Resistance to environmental stress in Drosophila ananassae: latitudinal variation and adaptation among populations

Authors


Bashisth N. Singh, Genetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221 005, India. Tel.: +91 542 670 2528; fax: +91 542 236 8174; e-mail: bnsingh@bhu.ac.in; bashisthsingh2004@rediffmail.com

Abstract

Geographical variation in traits related to fitness is often the result of adaptive evolution. Stress resistance traits in Drosophila often show clinal variation, suggesting that selection affects resistance traits either directly or indirectly. Multiple stress resistance traits were investigated in 45 natural populations of Drosophila ananassae collected from all over India. There was significant positive correlation between starvation resistance and lipid content. Significant negative correlations between desiccation and lipid content and between desiccation and heat resistance were also found. Flies from lower latitudes had higher starvation resistance, heat resistance and lipid content but the pattern was reversed for desiccation resistance. These results suggest that flies from different localities varied in their susceptibility to starvation because of difference in their propensity to store body lipid. Multiple regression analysis provided evidence of climatic selection driven by latitudinal variation in the seasonal amplitude of temperature and humidity changes within the Indian. Finally, our results suggest a high degree of variation in stress resistance at the population level in D. ananassae.

Introduction

Organisms have evolved various strategies to cope with environmental variations, including adaptation and phenotypic plasticity (Lynch & Gabriel, 1987; Stearns, 1989; Meyers & Bull, 2002). Extreme environmental conditions and stress may have significant negative effects on their physiology as well as on life-history traits. Analyses of such stress-inducing conditions are a major focus in furthering our understanding of ecological adaptation and the biological distribution of species. Genetic variation in stress tolerance results in adaptive changes that depend on the environmental conditions faced by organisms.

Clinal variations provide an opportunity to identify the traits and genes associated with environmental conditions, and when patterns are replicated and understood in terms of the effects on fitness, they provide strong evidence for selection (Endler, 1977). Many quantitative traits, including body size, development time, and heat and cold resistance, show latitudinal clines in Drosophila species (reviewed in Hoffmann et al., 2004; Hoffmann & Week, 2007). These studies have led to the conclusion that such clines represent the action of climate selection on these quantitative traits.

Geographical variations in the traits related to fitness are often the result of adaptive evolution. Particularly, strong support comes from clinal variation, which suggests a contribution of directional selection to differentiation among populations. Geographic gradients are of special interest in the study of climatic adaptation because the climate varies strongly with geographical variables. Although several environmental factors may impact on the physiology of individuals, temperature is thought to be one of the strongest, and thereby of great selective importance (Clarke, 2003; Hoffmann et al., 2003a). Despite the possibility of temperature having a major impact on species distribution and evolution, it is less clear which characteristics of the thermal environment are the most important agents of thermal selection.

It is widely accepted that thermal selection is of major importance and that Drosophila are likely to be exposed to stressful environments and they can therefore be expected to exhibit adaptation, although the mechanism of adaptation is uncertain (Feder, 1997; Gibbs et al., 2003; Hoffmann et al., 2003a). Multiple assays have been used in various laboratories testing thermal performance at both the high and low ends of the temperature scale (see Hoffmann et al., 2003a). For heat adaptation, knock-down resistance has been suggested to be important, and it may be correlated with natural adaptation to high temperature environments (Sorensen et al., 2001; Hoffmann et al., 2002). In nature, high temperature is correlated with desiccation but many studies have failed to show clinal variation in desiccation resistance (Hoffmann et al., 2001, 2002). Examples exist, however, of adaptive patterns in desiccation resistance (Karan et al., 1998; Hoffmann et al., 2003b; Sorensen et al., 2005). Starvation tolerance is variable among species and might be more pronounced in temperate than in tropical species (Parsons, 1983; Van Herrewege & David, 1997; Karan et al., 1998).

Most animals face periods of food shortage and are thus exposed to evolve adaptation, enhancing starvation resistance. Most of our knowledge on the genetic and physiological bases of these adaptations, their evolutionary correlates and trade-offs, and their patterns of variation within and among populations come from studies on Drosophila. Both the evolutionary change and the physiological plasticity involve increased accumulation of lipid, changes in carbohydrate and lipid metabolism, and reduction in reproduction. These changes are also typically associated with greater resistance to desiccation and oxidative stress (for review see Rion & Kawecki, 2007).

