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First published online August 17, 2006
Journal of Experimental Biology 209, 3293-3300 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02397
Selection for desiccation resistance in adult Drosophila melanogaster affects larval development and metabolite accumulation
Department of Biological Sciences, University of Nevada, Las Vegas, NV 89154-4004, USA
* Author for correspondence (e-mail: gefene{at}unlv.nevada.edu; gefene{at}gmail.com)
Accepted 20 June 2006
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
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Key words: Drosophila melanogaster, desiccation resistance, developmental time, critical weight, life history, water, carbohydrates
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Drosophilidae have been employed in stress resistance studies of both
natural populations and laboratory selected lines
(Gibbs et al., 1997
;
Gibbs et al., 2003;
Chippindale et al., 1998;
Hoffmann and Harshman, 1999;
Gibbs, 2002;
Marron et al., 2003). These
have shown that evolved differences in management of body water and energy
stores are correlated with stress resistance.
Stress (desiccation and starvation)-selected Drosophila
melanogaster are heavier than unselected controls immediately after
eclosion, and maintain this advantage during early adulthood
(Chippindale et al., 1998).
Soon after eclosion, desiccation-selected flies (D) have greater carbohydrate
content in comparison with starved controls, whereas the latter have
relatively high lipid content. It has been suggested that the increased
accumulation of glycogen in larvae of D flies is advantageous in contributing
to increased body water content (Gibbs et
al., 1997; Chippindale et al.,
1998) (but see Folk et al.,
2001), as glycogen binds three to five times its weight in bulk
water (Schmidt-Nielsen, 1997).
Carbohydrates also serve as the primary source of energy in adult flies during
desiccation stress (Marron et al.,
2003). Therefore, increased accumulation of carbohydrates during
larval feeding could result in enhanced desiccation resistance of the newly
eclosed fly. However, Chippindale et al.
(Chippindale et al., 1998)
collected adult flies at 0-6 h following eclosion, and therefore their
reported body metabolite contents may reflect both accumulated larval stores
and early adult feeding. These authors also reported significantly longer egg
to adult developmental times for desiccation-selected D. melanogaster
in comparison with control populations. Although selected for stress
resistance as adults, differences between newly eclosed selected and control
flies may indicate that selection affects their developmental physiology.
However, no significant relationship was found between selection for
desiccation resistance in adults and larval developmental time in other
reports (Hoffmann and Parsons,
1993; Bubliy and Loeschcke,
2005).
Adult insects do not grow, and their size is determined by the size at
which the last instar larva begins metamorphosis
(Nijhout, 2003). The timing of
metamorphosis is determined by reaching a critical developmental stage,
characterized by a small pulse of ecdysteroid secretion
(Nijhout, 1981), after which
completion of metamorphosis no longer depends on resource availability
(Bakker, 1959). The development
of D. melanogaster is divided into a flexible stage until larvae
commit to pupation early in the third instar, followed by a fixed growth
period until pupariation (Bakker,
1959). Reaching the critical developmental stage of committing to
pupation has been associated with the attainment of a critical body mass, or
`critical weight' (de Moed et al.,
1999).
In this study we examined the growth and developmental pattern of desiccation-selected D. melanogaster and compared it with those of starved and fed control populations. Previous studies varied in methodology, most notably in different time intervals used for determination of developmental time (counting eclosing adults every 6-24 h). We report more precise developmental time values under our experimental conditions by collecting emerging adults at hourly intervals. The main objectives of this study were to: (i) determine whether selected and control lines differ in larval developmental time; (ii) if these differences occur, determine whether they are also reflected in a delayed commitment to pupation in larvae of desiccation selected flies; (iii) establish whether a possible delay in the commitment of D larvae to pupation reflects lower growth rate earlier in development, or a shift in the critical mass associated with initiation of metamorphosis; and (iv) assess the contribution of longer larval development to the overall higher desiccation resistance of adult D flies.
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Materials and methods |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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400 females collected in New Jersey, USA in 1999. Flies were maintained
as a large outbred population in the laboratory until selection began. To
minimize the possibility of artifacts due to adaptation to a new environment,
the populations were maintained on a standard 3-week stock cycle for 12
generations before selection was started
(Chippindale, 2006). Pre-adult
stages were reared at densities of 60 larvae in vials containing 10 ml of
corn meal-sucrose-yeast medium. After 2 weeks, adult flies (approximately 4
days post-eclosion) were transferred to 5.5-l Plexiglas population cages
containing two Petri dishes of food. A cloth sleeve covered one end and
allowed access to the cage. The medium was changed every 2 days. After 4 days,
yeast paste was added to stimulate egg production. Approximately 1200 eggs
were collected after 7 days to found the next generation.
