Respiratory airflow in a wingless dung beetle
1 School of Physiology, Faculty of Health Sciences, University of the
Witwatersrand, 7 York Road, Parktown 2193, South Africa
2 Ecophysiological Studies Research Programme, School of Animal, Plant and
Environmental Sciences, University of the Witwatersrand, Johannesburg, Wits
2050, South Africa
* e-mail: duncanfd{at}physiology.wits.ac.za
Accepted 8 May 2002
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Summary |
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Key words: Circellium bacchus, discontinuous gas exchange cycle, respiration, spiracle, Scarabaeidae, dung beetle, subelytral cavity
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Introduction |
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Retrograde respiratory airflow is presumed to be typical for all insects
(Hadley, 1994;
Lighton, 1996
), and expelling
air into a sealed space below the elytra is assumed to be an adaptation, found
in many species of wingless beetle
(Cloudsley-Thompson, 1964
;
Ahearn, 1970
;
Draney, 1993
), to an arid
habitat. Because the cavity maintains its air at a high humidity, water loss
during respiration should be reduced because the tracheae will not be exposed
to dry air. However, the validity of this hypothesis requires an
anterior-to-posterior flow of respiratory gases through the body and
differential control of the spiracles, neither of which has previously been
demonstrated in these insects (Hadley,
1994
).
Spiracular control appears to be most precise in insects that inhabit dry
environments, where they limit water loss by opening their spiracles for only
limited periods during a discontinuous gas-exchange cycle (DGC) (for reviews,
see Kestler, 1985; Lighton,
1994
,
1996
;
Wasserthal, 1996
). The DGC is
a cyclic discontinuity in external gas exchange that typically consists of
three periods (Miller, 1981
;
Kestler, 1985
). There is a
closed (C) period, during which the spiracles are shut, preventing both
respiratory water loss and gas exchange. Oxygen levels in the tracheae drop,
while CO2 is largely buffered in the tissues and haemolymph. This
is followed by the flutter (F) period, during which slight, intermittent
opening of the spiracles allows some normoxic O2 uptake through the
spiracles by diffusion and convection, but little CO2 or water
vapour is lost. The final period, the CO2 burst (B) period, is
triggered when the accumulation of CO2 from respiring tissues
causes some or all of the spiracles to open widely. Rapid unloading of
CO2 should minimise the time that the spiracles are open and
therefore reduce water vapour loss. However, the role of the DGC as a
water-saving mechanism remains controversial
(Hadley, 1994
;
Shelton and Appel, 2000
;
Williams and Bradley, 1998
),
and some authors propose that it is more likely to be a response to a
high-[CO2] atmosphere than to water stress
(Lighton, 1996
). We have
demonstrated a relationship between the DGC and habitat aridity that is
independent of phylogeny in five species of African dung beetle
(Duncan and Byrne, 2000
) and
that most water loss occurs during the burst period of the DGC in
Circellium bacchus (Duncan,
2002
).
To investigate the route of external gas exchange, we chose a large
apterous beetle that would allow us simultaneously to measure CO2
and O2 exchange at the mesothorax, independently of exchange from
rest of the body, and in particular the subelytral cavity. Circellium
bacchus is a ball-rolling dung beetle that feeds on the dung of large
herbivores (elephant, buffalo and black rhinoceros) and is now restricted to a
few populations in the eastern Cape of South Africa, apparently because of
competition with heterothermic, winged dung beetles
(Chown et al., 1995). Although
the beetle is locally abundant, it is considered to be endangered because it
occurs at only seven widely separated localities in the Cape Province of South
Africa (Coles, 1993
). A permit
was obtained to collect 10 beetles from Addo Elephant Park, which has a low
annual rainfall of approximately 400 mm, spread throughout the year (mean
34±7.1 mm per month, mean ± S.D.; range 23-48 mm) (Sutherst and
Maywald, 1985). Circellium bacchus live for more than 2 years and
spend dry periods underground in sandy soil
(Tribe, 1976
). Females may
spend more than 4 months underground, tending a single brood ball, during
which time it is not known whether they feed
(Coles, 1993
). Given the dry
habitat and the lifestyle of this species, it is assumed to be under selection
pressure to reduce water loss. C. bacchus exhibits an extreme example
of the DGC (Duncan and Byrne,
2000
) and is a strict ectotherm, so metabolic measurements are not
compromised by heterothermy (Nicolson,
1987
).