Drosophila offer a unique opportunity to attain a comprehensive understanding and integration of the different facets of evolutionary responses to nutritional stress. The increasing recognition that responses to nutritional stress in organisms as diverse as yeast, nematodes, flies and mammals are regulated by highly conserved physiological and cellular mechanisms, such as insulin signalling and TOR nutrient sensing pathways, implies that those results may have more general application (Rion & Kawecki, 2007). As in many organisms, there is evidence in Drosophila that natural selection has occurred for traits related to resistance to environmental stress. The distribution of Drosophila species follows climatic variables and their distribution can be predicted by interspecific variation in resistance to abiotic stresses (Parsons, 1981). Intraspecifically, geographic variation in stress resistance often follows climatic predictions. For example, Drosophila melanogaster populations from temperate regions are more resistant to temperature and desiccation stress than populations from subtropical regions where these stresses are less common (Stanley & Parsons, 1981; David et al., 1983).

The Indian subcontinent, which covers a wide range of latitudes and altitudes, lends itself to the investigation of adaptation to climate. From south to north, the seasonal thermal amplitude shows a regular increase with progressively more marked cold and warm seasons. Seasonal variations strongly increase with latitude. The southern region is characterized by thermal stability and high humidity throughout the year. Going northward, summers become increasingly warmer and drier, suggesting a progressively stronger heat desiccation stress. Such a regular climatic pattern according to latitude does not exist between Europe and Africa or on the east coast of Australia and should produce clearer genetic trends if natural selection really does act on stress tolerance and adaptation (Karan et al., 1998).

Drosophila ananassae, a cosmopolitan and domestic species belonging to the ananassae subgroup of the melanogaster species group, is stenothermic and circumtropical in distribution. It occupies a unique status among the Drosophila species because of certain peculiarities in its genetic behaviour. It is of common occurrence in India. Behavioural studies also have revealed several interesting features in D. ananassae (Singh, 2000, 2010; Singh & Singh, 2008). Previous studies on D. ananassae have shown the effect of temperature on survival and longevity (Sisodia & Singh, 2002), on various life-history and fitness traits (Yadav & Singh, 2005), on fluctuating asymmetry (Vishalakshi & Singh, 2008), and the effect of short-term heat stress on survival and productivity (Sisodia & Singh, 2006). It also shows genetic and phenotypic variance under extreme temperatures (Sisodia & Singh, 2009), chill coma recovery and cold tolerance (Sisodia & Singh, 2010), and the role of natural selection and genetic drift on the degree of inversion polymorphism (Singh & Singh, 2007).

In this study, we have examined populations of D. ananassae collected from different ecogeographic regions of India. The objective was to explore the effect of different geographical parameters (latitude, altitude and longitude) and climatic variables (average annual temperature, average annual rainfall and relative humidity [RH]) on multiple traits: desiccation, starvation, lipid content and heat resistance in the Indian populations. An attempt has been made to find evidence of adaptive variation by comparing the populations of D. ananassae. A country such as India, with its wide range of diverse geoclimatic conditions, provides a very good platform for conducting such studies.

Materials and methods

Stocks investigated

Forty-five populations of D. ananassae collected from different localities in India, ranging from Jammu in the north to Kanniyakumari in the south, and Dwarka in west to Deemapur in east, were investigated (Fig. 1). During the course of collection, it was realized that in northern parts of India during hot and dry summer these flies dwindle in number because of drastic seasonal shifts in temperature and climatic conditions. However, in southern and north-eastern parts of India, where there is uniform distribution of temperature and where high humidity prevails, flies are available throughout the year for collection. All strains had been kept in the laboratory for less than 3 years before starting the experiments. These are the same populations that were analysed for chromosomal polymorphism by Singh & Singh (2007) and cold adaptation and chill coma recovery by Sisodia & Singh (2010). All the stocks were maintained on a standard culture medium (containing agar-agar, dried yeast, maize powder, crude sugar, nipagin, propionic acid and plain water) at approximately 24 °C temperature. Climatic data for the localities from where flies were collected were obtained from climatological tables (India Meteorological Department, New Delhi).

