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First published online May 1, 2006
Journal of Experimental Biology 209, 1837-1847 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02201
A new set of laboratory-selected Drosophila melanogaster lines for the analysis of desiccation resistance: response to selection, physiology and correlated responses
Centre for Environmental Stress Adaptation Research, La Trobe University, Bundoora, Victoria 3086, Australia
* Author for correspondence at present address: Department of Zoology, University of Florida, Gainesville, FL 32611, USA (e-mail: mtelonis{at}zoo.ufl.edu)
Accepted 8 March 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, laboratory selection, physiology, dehydration tolerance
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Schmidt-Neilson, 1990;
Williams et al., 1998). This
has led to a number of adaptations to minimize water loss, particularly in
desert species (reviewed in Addo-Bediako et
al., 2001; Gibbs,
2002; Hadley,
1994). The evolution of water balance in insects has been widely
investigated using the insect model Drosophila, which is amendable to
both natural and artificial selection for resistance to desiccation stress.
For example, in natural populations of D. melanogaster, desiccation
resistance often tends to be greater in populations from temperate zones
compared with tropical zones (Hoffmann and
Harshman, 1999), while in the laboratory, different
Drosophila respond to selection for desiccation resistance
(Hoffmann and Parsons, 1989a;
Hoffmann and Parsons, 1993;
Rose, 1996). By utilizing
laboratory selection to investigate the life-history, physiological and
genetic changes associated with selection responses, insights can be gained
into the evolutionary factors that shape adaptive responses, as well as the
types of traits that can increase desiccation resistance. These studies may
also aid in elucidating the genetic basis of physiological changes over longer
evolutionary timeframes, while comparative physiology between different
selected populations may highlight common resistance mechanisms and trait
interactions (Gibbs, 1999;
Harshman and Hoffmann, 2000;
Zera and Harshman, 2001).
In D. melanogaster, previous studies based on two different
populations have examined the physiological adaptations, correlated responses
and life-history tradeoffs associated with the desiccation selection response
(Chippindale et al., 1998;
Folk and Bradley, 2000;
Folk et al., 2001;
Gibbs et al., 1997;
Hoffmann and Parsons, 1989a;
Hoffmann and Parsons, 1989b;
Rose et al., 1990;
Rose et al., 1992;
Williams et al., 1998). More
recently, Bubliy and Loeschcke selected for desiccation resistance among other
stress traits in order to examine correlated responses with longevity
(Bubliy and Loeschcke, 2005)
but did not examine physiological responses to selection for desiccation.
Direct comparison of the Hoffmann and Parsons
(Hoffmann and Parsons, 1989a;
Hoffmann and Parsons, 1989b)
and Rose et al. (Rose et al.,
1990; Rose et al.,
1992) lines illustrates that enhanced desiccation resistance
following selection can be associated with different evolutionary trajectories
(reviewed in Hoffmann and Harshman,
1999). The three mechanisms by which insects can increase
desiccation resistance include (1) reducing the rate of water lost, (2)
increasing bulk water and (3) tolerating greater amounts of water loss
(dehydration tolerance) (Gibbs et al.,
1997; Gibbs and Matzkin,
2001; Hoffmann and Parsons,
1989a). While increased desiccation resistance in both sets of
selected lines has consistently been associated with reduced rates of water
loss, patterns of resource storage and partitioning as well as life history
trait associations have varied markedly. For example, while the Rose et al.
(Rose et al., 1990;
Rose et al., 1992) lines (from
now referred to as D and C lines) evolved increased wet weight (attributable
to extra water and carbohydrate stores) and reduced rates of water loss,
desiccation resistance in the Hoffmann and Parsons lines
(Hoffmann and Parsons,
1989a; Hoffmann and Parsons,
1989b; Hoffmann and Parsons,
1993) was primarily associated with both reduced water loss
and metabolic rates. Harshman and Hoffmann discussed several causes for these
dissimilar responses, in particular the use of different base populations,
selection regimes and degrees of adaptation to laboratory environments
(Harshman and Hoffmann, 2000).
