Evolution of water conservation mechanisms in Drosophila
1 Department of Ecology and Evolutionary Biology, 1041 E. Lowell St,
University of Arizona, Tucson, AZ 85721, USA
2 College of Veterinary Medicine, 105 Magruder Hall, Oregon State
University, Corvallis, OR 97331, USA
3 Department of Ecology and Evolution, State University of New York, Stony
Brook, NY 11794, USA
* Author for correspondence (e-mail: agibbs{at}arl.arizona.edu)
Accepted 13 January 2003
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Summary |
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Key words: cuticular lipids, discontinuous ventilation, Drosophila, metabolic rate, water loss
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Introduction |
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Comparative studies have found little consistent evidence that desert
insects store more water or are able to tolerate lower water levels
(Hadley, 1994a). The species
with the highest water contents are mesic insects. Although some arid-adapted
insects are able to tolerate loss of >50% of their water content
(Zachariassen et al., 1987
;
Hadley, 1994a
), they are not
consistently better at surviving dehydration stress. Indeed, the species able
to tolerate loss of the greatest fraction of its water is an aquatic beetle,
Peltodytes aquaticus (Arlian and
Staiger, 1979
), although it should be noted that this species
contains relatively large amounts of water to start with.
The most consistent difference between arid-adapted and mesic arthropods is
that the former species lose water relatively slowly. This pattern has been
documented for scorpions (Toolson and
Hadley, 1977), spiders (Hadley
et al., 1981
), beetles (Hadley
and Schultz, 1987
;
Zachariassen et al., 1987
),
ants (Hood and Tschinkel,
1990
; Johnson,
2000
) and fruitflies (Eckstrand and Richardson,
1980
,
1981
;
Gibbs and Matzkin, 2001
). A
few water-profligate desert insects use evaporative cooling for
thermoregulation, but these exceptional species have access to large
quantities of water (e.g. xylem-feeding cicadas;
Toolson, 1987
).
Drosophila species occur in a wide range of habitats, including
deserts, and differ in their ability to survive desiccation stress
(van Herrewege and David,
1997; Hoffmann and Harshman,
1999
; Gibbs and Markow,
2001
; Gibbs and Matzkin,
2001
). We have previously shown that enhanced desiccation
resistance in cactophilic Drosophila from North American deserts is
the result of reduced rates of water loss
(Gibbs and Matzkin, 2001
). In
the present study, we examine the mechanistic basis for reduced water-loss
rates. Drosophila lose water by three routes: excretion from the
mouthparts and anus, transpiration through the cuticle, and respiratory losses
by evaporation through open spiracles. (Females will also lose water in their
eggs, but not under the conditions of our experiments.) We directly quantified
total water loss and excretory water loss using flow-through respirometry.
Differences in cuticular lipids (composition, physical properties and amounts)
were assayed as a proxy for cuticular transpiration. Metabolic rates, activity
and ventilatory patterns were examined to assess the importance of respiratory
water loss.
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Materials and methods |
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All flies were maintained on the laboratory photoperiod (approximately 12
h:12 h light:dark) at 24°C, which was the temperature used for all assays.
Most species were reared in milk bottles on corn-meal medium, Drosophila
busckii was reared on WheelerClayton medium
(Carson, 1987), and the
cactophilic species were reared on banana medium containing powdered
Opuntia cactus. A small piece of the host cactus was provided to
cactophilic Drosophila, if necessary, to stimulate egg laying. Flies
used in assays were collected and separated by sex within several hours of
emergence to ensure that they were virgin. They were held in vials containing
corn-meal medium and live yeast for 610 days, then the sexes were
assayed separately.
Respirometry
Metabolic rates and rates of water loss were measured using flow-through
respirometry. Groups of 1020 flies were placed in 5 ml
glassaluminum chambers, which were then placed in a Sable Systems (Las
Vegas, NV, USA) TR-2 respirometer. Rates of carbon dioxide release and water
loss were measured with a Li-Cor (Lincoln, NE, USA) LI-6262 infrared
CO2 and water-vapor sensor. Most data for total water-loss rates
were reported previously (Gibbs and
Matzkin, 2001); additional species are included here. Metabolic
rates of individual flies were measured using the same conditions, except that
the chambers were only 1 ml in volume.
Cuticular lipids
We isolated cuticular hydrocarbons (HCs) from 18 species and examined their
composition and physical properties [melting points (Tm)].
