08

Coulometric respirometry 0.06

Coulometric respirometers usually work in an interrupted regime 0.04 (on–off) of electrolysis (e.g. Taylor, 1977; Heusner et al., 1982). By contrast, our coulometric respirometry (a volumetric 0.02 manometric system) was characterised by a continuously 0 (uninterrupted) O2-compensating system (Kuusik, 1977; Kuusik ) 0 20 40 60 et al., 1996; Tartes et al., 1999, 2002). This setup has also been –1

described by Lighton (2008). This respirometer ensures (ml h 2 1.5 continuous and adequate replacement of consumed O2 with . CO B V 0.18 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 1.4 V̇ −1 graphs as O2 in ml h . The system also records transient changes in the rate of release of CO2. In our respirometer, we did not use 0.16 the switching electrodes of electrolysis; instead, the electrolysis * 1.3 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 0.14 Contractions (V) photodiode by the photosensitive element of a transistor (Silicon 1.2 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 0 1.1 0 5 10 15 recording trace. The electrolysis current depends on the intensity of Time (min) the light falling on the phototransistor. The ethanol meniscus in the glass capillary serves as a shutter to screen the photosensitive area Fig. 1. Discontinuous gas exchange (DGE) recorded with flow-through respirometry in diapausing adult Leptinotarsa decemlineata. (A) A from light. The electrochemical equivalent of O2 generation has been reported as 209.5 µl O mA h−1 (Taylor, 1977). This value recording of two cycles of DGE in L. decemlineata possessing regular 2 opening–closing rhythms of the subelytral cavity (SEC). The horizontal line was used to convert the readings of the event recorder to O2 indicates the fraction recorded with higher resolution in B. (B) Higher resolution consumption values for our experimental conditions. recording of the indicated fraction of the gas exchange cycle. The upper trace is The coulometric respirometer allowed simultaneous recording the simultaneous recording by the infrared actograph (IRA); the abdominal – of O2 consumption, sudden O2 (air) uptake (known as PSI) by movements during opening closing of the SEC (double line), pumping convection into the tracheae at micro-openings of the spiracles, movements of active ventilation at CO2 burst (arrows) and weak abdominal discrete CO releases by bursts, abdominal pumping movements pulsations (asterisks) are indicated. All these movements are superimposed 2 on the heartbeats, which occur at a higher frequency. 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 Corp., USA). The empty chamber served to determine the baseline events, are not compensated and lead to corresponding rapid for the measurements. All measurements were performed in a changes in the electrolysis current, reflected as spikes on thermostat Sanyo incubator (Sanyo MR-254, Japan). recordings. Thus, our coulometric respirometer also served as an activity detector. Microphotography An Eclipse FN1 (Nikon, Japan) light microscope at a magnification

Flow-through CO2 respirometry of 40–200× and NIS Elements Imaging software version 4.30 CO2 release was measured using an IRGA. Ambient gas from (Nikon, Japan) were used for stacked photo-micrograph acquisition outside the laboratory was scrubbed of CO2 and water by passing it (automated multi-focus imaging, step size 1.4 µm with 20× lens, through columns containing soda lime and Drierite/Ascarote. The 14 µm with 4× lens). The number of stacked images ranged from CO2 channel was calibrated with commercially available span gas 115 to 255. (Linde AG, Höllriegelskreuth, Germany). We used an air flow rate of 33 ml min−1, controlled by an electronic flow meter (model Data acquisition and statistics 5067-0223, Agilent Technologies, USA). Respirometry was carried Computerised data acquisition and analysis were performed using out in dry air; the insect chamber (1.5 ml) was perfused with dry DAS 1401 A/D (analog–digital) hardware and TestPoint software (4–5% RH), CO2-free air. The humidity and temperature of air (Keithley, Metrabyte, Cleveland, OH, USA) with a sampling rate of entering the insect chamber were continuously measured using a 10 Hz. Four bipolar channels allowed simultaneous recording of

HygroPalm 3 for digital HygroClips probes (Rotronic Instrument four events. Data were analysed using Student’s t-test, with StatSoft Journal of Experimental Biology

