615
The Journal of Experimental Biology 200, 615–624 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
JEB0538
CO2 RELEASE PATTERNS IN DROSOPHILA MELANOGASTER: THE EFFECT OF
SELECTION FOR DESICCATION RESISTANCE
ADRIENNE E. WILLIAMS*, MICHAEL R. ROSE AND TIMOTHY J. BRADLEY
Department of Ecology and Evolutionary Biology, University of California, Irvine, CA 92697, USA
Accepted 8 November 1996
Summary
We used laboratory natural selection on insects as a
desiccation-resistant populations only. Maximum SER was
means of investigating the role of patterns of gas exchange
found to be strongly correlated with survival time in dry
in desiccation resistance. We used 15 populations of
air among selection treatments, but not among individuals
Drosophila melanogaster: five selected for desiccation
within a population. Access to dietary water resulted in
resistance, five control populations and five ancestral
lower SER values. These data demonstrate that gas
populations. Using flow-through respirometry, we found
exchange is physiologically controlled in Drosophila
that D. melanogaster from all populations produced
melanogaster and that the pattern of gas exchange can
change under selection. The relationship between these
irregular peaks of CO2 release. To quantify the height and
CO2 release patterns and classic discontinuous ventilation
frequency of these peaks, we used the standard error of a
linear regression (SER) through the recordings of CO2
is discussed.
release. The values for the SER were significantly larger in
the populations selected for desiccation resistance than in
the control and ancestral populations. Occasionally, highly
Key words: Drosophila melanogaster, selection, CO2, discontinuous
gas exchange cycle, desiccation resistance.
periodic peaks of CO2 release were observed in the
Introduction
Most terrestrial insects experience a ready availability of
oxygen and limited supplies of water. They seek to limit water
loss through a number of physiological specializations of the
excretory system, the cuticle and the respiratory system
(Edney, 1977; Hadley, 1994a). It has been suggested that the
control of gas exchange is one of these specializations. Insects
have long been known to ventilate the tracheae intermittently
(e.g. Punt, 1950; Schneiderman and Williams, 1953),
principally by the action of spiracular valves at the external
openings of the tracheae. This intermittent release of
respiratory gases has, at various times, been termed
discontinuous respiration, closed-flutter-open cycles, closedflutter-ventilation cycles and discontinuous ventilation.
Lighton and Garrigan (1995) have recently adopted a more
general descriptive term, ‘the discontinuous gas exchange
cycle’ (DGC). We employ this term here, since it avoids any
confusion between gas exchange and metabolic respiration.
The definition of the DGC also makes no assumption about the
diffusive or convective mechanism of gas exchange or about
the state of the spiracles during each phase of the cycle.
In its classic form, as seen in Platysamia cecropia pupae
(Schneiderman, 1960; Levy and Schneiderman, 1966a,b,c), the
DGC consists of three phases which correspond to the
condition of the spiracles: open, closed or fluttering. It has been
*e-mail: [email protected].
hypothesized that the DGC conserves water, since water loss
from the respiratory surfaces would occur only during periods
in which the spiracular valves are open. The simplicity and
clarity of the hypothesis that the DGC evolved as a means of
water conservation has led to its historical acceptance,
although direct experimental evidence is limited (for a
discussion of the limitations of the current evidence, see
Edney, 1977; Hadley, 1994b).
Recently, the role of the DGC in water conservation in
adult insects has been called into question, notably by
Hadley (Hadley and Quinlan, 1993; Hadley, 1994b), who
found that cyclic CO2 release in the lubber grasshopper
Romalea guttata occurs only when they are motionless and
that the DGC is disrupted when the insects are desiccated. It
has also been shown that measurements of water loss during
the open phase of the DGC in different insects indicate that
the vast majority of water loss is cuticular, not respiratory
(Machin et al. 1991; Lighton, 1992; Hadley and Quinlan,
1993). In contrast, Lighton et al. (1993) has found that ants
with more watertight cuticles show a higher percentage of
respiratory water loss during the open phase of the DGC.
These results suggest that the DGC may play an important
role in water balance in some, but not necessarily all, insects
where the DGC occurs.
616
A. E. WILLIAMS, M. R. ROSE
AND
T. J. BRADLEY
As gas exchange patterns have been studied in a greater
variety of insects, it has become clear that the classic DGC of
open, closed and flutter phases may be only one of many
existing patterns. Differences in the relative length of the
phases have been observed in ants (Lighton, 1988, 1990). In
grasshoppers, there is gas leakage during the closed phase, no
flutter phase and multiple bursts of CO2 release during the
open phase (Quinlan and Hadley, 1993; Hadley and Quinlan,
1993). Discrete ‘flutter bursts’ rather than flutter phases are
seen in tenebrionid beetles (Lighton, 1991). A periodic DGC
is absent in crickets (Quinlan and Hadley, 1982), in middleaged grasshoppers (Hamilton, 1964), in active insects (e.g.
Lighton, 1991) and at high temperatures (Hadley and Quinlan,
1993). Lighton and Berrigan (1995) even observed that the gas
exchange patterns of different castes within an ant species can
differ. They suggest that the DGC might be an adaptation, at
least in these species, to habitats high in carbon dioxide. It
would seem, therefore, that discontinuous gas exchange,
which was initially thought to be a universal adaptation for
water conservation in terrestrial insects, is actually quite
variable in its expression within and between species. Its
significance in the conservation of water has also been called
into question.