Figure 1.

 Map of India showing the localities from where Drosophila ananassae flies were collected. JU, Jammu; DH, Dharamshala; KG, Kangra; DN, Dehradun; HD, Haridwar; MD, Mansa Devi; GT, Gangtok; LK, Lucknow; GU, Guwahati; RP, Raidopur; CW, Chowk; DM, Dimapur; SH, Shillong; PN, Patna; AB, Allahabad; IM, Imphal; GY, Gaya; UJ, Ujjain; BP, Bhopal; IN, Indore; JR, Jamnagar; HW, Howrah; SD, Sealdah; KL, Kolkata; RJ, Rajkot; DW, Dwarka; AD, Ahemdabad; PA, Paradeep; BN, Bhubneswar; PU, Puri; SI, Shirdi; NA, Nashik; MU, Mumbai; VP, Visakhapatnam; VD, Vijaywada; PJ, Panaji; MA, Madgaon; GK, Gokarna; ML, Manglore; BL, Bangalore; YS, Yeswantpur; PC, Pondicherry; ER, Ernakulam; TR, Thiruvananthapuram; and KR, Kanniyakumari.

Desiccation resistance

Kennington et al.’s (2001) method of measuring desiccation resistance was followed. Desiccation resistance was measured on 4- to 5-day-old virgin flies, and up to 10 flies of each sex were measured for each population. Five replicates were carried out. To measure resistance, flies from each vial were transferred to a new vial containing a disc of dry filter paper and covered with muslin gauze secured with an elastic band. Desiccation vials were kept at 25 °C under constant light and were observed for the number of dead flies 7 h after the flies were originally transferred, and then at half-hourly intervals until all the flies had died.

Starvation resistance

Starvation resistance was measured on 4- to 5-day-old virgin flies and, as with desiccation resistance, up to 10 flies of each sex were measured for each population. Five replicates were carried out. To measure starvation resistance, flies from each vial were transferred to a new vial containing 7 mL of 1% agar and plugged with cotton to prevent desiccation. Starvation vials were kept at 25 °C under constant light and were observed for the number of dead flies 40 h after the flies were originally transferred, and then at 6-hourly intervals until all the flies had died.

Heat-shock survival

Flies were heat shocked in empty food vials. To prevent desiccation, the stoppers were moistened with tap water. The vials were placed evenly spaced in racks in incubators. One group was hardened at 37 °C for 1 h, followed by 1 h at 25 °C to allow the flies to recover before being heat shocked 1 h at 40 °C. The other group was directly exposed to 40 °C for 1 h. In each group, 10 flies per vial and five vials per sex and population were used. After the heat shock, flies were transferred to fresh food vials and allowed recovery for 24 h at 25 °C prior to scoring survival (ability to walk).

Lipid content

The method of Hoffmann & Parsons (1989) was used to measure lipid content. Flies (5–6 days old) were dried overnight at 55 °C temperature in groups of 20 and weighed on a Sartorius microbalance with an accuracy of 0.0001 g. Lipid was extracted by placing intact flies in diethyl ether overnight with gentle agitation. Flies were reweighed after they had been dried again. Females and males were treated in groups of 20 separately, and two groups were tested per sex per population.

Statistical analyses

Two-way factorial anova was undertaken to examine the effect of population, sex and their interactions on physiological traits. For this, Model I anova was used, in which both population and sex factors are fixed. For figures, linear regression analysis was carried out to find the relationship between each of the physiological traits and latitude of the origin (SigmaStat 2.0). The underlying assumptions of anova (homogeneity of variance) were tested by Bartlett’s test (Zar, 2005). Multiple regression analyses were carried out to examine the effect of latitude, altitude and longitude, as well as annual average temperature (Tave), rainfall (Rave) and RH on desiccation, starvation, heat shock and lipid content. For multiple regression, all the predictor variables are not correlated. For this, the software package Statistica was used. Principal Components Analysis (PCA) was performed using the Unsrambler X 10.0 package (CAMO Software, India Pvt Ltd, Bangalore, India).