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Table 1. Characteristics of the discontinuous gas exchange (DGE) cycle We compared body mass loss rate, interpreted as water loss rate in Colorado potato beetle Leptinotarsa decemlineata (N=12) (WLR), between beetles with normal opening–closing rhythms of Mean±s.e.m. 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 Body mass (g) 0.127±0.021 ̇ −1 we removed a small fraction of the elytra, we did not observe any VCO2 (µl g h ) 148.2±1.940 DGE period (s) 1149±76.343 haemolymph. Thus, water loss increased substantially when the DGE frequency (mHz) 0.871±0.0604 SEC was open. Comparison of WLRs in beetles before and after the ̇ −1 VO2 (µl g h ) 128.7±1.048 wings were removed during DGE showed that the WLR was ̇ ̇ significantly higher in beetles with removed wings (Student’s t-test VCO2 was measured by an infrared gas analyser; VO2 was measured by coulometric respirometry. WLR, t=−7.74, d.f.=10, P<0.05). v10 (StatSoft Inc., Tulsa, OK, USA). The significance level was set Coulometric measurements at 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 RESULTS (Fig. 3). This upward peak resulted in rapid uptake of air into the Flow-through measurements SEC from the atmosphere by the principle of PSI, indicating that Visual observation and recording of the opening–closing rhythms of there was sub-atmospheric air pressure inside the SEC. In some the SECs of many L. decemlineata beetles showed that the cavity was individuals the peak of air uptake was absent, suggesting that their opened by lowering the last abdominal segments, and closed by SEC was not tightly closed, i.e. it was leaking. pressing these segments upon the elytra, while the elytra were not After opening of the SEC by removal of the elytra, the large lifted. Commonly, the SEC was opened and immediately closed, and upward peaks were lost and a clear pattern of O-, C- and F-phases of one or two movements were done by the last abdominal segments. gas exchange appeared in the coulometric recording, suggesting The SEC was usually opened near the start of CO2 emission, while DGE cycles. The F-phase was recognised by spikes of micro- later it was kept in a closed state until the subsequent burst. opening of the spiracles and PSIs (Fig. 4). From these spikes, we Using flow-through CO2 respirometry, we studied how the state measured the duration of every micro-opening, which in the first of the SEC was associated with gas exchange patterns. The clear spikes of PSIs was 0.4–0.6 s, while in later spikes it was 1.5–1.8 s. DGC with open, closed and flutter phases was exhibited only in beetles with the regular opening–closing rhythms described above. The elytral locking mechanism The characteristics of DGE are given in Table 1. The IRA recording, Detailed visual examination and microphotography of L. made simultaneously with flow-through CO2 measurements, decemlineata elytra using a stereomicroscope revealed that the showed spikes due to opening–closing of the SEC (Fig. 1). The medial (sutural) margins of two elytra are asymmetrically designed: burst of CO2 started some minutes before the SEC opened, and the narrow sutural face of one elytron consists of two ridges, a dorsal continued after the closing of the cavity; this was why we concluded ridge and a ventral ridge with a groove between them. The ridge thatmostCO2 was released from the mesothoracic spiracles and not (key) of one elytron locks into the groove of the other. This lock- through the SEC. and-key construction forms the medial sutural lock of the two elytra In beetles whose SEC was continually semi-open or leaking, (Fig. 5). mainly CGE was recorded, i.e. during the interburst period A similar lock-and-key structure locks the elytra to the body. noticeable volumes of CO2 were released, while no C-phase was Along the inner surface of the outer margin of the elytron is a observed. In these beetles, irregular abdominal contractions were typical and no periods of active ventilation occurred (Fig. 2). 0.14

2.0 0.12 0.8 0.10 0.7 1.5 ) –1 0.08 0.6 (ml h

2 0.06 ) * 0.5 . O * * –1 1.0 V 0.4 0.04 (ml h 2

. CO

V 0.3 Contractions (V) 0.02

0.2 0.5 0 0 1020 30 40 50 0.1 Time (min) 0 0 0 4128 16 Fig. 3. Typical gas exchange cycle in adult L. decemlineata possessing Time (min) regular opening–closing rhythms of the SEC. Data were recorded with a coulometric respirometer. External passive suction (convective) uptake into

