Unconventional mechanisms control cyclic respiratory gas release in flying Drosophila
Department of Neurobiology, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany
* Author for correspondence (e-mail: fritz.lehmann{at}uni-ulm.de)
Accepted 11 July 2005
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Summary |
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Key words: flight, respiration, discontinuous gas exchange cycle, spiracle modeling, insect, fruit fly, Drosophila
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Introduction |
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In most running insects, as energetic demands increase the DGC typically
ceases, allowing respiratory gas exchange rates to increase likewise
(Full and Tullis, 1990;
Full et al., 1990
;
Jensen and Holm-Jensen, 1980
;
for reviews, see Lighton,
1994
,
1996
). In the desert ant
Pogonomyrmex rugosus, Lighton and Feener
(1989
) reported a
discontinuous breathing pattern while the unrestrained animal was walking
voluntarily at constant speed within a respirometric chamber. A similar
breathing pattern was found in blowflies Protophormia terraenovae
walking back and forth in a running tube
(Berrigan and Lighton, 1994
).
Although the first study was originally interpreted as a rare example in which
the environment constrained breathing behavior to avoid excessive water loss,
a recent study on walking energetics in ants suggests that DGC-like
respiratory behavior in walking animals may also result from Doppler-effects
occurring inside a running tube under flow-through respirometric conditions
(Lipp et al., 2005
).
In flying insects, flight-specific metabolic rate increases up to 15-fold
over resting values and spiracles open in order to allow gas exchange rates to
increase (Casey, 1980,
1989
;
Casey and Ellington, 1989
;
Harrison and Roberts, 2000
;
Hedenström et al., 2001
;
Lehmann et al., 2000
;
Miller, 1960
;
Moffatt, 2001
;
Wasserthal, 2001
;
Weis-Fogh, 1972
). In small
animals such as the fruit fly Drosophila, the uptake of oxygen into
and the release of carbon dioxide out of the tracheal system are thought to be
a diffusion-based process, and thus the spiracle opening area matches the
metabolic needs of the animal (Lehmann,
2001
; Weis-Fogh,
1964
). Applying the diffusive theory of respiration, adaptive
spiracle control in Drosophila may potentially establish constant
oxygen partial pressures near atmospheric partial pressure within the tracheae
(Lehmann, 2001
). Large insects
additionally ventilate their tracheal air system to satisfy the increased
oxygen needs of four distinct but interacting mechanisms: (i) contraction of
the abdomen (abdominal pumping; for example,
Harrison and Roberts, 2000
;
Komai, 2001
;
Miller, 1960
); (ii)
potentially, by muscle-induced deformations of large tracheae, as shown in
insects breathing under X-ray in a synchrotron
(Westneat et al., 2003
); (iii)
thoracic auto-ventilation resulting from the vibrations of the thorax during
wing flapping (Miller, 1966
);
and (iv) directed Bernoulli suction-ventilation due to differences in static
pressure distribution above two thoracic spiracles
(Miller, 1966
). In the
hawkmoth Manduca sexta, for example, auto-ventilation produces
pronounced pressure fluctuations in-phase with the 20 ms wing flapping cycle,
causing inhalation during the downstroke and exhalation during the upstroke of
respiratory gases on a stroke-by-stroke basis
(Wasserthal, 2001
).
In contrast to convective flow, diffusion-based respiratory gas exchange
mechanisms for flight have in common that the instantaneously measured gas
exchange rate is thought to reflect the animal's actual respiratory demands
because there is no bulk flow of respiratory gases into and out of the
tracheal system (Kestler,
1985). Experimentally, oxygen demands and the magnitude of
CO2 release rate from tethered Drosophila can be
controlled by flying the animal in a respirometric chamber under visually
controlled feed-back conditions (Dickinson
and Lighton, 1995
). In response to the vertical motion of a visual
pattern displayed in a surrounding panorama, the animal modulates the muscle
mechanical power output of its asynchronous flight muscles, and thus metabolic
rate. When visual lift stimuli are absent, the rate with which the fly
releases CO2 changes only slightly because flight muscle mechanical
power output, and thus metabolic rate, is not extensively modulated by the
fly's nervous system.