Selection for desiccation resistance was performed by removing food from
the cages 1-4 h after transferring the flies. A cheesecloth-covered dish
containing 200 g of silica gel desiccant was placed inside, and the open
end of the cage was loosely covered with plastic wrap to allow gas exchange
while reducing influx of water vapor from the surroundings. Initially, the
cages contained 7500 flies. They were checked hourly until 80-85% of the
flies had died. The desiccant was then removed and fresh food was provided for
the survivors. The flies were given several days to recover before egg
collection for the next generation.
Because desiccation selection required the removal of both food and water,
each selection line (D) was matched to a starved control line (S), whose cage
received two agar plates instead of desiccant. At each generation, each of
these stocks was starved for the same length of time. To control for the
effects of starvation stress, each pair of stressed populations had a matched,
unstressed, fed control population (F). Population sizes in all treatments
were maintained to provide an estimated 1000-1500 adults after selection.
Flies were selected as described above for the first 30 generations following
the initiation of selection. This was followed by 25-30 generations of less
severe selection before the experiments were carried out. During this time,
desiccation resistance was maintained by subjecting flies to desiccation for
24 h after transferring them to the cages, a treatment that kills nearly all
control flies (A.G.G., personal observation). It has been shown previously
that desiccation resistance of similar populations was not compromised even
after 35 generations of relaxed selection
(Passananti et al., 2004).
Three replicate populations (DA-DC,
SA-SC, FA-FC), sharing a common
ancestry, were maintained from each of the selected and control
populations.
Larval growth and development
Egg collection for each assay was carried out using selected and control
flies of the same age (usually 4-14 days), and of the same selection
generation. Adults from each of the nine fly populations were transferred to
empty 175-ml bottles for a 1 h. The bottles were covered with a 35x10 mm
Petri dish containing grape agar as a substrate for egg laying. Sets of 80
eggs were then collected and placed in food vials containing approximately 10
ml of cornmeal food. The vials were incubated in a controlled temperature
chamber at 24.5°C and constant light, and larvae were collected at 6-h
intervals, between 72 and 108 h after egg laying (AEL). Thirty larvae were
transferred to 1% agar vials for determination of commitment to pupation (see
below). Ten sets of three larvae were rinsed in water to remove food
particles, and immediately frozen at -20°C for future measurements of body
mass and water contents (N=30 for each of the nine populations, for
each time point). After thawing, the larvae were blotted dry on an absorbent
tissue and weighed to the nearest 0.001 mg (C30 Cahn microbalance, Cerritos,
CA, USA). The larvae were then dried at 60°C overnight for dry body mass
and water content determination. Wet and dry body mass of individual wandering
larvae was measured after male and female larvae from each of the experimental
populations (N=8 for each sex) were collected from the inner surface
of the food vials. Vials were checked every 1.5 h for emergence of wandering
larvae from the food. Gender identification of wandering larvae was based on
gonad morphology (Folk et al.,
2001).
Larvae from additional vials (N=60 for each population) were
frozen for determination of developmental stage as a function of larval age.
Larval instar was determined based on the morphology of the mouthparts and
anterior spiracles (Ashburner,
1989). We staged larvae at 3-h intervals from 72 and 84 h AEL,
after preliminary experiments indicated that all larvae were second instars at
72 h AEL, whereas practically all larvae had molted to third instar by 84 h
AEL.
Rates of commitment to pupation during development were measured by transferring 30 larvae from each population to vials containing non-nutritious agar at the same 6-h intervals (72-108 h AEL). The agar vials were then placed back in the incubator, and the eventual pupation rates reflected the rates of commitment to pupation at time of transfer. Additional sets of food vials with 80 larvae each (two vials for each of the nine populations) were used to determine pupation rates at 6-h intervals, ranging from 118 to 160 h AEL.
Larval feeding regimes
In order to quantify the contribution of third instar feeding to evolved
desiccation resistance, newly eclosed flies from the nine experimental
populations (three replicates each of D, S and F) were collected following one
of two larval feeding regimes. In one treatment, larvae were transferred from
food to agar vials at 96 h AEL after preliminary data (see Results) had shown
that larvae from all nine populations commit to pupation prior to this time
point. In the control treatment, larvae were allowed to feed ad
libitum throughout larval development.
