The role of the mesothoracic spiracles in respiration in flighted and flightless dung beetles
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
* Author for correspondence at present address: School of Animal, Plant and Environmental Sciences, University of the Witwatersrand, Johannesburg, Wits 2050, South Africa (e-mail: duncanfd{at}biology.biol.wits.ac.za)
Accepted 3 January 2005
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Summary |
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Key words: Scarabaeini, discontinuous gas exchange cycle, arid habitat, subelytral cavity
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Introduction |
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We tested the assumption that other flightless dung beetles would emit
proportionally more CO2 from the anterior mesothoracic spiracles
than the posterior subelytral spiracles, as evidence that they were using the
subelytral cavity in the same manner as C. bacchus. We compared the
relative contribution of both spiracle sets in two species of flightless dung
beetles Scarabaeus (Pachysoma) gariepinus Ferreira,
and Scarabaeus (Pachysoma) striatum Castelnau
(Scarabaeini) from arid habitats, with that of a flighted species
Pachylomerus femoralis (Kirby) (Scarabaeini), from a more mesic
habitat. Our expectation was that the winged P. femoralis would be
unable to seal its subelytral cavity as tightly as the flightless species and
would therefore emit CO2 from both the anterior and posterior body
halves. All three of the above species belong to the tribe Scarabaeini. These
results were compared with a previous study on the arid dwelling flightless
dung beetle C. bacchus from the tribe Canthonini, which is an extreme
example of anterograde CO2 emission from the mesothoracic spiracles
(Duncan and Byrne, 2002). The
Scarabaeini have been shown to be a monophyletic tribe and have a sister
relationship with the Canthonini (Philips
et al., 2004
), both tribes are in the same subfamily,
Scarabaeinae.
Many species of beetles lack wings and, therefore, potentially possess an
empty subelytral cavity. The function of this space in respiration and water
saving is under debate. Klok
(1994) found that flightless
dung beetles were more resistant to desiccation than winged species, although
Chown et al. (1995
), concluded
that these differences were insignificant when adjusted for body mass.
Nevertheless, in their experiments C. bacchus individuals were able
to survive twice as long under desiccating conditions than similar mass P.
femoralis. The air inside the subelytral cavity of flightless beetles is
known to have a high water content, and by keeping this cavity closed an area
of high humidity is created over the posterior spiracles (Zachiariassen, 1991;
Byrne and Duncan, 2003
). The
relative importance of respiratory water loss compared with cuticular
transpiration has been in dispute
(Zachariassen et al., 1987
;
Lighton 1994
,
1998
) but if there is
selection pressure to conserve water it is likely to act on all routes for
water loss, which includes the respiratory routes. Thus xeric insects should
have reduced respiratory and cuticular water loss. Gibbs
(2002
) showed that the
structure of cuticular lipids in Drosophila did not correspond to
interspecific differences in water loss rates and therefore concluded that
respiratory adaptations, along with a decrease in metabolic rate, coupled with
a reduction in locomotor activity, resulted in improved desiccation resistance
in desert species.
In addition to the subelytral cavity, a discontinuous gas exchange cycle
(DGC), seen during respiration at rest, is characteristic of many beetle
species from several sub-orders and differing habitats
(Lighton, 1991;
Davis et al., 1999
;
Duncan and Dickman, 2001
;
Duncan et al., 2002
;
Duncan, 2003
). The respiratory
patterns of these species are characterised by CO2 volleys during
an extended burst phase (B) of the cycle, which increase in prevalence as one
considers beetles from increasingly arid habitats
(Duncan and Byrne, 2000
).
Similar patterns have been found in centipedes from three orders and different
habitat types (Klok et al.,
2002
). This has been used as support for the hypothesis that the
DGC is an additional water saving strategy
(Kestler, 2003
), and is
bolstered by theoretical calculations that convective (active/forced)
ventilation loses less water than passive diffusion
(Kestler, 1985
; P. Kestler,
personal communication). Conversely, loss of the DGC at high temperatures
(Chappell and Rogowitz, 2000
)
or hypoxia (Chown and Holter,
2000
) has been used to question its value in respiratory
water-saving. However, the gradual change from DGC to continuous
CO2 release with increasing metabolic needs or lack of oxygen,
shows that the DGC persists as long as possible in situations of conflicting
needs. The match of spiracular opening to metabolic demands in flying
Drosophila, allowing a 23% reduction in water loss
(Lehman, 2001
), offers further
support for the adaptive significance of spiracular control in diminishing
desiccation stress.