C. bacchus has eight pairs of spiracles along its body
(Fig. 1), of which the single
pair of large, ventral mesothoracic spiracles occurs anteriorly in the
membrane connecting the prothorax and mesothorax, behind the coxal cavities of
the prothoracic legs. Here, they open into the space created by the
constriction between the prothorax and the mesothorax. The posterior half of
the body has a single pair of smaller metathoracic spiracles and six pairs of
even smaller abdominal spiracles, all of which open dorsally into the
subelytral cavity. Sealing a latex skirt between the two body sections
permitted measurement of gas exchange from each half of a live beetle enclosed
in a respiratory chamber (Fig.
2; Duncan, 2002).
Sampling gas from the anterior and posterior body allowed us to address three
questions with regard to the role of the subelytral cavity in respiration.
First, from which body half does the majority of gas exchange take place?
Second, in which direction do respiratory gases move through the beetle?
Third, which spiracles are involved in this gas flow?
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Materials and methods |
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Respirometry
A flow-through respirometry system was used to measure gas exchange in the
anterior and posterior halves of a live, intact beetle (for details, see
Duncan, 2002). The beetle to
be tested was placed in a Perspex respirometry chamber divided crosswise by a
sheet of latex (dental dam, 0.02 mm thick)
(Fig. 2). The head and
prothorax of the beetle were pushed into the anterior section through a small
hole in the centre of the latex sheet, which made a tight seal between the
prothorax and abdomen. The posterior section of the chamber was then bolted to
the anterior section. The latex sheet sealed the joint between the two
sections of the apparatus, giving each section a volume of approximately 250
ml. One inlet and one outlet pipe served each section of the chamber. The
entire configuration was tested for leaks, and the latex sheet was renewed for
each trial.
Gas emissions in each section of the chamber were measured separately and simultaneously using flow-through respirometry, with each section having its own source of air, flow controller and gas analysers. Briefly, air scrubbed of CO2 and H2O was drawn into each section at a flow rate of 50 ml min-1. The outlet from each section was led to a separate Licor CO2 analyser (LI-6262 and LI-6251; 0.1 p.p.m.) and then to the O2 sensor (Qubit Systems gas-phase O2 sensor, model S-102, with a resolution of 0.1 %) with its own flow control (calibrated Supelco flow meter). Readings of the concentration of CO2 and O2 in the separate chambers were taken every 5 s and recorded using a computerised data-acquisition software (Datacan V, Sable Systems). Recordings were made on individuals weighed to ±0.1 mg (Precisa 160A balance). The respiration pattern of each beetle was measured for a minimum of 6 h in dim red light. The beetles were occasionally observed to ensure that they remained quiescent during sampling. Only measurements from stationary beetles were used in the analysis. After measurements, the beetles were reweighed. Experiments were conducted in an air-conditioned laboratory at a temperature of approximately 23±1 °C.
Baseline drift of the analysers was corrected during analysis from
measurements taken at the beginning and end of each trial with the
respirometry chamber empty. All measurements were corrected to standard
temperature and pressure (STP). The CO2 recordings were converted
to the rate of CO2 production
(CO2; in ml
h-1), and O2 data were converted to the rate of
O2 consumption (in ml h-1). To measure O2
consumption within the flow-through system, an H2O/CO2
scrubber was placed between the CO2 analyser and the O2
analyser, which was upstream of the flow meter
(Withers, 1977
). This allowed
us to interpret a large drop in O2 concentration as O2
consumption. This assumption was checked by injecting small volumes of
CO2 and N2 into the system.
The DGC characteristics were calculated as follows. The DGC frequency (=burst frequency) was calculated by determining the number of peaks of CO2 emission per second, and the DGC duration was taken as one complete cycle (i.e. closed, flutter and burst periods). The mean rate of CO2 emission was calculated as the mean value over several complete discontinuous gas-exchange cycles. To measure the emission volume, the area under the curve was integrated against time.
