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Matching Spiracle Opening to Metabolic Need During Flight in Drosophila

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Science  30 Nov 2001:
Vol. 294, Issue 5548, pp. 1926-1929
DOI: 10.1126/science.1064821

Abstract

The respiratory exchange system of insects must maximize the flux of respiratory gases through the spiracles of the tracheal system while minimizing water loss. This trade-off between gas exchange and water loss becomes crucial when locomotor activity is increased during flight and metabolic needs are greatest. Insects that keep their spiracles mostly closed during flight reduce water loss but limit the flux of oxygen and carbon dioxide into and out of the tracheal system and thus attenuate locomotor performance. Insects that keep their spiracles completely open allow maximum gas exchange but face desiccation stress more quickly. Experiments in which water vapor was used as a tracer gas to track changes in the conductance of the respiratory system indicated that flying fruit flies minimize potential water loss by matching the area of the open spiracles to their gas exchange required for metabolic needs. This behavior maintained approximately constant pressure for carbon dioxide (1.35 kilopascals) and oxygen (19.9 kilopascals) within the tracheal system while reducing respirometric water loss by up to 23% compared with a strategy in which the spiracles are held wide open during flight. The adaptive spiracle-closing behavior in fruit flies has general implications for the ecology of flying insects because it shows how these animals may cope with environmental challenges during high locomotor performance.

The increased power output required for flapping flight places special demands and constraints on the respiratory system of flying insects (1). On the one hand, the respiratory system must permit the flux of oxygen (O2) and carbon dioxide (CO2) to and from flight muscles. On the other hand, the structures that permit respiratory exchange leave an animal susceptible to the loss of water vapor, thus increasing the danger of desiccation. The spiracles that occlude the outer openings of the insect tracheal system function as barriers that control the gas exchange between the network of air sacs, tracheas, and tracheoles and the outer environment. The terminal internal endings of the tracheal system, the tracheoles, are thought to be water filled, establishing a pressure that continuously drives water vapor out of the insect body when spiracles open for gas exchange (2–5). The potential threat of desiccation is greatest during flight, when the spiracles must remain open to sustain increased metabolic activity of the wing muscles. The metabolic cost of flight is not constant, however, but varies as an animal alters force production to carry loads or perform flight maneuvers (6–8).

In a diffusion-based respiratory system (9), the rate with which a gas is exchanged depends on two factors: the partial-pressure gradient between ambient and tracheal gas, and the cross-sectional area for diffusive flux (the area of the spiracle opening). The driving force on water vapor is assumed to be constant, so the flux of water depends only on the size of spiracle opening (10). The situation for the respiratory gases (CO2 and O2) is more complex, because the internal tracheal partial pressures might vary with metabolic rate. To limit water loss, an insect ought to match spiracle opening with its instantaneous metabolic demands. To test this hypothesis, I developed a method for indirectly measuring spiracle-opening area in tethered fruit flies,Drosophila melanogaster, flying within a respirometric chamber of a virtual-reality flight arena and estimated the concomitant changes in partial pressure of tracheal gases from simultaneous measurements of total flight force production, CO2 release, and water-loss rate (11, 12). The experiments were performed under visual closed-loop feedback conditions, in which the fly itself controls the angular velocity of a vertical dark stripe displayed in the arena by changing the stroke amplitude of its two wings during flight. Under these conditions, a fruit fly attempts to stabilize the azimuth position of the dark stripe in the frontal region of its visual field. While the animal actively controlled the dark bar, a superimposed background pattern of diagonal stripes was oscillated vertically around the animal.

In the fruit fly, 4 thoracic and 14 abdominal spiracles control the diffusive flux into and out of the tracheal system (Fig. 1, A and B). Although it is possible to record directly the mesothoracic spiracle opening area in flight of the large Hawaiian species D. mimica by video microscopy (Fig. 1C), it is difficult to monitor the opening and closing of all spiracles simultaneously during flight in smaller species ofDrosophila. The beating haltere, moreover, partly blocks the view on the opening of the large metathoracic spiracle. In addition, the small tracheole endings in fruit flies are inaccessible for direct pressure measurements of respiratory gases. I thus derived the average opening behavior of all 18 spiracles and the average partial pressure for tracheal CO2 and O2 during flight by estimating the conductance for tracheal gas flux from the measured water-loss rates of the fly.