Groups of 1040 flies were placed on a silica gel column in a Pasteur
pipette (Toolson, 1982), and
HCs were eluted with approximately 7 ml of HPLC-grade hexane. The solvent was
evaporated, and the lipid extracts were frozen until analysis. In some cases,
we quantified lipid amounts by adding 2 µg of n-icosane in a small
volume of hexane to the flies at the beginning of the column chromatography
procedure, as an internal standard.
We measured lipid Tm using Fourier transform infrared
(FTIR) spectroscopy (Gibbs and Crowe,
1991). Lipid extracts were dissolved in approximately 25 µl
hexane and deposited on CaF2 windows. After the solvent evaporated,
samples were placed in a temperature-controlled cell holder in the
spectrometer. The sample temperature was increased from <10°C to
>50°C in 2°C increments. We followed the progress of lipid melting
from the frequency of -CH2- symmetric stretching vibrations, which
increase from approximately 2849 cm1 to approximately 2854
cm1 as lipids melt. The midpoint of the phase transition
(Tm) was calculated from logistic equations fitted to
temperature-frequency plots.
We studied HC composition using a Hewlett-Packard (Palo Alto, CA, USA) 5890A gas chromatograph (GC) equipped with a DB-1 or DB-5 capillary column (30 mx0.32 mm i.d.; JW Scientific, Sacramento, CA, USA). Peaks were identified by comparison with the retention times of n-alkane standards and by reference to literature data on HC composition. For some species, we confirmed the identities of HCs by GCmass spectrometry (MS) using a Hewlett-Packard 5988 GCMS system at the Mass Spectroscopy Laboratory of the University of Arizona. Lipid amounts were determined by comparing peak areas with those for the n-icosane standard.
Activity
We used AD-1 activity meters from Sable Systems to assess the relative
activity of individual flies. These use a near-infrared (900 nm) light source,
which is reflected around the chamber to a detector. The chambers were covered
with a thick dark cloth to minimize interference from room lighting. Movement
was detected by fluctuations in the detector signal, with larger fluctuations
generally corresponding to greater activity. Individual flies were placed in
the same 5-ml chambers used for respirometry, with a flow rate of 100 ml
min1 dry air. Activity was recorded until at least 10 h
after the mean desiccation survival time, as determined in previous
experiments (Gibbs and Matzkin,
2001). With the exception of two cactophilic flies that survived
for >48 h, all flies were dead at the end of the experimental runs. At
least five individuals of each sex were assayed in each species.
Although the chambers were dark, a potential problem with these measurements is diurnal activity cycles. For each species, approximately half of the individuals were placed in the chambers at approximately 07.00 h local time, and the other half at approximately 18.00 h. Activity patterns were similar in both groups, suggesting that internal rhythms did not affect our results.
Data analysis
Statistical analyses were performed using Excel and JMP (SAS Institute)
software. To control for phylogenetic relationships, we used the Phylogenetic
Diversity Analysis Package (Garland et
al., 1992) to implement Felsenstein's method of independent
contrasts (Felsenstein, 1985
).
Because the species differed in body size, we regressed physiological
variables (e.g. metabolic rate) against mass and used the residuals from these
regressions in our phylogenetic analyses. For habitat comparisons, species
were classified as either cactophilic (xeric) or mesic. Although not all
cactophiles live in deserts, they and their host plants tend to inhabit dry
regions. Mesic species can sometimes be found in deserts but are less
desiccation resistant than cactophiles
(Gibbs and Matzkin, 2001
).
Thus, the cactophile/non-cactophile distinction is reasonable, both
ecologically and physiologically.
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Results |
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Cuticular lipids
Melting points (Tm) of cuticular lipids are provided in
Table 1. No differences between
males and females were detected. The highest Tm occurred
in mycophagous flies, which inhabit very moist forests. Species from the
subgenus Sophophora tended to have the lowest Tm values,
with cactophilic species being intermediate. Thus, a major determinant of
Tm appeared to be phylogeny rather than habitat. To
examine the relationship between Tm and water-loss rates,
we first calculated residuals of water-loss rate as a function of mass for 11
species for which we also had Tm data. We then calculated
phylogenetically independent contrasts
(Felsenstein, 1985) for the
residuals and Tm values. These were not significantly
correlated (r2=0.044, P=0.39), indicating that
high Tmdid not tend to reduce water loss.
