Genetic variation in thermal tolerance among natural populations of Drosophila buzzatii: down regulation of Hsp70 expression and variation in heat stress resistance traits



  • 1 Thermal adaptation was investigated in the fruitfly Drosophila buzzatii Patterson and Wheeler. Two natural populations originating from a high- and a low-temperature environment, respectively, were compared with respect to Hsp70 (heat shock protein) expression, knock-down resistance and heat shock resistance.
  • 2 Three main hypotheses were tested: (i) The expression level of Hsp70 in flies from the high-temperature habitat should be down-regulated relative to flies from the colder habitat. (ii) Flies having higher Hsp70 expression levels should be weakened most by a hardening treatment and go faster into coma, as Hsp70 level reflects stress intensity, and therefore display reduced heat knock-down resistance. (iii) Heat shock resistance should be increased in the population with highest Hsp70 expression because the level of Hsp70 is positively associated with this trait.
  • 3 The results generally matched the hypotheses. Hsp70 expression was reduced in the high-temperature population. Knock-down resistance was higher in the high-temperature population and survival after heat shock was lower in the high-temperature population.
  • 4 This study showed genetic differences in thermal tolerance between populations, indicating that high temperature in nature may be an important selective factor. Moreover, knock-down resistance in this study seems to be a more relevant trait than standard heat shock resistance for identifying thermal adaptation in natural populations.


Exposure to a sub-lethal temperature increases survival after an otherwise potentially lethal temperature. This increase in tolerance is termed hardening. Heat shock proteins (Hsps), found in every organism in which they have been investigated, have for a long time been considered as being at least partially responsible for the observed increase in tolerance to thermal and other environmental stresses following hardening (Lindquist 1986; Parsell & Lindquist 1993; Feder & Hofmann 1999). Here, we refer to stress as an ‘environmental factor causing a change in a biological system, which is potentially injurious’ (Hoffmann & Parsons 1991). Recently, a relationship between heat stress resistance and Hsp70 expression has been demonstrated from laboratory ‘natural’ and artificial selection experiments using Drosophila (Bettencourt, Feder & Cavicchi 1999; Sørensen et al. 1999). From these experiments, it has become clear that Hsp70 expression and heat stress resistance are complex traits that can evolve both in concert (Dahlgaard et al. 1998), and independently of one another (Bettencourt et al. 1999; Lansing, Justesen & Loeschcke 2000). Changes in the expression level of Hsp70 in selection lines of Drosophila over time have been shown to depend on both the selection regime and the life stage selected (Sørensen et al. 1999; Lansing et al. 2000).

Inducible Hsps protect primarily against temperatures high enough to kill Drosophila in several minutes to hours. At temperatures warm enough to induce Hsp expression but insufficient to kill Drosophila rapidly, inducible Hsps may have few benefits and a variety of deleterious consequences such as reduced fecundity (Krebs & Loeschcke 1994) and retarded development (Feder et al. 1992). Hence, evolution at high temperatures, with long-term Hsp induction, might favour decreased expression of inducible Hsps. Evolution under conditions where Hsps are induced before an extremely high temperature might favour increased expression of inducible Hsps. Recent results of independent investigations showed that when laboratory ‘natural selection’ was allowed (where fecundity is a component of the selection target) at either constant temperatures (28 °C and 30 °C) or high fluctuating temperatures (35 °C or 38·2 °C for 6 h and then 25 °C each day), the expression of Hsp70 is reduced (Bettencourt et al. 1999; Sørensen et al. 1999). This reduction, or down regulation of the induced expression level, may allow the organism to avoid the harmful effects that have been found to be associated with Hsp70 expression, e.g. reduced cell growth rates (Feder et al. 1992) and reduction in productivity (Krebs & Loeschcke 1994). Detrimental fitness effects may be avoided by flies inhabiting high-temperature habitats if the expression pattern of Hsp70 in nature can be changed by natural selection (Sørensen et al. 1999). There has, however, been uncertainty as to how often Hsps are induced in nature and how important they should be considered in an ecological sense (Feder & Hofmann 1999; Feder, Roberts & Bordelon 2000). Furthermore, it has been suggested that an increased understanding of the importance of Hsps and their relevance to adaptation and stress resistance should include studies of natural populations (Feder & Hofmann 1999; Feder et al. 2000).