Various laboratories are presently conducting studies that
attempt to clarify the role of the DGC by examining its
taxonomic distribution among arthropods, or by examining
differences in gas exchange patterns in insects from a variety
of habitats. An alternative approach is the use of selection
experiments. Such experiments can control precisely for
phylogeny and can reveal changes in physiology in response
to highly controlled selection criteria. We have been
conducting such experiments using the fruit fly Drosophila
melanogaster, which can be kept as large outbred populations
in the laboratory and is amenable to laboratory selection for
resistance to desiccation. Our experiment uses a series of 15
populations of D. melanogaster, five populations selected for
survival under desiccation, five control populations and five
ancestral populations. We describe the changes in CO2 release
patterns observed in these stocks of flies in response to
selection for desiccation resistance.
Materials and methods
Fly culture regime
We used 15 outbred populations of Drosophila
melanogaster selected and maintained in the laboratory of
M.R.R. (Rose, 1984; Rose et al. 1990; Graves et al. 1992). Five
replicate stocks of flies were the ancestor populations. These
stocks were formed in 1980 and maintained as large, outbred
populations. They were selected for postponed senescence and
are hence termed O (old) flies. They are currently on a 10-week
generation cycle.
In 1988, each replicate O stock was used to start two
additional populations, D (desiccated) flies, which were
subsequently selected for desiccation resistance, and C
(control) flies, maintained as controls for the D population.
Thus, the O1 stock is the direct ancestor of the D1 and C1
stocks, and the O2 stock is the ancestor of both the D2 and C2
stocks, etc. (Fig. 1).
The D populations were raised in vials until 4 days posteclosion, then selected for desiccation resistance by being
placed as large populations in acrylic cages containing 150 g
of Drierite desiccant wrapped in cheesecloth and containing no
water or food (since our fly food contains water). When
approximately 20 % of the flies remained alive (after
approximately 72 h), they were given food and water and
allowed to reproduce. The C and D populations differ only in
access to water: while flies from the D populations were
exposed to desiccating conditions, the C flies were maintained
as controls with access to a water source but not to food. The
C and D stocks were both on a generation time of
approximately 3 weeks. At the time of these experiments, the
C and D populations had undergone approximately 150
generations of selection.
All the stocks (O, C and D) were kept under 24 h light
regime and maintained on banana–molasses food as both
1980: O stocks created
1987: D and C stocks created
D1
O1
C1
D2
O2
C2
Ancestor
Fig. 1. The phylogenetic relationships between the 15
populations used in this experiment. The D populations were
selected for resistance to desiccation, and the C populations
were maintained concurrently with the D populations to act
as controls. The O populations are the ancestors to both D
and C populations.
D3
O3
C3
D4
O4
C4
D5
O5
C5
Effect of selection on gas exchange in Drosophila
larvae and adults. The stocks were raised and selected at
25 °C.
For the present study, all 15 stocks were raised identically
for two generations without overt selection pressure to
eliminate parental and grandparental effects. During these two
generations, the flies were treated in the following manner.
Larvae eclosed after 10 days in the hatching vials. The adult
flies were transferred into acrylic cages at day 14 and given
food and yeast paste to stimulate egg-laying. Eggs were
collected for the following generation and placed into 20 vials
containing standard food with 60–80 eggs per vial. Matching
replicates of stocks were reared on the same schedule: O1, C1
and D1 together, O2, C2 and D2 2 weeks later, etc.
Eggs for the experimental generation were collected from
parental stocks five times, providing five experimental groups
that would reach age 4 days post-eclosion every other day.
Only female flies were used from the experimental generation.
Females were used because only D females survive the
desiccation selection treatment. The D females are inseminated
by the males prior to selection.
For the separate hydration experiments, young adult females
were taken from the O1, C1 and D1 stocks during routine
maintenance. These flies were maintained in vials on
banana–molasses food until placed in the respirometer.
Patterns of CO2 release
Measurements were made on individual female flies in a
stream of dry, CO2-free air in a room maintained at 25±1 °C.
Peaks of CO2 release were measured by a Licor LI-6251
infrared CO2 analyzer with Sable Systems data aquisition
software. The noise level of this system is below 0.05 p.p.m.
at our level of measurement. The respirometry chambers were
1 ml plastic syringes cut down to 0.5 ml, with a small amount
of cotton placed at both ends to prevent the flies from leaving
the chamber and to maximize turbulence in the air flow.
A series of valves controlled the air flow to an empty control
chamber and six experimental chambers. For each experiment,
two flies from replicate stocks of ancestor, control and
desiccation-selected flies were placed individually in six
chambers (e.g. O1, O1, C1, C1, D1, D1). Air flow from only
one chamber was read at any one time. The air was passed
through a Drierite/Ascarite/Drierite column to dry it and
remove CO2, and was then drawn through one chamber at a
flow rate of 100 ml min−1 using a vacuum. The release of CO2
by the fly was averaged and recorded once per second. A pump
was used to provide dry, CO2-free air at a flow rate of
100 ml min−1 through all chambers not being analyzed. The
continuous flow of dry air over the insect created a strongly
desiccating environment during the experiment.
