ORIGINAL PAPER
Divergence of water balance mechanisms in two sibling species(Drosophila simulans and D. melanogaster): effects of growthtemperatures
Ravi Parkash • Dau Dayal Aggarwal • Divya Singh •
Chanderkala Lambhod • Poonam Ranga
Received: 27 May 2012 / Revised: 16 September 2012 / Accepted: 23 September 2012 / Published online: 19 October 2012
� Springer-Verlag Berlin Heidelberg 2012
Abstract Drosophila simulans is more abundant under
colder and drier montane habitats in the western Himalayas
as compared to its sibling D. melanogaster but the mecha-
nistic bases of such climatic adaptations are largely
unknown. Previous studies have described D. simulans as a
desiccation sensitive species which is inconsistent with its
occurrence in temperate regions. We tested the hypothesis
whether developmental plasticity of cuticular traits confers
adaptive changes in water balance-related traits in the sibling
species D. simulans and D. melanogaster. Our results are
interesting in several respects. First, D. simulans grown at
15 �C possesses a high level of desiccation resistance in
larvae (*39 h) and in adults (*86 h) whereas the corre-
sponding values are quite low at 25 �C (larvae *7 h; adults
*13 h). Interestingly, cuticular lipid mass was threefold
higher in D. simulans grown at 15 �C as compared with
25 �C while there was no change in cuticular lipid mass in
D. melanogaster. Second, developmental plasticity of body
melanisation was evident in both species. Drosophila sim-
ulans showed higher melanisation at 15 �C as compared with
D. melanogaster while the reverse trend was observed at
25 �C. Third, changes in water balance-related traits (bulk
water, hemolymph and dehydration tolerance) showed
superiority of D. simulans at 15 �C but of D. melanogaster at
25 �C growth temperature. Rate of carbohydrate utilization
under desiccation stress did not differ at 15 �C in both the
species. Fourth, effects of developmental plasticity on
cuticular traits correspond with changes in the cuticular
water loss i.e. water loss rates were higher at 25 �C as
compared with 15 �C. Thus, D. simulans grown under cooler
temperature was more desiccation tolerant than D. melano-
gaster. Finally, desiccation acclimation capacity of larvae
and adults is higher for D. simulans reared at 15 �C but quite
low at 25 �C. Thus, D. simulans and D. melanogaster have
evolved different strategies of water conservation consistent
with their adaptations to dry and wet habitats in the western
Himalayas. Our results suggest that D. simulans from low-
land localities seems vulnerable due to limited acclimation
potential in the context of global climatic change in the
western Himalayas. Finally, this is the first report on higher
desiccation resistance of D. simulans due to developmental
plasticity of both the cuticular traits (body melanisation and
epicuticular lipid mass) when grown at 15 �C, which is
consistent with its abundance in temperate regions.
Keywords Developmental plasticity � Water balance-
related traits � Cuticular lipid mass � Body melanisation �D. simulans � D. melanogaster
Abbreviations
D Desiccation resistant strains
I. F. Isofemale
J/mg Joules/mg
RWL Rate of water loss
Introduction
The sibling species Drosophila simulans and D. melano-
gaster are able to proliferate in temperate as well as trop-
ical regions but different climatic factors are likely to affect
Communicated by G. Heldmaier.
R. Parkash (&) � D. D. Aggarwal � D. Singh � C. Lambhod �P. Ranga
Department of Genetics, Maharshi Dayanand University,
Type IV/35, M.D.U., Campus, Rohtak 124001, India
e-mail: [email protected]
D. D. Aggarwal
e-mail: [email protected]
123
J Comp Physiol B (2013) 183:359–378
DOI 10.1007/s00360-012-0714-3
their distribution and abundance (Parsons 1975, 1983;
Hoffmann and Harshman 1999). Comparative analyses of
various quantitative traits have demonstrated lesser genetic
variability in D. simulans than D. melanogaster reared at
25 �C (Parsons 1983; Hoffmann and Parsons 1991; Powell
1997). Some studies on Australian populations have shown
higher desiccation sensitivity of D. simulans as compared
with its sibling species D. melanogaster (Parsons and
Stanley 1981; Hoffmann and Parsons 1991). Further, lab-
oratory selection experiments have shown lower genetic
response in D. simulans as compared with D. melanogaster
(Hoffmann and Parsons 1991, 1993a). In D. simulans,
mean (LT50) difference between desiccation resistant ver-
sus control lines is 3 h, but 10 h in case of D. melanogaster
(Hoffmann and Parsons 1993a). In spite of lower desicca-
tion potential of D. simulans, it occurs more abundantly
under drier habitats in temperate regions (Parsons 1983).
Thus, D. simulans might employ phenotypic plasticity as a
strategy to cope with colder and drier conditions in tem-
perate regions but this aspect has not been tested so far.
For Drosophila species and populations, desiccation
resistance has been mainly assessed in adult flies (Gibbs
and Matzkin 2001; Gibbs et al. 2003). The larval stages of
Drosophila species encounter desiccation stress directly
under field conditions but larval desiccation resistance and
larval water conservation strategies remain largely
unknown. A single study has shown that laboratory selec-
tion did not alter resistance to desiccation in the larvae of
D. melanogaster despite changes in adult flies (Hoffmann
and Parsons 1993b). However, associations between adult
and larval resistance to desiccation stress have not been
explored in wild populations of different Drosophila spe-
cies. Thus, it would be interesting to examine whether
evolved differences for adult desiccation resistance are
consistent at the larval stage of the sibling species D.
simulans and D. melanogaster.
Water conservation is critical to the ecological success
of diverse Drosophila species (Hadley 1994; Gibbs 2002;
Chown and Nicolson 2004). Survival under desiccation
conditions can be achieved by decreasing the rate of water
loss or by storing more amount of water or through toler-
ating greater loss of water before succumbing to death
(Hadley 1994; Gibbs et al. 1997; Gibbs 1999, 2002). Most
comparative studies have measured desiccation resistance
levels in various Drosophila species from temperate as
well as tropical parts of the world (Gibbs and Matzkin
2001; Gibbs et al. 2003; Matzkin et al. 2009). However,
detailed analysis of water budget has been investigated
mainly in laboratory selected desiccation resistant and
control lines of D. melanogaster (Gibbs et al. 1997; Folk
et al. 2001; Folk and Bradley 2005) but lesser attention has
been paid to D. simulans (Hoffmann and Parsons 1993b).
Further, water balance-related traits have been investigated
in Indian populations of D. melanogaster reared at 21 �C,
but not for D. simulans (Parkash et al. 2010). Thus, the
evolved physiological mechanisms for water balance in
larvae and adults of D. simulans are largely unknown.
For ectothermic insects, more than 80 percent of body
water loss occurs through the cuticle under desiccation
stress (Hadley 1994; Gibbs 2002). In diverse insect taxa,
changes in the composition or amount of cuticular lipids
significantly affect cuticular permeability (Edney 1977;
Toolson 1984; Hadley 1994; Rourke 2000). For example,
quantitative changes in epicuticular lipids are negatively
correlated with cuticular permeability in scorpions and
tenebrionid beetles (Hadley 1977; Toolson and Hadley
1979; Hadley and Schultz 1987); grasshopper—Melano-
plus sanguinipes (Rourke 2000); and drosophilid—Zapri-
onus indianus (Parkash et al. 2008). In contrast, laboratory
selected desiccation resistant lines (Gibbs et al. 1997) as
well as latitudinal populations of D. melanogaster (Parkash
et al. 2010) have shown no changes in the cuticular lipid
amount. Therefore, it is not clear whether changes in the
cuticular lipid mass form the physiological basis for des-
iccation resistance of D. simulans and D. melanogaster
when grown at different growth temperatures.
The association between cuticular permeability and
quantity of cuticular lipids has been demonstrated through
treatment of cuticular surfaces in dead insects with organic
solvents such as hexane or chloroform: methanol (Hadley
1989; Hadley and Quinlan 1989). For example, hexane-
treated cuticle of the cricket—Acheta domesticus showed
11-fold increase in cuticular permeability (Hadley 1989)
while there is 190-fold increase due to chloroform:metha-
nol (2:1) treatment of the cuticle of the spider—Latro-
dectus hesperus (Hadley and Quinlan 1989). In contrast,
the effects of organic solvents on cuticular permeability in
different Drosophila species have received lesser attention
so far. Further, changes in the body melanisation affect
cuticular transpiration i.e. darker flies of several Dro-
sophila species have shown relatively lower rate of water
loss as compared to flies with lighter body color (Rajp-
urohit et al. 2008; Parkash et al. 2010). In contrast, analysis
of water balance mechanisms in desert Drosophila species
has shown lack of changes in epicuticular lipid mass for
reduced body water loss as compared with mesic species
(Gibbs et al. 2003). Thus, it would be interesting to
examine the role of cuticular components (cuticular lipid
mass and/or body melanisation) in water balance mecha-
nisms which might vary between D. simulans and D. mel-
anogaster grown at different growth temperatures.
There is evidence of acquisition of carbohydrates as
energy reserves to alleviate the consequences of desicca-
tion stress in laboratory selected desiccation resistant
strains of D. melanogaster (Graves et al. 1992; Gibbs et al.
1997; Chippindale et al. 1998; Djawdan et al. 1998; Folk
360 J Comp Physiol B (2013) 183:359–378
123
et al. 2001; Folk and Bradley 2005). In contrast, higher
percentage of body lipid content has conferred greater
survival under desiccation stress in a new set of laboratory
selected desiccation resistant lines (Telonis-Scott et al.
2006). Similarly, large-sized insects (locusts and tsetse
flies) as well as small-sized mosquitoes store and use lipids
under dehydration stress (Loveridge and Bursell 1975;
Nicolson 1980; Benoit et al. 2010). Further, a single study
has investigated storage and utilization of energy metabo-
lites under desiccation stress in five Drosophila species but
this study did not include D. simulans (Marron et al. 2003).
It is likely that sibling species D. melanogaster and
D. simulans might store and utilize similar or varying
levels of energy metabolites to cope with desiccation stress
under wild conditions.