Developmental time analysis
Egg collection and incubation was carried out as described above. Newly
eclosed flies were collected at hourly intervals, and egg to adult
developmental time was calculated.
Desiccation resistance analysis
Following eclosion, the flies were transferred to empty vials individually,
and restricted to the lower half of the vials by a foam stopper. Silica gel
was then added above the stopper to maintain low humidity, and the vial was
sealed with Parafilm. The vials were placed back in the incubator and
mortality was recorded at hourly intervals, after which the dead flies were
sexed.
Metabolic fuel assays
Newly eclosed flies, from both feeding experimental groups, were collected
as described above and immediately frozen at -20°C. After thawing, the
flies were sexed, and individual flies were weighed (±0.001 mg) and
then homogenized in microfuge tubes containing 200 µl 0.05% Tween 20 (in
water) using a hand-held grinder. The tubes were then incubated at 70°C
for 5 min to prevent lipase activity. The samples were then centrifuged for 1
min at 16 000 g, and the supernatant was removed to new tubes
which were frozen until measurements.
Carbohydrates
Triplicates (10 µl) from each sample were loaded on 96-well microplates,
and 10 µl of Rhizopus amyloglucosidase (0.8 mg ml-1;
A-7255, Sigma-Aldrich Co., St Louis, MO, USA) were added to each to catalyze
the conversion of glycogen and trehalose into glucose
(Parrou and Francois, 1997).
The plates were then left overnight at room temperature. The following day, 90
µl of liquid glucose reagent (Pointe Scientific Inc., Canton, MI, USA) were
added to each sample, and absorbance at 340 nm was measured using a SpectraMax
Plus384 microplate reader (Molecular Devices, Sunnyvale, CA, USA).
Carbohydrate concentrations were determined using standards of known glycogen
concentration.
Triglycerides
Triglyceride content was measured using Serum Triglyceride Determination
Kits (TR 0100, Sigma-Aldrich Co.). Triplicate samples (30 µl each) were
placed in microplates, and 100 µl free glycerol reagent was added, before
absorbance was read at 540 nm. Then 25 µl of triglyceride reagent was
added, and the plates were allowed to sit in room temperature for 15 min,
before absorbance was read again at 540 nm. The amount of triglycerides was
calculated as the difference between free glycerol levels before and after the
use of the triglyceride reagent, using standards of known glycerol
concentration.
Protein
Supernatants were diluted with water at 1:1 and 1:2 ratios for the food
deprivation and ad libitum feeding treatments, respectively. To
measure protein content, 8 µl triplicates from each sample were loaded on a
microplate, and 200 µl of protein assay reagent (50 parts bicinchoninic
acid solution to 1 part 4% CuSO4) were added. The plates were then
incubated overnight at room temperature, and absorbance at 562 nm was measured
the following day. Protein concentrations were determined using standards of
bovine serum albumin (82516, Sigma-Aldrich Co.).
Water content
Newly eclosed flies were collected as described above following the two
larval feeding regimes, and frozen at -20°C for future measurements. The
flies were then thawed, and their wet mass was measured (±0.001 mg).
The flies were then dried at 60°C overnight before dry mass was
determined. Water content was calculated as the difference between the two
measurements.
Statistics
Unless stated otherwise, data were analyzed as follows. Larval wet and dry
mass were determined without sexing the larvae first, and therefore analyzed
by two-way ANOVA, with replicate populations treated as a random effect and
selection treatments as a fixed effect. Wandering larvae were sexed before
mass determination and therefore three-way ANOVAs were used with sex and
selection treatment as fixed effects and replicate populations as a random
effect. Developmental and desiccation times, body mass and water, and
metabolic fuel contents of newly eclosed flies were analyzed similarly.
Protein content was used as a covariate for comparisons of carbohydrate and
triglyceride contents. Dry body mass was used as a covariate for comparisons
of body water contents. Tukey's HSD tests were used for post-hoc
comparisons of means following ANOVA/ANCOVAs. In some of the assays (as stated
in the Results section), when high variability between replicate populations
prevented drawing clearer conclusions, data were pooled before further
between-treatments analyses.
Frequencies of second molt, commitment to pupation and pupation rates at
different time points during development were analyzed using a
2 test, with selection treatment and replicate population as
main effects. Statistical analysis was carried out using STATISTICA for
Windows software (version 7.1).
