Evolved patterns and rates of water loss and ion regulation in laboratory-selected populations of Drosophila melanogaster
Department of Ecology and Evolutionary Biology, University of California, Irvine, Irvine, CA 92697, USA
* Author for correspondence (e-mail: dfolk{at}uci.edu)
Accepted 15 May 2003
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Summary |
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Key words: water loss, ion regulation, hemolymph, Drosophila melanogaster, desiccation, Na+, K+, Cl-, excretion, osmoregulation
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Introduction |
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Mellanby (1939) proposed
that a vital function of insect hemolymph is to store water, which is then
available for distribution to the tissues during periods of water stress. One
of the evolutionary responses in our laboratory-selected,
desiccation-resistant populations is a striking increase in hemolymph volume
(
330 nl; a >6-fold increase in hemolymph volume relative to the
control populations). We propose that water from this large hemolymph pool can
buffer the tissues against water loss during periods of water stress, thus
supporting tissue homeostasis. The control populations, which have not been
subjected to selection for enhanced desiccation resistance, have a relatively
small hemolymph volume [
50 nl]. We expect that these populations may have
a reduced ability to prevent water loss from other body compartments during
desiccation.
During lengthy bouts of desiccation, rates of water loss in insects tend to
decline as water stress proceeds
(Loveridge, 1968; Edney,
1971
,
1977
;
Hadley et al., 1986
;
Noble-Nesbitt and Al-Shukur,
1987
; Hadley,
1994
). Previous studies have shown that our populations of
desiccation-resistant flies have a significantly lower mean rate of water loss
than the control populations (Gibbs et
al., 1997
; Williams et al.,
1998
). We wished to determine if the desiccation-resistant
populations had diverged from the control flies in the ability to modulate
water loss rates during extended periods of desiccation.
Sodium is the principle ion in the hemolymph of dipterans and generally
makes up approximately two-thirds of the cation pool
(Sutcliffe, 1963). The loss of
water during desiccation without a concomitant loss of Na+ would
lead to an increase in the Na+ concentration of the hemolymph. As
hemolymph osmotic concentration rises, water would tend to move out of the
cells. As a consequence, ion regulation of the hemolymph during desiccation
may be necessary to prevent intracellular water loss.
Most insect species regulate hemolymph osmotic concentration during
desiccation (Edney, 1977;
Hadley, 1994
). One component
of hemolymph osmoregulation in terrestrial insects during water loss is the
removal of inorganic ions, principally Na+, Cl- and, to
a lesser extent, K+, which are either permanently excreted or
transported to and sequestered within the tissues. Two general disadvantages
related to the strategy of ion excretion combined with hemolymph
osmoregulation are: (1) by necessity, water is lost during excretion and (2)
upon rehydration the insects are committed to replacing the excreted ions
via consumption. Although some insects have been shown to use
excretory osmoregulation, use of this strategy has been shown for only a small
number of species (Laird and Winston,
1975
; Hyatt and Marshall,
1978
; Albaghdadi,
1987
; Naidu and Hattingh,
1986
).
One strategy of hemolymph osmoregulation in terrestrial insects during
desiccation is the transport of ions out of the hemolymph and into some
tissues, where they may be stored in an osmotically inactive form. This
strategy has been demonstrated in the cockroach Periplaneta
(Wall, 1970; Tucker,
1977a
,b
;
Hyatt and Marshall, 1977
,
1985a
,b
).
Hemolymph osmoregulation in Periplaneta is achieved primarily by the
removal of Na+ and K+. Only a small fraction of the ions
are excreted; the greater proportion is sequestered within the fat body,
probably as urate salts. The advantages of this strategy over ion excretion
are: (1) water is not lost during ion storage and (2) upon rehydration the
ions are available for transport back into the hemolymph.
We present here the first study of water and ion distribution in Drosophila melanogaster during desiccation. We examined water levels and Na+ content of both the hemolymph and tissues. If the flies osmoregulate the hemolymph during desiccation using Na+ excretion, we predict that the Na+ content of the hemolymph will decline, while tissue Na+ will be unchanged (or perhaps reduced). Alternatively, if the flies use the `removal and sequestration' strategy, the hemolymph Na+ content should drop, while tissue Na+ increases.