In comparison to previous findings, we here report a novel type of gas release pattern in an insect by demonstrating that instantaneous CO2 release rate of tethered flying Drosophila may periodically oscillate with large amplitudes, even though the metabolic rate of the animal remains approximately constant. In general, oscillatory gas release patterns in insects flying at constant metabolic rate may result from at least two distinct major mechanisms: ventilation and changes in the gas exchange area of the spiracles. While the first mechanism results from compression of air sacs and tracheae, the second one relies on spiracle control strategies. We thus evaluated the underlying physiological mechanisms of cyclic breathing behavior at an integrative level of investigation by combining (i) CO2 release measurements of single fruit flies flying in a virtual-reality flight simulator with (ii) video-based in-flight tracking data of abdomen and proboscis movements, and (iii) employing an analytical model for tracheal gas diffusion through spiracles that allows simulations of tracheal CO2 partial pressure changes and gas release rates at various metabolic rates.
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Materials and methods |
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Infra-red video analysis
To evaluate abdominal pumping during flight of Drosophila, we
developed an automatic tracking technique that allowed simultaneous recordings
of abdominal length and width while the animal was flying inside the
respirometric chamber. For this purpose, we marked the underside of the fly's
abdomen with four small droplets of commercial yellow fluorescent dye and
illuminated these markers using a UV-light emitting diode. While measuring
wing kinematics and carbon dioxide release, we recorded in-flight movements of
the abdomen using a conventional 50 Hz video camera (frame rate=20 ms).
Subsequently, the positions of the four fluorescent markers were automatically
tracked using a commercial video analysis program (MaxTraq, Innovision,
Columbiaville, MI, USA). In-flight extensions of the fly's proboscis were
derived by analyzing light intensity changes on the video images in a
rectangular region (ROI) in front of the animal's head. To allow easy
adjustments of ROI size and shape for each fly, we employed self-written
software developed under Visual C++ and Matrox Imaging Library (Matrox,
Quebeck, Canada).
Theoretical modeling of spiracle function
To assess the consequences of synchronized spiracle opening activity for
cyclic gas release patterns, we modeled flight muscle-specific CO2
release out of the tracheal system, assuming diffusive gas exchange between
the muscle tissue, tracheal system and the ambient air
(Kestler, 1985). Due to the
large diffusive area of the four thoracic spiracles in Drosophila
(9862 µm2,
95% of total spiracle area;
Demerec, 1965
), we excluded the
14 smaller abdominal spiracles from the theoretical modeling
(Fig. 1A). We modeled the four
spiracles as working units that independently control tracheal partial
pressure of CO2, PTCO2,
around a threshold value. In a diffusion-based system,
PTCO2 depends on the number of gas
molecules per unit time, t, leaving the muscle tissue and entering
the tracheal system (dNT), and the outflow of gas
molecules through the open spiracles (dNA). This
relationship can be expressed as:
![]() | (1) |
![]() | (2) |
|
Spiracle function was modeled on the base of two previous findings in
flying Drosophila. (1) Optical recordings of the spiracle opening
area have shown erratic opening activity during flight, suggesting that
spiracles control gas flux by continuously varying their opening area. (2)
When the fly varied metabolic rate between minimum and maximum values,
tracheal partial pressure for CO2 and oxygen remained essentially
unchanged (Lehmann, 2001). The
latter finding is also supported by direct measurements of muscular partial
pressure of oxygen in resting and flying moths and honey bees. In both
insects, muscular partial pressure of oxygen during flight remains close to
resting values (approximately 8.57 kPa in the moth and 6.36 kPa in the
honeybee; Komai, 1998
,
2001
). According to these
data, we employed a binary function that models spiracle conductance for two
separate states: below (spiracle lids are predominantly closed) and above
(lids are predominantly open) a tracheal partial pressure threshold value
Ts. During oscillatory gas release, the `predominantly
closed' state allows tracheal partial pressure to rise over time, whereas the
`predominantly open' state removes CO2 out of the tracheal system
faster than the flight muscles deliver the gas, resulting in a decrease of
tracheal partial pressure. This simple relationship can be expressed as:
![]() | (3) |
![]() | (4) |
![]() | (5) |
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Results |
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Video analysis
To tackle the significance of abdominal pumping as a source of the measured
CO2 fluctuations in Drosophila, we monitored the geometry
(length and width) of the abdomen during flight using an automatic video
tracking technique (Fig. 3B).
