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Keywords:

  • altitudinal gradient;
  • desiccation resistance;
  • heat-shock protein;
  • knock-down resistance;
  • starvation resistance

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Multiple stress resistance traits were investigated in the cactophilic fly Drosophila buzzatii. Adults from seven populations derived from North-Western Argentina were compared with respect to traits relevant for thermal stress resistance and for resistance to other forms of environmental stress. The populations were collected along an altitudinal gradient spanning more than 2000 m in height, showing large climatic differences. The results suggest that knock-down resistance to heat stress, desiccation resistance and Hsp70 expression at a relatively severe stressful temperature best reflect thermal adaptation in this species. Furthermore, cold resistance seemed to be of less importance than heat resistance, at least for the adult life stage, in these populations. Clinal variation in thermal resistance traits over short geographical distances suggests relatively strong adaptive differentiation of the populations. This study provides the first evidence for altitudinal differentiation in stress-related traits, and suggests that Hsp70 expression level can be related to altitudinal clines of heat-stress resistance.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Geographical variation in traits related to fitness is often the result of adaptive evolution. Especially strong support comes from clinal variation which suggests a contribution of directional selection to the differentiation among populations. Geographic gradients are of special interest for the study of climatic adaptation because the climate strongly varies with geographical variables. Parallel clines have been identified for different species including several species of Drosophila and on different continents for some traits (e.g. size, allozyme frequencies and temperature resistance) (Hallas et al., 2002; De Jong & Bochdanovits, 2003) supporting a hypothesis of local adaptation, even for adaptation to environmental stress (Hoffmann et al., 2003a). Several environmental factors may impact on the physiology of individuals, but temperature is thought to be one of the strongest and thereby of great selective importance (Clarke, 2003; Hoffmann et al., 2003a).

Although temperature is likely to have 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. Annual mean temperature is closely related to latitude and altitude, but probably not as strong a driving agent of thermal selection as extreme upper and lower variables. A few studies have addressed this question looking at different environmental and thermal characteristics, e.g. maximum and minimum temperatures and rainfall parameters. A study of Drosophila melanogaster found heat knock-down resistance to be related to maximum temperatures in the warmest month and cold shock survival to be related to minimum temperatures in the coldest month (Hoffmann et al., 2002). Other studies have also suggested that maximum and minimum temperatures are more important for thermal adaptation than annual mean temperature (Davidson, 1988; McColl & McKechnie, 1999; Anderson et al., 2003).

Another important issue for successfully identifying adaptation is the relevance of the resistance traits examined. It is difficult to precisely determine the thermal environment that Drosophila adults and larvae experience in the field and therefore it is difficult to determine if a population is adapted to temperature. Drosophila buzzatii is closely dependent on cacti, especially of the Opuntia genus, for breeding and feeding and the temperature to which preadult individuals are exposed are thus better known (Sørensen et al., 2003). Probably, behavioural avoidance plays a crucial role for the adult life stage which makes the thermal exposure even more difficult to determine (Feder et al., 2000). However, it is widely accepted that thermal selection is of major importance and that Drosophila are likely to be exposed to stressful environments and thus adaptation by some mechanism is expected (Feder, 1997; Gibbs et al., 2003; Hoffmann et al., 2003a). Multiple assays have been used in various laboratories looking at thermal performance at both the high and low end of the temperature scale (see Hoffmann et al., 2003a). For cold adaptation, an ecologically important trait for identifying adaptive variation among populations of small insects has been suggested to be chill-coma recovery (Gibert et al., 2001; Hoffmann et al., 2002). Temperate species and populations show faster recovery to chill induced coma than do tropical species and populations (Gibert et al., 2001; Hoffmann et al., 2002). Measurements of adult survival have shown less clear patterns (reviewed in Hoffmann et al., 2003a). For example, survival to high temperature after heat exposure (Hsp induction) was strongly related to Hsp70 expression, which itself was inversely related to high temperature adaptation in adult D. buzzattii (Sørensen et al., 2001). For heat adaptation, knock-down resistance has been suggested to be important and to correlate with natural adaptation to high temperature environments (Sørensen 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). However, examples exist of adaptive patterns in desiccation resistance (Karan et al., 1998; Hoffmann et al., 2003b).

At the molecular level, candidate genes for adaptation are available for study. Clinal variation has been identified in allele frequencies in genes that are potentially important for adaptation to environmental stress (Bettencourt et al., 2002; Frydenberg et al., 2003). Especially, the heat-shock genes (HSPS) are thought to play an ecological and evolutionary role in adaptation (e.g. McColl et al., 1996), but genes with yet unknown function might also be important for the genetic basis of thermal adaptation (Sørensen et al., 2003; Norry et al., 2004). Not only allelic variation in the structural genes themselves but also their regulation seem to be related to climatic stress. For example, expression levels of the major inducible heat shock protein in Drosophila, Hsp70, have been related to temperature adaptation (Bettencourt et al., 1999; Sørensen et al., 2001).