Several studies have shown beneficial effects of thermal
acclimation (Hoffmann and Watson 1993; Bale 2002;
Hoffmann et al. 2003). Similarly, changes in longevity
under dehydration stress have also been reported due to
dehydration acclimation in arctic collembolan Onychiurus
arcticus (Holmstrup and Sømme 1998), in Folsomia can-
dida (Holmstrup et al. 2002), and for Cryptopygus ant-
arcticus (Elnitsky et al. 2008). However, the consequences
of desiccation acclimation for Drosophila species have
received lesser attention so far (Hoffmann 1990, 1991;
Bubliy et al. 2012). Further, only a single study has
reported the physiological basis of dehydration acclimation
in D. melanogaster (Bazinet et al. 2010). It has been sug-
gested that desiccation acclimation success depends upon
the natural habitats for Drosophila species (Hoffmann
1991). Thus, it would be interesting to compare the evolved
physiological mechanisms of dehydration acclimation for
sympatric populations of Drosophila species.
Drosophila simulans and D. melanogaster are cosmo-
politan species but their relative abundance varies in dif-
ferent localities on various continents. For example,
Australian populations of D. simulans are more abundant in
warmer places than D. melanogaster (Parsons 1975).
Likewise, D. simulans occurs more frequently in localities
around the Mediterranean Sea and also in the tropical
America (Powell 1997). In contrast, D. melanogaster is
often the dominant species in the West Africa with climatic
variation similar to tropical America (Tsacas 1980). Thus,
different climatic conditions might affect relative abun-
dance of these two sibling species. On the Indian subcon-
tinent, D. simulans and D. melanogaster occur in montane
localities of western Himalayas, but ecophysiological traits
of D. simulans for adaptations to diverse climatic condition
have not been investigated so far.
In the present study, we examined relative abundance of
D. simulans and D. melanogaster as a function of changes
in relative humidity along an altitudinal gradient in the
western Himalayas. We analyzed one highland population
(Fagu) of sibling species D. simulans and D. melanogaster
for desiccation-related traits. We investigated effects of
developmental phenotypic plasticity (15 vs. 25 �C growth
temperatures) on desiccation-related traits as well as
energy metabolites in the larvae as well as adults of
D. simulans and D. melanogaster. Further, we assessed
utilization of energy metabolites under desiccating condi-
tions in the sibling species reared at 15 and 25 �C. Finally,
we compared acclimation effects to low humidity for both
larval and adult stages of D. simulans and D. melanogaster.
Materials and methods
Collections and cultures
Sympatric populations of D. simulans and D. melanogaster
(n = 150–300) flies from each site) were collected in a
single trip in October, 2010 from 6 altitudinal localities of
the western Himalayas (Fig. 1). Percent species abundance
was estimated as the number of individuals of a particular
Drosophila species divided by the total number of indi-
viduals of all the different Drosophila species in the
samples collected from a given locality. Wild-caught
individuals of a highland locality (Fagu) were used to
initiate 20 isofemale lines (geographical variables: altitude
2,500 m, latitude 31�450N, longitude 77�010E, climatic
variables: Tmin 9.3, Tmax 17.9, Tave 13.6 8C, RH 42.9 %).
Cultures were maintained at low density (60–70 eggs per
vial of 40 9 100 mm size) on cornmeal–yeast–agar med-
ium at 15 and 25 �C. The identification of sibling species
was made on the basis of taxonomic differences in the
Relative humidity (%)
Perc
ent a
bund
ance
0
20
40
60
80
100
36 42 48 54 60 66
D. melanogaster
y = 70.65 - 0.80*x
D. simulans
y = 129.95 - 1.93*x
Fig. 1 Regression analysis of percent abundance as a function of
relative humidity (RH %) of the origin of six altitudinal populations
of D. simulans and D. melanogaster. Populations include (altitude;
RH %): (1) Chamba (996 m; 63.5 %); (2) Dharamshala: (1,219 m,
58.2 %); (3) Barog (1,680 m, 54.9 %); (4) Dalhousie: (1,959 m,
50.3 %); (5) Mashobra (2,236 m, 44.6 %); (6) Fagu (2,500 m,
40.3 %)
J Comp Physiol B (2013) 183:359–378 361
123
length of egg filaments and male genitalia (Ashburner
1989) and body melanisation (Eisses and Santos 1998). All
assays were performed on late 3rd instar larvae as well as
adult females (6-day post eclosion) grown at 15 and 25 �C.
We did not find induced diapause effects (i.e. degenerated
ovaries) in wild-caught flies as well as flies of D. simulans
and D. melanogaster grown at 15 �C. Climatic data for
thermal and humidity variables were obtained from
‘Climatological Tables’ published by the Indian Meteoro-
logical Department, Govt. of India, New Delhi (2010).
Isolation of third instar larvae
Five-day old single time mated females were allowed to lay
eggs in culture vials (37 9 100 mm) for 2 h on cornmeal–
yeast–agar medium at 15 and 25 8C. The vials were con-
tinuously inspected after 1-h interval. Sixty eggs were
collected with the aid of a camel hair brush and placed on a
black paper strip which was moistened with water. This
strip was finally placed in the food vials (37 9 100 mm).
We removed the strip having unhatched eggs after 12 h at
25 �C and after 24 h at 15 �C. Further, third instar larvae
were isolated from such food vials and sexed according to
gonad morphology (Ashburner 1989; Demerec and Kauf-
man 1996). We examined energy metabolites and water
balance-related traits in late third instar larvae of both the
species. Since, duration of development varied between
15 and 25 �C in both the species, we used 3rd instar larva
after 275 ± 2.03 h of egg laying at 15 �C, but after
160 ± 1.85 h of egg laying at 25 �C. However, between
species differences were nonsignificant for D. simulans and
D. melanogaster reared at 15 as well as 25 �C.
Trait analysis
We used 10 individuals of each replicate (10 replicates 9 20
isofemale lines each) of D. simulans as well as D. melano-
gaster to quantify body melanisation, epicuticular lipid mass,
desiccation resistance, multiple measures of water balance,
and levels of energy metabolites. However, multiple repli-
cates (*50) of each isofemale line were run simultaneously to
estimate the effects of organic solvents on cuticular perme-
ability as well as time series changes in the levels of energy
metabolite as a function of different durations of desiccation
stress. Further, for body melanisation, epicuticular lipid mass,
desiccation resistance, and multiple measures of water bal-
ance, and total body lipid content, we used individual larvae/
adult flies, and a group of 10 individuals were examined to
analyze the storage levels of energy reserves. For flies grown
at 15 and 25 �C, we tested desiccation-related traits at their
respective growth temperature i.e. at 15 and 25 �C, respec-
tively. Therefore, growth temperature and experimental
temperature were same in our experimental setup. For
analysis, we used mean of ten replicates for each isofemale
line of D. simulans as well as D. melanogaster.
Analysis of body melanisation
The progeny of each isofemale line was examined for dif-
ferences in body melanisation patterns on the abdominal
segments. The main difference between the sibling species
was the shape and width of melanisation band on the pos-
terior end of each abdominal segment i.e. triangular band
with small lateral terminal gaps in D. simulans, but smooth
stripes of pigmentation in D. melanogaster. For both the
species, body melanisation of individual female flies
(n = 20 I. F. lines 9 10 replicates per isofemale line) was
visually scored with Olympus stereo-zoom microscope
SZ-61 (http://www.olympus.com). It was estimated from
dorsal as well as lateral views of the female abdomen giving
values ranging from 0 (no melanisation) to 10 (complete
melanisation) for each of the 6 abdominal segments
(2nd–7th). Further, the relative size of each abdominal seg-
ment was calculated in relation to the largest 4th abdominal
segment which was assigned the value of 1.0. Since the
abdominal segments differ in size, these relative sizes (i.e.
0.86, 0.94, 1.0, 0.88, 0.67 and 0.38 for 2nd–7th segments,
respectively) were multiplied with segment-wise melanisa-
tion scores. Data on percent melanisation were calculated as
(R observed weighted melanisation scores of abdominal
segments per fly/R relative size of each abdominal seg-
ment 9 10 per fly) 9 100 (Parkash et al. 2008).
Assessment of epicuticular lipid mass
We assessed cuticular lipid mass in individual larva (20 I. F.
lines 9 10 replicates each) of both the species reared at 15
and 25 �C. Each larva was dried overnight at 60 �C to get dry
mass i.e. devoid of body water. Each dried larva was kept in
HPLC-grade hexane in 2 ml Eppendorf tube (http://www.
tarsons.in) for 3 min and thereafter, it was removed from the
solvent and was again dried at room temperature and finally
reweighed on a sartorius microbalance (Model-CPA26P;
0.001 mg precision; http://www.sartorious.com). We fol-
lowed similar steps for the determination of cuticular lipid
mass in individual adult flies. Cuticular lipid mass per cm2
was calculated as the difference in mass following solute
extraction divided by surface area (cm2).
Desiccation resistance
Desiccation resistance was measured as the time to lethal
dehydration effect under dry air. Third instar larvae were
separated out and placed individually in dry plastic vials
(40 9 100 mm) in which open end was covered with
muslin cloth. These vials were kept on top of another vial
362 J Comp Physiol B (2013) 183:359–378
123
containing 2 g of silica gel at the bottom. Finally, this
apparatus was made airtight with parafilm and kept in the
desiccator chamber (Secador electronic desiccator cabinet;
http://www.tarsons.in) which maintained 0–5 % relative
humidity. In the similar way, adult desiccation resistance
was measured. Number of immobile larvae or flies was
counted after every 1-h interval, and LT100 values in dry air
were recorded.
Basic measures of water balance
In order to estimate total body water content and dehy-
dration tolerance (%), 10 larvae/flies of each isofemale line
(20 I. F. lines 9 10 replicates each) were used. First,
individual flies were weighed on Sartorius microbalance
(Model-CPA26P; 0.001 mg precision) and then reweighed
after drying overnight at 60 �C. Total body water content
was estimated as the difference between mass before and
after drying at 60 �C. Further, after mild anesthesia (1 min)
with solvent ether, flies were weighed on a Sartorius
microbalance both before and after desiccation stress until
death. Dehydration tolerance was estimated as the per-
centage of total body water lost until death due to desic-
cation; and was calculated by the formula (wet body
mass - body mass at death)/(wet body mass - dry body
mass) 9 100 (Gibbs et al. 1997).