Although the data that have been reconstructed from single video images show
systemic changes in abdominal geometry, the changes were quite small and
consistently below approximately 100 µm
(Fig. 3E). The temporal
cross-correlation coefficient r, averaged over six flight sequences
all exhibiting pronounced and long-lasting gas release oscillations (sequence
length = 141±77 s, mean ± S.D.), shows that
superficially none of the abdominal length changes appear to be correlated
with the cyclic changes in gas exchange rate (maximum r=0.06 at phase
lag = 0.44 s, Fig.
3H).
As a second explanation for our experimental data, we considered whether
cyclic gas exchange is due to changes in haemolymph pressure caused by any
ventilatory function of the proboscis. In Drosophila, the proboscis
is relatively large and its volume amounts to approximately 1520% of
the head's volume (0.2 mm3;
Demerec, 1965
). Fruit flies
regularly extend and retract their proboscis during flight, a behavior that
might enlarge (inhalation) or compress (exhalation) the large paired frontal,
postgenal and postocular air sacs in the fly's head
(Lehmann et al., 2000
). We
quantified voluntary proboscis movements by mapping light intensity changes of
the video images in a defined region shown by the red box in
Fig. 3C. Cross-correlation
analysis between the derivative of proboscis motion and CO2 release
shows the following. (1) In single flies, up to 80% of the variance in
tracheal CO2 release fluctuations can be assigned to PER activity.
The mean correlation coefficient amounts to 0.31±0.24 (mean ±
S.D., N=6 sequences, 3 flies,
Fig. 3I), which is at least
fivefold higher than the maximum temporal correlation coefficient between
CO2 release and abdominal movements (0.31 vs 0.06). (2)
There is a small temporal shift of the maximum cross correlation value with
respect to zero phase lag, indicating that the proboscis starts moving
approximately 0.6 s before the fly changes its gas release rate
(
L, Fig.
3I).
Spiracle modeling
Besides the employment of convective strategies for breathing, oscillatory
gas release in diffusion based respiratory systems may also result from
changes in spiracle opening area, similar to the mechanism causing the
discontinuous gas exchange cycle in a resting insect
(Lighton, 1994;
Miller, 1981
;
Slama, 1994
;
Snyder et al., 1995
). In this
scenario, the proboscis extension reflex (PER) would only be correlated with
CO2 release rather than being the primary cause for its oscillatory
behavior. In insects, spiracle muscle control is CNS-mediated but the muscle
activity also depends on local concentrations of respiratory gases (for a
review, see Nikam and Khole,
1989
). In the locust Schistocerca gregaria, for example,
the mesothoracic closer muscle is innervated by two excitatory motorneurons
and a peripherally located neurosecretory cell
(Swales et al., 1992
). Due to
their small size it is difficult to record electrically from spiracle muscles
in flying Drosophila. The same holds for direct measurements of total
spiracle opening area, because the mesothoracic spiracle is partly covered
during flight by the beating haltere. For this reason, we cannot reject
per se the hypothesis that the CNS synchronously opens and closes
multiple thoracic spiracles via excitatory motorneurons.
|
Multiple variations of the analytical model parameters show that the
likelihood of the occurrence of oscillatory gas release depends at least on
two parameters: (i) the ratio between muscular partial pressure and the
spiracle threshold values for that gas
(Fig. 5A,B) and (ii) the ratio
between maximum conductance of each model spiracle and the simulated
conductance of the cytoplasm between muscle tissue and the tracheoles
(Fig. 5C,D). The first
prerequisite implies that cyclic CO2 release only occurs when the
tracheal partial pressure for CO2 is allowed to increase when
spiracles are held predominantly closed, and to decrease when the spiracles
are predominantly open over time. The analytical model thus predicts that
oscillatory release patterns disappear when tracheal partial pressure for
CO2 is consistently below or above the spiracle threshold value for
this tracheal gas (black traces, Fig.