Differences in Tm could result from changes in HC chain
lengths, unsaturation or branching patterns. Alkenes are the major HC
constituents of most Drosophila species studied to date, with
branched alkanes being the next most abundant class
(Bartelt et al., 1986;
Jallon and David, 1987
;
Toolson et al., 1990
). We did
not obtain detailed structural information on HC composition, but
Table 1 reveals that species
with longer chain lengths tended to have higher Tm. With
the exception of D. virilis, sophophorans had consistently shorter
chain lengths than members of the subgenus Drosophila. In addition, a limited
survey (seven species, including two cactophiles) indicated that flies from
mesic and arid habitats had similar quantities of surface lipids for their
size (not shown). Thus, desert Drosophila did not reduce cuticular
permeability by increasing the thickness of the lipid barrier.
Metabolic rates
Fig. 4 depicts the effects
of body size on metabolic rates of 30 Drosophila species. Metabolic
rates of female flies were over 50% higher in mesic species than in
cactophilic species (ANCOVA, P<0.012; adjusted least-squares means
were 4.84 µlCO2 h1 for mesic species and 3.08
µlCO2 h1 for cactophiles). By contrast,
metabolic rates of males did not differ as a function of habitat (ANCOVA,
P>0.3). To examine further the relationship between metabolic
rates and water-loss rates, both of which were positively correlated with body
size [ANCOVA, P<0.04 for metabolic rates of both sexes; see Gibbs
and Matzkin (2001) for
analysis of water-loss rates], we first calculated the residuals of these
parameters when plotted as a function of mass. The residuals were positively
correlated in both sexes (Fig.
5; r=0.57, P<0.001 for females;
r=0.39, P<0.04 for males).
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Because the cactophilic species tended to be closely related
(Fig. 1), we also analyzed the
relationship between metabolic rate and water-loss rate using phylogenetically
independent contrasts (Felsenstein,
1985). After controlling for phylogeny, positive correlations
between residuals were still detected, albeit with reduced statistical
significance (Fig. 6;
P=0.008 for females, P=0.093 for males).
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We also analyzed the relationship between metabolic rates and water-loss
rates separately for cactophilic and mesic species, an approach similar to
that taken previously (Zachariassen et
al., 1987; Zachariassen,
1996
; Addo-Bediako et al.,
2001
). Using simple ANCOVA, a marginally significant correlation
was detected only for mesic females (P=0.073; P>0.25 for
mesic males and for cactophilic flies of both sexes). This correlation
disappeared after phylogenetic relationships were taken into account using
independent contrasts (P>0.2 for all four sex-habitat
combinations).
Activity
Fig. 7 depicts
representative activity recordings for a mesic fly (Drosophila
melanogaster) and a desert fly (Drosophila mojavensis). The
overall activity patterns for these two flies are shown in
Fig. 8. The D.
melanogaster individual was active almost continuously for 6 h, then
exhibited no signs of activity thereafter. Presumably, this fly died at
67 h. The D. mojavensis individual was inactive for the first
12 h of the experiment, then became active for the next 14 h. The lack of
activity after approximately 26 h suggests that the fly died of desiccation
stress at this time.
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Similar activity patterns were observed in other species. In
Fig. 9, we have placed 15
species into three groups, depending upon their ability to survive desiccation
stress (Gibbs and Matzkin,
2001). Most individuals of the first group (four species with an
average survival time of <10 h) were active almost continuously until their
deaths. In the second group (six species that survived for 1024 h),
approximately 80% of flies were active up to approximately 8 h, then activity
decreased as flies became dehydrated. The third group included four
cactophilic species and Drosophila cardini, species for which the
average survival was >24 h. Except for the first hour after being placed in
their chambers, most of these flies were inactive for at least 10 h, then
became active for
15 hours (Fig.
9). Although our activity meters could not quantify the actual
amount of activity (e.g. distance traveled), these data indicate that lower
initial activity levels were associated with greater desiccation
resistance.
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Ventilatory patterns
Discontinuous gas-exchange cycles have been observed in numerous arthropod
taxa and can reduce respiratory water loss (Lighton,
1994,
1996
). We examined
CO2-release patterns in at least 17 females each from six
Drosophila species. We used females because they are larger than
males and have higher metabolic rates. In most cases, CO2 was
released continuously, with no evidence of spiracular control. A striking
exception was provided by the two cactophilic species examined; Drosophila
arizonae and D. mojavensis. Approximately 90% of individuals
from both species often released CO2 in cyclic bursts
(Fig. 10). Although we
observed periods of cyclic CO2 release in other species, these were
relatively rare and brief (Table
2).
