Osmotic regulation in adult Drosophila melanogaster during dehydration and rehydration
Department of Ecology and Evolutionary Biology, University of California, Irvine, CA 92612, USA
* Author for correspondence (e-mail: malbers{at}uci.edu)
Accepted 13 April 2004
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Summary |
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Key words: osmoregulation, hemolymph, desiccation, rehydration, Drosophila melanogaster
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Introduction |
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Several osmotic strategies have been observed in insects to deal with the
stress of desiccation; these include tolerance to osmotic variability
(Naidu and Hattingh, 1986;
Garrett and Bradley, 1994
;
Patrick and Bradley, 2000
),
osmotic regulation by sequestration (Hyatt and Marshall,
1977
,
1985
) and osmotic regulation
by excretion (Bradley, 1985
;
Hadley, 1994
). Although
numerous studies have been conducted examining osmoregulation in insects,
studies on small terrestrial insects are notably absent. An exception to this
statement is found in investigations regarding water vapor absorption in small
insects (Holmstrup et al.,
2001
).
We chose to study osmoregulation in a small insect that is of great
importance in the fields of genetics, evolution and molecular biology, namely
Drosophila melanogaster. The paucity of information regarding the
patterns and mechanisms of osmotic regulation in this species is regrettable,
given the numerous studies investigating the evolution and population genetics
of enhanced desiccation resistance in both wild and laboratory-selected
Drosophila populations
(Dobzhansky, 1952; Gibbs et
al., 1997
,
2003
;
Bradley et al., 1999
;
Hoffmann and Harshman, 1999
;
Nghiem et al., 2000
;
Pfeiler and Markow, 2001
;
Marron et al., 2003
).
In the current study, we have undertaken an analysis of patterns of osmoregulation in adult Drosophila during desiccation. Since some degree of desiccation is inevitable in these small insects as they fly about seeking mates, food sources and oviposition sites, the pattern of osmoregulation during water loss and subsequent rehydration are of considerable interest. We chose to study these phenomena in five replicate populations of Drosophila that have been maintained in the laboratory for over 250 generations (C populations). We also examined five replicate populations that have undergone selection for enhanced desiccation resistance (D populations) to determine if osmotic regulation or patterns of rehydration have evolved during this selection process.
Previous studies involving the populations of flies used in this experiment
have established that, relative to the C populations, the D populations have a
reduced rate of water loss both prior to and during a bout of desiccation
(Gibbs et al., 1997;
Williams et al., 1998
) and a
greater body water content (Gibbs et al.,
1997
). The majority of this additional water is found
extracellularly as hemolymph (Folk et al.,
2001
). It has also been demonstrated that hemolymph volume
decreases substantially in the C and D populations during desiccation and that
some ions are removed from this fluid compartment and excreted
(Folk and Bradley, 2003
). What
is not known is whether the flies allow their hemolymph osmolality (number of
solutes per kg of water) to increase substantially or whether they are
strictly regulating their internal fluids. The absence of regulation could
result in intolerable concentrations of osmolytes that lead to cellular and
metabolic malfunctioning. In this study, we measure the hemolymph osmolality
of flies of five D populations and five C populations before desiccation,
during desiccation and upon recovery from desiccation to determine the
osmoregulatory capabilities of the C and D populations under these
circumstances. In addition, we examine the ability of the C and D populations
to replenish hemolymph volume during recovery from desiccation.
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Materials and methods |
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Selection regime
The D populations were selected for enhanced desiccation resistance at
every generation. Fourteen days after egg collection (at approximately 4 days
post-eclosion), each C and D population was transferred from food vials to
separate large Plexiglas cages, one for each population. At this point,
selection was initiated. The D populations were placed in cages along with a
cheesecloth bag of desiccant (Drierite; W. A. Hammond, Drierite Company, Ltd,
Xenia, OH, USA) and no food or water. Cage entrances were sealed with plastic
wrap to retard the entrance of water vapor from the ambient environment. The C
populations were placed in identical cages but with a water source (a
non-nutritive agar plate), no food and no desiccant. When each D population
reached 80% mortality, selection was removed and food was presented to both
the D population and its paired control population. Therefore, the difference
in the treatment of the C and the D populations took place in the adult stage
and consisted only of the presence or absence of water.
