The role of discontinuous gas exchange in insects: the chthonic hypothesis does not hold water
1 Department of Ecology and Evolutionary Biology, 1041 E. Lowell Street,
University of Arizona, Tucson, AZ 85721 USA
2 School of Life Sciences, Arizona State University, Tempe, AZ 85287-4501,
USA
* Author for correspondence (e-mail: agibbs{at}arl.arizona.edu)
Accepted 29 June 2004
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Summary |
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Key words: discontinuous gas-exchange cycle, queen mating stage, Pogonomyrmex barbatus, respiratory water loss, seed-harvester ant
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Introduction |
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The paradigm that DGC are an adaptation to reduce respiratory water loss
has been challenged in recent years. Several studies reveal that respiratory
water loss comprises <15% of total water loss, even when the spiracles are
open (e.g. Lighton, 1992;
Quinlan and Hadley, 1993
;
Quinlan and Lighton, 1999
;
Bosch et al., 2000
;
Chappell and Rogowitz, 2000
;
Rourke, 2000
). Thus,
respiratory water loss is a relatively minor component of the overall water
budget, so that the pattern of gas exchange may not significantly affect
overall water balance. In addition, many insects cease performing DGC at high
temperatures or when dehydrated (Quinlan
and Hadley, 1993
; Chappell and
Rogowitz, 2000
; Rourke,
2000
), conditions under which water conservation should be most
important. These considerations have led many authors to conclude that the
standard explanation for discontinuous gas exchange is inadequate (Lighton,
1996
,
1998
;
Shelton and Appel, 2001
;
Chown, 2002
).
Discontinuous gas-exchange cycles are taxonomically widespread, but not
universal, and their evolutionary origin is unknown. Lighton
(1996,
1998
; see also
Lighton and Berrigan, 1995
)
noted the prevalence of DGC in fossorial insects, which inhabit microclimates
where CO2 levels may be relatively high. Consequently, Lighton
proposed the chthonic hypothesis, which suggests that DGC originated as a
mechanism to improve gas exchange while at the same time minimizing
respiratory water loss. Under the chthonic hypothesis, insects build up
CO2 when the spiracles are closed, creating an increased gradient
for outward diffusion when they open. The same amount of CO2 can
then be excreted during shorter periods of spiracular opening, thus reducing
the associated respiratory water loss. The chthonic hypothesis, therefore,
makes the specific prediction that the ratio of respiratory water loss to
CO2 release will be lower in insects performing DGC than in those
using other modes of gas exchange. The null hypothesis is that this ratio will
not be affected by the pattern of gas exchange.
Alternatively, DGC could reduce tracheal oxygen levels, thereby increasing
the gradient for O2 uptake in hypoxic habitats (which are often
hypercapnic). In either case, the chthonic model emphasizes minimizing
respiratory water loss while allowing effective gas exchange
(Lighton and Berrigan, 1995;
Lighton, 1998
). For example,
Lighton (1998
) notes that an
alternative mode of gas exchange would be to open the spiracles continuously.
Even in hypercapnic or hypoxic habitats, sufficient gradients for diffusive
gas exchange would eventually be established. However, he concludes that this
"... is not a viable strategy except in water-saturated
air." Thus, the chthonic hypothesis can be viewed as an extension
of the traditional water conservation hypothesis, that attempts to explain the
adaptive significance of DGC under the specific environmental conditions in
which these cycles may have evolved.
We tested the chthonic model by measuring the respiratory water
loss:CO2 release ratio in reproductive females (queens) of the seed
harvester ant Pogonomyrmex barbatus F. Smith (Formicidae:
Myrmicinae). Our central hypothesis was that this ratio would be lowest in
ants performing DGC, relative to those using other modes of gas exchange.
Pogonomyrmex barbatus is a soil-dwelling species that occurs in arid
regions of southwestern North America (Johnson,
2000a,b
).
Summer rains trigger the mating flights, and the sexuals fly to mating
aggregations that contain many thousands of individuals
(Hölldobler, 1976
). Each
female mates with several males, then tears off her wings and digs a burrow in
which she starts her colony. Water-loss rates more than double from the time
when the queen flies from her nest to several days after mating
(Johnson, 2000a
;
Johnson and Gibbs, 2004
). The
queen supports herself and rears her first brood of workers, which emerge
after 45 weeks at 30°C, solely using her body reserves
(Johnson, 2002
). Water
conservation during this period underground is critical to success of the new
colony (Johnson, 2000c
), so
P. barbatus provides an ideal species to test the chthonic
hypothesis.