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The pattern of cyclic ventilation also appeared to differ between mesic and cactophilic species. In mesic species, metabolic rates during cyclic and aperiodic periods were similar for a given individual. By contrast, cyclic ventilation in cactophilic Drosophila was associated with a marked increase in metabolic rate (Fig. 10A). Direct observations indicated that cyclic release occurred only when desert flies were active, whereas no such pattern was apparent for mesic species. It should be noted that CO2 release never fell to zero, suggesting that the spiracles were never completely closed or that at least one remained open at all times. Individuals from all species were observed to switch back and forth between cyclic and acyclic CO2 release.
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Discussion |
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A potential confounding factor in our experiments is adaptation to
laboratory culture. The stress resistance of Drosophila populations
may change over time (Hoffmann et al.,
2001) as a result of drift or relaxed selection. In our case, we
performed most of our experiments within 23 years of collection, when
the populations would have had little time to change. We also note that, even
after 15 years in culture, the cactophilic species Drosophila navojoa
remains more resistant to desiccation than non-cactophilic species
(Gibbs and Matzkin, 2001
).
Thus, differences between mesic and xeric species appear to be robust.
Drosophila lose water by three major routes (excretion, cuticular transpiration and via the spiracles). Excretory water loss accounted for <6% of total losses in both mesic and desert species and did not differ between these groups (Fig. 3). Thus, reduced excretion cannot account for the greater desiccation resistance of desert species. This leaves cuticular and respiratory routes as the foci of desiccation resistance.
Cuticular lipids
Distinguishing between cuticular and respiratory water losses is difficult
in any insect but particularly so in those as small as Drosophila. We
therefore must use indirect evidence. Surface lipids provide the primary
barrier to cuticular transpiration, so differences in the composition or
amounts of these should affect rates of water loss. Lipid melting points
(Tm) provide one indicator of water-proofing ability, as
solid-phase lipids are less permeable than melted ones
(Rourke and Gibbs, 1999).
Thus, one would predict an inverse relationship between water loss and
Tm. Structural differences that increase
Tm, such as longer chain lengths and reduced unsaturation
or methylbranching, should also be more prevalent in desert species.
Our GC analyses (Table 1), in combination with literature data on HC composition (Table 3), indicate no consistent relationship between chain length and habitat. Cactophilic and mycophilic members of the subgenus Drosophila had the longest-chain surface lipids (2939 carbons), despite living in habitats that are the most distinct from each other. Other members of Drosophila, and all members of the subgenus Sophophora, had relatively short-chain surface lipids (2333 carbons). Thus, phylogeny was a major factor affecting HC composition.
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Chain lengths are not the only factors determining HC melting points;
indeed, unsaturation and methylbranching have much greater effects on
Tm (Gibbs and Pomonis,
1995). We did not perform detailed analyses of HC composition but
were able to measure Tm values in species from a variety
of habitats (Table 1). Lipids
of species in the subgenus Drosophila, including mycophilic species from cool
moist forests, had longer chain lengths and melted above 32°C. By
contrast, sophophorans generally had short-chain, low-Tm
HCs. Neither Tm nor lipid amounts were correlated with
habitat or water-loss rates. It must be noted, however, that cuticular HCs
also have a second important function as contact sex-recognition pheromones
(Antony and Jallon, 1982
;
Markow and Toolson, 1990
;
Tompkins et al., 1993
;
Nemoto et al., 1994
).
Selection for reproductive success may have limited the evolution of better
cuticular waterproofing.
In summary, none of the expected relationships between water-loss rates and
cuticular lipids was detected. Similar conclusions have been obtained using
thermally acclimated D. mojavensis
(Gibbs et al., 1998) and
desiccation-selected populations of D. melanogaster
(Gibbs et al., 1997
). Although
lipid analyses can provide only an indirect indication, desert
Drosophila did not appear to have reduced cuticular permeability
relative to mesic species. This is not to say that cuticular transpiration was
a minor component of the flies' water budgets, only that it did not differ in
any systematic manner among species from arid and mesic habitats. By
elimination, the main mechanism by which desert fruitflies have reduced total
water loss must have been by lowering respiratory losses through the
spiracles.
Respiration and water loss
The significance of respiratory water loss in insects has become
controversial in recent years (Hadley,
1994b; Lighton,
1996
; Slama, 1999
;
Chown, 2002
). Comparative
studies have found a positive correlation between metabolic rates and
water-loss rates in xeric, but not mesic, species
(Zachariassen et al., 1987
;
Addo-Bediako et al., 2001
).