For example, Hoffmann and Parsons derived their selected and control lines
(Hoffmann and Parsons, 1989a)
from a mass-bred population founded by 30 inseminated field females following
three years of laboratory adaptation. By contrast, the D lines were derived
from a mass-bred population pre-selected for postponed age of reproduction (O
stocks) in 1980, five years following initial laboratory culture
(Service et al., 1985).
Despite inherent experimental differences, the studies have produced evidence
of some robust traits, evolutionary constraints and potential for multiple
evolutionary pathways available for desiccation resistance.
The primary aim of the present study was to establish a new set of desiccation-resistant selected lines of D. melanogaster for the analysis of the selection response in terms of physiology, correlated responses and life history traits, in addition to providing lines for understanding the genetic basis of the response to desiccation resistance selection, which remains poorly defined. Here, we present the physiological and correlated response data from the new lines selected for 26 generations. The traits assessed were primarily based on previous studies to form a comparative basis for understanding the mechanisms underlying artificially evolved resistance. We describe several components of water balance and partitioning, including measures of water loss rates, dehydration tolerance, bulk water levels, glycogen content and hemolymph volume. The selection response only partially overlapped with previous studies, including reduced water loss and increased wet mass; however, further dissection revealed that alternative mechanisms underlie the increased mass in these lines compared with the D lines. Here, increased desiccation resistance was primarily associated with an increase in dehydration tolerance, a physiological mechanism previously unobserved in selected studies, providing further evidence of the many ways D. melanogaster may evolve desiccation resistance, as well as an opportunity to further study a lesser understood adaptation to desiccation stress. We examined changes in development time and fecundity, as well as correlated responses with other climatic stresses; these traits overlapped with at least one previous study. The selection response was further characterized at the chromosome level by partitioning to each major chromosome the combined effects of dominant genes underlying desiccation resistance.
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Materials and methods |
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Directional selection: desiccation resistance
The selected and control lines were founded from a mass-bred population
comprising pooled isofemale lines collected from 10 locations along the
Australian east coast in 2000. For the selection regime, 1000 non-virgin
3–6-day-old flies (mixed sexes) were randomly allocated into vials of 50
using CO2 anesthesia and allowed 1 day for recovery prior to
desiccation. Flies were desiccated in empty glass vials covered in gauze in a
large glass chamber containing silica gel at a relative humidity (RH) of
<10%, and the final 10% of surviving flies were used to found the following
generation. The selected lines underwent this regime 26–29 times at the
time of most assays, unless noted otherwise. The control lines were not
subjected to any treatment and were maintained in comparable densities to the
selected lines on media in discrete two-week cycles. In this selection regime,
flies are starved as well as desiccated, although flies die from desiccation
stress well before they die from starvation after several days. The two
desiccation-resistant selected lines will be referred to as S1 and S2, and the
control lines as C1 and C2.
Physiological assays
For the following assays, fourth generation progeny (F4) from
selection 26 were tested to counter any paternal and grandparental effects of
desiccation. Batches of eggs were placed into vials containing 25 ml of fresh
media, and emerging flies were collected within 24 h. The flies were aged for
3 days, separated by sex using CO2 anesthesia, and held at a
density of 10 or 20 flies per vial for another day. Weight loss in groups and
individuals, mortality, water content and dehydration tolerance were conducted
following Hoffmann and Parsons (Hoffmann
and Parsons, 1989a) with minor modifications.
Detailed analyses of desiccation resistance
To assess desiccation resistance in the selected and control lines, a
mortality curve was generated by desiccating five replicates of 10 females and
10 males (sexes tested separately). The flies were placed in empty vials
sealed with gauze and were affixed with Parafilm® to another
vial containing approximately 10 g of silica desiccant. Vials were scored at
hourly intervals until all flies in a group had died. The time for half the
flies to die (LT50) was determined by linear interpolation.