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Materials and methods |
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Respirometry
We measured water-loss rates and metabolic rates for each ant using
flow-through respirometry. Ants were placed in 5 ml glass-aluminum chambers in
a darkened incubator at 30°C. Dry, CO2-free air was pumped
through the chambers at 100 ml min1 to a Li-Cor (Lincoln,
Nebraska, USA) LI-6262 infrared CO2 and water vapor sensor.
Chambers containing the ants were placed in the respirometer for an
acclimation interval of approximately 3 h, then data were recorded for 30 min.
Time-averaged data were recorded every 5 s and analyzed using Datacan V
software (Sable Systems; Las Vegas, Nevada USA). Ants were weighed to the
nearest 0.1 mg before and after each run. Estimates of water loss during the
run, as calculated from mass loss, were consistent with our flow-through
measurements.
We analyzed data using two separate two-way analysis-of-covariance models (ANCOVA). Both models included pattern of gas exchange and mating stage as independent variables, with starting mass as a covariate; observed patterns of gas exchange were discontinuous, cyclic and continuous. The dependent variable in each model was the respiratory water loss:CO2 release ratio and metabolic rate, respectively. Ants were assayed at different times after collection, so we also included date as a covariate in both models. Time in the laboratory did not have a significant effect in either model, indicating that our results were not affected by acclimatization to laboratory conditions. Thus, we dropped date from our final models. Statistical analyses were performed using JMP 4.0 software (SAS Institute, Cary, NC, USA).
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Results |
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Previous studies have quantified respiratory water loss during cyclic gas exchange by assuming that water loss when the spiracles are closed represents cuticular transpiration. Respiratory water loss is then calculated by subtracting cuticular water loss from total water loss. This procedure cannot be used for continuous breathers, however, because the spiracles never close completely. We therefore developed a new method to distinguish between cuticular transpiration and respiratory water loss that can be applied to any pattern of gas exchange. We plotted water-loss rate against CO2 release for each individual using the 5 s time-averaged values over the 30 min respiratory run. Regression analysis of these data yielded a significant positive relationship in each case (Fig. 2). The slope of each regression line estimates the hygric cost of gas exchange for that individual, i.e. the incremental increase in water loss associated with CO2 release.
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It must be noted that the CO2 and water-vapor detectors were arranged in series in our respirometer. Under our experimental conditions, a given parcel of air reached the CO2 sensor about 2 s before it reached the water vapor sensor. Thus, signals from these sensors were slightly out of phase, but correction for this effect (by using a weighted average of consecutive humidity readings) did not significantly affect the calculated regression lines. We therefore used the raw data in our analyses.
The chthonic hypothesis predicts that the ratio of respiratory water loss to CO2 release is lowest during DGC, that is, the slopes of the regression lines will be lower for individuals performing DGC than for those using other types of gas exchange. Our data did not support this prediction, because the slopes did not vary by pattern of gas exchange (ANCOVA, F2,52=0.83, P>0.4) or mating stage (F2,52=0.74, P>0.4), or for the interaction between these two effects (F4,52=0.19, P>0.9); starting mass was a non-significant covariate (F1,52=0.09, P>0.7). Across all three patterns of gas exchange, ants lost an average of 1.76 moles of water per mole of CO2 expelled (Fig. 3).
|
Respiratory water loss:CO2 release ratios varied through the gas exchange cycle. Inspection of individual cycles revealed that the ratio (i.e. the slope) was lowest shortly after spiracles opened, when high internal PCO2 would create a large gradient for CO2 release (Fig. 4). After CO2 release had peaked, the water loss:CO2 ratio increased later in the cycle, as internal CO2 levels declined.
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Metabolic correlates of gas-exchange pattern
Metabolic rates differed significantly across patterns of gas exchange
(ANCOVA, F2,52=12.88, P<0.0001;
Fig. 5), but not by mating
stage (F2,52=0.096, P>0.9) or the interaction
between these two main effects (F4,52=0.58,
P>0.6). A posteriori comparisons showed that metabolic
rates were lowest for individuals using discontinous gas exchange,
intermediate for individuals using cyclic gas exchange, and highest for
individuals using continous gas exchange (pair-wise TukeyKramer test,
P<0.05 for all comparisons)
(Fig. 5). Starting mass did not
affect metabolic rate (F1,52=0.030, P>0.8),
presumably because of the limited size range of individuals used in this
study.
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Discussion |
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A critical experimental problem is distinguishing respiratory water loss from cuticular transpiration across all modes of gas exchange. Cuticular transpiration is easily quantified during the closed phase of the gas-exchange cycle in insects performing DGC, and respiratory water loss can be measured by integrating water-loss peaks during the open phase. However, this approach does not work if insects breathe continuously. Our regression method allows us to estimate respiratory water loss no matter which gas-exchange pattern is used. The slope of water loss:CO2 release plots quantify how much water is lost per molecule of CO2. The chthonic hypothesis would be supported if these slopes were lowest for individuals that used discontinuous gas exchange.
