Flight-motor-driven respiratory air flow in the hawkmoth Manduca sexta
Institut für Zoologie I, Universität Erlangen-Nürnberg, Staudtstrasse 5, D-91058 Erlangen, Germany
*e-mail: ltwthal{at}biologie.uni-erlangen.de
Accepted April 3, 2001
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Summary |
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During shivering, ventilation pulses are generated by the flight muscles reminiscent of an autoventilation mechanism with tidal air flow. During steady flight, however, a unidirectional airstream arises with a mean negative (subatmospheric) pressure at the first (mesothoracic) spiracles and a mean positive pressure in the mesoscutellar air sacs. As a result of this pressure difference during flight, CO2 is emitted only at the posterior spiracles.
The suction force for the inspiration flow at the anterior spiracles is generated by the flight apparatus as a result of prevention of inspiration through the posterior thoracic spiracles. During the downstroke, the volume of the thoracic air sacs increases, while the posterior thoracic spiracles are automatically enclosed in the subalar cleft below the wing hinge and are probably closed. During the upstroke, the air sac volume decreases and the moth expires through the open posterior spiracles.
Key words: insect, respiration, air pressure, CO2 emission, trachea, spiracle, flight, muscle, gas supply, hawkmoth, ventilation, Sphingidae, Manduca sexta.
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Introduction |
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In insects such as locusts, hymenopterans, scarabaeid beetles and hawkmoths, the abdominal pumping movements observed during flight are assumed to contribute to respiratory gas exchange in the flight muscles (Snodgrass, 1935; Fraenkel, 1932a), but the effects of this abdominal pumping on tracheal ventilation of the thorax have rarely been measured. Our knowledge regarding respiration during insect flight is based mainly on the work of P. L. Miller (Miller, 1960; Miller, 1966; Miller, 1974; Miller, 1981) and Weis-Fogh (Weis-Fogh, 1964a; Weis-Fogh, 1964b; Weis-Fogh, 1967).
In locusts, haemolymph pressure data gave rise to the autoventilation model (Weis Fogh, 1964a; Weis-Fogh, 1967). Air is assumed to be sucked in and blown out equally through all the thoracic spiracles by deformations of the air sacs around and between the flight muscles as a consequence of wing movements. In locusts, a unidirectional flow between the anterior body and the abdomen caused by abdominal pumping was measured that contributed to the tidal flow of autoventilation (Miller, 1960; Weis Fogh, 1964a; Weis-Fogh, 1967).
In flying cerambycid beetles, the volume of air passively entering the exposed anterior spiracles and passing through the primary tracheae was analyzed and termed through-draught ventilation (Miller, 1966). In these beetles, this mechanism is assumed to be combined with autoventilation of the smaller secondary tracheae.
As powerful fliers, hawkmoths are capable of increasing their metabolic rate by up to 148-fold from rest to full flight (Bartholomew and Casey, 1978). They therefore have, like hummingbirds (Berger and Hart, 1972), one of the highest recorded metabolic rates. It is unclear how hawkmoths manage to meet the increased O2 demands during flight with their tracheal supply system, and the mechanism of air flow remains unresolved. Remarkably, the O2 content of the flight muscles during steady flight exceeds even the resting level (Komai, 1998). It is the aim of the present study to contribute to the understanding of this very efficient supply mechanism. Special care was taken to use healthy moths and to avoid invasive techniques and unphysiological conditions as far as possible.
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Materials and methods |
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Moths were suspended at the descaled mesoscutellum with elastic layers of Pattex (Henkel, Düsseldorf, Germany) from a rigid rod. Narcosis was avoided throughout all procedures. Instead, preparatory steps were carried out very gently, allowing recovery periods of several hours. To stimulate flight, the normal nocturnal activity rhythm was used; dimming of the room light was sufficient. The moths frequently flew for several hours without further encouragement, such as using a fan. The moths remained suspended for the entire experimental period over 35 days. They were fed ad libitum with a 1218% honey solution at the beginning and sometimes again at the end of the recording session, which lasted several hours. At the end of the experiments, the moths were still healthy and could be separated from the mounting rod. All moths were used first for tracheal pressure measurements and then for CO2 emission measurements. The experiments were performed in a Faraday cage at 20±1°C.