Each experiment was repeated with 2-6 individuals. To ensure that the results obtained were not sampling artefacts, between trials beetles were rotated within the chamber and the chamber within the apparatus. This removed any chance of inverse of air flow being due to slight pressure differences on the two sides of the latex sheet. The same apparatus, with slight modifications, was used for each experiment.
Direction of airflow
To determine the direction of gas movement in the beetle,
O2-enriched air containing 30 % O2 was used as the
tracer gas. The gas mixture was prepared using a Columbus Instruments gas
mixer and was stored in a Douglas bag. The gas mixture was first drawn into
the posterior section of the respirometer, and both sides of the chamber were
monitored for O2 and CO2 emissions, using the method
described above. The gas mixture was then drawn through the anterior section
while monitoring both ends of the chamber. The experimental apparatus was
otherwise as described above. The 30 % O2 gas mixture allowed the
beetle to continue to respire normally (compare Figs
3 and
5).
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Monitoring individual spiracles
Gas exchange was measured separately at each of the two mesothoracic
spiracles. Because these spiracles are situated in the soft intersegmental
membrane, it was not possible to seal tubes around each individual spiracle.
Instead, two tubes (1 mm diameter) were glued to the ventral prothoracic
suture such that each single spiracle opened directly into one tube (Figs
1,
2). The beetle was placed in
the respirometer as described above except that only the gas from the anterior
section was sampled by the flow-through system. This section had an additional
outlet so that the tube from each mesothoracic spiracle led directly to its
own CO2 and O2 analyser and was controlled by an
individual flow meter. To prevent an accumulation of CO2 in the
posterior section of the respirometer altering the normal resting respiratory
pattern, air scrubbed of CO2 and H2O was drawn through
the posterior section at a flow rate of 50 ml min-1. Beetles were
rotated within the chamber to ensure that the two mesothoracic spiracles were
being sampled independently. A small amount of cross-sampling did occur, which
confirmed that both tubes were collecting gas emissions, but was so small that
we were able to conclude that each spiracle was acting independently.
Statistical analyses
Data are represented as means ± standard deviation (S.D.). Sample
size (N) is indicated in the text as representing individual beetles
or, in the case of gas-exchange characteristics, 3-10 discontinuous
gas-exchange cycles per beetle. Statistical comparisons were made using
Student's t-tests. Regression analysis was performed using the
least-squares method.
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Results |
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We recorded a DGC respiratory pattern from the anterior body half
(Fig. 3A), which showed a burst
of CO2 emission approximately once per hour, lasting on average for
37±5 min (N=6), resulting in a mean DGC duration of
70±16 min (N=6) (Table
1). Lighton et al.
(1993) showed that accurate
interpretations of spiracular movements could be made from CO2
emissions, allowing us to conclude that the anterior mesothoracic spiracles
remained closed during the rest of the DGC. As with whole-animal recordings,
the closed and flutter periods could not be separated and are thus referred to
as the interburst period. Oxygen uptake occurred at the beginning of the
CO2 burst period (Fig.
3A). The peak of O2 uptake lasted 15±10.7 %
(N=6) of the duration of the CO2 burst period, and no
corresponding increase in the period of O2 uptake occurred with an
increase in CO2 burst duration.
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Weaker cyclic CO2 emission was recorded from the posterior spiracles, but a DGC was not apparent (Fig. 3B). No distinct periods could be seen. In most cases, but not all, the small increments of CO2 emission from the posterior spiracles occurred during the corresponding burst period of the anterior spiracles. The rate of CO2 emission was 1.5-7 times lower than that from the anterior spiracles (t0.05,10=3.36, P<0.01) (Table 2). Of the total CO2 emitted, 79.4±4.48% (N=6) was expelled through the anterior mesothoracic spiracles. We assume that there is little movement of the posterior spiracles, which open into the subelytral cavity, because only a small coefficient of variation of CO2 emission was seen; 0.5±0.15 compared with 1.3±0.32 for the anterior spiracles (t=5.47, P<0.001). No measurable O2 uptake was recorded for the posterior body half, and fluctuations in the rate of O2 consumption can be attributed to baseline drift rather than to actual consumption.