Bosch et al. (1988) and Brennecke, Gellner & Bosch (1998) studied various Hydra species and found the expression of some Hsps to be strongly reduced in a number of cases. The species with reduced expression of Hsps were less tolerant to heat stress and occurred only in stable environments. It is not obvious, however, whether this is adaptive or if these species lost the inducible thermal tolerance accidentally and were thus able to survive only in the more stable environments. By observing the differences among populations within a species we may be able to conclude more clearly the adaptive significance of different Hsp70 expression levels.

In order to test hypotheses on associations between environmental variation and Hsp70 expression as well as thermal resistance traits in nature, it is important that the environment in question is well characterized. Drosophila buzzatii Patterson and Wheeler is one Drosophila species that has a well-defined ecology. This species has been found to be strongly associated with cacti of the genus Opuntia(Barker & Mulley 1976). Breeding and adult feeding take place on or nearby cacti and preadult development occurs inside rotting cladodes. The strong association between D. buzzatii and Opuntia means that the thermal environment shaping the phenotypes can be considered to be that of the cacti. Previous studies of laboratory populations of this species have shown a positive correlation between the climate of origin and thermal resistance (Loeschcke, Krebs & Barker 1994), indicating adaptation to the thermal climate.

Here we report on heat hardening, thermal resistance and Hsp70 expression in two natural populations of D. buzzatii from Argentina. The two populations originated from habitats with very different temperature profiles. One of the populations was collected from a ‘lowland’ site where temperatures during the day can get high (known to be lethal within a few hours in the laboratory), and the other from a ‘mountain’ site that only very rarely experiences lethal temperatures (Table 1). Differences in several traits have been found in field flies from these two populations, suggesting there has been local adaptation to the different environments (Dahlgaard, Hassson & Loeschcke 2001). These traits were consistently different after the populations were brought back into the laboratory, indicating a genetic basis for these adaptations. Activity rhythm (measured as egg-laying) differed between the populations in the laboratory and corresponded to measures of activity in the field. Perhaps the behavioural difference arose as a means of thermal adaptation through avoidance of stressful temperatures (Dahlgaard et al. 2001). These populations thus provide a good system for studying thermal adaptation in nature with a focus on thermal resistance, heat hardening and associated Hsp70 expression.

Table 1.  Monthly averages of mean, maximum mean and minimum mean temperatures from three weather stations in Argentina. The sample site of La Rioja is located close to the lowland population of Catamarca. The sample site of the mountain population, Tilcara, is located between Salta and La Quiaca and temperature values for Tilcara are assumed to fall about midway between temperature values for these two sites. The data were obtained from the Argentinean Meteorological Services web-site (
Mean (°C)La Rioja282623201612121418232527
 La Quiaca13131210 6 4 4 6 9101213
Max. mean (°C)La Rioja353330282420202326303335
 La Quiaca202020201715151719202120
Min. mean (°C)La Rioja2120181510 5 4 710151820
 Salta16151411 7 4 3 5 8111515
 La Quiaca 8 7 6 2−3−6−7−5 0 2 5 6

Results from laboratory ‘natural’ selection experiments allow the investigation of predictions concerning the variation in Hsp70 expression and levels of heat stress resistance between the two populations. The three main hypotheses tested here are: (i) the expression level of Hsp70 in flies from the high-temperature habitat (the lowland site) should be down-regulated (i.e. reduced) relative to flies from the colder mountain site; (ii) if Hsp70 is a valid indicator of damage due to stress, flies with the highest Hsp70 levels prior to a subsequent thermal challenge should show the least resistance to this challenge. Therefore flies having higher Hsp70 levels should display reduced heat knock-down resistance since Hsp70 in itself seems to have little positive effect on this trait (unpublished results); and (iii) survival after severe heat shock should be increased in the population with highest Hsp70 expression level because the level of Hsp70 expression is positively associated with this trait (Krebs & Feder 1997; Dahlgaard et al. 1998).