For calculation of the rate of water loss, we followed
Wharton’s method (1985). Total body water content
(m) was calculated as the difference between wet or fresh
(f) and dry mass (d) i.e. m = f - d. Individual larvae/flies
were weighed and placed at 0–5 % relative humidity for a
specified time at 1-h interval (1–8 h), and reweighed. The
rate of water loss was derived from the slope of regression
line on a plot of 1n(mt/m0) against time according to
Wharton’s exponential equation (Wharton 1985)
mt = m0e-kt, where mt is the water mass at time t, and m0 is
the initial water content. Rate (kt) is the slope of the
regression line and was expressed as % per hour.
Effect of organic solvent on cuticular water loss
Changes in cuticular permeability due to organic solvent
were tested on larvae as well as adult flies (20 I. F.
lines 9 50 replicates). The assays were conducted by
treating over-etherized (dead) larvae with 5 ml of hexane
and then were gently vortexed 5 times each for 30 s. Lar-
vae were then blotted dry on tissue paper, weighed and
placed in a desiccator chamber (Secador electronic desic-
cator cabinet; http://www.tarsons.in) which maintained
0–5 % relative humidity. The effect of hexane on rate of
water loss was monitored at 30-min interval for D. simu-
lans reared at 15 and 25 �C. However, changes in water
loss due to hexane treatment were recorded at 2-h interval
(due to lack of any effect) in D. melanogaster. For control
groups, no solvent treatment was given and cuticular water
loss was determined after every 1-h interval. We applied
similar methods to determine the effects of hexane on adult
flies of D. simulans and D. melanogaster.
Assessment of extractable hemolymph content in larvae
and adults
Individual larva was placed on a paper towel and cleaned
with distilled water followed by air drying for 2 min. The
dry larva was carefully pinned to a microdissection dish at
its anterior and posterior ends with microdissection pins,
and a narrow incision was made through the cuticle with a
third pin while observing through a stereo-zoom micro-
scope (SZ-61; http://www.olympus.com). The leaking
extractable hemolymph was absorbed with an absorbent
tissue moistened with an isotonic saline solution (Folk
et al. 2001). Hemolymph content was estimated as reduc-
tion in mass following hemolymph blotting (Cohen et al.
1986; Hadley 1994). We followed similar steps for esti-
mation of extractable hemolymph content in adult flies.
Tissue water was estimated after subtracting exsanguinated
mass before and after drying. From same data, we also
calculated hemolymph water content by subtracting tissue
water from total body water content.
Assessment of desiccation acclimation responses
To measure pre-treatment duration, 10 larvae/adult indi-
viduals of each replicate (20 I. F. lines 9 10 replicates
each) were subjected to desiccation stress at *0–5 %
relative humidity. The initial body water content in each
replicate group was recorded. The time period in which
flies lost *15–17 % body water was considered as the pre-
treatment time duration. Further, for the recovery period,
individuals were placed on laboratory food till the original
mass was regained. Such individuals were subjected to
desiccation stress until death in order to test the increased
desiccation resistance due to acclimation. Thus, absolute
acclimation capacity (increased desiccation survival hours)
was calculated by subtracting the desiccation resistance
(h) of non-acclimated (control) from desiccation resistance
(h) of acclimated individuals. Control and treatment
experiments were run simultaneously under identical
experimental conditions.
Analysis of body lipid content
Individual larvae or adult flies were dried in 2 ml Eppendorf
tubes (http://www.tarsons.in) at 60 �C for 48 h and then
weighed on Sartorius microbalance (Model-CPA26P;
0.001 mg precision; http://www.sartorious.com). Thereafter,
J Comp Physiol B (2013) 183:359–378 363
123
1.5 ml di-ethyl ether was added in each Eppendorf tube and
kept for 24 h under continuous shaking (200 rpm) at 37 �C.
Finally, the solvent was removed and individuals were again
dried at 60 �C for 24 h and reweighed. Lipid content was
calculated per individual by subtracting the lipid-free dry
mass from initial dry mass per larva/fly.
Estimation of trehalose and glycogen
For trehalose and glycogen content estimation, 10 larvae or
adult flies of each isofemale line were homogenized in a
homogenizer (Labsonic@ M; http://www.sartorious.com)
with 300 ll Na2Co3 and incubated at 95 8C for 2 h to
denature proteins. An aqueous solution of 150 ll acetic
acid (1 M) and 600 ll sodium acetate (0.2 M) was mixed
with the homogenate. Thereafter, the homogenate was
centrifuged (Fresco 21, Thermo-Fisher Scientific, Pitts-
burgh, USA) at 12,000 rpm. (9,6609g) for 10 min. This
homogenate was used for independent estimations of tre-
halose and glycogen as given below.
For trehalose estimation, aliquots (200 ll) were placed
in two different tubes; one was taken as a blank whereas
the other was digested with trehalase at 37 �C using the
Megazyme trehalose assay kit (K-Treh 10/10, http://www.
megazyme.com). In this assay, released D-glucose was
phosphorylated by hexokinase and ATP to glucose-6-
phosphate and ADP, which was further coupled with
glucose-6-phosphate dehydrogenase and resulted in the
reduction of nicotinamide adenine dinucleotide (NAD).
The absorbance by NADH was measured at 340 nm (UV-
2450-VIS, Shimadzu Scientific Instruments, Columbia,
USA).The pre-existing glucose level in the sample was
determined in a control reaction lacking trehalase and
subtracted from total glucose concentration.
For estimation of glycogen, a 50 ll aliquot was incu-
bated with 500 ll Aspergillus niger glucoamylase solution
(8.7 U/ml in 200 mM of acetate buffer) for 2 h at 40 �C
with constant agitation and the suspension was centrifuged
at 4,000 rpm. (1,0739g) for 5 min. It mainly hydrolyzed
alpha-(1,4) and alpha-(1,6) glycosyl linkages and was
suited for the breakdown of glycogen. Glucose concentra-
tion was determined with 20 ll of supernatant from the
suspension and added with 170 ll of a mixture of G6-DPH
(0.9 U/ml); ATP (1.6 mM); and NADP (1.25 mM) in tri-
ethanolamine hydrochloride buffer (380 mM TEA–HCl
and 5.5 mM of MgSO4) and 10 ll of hexokinase solution
(32.5 U/ml in 3.2 M ammonium sulfate buffer), and
absorbance was measured at 340 nm.
Protein assay
Protein levels were determined using the bicinchoninic
acid method as followed by Gibbs and coworkers (Marron
et al. 2003). For protein assay, 10 female flies per isofe-
male line (n = 10 replicates 9 20 I. F. lines of each spe-
cies) were homogenized in 3 ml distilled water and
centrifuged at 10,000 rpm for 5 min. Further, 50 ll of
aliquot was taken from supernatant and treated with
2 ml of Sigma BCA reagent and incubated at 25 �C for
12 h. Absorbance was recorded at 562 nm and protein
concentration was determined by comparing with standard
curve.
Utilization of energy metabolites
We measured each energy metabolite (carbohydrates, body
lipids or proteins) in multiple replicate sets of isofemale
lines (20 I. F. lines 9 10 replicates each) before and after
its utilization under desiccation stress until death in
D. simulans and D. melanogaster reared at 15 and 25 �C.
Larvae/flies were subjected to different durations of des-
iccation stress (at 5-h interval). Further, rate of utilization
of each metabolite was calculated as the regression slope
value as a function of desiccation stress duration (Marron
et al. 2003).
Statistical analyses
For each trait, mean values (±SE; 20 isofemale lines,
10 replicates each) were used for illustrations and tables.
Effects of developmental temperatures (15 vs. 25 �C) on
desiccation-related traits, energy metabolites, body
weight, basic measures of water balance and dehydration
tolerance were compared with mixed model ANOVA
(temperature: fixed effect; isofemale line: random effect).
However, we used partly crossed and partly nested
ANOVA (isofemale lines nested into species) to assess
% variance due to species, growth temperature, isofemale
lines and their interaction effects for desiccation resis-
tance, cuticular lipid mass and carbohydrate contents.
Pearson’s correlation coefficients were calculated on the
basis of isofemale line data (10 I. F. lines 9 10 replicates
each). To assess the changes in levels of energy metab-
olites as a function of different durations of desiccation
stress i.e. rate of metabolite utilization, we followed
Gibbs and coworkers (Marron et al. 2003). Further, the
slope values of differential utilization of energy metabo-
lites at 15 and 25 �C in D. simulans as well as D. mel-
anogaster were compared with t test (Zar 1999). For
multiple comparisons, alpha value was adjusted with
Bonferoni corrections. Energy contents due to carbohy-
drates, lipids, and proteins of larval as well as adults were
calculated using standard conversion factors (Schmidt-
Nielsen 1990; Marron et al. 2003). Statistica (Statsoft
Inc., Release 5.0, Tulsa, OK, USA) was used for calcu-
lations as well as illustrations.
364 J Comp Physiol B (2013) 183:359–378
123
Results
Data on percent abundance of wild-caught flies of D. sim-
ulans and D. melanogaster from 6 altitudinal localities
(996–2,500 m) as a function of relative humidity of origin
of populations are shown in Fig. 1. Drosophila simulans is
more abundant (*48 %) in highland localities but occurs
less frequently in lowland localities (*10 %). In contrast,
D. melanogaster is more abundant in lowland localities
(*20 %) than D. simulans (Fig. 1). The highland locali-
ties are moderately colder and drier (Tave = 13.5 �C;
RH = 40.3 %) while lowland localities are warm and less
desiccating (Tave = 25.6 �C; RH = 62.2 %). Therefore,
significant reduction in Tave. (*1.6 8C per 200 m) as well
as relative humidity (*3 % per 200 m) along an eleva-
tional gradient may act as selection factors for affecting
species relative abundance. Thus, D. simulans is better
adapted under colder and drier conditions in highland
localities as compared with its sibling species
D. melanogaster.
Comparison of plastic effects for desiccation-related
traits in sibling species
Changes at the larval stage
Data on intra-specific differences in desiccation resistance
and energy metabolites due to growth temperatures i.e.