Measuring hemolymph osmolality
Hemolymph osmolality was measured in individual flies of all 10 populations
(N=10). Hemolymph samples were collected by piercing the lateral
thoracic segment of individual flies, under oil, with a pulled micropipette
(micropipette puller; Narishig Scientific Instruments Lab, Setagaya-Ku, Tokyo,
Japan). Through capillary action, hemolymph was drawn into the micropipette.
Oil was collected in the micropipette before and after hemolymph collection to
avoid evaporative water loss from the hemolymph sample. The samples were
immediately expelled via mouth pipetting into oil wells of a
calibrated nanoliter osmometer (Clifton Technical Physics, Hartford, NY, USA)
under a dissecting microscope (500x), and osmolality (mOsm) was
determined by melting point depression
(Bradley and Phillips, 1975).
Measured hemolymph samples ranged from volumes of
0.05 to 1.4 nl. No
melanization of the hemolymph was observed subsequent to collection.
Hemolymph osmolality during desiccation
Hemolymph osmolality was determined in 10 individual female flies in each
population at various time intervals during a bout of desiccation stress. The
D flies were desiccated for 8, 16, 24 and 48 h and the C flies for 8 and 16 h.
Five flies were placed in a 40 ml glass vial containing approximately 5 g of
indicator Drierite. Flies were allowed to occupy the lower three-quarters of
the chamber and were isolated from the Drierite by a thin foam plug. Entrances
to the desiccating chambers were sealed with Parafilm (American Can Company,
Greenwich, CT, USA). After the allotted desiccation period, live flies were
removed from desiccating chambers and directly submerged in oil. Hemolymph
samples were drawn and osmolality was measured as described above.
Recovery
We also examined hemolymph osmolality and hemolymph volumes following
recovery from a bout of desiccation. After 8 h of desiccation in the C
populations and 24 h desiccation in the D populations, live flies were removed
from desiccating chambers and placed in recovery chambers. Recovery chambers
were 40 ml vials containing a Kimwipe (Kimberly-Clark, Roswell, GA, USA)
saturated with 1.5 ml of one of three recovery fluids: (1) distilled water,
(2) a saline solution (25 mmol l-1 KCl, 135 mmol l-1
NaCl) or (3) a saline+sucrose solution (5% sucrose, 35 mmol l-1
KCl, 135 mmol l-1 NaCl). The flies were allowed to recover in these
chambers for 24 h (five flies per vial). Hemolymph osmolalities were then
obtained as described above and the hemolymph volumes were estimated
gravimetrically using the blotting technique described by Folk et al.
(2001).
Data analysis
Initial hemolymph osmotic concentrations and those of dehydrated flies were
plotted against proportional dehydration of the hemolymph. Proportional
dehydration of the hemolymph is defined by the expression
Vi/Ve
(Hadley, 1994), where
Vi is the initial volume of the hemolymph (i.e. the
hemolymph volume prior to desiccation) and Ve is the
volume of hemolymph remaining after a given amount of time in the desiccating
chamber. Volumes were obtained in a previous study examining rates of water
loss and ion regulation in the C and D populations
(Folk and Bradley, 2003
).
Using a gravimetric blotting technique, these authors determined the average
extractable hemolymph volume in individual flies of each population before
desiccation and after 8, 16, 24 and 48 h of desiccation (8 and 16 h in the C
flies).
To determine the degree to which flies of each population osmoregulate, a
theoretical regression line was constructed. This theoretical line was the
osmolality for a given hemolymph volume if no ion regulation were to occur.
The theoretical osmolality (osmolalitye) was defined by:
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To quantify the extent of osmoregulation, a modification of a method
proposed by Riddle (1985) was
used. The ratio of observed osmolality slope/theoretical osmolality slope
served as an index of the extent of osmoregulation. If this ratio is equal to
1, there is no evidence of osmoregulation. The closer this value is to 0, the
greater the extent of osmoregulation. This index allows comparison between the
C and D populations because it ignores differences in initial hemolymph
osmolality or volume (Hadley,
1994
).