We also measured whole-body potassium and chloride content during
desiccation. Potassium is the principal intracellular cation and is found only
in very low concentrations within the hemolymph of dipterans
(Sutcliffe, 1963). We
anticipated that because of the increased capacity of the
desiccation-resistant populations to prevent cell shrinkage during
desiccation, the maintenance of cellular ion composition would also be
enhanced. If true, the K+ content of the tissues should not change,
considering net tissue water loss is minimized. Chloride, the principle
inorganic anion in dipteran hemolymph, generally comprises
20% of the
total anion pool (Sutcliffe,
1963
). We propose that in order to maintain electroneutrality, as
well as osmolarity, loss of Na+ and/or K+ content may be
accompanied by loss of Cl-.
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Materials and methods |
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Details of the maintenance protocol and selection regime are provided in
Folk et al. (2001). Briefly,
multiple batches of eggs (6080 eggs per batch) were collected from each
population, and each was placed into a food vial. The insects were allowed to
metamorphose and mature for 14 days, at which time the D populations were
subjected to the selection protocol until 80% mortality was reached. During
selection, the D populations were deprived of food and water, while the C
populations were provided water. Following selection, all surviving flies were
allowed to recover on moist food for 3 days. Eggs were then collected for
rearing of the next generation. (Approximately the same number of females from
all C and D populations was available for establishment of each new
generation.) Selection for enhanced desiccation has been imposed for more than
250 generations.
Prior to all experiments, flies from the C and D populations were removed from the pressure of selection for two generations. By rearing C and D flies for two generations under identical conditions, and without the stress of selection, we removed grandparental and parental effects that derived from the selection regime. We were then able to attribute divergences between the C and D populations to genetic differences. Additionally, to eliminate the effects of gender, only females were studied. All experiments were performed on females that were (on average) 4-day-old, mated adults.
Desiccation protocol
Five or six flies were briefly anesthetized with CO2 and placed
in a 30 g vial. A foam stopper was placed 3 cm into the vial, and
4.5 g of Drierite (calcium sulfate desiccant) was poured on top of the
stopper. The open end of the vial was then tightly covered with Parafilm.
Flies were allowed to desiccate undisturbed within the vials for 8 h, 16 h, 24
h (D flies only) or 48 h (D flies only).
Estimation of extractable hemolymph volume and gravimetric
measurement of water content in exsanguinated flies
Mature females were anesthetized with CO2 and then weighed using
a Cahn 29 automatic electrobalance (Cerritos, CA, USA). Flies were kept under
CO2 anesthesia while the abdomen was gently torn with fine-tipped,
surgical forceps. A Kimwipe (i.e. low-lint, laboratory-grade tissue paper)
moistened with isotonic saline (250 mosmol l-1;
Singleton and Woodruff, 1994)
was used to absorb the extractable hemolymph. By slightly moistening the
Kimwipe, tissue from the fat body was less likely to stick to it. To obtain
the total wet mass of the exsanguinated flies (i.e. of the
tissuegutcuticle), each fly was re-weighed immediately following
hemolymph extraction. The hemolymph volume was then estimated by subtracting
the wet mass of the exsanguinated flies from the whole-body wet mass (for a
brief review of estimating hemolymph volume by use of the blotting technique,
see Hadley, 1994
). Hemolymph
volume and total wet mass of the exsanguinated flies were measured in 20
individuals from each C and D population at the following time intervals:
prior to desiccation (designated as 0 h) and after 8 h, 16 h, 24 h (D flies
only) and 48 h (D flies only) of desiccation. Because control flies survive
for an average of 23 h when desiccated, measurements of these flies were not
made at 24 h and 48 h. The exsanguinated flies were dried overnight at
60°C and reweighed to obtain dry mass. Water content of the exsanguinated
flies was estimated by subtracting dry mass from total wet mass.