(15 and 25 �C) in larval as well as in adult stage of
D. simulans and D. melanogaster are shown in Table 1. In
D. simulans, we observed *sixfold higher desiccation
resistance at 15 �C as compared to 25 �C (15 �C =
38.58 ± 0.73 h; 25 �C = 6.49 ± 0.25 h; F1,19 = 247.58;
P \ 0.001; Table 1). However, corresponding differences
were only 2.5-fold in case of D. melanogaster (15 �C =
30.11 ± 0.54 h; 25 �C = 11.54 ± 0.28 h; F1,19 = 208.58;
P \ 0.001). Further, epicuticular lipid mass also showed
plastic responses due to thermal variables i.e. in D. simu-
lans, there is *threefold increase at 15 �C as compared
with 25 �C (15 �C = 28.50 ± 0.41 lg cm-2; 25 �C =
10.62 ± 0.34 lg cm -2; F1,19 = 109.81; P \0.001; Table 1)
but such changes in epicuticular lipid mass were statistically
nonsignificant in D. melanogaster (F1,19 = 0.63 ns). The dry
mass specific levels of trehalose content increased *2.3-fold
in D. simulans at lower temperature (15 �C = 0.236 ±
0.003 mg mg-1 dry mass; 25 �C = 0.104 ± 0.002 mg
mg-1 dry mass; F1, 19 = 395.22; P \ 0.001) but such dif-
ferences were *1.4-fold in D. melanogaster (15 �C =
0.183 ± 0.004 mg mg-1 dry mass; 25 �C = 0.133 ±
0.002 mg mg-1 dry mass; F1,19 = 211.05; P \ 0.001;
Table 1). However, in both the sibling Drosophila species,
we did not find significant changes in the levels of dry mass
specific glycogen content due to differences in growth
temperatures (D. simulans: F1,19 = 0.69 ns; D. melano-
gaster: F1,19 = 2.39 ns). Further, protein content also
showed lack of plastic responses in D. simulans as well
as in D. melanogaster (D. simulans: F1,19 = 1.93 ns;
D. melanogaster: F1,19 = 1.03 ns; Table 1). In contrast,
lipid content was higher at 25 �C growth temperature as
compared with 15 �C in larvae as well as adults of both the
sibling species (Table 1).
Changes at the adult stage
We observed similar trends but different mean trait values
for adult flies as compared with larvae in D. simulans as
well as in D. melanogaster (Table 1). For adult flies, des-
iccation resistance increased *6.7-fold in D. simulans, but
*threefold in D. melanogaster at 15 �C growth tempera-
ture as compared with 25 �C (Table 1). In D. simulans,
body melanisation and cuticular lipid mass increased
*4.30-fold and *2.8-fold, respectively, due to thermal
plastic effects (melanisation: 15 �C = 86.37 ± 2.01 %;
25 �C = 20.06 ± 0.51 %; F1,19 = 501.86; P \ 0.001;
epicuticular lipids: 15 �C = 26.88 ± 0.37 lg cm-2;
25 �C = 10.45 ± 0.27 lg cm-2; F1,19 = 468.59; P \0.001). However, there was only *twofold increase in
body melanisation in D. melanogaster (melanisation:
15 �C = 70.08 ± 1.60 %; 25 �C = 34.19 ± 0.56 %;
F1,19 = 356.17; P \ 0.001), but no significant changes
in the cuticular lipid mass (15 �C = 13.62 ± 0.23 h;
25 �C = 13.24 ± 0.29 h; F1,19 = 1.52 ns; Table 1). Fur-
ther, trehalose content was significantly higher (2.3-fold) at
15 �C (0.221 ± 0.004 mg mg-1 dry mass) than at 25 �C
(0.099 ± 0.002 mg mg-1 dry mass) in D. simulans
(F1,19 = 195.43; P \ 0.001) but only 1.4-fold in D. mel-
anogaster (15 �C = 0.174 ± 0.004 mg mg-1 dry mass;
25 �C = 0.126 ± 0.003 mg mg-1 dry mass; F1,19 =
157.89; P \ 0.001; Table 1). In contrast, our results did not
evidence significant changes in the levels of glycogen
(D. simulans: F1,19 = 3.58 ns; D. melanogaster: F1,19 =
0.39 ns) and protein content (D. simulans: F1,19 = 1.45 ns;
D. melanogaster: F1, 19 = 0.96 ns; Table 1). Thus, plastic
responses for larval as well as adult desiccation-related
traits differ between these two sibling Drosophila species.
Analysis of trait variability
We used partly crossed and partly nested ANOVA model
for partitioning %variance in three desiccation-related
traits (desiccation resistance, cuticular lipid mass and total
carbohydrate content) in 20 isofemale lines (20 I. F.
lines 9 10 replicates) of 2 sibling species (D. simulans and
D. melanogaster) grown at 15 and 25 �C (Table 2). Inter-
J Comp Physiol B (2013) 183:359–378 365
123
Ta
ble
1D
ata
(mea
n±
SE
)o
nec
op
hy
sio
log
ical
trai
ts—
des
icca
tio
nre
sist
ance
ho
urs
,cu
ticu
lar
lip
idm
ass;
and
dry
mas
ssp
ecifi
cen
erg
ym
etab
oli
tes—
treh
alo
se,
gly
cog
en,
lip
ids
and
pro
tein
con
ten
tin
a3
rdin
star
larv
aan
db
adu
ltfl
ies
(n=
20
I.F
.li
nes
91
0re
pli
cate
s)o
fD
.si
mu
lan
san
dD
.m
ela
no
ga
ster
gro
wn
at1
5�C
asw
ell
as2
5�C
(pla
stic
effe
cts)
.T
rait
val
ues
for
each
spec
ies
gro
wn
at1
5an
d2
5�C
wer
eco
mp
ared
asra
tio
(fo
ld-d
iffe
ren
ces)
and
wit
hm
ixed
mo
del
AN
OV
A(F
val
ues
)
Tra
its
D.
sim
ula
ns
D.
mel
an
og
ast
er
15
�C2
5�C
Rat
ioF
1,1
91
5�C
25
�CR
atio
F1,1
9
(a)
Th
ird
inst
ar
larv
a
1.
Des
icca
tio
nh
ou
rs3
8.5
8±
0.7
36
.49
±0
.25
5.9
42
47
.58
**
*3
0.1
1±
0.5
41
1.5
4±
0.2
82
.60
20
8.5
8*
**
2.
Ep
icu
ticu
lar
lip
ids
(lg
cm-
2)
28
.50
±0
.41
10
.62
±0
.34
2.6
81
09
.81
**
*1
4.0
6±
0.3
91
3.9
7±
0.3
11
.01
0.6
3n
s
3.
Tre
hal
ose
(mg
mg
-1d
rym
ass)
0.2
36
±0
.00
30
.10
4±
0.0
02
2.2
73
95
.22
**
*0
.18
3±
0.0
04
0.1
33
±0
.00
21
.37
21
1.0
5*
**
4.
Gly
cog
en(m
gm
g-
1d
rym
ass)
0.0
70
±0
.00
20
.06
8±
0.0
02
1.0
20
.69
ns
0.0
74
±0
.00
30
.07
1±
0.0
02
1.0
42
.39
ns
5.
Lip
ids
(mg
mg
-1d
rym
ass)
0.1
77
±0
.00
30
.28
3±
0.0
04
1.5
91
86
.23
**
*0
.18
6±
0.0
02
0.2
25
±0
.00
51
.20
15
4.2
7*
**
6.
Pro
tein
s(m
gm
g-
1d
rym
ass)
0.1
52
±0
.00
20
.14
9±
0.0
03
1.0
21
.93
ns
0.1
55
±0
.00
40
.15
1±
0.0
03
1.0
31
.21
ns
(b)
Ad
ult
1.
Des
icca
tio
nh
ou
rs8
4.4
5±
1.7
81
2.5
1±
0.2
96
.75
39
2.5
5*
**
65
.36
±1
.46
22
.10
±0
.34
2.9
53
42
.63
**
*
2.
Mel
anis
atio
n(%
)8
6.3
7±
2.0
12
0.0
6±
0.5
14
.30
50
1.8
6*
**
70
.08
±1
.60
34
.19
±0
.56
2.0
43
56
.17
**
*
3.
Ep
icu
ticu
lar
lip
ids
(lg
cm-
2)
26
.88
±0
.37
10
.45
±0
.27
2.7
64
68
.59
**
*1
3.6
2±
0.2
31
3.2
4±
0.2
91
.02
1.5
2n
s
4.
Tre
hal
ose
(mg
mg
-1d
rym
ass)
0.2
21
±0
.00
40
.09
9±
0.0
02
2.2
31
95
.43
**
*0
.17
4±
0.0
04
0.1
26
±0
.00
31
.38
15
7.8
9*
**
5.
Gly
cog
en(m
gm
g-
1d
rym
ass)
0.0
79
±0
.00
20
.08
2±
0.0
02
1.0
33
.58
ns
0.0
80
±0
.00
20
.07
9±
0.0
02
1.0
10
.39
ns
6.
Lip
ids
(mg
mg
-1d
rym
ass)
0.1
82
±0
.00
30
.27
1±
0.0
05
1.4
01
98
.66
**
*0
.19
2±
0.0
03
0.2
26
±0
.00
31
.18
12
3.8
9*
**
7.
Pro
tein
s(m
gm
g-
1d
rym
ass)
0.1
59
±0
.00
20
.15
4±
0.0
03
1.0
31
.45
ns
0.1
57
±0
.00
20
.16
0±
0.0
04
1.0
20
.96
ns
Dat
aw
ere
arcs
ine
tran
sfo
rmed
for
AN
OV
A
ns
No
nsi
gn
ifica
nt
**
*P
\0
.00
1
366 J Comp Physiol B (2013) 183:359–378
123
estingly, the results of ANOVA for all the three desicca-
tion-related traits showed similar levels of variability in
larvae and adult stages. The percent variance for desicca-
tion resistance and carbohydrate content was *12–18 %
due to species, but *60–70, *8–13, and 7–10 % due to
growth temperatures, isofemale lines and interaction
effects, respectively. However, for cuticular lipid mass,
both larvae and adult flies have shown *28–32, 50–53,
8–10 and 3–6 % variability due to species, growth tem-
peratures, isofemale lines and their interactions, respec-
tively. Thus, we found major differences in desiccation-
related traits of sibling Drosophila species due to growth
temperatures, while inter-specific differences were rela-
tively lower (Table 2).