The discontinuous gas exchange cycle has three distinct periods; closed
(C), flutter (F) and burst (B), each contributing to reducing water loss
(Kestler, 1985,
2003
; and P. Kestler personal
communication). The closed period, when all the spiracles are shut, prevents
any gas exchange with the atmosphere
(Bridges et al., 1980
). Any
closing saves water (the `closing strategy'), as only CO2 and
O2 are exchanged rapidly at the next opening; which is not the case
for water vapour (the `partial pressure strategy';
Kestler, 2003
). The closed
period is followed by the F period, during which the spiracles open, either
repetitively in micro-openings (Kestler,
1985
) or in more regular miniature inspirations as found in locust
(Hustert, 1975
) and
tenebrionid beetles (Lighton,
1991
; Duncan,
2003
), or by a more or less wide opening with vibrating flutter
movements according to the O2 demand. Minimising the opening
frequency minimises water loss by the closing strategy in combination with the
partial pressure strategy. Simultaneous diffusion and convection during
expiratory openings causes CO2 to be released faster than
H2O is lost (Kestler,
1985
).
Four species, from two closely related tribes
(Philips et al., 2004) and in
the same subfamily, that have widely separate distributions
(Fig. 1B) and different flight
abilities, were chosen to examine the role of the mesothoracic spiracles in
respiration. Circellium bacchus is a large, flightless, slow-moving
beetle that has a restricted distribution in the xeric Valley Bushveld
(Acocks, 1988
) of the Eastern
Cape Province of South Africa (Coles,
1993
). It has the ability to roll dung balls for food caching, but
this behaviour is largely restricted to immature `teneral' adults, while
mature adults feed at the middens of large herbivores (C. H. Scholtz, personal
communication). Beetles of the subgenus Pachysoma are small to medium
flightless species, found on the opposite coast, in a narrow sandy strip of
the Western Cape Province, where the rainfall is very low
(Fig. 1B), but the relative
humidity varies between 79% to 88%. In addition, beetles forage on dry rodent
pellets with which they provision a chamber below the moisture line in the
sand where the dung rehydrates, providing food for about a week underground
(Scholtz, 1989
). Finally,
P. femoralis is a widespread species, occurring across the central
summer rainfall region of southern Africa. It is a large, powerful flier and
competes strongly for dung, which it either rolls or butts away from the pat
(Byrne et al., 2003
). All four
species have eight pairs of spiracles, one pair that open under the
mesothorax, and seven under the elytra (one pair of metathoracic and six pairs
of abdominal) (Fig. 1A). All
species are diurnal.
Given the general ecological pattern of changing DGC patterns, from CFO
cycles to CV cycles, found in dung beetle species adapted to arid habitats
(Duncan and Byrne, 2000) and,
specifically, the extreme example found in C. bacchus, coupled to an
anterograde respiratory airflow, both of which are assumed to be water saving
adaptations (Duncan and Byrne,
2002
; Duncan,
2002
), we would predict that different dung beetle species, from
different tribes and different regions that are nevertheless both arid, are
very likely to share similar respiratory adaptations.
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Materials and methods |
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To measure the relative amounts of CO2 released from the
mesothoracic and subelytral spiracles, beetles were placed in a perspex
respirometry chamber, divided crosswise by a sheet of latex. 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 an airtight seal between the
prothorax and the abdomen. (For evidence that the seal was airtight see fig. 5
in Duncan and Byrne, 2002. For
all beetles measured, the seal was tested by pumping a different gas
concentration though one chamber and measuring it from both.) Each section of
the chamber had a volume of approximately 100 ml. A flow-through respirometry
system was used to measure CO2 emission from an inactive beetle at
room temperature (25±2°C), with simultaneous sampling from the
anterior body, including the head and the mesothoracic spiracles, and the
posterior body comprising the elytral case; which covers the metathoracic and
abdominal spiracles (for details see Duncan,
2002
,
2003
;
Duncan and Byrne, 2002
). One
inlet and one outlet served each compartment of the chamber. The air pressure
in each compartment was monitored by manometers to ensure that there was no
difference in pressure between the chambers throughout the experiment. The
latex sheet was renewed for each trial.