Ion measurements
Sodium and potassium
Eight samples of whole flies (Na+ and K+
measurements) and eight samples of exsanguinated flies (Na+
measurements only) were prepared from each population prior to desiccation
(designated as 0 h) and after 8 h, 16 h, 24 h (D flies only) and 48 h (D flies
only) of desiccation. Each sample consisted of three flies that had been
solubilized overnight in 100 µl of concentrated HNO3 (containing
0.02 p.p.m. Na+, 0.05 p.p.m. Cl- and <0.002 p.p.m.
K+) at room temperature (2123°C). Following
solubilization of the flies, 2.9 ml of doubly distilled water
(ddH2O) was added. Sodium and potassium concentrations of the
samples were determined using atomic absorption spectrophotometry (AA-125
series; Varian Analytical Instruments, Springvale, Australia). The mean
Na+ content of either the intact flies or the exsanguinated flies
was calculated from the Na+ concentration of the sample. The
Na+ content of the hemolymph was estimated by subtracting
Na+ content of the exsanguinated flies from whole-body
Na+ content.
Chloride
Whole-body chloride was determined using a colorimetric assay
(Gonzalez et al., 1998). Each
sample was made up of three flies that had been solubilized overnight in 50
µl of concentrated HNO3 at 21-23°C. Following liquefaction
of the flies, 0.95 ml ddH2O was added to each sample. Eight samples
were prepared from each population at the time intervals described in the
previous section. Chloride was quantified through a two-step assay: (1)
thiocyanate ions were released from mercuric thiocyanate through the formation
of mercuric chloride and (2) in the presence of ferric ions, a colored
compound (ferric thiocyanate) was produced in proportion to the Cl-
content in each sample. The absorbance of each sample was measured
spectrophotometrically at 480 nm. Chloride concentration of the fly samples
was then calculated from standard curves that were constructed from samples of
known Cl- concentrations.
Statistical analyses
The changes in hemolymph volume, water and Na+ content of the
tissuegutcuticle and whole-body K+ were compared
between the C and D groups prior to and throughout desiccation using a linear
mixed-effects statistical model, in which the pairing of each
Cn with Dn was treated as a block
effect. The model included C and D treatments and the paired-population blocks
as fixed effects, and time and within-population variance as random effects.
The analyses were performed using the statistical program, R. Within the C and
D treatment groups, we performed one-way analyses of variance with Bonferroni
post-hoc comparison of means to determine the time period(s) at which
significant changes in each dependent variable occurred. (To obtain
Na+ content of the hemolymph, we had subtracted the mean
Na+ content in exsanguinated flies from the mean Na+
content in whole flies for each Cn or
Dn population at each time interval. Hence, we could
include in the statistical analyses only the five population means from both
treatments at each time interval. As a result, we used only treatment and time
when analyzing changes in hemolymph Na+ content.) Least-squares
linear regression was used to examine the relationship between chloride
content and hemolymph volume during desiccation. Each point in the regression
analyses represented the estimated mean from all five of the C or D
populations. The significance level for all analyses was 0.05.
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Results |
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The D flies experienced loss of hemolymph throughout the entire desiccation
period, and the volume was ultimately reduced by >80%. The mean rate of
hemolymph loss during the initial 8 h of desiccation was 14 nl h-1.
Between 8 h and 24 h, this rate was reduced by 50% to 6 nl h-1
and dropped to
2.5 nl h-1 during the 2448 h period.
While the volume of hemolymph in the D flies declined continually throughout
desiccation, the water content of the tissuegutcuticle was
maintained during the initial 24 h (Fig.
2). At 2448 h, the D flies had a significant reduction in
water from the tissuegutcuticle (P=0.0001). The overall
rate of water loss in the D flies during 2448 h totaled
10 nl
h-1 (
2.5 nl h-1 and 7.5 nl h-1 from the
hemolymph and tissuegutcuticle, respectively).