Materials and methods

Origin of flies

The sample sites for populations of D. buzzatii used in this study are located in north-western Argentina: a lowland population near Catamarca (28°29′ S, 65°39′ W; 590 m above sea level) and a mountain population near Tilcara (23°35′S, 65°24′W; 2460 m above sea level). Temperatures at the two localities differ greatly, with Catamarca having a generally warmer climate. Temperature data from three weather stations were obtained from the web-site of the national meteorological service of Argentina. These were La Rioja (29°23′ S, 66°49′ W; 429 m above sea level), Salta (24°51′ S, 65°29′ W; 1221 m above sea level) and La Quiaca (22°06′ S, 65°36′ W; 3459 m above sea level). La Rioja is located closely to the Catamarca population and Tilcara is located about midway between Salta and La Quiaca (both geographically and altitudinally). The data shown in Table 1 include monthly means, maximum means and minimum means averaged over collections from 1961 to 1990. On Tenerife, the Canary Islands, Dahlgaard & Loeschcke (1997) have shown that sun-exposed cladodes can reach temperatures above 45 °C. However, the relation between air and cladode temperature is not known. We have measured 8–10 cladode and air temperatures every hour over consecutive days on Tenerife. Here a surprisingly good correlation between air temperature and the temperature of cladodes was found (Pearson’s correlation r = 0·89, P < 0·01). We therefore feel confident that not only air temperature but also the temperature of cladodes in Catamarca is higher than in Tilcara. Collection of D. buzzatii populations was carried out in March 1997 over 2 days, under dry and sunny weather conditions. The collection areas covered between 1000 and 2000 m2 at each location. Twenty-one buckets containing fermented chopped bananas were positioned in the shade next to pieces of rotting Opuntia cacti. Buckets were examined regularly and flies were collected with a net. Specimens of D. buzzatii were brought back to the laboratory. Wild caught females were placed in vials to establish isofemale lines which thereafter were maintained in three vials per line (9 lines from Tilcara and 35 from Catamarca) for nine generations, with 60 flies per line. Subsequently lines of each population were mixed to found two separate mass populations. Both isofemale lines and mass populations were maintained in a controlled temperature room at 25 ± 1 °C and 60% relative humidity in a 12/12 h light–dark cycle.

Maintenance of stocks

Each mass population was maintained in 15 bottles (200 ml) with 7 ml of instant Drosophila medium (Carolina Biological Supply, Burlington, NC, USA), with ≈20 pairs per bottle which were allowed to oviposit for 24 h before being discarded. Random mating was obtained by mixing adults among bottles in every generation.

Hsp70 expression after hardening

Flies less than 24 h old were collected, sexed under light CO2 anaesthesia and transferred to food vials at a density of 25 flies per vial. The flies were transferred to fresh vials after being kept for 2 days. On the fourth day, when flies were between 4 and 5 days old, the Hsp70 induction experiment was conducted. The hardening took place in glass vials in waterbaths. Each vial had a short foam stopper at the bottom to prevent immobilized flies from getting stuck in the vial. The top-stoppers were moistened with tap water and inserted fully to avoid desiccating the flies. The vials were placed in racks and spaced evenly to ensure homogeneous heating. The racks were placed in preheated waterbaths, with the water level exceeding the top-stoppers lower end. Two hardening temperatures were used: 37 °C and 39 °C for 1 h (preliminary tests revealed some, but low expression levels at 35 °C). The accuracy of the waterbath thermostats has been measured to approximately ±0·1 °C. Each treatment included the two populations, both sexes and five replicates, giving a total of 20 vials per treatment. After the treatment, flies were kept at 25 °C for 1 h and then stored at −70 °C. Later, flies in each vial were homogenized and the level of Hsp70 was measured using a monoclonal inducible Hsp70-specific antibody (7.FB). The enzyme-linked immunosorbent assay (ELISA) was conducted in five replicate microwell plates according to the protocol described in Dahlgaard et al. (1998) and Sørensen et al. (1999), after having verified the linearity of the response with increasing Hsp70 concentration. A standard was used in all plates to correct for plate variation. This standard was prepared from a mixture of males and females from both populations, hardened at 37 °C for 1 h. The standards were diluted three times compared with the samples.