At the start of each respirometry run, one female fly at age
4 days post-eclosion was anesthetized using CO2 gas, placed
in an experimental chamber and allowed 20 min to recover,
after which the respirometry recording was activated. This
process was repeated with subsequent flies placed in the
respirometer at timed intervals so that each fly was subjected
to identical treatment prior to recording. Each cycle of data
617
acquisition recorded from the empty control chamber for two
separate 6 min periods and from each experimental chamber
containing a fly once (for 18 min). This cycle was repeated
every 2 h until all flies were dead. Since the flies from the D
populations had significantly longer survival times, the
recording procedure was changed after the death of the O and
C flies to measure values for the two D flies more frequently
until their death. Repeating the recordings in this manner
allowed us to examine the CO2 release patterns of the flies as
they spent increasingly more time in the desiccating
environment of the respirometry chamber.
Each set of replicates (e.g. O1, O1, C1, C1, D1, D1) was run
five times over 5 days. The order of the flies in the eight
chambers was systematically varied to eliminate any chamber
effects. By the end of the experiment, we had collected data
for 8–10 flies from each of the 15 populations.
Effect of hydration
To test whether the ventilatory patterns of our Drosophila
melanogaster populations are affected by hydration state, an
additional experiment was carried out. Adult flies were
gathered from O1, C1 and D1 populations as representative
samples of our 15 populations. These flies were divided into
two treatments: hydrated or dehydrated (N=15 for each
treatment per population). The release of CO2 from the flies
was then measured. Individual females were placed in
modified respirometry chambers made from 3 ml syringes. The
flies either had access to a wet filter-paper wick (hydrated
treatment) or were separated from the wick by a sponge barrier
and dry air flow (dehydrated treatment). This protocol served
to equalize the humidity of the excurrent air samples and
matched any effects of the water-filled wick acting as a CO2
sink or source. At the start of each run, six flies from one
population were anesthetized and placed individually in six
chambers: three hydrated and three dehydrated. CO2 release
was measured as described above, with repeats continuing until
the death of the dehydrated flies.
Data analysis
Measurements of survival time in the respirometer,
metabolic rate and gas exchange pattern were obtained for
8–10 flies from each of the 15 stocks. We recorded CO2 release
for each fly for 18 min once every 2 h from the time they were
placed in the respirometer until death. A precise determination
of death is difficult in small insects, but we found that the
disappearance of peaks on the CO2 recording corresponded
with the loss of visible movement in the fly and was quickly
followed by a decrease in CO2 release to 0 p.p.m. We felt
confident that the change in CO2 release from large peaks to
uniform release could be used to indicate death. Zero CO2
levels in the control chamber were used as a baseline zero
recording for each repeat. Although each recording interval
lasted 18 min for each fly, we used only the last 16 min in our
data analysis to reduce any effect caused by the switch from
flushing to sampling air flow.
Behavior often has a strong effect on insect gas exchange
618
A. E. WILLIAMS, M. R. ROSE
AND
T. J. BRADLEY
(e.g. Kestler, 1991; Hadley, 1994a). In experiments with other
insects, data on DGCs are often collected when the animal is
motionless, thus removing the effects of increased CO2
production due to muscular ventilation of the tracheae and air
sacs. To a large extent, however, Drosophila melanogaster are
never motionless. Flies from the C and O populations often
walked and groomed continuously (but did not fly) until they
were severely desiccated. In spite of this limitation, our
observations established that large, noticeable peaks of CO2
release in active flies were not correlated with isolated
behavioral events such as struggling to exit the chamber,
righting or bouts of flight. Non-quantitative observations
indicated that flies from the D populations were much more
quiescent. Using the D flies, we were able to establish that
peaks of CO2 release were not due to any visible activity.
Large, occasionally rhythmic releases of CO2 were observed
in flies that appeared entirely quiescent.
The peaks of CO2 release recorded from D. melanogaster
were not periodic or of regular height. While fast-Fourier
transformations and autocorrelation analyses are useful for
assessing periodicity, we were more interested in quantifying
peak height and frequency in our non-periodic data. We
therefore used the standard error of a regression through the
data to provide an objective measure of peak height and
frequency. The recordings of CO2 release were fitted using a
linear regression, and we calculated the standard error of that
regression (SER) as a measure of the ventilatory pattern.
Repeated measurements from the empty control chamber gave
an average SER of 0.01326 µl CO2 h−1. This value was
subtracted from all other measurements of SER to remove the
signal noise of the respirometry equipment.
The statistical ‘units’ in the comparisons of O, C and D
selection regimes are the 15 populations. We compared the
SER values of these populations in two ways: by comparing
the largest peak size and frequency that the populations are
capable of generating (maximum SER), and by comparing the
average SER present at different times in the desiccating
environment of the respirometry chamber (average SER).