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A large difference in the absolute magnitude of CO2 emission rates from the anterior and posterior spiracles was noted (Fig. 3). The length of the DGC period of the anterior spiracular emissions was used to calculate the CO2 output from both anterior and posterior spiracles. The anterior spiracular rate of CO2 emission was 77.5±0.07% (N=6) of total CO2 output, while the rate of CO2 emission from the posterior spiracles was 22.5±0.07% (N=6) of total CO2 output. The anterior and abdominal spiracle rates of CO2 emission, averaged over the entire measurement (i.e. several DGCs) for each beetle, showed that the anterior rate of CO2 emission as a proportion of the total rate of CO2 emission increased as the metabolic rate rose (Fig. 4), with the posterior spiracular contribution declining from 53 to 4% of the anterior spiracular output.
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Therefore, from the above results, the anterior spiracles appear to be the main site of gas exchange during respiration at rest. Little gas exchange was seen to take place via the subelytral cavity, and the anterior-to-posterior airflow expected in apterous beetles was not observed in C. bacchus.
Direction of airflow
Drawing air containing 30% O2 over the posterior body half
revealed that, during the early part of the burst phase, O2 was
withdrawn from this section of the respirometer and then expelled into the
anterior chamber by the mesothoracic spiracles when they opened to release
CO2 (Fig. 5A). When
the same gas mixture was drawn into the anterior respiratory chamber, we
detected no airflow from the thorax to the abdomen
(Fig. 5B). The elevated
O2 levels had no noticeable effect on the respiration patterns of
the beetles other than slightly increasing the period of the DGC when used in
the anterior section of the respirometer
(Fig. 5A,B). The metabolic
rates of these beetles (372.16 µl h-1, N=2) showed that
they were within the range of metabolic rates recorded in the rest of the
study (322.57 µl h-1, N=6). CO2 emission
rates from the anterior and posterior regions did not indicate an altered
metabolic rate, and the emission from the posterior spiracles was within the
normal pattern seen in all the other trials. The prolonged drop in the
O2 concentration at the anterior spiracles
(Fig. 5B) is assumed to be a
result of the elevated levels of O2 taking longer to reach
equilibrium than when at normal concentrations (cf.
Fig. 3A). Why this should be so
is unclear, and this phenomenon warrants further experimental
investigation.
From Fig. 5A, it appears that O2 can diffuse rapidly through the beetle, in through the posterior spiracles and out through the anterior spiracles, along its concentration gradient. The anterior and posterior spiracles may be opening in a coordinated fashion because the O2 consumption peak corresponds closely with the start of the CO2 burst phase. Airflow in this experiment is from posterior to anterior, contrary to the assumption of CO2 emission from the posterior spiracles into the subelytral cavity.
Carbon dioxide emission rates from individual spiracles
Measuring gas exchange separately at individual mesothoracic spiracles
revealed that the right spiracle was the main route for CO2
emission and O2 uptake (Fig.
6). Sampling tubes were successfully attached to three beetles,
all of which respired through the right mesothoracic spiracle. From these
individuals, we calculated that 89.2±12.0 % (N=3) of the total
CO2 output from the anterior body half, which amounts to 76 % of
the total body output, occurred through the right mesothoracic spiracle
(Table 3).
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Discussion |
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Airflow
Several lines of evidence support the hypothesis that ventilation in C.
bacchus at rest involves tidal or anterograde air flow. Respiration
during activity is a more complex subject, and our results may or may not be
applicable to it.
First, the mesothoracic spiracles are responsible for approximately 80% of
the CO2 emission and most of the O2 uptake that we
measured in C. bacchus at rest in a normal atmosphere. Oxygen in an
enriched atmosphere was seen to be consumed from the posterior chamber, which
could be due to diffusion into the body via the abdominal spiracles
under these circumstances. The anterior thoracic spiracles are visibly larger
than their posterior counterparts in C. bacchus, and in many other
insects (Richter, 1969;
Lighton et al., 1993
;
Zachiariassen, 1991), and have been shown to be responsible for 73% of
CO2 exchange in Cataglyphis bicolor
(Lighton et al., 1993
) and for
70% of water loss in a flightless, arid-habitat tenebrionid, Phrynocolus
petrosus (Zachiariassen, 1991). Permanent apertures cut into the elytra
of Onymacris plana, a desert tenebrionid, caused no change in the
cyclic respiratory pattern (Bartholomew et
al., 1985
), which is now explicable if the majority of respiration
takes place through the mesothoracic spiracles in that species.