Reducing metabolic rates will help to conserve water by reducing the need for
gas exchange, but this is not a sufficient condition; insects must also be
able to regulate spiracular opening. Water-loss rates will remain high if the
spiracles are open continuously, no matter how low the metabolic rate is.
Unfortunately, spiracular regulation has been studied in only a limited set of
species (Lighton, 1996
).
Several studies have compared water-loss rates when the spiracles are open and
when they are closed. Respiratory water loss typically accounts for <10% of
total losses (Quinlan and Hadley,
1993
; Williams and Bradley,
1998
), implying that cuticular transpiration must be the major
loss route.
Other authors have argued that the relative importance of respiratory water
loss is greater in xeric species because of greatly reduced cuticular
permeability (Lighton et al.,
1993; Zachariassen,
1996
). Thus, a correlation between metabolic rate and water loss
may be observed only in insects from arid environments, even when their
metabolic rates are similar to those of mesic species
(Zachariassen et al., 1987
;
Addo-Bediako et al., 2001
). One
difference between this work and ours is that our Drosophila species
spanned only a fivefold range in body size, whereas Zachariassen et al.
(1987
) studied beetles
differing by more than two orders of magnitude, and Addo-Bediako et al.
(2001
) used literature data
spanning an even larger size range. We were therefore limited to a relatively
narrow range of metabolic rates, which would have reduced our ability to
detect habitat-related differences.
Another important difference between our experiments and previous work is
that we used groups of flies and were unable to control for activity. We
measured metabolic rates 25 h after flies had been placed in their
respirometry chambers, at a time when cactophilic species tended to be much
less active (Fig. 9). Our
observations of individual flies suggested that metabolic rates increased more
than twofold as a result of activity (Fig.
10). Thus, differences in locomotor activity can fully account for
inter-specific differences in metabolic rates, and greater tracheal
ventilation could have increased water loss. By contrast, Zachariassen et al.
(1987) studied individual,
inactive beetles, and most of the studies surveyed by Addo-Bediako et al.
(2001
) measured standard
(resting) metabolic rates, so that activity-related water loss should not have
been a factor.
Selection experiments have suggested that reduced locomotor activity and
metabolic rate contribute to desiccation resistance
(Hoffmann and Parsons, 1993).
Direct measurements from individual Drosophila reveal that total
water loss more than doubles as a consequence of tracheal ventilation during
flight (Lehmann et al., 2000
;
Lehmann, 2001
). Thus,
respiratory losses may be very high in active flies. Unfortunately, water loss
from individual flies in our experiments was so low that we could not measure
it reliably against the background noise and drift in our respirometry system.
In some cases, we noted that flies appeared to lose water more rapidly when
they became active, but we were unable to quantify the increase with any
confidence. Carbon dioxide readings never reached zero, indicating that at
least one spiracle remained open at all times, which would also reduce our
ability to distinguish respiratory losses. Thus, we were unable to reliably
detect increases in water loss caused by spiracular opening and therefore
could not determine whether water loss from individual flies increased
significantly when they became active.
We note that the cyclic CO2 release we observed in desert
Drosophila represents an unusual ventilatory pattern in insects.
Discontinuous gas exchange in other species generally occurs when insects are
quiescent (Lighton, 1996). The
spiracles close completely on each cycle, as indicated by the cessation of
CO2 release. Carbon dioxide release in our experiments was cyclic
but continuous. In addition, the minimal rate of CO2 release during
cyclic ventilation was often greater than that observed in inactive flies
(Fig. 10). In these cases,
cyclic CO2 release may reflect abdominal pumping to aid convective
gas exchange and oxygen delivery when demand is high
(Weis-Fogh, 1964
;
Lehmann, 2001
).
Summary
Water conservation is critical to the ecological success of desert
Drosophila. We have been able to exclude one route for water loss
excretion as a major component of overall water balance. We
were unable to distinguish between cuticular transpiration and respiration as
well as we would like, so our conclusions regarding these remain tentative.
None of the expected differences in cuticular lipids was detected, whereas
three parameters associated with respiratory water loss (metabolic rate,
activity and spiracular control) differed between desert and mesic species.
Thus, the overall evidence indicates that desert Drosophila conserve
water by reducing its loss from the tracheal system.
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Acknowledgments |
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