Both compartments had independent air sources, scrubbed of CO2 and H2O vapour by a Drierite/Ascarite column, which were drawn through at 50 ml min1 (controlled by separate calibrated Supelco flow meters; Bellefonta, PA, USA) and into individual Licor CO2 analysers (a differential non-dispersive gas analyser, LI-6262, resolution 0.1 ppm; Licor, Lincoln, NE, USA). The length of the tubes to each analyser was identical and was kept to a minimum. Samples of the volume of CO2 emitted were taken every 5 s and recorded using computerised data acquisition software (Datacan V, Sable Systems, Henderson, NV, USA). Measurements were made on individual beetles that had been weighed to ±0.1 mg (Precisa 160A balance; Instrulab, Midrand, South Africa). The beetles' respiration patterns were recorded for a minimum of 6 h during the night, with the same conditions being used for all the species. Beetles were re-weighed after the respiratory measurements.
Baseline drift of the analysers during recording was corrected from
measurements taken at the beginning and end of each trial with the
respirometry chambers empty. The zero drift over the time of the experiment
was a continuous function, not cyclic. All measurements were corrected to
standard temperature and pressure (STPD). The CO2 recordings were
converted to rate of CO2 emission
(CO2) in ml
h1. The DGC characteristics were calculated as follows: the
DGC frequency (= burst frequency) was calculated by determining the number of
peaks of CO2 per second, and the DGC duration was considered as one
complete cycle (closed, flutter, burst). The mean rate of CO2
emission was taken as the mean value over several complete DGC cycles and the
emission volume of CO2 was obtained by integration of the area
under the curve against duration in hours.
Data are presented as means ± S.D. Sample size (N) is indicated in the text as either representing individual beetles or in the case of gas exchange characteristics, ten to twenty discontinuous gas exchange cycles per beetle. Unless otherwise noted, statistical comparisons were made either with the Student's t-test or analysis of variance (ANOVA). Significant ANOVAs were followed with the Newman-Keuls multiple range test. Regression analysis was done by the least squares method and regression lines were compared using ANCOVA.
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Results |
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In P. femoralis and S. gariepinus the rate of CO2 emission from the subelytral spiracles increased with rising metabolic rate (Fig. 3A,B) and the slopes were identical, with the common slope being 0.86. Conversely, in C. bacchus and S. striatum the mesothoracic emission rate increased with increasing metabolic rate (Fig. 3C,D), with both species having a common slope of 0.87 (ANCOVA).
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Metabolic rates
The metabolic rate from the four beetle species formed a series with C.
bacchus having the lowest, followed by the other two wingless species and
P. femoralis having the highest
(Table 1). The mass specific
metabolic rates showed a significant difference between the flighted P.
femoralis and flightless C. bacchus
(Table 1).
Respiratory patterns
The CO2 emission trace for P. femoralis
(Fig. 2A) shows the three
periods of the DGC in a closed, flutter, open (CFO) cycle, as compared with
the closed, flutter, ventilation (CFV) cycle seen in the other three species
(Fig. 2BD). The burst
periods differs in that P. femoralis shows a smooth trace of
CO2 release without CO2 volleys, while the other three
species reveal strong CO2 volleys
(Fig. 2).
The frequency of DGC is significantly faster in P. femoralis than in the flightless species (Table 1). In P. femoralis and S. gariepinus the posterior spiracles contribute more to the emission of CO2 in all the DGC periods, but in S. striatum there is no significant difference between the contribution of the mesothoracic spiracles and elytral case (Table 2). Scarabaeus gariepinus and C. bacchus have the longest closed period, occupying almost half of the DGC duration. In all the flightless species ventilative bursts, which are seen as volleys of CO2 release within the burst period, are present.
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Discussion |
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Anterior and posterior spiracle use
Direct observation of spiracular movements and abdominal pumping are needed
to interpret the respiratory strategies used by these beetles more precisely.
These are difficult to view in a live specimen because of the tight elytra and
spiracular sieve plates, but a reasonable interpretation of their movements
can be made by proxy through their patterns of CO2 release in a
divided through-flow respirometry system. What is revealed is a pattern of
breathing in which the flightless species are more similar to each other,
despite being members of different tribes, but still remain specifically
different from each other, even within the same genus. Each species remains a
unique expression of the sum of adaptations which characterise it as a
species.
Pachylomerus femoralis appears to be a predominantly diffusive
burster of CO2, using outward diffusion mainly through the
posterior spiracles, under relatively unsealed elytra, whereas C.
bacchus restricts resting CO2 emission to the anterior
mesothoracic spiracles. Intermediate states of these two extremes are shown by
the Scarabaeus species, where S. gariepinus directs 20% of
its CO2 output to the mesothoracic spiracles, and S.
striatum increases this to 46% of the total production. This progressive
replacement of diffusion by a convective forward flow, and a replacement of
diffusive outflow from the subelytral cavity by active emission through the
mesothoracic spiracles is an anterograde version of the active ventilation
strategy proposed by Kestler
(2003). That it has not been
absolutely adopted by all of the flightless species tested here, suggests that
there may be a trade-off involving reduction of activity levels associated
with a decline in metabolic rate.