Statistics
We tested the difference in the means of hemolymph osmolality for each
population (N=5) prior to desiccation between the C populations and
the D populations using a paired Student's t-test to determine
whether the populations were at the same hemolymph osmolality at the onset of
desiccation. We determined whether the hemolymph osmotic concentration of the
C and D populations increased as a result of desiccation by performing a
linear regression test, which established if the observed slopes differed from
zero. To evaluate osmoregulatory abilities of flies within a given selection
treatment, a paired Student's t-test was performed comparing the five
Cn (or Dn) slopes of the observed
regression lines to the five slopes of the theoretical regression lines. To
determine whether the C populations were osmoregulating differently from the D
populations, we compared the ratios of the slopes of the observed regression
line with the slopes of the theoretical regression lines. This ratio,
calculated for each population, was used to evaluate the extent of
osmoregulation. The ratio did not satisfy the assumptions of a parametric
test; therefore, a one-tailed Wilcoxon signed rank test was used to determine
whether the D populations exhibit a greater extent of osmoregulation than
their paired control populations. We performed two analysis of variance
(ANOVA) tests with Bonferroni/Dunn post-hoc tests on hemolymph volume
and another two for hemolymph osmolality to determine differences between
initial values, values after the prescribed desiccation and values after
recovery on water, saline or saline+sucrose. One ANOVA detected differences
within the C populations and the other within the D populations. Hemolymph
volume data obtained from Folk and Bradley
(2003) were used for the
pre-desiccation volume and volume after desiccation.
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Results |
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Hemolymph osmolality during desiccation
Throughout desiccation, the hemolymph osmolality in flies from both
selection treatments increased gradually as hemolymph volume decreased, that
is to say as proportional dehydration of the hemolymph increased (Figs
1,
2). The linear relationship for
each selection treatment is the regression line of mean values of each
population (C15 or D15). The positive
slopes of these mean lines were found to be statistically significantly
different from zero (P<0.05). The point that represents the
largest value on the x-axis is a measured value during a non-lethal
prescribed bout of desiccation and does not represent the hemolymph osmotic
concentration at death.
|
|
The observed hemolymph osmotic concentration of the C and D populations during dehydration (Figs 1, 2) is plotted adjacent to slopes representing the theoretical osmolalities that would arise if no osmotic regulation occurred. The observed increase in hemolymph during dehydration is substantially lower (P<0.001) than the theoretical increase for both C and D populations. This discrepancy between observed and theoretical slopes is a clear indication that all populations were osmoregulating.
The ratio generated by dividing the observed change in hemolymph osmotic concentration by the theoretical concentrations can be used as a measure of the extent of osmoregulation that occurred during dehydration (Table 2). There is some variation in this ratio among populations within selection treatments; however, when we compare the paired populations (i.e. compare Cn with Dn) each D population has a lower ratio than its paired control population. Therefore, at least with regard to this parameter, selection for enhanced desiccation resistance has led to a greater capacity for osmoregulation (P<0.05).
|
Recovery (hemolymph volume)
Simple visual observations of the flies under a dissecting scope following
24 h of recovery suggested that the responses during recovery were not
entirely uniform. A few flies appeared to have drunk nothing at all and were
still in a rather dehydrated state while other individuals had very little
hemolymph despite a largely distended gut. Nonetheless, most flies were able
to increase their hemolymph volume during recovery. Pre- and post-desiccation
values are included in Figs 3,
4,
5,
6 to show what is happening to
hemolymph volume and hemolymph osmolality during desiccation.
|
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Following a drop in hemolymph volume during an 8 hdesiccation bout, the C populations increased hemolymph volume after rehydration on the three recovery solutions (Fig. 3). Hemolymph volumes averaged 84±9 nl on water, 99±11 nl on saline and 101±7 nl on saline+sucrose. The type of recovery fluid had no significant effect on the mean hemolymph volume following the 24 h rehydration period (P<0.05). Flies that recovered on saline or saline+sucrose had a significantly higher hemolymph volume than when they were in their pre-desiccated state (P<0.05).