Weight loss measured in groups
Using flow-through respirometry to detect rates of water loss in D.
melanogaster, Gibbs et al. reported that water loss was high in the first
two hours of desiccation before stabilizing for the next few hours
(Gibbs et al., 1997).
Consequently, in the present study, weight loss due to short-term desiccation
was measured in groups of 20 females desiccated for 3 h (7–8
replicates). Groups were weighed on a Satorius microbalance to the nearest 0.1
mg before and after desiccation. Weight loss, expressed as the initial
percentage of weight lost, was used as an estimate of water loss rates,
although it may also reflect weight lost through metabolism and, to a much
lesser extent, defecation (Gibbs,
2002).
Water content, weight loss and mortality of individual flies
To assay total water content and dehydration tolerance, individual C and S
females were placed in vials as described above, and mortality was scored at
hourly intervals. Females were weighed as described above immediately prior to
desiccation and again after drying overnight at 60°C. Water content of the
females was estimated from the difference between wet and dry weights.
Dehydration tolerance was assessed as the percentage of total water lost at
death (wet weight – weight at death)/(wet weight – dry
weight).
Water partitioning, carbohydrate and lipid content
Altered patterns of resource storage, including water partitioning
(Chippindale et al., 1998;
Folk et al., 2001) and
whole-body lipid content (Djawdan et al.,
1998; Hoffmann and Parsons,
1989b), were examined in previous studies of desiccation-resistant
selected lines. For example, Chippindale et al. observed that, relative to
their controls, the D selected lines stored extra water and had high levels of
the energy storage carbohydrate glycogen
(Chippindale et al., 1998) and
that the extra water was partitioned to the hemolymph
(Folk et al., 2001). In the
present study, we tested glycogen, hemolymph and gross lipid content in
females. To measure hemolymph volume, blotting assays were conducted after
Folk et al. (Folk et al.,
2001) with slight modifications. Five replicate groups of 10
four-day-old females from each of the four populations were anaesthetized with
CO2 and weighed as a group. The abdomen of each was gently torn
with surgical forceps, and hemolymph was blotted from the opening with a piece
of Kimwipe® slightly moistened with isotonic saline. Within a
maximum of 10 min, the 10 blotted flies were reweighed as a group and dried
for 1 h at 60°C and weighed a third time. Hemolymph volume was estimated
from the reduction in mass following blotting.
Glycogen levels were determined following the methods described by Clark
and Keith (Clark and Keith,
1988) using the Sigma Glucose assay kit (product number GAGO-20;
Castle Hill, NSW, Australia). Five replicates of 10 females per line were
weighed to the nearest 0.1 mg and homogenised (on ice) in 250 µl of
homogenisation buffer (0.01 mol l–1
KH2PO4, 1 mmol l–1 EDTA, pH 8). The
homogenates were centrifuged at 17 949 g for 2 min at 4°C.
The supernatant was then removed and placed in a microcentrifuge tube on ice.
The pellet was resuspended in 100 µl of homogenisation buffer and
recentrifuged, and the second supernatant was added to the first. The test
reagent contained 0.1 U ml–1 amyloglucosidase, 5 U
ml–1 glucose oxidase, 1 U ml–1 peroxidase
and 0.04 mg ml–1 o-dianisidine dihydrochloride. The
reagent is buffered by salts contained in the Sigma preparation of glucose
oxidase and peroxidase (Clark and Keith,
1988). Aliquots of 10 µl of homogenate were added to 1.5 ml of
test reagent and incubated at 37°C for 30 min. Optical density was read at
450 nm, and concentration of glycogen was estimated from a standard curve
prepared using glucose standards.
Gross lipid content in females was measured following Hoffmann and Parsons
(Hoffmann and Parsons, 1989b)
with minor modifications. Briefly, females were dried at 60°C for 48 h in
groups of 20 and weighed as described above. Lipid was extracted by placing
whole flies in ether for 24 h. Flies were reweighed after drying again for 48
h. Five replicates of 20 flies from each line were extracted.