Metabolic rates
Low metabolic rates have been previously reported for flightless dung
beetles (Davis et al., 1999),
arid dwelling carabids and tenebrionids
(Zachariassen et al., 1987
)
and arid adapted Drosophila species
(Gibbs et al., 2003
).
Pachylomerus femoralis is a pugnacious competitor, described as a
facultative endotherm (Chown et al.,
1995
). Given the high energy requirements of competing for and
rolling dung balls (Bartholomew and
Heinrich, 1978
), coupled with the energetic demands of long
distance foraging flights (Yborrondo and
Heinrich, 1996
), this is predictable. Circellium bacchus
is a slow moving species that has been shown to be strictly ectothermic
(Nicolson, 1987
), and may have
been restricted to the arid bush of the Eastern Cape through competition with
Scarabaeini such as P. femoralis in more open moist habitats
(Chown et al., 1995
). By
comparison, it is reasonable to assume that given their small size, both
Scarabaeus species are ectothermic, but after initially basking flat
on the sand with their legs outstretched, they run swiftly while foraging for
dung pellets on the surface (Scholtz,
1989
). Their metabolic rates are intermediate between the two
larger species and may reflect a combination of lower metabolic demands of
flightlessness and behavioural thermoregulation in an open, sunny habitat.
Energetic constraints have been suggested to limit habitat use in
Afrotropical dung beetle guilds (Krell et
al., 2003). Even though the Scarabaeus species live in an
apparently more water-stressed habitat than C. bacchus, they probably
have access to a more predictable water supply in the daily fog that sweeps
the area (Seely, 1978
), the
high air humidity, and moisture deep in the sand
(Seely, 1978
) where they
rehydrate their dung supplies (Scholtz,
1989
). On the sand surface, rodent pellets would be expected to
persist in the absence of other competing species making their food supply
reasonably stable. By contrast, C. bacchus can only utilise fresh
dung and lives in a region of unpredictable rainfall with low soil moisture.
The metabolic rates of the experimental species are, therefore, explicable in
terms of their respective habitats and lifestyles and may indirectly
contribute to a reduction in water loss as lower needs for gas exchange allow
tactically longer closing periods between expiratory openings
(Kestler, 2003
).
Respiratory patterns
The CFO type of discontinuous gas exchange cycles seen in the mesic P.
femoralis suggest a loss of the ventilation period. The CFV form of the
discontinuous gas exchange cycle is typical for most adult insects except ants
(Lighton, 1996). However,
over-hydrated Periplaneta americana show a loss of pumping in the
ventilation period (Kestler,
1985
). Diffusion leads to higher water loss than pure convection
as water molecules diffuse faster than both O2 and CO2
(Kestler, 1985
).
Pachylomerus femoralis therefore could have lost the active
ventilation strategy to save energy at the expense of increased water loss. By
contrast, all three arid-adapted species use both strategies of water
retention, despite potential energy costs. From the 6 h of measurement the
total mass loss per hour was significantly greater in P. femoralis
(0.034±0.015 g h1) than in either S.
gariepinus (0.005±0.004 g h1) or S.
striatum (0.004±0.003 g h1)
(F(2,12)=16.4, P>0.05), indicating a
higher water loss rate for the mesic species. P. Kestler (personal
communication) has shown that a combination of diffusion and convection saves
more water than diffusion alone, but less than pure convection, which is not
possible in insects due to the short spiracular diffusion path that leads to a
rapid outward diffusion of CO2 during expiration.
In conclusion, the closely related dung beetle species in this study show limited physiological variations in their respiratory patterns. Nevertheless, the arid-dwelling species from different tribes and dissimilar habitats on opposite coasts of the continent, show increasing use of the anterior mesothoracic spiracles for CO2 expiration (which could be anterograde), coupled with more convection than diffusion in the discontinuous gas exchange cycle, than the centrally distributed mesic species. The patterns discovered represent a continuum from extreme anterior mesothoracic spiracle respiration to expiration from all the spiracles, rather than a strict dichotomy between the two, suggesting that control of breathing in these insects is closely adapted to the demands of their respective habitats.
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Acknowledgments |
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