Knock-down resistance

This technique was first used and described by Huey et al. (1992), and is different from the more traditional heat shock assay, where survival is the trait scored. In the knock-down assay, all flies survive and it is the time taken to be knocked down (i.e. become unconscious) which is measured. For testing heat knock-down resistance, adults (less than 1-day-old) were collected (males and females together) from both populations without the use of anaesthesia. Prior to testing, all flies were kept for 4 days at a density of ≈50 individuals per vial (equal number of both sexes). Flies were transferred to fresh vials on the second day of holding. The test was carried out in a knock-down tube at 40·2 ± 0·2 °C. The tube had seven baffles and was 82·5 cm long (including a 76 cm path for the flies to fall down) with an internal diameter of 9·4 cm. Heated water was circulated through a water jacket, which maintained the temperature required within the tube. For each run, ≈50 flies, equal number of males and females, were placed into a cylindrical container (length 7 cm, diameter 4 cm) positioned at the top of the tube made from a rough mesh to allow air circulation. The flies were allowed to settle for 30 s before the bottom of the container was opened. The tube was set directly beneath an overhead light. Flies stayed at the top because of positive phototaxis and negative geotaxis until they succumbed to the heat and rolled down the series of baffles reaching a collecting vial. This vial was replaced at 30-second intervals, and each individual fly was assigned the knock-down time corresponding to the 30-second interval of the respective collecting vial. Two traits were tested: basal knock-down resistance without hardening and knock-down resistance after hardening at 39 °C for 1 h. After the hardening treatment at 39 °C and one hour at 25 °C the flies had generally recovered, but Tilcara males had not regained full mobility. Since mobility is important for knock-down resistance, all the experimental groups hardened at 39 °C were allowed to recover for 2 h before being tested in the knock-down tube. Five separate runs were carried out for each of the four experimental groups (two populations × two treatments), alternating between groups for each successive run. Finally, flies collected in each 30-second interval were sexed and counted under CO2 anaesthesia.

Heat shock resistance

Heat stress resistance after hardening to 37 °C or 39 °C for 1 h was measured as survival after exposure to 41·1 °C for 1 h. Between hardening and heat shock a 1-h recovery period was allowed at 25 °C. The hardening and heat shock processes were carried out following the protocol used for Hsp70 expression described above. Five replicate vials of 25 flies of each sex, treatment and population were used. Heat stress resistance without prior hardening was also investigated in 10 replicate vials of each sex and population. Here the vials with flies were exposed directly to 41·1 °C for 1 h. In both experiments the flies were transferred to fresh food vials and allowed 24 h at 25 °C after the heat shock to recover before being scored as either dead or alive. Flies were considered alive if they were able to walk after being touched lightly with a brush.


To test for differences between populations and treatments anovas were performed with effects of population, sex and treatment kept as fixed. The analysis of heat knock-down was based on run means (five for each of four experimental groups) rather than indi-vidual scores to avoid a bias due to deviation of the distribution from normality. Since survival after heat shock was measured in ratios, the data were arcsin-square-root transformed to improve homogeneity of variances. The results were analysed using the GLM procedure in the SPSS program package (SPSS 1998).


Hsp70 expression

The results of the anova on Hsp70 expression are presented in Table 2 and the means are presented in Fig. 1. The hardening temperature had a significant effect on Hsp70 expression. Hardening at 37 °C yielded small differences in Hsp70 expression between the two populations. However, hardening at 39 °C resulted in an increased Hsp70 expression for both males and females of the Tilcara population (mountain) compared with the Catamarca population (lowland). This is seen as a significant population by hardening temperature interaction. The sex effect was not significant. Both population by sex and temperature by sex interactions were significant indicating differences in the reaction norms of Hsp70 expression.