Correlated stress responses
To examine correlated abiotic stress responses associated with selection
for desiccation resistance, we assayed the S and C lines for starvation
resistance, cold and heat mortality. For starvation resistance, three
replicates of 10 four-day-old females were tested using the two-vial method
described for desiccation, substituting the desiccant with cotton wool soaked
in 10 ml of water. Flies were scored at 8-h intervals until at least 50% in
each vial were dead.
Cold mortality was examined by exposing S and C flies to subzero conditions until vials reached around 50% mortality. To determine this point, females were submerged in a –2°C cold bath filled with ethylene glycol for increments of 30 min ranging from 30 min to 2.5 h. Mortality was scored after 24 h. Initially three replicate vials of 10 flies were stressed at each time point, and the assay was repeated using 10 replicates of 10 females per line at 2.5 h. These assays were treated as separate blocks.
For heat stress, females were assessed for survival by subjecting three replicates of 10 females to a 39°C water bath for 30 min and scoring mortality after 24 h.
Correlated life history changes
The effect of selection for desiccation resistance on development time and
early fecundity was assessed after 26 generations of selection. For
development time, 20 replicates of 10 eggs were placed on 25 ml of fresh
media. Egg to adult development was scored at 6-h intervals until all flies
had eclosed; males and females were recorded separately to test for sex
specific differences. Early fecundity was examined by placing pairs of virgin
females and males (0–1 days old) into empty vials containing a small
plastic spoon filled with treacle media coated with a 10% yeast suspension.
Egg production within a 24-h period was recorded at the same time daily for 5
days, and early fecundity was calculated as the number of eggs produced,
averaged over 15–18 replicates per line.
Chromosomal analysis
Desiccation resistance was mapped at the chromosome level in the resistant
selected lines. The selected lines were each crossed to a line derived from
the control lines (representing the same genetic background) that were briefly
and weakly selected for six generations for desiccation susceptibility. We
have also utilized this combination of lines to identify quantitative trait
loci affecting survival to desiccation stress (K. M. Guthridge, M.
Telonis-Scott, R. J. Hallas and A. A. Hoffmann, unpublished).
Indirect selection: desiccation susceptibility
Selection for desiccation susceptibility was carried out as described by
Quintana and Prevosti (Quintana and
Prevosti, 1990) with minor modifications. Thirty isofemale lines
were established from the mass-bred population described above. From each
isofemale line, 30 inseminated 4–7-day-old females were sorted and
desiccated (as described above) until all lines reached 100% mortality. From
the original 30 isofemale lines, the eight lines with the lowest desiccation
resistance were chosen (for their siblings) to found the next generation;
these lines all produced abundant offspring (minimizing the likelihood that
they carried deleterious alleles with large effects). Untreated siblings were
crossed (30 crosses between the eight isofemale lines) and offspring were
reselected as described above. This fairly weak selection process was repeated
for six generations and eventually resulted in eight isofemale lines with a
lowered level of desiccation resistance. The two most desiccation-susceptible
lines were used for the chromosome mapping crosses and were not included in
the physiology assays.