Maximum SER provides an estimate of the size of the largest
peaks of CO2 a fly can produce, while average SER is useful
for describing changes in the peak height with time or
desiccation. We measured the maximum SER for a population
by averaging the maximum SERs of the 8–10 flies used from
that population. To find the maximum SER for a single fly, we
calculated the SERs for the full recording interval for each of
the 6–50 recording intervals obtained for each fly (the number
of intervals was dependent on the survival time). The highest
SER from all the intervals for that fly was averaged with the
maximum SERs from the other flies in the same population to
obtain the population maximum SER. The five population
SERs from each selection regime (O1–5, C1–5, D1–5) were
also averaged to produce treatment maximum SERs for the O,
C and D populations. The average SER values were gathered
at each time interval by taking the SER values from all live
flies within a population, and using the mean SER to represent
that population at that time.
Statistics
Comparisons of survival time and maximum SER between
selected stocks were carried out using a single-factor analysis
of variance (ANOVA) on 15 population means (d.f.=2,
N=15). Correlations between survival time and SER were
calculated using a linear regression (d.f.=1, N=15). The same
correlations within individual populations were also checked
with linear regressions (d.f.=1, N=8–10). Comparisons of
metabolic rate at all the times between hours 2 and 10 were
calculated using a 3(stock) × 5(time) repeated-measures
ANOVA on all 15 population means (d.f. stock=2, d.f.
time=4, d.f. interaction=8; N=75). The SER comparisons
were analyzed similarly at all the times between hours 0 and
10. After finding a significant effect of selected stock for both
SER and metabolic rate, single-factor ANOVAs were then
calculated on the grouped data to allow for post-hoc
comparisons. The comparison between hydrated and
dehydrated SER on three combined populations was analyzed
using single-factor ANOVAs (d.f.=1, N=45). All post-hoc
comparisons were analyzed using the Student–Newman–
Keuls test at the 0.05 significance level.
Results
Patterns of CO2 release in Drosophila melanogaster
The selection regime for each experimental treatment (O,
C, D) has been repeated in each of five populations. We
defined the maximum SER value as the average of the
maximum responses observed in all populations from a single
selection treatment (see Materials and methods). These
treatment means ± S.E.M. give the differences in SER between
the O populations (0.48±0.02 µl CO2 h−1), the C populations
(0.70±0.03 µl CO2 h−1) and the D populations (1.38±
0.14 µl CO2 h−1).
Fig. 2 gives examples of the CO2 release patterns observed
in the O, C and D flies. We illustrate patterns that provide a
calculated SER near the maximum SER. In flies from the O
populations (Fig. 2A) and the C populations (Fig. 2B), these
patterns have irregular peaks of moderate height. The peaks
from the flies of the D populations (Fig. 2C,D) are larger. Note
that in the D populations, large values of SER are possible
either from irregular peaks with heights 1–3.5 times those of
the lowest CO2 levels (Fig. 2C) or from highly regular peaks
with heights 1–1.5 times the lowest CO2 level (Fig. 2D). In our
recordings from 50 D flies from five populations, the irregular
peaks were the most common pattern observed. Regular,
periodic peaks such as those shown in Fig. 2D were never seen
in the C or O populations.
As discussed in Materials and methods, Drosophila
melanogaster, especially in the five O and five C populations,
were continuously active in the respirometry chamber. It is
very likely that the noncyclic nature of CO2 release in these
two treatments is at least partially due to their continual
walking and grooming. We made visual observations of the
flies in the respirometer at the same time as recording CO2
Effect of selection on gas exchange in Drosophila
14
A
B
12
O fly
SER = 0.52
C fly
SER = 0.75
10
619
8
Rate of CO2 release (µl h−1)
6
4
2
0
0
2
4
6
8
10
12
0
2
14
C
D
12
D fly
SER = 1.54
D fly
SER = 1.47
10
4
6
8
10
12
4
6
8
10
12
8
6
4
2
0
0
2
4
6
8
10
12
0
Time (min)
2
Fig. 2. Samples of ventilatory patterns observed in female flies from the O populations (A), the C populations (B) and the D populations (C).
The SER (standard error of the linear regression) is a measure of the height and frequency of the CO2 peaks. These recordings were chosen
because they have SER values near the maximum SER recorded for each treatment (see Materials and methods). (D) The flies from the D
populations also showed bouts of periodic CO2 release.
release and found that large peaks of CO2 release, such as the
spikes above 10 µl CO2 h−1 in Fig. 2C, were not correlated with
isolated bouts of activity.
1.4
Maximum SER (µl CO2 h−1)
Survival time and maximum CO2 peaks
Fivefold replication of the populations allows us to test for
statistically significant differences between treatments. On
the basis of survival time in the stream of dry air in the
respirometer, the desiccation-selected populations can resist
desiccation better than the control or ancestral flies (P<0.01).
If survival time is plotted against maximum SER for the 15
populations, there is a significant positive correlation
(r=0.847, P<0.01). Post-hoc tests indicate that the survival
time and maximum SER of the D populations are
significantly different from those of the O and C populations
(P<0.05), but the O and C treatments are not significantly
different from each other with respect to either characteristic
(Fig. 3).
While the correlation between maximum SER and survival
time is highly significant at the treatment level, it is not seen
within single populations (Fig. 4). Correlations between
maximum SER and survival time for values from individuals
from populations C5, D5 and O5, for example, have r values
of 0.33 for C5, 0.23 for D5 and 0.03 for O5. None of the 15
stocks (N=8–10 individuals for each) showed a significant
within-population correlation between survival time in the
respirometer and maximum SER.