Second, the proportion of CO2 emission from the anterior
spiracles rises as the total rate of CO2 emission increases
(Fig. 4), indicating that the
contribution of the mesothoracic spiracles becomes more important in
respiratory exchange as
CO2 increases;
notably, the DGC is maintained. Virtually no increase in the outflow of
CO2 from the abdominal spiracles is seen, despite the presence of
seven additional pairs of spiracles under the elytra, which are open during at
least part of the DGC, when they are presumably available for gas exchange, as
shown by the small amounts of CO2 released into the posterior
chamber and the ingress of O2 from an O2-enriched
atmosphere.
Third, when O2-enriched air was flooded over the posterior half of the body, an anterograde flow of oxygen was recorded during the burst phase of the DGC. Opening of the anterior spiracles and the subsequent loss of water vapour from the tracheae, coupled with opening of the subelytral spiracles, could allow bulk flow of O2 into the body and diffusion through the longitudinal tracheal trunks, which then exists through the wide-open anterior spiracles. However, no CO2 burst was seen from the abdomen during either this trial or normal respiration, which suggests that the subelytral spiracles remain closed during at least part of the DGC or that their exchange capacity, in combination with that of the elytral cavity, is limited. Recordings of the action of the spiracles and abdominal pumping movements will be required to resolve this paradox. The rigid ventral surface of the abdomen reveals no movements during respiration, but strong pumping actions of the dorsal surface have been noted. These are certainly important under stress and may be responsible for the peaks of CO2 emission seen during the burst phase of the DGC. Nevertheless, we conclude from the above that the majority of air flow in and out of C. bacchus is through the anterior mesothoracic spiracles and that the subelytral spiracles contribute little to CO2 output at rest. Our results do not preclude the possibility that air enters via the subelytral cavity, where it will become saturated before entering the posterior spiracles. However, as the air leaves via the anterior spiracles, water will have to be retrieved from it or be lost.
Use of a single spiracle
A surprising finding was that the right mesothoracic spiracle is the main
site of CO2 emission and oxygen uptake. The dextral preference of
the three specimens examined is probably be due to chance because beetles were
collected on an ad hoc basis from a large population and are unlikely
to be close relatives since the species wander long distances in search of
dung and have a low reproductive rate
(Coles, 1993). Given the
identical appearance of the left-hand spiracle, we would expect other
individuals preferentially to use this spiracle. Unilateral dominance of
abdominal spiracles has been observed in blaberid cockroaches under various
conditions, but never involving the thoracic spiracles or reversal of flow
through them (Miller, 1982
).
The right mesothoracic spiracle looks identical to its left-side partner but,
with a surface area of 3.73 mm2, it is more than four times larger
than any of the posterior spiracles. Spiracle size, position and number are
highly variable within the Scarabaeoidea, which is considered to be a
reflection of the varied environmental pressures experienced by the different
groups (Richter, 1969
;
Browne and Scholtz, 1999
). Our
own observations within the Scarabaeidae show C. bacchus to have
unusually large thoracic spiracles. Activity, or an increase in metabolic
rate, would presumably result in opening of the left spiracle, doubling the
diffusive capacity available for respiration. A single, permanently opened
mesothoracic spiracle was found to be sufficient for the gas-exchange needs of
an inactive ant, Cataglyphis bicolor, which can potentially increase
gas-exchange rates sevenfold by using its full complement of thoracic
spiracles (Lighton et al.,
1993
). Walking beetles would probably benefit from
autoventilation, but ball-making in a hypercapnic dung heap, or in the hot
sun, might necessitate active ventilation. Abdominal pumping versus
convection for ventilation warrants further investigation in this species.