In queens of P. barbatus, the ratio of respiratory water loss to
CO2 release did not vary as a function of gas-exchange pattern. No
matter which pattern was used, 1.52 molecules of water were lost with
each molecule of CO2. This value is higher than that observed in
the carpenter ant Camponotus vicinus
(Lighton and Garrigan, 1995),
presumably due in part to the higher experimental temperature (and therefore
higher tracheal water-vapor pressure) in our experiments. More importantly,
the absence of differences between ants using different patterns of gas
exchange contradicts an important prediction of the chthonic model. We note,
however, that DGC could reduce respiratory water loss from P.
barbatus, if the ants opened their spiracles for shorter periods of time.
During the early portion of the open phase, high CO2 levels in the
tracheal system resulted in a steeper gradient for CO2 flux and a
lower ratio of water:CO2 release
(Fig. 4). Late in the open
phase, the CO2 gradient had decreased, but the gradient for water
loss would have remained the same. Water loss:CO2 release ratios
were relatively high during this period. If ants had closed their spiracles
sooner, the amount of water lost per CO2 would have been lower.
Thus, P. barbatus queens appear to open their spiracles longer than
they `should' to conserve water. We conclude that these ants do not regulate
gas exchange so as to conserve water, and that DGC do not reduce respiratory
water loss relative to other patterns of gas exchange. Similarly, Kanwisher
(1966
) noted that
Hyalophora pupae had longer open phases than necessary for effective
gas exchange.
One could argue that our results do not directly test the chthonic hypothesis, because our respirometry measures were not performed under hypercapnic conditions. Unfortunately, high CO2 levels saturate the LI-6262 detector and interfere with its ability to detect water vapor (M. C. Quinlan, personal communication), so we were unable to perform these experiments. We predict that, if such measurements were technically feasible, the ratio of respiratory water loss to CO2 release would be higher, because the gradient for water loss would be unchanged, whereas the gradient for CO2 release would decline. Assuming that metabolic rates remain the same, the frequency of cyclic gas exchange would increase as the net amount of CO2 lost during the open phase decreased. The more important issue is whether respiratory water loss:CO2 release ratios would differ among ants using different patterns of gas exchange. We find no reason to think that the chthonic hypothesis would be better supported under hypercapnia, because only the magnitude of the CO2 gradient would have changed from our acapnic experimental conditions.
We conclude that selection to reduce respiratory water loss in burrowing
insects cannot explain the origin of DGC. What then is the explanation? An
alternative formulation of the chthonic model proposes that DGC serves to
decrease tracheal oxygen levels, thereby increasing the gradient for
O2 uptake when the spiracles open (Lighton,
1996,
1998
). Our experiments do not
test this idea directly, but we note that CO2 production and
O2 consumption are tightly coupled via aerobic
respiration. Both gases are transported during the gas exchange cycle, and gas
exchange in other ants occurs primarily by diffusion rather than convection
(Lighton and Berrigan, 1995
).
Thus, O2 uptake and water loss will also be tightly coupled. In
addition, insects in general are still able to maintain their metabolic rates
in hypoxic conditions (Hoback and Stanley,
2001
) and may lose the ability to perform DGC under hypoxia
(Chown and Holter, 2000
), when
it would be most valuable for O2 uptake. Discontinuous gas exchange
has also been proposed as a mechanism to reduce parasitism
(Shelton and Appel, 2001
) or
to protect insects from oxidative stress by reducing internal O2
levels (Bradley, 2000
), but
these hypotheses have not been tested.
Alternatively, DGC may not be adaptive, but may simply arise from the
interaction of CO2 and O2 gas exchange setpoints and
their effects on spiracular regulation
(Chown and Holter, 2000). One
implication of this hypothesis is that cycle frequency increases with
metabolic rate, such that gas exchange becomes continuous at the highest
metabolic rates. Consistent with this, continuous breathers had the highest
metabolic rates, those exhibiting rapid cycling had intermediate metabolic
rates, and ants performing classical discontinuous gas exchange had the lowest
metabolic rates (Fig. 5). This
non-adaptive explanation for the origin of DGC does not preclude its retention
and modification for adaptive reasons. Discontinuous gas exchange will still
aid in water conservation, because water loss is reduced when the spiracles
are closed. Even so, DGC is not necessary for survival in arid environments
(Lighton and Berrigan, 1995
;
Quinlan and Lighton,
1999
).
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Acknowledgments |
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