1.6
D
1.2
1.0
0.8
0.6
C
0.4
O
0.2
5
10
15
20
25
Survival time (h)
30
Fig. 3. Effect of selection for desiccation resistance on survival time
and ventilatory pattern, where the height and frequency of peaks of
CO2 release are quantified as the standard error about a linear
regression through the data (SER) (see Fig. 2). Values are treatment
means ± S.E.M., N=5. All five D populations show a significant
increase in survival time in the respirometer and an increase in
maximum SER relative to the control or ancestor flies (P<0.05). Posthoc tests indicate that the O and C treatments are not significantly
different from each other.
620
A. E. WILLIAMS, M. R. ROSE
AND
T. J. BRADLEY
1.4
O5
C5
D5
1.6
O
C
D
1.2
SER (µl CO2 h−1)
Maximum SER (µl CO2 h−1)
2.0
1.2
0.8
1.0
0.8
0.6
0.4
0.2
0.4
0
0
0
0
10
30
20
Survival time (h)
40
10
20
Time (h)
30
40
50
Fig. 4. Lack of relationship between maximum SER (standard error
of a linear regression through the data, see Fig. 2) and survival time
within populations. While there is a strong correlation between these
factors among treatments, there were no significant correlations
within any of the 15 populations. Three populations, O5, C5 and D5,
are shown here as examples. Each point represents the data from an
individual fly. The r values for these correlations are 0.03 for O5, 0.33
for C5, and 0.23 for D5.
Patterns of CO2 release under increasing desiccation
If discontinuous gas exchange is a physiological response to
desiccation, one might expect the CO2 release pattern to
change as the insect desiccates. Fig. 5 illustrates the SER of
the CO2 release versus time in the respirometer. We analyzed
the mean SER of the desiccation-resistant, control and ancestor
populations over the first 10 h, the period during which all 15
populations were alive. Mean SER is significantly different
between selection treatments (P=0.04) and with time (P<0.01).
There is also a strong interaction between stock and time
(P<0.01), indicating that there is a difference in how the
treatment groups respond to desiccation over time.
All flies show similar SER values at hour 2. The C and D
stocks show identical and increasing mean SER values until
hour 8. At that time, these populations diverge in their
response. The C flies show a decrease in SER for several hours
prior to death. The D flies show higher and somewhat more
variable SER values for many hours until levels decline shortly
before death. The O flies showed no increase in SER above
initial levels, but a similar decrease near death. Post-hoc tests
indicate that SER values in the O flies are significantly
different from those in the C and D flies during hours 0–10.
Effect of hydration on ventilatory pattern
The above results demonstrate that the flies exhibit nonuniform CO2 release in the respirometer under our
experimental conditions. In addition, flies which have
undergone selection for desiccation resistance show an
Fig. 5. Effect of desiccation on ventilatory pattern (SER, see Fig. 2),
where values are treatment means ± S.E.M., N=5. D flies survive for
longer periods in the desiccating environment of the respirometer. An
examination of average SER during the first 10 h (when all flies were
alive) shows that SER changes significantly with time (P<0.01) and
with selection treatment (P=0.04). The post-hoc tests indicate that
SER is reduced in the O flies compared with the other treatments
(P<0.05).
increase in maximum SER compared with flies that have not
undergone such selection. One possible explanation for these
results is that the CO2 release pattern observed in the
respirometer is a specific response to the desiccating
conditions. If so, access to water during respirometry should
cause a reduction in SER. We therefore examined the CO2
release pattern of flies in the respirometer under conditions in
which they were or were not allowed access to free water in
the form of a wet wick. We investigated whether flies reduce
levels of discontinuous gas exchange in the presence of water,
a hypothesis that makes no assumptions regarding the selection
treatments of the flies. We used flies from the O1, C1 and D1
populations as a representative sample of our 15 populations.
Flies provided with a water source maintained a lower SER
(0.44±0.05 µl CO2 h−1, mean ± S.E.M.) than flies in desiccating
conditions (0.78±0.08 µl CO2 h−1, P<0.01). Sample CO2
release patterns of a hydrated and dehydrated fly are shown in
Fig. 6.
Rate of CO2 release
We wished to determine whether the larger peaks in CO2
production in the D populations were associated with an
increased overall rate of CO2 release. We found that the mean
rate of CO2 release differed significantly with selection
treatment (P=0.019) (Fig. 7) but post-hoc tests indicate that it
is the C populations rather than the D populations that have a
slight but significantly higher average CO2 release
(4.7 µl CO2 h−1 for the C flies compared with 3.9 µl CO2 h−1 for
the O flies and 4.1 µl CO2 h−1 for the D flies, P<0.05).
Correction for dry mass removed any significant difference in
Effect of selection on gas exchange in Drosophila
621
Fig. 6. Sample ventilatory patterns
of flies from the O1 population
when a water source was available
in the respirometer (hydrated) and
when it was not (dehydrated). Both
recordings were obtained after the
flies had been in the respirometer
for 4 h.