Fig. 2. Cyclic gas exchange recorded with flow-through respirometry in the SEC caused large upward peaks followed by CO2 bursts (asterisks); flutter L. decemlineata ̇ beetle. The lower trace shows the VCO2 for a beetle in which phase begins after the CO2 burst. Note the upward and downward spikes due the SEC is continually semi-open. Irregular abdominal contractions on the to abdominal active ventilation movements during the CO2 burst. Between simultaneous recording by the IRA are visible (upper trace). bursts are clear spikes resulting from weak abdominal pulsations. Journal of Experimental Biology

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0.6 0.18 was also demonstrated in flightless dung beetles by Duncan (2002, 2003) and Byrne and Duncan (2003). 0.4 Our observations in L. decemlineata demonstrated characteristic 0.16 convective uptake or passive suction inflow, i.e. inspiration (PSI) of 0.2 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 0.12 –1 closure at the start of the CO2 burst has also been recorded in 0 H. abietis beetles (Sibul et al., 2004a,b). The decrease in air pressure (ml h 2

. O 0.08 in the SEC indicated oxygen depletion into the tracheae. Similar V –0.2 oxygen uptake from the SEC into the tracheae has been described in Contractions (V) VCF dung beetles, but here the leaky elytra would enable air to enter the 0.04 –0.4 SEC by passive mass flow, without opening of the SEC by lifting the elytra (Duncan et al., 2010). 0 –0.6 The flightless beetles with fused elytra are able to hermetically 0 28124 610 seal the SEC and to open it again by lifting the elytra (Gorb, 1998; Time (min) Frantsevich et al., 2005). Our visual examination of L. decemlineata elytra under the stereomicroscope suggests that these beetles are Fig. 4. A gas exchange cycle recorded with coulometric respirometry in a L. decemlineata beetle after removal of its elytra. The lower trace shows able to close their SEC, if not hermetically then at least tightly, by ̇ means of special structures: the sutural (medial) ridges of two elytra VO2, with the actively ventilated O-phase (V-phase), closed phase (C-phase) and flutter phase (F-phase) indicated. Note the upward spikes during the forming a lock. A tight seal between the elytra and the body is F-phase caused by micro-opening of the spiracles and passive suction possible because of the close fit of the ridge on the edge of the inspiration into the tracheae. The upper trace is a simultaneous recording with elytron and the groove along the body side at the pleural area. A an IRA, showing active ventilation during the O-phase; the more frequent similar elytron-to-elytron locking system has been described in spikes are the heartbeats and abdominal pulsations. some other winged beetles, but elytra-to-body locking in flying and flightless beetles is performed by microtrichia (Gorb, 1998; soft rounded ridge. Along the body side on the pleural area there Frantsevich et al., 2005). is a groove. Elytron-to-body locking occurs by pressing the soft Water loss increased by 62% in L. decemlineata beetles when the elytron ridge into the groove on the pleural area of the body side SEC was opened by removing a section of an elytron. Therefore, we (Fig. 6). These interlocking structures probably allow the SEC to hypothesise that L. decemlineata beetles with regular opening– be closed tightly. closing rhythms of the SEC are able to close this cavity tightly or We suggest that the elytra provide complete cover and also even hermetically, and that it serves as a water-saving device. A protection for the delicate hindwings and abdomen against external similar water-conserving role of the SEC has been described in disturbances, but also reduce overall water loss in individuals that flightless dung beetles, in which water loss increased by 74% when are able to close the SEC tightly (Fig. 7). the SEC was open (Duncan, 2003). After removing the elytra and wings from L. decemlineata, all DISCUSSION three DGE phases appeared, including the F-phase when monitored The Colorado potato beetle, L. decemlineata, uses several strategies by the coulometric respirometer, which recorded PSIs into the for hibernating in conditions of different humidity and one of these tracheae and air uptake from the atmosphere into the SEC. This is to tightly close its SEC to restrict water loss. In our experiments, pattern of flutter looked very similar to the flutter described in the opening–closing rhythms of the SEC were visually observed M. brassicae pupae (see Jõgar et al., 2014). The coulometric and recorded by an IRA and coulometric respirometer. During measurements in L. decemlineata revealed that only for a short time the DGE cycles, the SEC was opened and immediately closed at at the beginning of the F-phase was the gas exchange purely or the start of the CO2 burst emission. Therefore, we suggest from predominantly convective, presuming no efflux of CO2 and water our whole-body measurements in L. decemlineata beetles, as vapour, but that later gas exchange was instead diffusive. Our results representative of insects with flying ability, that most of the CO2 was are consistent with those of Wobschall and Hetz (2004) that in a expelled from the body via the mesothoracic spiracles, and not from diapausing lepidopteran pupa during the initial period of F-phase the SEC. That these spiracles are the main outlet for CO2 emission only the brief spiracular openings allow O2 uptake mainly by