The wingbeat was recorded by projecting the shadow of the left or right or both moving wings onto one or two silicon photocells (Conrad 55 mmx20 mm or Telefunken BPY 10 mmx3 mm) installed on the bottom of the Perspex specimen chamber. For high-resolution analysis of wing position in relation to the course of a tracheal pressure pulse, the shadows produced by the wing were projected on two vertically arranged sensors. One sensor was shaded by the wing during upstroke, the other one was shaded during downstroke. Both sensors show the complete wingbeat cycle, but the arrangement was optimal when the curves of both sensors were symmetrical. Only the curve of the sensor shaded during the downstroke was used for the documentation of wing movement (see Fig.6). For this high temporal resolution, the sampling rate was increased to 40kHz; in contrast, 400Hz was used during most long-term measurements. The photocell voltage signal showed a delay of 2.53ms relative to the pressure pulse which was accounted for in the analysis of the recordings (e.g. in Fig.6). For observations of thoracic deformation and spiracular activity, the left side of the thorax was descaled and illuminated using an industrial strobelight (Drello, Mönchen Gladbach, Germany).
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For CO2 measurement, the air flow from either the anterior (Fig.1A) or the posterior compartment (Fig.1B) was conveyed directly to an infrared gas analyzer URAS 3G (Hartmann & Brown) while the air from the other compartment was conveyed to a CO2-absorbing vessel containing NaOH. The pressure within both compartments was normally adjusted to the same value (between 10 and 100Pa). The detection limit was approximately 0.05µls-1 in the smaller specimen chamber and 0.1µls-1 in the larger one; the response time of the system was 1.4±0.2s. The baseline of the CO2 analyser and system was checked for drift after each experiment without the moth. In some experiments, the pressure of the posterior chamber was slightly increased to avoid an artificially high pressure at the anterior spiracles that would have prevented CO2 emission at SpI and, thus, might have caused unnaturally high CO2 outputs at SpII. A higher pressure in the posterior chamber should produce increased outflow through the anterior spiracles. The CO2 output was calibrated for each pressure regime and specimen chamber after the experiments using a motor-driven 50ml syringe simulating the release of a constant volume of pure CO2 at different rates (0.120µls-1). Data were acquired using an eight-channel MacLab interface, and calculations were performed using Chart 3.63 software on Power Macintosh computers.
Spiracle morphology
After descaling and cleaning, the thoracic spiracles were examined using a binocular microscope and photographed using a custom-made light scanning microscope and a field-emission scanning microscope at 2kV accelerating voltage (Hitachi S 800). For scanning electron microscopy, specimens were air-dried, gold-coated for 3min under argon plasma at 25mA and 2kV (Hummer JR) and glued with colloidal silver to aluminium stubs.
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Results |
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A single wingstroke in Manduca sexta lasts between 32 and 41ms. Analysis of pressure changes during a single wing stroke showed that the intratracheal pressure minimum at both measuring sites coincided with the second half of the downstroke (Fig.6). The pressure maximum occurred during the last third of the upstroke. The maximum and minimum pressures at SpI and the mesoscutellar air sac often coincide but may also be slightly shifted in time, with the maximum at the mesoscutellar air sac occurring 1.22.5ms earlier than that at SpI. The pressure minima of the mesoscutellar air sac and anterior spiracles could be shifted in both directions by 0.81.3ms.
CO2 emission
To investigate whether the observed pressure difference between SpI and the mesoscutellar air sac affects the pattern of air flow in the thorax, CO2 measurements in the split-specimen chamber were performed with the same air flow speed and pressure in both chambers. No CO2 emission was recorded from the anterior spiracles, which opened into the anterior chamber (Fig.1A, Fig.7A). All recorded CO2 emission was from the posterior spiracles (Fig.1B, Fig.7B). Even when the pressure in the posterior chamber was higher (P up to 25Pa) than in the anterior chamber, the CO2-containing air stream was not reversed (Fig.8, Fig.9). Only when the pressure in the posterior chamber was artificially raised to more than 25Pa above that of the anterior chamber was CO2 expired at the anterior spiracles. The moths were only capable of short periods of flight under these imposed pressure differences (Fig.9). Under these conditions, CO2 emission increased at the anterior spiracles during pauses between flights and decreased again during flight periods with a latency of 11.5s. A complete reversal of CO2 output from the anterior spiracles could not be achieved by the application of a higher pressure to the posterior chamber.