Rate of CO2 release (µl h−1)
12
10
Dehydrated fly: hour 4
8
6
Hydrated fly: hour 4
4
2
0
0
2
4
6
8
10
12
14
Time (min)
the rate of CO2 release between treatments during hours 2–10.
Because the dry mass of the flies changed during desiccation
in the respirometer, data are presented in Fig. 7 as µl CO2 h−1
per fly, not per milligram dry mass. There was no significant
change in the rate of CO2 release with time (P=0.070) during
hours 2–10 when all populations were alive. The interaction
term was significant, indicating that the treatment groups
respond differently as time in the respirometer increases
(P<0.01, see Fig. 7).
1957; Miller, 1964), measurement of water loss during
different phases of the DGC (Kestler, 1985; Lighton, 1988;
Machin et al. 1991; Hadley and Quinlan, 1993) and
comparisons of the DGC in mesic versus xeric species
(Lighton, 1992; Quinlan and Hadley, 1993). We have used an
additional experimental approach, that of laboratory natural
selection, as a tool for exploring the association of ventilatory
pattern with water conservation. The advantages of the use of
laboratory natural selection are as follows: (1) the number of
groups or replicate populations that can be compared is subject
to the control of the experimenter, (2) the number of subjects
from each group can be maximized since populations are
maintained in the laboratory, (3) the phylogenetic relationships
between all groups are known exactly, and (4) the selection
pressures can be defined and replicated and are thus much
better understood than historical conditions for wild-caught
species.
Our selection study began with five replicate populations of
the ancestral O treatment. By deriving one desiccation-selected
(D) and one control (C) population from each ancestral (O)
Discussion
Effects of selection on discontinuous gas exchange
Since the discovery and characterization of the DGC (see
Introduction), a number of experimental approaches have been
used to examine the role of discontinuous gas exchange in
water conservation. These include studies examining changes
in spiracular opening rates under humid or dry conditions
(Miller, 1964; Krafsur, 1971), water loss measurements with
the spiracles forced open using CO2 (Mellanby, 1934; Bursell,
Fig. 7. Effect of desiccation on rate of CO2 release. Average
CO2 release rate over hours 2–10 (when all flies were alive)
differs significantly with selection treatment (P=0.02), with
the C flies having slightly higher rates than the D and O flies.
Values are treatment means ± S.E.M., N=5.
Rate of CO2 release (µl h−1 fly−1)
7
6
5
4
3
2
O
C
D
1
0
0
5
10
Time (h)
15
40
622
A. E. WILLIAMS, M. R. ROSE
AND
T. J. BRADLEY
population, we produced a balanced phylogeny; each D
population is more closely related to its control and ancestor
populations than to other D populations. This maximizes our
capacity to distinguish the effects of selection from the effects
of shared phylogeny. All five D populations show a three- to
fourfold change in desiccation resistance, expressed as hours
of survival in dry air, compared with both the ancestral and
control populations. Since this differentiated desiccation
resistance is expressed following exposure to identical rearing
conditions for two generations (see Materials and methods), it
is clearly a result of genetic differentiation and not
environmental acclimation.
We used these stocks to search for correlations between
desiccation resistance and the pattern of CO2 release. If control
of CO2 release has no effect on resistance to desiccation, we
would expect no change in CO2 release pattern in the D
treatment. We found, however, that the D populations do show
an increase in the size and/or frequency of CO2 peaks as
measured by the standard error of the regression through the
CO2 release recordings (SER). This increase occurred
independently five times under the same selection regime, and
similar increases did not appear in the five control or the five
ancestral stocks. These results indicate that CO2 release pattern
can vary with selection. The fact that the pattern varies in
different populations under identical conditions indicates a
genetic control of the DGC.
This link between increased SER and increased desiccation
resistance could occur in two ways. Selection for resistance to
desiccation might act directly on the control of gas exchange
because of its value in reducing water loss. Alternatively, CO2
release might only be linked to the trait that selection operated
upon, either through pleiotropy or through a physiological
effect. For example, selection for desiccation resistance may
increase quiescence under stress, which allows greater
periodicity in CO2 release. Our finding that the SER increased
in the D populations establishes that selection for desiccation
resistance affects gas exchange. Whether the DGC is the target
of selection could be elucidated using additional experiments,
such as by relaxing selection in the D populations or by
selecting for both high activity and desiccation resistance in
other populations of Drosophila melanogaster.
Pattern of CO2 release and hydration
Because of our interest in the effect of desiccation on
patterns of CO2 release, and the above observation that
desiccation-resistant flies show enhanced SER values, we
chose an additional method of testing the relationship between
CO2 release peak size and hydration state by examining the
pattern of CO2 release of hydrated flies in the respirometer.
When insects were placed in the respirometer with a water
source, the SER was significantly reduced compared with that
of flies from the same populations that did not have access to
water.
Only a few previous studies have compared the patterns of
gas exchange in hydrated and dehydrated insects. Hamilton
(1964) found that locusts respired discontinuously only when
very young (and water loss was high) and when starved (no
water source). He concluded that the DGC was initiated to save
water. Loveridge (1968) showed that dehydrated locusts
ventilated the abdomen more slowly, and argued that this
served to save water. These early studies showed a link
between hydration and patterns of gas exchange – apparently
so convincingly that little similar work followed for many
years. Recent studies have provided more ambiguous results.