Figure 1

Location of spiracle openings, and spiracle closing and opening behavior during flight, in the fruit flyDrosophila. (A) sp1, mesothoracic spiracle; sp2, metathoracic spiracle; sp3 to sp9, abdominal spiracles. (B) The oval thoracic spiracular openings are bordered by a thick sclerite and protected with hairs. Narrow flexible spiracular lids cover the tracheal entrance. Spiracles open by the elasticity of their cuticular structures and are held actively closed by the spiracle-closing muscle (32). Images are taken from D. virilis. (C) Closing and opening behavior of the right mesothoracic spiracle in D. mimica Hardy (3.06 mg wet body mass) during tethered flight and rest (33).

The tracheal partial pressure of CO2 and O2 may be expressed by using a geometric model for tracheal diffusion (13) in which the respirometric flux of a gas through the spiracles, , is the product of conductance for diffusion, G, and the driving force given by the difference of tracheal partial pressure, P T, and partial pressure in the ambient air, P A:Embedded Image(1)During flight, the conductance for CO2 and O2 is expected to vary with the changes in the open area of the spiracles. If certain assumptions are met, mean conductance for respirometric gas flux within a short period of time (100 ms) can be derived from the conductance of water vapor because both gases follow the same diffusive path. Assuming that the water-filled tracheole ends establish a constant driving force, conductance for water was derived from the measured water flux according to Eq. 1(14). The functional geometry of the tracheal system is given by the relationEmbedded Image(2)in which A T is the representative cross-sectional area and L T the characteristic length of the tracheal system, D is the effective diffusion coefficient of the gas, and β is the capacitance coefficient. Given these geometric parameters, the conductance for CO2 and O2 within the tracheal system may then be estimated by replacing D and β in Eq. 2 with values appropriate for CO2 and O2 (15,16). Assuming that maximum tracheal conductance matches the need for respiratory gas exchange at maximum locomotor performance, the total opening area of all spiracles,A S, can be approximated by setting maximum total spiracle area, A max, of 5949 μm2(17) equal to the extreme 1% of all values ofA T within a flight sequence in which metabolic needs are the greatest. Overall opening area in the flying insect may be eventually calculated byEmbedded Image(3)Tethered flying fruit flies typically modulated their total flight force production in response to the vertical oscillating diagonal stripe pattern in an attempt to minimize the slip of the retinal image (Fig. 2A, green and black traces). In freely flying flies this optomotor behavior would stabilize the vertical position of the animal in space. The modulation of flight force was accompanied by regular changes of both CO2release and respiratory water loss (Fig. 2A, red and blue traces). While the fly was responding to the motion of the moving pattern, CO2 flux varied by 35 ± 11% of its mean value, which reflected the changes in energy expenditures during flight (Table 1). The modulation of water loss rate by 26 ± 14% indicates either that (i) Drosophila does not hold its spiracles continuously open during flight as reported in previous studies on large beetles (18); (ii) an increase in strain amplitude of the asynchronous flight power muscles intensifies thoracic autoventilation and thus enhances water loss in proportion to flight force production; or (iii) changes in wing kinematics modulate induced air-flow velocity (19), which in turn alters cuticle water-loss rate due to changes in the boundary-layer condition around the animal. Experiments in which cuticle water-loss rates were determined in unrestrained flies, however, reveal that mean cuticle water-loss rate is relatively low (6.4 ± 1.4 μl g−1 body mass hour−1) and did not significantly increase with increasing flow velocity of the ambient air over a wide range of different values (linear regression, ttest on slope, P > 0.2) (Fig. 3). It thus seems unlikely that changes of water-loss rate during flight are due to alterations in cuticle water loss. The same holds for possible alterations in thoracic autoventilation of the tracheal system because the oscillations in length of the thoracic box in Drosophilarange only between 2 and 5% throughout a complete contraction-extension cycle of the flight muscles (20). Moreover, in small insects such as fruit flies, thoracic autoventilation and tracheal ventilation due to the Bernoulli effect (18) are thought to be greatly attenuated by the low Reynolds number for tracheal air flow (21).