Results

Analysis of physiological traits

Analysis of variance was performed to compare the resistance to desiccation, starvation, heat shock with and without hardening and lipid content of both sexes of different geographic populations of Dananassae (Table 1). Highly significant differences were found among populations and between sexes in Dananassae. For convenience, we chose five populations from extreme north India (JU, DH, KG, DN and MD) as northern populations and five populations from extreme south India (KR, TR, ER, PC and YS) as southern populations for further analysis. Figure 2 shows comparison for physiological traits between the northern and southern populations. Desiccation resistance level was higher for northern populations whereas starvation resistance was higher in southern populations. Heat-shock resistance with and without hardening as well as lipid content was also higher in the south Indian populations. All the 45 populations were used to test for correlations between desiccation and each of other four physiological traits. Correlation coefficients were negative between heat shock (without hardening) and desiccation resistance (−0.79 and −0.82 for males and females, respectively). A negative correlation was found between heat shock (with hardening) and desiccation (−0.69 and −0.78 for males and females, respectively), and for desiccation resistance and lipid content (−0.75 and −0.81 for males and females, respectively). A positive correlation was found between lipid content and starvation resistance (0.86 and 0.82 for males and females, respectively). All the values of the correlation coefficients were highly significant, whether that be positive or negative.

Table 1.   Analysis of variance comparing differences among populations and between sexes for multiple stress resistance in populations of Drosophila ananassae.
TraitsSourced.f.Mean squareF
  1. ***< 0.001.

DesiccationPopulations4465.72912.77***
ResistanceSex1294 296.294 087 448.5***
LT 50 in hPopulation × Sex446683.7092 829.16***
Error3600.072 
StarvationPopulations441706.10162.61***
ResistanceSex11 536 969.614 688.16***
LT 50 in hPopulation × Sex4444 662.75426.82***
Error360104.64 
Heat shock (with hardening)Populations4410.048.43***
Sex1242203.36***
Population × Sex4418.3415.41***
Error3601.19 
Heat shock (without hardening)Populations4480.5555.55***
Sex1132.3091.24***
Population × Sex4437.9026.13***
Error3601.45 
Lipid contentPopulations440.0001110***
Sex10.0000867.81***
Population × Sex440.0000888***
Error3600.000011 
Figure 2.

 Population means for the traits compared between the south and north Indian populations of Drosophila ananassae.

Genetic variation in relation to latitude of origin

Environmental stress was influenced by the latitude of origin. All the 45 natural populations from the different geographic regions of the country (covering the regions from Kashmir to Kanniyakumari and Gujarat to Nagaland) were analysed for multiple stress responses. We investigated the relationship between resistance of physiological traits and latitude of origin in both sexes. A significant positive linear regression was found for desiccation resistance for both the sexes (Fig. 3 males: = 0.312 ± 0.26, R2 = 0.77 < 0.001; females: b = 0.291 ± 0.06, R2 = 0.76 < 0.001). For both sexes, a significant negative linear regression was found for the regression of starvation resistance on latitude (Fig. 4 males, = −0.39 ± 0.01, R2 = 0.98, < 0.001; females, b = −0.34 ± 0.012, R2 = 0.97, < 0.001). For heat shock with hardening resistance, a significant negative relationship was found for both sexes (Fig. 5, males, = −0.289 ± 0.14, R2 = 0.87, < 0.01, females, = −0.458 ± 0.23, R2 = 0.79, < 0.001). For heat shock without hardening, a significant negative relationship was found for both sexes (Fig. 6 males, = 0.47 ± 0.15, R2 = 0.85, < 0.001, females, = 0.44 ± 0.15, R2 = 0.85, < 0.01), and for lipid content, a significant negative relationship was found (Fig. 7 males, = −0.245 ± 0.05, R2 = 0.58, < 0.001; females, b = −0.0245, R2 = 0.376, < 0.001).

Figure 3.

 Relationship between desiccation resistance and latitude for 45 populations of Drosophila ananassae. Females (○) and males (•).

Figure 4.

 Relationship between starvation resistance and latitude for 45 populations of Drosophila ananassae. Females (○) and males (•).

Figure 5.

 Relationship between heat shock without hardening and latitude for 45 populations of Drosophila ananassae. Females (inline image) and males (inline image).

Figure 6.

 Relationship between heat shock with hardening and latitude for 45 populations of Drosophila ananassae. Females (□) and males (inline image).