The patterns and rates of water loss in the control flies were distinct
from those observed in the desiccation-resistant flies. Approximately
two-thirds of the hemolymph volume of the C flies was lost during the initial
8 h (P=0.001; Fig. 1),
after which the volume was not significantly reduced. In contrast to the D
flies, the C flies lost significant volume from the
tissuegutcuticle by 8 h (P<0.0001;
Fig. 2). The mean net rate of
water loss in the C flies during the initial 8 h was 39 nl h-1 (4
nl h-1 and 35 nl h-1 from the hemolymph and
tissuegutcuticle, respectively), which was 3 times that
observed in the D flies during the same time period. Net water loss rate
dropped by
90% to a mean rate of
4 nl h-1 during
816 h.
Hemolymph sodium
The desiccation-resistant flies had a higher rate of Na+ removal
from the hemolymph, which was a consequence of the greatly increased
Na+ content (P=0.002). The hemolymph Na+
content of the D populations was significantly reduced due to the excretion of
Na+ between 8 h and 16 h (P=0.004;
Table 1). Despite this
excretory event, the hemolymph Na+ content of desiccated D flies
remained more than twice as high as that of fully hydrated C flies. Hemolymph
Na+ content of the D flies appeared to be regulated; yet
Na+ concentration was not strongly controlled
(Table 1). Hemolymph
Na+ concentration increased by 40% during the first 8 h of
desiccation and was reduced by >50% following the Na+ excretion
event (816 h). Between 16 h and 48 h, the Na+ content was
stable and, as a result, Na+ concentration increased as hemolymph
volume continued to decline. By 48 h, Na+ concentration exceeded
pre-desiccation values by >60%.
|
Hemolymph Na+ content of the C flies dropped significantly (84%
reduction) during the initial 8 h (P=0.04;
Table 1) and was maintained at
a very low level during 816 h. The hemolymph Na+
concentration in the C flies declined >50% during the initial 8 h, and by
16 h it had been reduced by 75%, following the removal of much of the
Na+ from the hemolymph.
Sodium content in exsanguinated flies
The rate of loss of Na+ from the tissuegutcuticle
of the C flies was significantly higher (2-fold increase) than that
observed in the D flies (P=0.007). The Na+ content of the
tissuegutcuticle of the D flies was maintained during the
initial 24 h of desiccation (Fig.
3); then, between 24 h and 48 h, a significant amount (14%) of
Na+ was removed (P=0.01) and subsequently excreted. The
exsanguinated control flies had a significant reduction (16%) in
Na+ following 8 h (P=0.01;
Fig. 3); the Na+
content was unchanged following this reduction (P=0.52).
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Whole-body potassium
Potassium content did not change significantly in the D flies throughout
the entire desiccation period (P=0.13;
Fig. 4). By contrast, the
control flies had an excretory loss of 17% K+ following 8 h of
desiccation (P=0.01; Fig.
4), following which no significant loss of K+ was
detected (P=0.31).
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Whole-body chloride
Both control and desiccation-resistant flies had significant losses of
whole-body Cl- during desiccation. The mean rate of Cl-
reduction in the C flies was 0.22 nmol h-1 (P=0.001,
r2=0.99, y=-0.223x+8.323;
Fig. 5), which was >100%
higher than the rate of Cl- reduction in the D flies
(P=0.023, r2=0.94,
y=-0.094x+9.875; Fig.
5). At the end of the desiccation periods (16 h and 48 h for the C
and D flies, respectively), pre-desiccation levels of Cl- had been
reduced by 43% in both groups.
|
A significant positive relationship between Cl- content and hemolymph volume was found in both the C populations (regression not shown; P=0.01, r2=0.65, y=0.081x+4.205) and the D populations (P=0.001, r2=0.88, y=0.0163x+5.123; Fig. 6), suggesting that the decline in Cl- was associated with the loss of hemolymph.
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Discussion |
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Sodium regulation during desiccation
The Na+ content of the hemolymph of a hydrated D fly was
>7-fold higher than that of a hydrated C fly
(Table 1). Hemolymph water loss
coupled with unregulated Na+ content could result in exceedingly
high Na+ concentrations within the hemolymph of the D flies. The
osmotic concentration of Na+ within the hemolymph could increase to
>400 mosmol l-1 following 48 h desiccation if Na+
content remained unadjusted. Yet, the highest hemolymph Na+
concentration that we observed in the D flies was 114 mosmol l-1,
indicating that Na+ content was regulated during desiccation.