Differences in basic measures of water balance
and dehydration tolerance
Divergence at larval stage
We observed a significant increase in wet and dry mass
as well as total body water content (1.5-fold) at 15 �C
as compared with 25 �C in D. simulans (wet mass:
F1,19 = 271.82; P \ 0.001; dry mass: F1,19 = 224.70;
P \ 0.001; body water content: F1,19 = 259.02;
P \ 0.001; Table 3). Similarly, differences for body
weight as well as total water content were highly signifi-
cant in D. melanogaster at 15 versus 25 �C (wet mass:
F1,19 = 219.83; P \ 0.001; dry mass: F1,19 = 236.27;
P \ 0.001). Interestingly, hemolymph content increased
*3.9-fold and *2.8-fold in the larvae of D. simulans and
D. melanogaster, respectively (Table 3). Significant dif-
ferences were also observed for hemolymph water content
in D. simulans and D. melanogaster (Table 3). In contrast,
we did not find plastic changes for tissue water content in
both these sibling Drosophila species at 15 versus 25 �C
growth temperatures (D. simulans: F1,19 = 42.56 ns;
D. melanogaster: F1,19 = 1.23 ns). Further, larvae of
D. simulans as well as D. melanogaster have shown greater
body water loss until death (dehydration tolerance),
F1,19 = 139.55; P \ 0.001; Table 3).
Divergence at the adult stage
A comparative analysis of body mass has shown consistent
differences in multiple measures of body weight as well as
body water content in D. simulans (wet mass: F1,19 =
254.63; P \ 0.001; dry mass: F1,19 = 199.50; P \ 0.001;
body water content: F1,19 = 230.47; P \ 0.001) and
D. melanogaster (wet mass: F1,19 = 196.21; P \ 0.001;
Table 2 Analysis of variance (n = 20 IF 9 10 replicates each) for explaining trait variability due to species (S) growth temperatures (T),
isofemale lines (nested in species), and their interactions in (a) larvae as well as (b) adults of D. simulans and D. melanogaster
df Species (S) Temperature (T) IF lines S 9 T IF 9 T Error
1 1 38 1 38 720
(a) Larva
1. Desiccation resistance MS 16942.94 72724.98 218.45 4156.01 97.90 0.62
F 77.56 742.85 352.33 42.45 157.90
% Var 15.94*** 68.42*** 7.81*** 3.91*** 3.50*** 0.42
2. Cuticular lipid mass MS 22769.04 42113.01 206.04 4137.61 97.80 0.57
F 110.51 430.60 361.47 42.31 171.58
% Var 28.12*** 52.01*** 9.67*** 5.12*** 4.59*** 0.51
3. Carbohydrate content MS 24449.36 98114.39 320.51 8330.89 189.04 0.92
F 76.28 519.01 348.38 44.07 205.47
% Var 16.20*** 65.01*** 8.07*** 5.52*** 4.76*** 0.44
(b) Adult flies
1. Desiccation resistance MS 21695.87 130068.18 372.33 8653.37 101.41 0.32
F 58.27 1282.59 1163.53 85.33 316.91
% Var 12.16*** 72.90*** 7.93*** 4.85*** 2.16*** 0.13
2. Cuticular lipid mass MS 22898.26 37753.56 170.01 2952.01 65.73 0.95
F 134.69 574.37 178.96 44.91 69.19
% Var 31.26*** 51.54*** 8.82*** 4.03*** 3.41*** 0.94
3. Carbohydrate content MS 32718.30 104573.32 587.73 7583.82 137.77 2.01
F 55.67 759.04 292.40 55.05 68.54
% Var 18.81*** 60.12*** 12.89*** 4.36*** 3.01*** 0.87
ns = Nonsignificant
*** P \ 0.001
J Comp Physiol B (2013) 183:359–378 367
123
dry mass: F1,19 = 230.37; P \ 0.001; body water: F1,19 =
183.96; P \ 0.001) when grown at 2 different temperatures
i.e. 15 and 25 �C. Hemolymph content and hemolymph
water content have shown *3.2-fold and *4.7-fold
increase at 15 than at 25 �C in D. simulans but such dif-
ferences were *twofold in case of D. melanogaster.
However, tissue water did not change significantly due
to variation in growth temperatures in D. simulans as well
as in D. melanogaster (D. simulans: F1,19 = 1.86 ns;
D. melanogaster: F1,398 = 2.35 ns). Further, for both the
sibling species, we observed *18 % higher dehydration
tolerance at 15 �C as compared with 25 �C (D. simulans:
F1,19 = 268.94; P \ 0.001; D. melanogaster: F1,19 =
118.81; P \ 0.001).
Comparison of rate of water loss in two sibling species
We used nine sets of independent experiments to determine
changes in the rate of body water loss in control versus
larvae/flies exposed to different durations (1–8 h) of des-
iccation stress in D. simulans and D. melanogaster reared
at 15 as well as at 25 �C; and the data are shown in Fig. 2.
A comparison of slope values (Wharton’s method) has
shown a significant increase in the rate of water loss in
larvae (Fig. 2a, b) as well as in adult flies (Fig. 2c, d) for
both the species grown at 25 �C than at 15 �C. At the inter-
specific level, rate of water loss was significantly higher in
D. simulans larvae/adults grown at 25 �C than D. mela-
nogaster and these results are consistent with their desic-
cation resistance levels. In contrast, for larvae/adults grown
at 15 �C, the slope values are lower for D. simulans than
for D. melanogaster. These observations on the rate of
water loss suggest greater desiccation resistance of
D. simulans than D. melanogaster when grown at 15 �C.
We found *3.2-fold increase in the rate of water loss in
the larvae of D. simulans grown at 25 �C as compared with
15 �C, while corresponding increase in the rate of water
loss was *twofold in D. melanogaster (Fig. 2a, b). In
Table 3 Data (mean ± SE) on different measures of water balance and dehydration tolerance in (a) 3rd instar larva and (b) adult flies of
D. simulans and D. melanogaster grown at 15 and 25 �C
Traits D. simulans D. melanogaster
15 �C 25 �C Ratio F1, 19 15 �C 25 �C Ratio F1, 19
(a) 3rd Instar larva
1. Wet weight (mg fly-1) 2.012 ± 0.04 1.324 ± 0.02 1.51 271.82*** 1.998 ± 0.03 1.456 ± 0.02 1.37 219.83***
2. Dry weight (mg fly-1) 0.607 ± 0.009 0.398 ± 0.005 1.52 224.70*** 0.597 ± 0.008 0.438 ± 0.005 1.36 236.27***
3. Total water content
(mg fly-1)
1.405 ± 0.03 0.926 ± 0.02 1.50 259.02*** 1.401 ± 0.02 1.019 ± 0.03 1.38 153.64***
4. Hemolymph content
(mgfly-1)
0.726 ± 0.01 0.183 ± 0.004 3.96 316.59*** 0.694 ± 0.006 0.247 ± 0.004 2.80 274.31***
5. Hemolymph water
content (mg fly-1)
0.577 ± 0.007 0.107 ± 0.003 5.39 268.43*** 0.566 ± 0.005 0.186 ± 0.002 3.04 225.58***
6. Tissue water content
(mg fly-1)
0.828 ± 0.01 0.819 ± 0.009 1.01 2.56 ns 0.835 ± 0.01 0.832 ± 0.01 1.01 1.23 ns
7. Dehydration tolerance (%) 66.49 48.44 1.37 184.30*** 61.15 52.66 1.16 139.55***
(b) Adult
1. Wet weight (mg fly-1) 1.634 ± 0.03 1.087 ± 0.02 1.50 254.63*** 1.624 ± 0.04 1.202 ± 0.03 1.35 196.21***
2. Dry weight (mg fly-1) 0.492 ± 0.006 0.334 ± 0.003 1.47 199.50*** 0.486 ± 0.008 0.372 ± 0.005 1.30 230.37***
3. Total water content
(mg fly-1)
1.142 ± 0.01 0.754 ± 0.008 1.51 230.47*** 1.138 ± 0.03 0.830 ± 0.01 1.37 183.96***
4. Hemolymph content
(mg fly-1)
0.596 ± 0.006 0.185 ± 0.003 3.22 165.98*** 0.569 ± 0.007 0.227 ± 0.003 2.50 214.66***
5. Hemolymph water
content (mg fly-1)
0.484 ± 0.004 0.103 ± 0.002 4.69 286.33*** 0.476 ± 0.005 0.174 ± 0.002 2.09 260.41***
6. Tissue water content
(mg fly-1)
0.658 ± 0.003 0.651 ± 0.003 1.01 1.86 ns 0.662 ± 0.008 0.656 ± 0.007 1.01 2.35 ns
7. Dehydration tolerance (%) 65.74 47.01 1.40 268.94*** 60.13 52.09 1.15 118.81***
Data are based on 10 replicates of 20 isofemale lines of each species. F values of ANOVA were used for trait values comparison at 15 and 25 �C
Percent data were arcsine transformed for ANOVA
ns Nonsignificant
***P \ 0.001
368 J Comp Physiol B (2013) 183:359–378
123
contrast, for adult flies, there was *fourfold higher rate of
water loss in D. simulans, but *3.2-fold in D. melano-
gaster at 25 �C. At inter-specific level, we observed
reduced rate of water loss in D. simulans and D. melano-
gaster at 15 �C, but a reverse trend was evident at 25 �C
growth temperature.
Effects of organic solvents on rate of water loss
For both the sibling Drosophila species grown at 15 and
25 �C, the larvae and adult flies were treated with hexane;
and time series changes in percent body water content were
shown for D. simulans (Fig. 3a, b) and D. melanogaster
(Fig. 3c, d). Slope values for RWL in control and treated
individuals are shown in Table 4. For D. melanogaster,
there is no difference in the percent body water loss and
also in slope values of control and treated larvae or adults
at 15 �C as well as at 25 �C (Table 4; Fig. 3c, d). Thus, an
organic solvent has no effect on the cuticular permeability
of larvae and adult flies of D. melanogaster (Table 4). In
contrast, the rate of water loss in D. simulans larvae
increased *27-fold and eightfold at 15 and 25 �C,
respectively, after hexane treatment (Table 4). In contrast,
the adults of D. simulans showed only *twofold increase
in the rate of water loss at 25 �C as compared with 15 �C.
However, if adults of D. simulans had only cuticular lipids
as a barrier, we expect flies to loose body water in few
hours (\2–3 h), but this trend was not observed in the
present study. Thus, our results suggest that two different
cuticular components i.e. cuticular lipid mass and body
melanisation confer higher desiccation tolerance by low-
ering the rate of water loss in adult flies of D. simulans.