Figure 7.

 Relationship between lipid content and latitude for 45 populations of Drosophila ananassae. Females (□) and males (inline image).

Analyses with climatic variables

Association among three the geographical parameters (latitude, altitude and longitude) and also for the three climatic factors (Tave, average annual temperature; Rave, average annual rainfall, and RH) and five physiological traits were assessed using multiple regression (Table 2). For desiccation, starvation, heat shock with and without hardening, and lipid content, both latitudinal and altitudinal associations were significant and coefficients of determination R2 were high. Climatic variables like average annual temperature and RH significantly affected all five physiological traits. Coefficients of determinations were also high for all five physiological traits. Principal Components Analysis (PCA) was applied to all the climatic factors and physiological traits on all 45 populations. PC1 accounted for 56% of the variation and PC2 for 14% of the variation (Fig. 8). Latitude and desiccation were positively correlated whereas heat shock with and without hardening, starvation and lipid content were negatively correlated with latitude. RH, rainfall and average temperature were also negatively correlated with desiccation and latitude.

Table 2.   Multiple regression analysis of five physiological traits as a simultaneous function of: (A) latitude, altitude and longitude; (B) Tave, Rave and relative humidity of origin of populations of Drosophila ananassae.
  1. RH, relative humidity.

  2. *< 0.05; **< 0.01; ***< 0.001.

(A) TraitsIntercept ± SEb1 ± SE (latitude)b2 ± SE (altitude)b3 ± SE (longitude)R2
Desiccation45.79 ± 0.28*0.434 ± 0.28**0.414 ± 0.0040.123 ± 0.230.56
Starvation175.50 ± 0.73***−0.333 ± 0.92*−0.184 ± 0.08*−0.144 ± 0.920.66
Heat shock (with hardening)78.72 ± 0.97***−0.67 ± 0.36 *−0.24 ± 0.05*−0.116 ± 0.070.40
Heat shock (without hardening)42.22 ± 0.58*−0.388 ± 0.23*−0.26 ± 0.02*0.191 ± 0.030.43
Lipid content578.84 ± 0.47*−0.56 ± 0.31*−0.201 ± 0.01*1.92 ± 0.050.45
(B) TraitsIntercept ± SEb1 ± SE (Tave)b2 ± SE (Rave)b3 ± SE (RH)R2
Desiccation46.64 ± 0.64**−0.82 ± 0.38*−0.018 ± 0.002−0.214 ± 0.02*0.38
Starvation805.36 ± 0.56***0.48 ± 0.89*−0.558 ± 0.050.302 ± 0.31*0.32
Heat shock (with hardening)42.09 ± 0.04*0.72 ± 0.02*−0.09 ± 0.030.267 ± 0.02*0.34
Heat shock (without hardening)56.09 ± 0.05*0.79 ± 0.38*0.240 ± 0.020.184 ± 0.03*0.28
Lipid content38.49 ± 0.16*0.41 ± 0.06*0.195 ± 0.030.447 ± 0.05*0.36
Figure 8.

 (a, b) Graphic presentation of the first two principal components from a PCA analysis of six climatic variables and five physiological traits from 45 populations of Drosophila ananassae. (a) explanation of abbreviations has already been given in Fig. 1. (b) LC, lipid content; ST, starvation; HS, hard; heat shock with hardening; HS, WT hard; heat shock without hardening; and DS, desiccation.

Discussion

In this study, desiccation resistance varies among populations of Dananassae. Populations from high latitudes survive longer than those from low latitudes. Sorensen et al. (2005) found a negative relationship between desiccation resistance and altitude in Dbuzzatii. Karan et al. (1998) measured desiccation and starvation tolerance along latitudinal transects in three drosophilid species (Dananassae, Dmelanogaster and Zaprionus indianus) of the Indian subcontinent. In each case, significant latitudinal clines were observed, with desiccation tolerance increasing and starvation tolerance decreasing with latitude. However, Arthur et al. (2008) found a lack of clinal variation for desiccation resistance in Drosophila simulans, and Davidson (1990) found no geographic variation in desiccation resistance between populations of Dsimulans collected from northern and southern Australia. Similarly, populations of Dmelanogaster (Hoffmann et al., 2001) and Dserrata (Hallas et al., 2002) from eastern Australia do not exhibit clinal pattern for desiccation resistance. These results contrast with the findings of this study and those reported earlier in Indian drosophilids (Karan & Parkash, 1998; Karan et al., 1998) where consistent clines occur, with desiccation resistance increasing with latitude. It appears that selective factors acting on desiccation resistance are inconsistent across continents, a situation that may be explained by the climatic conditions of the continent.