The D populations showed a different response following the recovery treatment. Hemolymph volumes were 185±44 nl, 177±41 nl and 273±47 nl for water, saline and saline+sucrose, respectively (Fig. 4). There were no statistically significant differences in hemolymph volume between the flies hydrated on water, saline or saline+sucrose (P>0.05). These volumes also did not differ from either pre- or post-desiccation hemolymph volumes (P>0.05), although when comparing hemolymph volumes of pre- and post-desiccation, there was a statistically significant difference (P<0.05).
Recovery (hemolymph osmolality)
Hemolymph osmolality following recovery was not dependent on the recovery
treatment. The hemolymph osmolalities in flies of the C populations were
298±19 mOsm, 334±18 mOsm and 329±4 mOsm after recovery on
water, saline and saline+sucrose, respectively
(Fig. 5). These values were not
statistically different from pre-desiccated hemolymph osmolalities
(P>0.05).
Following recovery, the osmolality of the hemolymph in the D populations was measured as 296±22 mOsm, 315±14 mOsm and 305±24 mOsm after rehydration on water, saline or saline+sucrose, respectively (Fig. 6). These values are not statistically distinguishable from the pre-desiccation values (P>0.05).
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Discussion |
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Hemolymph osmolality prior to desiccation
Insects exhibit a much larger variation in the concentration of the
extracellular fluid, both individually and in response to environmental
variation, than do vertebrates (Buck,
1953; Jeuniaux,
1971
; Bosquet,
1977
). It has been argued that the selectively permeable sheath
that protects the insect central nervous system permits a greater tolerance of
both osmotic and ionic variability in the hemolymph
(Treherne and Pichon, 1973
;
Jones, 1977
;
Ashhurst, 1985
).
In the present study, the control populations have a mean osmotic
concentration prior to desiccation of 353 mOsm. This value is considerably
higher than the single other measurement of 251±9 mosmol l-1
from individual D. melanogaster
(Singleton and Woodruff,
1994). Other values from adult dipterans include 400 mOsm for
blowflies (Phillips, 1969
) and
354 mOsm for the mosquito Aedes aegypti
(Williams et al., 1983
).
Interestingly, the osmotic concentration of the hemolymph of the control populations is higher than that of the D flies under the same conditions. The hemolymph osmolality in the O populations, the ancestors of both the C and D populations, is unknown. It is therefore unclear whether the C populations have experienced an increase in hemolymph concentration relative to their ancestor following reproductive isolation from the D populations or whether the D populations have evolved a lower hemolymph osmolality in response to selection for enhanced desiccation resistance.
Hemolymph osmolality during desiccation
As water is lost during desiccation, the hemolymph osmolality increases in
all populations tested. This increase in hemolymph osmolality is still well
below the theoretical osmolality values, which are expected in the absence of
osmoregulation, demonstrating a strong capacity for osmoregulation in D.
melanogaster. Other insects have a similar pattern of osmotic regulation,
including, for example, the orthopteran Carausius morosus
(Nicolson et al., 1974) and
the coleopterans Stips stali
(Naidu and Hattingh, 1986
) and
Onymacris plana (Nicolson,
1980
). Phillips
(1969
) found that the dipteran
Calliphora erythrocephala increased its hemolymph osmotic
concentration during dehydration by 25% after two days of water deprivation,
concomitantly increasing its urine concentration 15-fold. Wall
(1970
) reported a similar
trend in the cockroach Periplaneta americana during dehydration.
In examining populations that have undergone 250 generations of selection
for enhanced desiccation resistance, we found that their capacities for
osmotic regulation have been marginally improved in response to this selection
regime. This difference in osmotic regulatory capacity is not the
physiological trait that is considered the most important for enhanced
desiccation resistance, however. More important evolved physiological
differences between the C and D populations are a reduced rate of water loss
(Gibbs et al., 1997;
Williams et al., 1998
) and an
increase in water content (Gibbs et al.,
1997
; Folk and Bradley,
2003
).