5B). At high metabolic rates, for example, all four model
spiracles stay predominantly open (between 0.5 and
1.0GS,max) because the tracheal partial pressure of the
respiratory gas remains above the opening threshold value
(PMCO2/Ts1.1,
Fig. 5A,B). Under these
conditions, any changes in gas release rates reflect changes in metabolic rate
or differences in partial pressure between the tracheal system and the ambient
air, but not changes in the spiracle's own diffusive exchange area. In
contrast, the changes in gas release pattern are more subtle when changing the
ratio between cytoplasm and maximum spiracle conductance
(Fig. 5C). At the given
parameter settings, small ratios below 0.1 suppress the likelihood of cyclic
release patterns because CO2 gets completely removed out of the
tracheal system even at the leaky (`predominantly closed') spiracle state. In
contrast, higher conductance ratios between 0.2 and 0.8 consistently produce
pronounced temporal fluctuation in gas release rate. With increasing
GS,max/Gc ratio >0.1, however, the
amplitude of the data's main FFT frequency component (i.e. equal to spiracle
hysteresis
) simultaneously decreases, and the broader distribution of
frequency components in the time domain indicates that the gas release rates
become increasingly noisy (Fig.
5D).
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Discussion |
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Modeling synchronized behavior of spiracle muscles
In the past, several researchers have attempted to model respiratory
processes. At the single cell level, Thumfort et al.
(2000) recently modeled oxygen
diffusion by computer simulation in three dimensions and applied this model to
a case study. They found that generating a one-dimensional representation of
the three-dimensional surface of the cell is a close approximation to the more
complex three-dimensional model with systematic differences below 10%.
Research on diffusive models of the entire tracheal system of insects was
pioneered by a study of Weis-Fogh
(1964
), who developed an
extensive set of equations for steady state diffusion in an isotropic tissue,
tracheal gas transport and exchange (airtube diffusion). Weis-Fogh, for
example, also considered different topologies of diffusive systems and
estimated their effect on gas exchange. Later, Kestler
(1985
) also modified these
models towards ventilation and focused on different gas exchange models
describing respiratory flux between the tracheoles and mitochondria. Lehmann
(2001
) applied Kestler's model
to diffusive gas exchange and respiratory water loss through the spiracle in
tethered flying Drosophila and demonstrated how the water loss rate
can be used to derive total spiracle opening area in the animal in
vivo. Snyder et al.
(1995
) proposed an elaborate
model for cyclic ventilation in insects that also covers the three phases of
cyclic ventilation. The authors basically reported that volume expansion of
the trachea, and not an increased cross-sectional area of the spiracles
per se, is the important adaptation to normobaric hypoxia. However,
although all elegant, none of the elaborate studies above have considered the
potential complex temporal interactions in diffusive gas exchange between
multiple spiracles.
The simplified analytical model of tracheal diffusion presented here offers
an alternative explanation for the experimental data achieved in the flying
fruit fly. The model proposes that the temporal switching between non-cyclic
and cyclic breathing patterns can be explained by phase transitions between
(i) times during which opening activity of the four autonomously working
thoracic spiracles in the fly synchronize (cyclic breathing) and (ii) times at
which the spiracle-controlled total diffusive areas are temporally
out-of-phase (non-cyclic breathing). Interestingly, recent data on respiration
in resting ants Camponotus seem to support this scenario also
occurring during the discontinuous gas exchange cycle
(Lipp et al., 2005). Multiple
studies have shown that resting ants typically release a single peak of
CO2 during the DGC's opening phase (`O'-phase; Lighton,
1992
,
1994
,
1996
;
Lighton and Feener, 1989
),
which is consistent with the idea of synchronized spiracle opening activity,
assuming that multiple spiracles participate in gas exchange. In comparison,
the recent study on Camponotus gas release measured with
high-temporal resolution showed that resting animals also release
CO2 as multiple `O'-peaks within a single DGC cycle
(Lipp et al., 2005
). One
explanation for the latter finding could be that it is a consequence of
desynchronized opening activity of at least two spiracle muscles, which
compares to the non-cyclic gas release pattern in our flying insect. The
temporal distribution of the gray areas in
Fig. 1B,C shows that in flying
Drosophila, the desynchronized spiracle opening condition seems to be
more common and, in addition, not all tested flies have shown cyclic breathing
patterns. In most recordings of flying fruit flies, instantaneous gas exchange
rate thus matches the actual metabolic needs of the animal, as reported
previously (Lehmann,
2001
).