Machin et al. (1991) found that dehydrated cockroaches
ventilate more regularly and more often. They attributed this
to a decreased capacity for CO2 storage due to declining blood
volume. Hadley and Quinlan (1993) found that desiccated
grasshoppers have a less organized DGC than that of hydrated
individuals, which led Hadley (1994b) to suggest that the DGC
is not important for water conservation in this species.
Our work on Drosophila melanogaster adds information
about a small dipteran to this body of data. The large surface
area to volume ratio of Drosophila melanogaster makes them
particularly vulnerable to water loss, so we might expect
ventilatory control to be important in this species. Our findings
support this expectation, since Drosophila melanogaster show
a CO2 release pattern that changes with desiccation.
Do Drosophila melanogaster show a DGC?
The opening and closing of spiracles during the DGC has
historically been demonstrated by measuring CO2 release and
O2 uptake. In a classical DGC, levels of CO2 release fall to
zero during the closed phase and rise in single bursts during
the periodic open phases. Drosophila melanogaster show
mostly non-periodic release of CO2 (see Fig. 2) without any
reduction to zero release between peaks (see Fig. 2D). Does
this mean that the gas exchange pattern observed is not a DGC?
D. melanogaster are too small to measure O2 uptake in single
individuals, so we cannot obtain temporally correlated data on
CO2 release and O2 uptake with the technology available.
Our evidence indicates that the CO2 release pattern of fruit
flies is responsive to the environment and to selection, and is
apparently adaptive. As yet, we have little information
regarding the mechanism of gas exchange control in these
insects. Drosophila melanogaster have thoracic and abdominal
spiracles, and large thoracic and cranial air sacs. The CO2
peaks observed in the present study may be due to spiracular
control or to forced ventilation through partially open spiracles.
CO2 release measured during the ‘closed’ phase of the periodic
pattern seen in the D flies may represent leakage through
partially open spiracles or leakage through the cuticle while the
spiracles are closed.
Our limited knowledge of the respiratory physiology of this
insect limits our understanding of what aspect of its physiology
has responded to selection for resistance to desiccation. Casual
observation of the D flies indicates they are less active than the
C and O flies. It may be that this selection causes a decrease
in activity and that quiescent flies show increased spiracular
control. In addition, the D flies may have developed
morphological differences such as changes in the spiracular
muscles, or physiological differences such as increased
Effect of selection on gas exchange in Drosophila
capacity for buffering CO2, increased tolerance for CO2, or
neural pathways that better control periodic release.
Even if the gas exchange patterns seen in Drosophila
melanogaster are not regarded as discontinuous gas exchange
because they are too far removed from the classical patterns,
they are clearly capable of change under the influence of
selection and they respond to hydration levels in the flies. Our
future work will seek to elucidate the source of improvement
of gas exchange pattern in the D populations.
In conclusion, we have established that ventilatory control
increases after many generations of selection under desiccating
conditions. We have also found changes in respiration within
a single generation that show a strong link between the SER
and hydration state. It is clear, however, that insects use many
other methods to conserve water, including habitat choice,
behavior, cuticular lipid type, absorption of water from
excretory products and adjustment of hemolymph volume. The
large variability in gas exchange patterns reported across
different insect species is logical given this range of water
conservation options. Only insects with potentially high rates
of respiratory water loss would be expected to show a strong
DGC. Our current data support the traditional hypothesis that
the DGC has evolved as a mechanism to conserve water. We
should be able to provide additional insights from future
studies of the mechanism of CO2 release in these insects and
a determination of the relationship between water loss and the
SER.
The authors would like to thank A. F. Bennett, A. Gibbs, R.
K. Josephson and two anonymous referees for their comments
on early manuscripts. We also thank A. Gibbs for providing
additional equipment for these experiments. This research was
carried out with support from NSF grant IBN 9507435 and
NIH grant AG 09970.
References
BURSELL, E. (1957). Spiracular control of water loss in the tsetse fly.
Proc. R. ent. Soc. Lond. A 32, 21–29.
EDNEY, E. B. (1977). Water Balance in Land Arthropods. Berlin:
Springer-Verlag.
GRAVES, J. L., TOOLSON, E. C., JEONG, C., VU, L. N. AND ROSE, M.
R. (1992). Desiccation, flight, glycogen and postponed senescence
in Drosophila melanogaster. Physiol. Zool. 65, 268–286.
HADLEY, N. F. (1994a). Water Relations of Terrestrial Arthropods.
San Diego: Academic Press.
HADLEY, N. F. (1994b). Ventilatory patterns and respiratory
transpiration in adult terrestrial insects. Physiol. Zool. 67, 175–189.
HADLEY, N. F. AND QUINLAN, M. (1993). Discontinuous carbon
dioxide release in the eastern lubber grasshopper Romalea guttata
and its effect on respiratory transpiration. J. exp. Biol. 177,
169–180.
HAMILTON, A. G. (1964). The occurrence of periodic or continuous
discharge of carbon dioxide by male desert locusts (Schistocerca
gregaria Forskål) measured by an infrared gas analyzer. Proc. R.
ent. Soc. Lond. B 160, 373–395.