The mechanistic details of osmoregulation in Drosophila are yet to
be worked out. Folk and Bradley
(2003) found that as the flies
lose water, both C and D populations excrete sodium, potassium and chloride.
The quality of these excreted solutes, as reported in their study, does not
account for all of the osmolytes removed from the hemolymph to maintain
hemolymph osmolalities as were measured during desiccation. Further studies
are required to determine what additional solutes are removed and to where
they are transferred. Of the organs engaged in osmoregulation in
Drosophila, only the Malpighian tubules have been examined in
mechanistic detail (Maddrell and
O'Donnell, 1992
; Dow et al.,
1994
; O'Donnell and Maddrell,
1995
; O'Donnell et al.,
1996
; Linton and O'Donnell,
1999
; O'Donnell and Spring,
2000
; Rheault and O'Donnell,
2001
). It would be valuable to determine the relative roles of the
osmoregulatory organs in Drosophila, particularly the rectum, which
Phillips (1969
) demonstrated
is the site of urine concentration in adult dipterans. The specific osmolytes
that are important in the various fluid compartments (intracellular fluid,
hemolymph, urine) are also unknown.
Recovery
The full selection regime of these fly populations involves not only
resistance to desiccation but also the capacity for recovery. We were
therefore interested in the capacity of the flies to resist desiccation and
their capacity for osmotic recovery. We therefore examined recovery on various
fluids.
Recovery in the control populations
Recovery on distilled water. Following an 8 h bout of desiccation,
the C populations lost almost 60% of their hemolymph volume yet did not
increase their hemolymph osmotic concentration significantly. During a 24 h
recovery period on distilled water, the C populations were able to increase
hemolymph volume to pre-desiccation values. Hemolymph osmolality after
rehydration on this fluid was also returned to pre-desiccation values. It
follows that the flies must have obtained osmolytes from the body compartment
in order to replace hemolymph volume at the appropriate osmotic concentration.
Folk and Bradley (2003)
examined the changes in ion content of the C populations under identical
conditions of desiccation. They found that the flies excrete some sodium
during desiccation but retain approximately 85% of whole body sodium content,
83% of potassium and 60% of chloride. A detailed study of the location of
these ions following desiccation and the degree to which they are mobilized
upon rehydration has yet to be carried out. Diptera normally have a fairly
sodium-rich hemolymph compared with other insects
(Sutcliffe, 1963
). It might be
expected that ion mobilization, particularly that of sodium, would be a major
aspect of hemolymph reconstitution during recovery in Drosophila. The
fat body in Periplaneta americana acts as a sink for sodium and
potassium ions from the hemolymph during dehydration
(Hyatt and Marshall, 1977
).
Upon rehydration on deionized water, these ions are removed from the fat body
and replaced in the hemolymph (Hyatt and
Marshall, 1985
). In Drosophila, however, a role for other
osmolytes, including amino acids, organic acids and peptides, in the
rehydration process cannot at this time be ruled out. Dipterans and other
species of insects have been shown to break down proteins into osmotically
active amino acids in response to perturbations in hemolymph osmotic
concentration (Collett,
1976a
,1976b
;
Woodring and Blakeney, 1980
).
Further studies will be required to determine hemolymph composition before and
after recovery in D. melanogaster as well as the source of hemolymph
osmolytes.
Recovery on a saline or a saline+sucrose solution. In the C flies,
rehydration on a saline solution isosmotic to the hemolymph resulted in an
increase in hemolymph volume following a decline during the 8 h desiccation
period. The restored hemolymph volume actually surpassed pre-desiccation
volume and was statistically indistinguishable from that of flies rehydrated
on distilled water. As in the flies that recovered on distilled water, the
hemolymph osmolality was returned to the original pre-desiccated values after
rehydration on the saline solution. It is clear that Drosophila can
fully rehydrate and maintain osmotic concentration using only saline without a
supplemental energy source. Hemolymph volume subsequent to a bout of
dehydration and recovery has been shown to increase beyond levels of
pre-stressed values in the orthopteran Chortoicetes terminifera
(Djajakusumah and Miles,
1966).