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Discussion |
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The unidirectional air stream observed in steady flight is not operative during shivering, when the mean tracheal pressure at the anterior spiracles and the posterior air sac oscillates around atmospheric pressure and the amplitude of the pressure pulses at the anterior and the posterior spiracles was only one-third to one-quarter of the amplitude during steady flight (Fig.3). During shivering, the mechanism is similar to autoventilation with a two-way (tidal) flow, as observed in Schistocerca gregaria (Miller, 1960; Weis-Fogh, 1967). In locusts, wing movements and the corresponding up and down movements of the nota result in air moving into and out of the anterior and posterior thoracic spiracles. In Manduca sexta, this flow becomes directional only with the greater downstroke amplitude that occurs during steady flight (Fig.4, Fig.5). At the anterior spiracles, as wingbeat amplitude increases, there is an increase in pressure amplitude correlated with a decrease in the mean pressure (Fig.5A). At the mesoscutellar air sac, an increase in wingbeat amplitude is correlated with an increase in the mean pressure (Fig.5B).
Komai (Komai, 1998) states that a hawkmoth has no shunt mechanism in the primary tracheae and that it does not use unidirectional ventilation flow and it is not known whether other large insects use unidirectional flow. However, the experiments in which an artificially high pressure was imposed on the posterior spiracles from outside and induced emission of CO2 through the anterior spiracles (Fig.8, Fig.9) provide further evidence for a unidirectional air flow. The minimum counter-pressure in the posterior chamber (P) necessary for an air flow reversal to overcome the internal pressure gradient produced by the moth was approximately 25Pa. This value corresponds to the lowest measured mean pressure gradient between the anterior spiracle and mesoscutellar air sac. This reversal of air flow must take place via longitudinal tracheae. Such a connection between the mesothoracic and metathoracic spiracles has been described in Manduca sexta (Eaton, 1988). Lateral primary tracheae with a simple spirally coiled intima could be traced from a complete histological series of transverse sections through the thorax and anterior abdomen of a smaller hawkmoth species Proserpinus proserpina (L. T. Wasserthal, unpublished data). These tracheal stems clearly connect the anterior spiracles with the air sacs of the mesothorax, the metathorax and the first abdominal segment and, thereby, the spiracles of these segments. Gas exchange during flight, under continuous mean negative pressure at the anterior spiracles, can only function with a corresponding mean positive pressure at the posterior spiracles.
Generation of unidirectional air flow by a thoracic suction pump
The intratracheal pressure maximum occurred at the end of the upstroke, and the haemocoel pressure measured in the locust was also greatest at this point (Weis-Fogh, 1967). This does not support the suggestion that the air sacs are compressed by contraction of the dorsal longitudinal muscles (DLMs) during the downstroke in Agrius convolvuli (Komai, 1998). According to the generally accepted model of indirect flight muscle systems (Pringle, 1975; Chapman, 1998), the thorax of hawkmoths is deformed by the action of the flight apparatus as follows. In Manduca sexta, wing upstroke is correlated with a slight flattening of the thorax caused by contraction of the dorsoventral muscles (DVMs), thus compressing the large air sacs lying between the tergites and the DLMs. These air sacs are continuous with the branching intramuscular air sacs (Fig.2). During the downstroke, the thorax becomes slightly arched and shortened by contraction of the DLMs, increasing the volume of the posterior and dorsal thoracic air sacs. Thus, during the downstroke, the intratracheal pressure is negative (Fig.6). During the downstroke, the posterior thoracic spiracles are covered by the intersegmental cleft and are probably closed by the valve flap fitting against the opposite cuticle, while the anterior spiracles remain open (Fig.11B, Fig.12), so air can be inspired unhindered only through the anterior spiracles and will be sucked posteriorly and dorsally into the large air sacs. Thus, a relatively simple mechanism involving volume changes of the thoracic air sacs together with the prevention of air inflow into the posterior thoracic spiracles is responsible for the retrograde air flow through the pterothorax. Whether the closing muscle of the posterior thoracic spiracle (Eaton, 1988; Nikam and Khole, 1989) is also involved in the closing mechanism during the downstroke needs to be investigated. Active closing and opening could be observed to result from passive wing bending in some individuals. It is probable that contraction of this closing muscle is synchronized with contraction of the DLMs, but a passive mechanism of valve closure may also be involved because the valve flap has no perforations and is unlikely to remain open against inspiratory suction, especially when the flap is close to the opposite cuticle. The role of the first abdominal spiracle, which communicates with the thoracic tracheal system, in the air supply mechanism is unclear. The first abdominal spiracles have an even denser peritrema filter structure than the anterior thoracic spiracles, so they are likely to serve for inspiration rather than for expiration.