KESTLER, P. (1985). Respiration and respiratory water loss. In
623
Environmental Physiology and Biochemistry of Insects (ed. K. H.
Hoffmann), pp. 137–183. Berlin: Springer Verlag.
KESTLER, P. (1991). Cyclic CO2 release as a physiological stress
indicator in insects. Comp. Biochem. Physiol. 100, 207–211.
KRAFSUR, E. S. (1971). Behavior of thoracic spiracles of Aedes
mosquitoes in controlled relative humidities. Ann. ent. Soc. Am. 64,
93–97.
LEVY, R. I. AND SCHNEIDERMAN, H. A. (1966a). Discontinuous
respiration in insects. II. The direct measurement and significance
of changes in tracheal gas composition during the respiratory cycle
of silkworm pupae. J. Insect Physiol. 12, 83–104.
LEVY, R. I. AND SCHNEIDERMAN, H. A. (1966b). Discontinuous
respiration in insects. III. The effect of temperature and ambient
oxygen tension on the gaseous composition of the tracheal system
of silkworm pupae. J. Insect Physiol. 12, 105–121.
LEVY, R. I. AND SCHNEIDERMAN, H. A. (1966c). Discontinuous
respiration in insects. IV. Changes in intratracheal pressure during
the respiratory cycle of silkworm pupae. J. Insect Physiol. 12,
465–492.
LIGHTON, J. R. B. (1988). Simultaneous measurement of oxygen
uptake and carbon dioxide emission during discontinuous
ventilation in the tok-tok beetle, Psammodes striatus. J. Insect
Physiol. 34, 361–367.
LIGHTON, J. R. B. (1990). Slow discontinuous ventilation in the Namib
dune-sea ant Camponotus detritus (Hymenoptera, Formicidae). J.
exp. Biol. 151, 71–78.
LIGHTON, J. R. B. (1991). Ventilation in Namib Desert tenebrionid
beetles: mass scaling and evidence of a novel quantized flutterphase. J. exp. Biol. 159, 249–268.
LIGHTON, J. R. B. (1992). Direct measurement of mass loss during
ventilation in two species of ants. J. exp. Biol. 173, 289–293.
LIGHTON, J. R. B. AND BERRIGAN, D. (1995). Questioning paradigms:
caste-specific ventilation in harvester ants, Messor perganei and M.
julianus (Hymenoptera: Formicidae). J. exp. Biol. 198, 521–530.
LIGHTON, J. R. B. AND GARRIGAN, D. (1995). Ant breathing: testing
regulation and mechanism hypotheses with hypoxia. J. exp. Biol.
198, 1613–1620.
LIGHTON, J. R. B., GARRIGAN, D. A., DUNCAN, F. D. AND JOHNSON, R.
A. (1993). Spiracular control of respiratory water loss in female
alates of the harvester ant Pogonomyrmex rugosus. J. exp. Biol.
179, 233–244.
LOVERIDGE, F. P. (1968). The control of water loss in Locusta
migratoria migratoroides R. and F. II. water loss through the
spiracles. J. exp. Biol. 49, 15–29.
MACHIN, J., KESTLER, P. AND LAMPERT, G. J. (1991). Simultaneous
measurement of spiracular and cuticular water losses in Periplaneta
americana: implications for whole-animal mass loss studies. J. exp.
Biol. 161, 439–453.
MELLANBY, K. (1934). Site of loss of water in insects. Proc. R. Soc.
Lond. B 116, 139–149.
MILLER, P. L. (1964). Factors altering spiracle control in adult
dragonflies: water balance. J. exp. Biol. 41, 331–343.
PUNT, A. (1950). The respiration of insects. Physiol. comp. Oecol. 2,
59–74.
QUINLAN, M. C. AND HADLEY, N. F. (1982). A new system for
concurrent measurement of respiration and water loss in
arthropods. J. exp. Zool. 222, 255–263.
QUINLAN, M. C. AND HADLEY, N. F. (1993). Gas exchange, ventilatory
patterns and water loss in two lubber grasshoppers: quantifying
cuticular patterns and respiratory transpiration. Physiol. Zool. 66,
628–642.
624
A. E. WILLIAMS, M. R. ROSE
AND
T. J. BRADLEY
ROSE, M. R. (1984). Laboratory evolution of postponed senescence in
Drosophila melanogaster. Evolution 38, 1004–1010.
ROSE, M. R., GRAVES, J. L. AND HUTCHINSON, E. W. (1990). The use
of selection to probe patterns of fitness characters. In Insect Life
Cycles (ed. F. Gilbert), pp. 29–42. London: Springer-Verlag.
SCHNEIDERMAN, H. A. (1960). Discontinuous respiration in insects:
role of the spiracles. Biol. Bull. mar. biol. Lab., Woods Hole 119,
494–528.
SCHNEIDERMAN, H. A. AND WILLIAMS, C. M. (1953). Discontinuous
carbon dioxide output by diapausing pupae of the giant silkworm
Platysamia cecropia. Biol. Bull. mar. biol. Lab., Woods Hole 105,
123–143.