Fig. 5. The lock-and-key structure ABof elytra in adult L. decemlineata. (A,B) Cross-sections of a left (A) and right (B) elytron forming a lock by co- D option of the medial dorsal side (D) D and ventral side (V), and the groove (G) between them. The locking occurs CA R by pressing the ridge (R) of the left G CA elytron, as a key, into the groove of the right elytron. A canal filled with V haemolymph is seen inside the edge of the elytron (CA). V 100 µm 100 µm Journal of Experimental Biology

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D OEE CE

SRR

100 µm 500 µm

Fig. 7. The elytra to body locking device in locked condition on the B abdominal side of adult L. decemlineata. The tight seal between the elytra and body allows closure of the SEC in adult L. decemlineata. The outer edge of the elytra (OEE) is indicated by double-headed arrows.

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 SRR role in L. decemlineata respiration. This has previously been demonstrated in several other insects (Kestler, 1980, 1985; Sláma, CE 1988; Wobschall and Hetz, 2004; Jõgar et al., 2011, 2014). The 500 µm 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. C 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 TER 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. SPA Competing interests STE The authors declare no competing or financial interests.

Author contributions 500 µm 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 Fig. 6. The elytra-to-body locking structures in adult L. decemlineata. microphotographs. I.H.W. and M.M. revised the manuscript. K.H. and I.S. collected (A) Cross-section of the inner surface of the left elytron margin. Indicated are data from the experiments. All authors approved the manuscript. the soft rounded ridge (SRR) and cuticular edge (CE). A canal filled with haemolymph is seen inside the edge of the elytron (CA). D, dorsal side; V, Funding ventral side. (B) The soft rounded ridge (SRR), indicated by the double-headed The research was supported by the Estonian Science Foundation (grant nos 9449 arrow, and cuticular edge (CE) along the inner surface of the outer margin of and 9450), institutional research funding IUT36-2 of the Estonian Ministry of the elytron. (C) Abdominal side with tergites (TER), sternites (STE) and soft Education and Research, and research funding 3-2_8/4304-1/2015 of the pleural area (SPA); the groove is indicated by the double-headed arrow. Note Environmental Investment Centre. that the rounded ridge of the elytron margin fits in this groove. References Ahearn, G. A. (1970). The control of water loss in desert tenebrionid beetles. J. Exp. convection or mass flow, which conserves water; later O2 uptake Biol. 53, 573-595. occurs mainly by diffusion, not conserving water. Byrne, M. J. and Duncan, F. D.(2003).The role of the subelytralspiracles inrespiration in the flightless dung beetle, Circellium bacchus. J. Exp. Biol. 206, 1309-1318. Our measurements showed that the release of CO2 was always accompanied by active ventilation or pumping in Chown, S. L. (2002). Respiratory water loss in insects. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 133, 791-804. L. decemlineata beetles. Kestler (1980, 1985, 2003; P. Kestler, Die Chown, S. L. (2011). Discontinuous gas exchange: new perspectives on diskontinuierliche Ventilation bei Periplaneta americana L. und evolutionary origins and ecological implications. Funct. Ecol. 25, 1163-1168. Journal of Experimental Biology

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