When the control populations were rehydrated on a saline+sucrose solution, hemolymph volume was higher than pre-desiccation values. Clearly, Drosophila can restore water lost from the hemolymph by the consumption of fluids of variable composition. Hemolymph osmolality was also restored after recovery on the saline+sucrose solution.
Recovery in the populations selected for enhanced desiccation resistance
Following a 24 h bout of desiccation, the D populations had lost on average
66% of their hemolymph volume and increased their hemolymph osmolality by
100 mOsm. When provided with any of the three recovery fluids, the D
flies were able to return to a hemolymph volume statistically
indistinguishable from the initial hemolymph volume. The final hemolymph
volumes achieved were intermediate to the pre-desiccation and post-desiccation
levels.
Although the flies did not fully recover lost hemolymph, they did manage to regain their original hemolymph osmolality after recovery on distilled water. Like the C populations, the D populations can replace substantial volumes of hemolymph at the appropriate osmolality while imbibing only distilled water, as well as by drinking isosmotic saline or saline+sucrose solutions. Clearly, their capacities for osmotic regulation of the hemolymph are substantial and the flies are capable of dealing with a variety of environmental conditions.
Osmoregulation in Drosophila
Drosophila face intermittent desiccation in their normal
environment. Competition for mates and searching for food and oviposition
sites inevitably lead the insects away from dietary sources of water. Their
exceptionally small surface area to volume ratio exacerbates water loss, with
rates of water loss of 2030 µl g-1 h-1 being
reported for control populations in the laboratory
(Gibbs et al., 1997;
Williams et al., 1998
) under
non-flying conditions. Lehmann
(2001
) measured the rate of
water loss in D. melanogaster during flight and found that, as
metabolic demand increased, spiracles must be open more frequently and the
rate of water loss increases accordingly (60140 µl g-1
h-1).
The present study was designed to determine the degree of osmotic regulation that occurs in Drosophila during the periods of water loss, as well as during the rehydration events that must occur when the insects again encounter a source of water. We found that Drosophila display surprisingly strict osmotic regulation under conditions of dehydration, being able to regulate osmotic concentration when over two-thirds of the hemolymph volume has been lost. Similarly, recovery of hemolymph volume can be achieved with a variety of recovery fluids, including distilled water. Neither external sources of sodium nor energy in the form of sugar are required. This implies that Drosophila could rehydrate in the wild using a variety of sources of water such as rainwater or dew, nectar and the fruit juices associated with their oviposition sites.
Hoffmann (1990) reported an
acclimation response in D. melanogaster when subjected to a
non-lethal dry environment. Subsequent to this temporary bout of desiccation,
the flies became more resistant to a further desiccation stress. We now know
that this species osmoregulates; therefore, it would be interesting to
determine the pattern of osmoregulation during the second bout of desiccation.
Potentially, this increase in desiccation resistance could result from a
higher tolerance to the elevation of hemolymph osmotic concentration (due
perhaps to the presence of heat shock proteins;
Lindquist, 1986
), a decrease
in cuticular water loss, an increase in body water during the recovery phase
or another physiological mechanism. When Hoffmann
(1991
) carried out these
experiments on various field-collected Drosophila species of
differing habitats, he found that the acclimation response was well
established in Drosophila with the exception of the species D.
birchii, which is found exclusively in tropical rainforests. It would be
interesting to measure physiological mechanisms behind this result and to
determine other osmoregulatory differences in this tropical species.
In the course of examining osmotic regulation in Drosophila, very interesting new questions have arisen. Of the populations of Drosophila examined in this study (both control and selected), all are able to replace hemolymph lost during desiccation using only distilled water while osmoregulating, suggesting that the flies can restore this hemolymph using internally stored or produced osmolytes. Clearly, this result deserves further study. The processes by which these two osmotic strategies, osmotic regulation and volume homeostasis, are maintained in Drosophila are of considerable interest, given the extensive physiological, molecular, genetic and now genomic techniques available for their investigation.
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Acknowledgments |
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