Kennington et al. (2001) have shown that genetic bases of differences in desiccation and starvation resistance among temperate and tropical populations of Dmelanogaster are complex. It is possible that adaptation to laboratory culture has led to an underestimation of the true genetic effects underlying differentiation between natural populations. The level of resistance to desiccation tends to be relatively high in populations from temperate areas compared to tropical ones, whereas the reverse is the pattern for starvation resistance (for review see Hoffmann & Harshman, 1999).

This study revealed a negative association between starvation resistance and latitudes in our populations. Populations from south India were more resistant to starvation than populations from north India. Arthur et al. (2008) have also reported a weak linear cline for female starvation resistance in Dsimulans. A weak association between starvation resistance and latitude has been found in female Dmelanogaster from eastern Australia (Hoffmann et al., 2001) and Drosophila kikkawai from India (Karan & Parkash, 1998). In all the mentioned cases, starvation resistance decreases with latitude. In contrast, there is no clinal pattern for starvation resistance in populations of Dmelanogaster collected from a latitudinal gradient in South America (Robinson et al., 2000), which again suggests that selection on starvation resistance may be inconsistent across continents. We found a significant positive correlation between starvation resistance and lipid content among Indian populations of Dananassae. Lipid content decreases with latitude. South Indian populations have a higher lipid content compared to north Indian populations. These data raise questions about the role of diet in maintaining fitness variations within and among populations. One possible explanation could be that flies from south Indian populations feed on a high carbohydrate diet, such as banana, mango and grapes, and the consumption of excess carbohydrate relative to metabolic requirements will lead to the deposition of high levels of body fat, whereas north Indian populations feed on fruits such as apple and plum. It is the case that north Indian fruits have less carbohydrate content than south Indian ones. Andersen et al. (2010) have studied the effect of protein and carbohydrate composition of larval food on thermal stress in Dmelanogaster. They report that flies reared on a high protein medium have higher heat and desiccation resistance than those reared on a carbohydrate-enriched medium. Deposition of a high level of body fat reduces viability in larval insects (Raubenheimer & Simpson, 1999), and the same is true for other animals, including humans (Simpson & Raubenheimer, 2005). Chippindale et al. (1996) scored lipid and starvation levels in different sets of lines selected for starvation or change in life-history traits. They report a correlation close to one between starvation and lipid level when all lines were considered. Ballard et al. (2008) have reported that starvation resistance is positively correlated with body lipid proportion in wild-caught Dsimulans populations.

In our study, a linear cline for heat shock with and without hardening was found in Dananassae. Populations from south India tend to be more resistant to heat shock, a finding that is consistent with the higher levels of heat stress likely to be encountered at low latitudes. In contrast, there is absence of variation within and between populations for heat resistance in Australian populations of Dsimulans (Arthur et al., 2008). This finding is consistent with studies on the restricted rain forest species Drosophila birchii (Griffiths et al., 2004). A lack of variation for heat resistance (measured as heat knock-down time) among and within populations of Dbirchii was also found. However, the absence of variation for heat resistance in Dsimulans contrasts with the findings for its widespread sibling species Dmelanogaster, which exhibits a linear cline along a similar latitudinal gradient (Hoffmann et al., 2002) and a higher level of within-population variation. Our results are similar to those of Hoffmann et al. (2002) in Dmelanogaster.