The posterior thoracic spiracle as a valve for the expiratory air stream
The morphology of the posterior thoracic spiracle and its remarkable differences from the anterior spiracle have generated relatively little interest. The location of the posterior thoracic spiracle in the subalar cleft immediately below the wing hinge indicates that it is coupled to wing movements. In contrast to the extremely dense filter apparatus of the anterior spiracle (and of the first abdominal spiracle), the absence of any filter structures suggests that the posterior thoracic spiracle is adapted mainly for expiration. In contrast to all the other spiracles, it has no inner valve. It is only present after metamorphosis; caterpillars and pupae lack a functional spiracle on the metathoracic segment. The spinose margin of the valve lip and the spinose perispiracular cuticle could serve to prevent tight adhesion when the valve lip touches this soft cuticle.
Although the anterior spiracles are also open during the upstroke, expiration through the posterior spiracles may be easier because of their vicinity to the large posterior air sacs and because of the absence of filter structures around SpII, both of which should result in less resistance to the air stream.
Mechanisms of tracheal ventilation in flying insects
In other insect orders with similar fundamental differences between the anterior and posterior thoracic spiracles, a similar unidirectional air stream might occur during flight. While, in resting insects, observations of spiracular closing and opening behaviour have led to the conclusion that respiratory air flow is unidirectional during abdominal pumping movements (for references, see Mill, 1985), only few studies using split-chamber experiments have measured the effects of such directional air streams (Fraenkel, 1932b; Bailey, 1954; Wasserthal, 1996). For flying insects, three mechanisms of tracheal ventilation have been described: autoventilation with tidal flow, abdominal pumping and passive stream by the Fahrtwind. The latter has been measured in cerambycid beetles and was termed the through-draught mechanism (Miller, 1966). This retrograde air flow is suggested to be caused by the air stream passively entering the exposed anterior spiracles. A similar passive inflow of air into the anterior lepidopteran spiracles is unlikely because of the presence of dense scale layers and the peritrema filter apparatus. In flying locusts, a retrograde air stream produced by abdominal ventilatory movements is superimposed on the two-way autoventilation system (Miller, 1960; Weis-Fogh, 1967). In this case, all the thoracic spiracles are open during flight. The rate of flow through the spiracles is identical in both directions, so that no direction-sensitive valves are involved. The tidal flow of autoventilation mainly involves spiracles 2 and 3, while during abdominal pumping air is assumed to supply mainly the head via spiracles 1 (inspiration) and 510 (expiration) (Miller, 1960; Weis-Fogh, 1967). In Schistocerca gregaria, the haemolymph pressure amplitude near a ventral thoracic air sac was 100150Pa generated by the flight motor and 300500Pa generated by abdominal pumping (Weis Fogh, 1967). At 50450Pa, the pressure pulse amplitudes in Manduca sexta are within the range of values for the locust, but they are produced only by the the flight motor with no measurable contribution from abdominal pumping. Abdominal ventilatory movements have been described in other hawkmoth species (Sphinx ligustri and Deilephila elpenor) during flight (Fraenkel, 1932a). It is likely that the effects of these movements are restricted to the abdomen, as in the giant silk moth Attacus atlas at rest, where they are correlated with CO2 bursts recorded from the abdominal chamber (Wasserthal, 1996).
Concluding remarks
The present results in Manduca sexta reveal a new mechanism for the supply of oxygen to the flight muscles and extend the spectrum of respiratory air supply mechanisms discussed above. The flight motor itself, by increasing thoracic volume during the downstroke and the simultaneous automatic closure of the metathoracic spiracle in the subalar cleft, produces a retrograde airflow through the pterothorax, with CO2 output occurring only at the posterior spiracles. The flight-motor-driven retrograde air flow is reminiscent of a turbo engine that sucks fresh air in at the anterior openings and releases the used air through the posterior openings. This efficient respiratory air supply may provide part of the explanation for the increase in O2 levels in sphingid flight muscles during steady flight (Komai, 1998) and the physiological basis for their powerful and long-lasting flight characteristics and hovering ability.
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Acknowledgments |
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References |
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