To examine the effect of each resistant chromosome on resistance in heterozygous form (excluding chromosomes 4 and Y), crosses were set up between S1 and S2 and the two most susceptible of the indirectly selected lines (data not shown). Prior to crossing, heterozygosity in the selection/susceptible lines was decreased by full sib mating for 16 generations (lines that maintained the resistant/susceptible desiccation phenotypes were maintained). The crosses were designed to isolate each chromosome in turn in heterozygous form in the desiccation-susceptible background. Four lines were used in the crosses, representing two replicates of each desiccation-resistant and -susceptible background. The three major chromosomes (X, 2 and 3) from the selection and susceptible lines were identified using three polymorphic microsatellite repeats that distinguished the inbred lines: for the X chromosome a repeat from the period locus was used (GenBank accession number AE003425), MS:AC004516 was used for chromosome 2 (AC004516) and MS:AC008193 for chromosome 3 (AC008193). For brevity, the lines established from the resistant selected lines with the high levels of resistance are referred to as H, and those lines from the susceptible selected lines with a low level of resistance are L. Resistant H males were mated to virgin L females and the reciprocal cross was also set up. The F1 males from each cross were backcrossed to virgin H females, yielding eight classes of progeny (Fig. 4). F2 females from each cross were phenotyped for desiccation resistance (as described for the selection regimes) and genotyped using a LiCor IR2 DNA analyzer (Lincoln, NE, USA). Fecundity varied markedly between the crosses derived from the four inbred replicate selected lines, at least 15–20 F2 females were assayed from each of the crosses derived from S1, and 48–50 females were assayed from each of the crosses derived from S2.
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Mortality and weight change of individual females
Data and analyses for mortality and weight changes of individual females
are presented in Table 2. When
flies were desiccated individually, there was a highly significant line term
in the ANOVA (P<0.001), as individual S1 and S2 females survived
twice as long as control females, and this was significant by a planned
contrast (P<0.001). There was variation among the controls that
was significant when tested with a planned contrast (P<0.05); C2
survived desiccation approximately 23% longer than C1 (an outcome not observed
when groups of flies were tested). The data for body weight of individuals was
consistent with the data collected in groups
(Table 1), with a significant
line term in the ANOVA (P<0.001), also evident when tested with a
planned contrast (P<0.001). As in groups, S1 weighed around 8%
more than S2.
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We measured the percentage of total water lost at death to determine whether the selection response was associated with an increase in dehydration tolerance. There was a significant line term in the ANOVA (P<0.001), as the selected lines tolerated on average 10% more total water loss than the controls, and this was also significant when tested with a planned contrast (P<0.001).
To examine variation in water content, individuals were dried until they stopped losing weight. The line term was significant in the ANOVA (P<0.05), as well as a planned contrast (P<0.05). This variation may be attributable to differences between the control lines (planned contrast, P<0.05). There was a significant line effect for dry weight (P<0.0001), and the differences between the control and selected lines were significant when tested with a planned contrast (P<0.001), in addition to significant differences between the selected lines (P<0.05).
Water partitioning
Females were assayed for glycogen levels and weighed before and after
hemolymph blotting to examine their potential for extracellular water storage.
The proportional quantities of hemolymph volume, carbohydrate per fly and
gross lipid content in groups of 20 flies are given in
Table 3. The average hemolymph
content per fly was 13%, while the average glycogen content per fly was 17%.
There was no significant difference between the selected and control lines for
hemolymph and glycogen content. Whole-body lipid content tended to be higher
in the selected lines, and there was a significant line term in the ANOVA
(P<0.001) as well as significant variation between the selected
and control lines when tested with a planned contrast (P<0.001).
Multiple comparisons also revealed intra-line variation for both the control
comparisons (P<0.001) and selected line comparisons
(P<0.001).
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Life history traits
To determine the effect of selection for desiccation resistance on
development time, egg to adult development was quantified for both males and
females (Fig. 2A) and was found
to increase in response to selection. Females eclosed earlier than males, and
there was a significant line and sex term in the ANOVA
(F3,72=27.48, P<0.001 and
F1,72=12.80, P<0.001), respectively.
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Chromosome resistance
The ANOVA comparing LL (desiccation-susceptible) females with LH females
(heterozygous for resistant selected line chromosomes) showed that when
isolated in heterozygous form, chromosome 3 significantly contributed to the
variation between the genotypes (P<0.001), with a significant
interaction effect between chromosomes 2 and 3 (P<0.001)
(Table 4).