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Second molt frequencies at different time points, presented in Fig. 3, show that larvae from all three selection treatments reach 50% molt rate between 81 and 84 h AEL. No significant differences in frequencies of second molt were found among the three populations at either 81 or 84 h AEL (P>0.05).
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Timing of commitment to pupation was expressed by the pupation rates of larvae transferred from food to agar vials at 6-h intervals throughout larval development. Pupation rates were relatively low (<10%), and similar among the three fly lines, when larvae were transferred to agar up to 78 h AEL (Fig. 4). Rates of commitment to pupation increased rapidly afterwards, and when transferred to agar 96 h AEL pupation rates exceeded 95% in larvae of all three experimental lines. Interestingly, pupation rates of D larvae were significantly lower than those of S and F larvae when transferred to agar 90 h AEL (P=0.02; Fig. 4).
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Larval feeding regimes
Developmental time
Egg to adult developmental time of both males (241.3±1.1 h,
N=35) and females (235.6±0.9 h, N=52) was
significantly longer in D larvae compared with S (236.5±0.8 h,
N=58; 231.9±0.8 h, N=66) and F (235.2±0.8 h,
N=69; 231.6±0.8 h, N=69) control groups following
ad libitum larval feeding (F=17.6; P<0.001). No
significant differences were found between replicate populations of each of
the selection treatments (F=2.0; P>0.05). Developmental
time of male flies was significantly longer than that of females
(F=40.7; P<0.001).
Desiccation resistance
Significant differences in desiccation resistance of newly eclosed flies
were detected under both larval feeding regimes in the order D>S>F
(Table 1) (F=31.8;
P<0.001). As in developmental time measurements, no differences
were found between replicate populations of each of the selection treatments
(F=0.1 and 0.2 for the two feeding regimes; P>0.05).
Transfer of D larvae to agar vials 96 h AEL resulted in a decrease of 13-14 h
in desiccation resistance time compared with 7.5-9.3 h in S and F flies.
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Body mass and metabolite content
Table 2 summarizes wet mass
and metabolic fuel contents of newly eclosed flies. When allowed to feed
throughout larval development, the newly eclosed D flies had significantly
higher wet mass in comparison with starved and fed controls (F=108;
P<0.001). Note that a similar, albeit statistically insignificant
pattern was seen in wandering larvae (see above). Female flies were
significantly larger than males (F=811; P<0.001). No
significant differences were found between selection treatments in
carbohydrate contents when using replicate populations as a random effect
(F=2.1; P=0.23). However, D flies consistently contained
more carbohydrates than their replicate controls. The mean (± s.e.m.)
values of pooled samples were 35.6±1.2 µg for D (35.7±2.5,
37.9±1.7 and 33.2±1.7, for DA-DC,
respectively), 29.3±1.3 µg for S (27.2±2.3, 34.9±2.1,
25.8±1.4) and 30.4±1.1 µg for F (24.3±1.3,
33.3±1.6, 33.6±1.7). Pooling replicate populations' data
resulted in significantly higher carbohydrate levels for newly eclosed D flies
in comparison with S and F (F=7.9; P<0.001). No
differences were found in triglyceride contents of selected and control flies
(F=1.4; P>0.05). Males and females did not differ
significantly in either triglyceride or carbohydrate contents (F=3.3
and 0.2 respectively; P>0.05).
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Mean body mass of newly eclosed female flies was significantly higher than that of males even when larvae were deprived of further feeding at 96 h AEL (F=29.9; P<0.05), but the effect of sex on triglyceride and carbohydrate contents was not significant (F=1.5; P>0.05). This larval feeding regime resulted in similar mean body mass values (F=0.01; P=0.99), as well as similar triglyceride and carbohydrate contents (F=1.1 and 1.5 respectively; P>0.05) for newly eclosed selected and control flies (Table 2).
Water content
Mean wet and dry body mass and water contents are summarized in
Table 3. Significant
differences in wet body mass of newly eclosed flies were found in the order
D>S>F (F=12.4; P<0.05). Selection treatment also
had a significant effect on dry body mass (F=17.0;
P<0.05). However, no significant difference in dry body mass was
found between newly eclosed D and S flies, whereas both were significantly
larger than F (
=0.05). As in wet body mass data, differences in water
content were in the order D>S>F (F=7.2;
P<0.05).