Table 2.  Mean squares from anovas for: Hsp70 expression after hardening at 37 °C or 39 °C for 1 h (column 1), knock-down time with or without a hardening treatment at 39 °C for 1 h (column 2) and survival after heat shock after hardening at either 37 °C or 39 °C for 1 h (column 3) in Drosophila buzzatii males and females from Tilcara and Catamarca. See Materials and methods for details of treatments. Degrees of freedom in parentheses
Source (df)Hsp70 expressionKnock-down timeSurvival after heat shock
  • P = 0·06,

  • *

    P < 0·05,

  • **

    P < 0·01,

  • ***

    P < 0·00.

Population (1) 49764***212411*** 0·238*
Sex (1)    58   912 0·025
Hardening treatment (1)202953***  393014·39***
Population × Sex (1) 11866**  1664 0·135
Population × Hardening treatment (1) 47197***  4836 0·169
Sex × Hardening treatment (1) 22987***  6199 0·100
Population × Sex × Hardening treatment (1)   427    33 0·661***
Error (32)  1446  2907 0·046
Figure 1.

Hsp70 expression in percentage of standard (mean ± SE) in adult Drosophila buzzatii males (circles) and females (triangles). The Hsp70 expression level after hardening at either 37 °C or 39 °C is given for both the Catamarca (open symbols) and Tilcara population (filled symbols).

Knock-down resistance

The results of the anova on knock-down time are presented in Table 2. Figure 2 shows the mean knock-down time (±SE) for non-hardened and hardened males and females from both populations. The Catamarca population had a significantly longer knock-down time than the Tilcara population. Sex, hardening treatment and their interaction with one another and with population did not significantly affect knock-down resistance.

Figure 2.

Knock-down times in seconds (mean ± SE) for adult Drosophila buzzatii males (circles) and females (triangles). The knock-down times are given for non-hardened flies and for flies hardened at 39 °C for 1 h from both the Catamarca (open symbols) and the Tilcara population (filled symbols).

Heat shock resistance

The results of the anova on survival after heat shock are presented in Table 2. Figure 3 shows mean survival rate (±SE) for males and females from both populations hardened at either 37 °C or 39 °C for 1 h. After hardening at 37 °C no difference was found either between the sexes or between the populations. After hardening at 39 °C, survival after heat shock was lower for all groups. At this temperature, females from the Tilcara population had a survival rate exceeding the corresponding rate of the Catamarca females, whereas no difference was found in the survival rate for males. This is seen as a marginally significant population by hardening temperature interaction.

Figure 3.

Survival rate to heat shock after hardening (mean ± SE) in adult Drosophila buzzatii males (circles) and females (triangles) for both the Catamarca (open symbols) and Tilcara population (filled symbols). The y-axis scale is arcsin-square-root transformed.

With no hardening the Tilcara population survived better than the Catamarca population in both sexes and within populations females survived better than males (anova, population effect: F1,35 = 4·7, P < 0·05; sex effect: F1,35 = 16·4, P < 0·001; population × sex effect: F1,35 = 0·87, P = 0·36; data not shown).


The hardening treatments performed at both 37 °C and 39 °C induced high levels of Hsp70. A treatment of 35 °C under the same circumstances also induced Hsp70, but at low levels (about 20% of standard), indicating that 35 °C is closer to the threshold temperature for induction of Hsp70 in both populations. Hardening at 37 °C led to similar levels of Hsp70 expression in the two populations. However, hardening at 39 °C led to increased levels of Hsp70 in both males and females of the Tilcara population (mountain) compared to the Catamarca population (lowland). Assuming that Hsp70 expression is correlated to resistance towards stressful temperatures in nature, this result does not seem intuitive, as the Catamarca population is from the habitat with higher mean temperatures (Table 1). As both Hsp70 expression (Krebs & Bettencourt 1999) and thermotolerance (Krebs, Feder & Lee 1998) decline with adult age, population differences could be due to different rates of ageing in the two populations. However, in the laboratory the flies are maintained at the same constant temperature and only very small differences in development time were observed between the two populations. Therefore we consider it unlikely that differences in Hsp70 expression are caused by differences in physiological age. Increased heat shock protein expression in the Tilcara population after exposure to 39 °C may be interpreted as an emergency response and a greater sensitivity to increasing temperatures for this population. This population has rarely or never been exposed to very high temperatures in nature (Table 1). Consequently when exposed to extreme temperatures in the laboratory, the Tilcara population experiences the imposed high temperature as more stressful than the Catamarca population. This is also what we predicted on the basis of results from laboratory selection lines, where selection mimicked natural selection in a high-temperature habitat (Sørensen et al. 1999). Those data suggested that long-term exposure to elevated temperatures results in selection for reduced Hsp70 expression as a consequence of fitness costs associated with expressing Hsp70 (Feder et al. 1992; Krebs & Loeschcke 1994). This is supported by the observations that increased basal resistance can evolve without a simultaneous increase in the expression level of Hsp70 (Bettencourt et al. 1999; Lansing et al. 2000) indicating that there might be selection for basal resistance at the expense of inducible resistance.