Comparison of rate of metabolite utilization in sibling
species under desiccation stress
In the present study, we examined the actual utilization of
different energy metabolites (carbohydrates or lipids or
proteins) in larvae and adult flies as a function of different
durations of desiccation stress; and the slope values were
Desiccation stress duration (hours)
In (
mt
/ m0)
-0.60
-0.45
-0.30
-0.15
0.00
0 2 4 6 8
15 °C = 0.0 - 0.011*x
D. melanogaster
25 °C = 0.0 - 0.030*x
Desiccation stress duration (hours)
In (
mt
/ m0)
-0.60
-0.45
-0.30
-0.15
0.00
0 2 4 6 8
D. simulans
15 °C = 0.0 - 0.017*x
25 °C = 0.0 - 0.055*x
Desiccation stress duration (hours)
In (
mt
/ m0)
-0.60
-0.45
-0.30
-0.15
0.00
0 2 4 6 8
D. melanogaster25 °C = 0.0 - 0.055*x
15 °C = 0.0 - 0.022*x
Desiccation stress duration (hours)
In (
mt
/ m0)
-0.60
-0.45
-0.30
-0.15
0.00
0 2 4 6 8
D. simulans
15 °C = 0.0 - 0.007*x
25 °C = 0.0 - 0.039*x
(A)
(D)
(C)
(B)
Larvae Adult flies
Fig. 2 A comparison of water loss rate (according to Wharton’s
method) in larvae and adult flies of D. simulans and D. melanogastergrown at 15 and 25 �C. The water loss rate was derived from the slope
(b) of In(mt/m0) as a function of different durations of desiccation
stress at \5 % relative humidity. Slope values for rate of water loss
vary significantly between species at larval as well as adult stage,
when grown at 15 and 25 �C
J Comp Physiol B (2013) 183:359–378 369
123
compared for each species at two different growth tem-
peratures (15 and 25 �C; Table 5). Interestingly, larvae and
adults of both the sibling species did not utilize lipids as
well as proteins under desiccation stress (P [ 0.34 ns;
Table 5). However, the levels of carbohydrates decreased
significantly with increase in the duration of desiccation
stress in both the species reared at 15 �C as well as at 25 �C
(P \ 0.001; Table 5). Further, the rate of utilization of
Duration of desiccation stress (hours)
Adu
lt w
ater
con
tent
(%
)
20
40
60
80
0 20 40 60 80 100
Adults
Duration of desiccation stress (hours)
Lar
val w
ater
con
tent
(%
)
20
40
60
80
0 10 20 30 40 50
ControlHexane washed
at 15 °C at 25 °C
Larvae
Duration of desiccation stress (hours)
Lar
val w
ater
con
tent
(%
)
20
40
60
80
0 10 20 30 40 50
Larvae
Duration of desiccation stress (hours)
Adu
lt w
ater
con
tent
(%
)
20
40
60
80
0 20 40 60 80 100
Adults
(A) (C)
(B) (D)
D. simulans D. melanogaster
Fig. 3 A comparison of changes in larval (a, c) and adult (b, d) body
water loss in control (untreated) and organic solvent-treated larvae
or adult flies as a function of desiccation stress for D. simulans(a, b) and D. melanogaster (c, d) at 15 and 25 �C. Body water loss was
measured after every 2-h interval for control larvae/flies of D. simu-lans, but for control as well as hexane-treated individual of
D. melanogaster. However, rate of water loss after hexane treatment
was assessed after every 30-min interval in D. simulans. For
D. simulans, there are significant differences due to hexane treat-
ment of adult flies while no such differences were evident for
D. melanogaster
Table 4 Data (slope ± SE) for rate of water loss (Wharton’s method) in control (dead over-etherized larvae or flies) and hexane-treated dead
larvae/flies of D. simulans and D. melanogaster
D. simulans D. melanogaster
15 �C 25 �C 15 �C 25 �C
1. Third instar larva
Control 0.017 ± 0.0003 0.055 ± 0.0015 0.022 ± 0.0006 0.046 ± 0.0013
Hexane treated 0.462 ± 0.0154 0.460 ± 0.0131 0.021 ± 0.0007 0.045 ± 0.0011
t test (P value) \0.001 \0.001 0.58 0.31
2. Adult
Control 0.007 ± 0.0001 0.039 ± 0.0011 0.011 ± 0.00031 0.030 ± 0.0008
Hexane treated 0.015 ± 0.0004 0.257 ± 0.0073 0.011 ± 0.00034 0.031 ± 0.0009
t test \0.001 \0.001 0.86 0.47
Rate of water loss in control versus hexane-treated larvae/flies was compared with t test
ns Nonsignificant, ***P \ 0.001
370 J Comp Physiol B (2013) 183:359–378
123
carbohydrates did not differ between species grown at
15 �C. In contrast, D. simulans utilized carbohydrates at a
higher rate at 25 �C as compared with D. melanogaster
(P \ 0.001; Table 5). Further, an intra-specific comparison
at 15 versus 25 �C showed a significantly higher rate of
carbohydrate utilization at 25 �C in larval as well as in
adult stage of both the species (Table 5; P \ 0.001). Thus,
at 25 �C growth temperature, low levels of storage of
carbohydrates but a significantly higher rate of utilization
in D. simulans are consistent with its lower desiccation
potential as compared with its sibling species D. melano-
gaster (Table 5).
Correlation between energy budget and desiccation
resistance in larvae and adults
Data on species-specific differences in the storage of
energy metabolites and desiccation resistance in D. simu-
lans and D. melanogaster reared at 15 and 25 �C are shown
in Table 1. We calculated energy budget due to carbohy-
drates (which are actually consumed under desiccation
stress) using standard conversion factors (Schmidt-Nielsen
1990) and the data on larvae and adults are shown in Fig. 4.
For larvae as well as adults, the energy budget due to stored
carbohydrates is higher at 15 than at 25 �C (Fig. 4a, d).
Energy budget of D. simulans is higher than D. melano-
gaster at 15 �C but the reverse is true at 25 �C (Fig. 4a, d).
Further, for each species, storage levels of carbohydrates
are about 40–50 % lower at 25 �C than at 15 �C. We found
significant correlations between carbohydrates energy
budget (J/mg) and desiccation resistance for larvae
(Fig. 4b, c) as well as for adults (Fig. 4e, f) of D. simulans
and D. melanogaster grown at 15 and 25 �C. Thus, there
are significant correlations between carbohydrate energy
budget and desiccation resistance.
Effects of growth temperature on acclimation potential
We tested whether the sibling species D. simulans and
D. melanogaster show similar or different desiccation
acclimation responses to prior treatment of desiccation
stress when compared across two growth temperatures (15
vs. 25 �C). Interestingly, both the larvae and adult flies
showed similar trends for acclimation effects but varied in
trait values in these two Drosophila species. In D. simu-
lans, both larvae and adults reared at 15 �C showed
significant increase in desiccation resistance (net increase
in desiccation resistance due to acclimation in larvae:
11.25 ± 0.28 h; adults: 15.12 ± 0.38 h) while corre-
sponding values for D. melanogaster were lower (larvae:
8.10 ± 0.19 h; adults: 11.49 ± 0.31 h; Fig. 5a, d). In
contrast, D. melanogaster showed higher acclimation
response than D. simulans at 25 �C (larvae: D. simu-
lans = 2.01 ± 0.10 h; D. melanogaster = 3.16; P \ 0.001;
adults: D. simulans = 2.30 ± 0.13 h; D. melanogas-
ter = 4.01 ± 0.19 h; P \ 0.001; Fig. 5a, d). However, we
did not find any significant change in the cuticular lipid
mass as a consequence of desiccation acclimation in both
the species at larval as well as at adult stage (Fig. 5b, e).
Interestingly, we observed no significant reduction in rate
of water loss due to acclimation in D. simulans as well as in
D. melanogaster across both the growth temperatures
(Fig. 5c, f; P [ 0.21 ns). Thus, our results suggest
that larvae and adults both respond to acclimation for
Table 5 Comparison of rate of metabolite utilization (regression slope values as a function of different durations of desiccation stress) at 15 and
25 �C in D. simulans and D. melanogaster
Metabolites D. simulans D. melanogaster
15 �C 25 �C t test 15 �C 25 �C t test
(a) Larvae
1. Carbohydrates -4.17 ± 0.13*** -8.55 ± 0.28*** *** -4.23 ± 0.11*** -6.28 ± 0.20*** ***
2. Lipids -0.08 ± 0.19 ns -0.12 ± 0.15 ns ns -0.04 ± 0.07 ns -0.09 ± 0.14 ns ns
3. Proteins -0.05 ± 0.26 ns -0.15 ± 0.34 ns ns -0.13 ± 0.51 ns -0.03 ± 0.07 ns ns
(b) Adult flies
1. Carbohydrates -1.52 ± 0.07*** -4.26 ± 0.13*** *** -1.56 ± 0.06 -2.94 ± 0.09*** ***
2. Lipids –0.06 ± 0.18 ns –0.14 ± 0.22 ns ns –0.05 ± 0.09 ns –0.08 ± 0.22 ns ns
3. Proteins –0.02 ± 0.09 ns –0.05 ± 0.15 ns ns –0.11 ± 0.25 ns –0.13 ± 0.16 ns ns
Slope values were compared with t test at two different temperatures (15 vs. 25 �C) in larvae as well as adult flies of both the species
Slope values represent rate of metabolite utilization as a function of time (negative sign indicates that metabolite level decreased with time under
desiccation stress). Asterisk with slope values denotes significant decrease in metabolite level with time. Unit is lg/h
ns Nonsignificant
***P \ 0.001
J Comp Physiol B (2013) 183:359–378 371
123
desiccation stress. It is also evident that acclimation
potential varies according to growth temperatures in
D. simulans and D. melanogaster.
Discussion
In the present study, we found significant differences
in desiccation-related traits in the sibling species
D. simulans and D. melanogaster. Interestingly, there are
developmental plastic effects for both the cuticular traits
(cuticular lipid mass and body melanisation) in D. simu-
lans, but only for body melanisation in D. melanogaster.