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No differences were found between selection treatments in wet body mass (F=0.5; P=0.6), dry body mass (F=0.8; P=0.5) or water content (F=0.1; P=0.9) of newly eclosed flies when larvae were transferred to non-nutritious agar vials at 96 h AEL.
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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)]. Selection for desiccation resistance in laboratory-reared
populations has been performed on adult flies, and previous studies have
largely focused on the evolved responses of adult flies to stressful
environment. Nevertheless, there is some evidence that selection for
desiccation resistance of adult flies results in altered larval developmental
physiology (Chippindale et al.,
1998).
Our results show longer developmental time for D larvae in comparison with
that of their controls. This is in agreement with the findings of Chippindale
et al. (Chippindale et al.,
1998), but not with other studies
(Hoffmann and Parsons, 1993;
Bubliy and Loeschcke, 2005).
However, previous studies differed in methodology, including nutrition,
temperature, selection regime (duration of exposure to stress, number of
generations) and larval rearing density. Differences in these environmental
factors result in varying patterns of responses to selection in life history
traits (Prasad and Joshi,
2003), and could be responsible for the apparent inconsistent
responses of larval developmental time to desiccation selection. Moreover, we
assayed eclosion at hourly intervals and were able to detect a 5-6 h
difference in developmental time between selected and control flies. These
differences could have been masked in previous studies where developmental
time was determined by collecting eclosing adults every 6 h
(Bubliy and Loeschcke, 2005) or
24 h (Hoffmann and Parsons,
1993).
The size of adult insects is determined by larval developmental time and
growth rate. Prolonged development results in increased food consumption
(assuming unchanged growth rate), thus increasing resources for the adult
insect. Wet and dry mass measurements at 6-h intervals throughout larval
development (Figs 1,
2) indicate that the higher
body mass of newly eclosed D flies results from their extended larval
developmental time, which is not coupled with increased growth rate. It is
important to note that despite their shorter development times, body mass of
newly eclosed females is significantly higher than that of males (Tables
2,
3). This could be explained by
a higher growth rate of females during late developmental stages
(Partridge et al., 1994).
Despite their longer egg to adult developmental time, D larvae did not have a delayed second molt (Fig. 3). Our data show that larvae of both desiccation selected and control lines molt from second to third instar 81-84 h AEL on average. Egg to pupa (Fig. 5) and egg to adult developmental times show similar differences between selected and control lines, suggesting a similar length of pupal stage for all three lines. Thus, the observed differences in total developmental time between selected and control populations result from an extended third larval instar stage.
Larvae of D. melanogaster reach a `critical stage' early in the
third instar, after which they will pupate and then eclose even if prevented
from further feeding (Bakker,
1959). It was therefore interesting to determine whether the
longer developmental time of D larvae coincides with a delay in reaching the
irreversible developmental stage of commitment to pupation. Our results show
(Fig. 4) that D larvae have
significantly lower rates of commitment to pupation 90 h AEL in comparison
with control populations. The critical stage for pupation is associated with
attaining a relatively constant `critical weight' for pupation
(Robertson, 1963).
Interestingly, no significant difference was found in either wet or dry mass
of D, S and F larvae 90 h AEL (P=0.32 and 0.33, respectively).
Therefore, it appears that selection for desiccation resistance in adult
D. melanogaster has resulted in a shift in the `critical weight'
associated with the commitment of the larvae to complete development and
pupate. Observed differences in total developmental times, together with the
fact that the growth period following commitment to pupation is fixed in
D. melanogaster (Santos et al.,
1997), support the notion of a shift in the critical stage as a
result of selection for desiccation resistance. It is interesting to note that
differences in the `critical weight' have been shown to occur between natural
populations of D. melanogaster from different geographical regions,
highlighting the existence of genetic variation in this developmental trait
(de Moed et al., 1999). An
increase in body size threshold and an extended last larval instar have also
been shown to occur following the introduction of the dung beetle
Onthophagus taurus to a new habitat, and were shown to result from an
evolutionary modification of sensitivity to juvenile hormone
(Moczek and Nijhout,
2002).
In this study, wet body mass of newly eclosed D flies was significantly
higher than that of control populations. However, no significant difference
was found between the dry mass of D and S flies. This is in agreement with a
previous report in which wet body mass of newly eclosed D flies was higher
than that of starved controls, but no differences were found in dry mass
(Chippindale et al., 1998).
