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: rpgenetics@ymail.com

D. D. Aggarwal

e-mail: ddgenetics@ymail.com

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

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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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