For D. simulans, plastic responses for both the cuticular
traits are consistent with higher desiccation potential as
compared with D. melanogaster. Further, intra-specific
differences in desiccation resistance due to developmental
plasticity match significant increase in the body mass (wet
Car
bohy
drat
es e
nerg
y b
udge
t (
J/m
g)
0
1
2
3
415 °C
P < 0.001P < 0.001
25 °C
Desiccation resistance (hours)
Car
bohy
drat
es e
nerg
y b
udge
t (
J/m
g)
0.6
1.6
2.6
3.6
0 10 20 30 40 50
r = 0.92 ± 0.07
r = 0.87 ± 0.09(P < 0.001)
(P < 0.001)
D. simulans
15 °C25 °C
Desiccation resistance (hours)
Car
bohy
drat
es e
nerg
y b
udge
t (
J/m
g)
0.6
1.6
2.6
3.6
0 10 20 30 40 50
r = 0.89 ± 0.09
r = 0.85 ± 0.12(P < 0.001)
(P < 0.001)
D. melanogaster
Car
bohy
drat
es e
nerg
y b
udge
t (
J/m
g)
0
1
2
3
4
P < 0.001 P < 0.001
Desiccation resistance (hours)
Car
bohy
drat
es e
nerg
y b
udge
t (
J/m
g)
0.6
1.6
2.6
3.6
0 20 40 60 80 100
r = 0.94 ± 0.04(P < 0.001)
r = 0.90 ± 0.08(P < 0.001)
D. simulans
Desiccation resistance (hours)
Car
bohy
drat
es e
nerg
y b
udge
t (
J/m
g)
0.6
1.6
2.6
3.6
0 20 40 60 80 100
r = 0.96 ± 0.03
(P < 0.001)
r = 0.83 ± 0.13(P < 0.001)
D. melanogaster
(A)
(F)
(E)
(D)
(C)
(B)
Larval traits Adult traits
D. simulans D. melanogaster D. simulans D. melanogaster
Fig. 4 A comparison of energy budget due to stored carbohydrates of
D. simulans and D. melanogaster grown at 15 and 25 �C (a, d). There
are positive correlations between desiccation resistance and
carbohydrate energy budget in larvae (b, c) as well as adults
(e, f) of D. simulans (b, e) and D. melanogaster (c, f)
372 J Comp Physiol B (2013) 183:359–378
123
and dry mass), body water content and hemolymph water
in both the sibling species D. simulans and D. melano-
gaster reared at 15 �C as compared to 25 �C. We also
observed significant effect of growth temperatures on the
storage and utilization of carbohydrates under desiccation
stress. For example, lower storage level of carbohydrates
but a higher rate of utilization was observed in D. simu-
lans when compared with D. melanogaster grown at
25 �C whereas the reverse trend was evident at 15 �C.
Finally, we found that absolute desiccation acclimation
capacity was quite low in D. simulans reared at 25 �C and
this might reflect its future vulnerability under global
climate warming.
Role of cuticular lipids in D. simulans
Insect’s cuticle is a complex structure and its components
might vary between species and populations (Willmer et al.
2000). Several studies have shown variable cuticular per-
meability due to changes in the composition or amount of
cuticular lipids in diverse insect taxa (Edney 1977; Toolson
1984; Hadley 1994; Rourke 2000). In contrast, no associ-
ations were found between cuticular lipid quantity and rate
of water loss in laboratory selected desiccation resistant
strains of D. melanogaster (Gibbs et al. 1997); as well as in
xeric versus mesic Drosophila species (Gibbs et al. 2003);
and in geographical populations of D. melanogaster
0
5
10
15
20
P < 0.001P < 0.001
15 °C
25 °C
C
han
ges
in c
uti
cula
r li
pid
mas
s
(µg
cm -2 )
0
4
8
12
P = 0.23 ns P = 0.34 ns
Ch
ange
s in
slo
pe
valu
es f
or r
ate
of w
ater
loss
-0.06
-0.04
-0.02
0.00
P = 0.31 ns P = 0.28 ns
0
5
10
15
20
P < 0.001P < 0.001
C
han
ges
in c
uti
cula
r li
pid
mas
s
(µg
cm -2 )
0
4
8
12
P = 0.68 ns P = 0.32 ns
Ch
ange
s in
slo
pe
valu
es f
or r
ate
of w
ater
loss
-0.06
-0.04
-0.02
0.00
P = 0.21 nsP = 0.25 ns
(A)
(F)
(E)
(D)
(C)
(B)
Acclimated larvae Acclimated adult flies
Incr
ease
d d
esic
cati
on r
esis
tan
ce (
hou
rs)
(Ab
solu
te a
ccli
mat
ion
cap
acit
y)
Incr
ease
d d
esic
cati
on r
esis
tan
ce (
hou
rs)
(Ab
solu
te a
ccli
mat
ion
cap
acit
y)
D. simulans
D. melanogasterD. simulans
D. simulans
D. simulans
D. simulans
D. simulans
D. melanogaster
D. melanogasterD. melanogaster
D. melanogasterD. melanogaster
Fig. 5 Changes in desiccation-related traits due to acclimation of
larvae and adult flies of two Drosophila sibling species (D. simulansand D. melanogaster) grown at 15 and 25 �C. Changes in trait values
in larvae (a–c) and adult flies (d–f) are shown for desiccation
resistance (a, d); and cuticular lipid mass (b, e) and rate of water loss
(c, f). For a, d; absolute acclimation capacity = desiccation hours of
acclimated - non-acclimated adults
J Comp Physiol B (2013) 183:359–378 373
123
(Parkash et al. 2008, 2010). However, in case of D. simu-
lans, no previous study has examined developmental
plastic effects (due to growth temperatures) on cuticular
lipid mass as well as corresponding changes in the rate of
water loss. In the present work, for D. simulans, we
observed *2.5-fold increase in the cuticular lipid mass of
larvae as well as adults reared at 15 �C as compared with
25 �C, but no such changes were observed in D. melano-
gaster. Therefore, our results suggest the role of cuticular
lipids in D. simulans but not in D. melanogaster. In con-
trast, developmental plastic effects for cuticular melanisa-
tion are evident in both the sibling species. Thus, we found
decrease in cuticular permeability due to two components
(cuticular lipids mass and melanisation) in adults of
D. simulans. Unlike hexane-treated D. simulans larvae
which lost *60 % body water in just 2 h, hexane-treated
adult flies took *40 h to loose similar amount of body
water. This difference could be due to the effect of body
melanisation on cuticular permeability in adults of D. sim-
ulans. As evident in Fig. 5b, we may argue that cuticular
lipids and body melanisation both contribute equally to the
total desiccation survival hours (*86 h) for adults of
D. simulans.
Plastic changes for hemolymph content
and dehydration tolerance
In insects, hemolymph is a major source for changes in the
level of body water to support longer survival under dehy-
dration stress (Hadley 1994; Chown and Nicolson 2004; Folk
et al. 2001; Folk and Bradley 2005). A significant increase
(*sixfold) in hemolymph content was observed in labora-
tory selected desiccation resistant lines of D. melanogaster
as compared with control (Folk et al. 2001; Folk and Bradley
2005). In contrast, several studies on wild populations of
various Drosophila species have not considered changes in
hemolymph content to enhance survival under desiccation
stress (Gibbs and Matzkin 2001; Gibbs et al. 2003; Parkash
et al. 2010). In the present study, we found changes in
hemolymph content as a consequence of developmental
plastic effects (i.e. 15 vs. 25 �C). Interestingly, we observed
similar trends for changes in the hemolymph content in lar-
vae and adult stages of both the sibling species. Hemolymph
content increased*fourfold at 15 �C growth temperature in
larvae as well as in adults of D. simulans as compared with
25 �C. In contrast, there was *twofold increase in hemo-
lymph water content in D. melanogaster larvae as well as in
adult flies reared at 15 �C as compared with 25 �C. Thus, the
two sibling Drosophila species have shown changes in
hemolymph content consistent with their different levels of
desiccation resistance potential.
Further, most arthropods can tolerate *30–50 % loss of
body water but some taxa adapted to drier habitats have
evidenced higher dehydration tolerance i.e. *40–60 %
body water loss before succumbing to death (Hadley 1994;
Willmer et al. 2000; Benoit et al. 2005). Desert Drosophila
species have shown greater desiccation resistance, lower
rates of water loss, but no consistent differences in dehy-
dration tolerance as compared with mesic species (Gibbs
and Matzkin 2001). In contrast, enhanced survival of lab-
oratory selected desiccation resistant lines is significantly
associated with greater dehydration tolerance as compared
with control (Telonis-Scott et al. 2006). Therefore, dehy-
dration tolerance may not have evolved similarly in field
populations versus laboratory selected desiccation resistant
lines of D. melanogaster (Gibbs et al. 1997; Hoffmann and
Harshman 1999). In the present study, we found increased
dehydration tolerance at 15 �C when compared with 25 �C
in D. simulans and D. melanogaster which is consistent
with intra-specific differences in desiccation resistance.
Therefore, dehydration tolerance has evolved as a common
physiological mechanism to support survival under desic-
cation stress at a lower growth temperature in the sibling
Drosophila species D. simulans and D. melanogaster.
Inter-specific differences in the storage and utilization
of energy metabolites
The acquisition of greater energy reserves has been asso-
ciated with increased survival under dehydration stress
(Gibbs 2002; Chown and Nicolson 2004). Laboratory
selected desiccation resistant lines (D) have shown higher
storage of carbohydrates as compared with control (Graves
et al. 1992; Gibbs et al. 1997; Djawdan et al. 1998;
Chippindale et al. 1998; Folk et al. 2001; Folk and Bradley
2005). In contrast, a new set of laboratory selected desic-
cation resistant lines have shown increased lipid content in
selected (D) lines when compared with control (Telonis-
Scott et al. 2006). Therefore, results of laboratory selection
experiments are not consistent whether carbohydrates or
lipids support survival under desiccation stress. Further,
wild Drosophila species from xeric and mesic habitats vary
in desiccation resistance, despite lack of differences in the
storage of energy metabolites (Marron et al. 2003). How-
ever, increased desiccation tolerance of cactophilic Dro-
sophila species has been associated with reduced rate of
metabolite utilization (Marron et al. 2003). Further, no
previous study has examined changes in the storage levels
as well utilization of energy metabolites due to thermal
plastic effects. In the present study, we observed higher dry
mass specific levels of carbohydrates in D. simulans and
D. melanogaster reared at 15 than at 25 �C, which is in
agreement with differences in their desiccation resistance
at 2 different growth temperatures. Interestingly, the rate of
metabolite utilization was significantly lower at 15 than
at 25 �C in both the species. However, we did not find
374 J Comp Physiol B (2013) 183:359–378
123
inter-specific differences in the rate of metabolite utiliza-
tion at 15 �C, but a significantly reduced rate was evident
at 25 �C in D. melanogaster than in D. simulans. Thus,
these two sibling Drosophila species have stored higher
levels of carbohydrates, but utilized them with reduced rate
at lower temperature (15 �C) to alleviate the effects of
desiccation stress. In contrast, low storage level and higher
rate of utilization of carbohydrates at 25 �C are consistent
with significantly lower desiccation resistance for 2 sibling
species. Our results suggest that storage as well as rate of
utilization of energy metabolites is constrained by growth
temperatures in D. simulans and in D. melanogaster.