Therefore, it appears that selecting adult flies for desiccation resistance
affects water management during earlier developmental stages, resulting in
higher body water content of newly eclosed flies
(Table 3).
Although larval development of D. melanogaster was clearly
affected by adult selection, it was still unclear whether the observed
developmental changes contributed to the increased fitness of the selected
flies under desiccation conditions. Results from the desiccation assay
emphasize the contribution of the third instar larval feeding to stress
resistance performance of adult flies. As was expected for all three
experimental fly lines, our results indicate that newly eclosed flies are
considerably more desiccation resistant when fed ad libitum during
larval stages in comparison with newly eclosed flies from the food deprivation
treatment. However, newly eclosed D flies are significantly more resistant to
desiccation than their controls, even when prevented from completing the full
course of normal larval feeding (Table
1, 96 h AEL). This highlights a component of stress resistance
that is independent of metabolite acquisition during larval development, as no
differences in either triglyceride or carbohydrate contents were found among
flies from selected and control lines. This component of evolved resistance to
desiccation in D flies is probably a result of relatively low rates of water
loss of desiccation-selected flies (Gibbs
et al., 1997). Still, our results show that the extended third
instar larval development of D larvae contributes even further to the overall
higher desiccation resistance in comparison with control groups. The
contribution of late third instar larval feeding to desiccation resistance is
50% higher in D flies compared to their controls (13.0-13.7 h and 7.5-9.3 h,
respectively). Overall, the feeding period extending from 96 h AEL through to
the wandering stage results in a 60% increase in desiccation resistance of
newly eclosed D flies and 40-50% in newly eclosed control flies, as reflected
by comparing ad libitum larval feeding with the starvation
treatment.
Increased desiccation resistance in selected D. melanogaster has
been associated with higher carbohydrate contents
(Djawdan et al., 1998;
Chippindale et al., 1998;
Folk et al., 2001) (but see
Hoffmann and Harshman, 1999).
Glycogen storage has been suggested as a mechanism of increasing intracellular
water contents (Gibbs et al.,
1997), but other evidence supports carbohydrate storage in the
haemolymph, possibly in the form of trehalose
(Folk et al., 2001). Results in
this study show a trend for higher carbohydrate accumulation during pre-adult
development of D flies in comparison with their controls
(Table 2). Despite the
variability among replicate populations, the trend shown is in agreement with
previously reported carbohydrate contents of newly eclosed flies (3 h post
eclosion) (Chippindale et al.,
1998). This correlates with their longer developmental time and
enhanced resistance of adults to desiccation stress
(Table 1). The relatively high
variability associated with the carbohydrate assay could explain the lack of
statistical difference when replicate populations were treated as a random
effect, but pooling selection treatment data resulted in significantly higher
carbohydrate contents in newly eclosed desiccation selected flies.
Interestingly, newly eclosed flies from all three experimental lines had similar carbohydrate contents when resource accumulation was prevented from 96 h AEL onwards (Table 2). This indicates that the higher carbohydrate content of newly eclosed D flies results from increased carbohydrate accumulation in the late third instar larval stage (assuming catabolism of comparable ratios of metabolic fuels during the pupal stage).
The lack of statistical difference between starved (S) and fed (F) control
flies in rates of commitment to pupation
(Fig. 4) suggests that
desiccation stress rather than food deprivation is the selective force
responsible for the shift in larval `critical weight' of the D flies. This
developmental change results not only in increased body size, but also in
higher carbohydrate (Table 2)
and water contents (Table 3).
Hence, increased carbohydrate accumulation as a result of an extended third
instar stage may provide the necessary mechanism for increasing larval water
storage, thus increasing the fitness of newly eclosed D flies under
desiccating conditions. The carbohydrate content of newly eclosed D flies was
6 µg higher than that of their controls
(Table 2). Glycogen can only
bind three to five times its own mass water, and therefore water bound to
accumulated intracellular glycogen cannot fully account for the higher water
content of newly eclosed D flies (Table
3). This may suggest that the extra water accumulated during
larval development of D flies is stored both as intracellular glycogen-bound
water and in the haemolymph (Folk et al.,
2001).
In conclusion, this study shows that selecting adult flies for desiccation resistance results in major developmental changes. A shift in the `critical weight' of the larvae contributes to an extended third larval instar, which in turn provides the newly eclosed adult fly with increased body mass and carbohydrate and water contents. These developmental changes significantly affect adult fitness under stressful conditions.
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Acknowledgments |
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