Fig. 4 presents the average
desiccation resistance of the eight genotypes generated by the crosses
described in the Materials and methods. Desiccation resistance was highest
when all chromosomes were heterozygous, while individuals heterozygous for
chromosome 2 (SS RS SS) or both X and 2 (RS RS SS) showed no increase in
resistance, with a very similar phenotype to the susceptible homozygotes (SS
SS SS), although both chromosomes X and 2 increased resistance when in
combination with chromosome 3. Both chromosome 3 (SS SS RS) and the X
chromosome (RS SS SS) increased desiccation resistance in the susceptible
background, although we did not detect significant effects for the X
chromosome in the ANOVA (Table
4).
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Discussion |
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).
There was sexual dimorphism for desiccation survival at the onset of our
selection regime, and males were invariably culled before the 90% mortality
mark, as also reported by Chippindale et al.
(Chippindale et al., 1998),
resulting in only inseminated females founding subsequent generations. Despite
the different fitness requirements of the sexes, the presence of resistant
males implies that there is a partially common genetic basis between the sexes
underlying the selection response.
Given the fitness advantage of females over males for desiccation
resistance, we were most interested in characterizing the physiological basis
of the selection response in mature females.
Table 5 presents a summary of
genetic associations between traits involved in selection for desiccation
resistance, both for the lines described in this study and those previously
published. Here, selection was associated with an increase in wet weight. One
obvious explanation for increased weight is that larger flies have a smaller
surface area across which to lose water. This outcome is consistent with lines
directly selected for desiccation resistance
(Gibbs et al., 1997) and in
lines selected indirectly in response to very mild desiccation stress
(Kennington et al., 2003), but
not in other direct-selection lines (Bubliy
and Loeschcke, 2005; Hoffmann
and Parsons, 1989b). Size and desiccation resistance covary in
natural populations of Drosophila; van Herrewege and David observed
that temperate species were on average heavier and survived desiccation longer
than their tropical counterparts (van
Herrewege and David, 1997). At first glance, the different sets of
desiccation-selected lines have converged on similar size phenotypes, but
further examination of the mechanisms underlying the selection responses
reveals very different adaptations. For example, relative to their controls,
the D lines are larger, due primarily to post-pupation increases in bulk
water, shown to be partitioned to the hemolymph (extracellular accumulation)
rather than associated with augmented glycogen levels (intracellular
accumulation) (Chippindale et al.,
1998; Folk et al.,
2001). Conversely, we observed that resistant females do not
sequester extra water as a resistance strategy; accumulated water could not
account for the increase in wet mass, and consistent with this observation is
the lack of association with hemolymph and glycogen content in resistant
females compared with their controls.
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The primary mechanism by which Drosophila survive desiccation is
increased water retention, a response consistently demonstrated across
multiple studies, including natural adaptation in desert species
(Gibbs, 2002;
Gibbs et al., 2003),
artificial selection experiments (Hoffmann
and Harshman, 1999) and a mutagenesis study
(Telonis-Scott and Hoffmann,
2003). We also observed decreased water loss rates in the selected
lines, although this was not consistent in comparisons between all control and
selected lines, and the selected lines also varied (S2 lost approximately 30%
less water than S1 in the same 3-h period). In contrast to previous selection
studies (see Table 5), as well
as data from xeric Drosophila
(Gibbs and Matzkin, 2001), the
selected populations also endured desiccation longer by increasing dehydration
tolerance by around 10%, measured as total water loss prior to death. Some
mechanisms that insects may employ to tolerate low water content include
compartmentalizing water, regulating osmotic effects, rendering a higher
proportion of water osmotically inactive or, in the severest form of
dehydration, some insects undergo anhydrobiosis
(Danks, 2000). These selected
lines provide an opportunity to further explore the physiological basis of
enhanced dehydration tolerance in D. melanogaster from a laboratory
evolution perspective. In terms of the primary physiological adaptations
insects may evolve in response to desiccation stress, the selected females do
not accumulate extra water but show increased water conservation in addition
to tolerating greater water loss during desiccation.