In dehydrated Periplaneta, Na+ is removed from the
hemolymph and subsequently stored in the fat body
(Wall, 1970; Tucker,
1977a
,b
;
Hyatt and Marshall, 1977
,
1985a
,b
).
We examined Na+ content of the hemolymph and of exsanguinated flies
to determine if Na+ was removed from the hemolymph and stored in
another compartment. Sodium was removed from the hemolymph in both C and D
flies, yet the Na+ content of the other compartments did not
increase; hence we found no evidence of Na+ sequestration
(Table 1;
Fig. 3). Instead, our data
suggest that after Na+ was removed from the hemolymph, it was
permanently excreted.
Sodium content of the tissuegutcuticle in the D flies did not
change significantly until 2448 h. To determine if the drop in
Na+ was due merely to gut clearance, we estimated the
Na+ content of a full gut. Total gut volume was estimated to be
0.1 µl (based on morphometric data from
Miller, 1994
). The
Na+ concentration of the food was
10 mmol l-1.
Given these values, we estimate that only
1 nmol of Na+ is
contained within a full gut. Clearing of the gut, therefore, could not account
for the loss of Na+. We conclude that a reduction in Na+
content must have occurred within some tissues.
Relative to the D flies, the C flies had a much lower hemolymph volume and Na+ content when fully hydrated (Table 1; Fig. 1); yet, during the initial 8 h of desiccation, the C flies lost a high proportion of both. The loss of Na+ from the hemolymph was not followed by an increase in Na+ content of the tissuegutcuticle (Fig. 3), indicating that the control flies also excreted Na+ during desiccation.
Potassium regulation
Potassium is the predominant intracellular cation and is found at
relatively low concentrations (25 mmol l-1) in the hemolymph of
Drosophila melanogaster (Van der
Meer and Jaffe, 1983). The control flies lost substantial
whole-body K+ within the first 8 h of desiccation. Overall, our
data suggest that the initial 8 h period was physiologically stressful in the
C flies: they lost 28% of the water and 16% of the Na+ content from
the tissuegutcuticle, as well as 17% of whole-body K+
(Figs 2,
3,
4). We propose that the
reduction in K+ occurred in parallel with the acute loss of water
and Na+ from the tissues and as a means of regulating osmotic
concentration of the tissues.
In response to selection for enhanced resistance to desiccation, the D populations have evolved osmoregulatory capacities divergent from those observed in the C flies. Despite the loss of volume from the tissuegutcuticle in the latter 24 h, the whole-body potassium content of the D flies did not significantly decline during the entire 48 h of desiccation (Fig. 4).
Regulation of chloride
Both the C and D groups of flies lost substantial whole-body Cl-
during desiccation (Fig. 5).
Several lines of evidence suggest that Cl- was lost principally
from the hemolymph, at least in the D flies. (1) Loss of Cl- was
strongly associated with a reduction in hemolymph volume (P=0.001;
Fig. 6). (2) The water,
K+ and Na+ content of the tissuegutcuticle
were not reduced during 24 h of desiccation (Figs
2,
3,
4). Therefore, loss of
Cl- from any body compartment other than the hemolymph during
024 h is doubtful. (Between 24 h and 48 h, the D flies had a small, but
significant, loss of Na+ from the tissuegutcuticle.
Chloride ions may have been lost concurrently with Na+ from the
tissuegutcuticle between 2448 h.) (3) Following the
single Na+ excretion event, hemolymph Na+ and
Cl- content were maintained at almost equimolar levels
(Fig. 7). (4) Finally,
intracellular Cl- concentration is usually quite low in relation to
extracellular Cl- (Alberts et
al., 1994). For these reasons, we propose that most of the
Cl- measured in the whole flies was located in the hemolymph and
that regulation of both Na+ and Cl- plays a role in
hemolymph osmoregulation in the D flies.
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During the initial 8 h period, the C flies had significant losses of K+ and Na+ from the tissuegutcuticle, and some Cl- may have been lost concomitantly from these sites. For this reason, we are unable to conclude that the reduction in Cl- was a major component of hemolymph osmoregulation in the control flies.