Acclimation potential of sibling Drosophila species
Ectothermic organisms are capable of increasing their
stress resistance level due to prior exposure of few or
more bouts of thermal stresses (Bale 2002; Hoffmann
et al. 2003). However, drought acclimation has been
found beneficial to the arctic collembolan O. arcticus
(Holmstrup and Sømme 1998), for F. candida (Holmstrup
et al. 2002), in Belgica antarctica (Benoit et al. 2007) and
in Cryptopygus antarcticus (Elnitsky et al. 2008). In
contrast, in Drosophila species, there are few studies
which have shown increase in desiccation resistance due
to prior treatment of non-lethal level of desiccation stress
in two Australian populations of D. melanogaster and
D. simulans (Hoffmann 1991); and in one Canadian
population of D. melanogaster (Bazinet et al. 2010).
However, these studies, did not consider possible effects
of different growth temperatures which may reflect ther-
mal conditions prevalent in temperate and tropical regions
on the Australian continent.
Two pairs of sibling species grown at 25 �C (D. serrata
vs. D. birchii; and D. melanogaster vs. D. simulans from
Australia) have shown species-specific differences in the
acclimation to desiccation stress (Hoffmann 1991). In that
study, both D. melanogaster and D. simulans from Cairns
(Australia) showed increased desiccation resistance due to
acclimation to desiccation stress (D. melanogaster:
LT50 = 14.41 h in control vs. LT50 = 15.84 in acclimated;
D. simulans: LT50 = 10.14 h in control vs. 12.80 h in
acclimated; Hoffmann 1991). Thus, higher acclimation
capacity (2.5 h) was evident in adults of D. simulans as
compared with *1.4 h in D. melanogaster (Hoffmann
1991). However, the effects of acclimation at ecologically
relevant growth temperatures in pre-adult as well as in
adult stages have not been considered in any Drosophila
species so far. In the present study, we found higher
acclimation capacity in D. melanogaster (*4 h) than in
D. simulans (*2 h) when grown at 25 �C. In contrast, for
D. simulans, a significant increase in desiccation resistance
due to acclimation was observed at 15 �C in larvae
(11.25 h) as well as in adults (15.12 h). Further, in
D. melanogaster reared at 15 �C, the corresponding
increase in desiccation resistance due to acclimation was
8.1 h in larvae and 11.5 h in adults. Thus, for both the
sibling species grown at 15 �C, there is a significant
increase in the ability to resist desiccation stress after
acclimation. Our results suggest that contrasting levels of
acclimation capacity in two sibling species are constrained
by their basal levels of desiccation resistance at different
growth temperatures.
In the temperate and tropical regions, Drosophila spe-
cies are expected to experience drier conditions due to
global climatic change (Hoffmann 2010). For example, a
rainforest species D. birchii with its low desiccation
resistance level is likely to suffer due to changes in pre-
cipitation under global climatic change (Kellermann et al.
2009; Hoffmann 2010). However, there are few studies
which have considered acclimation potential of widespread
Drosophila species (Hoffmann 1991). In the present study,
D. simulans larvae and adults have shown lower desicca-
tion potential as well as lower acclimation response at
higher temperature as compared with D. melanogaster.
Thus, D. simulans can be vulnerable to drought conditions
in lowland localities while D. melanogaster might cope
with such changes. For possible mechanistic basis of
acclimation responses, we found no evidence of changes in
the rate of water loss in both the sibling species. Thus,
acclimation to drought conditions is adaptive in both these
species but their potential varies according to growth
temperatures. For D. simulans, our results differ from
Australian populations which may reflect the possible dif-
ferences in genetic variation for desiccation stress as well
as for selection responses due to continental differences in
the ambient levels of humidity conditions. However, fur-
ther studies are required to focus on the probable vulner-
ability of D. simulans in warmer and drier habitats in
different parts of the globe.
Species-specific desiccation resistance and distribution
patterns
For ectothermic organisms, differences in desiccation
resistance match geographical distribution of species
(Willmer et al. 2000). For example, temperate populations
of Drosophila species are desiccation resistant than tropi-
cal, which is consistent with colder and drier conditions in
the temperate but hot and humid in the tropics (Hoffmann
and Harshman 1999). However, based on earlier reports,
higher abundance of D. simulans in temperate regions
appears a mismatch due to its sensitivity for desiccation
stress (Parsons 1983). Some studies on Australian popu-
lations of the sibling species D. melanogaster and D. sim-
ulans have shown differences in the level of geographical
J Comp Physiol B (2013) 183:359–378 375
123
variation for desiccation resistance (Hoffmann and Harsh-
man 1999). For example, in contrast to a shallower cline of
desiccation resistance in D. melanogaster (Hoffmann et al.
2001), a lack of clinal variation for desiccation resistance
was evident in D. simulans (Arthur et al. 2008). Thus, it is
not clear how D. simulans (a desiccation sensitive species)
adapts to drier habitats in the temperate regions. This might
be because all the comparative studies have analyzed
the stress-related traits in these sibling Drosophila species
reared at 25 �C (Parsons 1983; Hoffmann and Parsons
1991) but the species-specific thermal ranges are not
identical for D. melanogaster and D. simulans (i.e.
12–27 �C for D. simulans vs. 13–30 �C for D. melano-
gaster). Further, no previous study has investigated
desiccation resistance of D. simulans reared at lower
temperatures i.e. 12–17 �C. Thus, the comparison of
D. melanogaster and D. simulans at 25 �C is likely to bias
their desiccation resistance potential.
Our results are consistent with significant changes in
desiccation resistance due to developmental plasticity of
both the cuticular traits of D. simulans but for body mel-
anisation only in D. melanogaster. To the best of our
knowledge, change in the amount of cuticular lipids in
D. simulans due to developmental plasticity has not been
investigated earlier. Such an observation is important
because no other Drosophila species has evidenced chan-
ges in both the cuticular traits (i.e. cuticular lipid mass and
cuticular melanisation). In the present study, significant
changes in cuticular traits of D. simulans reared at 15 �C
are consistent with its greater potential for desiccation
resistance and its ability to cope with colder and drier
habitats in the western Himalayas. Thus, future studies on
D. simulans should consider changes in desiccation-related
traits by rearing flies at cooler temperatures matching the
thermal conditions of the sites of origin of D. simulans
populations from different continents.
Conclusions
In the present study, we investigated changes in the
cuticular traits (cuticular lipid and body melanisation) and
their possible effects on water balance-related traits in two
sibling species—D. simulans and D. melanogaster. We
grew both these species at ecologically relevant thermal
conditions (i.e. 15 and 25 �C), which are likely to be
encountered by them in highland versus lowland localities.
At a lower temperature (15 �C), we observed an increase in
desiccation resistance in the larvae as well as in adults of
D. simulans due to developmental plasticity as compared
with 25 �C. In D. simulans, cuticular lipid mass as well as
body melanisation increased significantly (fourfold and
threefold, respectively) at 15 �C, and these changes were
consistent with significant reduction in the rate of water
loss. Similarly, larvae as well as adults of D. simulans
grown at 15 �C have shown *50 % increase in water
content, sixfold higher hemolymph water, *18 % greater
dehydration tolerance. These observations match relative
abundance of D. simulans under colder and drier habitats in
the western Himalayas. Further, D. melanogaster grown at
15 �C did not evidence any change in cuticular lipid mass
in larvae as well as in adult flies as compared with 25 �C.
However, adults of D. melanogaster reared at 15 �C
increased *twofold melanisation as compared with 25 �C.
These observations are consistent with inter-specific dif-
ferences in desiccation potential of these two sibling
Drosophila species. The results on desiccation resistance in
D. simulans and D. melanogaster grown at 25 �C are in
agreement with previous reports. The comparative data on
water balance-related traits have shown higher level of
water content, hemolymph water and dehydration tolerance
in D. melanogaster as compared with D. simulans reared at
25 �C. Thus, we observed superiority of D. melanogaster
over D. simulans grown at 25 �C, but a reverse trend was
evident at 15 �C. The present data suggest that the two
sibling species have evolved different strategies for water
conservation which match their abundance under con-
trasting habitats.
For energy metabolites, we found higher storage of
carbohydrates in D. simulans larvae as well as in adults
grown at 15 �C as compared with D. melanogaster but a
reverse trend was observed when both the sibling species
were reared at 25 �C. Interestingly, rate of utilization of
carbohydrates did not vary between the two sibling species
reared at 15 �C. However, we found higher rate of carbo-
hydrate utilization under desiccation stress for flies grown
at 25 �C as compared with 15 �C. Both the species did not
show utilization of lipid and proteins under desiccation
stress. Further, lower storage level but higher rate of uti-
lization of carbohydrates in D. simulans reared at 25 �C is
in agreement with its lower desiccation resistance poten-
tial. Finally, we found higher levels of acclimation effects
to low humidity in D. simulans grown at 15 �C as com-
pared with 25 �C. In contrast, for flies grown at 25 �C,
acclimation effects were lower, but superior in D. mela-
nogaster as compared with D. simulans. Thus, we have
observed that acclimation responses in the two sibling
species are constrained by their basal level of desiccation
resistance. The quite low acclimation potential of D. sim-
ulans reared at 25 �C reflects its likely vulnerability under
warmer and drier conditions in lowland localities of the
western Himalayas. Future studies are required to investi-
gate possible effects of climatic change on the relative
abundance of D. simulans in warmer regions.
376 J Comp Physiol B (2013) 183:359–378
123
Acknowledgments We are indebted to three anonymous reviewers
for several helpful comments which improved the MS. Financial
assistance from Council of Scientific and Industrial Research, New
Delhi [Emeritus Scientist project no. 21(0847)11 EMR-11] is grate-
fully acknowledged. Divya Singh, Chanderkala Lambhod and Poo-
nam Ranga are thankful to University grants commission, New Delhi,
for award of Rajiv Gandhi National Fellowship (RGNF).
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