One of the most interesting predictions of our analytical model is that cyclic CO2 release patterns do not necessarily require large changes in diffusive area of a single spiracle during control behavior, as indicated by the small (±3%) tracheal partial pressure changes in the example shown in Fig. 4B. Under certain conditions, these small changes in diffusive area, in conjunction with concomitant small changes in tracheal partial pressure, appear to be sufficient to modulate total CO2 release rates of up to ±50% peak-to-peak of the mean value, when all four model spiracles open and close at similar phase, as shown in Fig. 4C. The most critical prerequisite for the occurrence of CO2 release cycling of the analytical model seems to be that tracheal pressure of CO2 decreases (increases) when the model spiracle opens between 50% and 100% (0 and 50%) of the maximum diffusive area, as mentioned above. If metabolic rate causes tracheal partial pressure for CO2 to increase above the spiracle opening threshold (Ts), cyclic respiration vanishes and the modeled instantaneous CO2 release rate matches instantaneous metabolic activity. This observation could potentially explain why, in the experiments performed with flying Drosophila, oscillatory gas release patterns only occurred at flight forces and metabolic rates well below maximum locomotor capacity.
Due to the lack of elaborate data for Drosophila's respiratory
system, including muscular partial pressure estimates, gas conductance of the
cytoplasm and flow conditions inside the tracheae, it is difficult to model
gas release using exact physiological values
(Kestler, 1985). The proposed
analytical model, including all parameter settings, should thus be considered
as a rough hypothesis on how tracheal gas release can potentially be shaped by
stochastic spiracle opening and closing processes. Moreover, our simplified
analytical model makes some inherent assumptions about spiracle control
strategies of the living organism (e.g. binary random function for spiracle
opening/closing behavior). The results derived from the analytical model,
including any comparison with data recorded in the flying animal, should thus
be treated with caution. Nevertheless, considering all limitations and
problems of our simplified analytical model for insect respiration, the
coincidence between the data produced by the simulation and the flying fly is
marked, and thus might highlight a fundamental inherent property of spiracle
function in diffusion-based tracheal systems of small insects.
Conclusions
In sum, this study proposes that periodically oscillating gas release
patterns in flying Drosophila might result from at least two
unconventional respiratory mechanisms: firstly, the proboscis appears to serve
as a pumping organ for ventilation, and secondly, gas release oscillations may
come about by synchronized opening activity of the large thoracic spiracles
similar to the DGC. Interestingly, in the fruit fly both mechanisms are
thought to have little significance for flight muscle function, and
ventilation is apparently not required to satisfy the high oxygen demands even
at maximum locomotor capacity (Weis-Fogh,
1964; Lehmann,
2001
). Instead, a possible advantage of the proboscis extension
reflex for tracheal ventilation might be to actively promote local oxygen
supply of the animal's head. In dipteran flies, the retina and optic lobes may
require at least 20% of the resting metabolic rate due to the highly
specialized and large visual system of flies
(Laughlin, 1987
). Since there
are no spiracles in the head, respiratory gases must pass through small
tracheae inside the approximately 80 µm diameter neck connective
(Demerec, 1965
). If correct,
the hypothetical benefit of the proboscis-induced ventilation for breathing
might be to circumvent this bottleneck for diffusive respiration, in order to
ensure evacuation of CO2 from and the supply of oxygen to
the fly's brain. Eventually, this behavior might balance tracheal
partial pressures of respiratory gases locally within the body compartments of
Drosophila during certain flight conditions.
List of symbols and abbreviations
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Acknowledgments |
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