We found that the pattern of Hsp70 expression was not identical for the sexes (Fig. 1). For males of both populations it seems that the increase in Hsp70 expression with temperature is less steep than that for females of the corresponding population. This might indicate that maximum Hsp70 expression occurs at a lower hardening temperature in males than in females. The levels of Hsp70 expression (low at 35 °C and higher at 37 °C and 39 °C) measured by our laboratory in D. buzzatii resemble those of D. mojavensis more so than those of two temperate species, D. melanogaster and D. simulans, measured by Krebs (1999). Drosophila mojavensis is a species that inhabits a variety of cacti hosts in the Sonoran Desert. This species therefore experiences high temperatures just as D. buzzatii does. The similarity in Hsp70 expression pattern thus seems to correspond to the climate of origin of the populations.

The results of Hsp70 expression measurements presented here underline the importance of determining the Hsp70 expression reaction norm in contrast to measuring at just a single hardening temperature. From selection experiments it seems that Hsp70 expression reaction norms might also be displaced (Bettencourt et al. 1999). When choosing just a single hardening temperature, one might pick a temperature where reaction norms cross and populations consequently seem alike even when large differences exist at other parts of the reaction norm.

The comparison of survival in non-hardened flies after heat shock in a waterbath could be viewed as a test of the basal heat resistance level. In this test the Tilcara population in general survived better than that from Catamarca, and within populations females survived better than males. In the waterbath, heating is rapid which limits the induction of Hsps as the expression is repressed at the extreme ‘high end’ of the temperature scale. Western blotting performed after a 1-hour treatment at stressful temperatures followed by a 1-hour recovery period at 25 °C revealed no detectable level of Hsp70 after exposure to 42 °C and only very little after exposure to 41 °C (J. G. Sørensen and J. Dahlgaard, unpublished data). This suggests that Hsp70 is probably not responsible for the increased survival in the Tilcara population or in females compared with males when tested without hardening.

For heat shock after hardening, the Tilcara population showed significantly higher levels of survival than the Catamarca population. This population difference was most pronounced after hardening occurred at 39 °C where females from the Tilcara population had higher survival. The highly significant three-way interaction (population × sex × hardening temperature) and the significant population effect in the anova were due to the increased survival at 39 °C of females from Tilcara. Increased survival in the Tilcara population after hardening and exposure to a potentially lethal temperature was probably a side effect of the high level of induced Hsp70 in this population. The reason that Tilcara males did not experience a survival benefit from their increased Hsp70 expression as females did was probably due to the fact that hardening at 39 °C itself was stressful to the males. After the period of 1 h at 25 °C before the heat shock treatment, it was observed that in general flies had recovered, but Tilcara males had not regained full mobility. This suggests that they suffered considerably from the hardening for a longer period of time. The cumulative effect of the first and the second heat exposure could therefore be more severe for males of the Tilcara population. Thus, as Tilcara males were probably in a worse condition than Catamarca males, a relatively poorer survival would be expected. On the other hand, the fact that survival in Tilcara and Catamarca males did not differ significantly may actually indicate a hardening response in the Tilcara males.