The Indian localities are highly variable in latitude and altitude, and there are significant seasonal variations moving from south to north. On the Indian subcontinent, thermal variations are also linked with changes in RH, along both altitudinal and latitudinal gradients. Several localities with higher elevations all along the south–north transect have lower RH and ambient temperature than many other localities. By contrast, in the Indian tropical peninsula, low altitude localities are characterized by ambient temperatures of 25–30 °C and high RH. Thus, Drosophila species and ectothermic insects face locality-specific environmental stress (Parkash & Munjal, 1999; Parkash et al., 2008). Sorensen et al. (2005) investigated multiple stress resistance in Dbuzzatii. Their results suggest that knock-down resistance to heat stress, desiccation resistance and Hsp 70 expression at a relatively severe stressful temperature are good examples of thermal adaptation in this species. They found no relationship between heat-shock survival and altitude. This resistance trait may not be very relevant ecologically as temperatures are not expected to change from 25 to 40 °C without warning. Even a gradual warming over a few hours gives the flies time to respond with countermeasures (i.e. to induce the stress response), which would be a different resistance trait. A strong effect of Hsp 70 expression on survival has been observed in many studies (Dahlgaard et al., 1998). Thus, it is possible that if heat-shock survival was measured after hardening to 38 °C, a positive relationship with altitude would have been observed because of the effect of expression levels of Hsp 70 at that temperature (Sorensen et al., 2001). In south India, the yearly average temperature ranges from 35 to 40 °C so in both assays (with and without hardening) populations from these places are more resistant to heat compared to north Indian populations where average annual temperature ranges from 13 to 20 °C. Collinge et al. (2006) have found no significant difference in heat resistance between high and low altitudinal sites in Dmelanogaster from eastern Australia. Although there is latitudinal variation for this trait (Hoffmann et al., 2002), selection on heat resistance may be weaker than on cold resistance (Gaston & Chown, 1999). This reflects the facts that climatic conditions at the higher end of the temperature range vary less than those at the lower end, which is reflected in patterns of variation among and within species for lower and upper thermal limits when species are collected along thermal transects (Addo-Bediako et al., 2000; Chown & Nicolson, 2004). Bubliy & Loeschcke (2005) have reported that the lines selected to survive severe heat shock after heat hardening probably evolved a physiologically different resistance mechanisms involving induced Hsps.

The present investigation provides evidence of variability in physiological traits because of changes in geographical and climatic variables across the Indian subcontinent. From the multiple regression analysis, the coefficient of determination (R2) of trait variability was higher as a simultaneous function of both the geographical parameters (altitude and latitude) and the climatic parameters (Tave and Rave). Geographical and climatic parameters are related and we may infer that annual average temperature and RH of the site of origin of Dananassae populations are the potential selective factors for the observed changes in physiological traits. Sarup et al. (2009) studied local adaptation of stress-related traits in Dbuzzatii and Dsimulans and observed that heat-shock resistance after hardening, desiccation, starvation resistance and Hsp expression seem to be the traits that are under the strong selection for local adaptation, as they show clinal variation and differences between populations in both species. In this study, desiccation resistance, starvation resistance, lipid content and heat shock with and without hardening seem to be the traits that are under strong selection for local adaptation, as they show clinal variation and differences among populations in both the sexes. Although climate, and especially temperature in these populations, generally varies with latitude and altitude, selection pressures in the field and mechanisms used for adaptation are very complex. Each population is faced with a unique combination of temperature and humidity varying over the day and over the year. Clear adaptive patterns were observed for desiccation, starvation, lipid content and heat shock with and without conditioning. To a greater extent, these results are in concordance with latitudinal patterns of tolerance to climatic stress (Karan et al., 1998; Hoffmann et al., 2002, 2003a; Sorensen et al., 2005), supporting the hypothesis of climatic adaptation. Overall, the present results provide further insight into the ecological importance of environmental stress and adaptation, and into the traits that are responding to environmental pressure.

Acknowledgments

Financial assistance in the form of Fast Track Young Scientist Research Project from the Department of Science and Technology, New Delhi to SS is gratefully acknowledged. The authors thank V. Loeschcke and J. G. Sorensen for their valuable suggestions during the course of this study and Pranveer Singh for his diversified collections of Dananassae flies. The authors also thank both the anonymous reviewers for their helpful comments on the original draft of the manuscript, Thomas Flatt, for his suggestions regarding statistical analysis of data, Rajiva Raman, Department of Zoology and Anita Singh, Department of English, Banaras Hindu University for checking language of the manuscript, R Bhanu Prakash and B V Suresh Kumar, camo Software for their help in Principal Components Analysis (PCA) of our data and Stephen Jones for editorial corrections in the manuscript.

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