The role of the abdominal spiracles of C. bacchus in respiration
could be masked by the presence of the subelytral cavity, which will dampen
small oscillations in gas exchange taking place under the elytra. Although a
flutter phase was not seen, it may still be taking place in the abdominal
spiracles. Duncan and Dickman
(2001) recorded a flutter
phase from a flightless carabid Cerotalis sp. and Carenum
sp. from the Simpson desert, and Duncan and Byrne
(2000
) demonstrated flutter
phases in winged dung beetles from mesic habitats, which would be expected not
to have such tightly sealed elytra as C. bacchus. Lighton et al.
(1993
) found that the
abdominal spiracles in Cataglyphis bicolor play a major role during
the early flutter phase of the DGC, when they allow bulk flow of oxygen into
the spiracles with minimal water loss of water because of their limited
diffusive capacity. Allowing for the possibility of temporal coordination of
the subelytral and mesothoracic spiracles in C. bacchus, the hypoxic
trigger point for opening of the abdominal spiracles could be reached earlier
in the subelytral cavity if it were to become hypoxic more rapidly than the
mesothorax. The flutter phase would then be initiated in the subelytra when
the negative pressure gradient is large, aiding the bulk flow of
O2, which was clearly seen to be taken up from an
O2-enriched atmosphere by the posterior spiracles. Ingress of
O2 would then inhibit the flutter phase in the subelytral
spiracles, and CO2 accumulation would eventually stimulate the
anterior spiracles to open. Restricting the CO2 output to one large
exit point may save water by reducing the area of the system exposed to the
atmosphere.
Considerations for respiratory water saving in insects
Two hypotheses arise from these results, both of which probably reflect on
water-saving adaptations in insects. First, the use of a single spiracle for
respiration at rest could reduce water loss by restricting exchange to a
single site. Consequently, only a small area of the respiratory passages would
be open to the atmosphere for short periods during the DGC, as has been found
in quiescent cockroaches (Miller,
1982) and moth pupae that do not have access to free water
(Levy and Schneiderman, 1958
).
Second, the mesothoracic spiracles of C. bacchus are large, as they
are in desert tenebrionids (Draney,
1993
), and have a well-developed sieve plate across their opening.
If the sieve plate functions as a water-retention mechanism
(Schneiderman, 1960
;
Schmitz and Wasserthal, 1999
),
then a tidal airflow through a single opening, as found in mammals
(Schmidt-Nielsen, 1997
), into
and out of a humid subelytral cavity could also contribute to reduced water
loss. Many beetles, including C. bacchus, have a highly developed
system of large air sacs attached to the tracheae which, coupled with
abdominal pumping, could be involved in moving air back and forth through the
body through longitudinal tracheal tracts
(Wasserthal, 1996
). Surface
activity of C. bacchus is closely tied to sunny periods after
rainfall (Coles, 1993
), which
will expose beetles to variable humidities during extreme activity. The rest
of their time is spent underground in sandy soils, either brooding a single
offspring or simply waiting for rain. The humidity experienced underground is
unknown, but humidity recordings for Addo Elephant Park show an annual mean
maximum and minimum relative humidity of 79±6.1 and 48±2.9%
respectively (means ± S.D., N=30 years) (Sutherst and Maywald,
1985). A mechanism to reduce water loss under these circumstances would be
selectively advantageous and give C. bacchus an advantage over other
large flighted dung beetles, which are conspicuously absent from this
habitat.
The role of the DGC as a water-saving adaptation remains controversial
(Lighton, 1996;
Davis et al., 1999
) and
inconclusive (Hadley, 1994
;
Zachariassen, 1991
;
Ahearn, 1970
). However, the
extreme DGC in C. bacchus described here, coupled with its large body
size and low metabolic rate, correlate strongly with its dry habitat
distribution (Duncan and Byrne,
2000
) and can be interpreted as a survival trade-off against
limited competitiveness in more mesic environments
(Chown and Gaston, 1999
). Our
results indicate that, although many insect species may show a typical DGC
respiration pattern at rest, the spiracles involved in gas exchange may
differ, as will the resultant effect on water conservation. Forward airflow
through the respiratory system of apterous beetles will force insect
physiologists to rethink the way in which the subelytral cavity could function
to reduce respiratory water loss.
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Acknowledgments |
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