When knock-down resistance to heat was tested, Catamarca flies performed better than Tilcara flies. These results differ from the heat shock survival results, supporting observations in D. melanogaster that heat knock-down resistance and heat shock resistance are different traits (Hoffmann et al. 1997). Clearly the Tilcara population does not benefit with respect to knock-down resistance from its increased Hsp70 expression, as it did for survival to heat shock. Other hsp genes that can explain differences in knock-down resistance have been identified. Changes in expression of heat shock genes (hsp68 and hsr-Omega) have been shown to correlate with selection for knock-down resistance in D. melanogaster (McColl, Hoffmann & McKechnie 1996).

The pattern of knock-down resistance correlates well with the thermal history of the two populations investigated here. Flies from the warm lowland population show significantly extended knock-down duration. The differences found in this trait, like several others (Dahlgaard et al. 2001), suggests that natural selection might be responsible for the differences between the populations and that the differences are due to thermal adaptation.

Survival after heat shock was highest in the Tilcara population, which was from the colder environment. This may imply that this trait is not under strong selection in natural environments or that adaptive changes in Hsp70 expression in nature result in correlated changes in survival after heat shock that at first appear puzzling. Evidence from earlier laboratory experiments suggests that heat-adapted lines should indeed be more resistant to heat shocks without hardening (Cavicchi et al. 1995). The Tilcara (mountain) population, however, performed surprisingly well in the waterbath heat shock experiment without hardening.

The tolerance differences found between the Catamarca and the Tilcara population might be due to the fact that the mountain location also represents a stressful environment. While it is not as hot an environment as Catamarca is, it might be a more variable one, where populations risk exposure to other climatic extremes such as cold. This could have led to the Tilcara population becoming more generally resistant. In the lowland, it may be that high temperature is the most important environmental selective factor to which the Catamarca population is specifically adapted by having a high level of knock-down resistance.

Stratman & Markow (1998) compared the heat stress resistance of four species of desert Drosophila. Using a heat shock assay they found large differences between the species that in their natural habitats have overlapping ranges and experience the same thermal environment. The least resistant desert species had a resistance level comparable with the non-desert species included in the study. If knock-down resistance was important as an indicator of adaptation to natural temperature stress as suggested here, measurement of this trait would have shown a higher resistance in all desert species compared with the non-desert species. Furthermore, differences in expression of Hsp70 in the desert species could have indicated whether the differences observed in heat shock resistance could be due to differences in dependence on basal vs inducible resistance as a means of adaptation.

In D. buzzatii as well as in D. melanogaster, it appears that whether or not you will find a warm adapted population in nature also to be heat resistant in the laboratory depends on the heat resistance trait measured. Our results suggest that the knock-down trait might be a better indicator of adaptation to natural high-temperature environments than the traditionally used heat shock assay. Until more is known about the relationship between the different heat resistance traits both should be examined during identification of heat resistant lines or populations.

The populations studied here are separated by a distance of 550 km. However, our data suggest that the altitude difference of 1900 m and thereby the differences in the thermal environment experienced at each locality appear to be responsible for the strong ecological and genetic differentiation that exists between the populations. This suggests that relevant thermal resistance traits are important and under strong selection in nature.

We have shown that Hsp70 expression is decreased and that knock-down time is increased in the high-temperature Catamarca population as compared to the Tilcara population. Our results suggest that differences in various temperature-related traits occur between populations in nature. Furthermore, because direct or correlated changes in nature are similar to those from artificial selection experiments in the laboratory, it is suggested that these changes are likely to be adaptive under natural conditions.


We are grateful to Dr Susan Lindquist for kindly providing the 7.FB antibody, to E. Hasson and J. S. F. Barker for collecting the flies, to Miriam Hercus, M. E. Feder and an anonymous reviewer for helpful comments on the manuscript. We also thank Trine Gammelgaard for technical help, the Argentinean National Meteorological Service for providing the temperature data and the Carlsberg foundation for financial support (No. 990338).

Received 13 September 2000; accepted 20 October 2000