Patterns and rates of water loss
The control flies experienced a net loss of 30% of water from the
tissuegutcuticle during the initial 8 h of desiccation. Although
we were unable to separate the volume of water lost from the tissues from that
removed from cuticle or gut, the following evidence supports the idea that
water was, indeed, lost from the tissues early in desiccation: (1) The C flies
had a relatively small hemolymph volume even when hydrated, thus the ability
to replace tissue water with that stored in the hemolymph was presumably
minimal. (2) The substantial loss of K+ suggests that intracellular
osmoregulatory adjustments were made in response to acute cellular water loss.
It is feasible that the flies also lost some water from the cuticle and gut.
The importance to water balance of the water absorbed by the cuticle remains
controversial (Loveridge,
1968
; Winston and Beament,
1969
; Machin et al.,
1985
; Machin and Lambert,
1987
). The significance of water storage within the gut of
drosophilids is unclear. We estimated gut volume of the flies to be
100
nl. Assuming that the gut was filled with food containing 80% water, clearing
of the gut would only account for
30% of the water lost from the entire
tissuegutcuticle. Therefore, we propose that substantial water
was lost from the tissues.
After the initial 8 h period, hemolymph volume remained unchanged in the C
flies (Table 1). At this time,
the volume was low (19 nl) and may have reached a critical level, which if
further reduced could be physiologically detrimental, if not fatal. The
hemolymph has multiple circulatory and osmoregulatory functions
(Chapman, 1998). If hemolymph
levels are reduced below some critical level, distributive and regulatory
functions of the hemolymph may be compromised. We suggest that the adverse
effects deriving from a severe reduction in hemolymph volume may play a role
in defining the lower limits of hemolymph volume, necessitating that
additional water loss comes from the intracellular compartment.
The patterns and rates of water loss during desiccation in the D flies is distinct from that observed in the C flies. During 24 h of desiccation, water was only lost from the hemolymph, and the tissue water content was conserved. Interestingly, whole-body K+ content did not significantly decrease during the 48 h period in the D flies; therefore, it remains unclear if the water lost from the tissuegutcuticle was principally lost from tissues.
During desiccation, the rate of absolute water loss declined in both C and
D groups. The C and D flies had net water loss rates in the range of
439 nl h-1 and 6-14 nl h-1, respectively. The
phenomenon of diminishing water loss rates as time of exposure to drying
conditions increases has been observed in various insects and has been
hypothetically attributed to: (1) a decline in activity as the insect
familiarizes itself with its new conditions, resulting in a lower metabolic
rate and a reduction in spiracular water loss; (2) a reduction in hemolymph
volume leading to an increase in ion concentration, which may cause structural
changes in cuticular proteins and a reduction in cuticular permeability; and
(3) hormonal control of water movement across the integument
(Treherne and Willmer, 1975;
Edney, 1977
; Noble-Nesbitt and
Al-Shukur,
1988a
,b
).
The mechanistic explanation for diminishing rates of water loss in our
populations remains unclear.
Conclusions
We have shown that flies selected for enhanced desiccation resistance have
evolved a large hemolymph pool, which acts to protect intracellular volume
during periods of water stress. Gibbs and Matzkin
(2001) have shown that
desiccation resistance in cactophilic species of Drosophila is not
correlated with total body water content. The lack of increasing total body
water in the desert species may reflect a trade-off between locomotor capacity
and the storage of water for desiccation resistance
(Lehman and Dickinson, 2001
).
The precise role of water compartmentalization in desert flies remains
unclear. The findings reported herein suggest that this would be a valuable
area to explore.
The capacity to regulate the sodium, chloride and potassium levels of the
hemolymph in conjunction with volume loss has been observed to varying degrees
in insects, including desert beetles
(Coutchie and Crowe, 1979;
Naidu and Hattingh, 1986
). We
demonstrate for the first time that drosophilids have the capacity to adjust
the inorganic solute content of the hemolymph when volume declines during
desiccation and the degree to which those patterns have been affected by
selection.
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Acknowledgments |
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