Table 5 illustrates that these
particular adaptive responses in concert are unique to this set of lines,
further emphasizing that the abundant variation for desiccation resistance in
D. melanogaster may lead to many potential evolutionary pathways.
Evidence suggests that in D. melanogaster, body size, water
content and carbohydrate content are genetically correlated
(Clark and Doane, 1983;
Clark et al., 1990;
Folk et al., 2001). In the
present study, we tested whole-body lipids and found an overall trend for
increased lipid storage in the selected lines. Clark et al. observed that body
size tended to decrease as lipid content increased in D. melanogaster
artificially selected for lipid storage
(Clark et al., 1990);
interestingly, S2 stored more lipids and weighed significantly less than S1.
Previous studies have demonstrated that water volume and lipid storage are
inversely proportional (Clark and Doane,
1983; Clark et al.,
1990; Folk et al.,
2001); for example, Folk et al. proposed that, in response to
selection, the D flies accumulated more water and therefore may be constrained
to reduce lipid content (Folk et al.,
2001). Differences in lipid levels between the two different sets
of selected lines are not unexpected given the different control treatments
employed, as the D control lines (C lines) accumulated more lipids, most
likely owing to an evolutionary history of mild starvation stress
(Chippindale et al., 1998). The
selected lines developed here may be constrained to evolve increases in water
volume due to increased lipid storage and/or correlations with other
biochemical characters involved in dehydration tolerance.
Correlated stress responses
The association between starvation and desiccation resistance following
artificial selection has been well documented in D. melanogaster
(Hoffmann and Parsons, 1993;
Harshman et al., 1999), as
well as in natural populations of temperate Drosophila
(van Herrewege and David,
1997). We also report a strong positive correlation between
starvation and desiccation resistance following selection. Previous studies
suggested that this association might be in part due to shared patterns of
glycogen storage (Hoffmann and Harshman,
1999) although this seems an unlikely scenario here. In the
present study, the positive correlation between starvation and desiccation
might be associated with increased lipid reserves; starvation resistance in
D. melanogaster is correlated with increased lipids, evident from
data collected from adipose60 mutants (Clarke and Doane, 1983) as
well as selected lines (Chippindale et al.,
1996; Harshman et al.,
1999). However, this hypothesis requires further rigorous
physiological examination; the effect of mild starvation stress on the
selected lines in contrast to the untreated controls needs to be considered,
although Hoffmann and Parsons did not observe augmented lipids following
selection via an identical selection regime
(Hoffmann and Parsons,
1989b).
We did not observe a correlation with heat mortality and increased
desiccation resistance, in contrast to Bubliy and Loeschcke, who suggest that
heat shock proteins may underlie the positive correlation between the traits
(Bubliy and Loeschcke, 2005).
However, we did observe a negative correlation with cold mortality and
desiccation resistance in S1. There is no physiological data to suggest a
tradeoff for cold tolerance in desiccation-resistant females, although
Hoffmann et al. reported the same tradeoff in females selected for starvation
resistance and cold resistance, respectively, potentially related to lipid
metabolism (Hoffmann et al.,
2005). As the starvation lines from the latter study were derived
from the controls here, there may be some common mechanism related to lipid
metabolism underlying both starvation and desiccation resistance and
contributing to the tradeoff with cold survival. However, the complex
antagonistic pleiotropy observed here is difficult to interpret given the
response in only one selected line. Clearly, the replicate lines have
exhibited variation in responses, despite efforts to maintain identical
treatments. This can occur when two replicates (present study) or five
replicates (Bradley and Folk,
2004) are compared and may reflect levels of genetic variation in
the base stocks expressed through replicate lines.
Life history tradeoffs
Evidence from populations of D. melanogaster selected for stress
resistance suggests that energy allocation may influence tradeoffs between
survival and reproduction (Borash and Ho,
2001). Studies have directly tested this hypothesis and shown that
there is a reproductive cost to stress resistance for oxidative stress
(Wang et al., 2001) and both
oxidative and starvation stress (Salmon et
al., 2001) as well as resistance to cold
(Watson and Hoffmann, 1996).