Figure 2

Alterations in flight performance and concomitant changes in respiratory behavior of a single D. melanogaster fruit fly. (A) Representative recording showing water-loss rate (blue) and CO2 release (red) while the tethered animal varied its flight force (black) in response to the vertical motion of a stripe pattern (green) displayed in the flight arena. Negative angular velocities of the visual stimulus indicate that the stripe pattern moves downwards (gray areas). Both respirometric values are given in units that take into account the body mass of the fly. (B) Spiracle-opening behavior (green) and tracheal partial pressure for CO2 (red) in flying flies were determined with a geometric model for tracheal diffusion. Assuming that spiracles are held continuously open during flight, tracheal partial pressure would vary in-phase with flight-power requirements (blue).

Figure 3

Total water-loss rate (○) and cuticle water loss (•) in unrestrained D. melanogaster plotted against free-stream velocity of the ambient air. A total of 114 flies were exposed for 4 hours to different flow velocities of dry air within a small respirometric chamber in which water-loss rate and CO2 release were simultaneously measured by flow-through respirometry. Total water-loss rate was determined from weight loss. To yield cuticle water-loss rates, spikes of water loss due to discontinuous gas exchange, droplets, and proboscis extensions were estimated by flow-through respirometry and then subtracted from total loss. The gray areas represent the expected ranges of induced mean air velocity produced by the beating wings during flight (A) near the stroke plane and (B) in the “far” wake behind the animal, while the fly modulates its aerodynamic forces between minimum and maximum values (19).

Table 1

Alterations in flight performance, gas-exchange rates, spiracle activity, and partial pressure of CO2 and O2 with maximal and minimal power output. Modulation was defined as the ratio between the difference and half the sum of maximum and minimum performance. Force, total flight force production;*CO2, flight-specific flux of CO2 through spiracles; *H2O, flight-specific rate of water loss; A S, total spiracle opening area; P T,CO2, tracheal partial pressure for CO2;P P,CO2, predicted tracheal partial pressure for CO2 based on the assumption that the spiracles are held continuously open during flight; andP T,O2, partial pressure for tracheal O2. The data were collected from 13 females. Two flight sequences were typically recorded from each animal, representing a mean flight time of 13 ± 6 min. Mean body mass of the animals = 1.05 ± 0.13 mg. All values shown are the means ± SD.

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Instead, in response to a drop in flight power requirements, fruit flies appear to actively reduce average opening area of all 18 spiracles by up to 77 ± 11% of their maximum area. At a flight force that is equal to body weight, the flux of water is reduced by 11.6 ± 5.6% or 12.1 ± 5.8 μl g−1 body mass hour−1 compared with the maximum value. Although the fly is modulating flight force production by 88 ± 17%, tracheal partial pressure for CO2 and O2 as calculated from Eq. 1 appear not to be correlated with flight force production or metabolic rate, yielding means of 1.35 ± 0.16 and 19.9 ± 0.16 kPa, respectively (Fig. 2, A and B). These results are consistent with the hypothesis that Drosophila limits water loss during flight by actively matching the extent of spiracle opening to its metabolic needs (22, 23). Moreover, the high partial pressure for tracheal oxygen strongly supports the assumption that diffusion alone can account for gas exchange during flight in fruit flies (24).

In contrast to findings in other insects, this study shows that fruit flies appear to control the diffusive flux for respiratory gases through their spiracles by modulating the opening area of the spiracles. This modulation in mean cross-sectional area of the diffusive path results in nearly constant mean partial pressure for CO2 and O2 in the tracheal system, even though metabolic demands vary substantially during flight. Although this finding may not explain the mechanism whereby each spiracle contributes to overall tracheal conductance during flight (25), it demonstrates how small insects may cope with environmental challenges during increased locomotor performance. By whatever mechanism, in the diffusion-based respiratory system of Drosophila, the adaptive spiracle-closing behavior should lower the risk of desiccation for animals flying under dry environmental conditions.

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