For desiccation resistance, tradeoffs between resistance and early fecundity
were reported in one study (Hoffmann and
Parsons, 1989a) but not in another
(Chippindale et al., 1993).
Zera and Zhao demonstrated that lipid accumulation (in the form of
triglycerides) is a key component in the tradeoff between increased early
reproduction and reduced flight capability in the short-winged morph of
Gryllus firmus (Zera and Zhao,
2003). The negative association between lipid biosynthesis and
early ovarian development may occur for starvation-selected lines as well. We
observed significant variation between the control and selected lines for
total egg number over five days, with egg number reduced in the selected
lines, in addition to significant variation in intra-group comparisons.
Hoffmann and Parsons suggested that the decline in early fecundity in their
lines was related to reduced metabolic rates because they found no changes in
lipid levels (Hoffmann and Parsons,
1989a). It is possible that the correlated fecundity response in
the present study may be associated with lipid levels, potentially of the same
nature as those utilized for starvation resistance, however more detailed
physiological assays are required, and basal metabolic rate also needs to be
considered. Selected flies of both sexes tended to take longer to develop;
this result was reported by Chippindale et al.
(Chippindale et al., 1998) but
not by Hoffmann and Parsons (Hoffmann and
Parsons, 1993) or Bubliy and Loeschcke
(Bubliy and Loeschcke, 2005),
although these lines were selected for a shorter duration and the expression
of different patterns of resource acquisition may require longer selection.
Prolonged development time has also been documented in lines selected for
starvation resistance in both long-term laboratory-adapted lines
(Chippindale et al., 1996) and
recently derived field lines (Harshman et
al., 1999), suggesting a general association between starvation
resistance, increased lipid and increased development time
(Harshman et al., 1999). If
increased lipids contribute to both the correlation between starvation
resistance and other life history traits, it is possible that we may have
selected for an overlapping pathway of general stress resistance in these
desiccation-resistant lines.
Chromosome mapping
The outcome of the chromosome mapping crosses suggests that autosomal
(chromosome 3) and (to a lesser extent) X-linked genes improve desiccation
resistance in a desiccation-susceptible background derived from the same
control population as the resistant-selected lines. A direct comparison of the
selected and untreated control lines would provide information as to which
chromosomes carry genes to improve desiccation resistance, however our
comparison of both resistant and susceptible lines derived from the same
background is likely to provide similar information. Deleterious alleles might
contribute to decreased resistance in susceptible lines compared with the
controls, but we only undertook weak selection for susceptibility for a short
duration (six generations) among family groups that all produced offspring,
making it less likely that deleterious genes contributed to the selection
response. We also did not find any reduction in fitness of the susceptible
lines that might have indicated the presence of such genes. In concurrence
with our results, Hoffmann and Parsons found that the response to selection
for desiccation resistance was not sex specific and reported that both
autosomal and X-linked genes acted mostly additively
(Hoffmann and Parsons,
1989b).
The interactions between chromosomes detected in the present study suggest
some epistasis among genes related to stress resistance; widespread epistasis
was also observed at the gene expression level during starvation stress
(Harbison et al., 2005) and
may be expected for complex traits such as stress resistance.
In summary, our results highlight the diversity of mechanisms that can underlie responses to selection for desiccation resistance. We have found a new mechanism (tolerance of degree of total water loss) not detected in previous studies and confirmed the central role of water conservation in selection responses for increased desiccation resistance. Correlated responses in life history traits show overlap with those detected in other studies. Altered desiccation resistance is associated with genes on the third chromosome but there are also interactions with genes on chromosome 2.
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Acknowledgments |
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|
Footnotes |
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Present address: Centre for Environmental Stress Adaptation Research,
Department of Genetics, The University of Melbourne, Victoria 3010,
Australia 
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References |
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