Here, we investigated an altitudinal gradient spanning more than 2000 m in altitude. Compared with latitudinal clines, altitudinal clines can result from climatic differences over much shorter geographical distances (Dahlgaard et al., 2001; Sørensen et al., 2001). If clinal variation is observed on an altitudinal scale, it would suggest strong natural selection. Furthermore, the geographical distances among populations are much smaller than would be the case in latitudinal gradients with comparable climatic differences. Thus, relatively small differences in light regimes are found among populations. This is an advantage in interpreting results as many Drosophila species exhibit various adaptive plastic reponses to day-length (e.g. Goto & Kimura, 1998). By measuring several stress resistance traits, we expect to identify the selectively important climatic factors under natural conditions, i.e. the traits showing clinal variation with altitude. More specifically, we expect the heat induced Hsp70 expression level to be positively correlated with altitude of origin of population (Sørensen et al., 2001). As desiccation and high temperature covary in the wild, desiccation, like high temperature resistance, is expected to have a negative relation with altitude. Starvation resistance can be expected to increase with altitude if the feeding resources for D. buzzatii are much less abundant in highland than in lowland populations (Hasson et al., 1995). In addition, several measures of resistance to heat stress, as opposed to chill coma recovery, may be expected to be negatively correlated with altitude as suggested by a study on two populations from different altitudes (Sørensen et al., 2001).

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Populations used

Adult flies were collected over banana bait at seven localities of north-western Argentina in mid-April 2003. Cultures in mass were set up using 40–100 wild flies for each locality sampled. Inseminated females of the laboratory G2 generation were transferred from mass cultures to individual vials to set up isofemale lines. Drosophila buzzatii was identified by examining the male genitalia (Vilela, 1983). After seven laboratory generations (when all lines were checked for species), about 20 isofemale lines of D. buzzatii per population were crossed, and a mass population was established for each locality sampled. All stocks were maintained at 25 °C, with five bottles per population and 50–100 flies per bottle on instant Drosophila medium (Carolina Biological Supply, Burlington, NC, USA). The collected populations of D. buzzatii were: Tafi (26°36′S, 65°52′W; 2300 m above sea level), Quilmes (26°28′S, 66°02′W; 1855 m above sea level), Cafayate (26°07′S, 65°58′W; 1660 m above sea level), Trancas (26°08′S, 65°11′W; 879 m above sea level), San Luis (33°25′S, 66°25′W; 709 m above sea level), Chumbicha (28°53′S, 65°16′W; 401 m above sea level), Santiago de Estero (27°48′S, 64°18′W; 202 m above sea level). The population from Quilmes was only available for the tests of knock-down resistance, chill coma recovery and Hsp70 expression at 38 °C.

Climate of population origin

Data on average monthly temperatures and rainfall is available for a number of locations from the Argentinean Meteorological Services web-site (http://www.meteofa.mil.ar). These data show a clear relation between altitude and temperature calculated as monthly means, maximum means and minimum means (see also Sørensen et al., 2001). The data suggests that the monthly maximum means vary more with altitude than the monthly minimum means. From these data, it is expected that natural selection for heat resistance would be more important than cold resistance. Moreover, the data shows a generally dry winter period, where the rainfall is below 10 mm per month during 3–5 months. Marked differences (up to twofold) exist between the levels of rainfall during the remaining part of the year, but with no clear relation to altitude. Climatic patterns thus provide the possibility for both altitudinal adaptation for some stress traits and adaptation to local conditions for other traits.

Experimental protocols

For all experiments flies less than 24 h old were collected, sexed under light CO2 anaesthesia and transferred to vials with an agar–sugar–yeast food medium. On day 2 they were transferred to fresh food vials. Flies were aged to between 4 and 5 days old before being used for the experiments unless otherwise stated.

Desiccation

At age 4–5 days flies were transferred to empty food vials, with 10 flies per vial and five vials per sex and population. Six populations were tested for desiccation resistance (all except Quilmes). The vials were placed in a desiccator for 39 h, where after the vials were scored for living flies. Flies able to walk were considered alive.

Starvation

Freshly emerged flies (less than 18 h) were sexed under CO2 anaesthesia and transferred to agar vials (to provide access to water), with 10 flies per vial and five vials per sex and population. Six populations were tested for starvation resistance (all except Quilmes). The flies stayed in the vials for 5 days (120 h) and were then scored for survival (ability to walk).

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 preheated water baths with the water above the lower end of the stoppers. One group was hardened at 36.5 °C for 1 h; followed by 1 h at 25 °C to allow the flies to recover before being heat shocked 1 h at 41.5 °C. The other group was directly exposed to 40.5 °C for 1 h. In each group, 20 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 before survival (ability to walk) was scored.

Knock-down resistance

The resistance to heat knock-down was measured without prior heat hardening. The experiment was carried out in a knock-down tube (67 cm long, 10 cm diameter) at a constant temperature of 37.0 ± 0.1 °C (Huey et al., 1992). Flies were placed in the knock-down tube, and collected at the bottom of the tube every 30 s. We used males only. Knock-down time was recorded for approximately 30 males per population.

Hsp70 expression

Induction of Hsp70 took place as described for heat-shock survival. Two temperatures were used: 36.5 °C and 38 °C for 1 h succeeded by 1 h recovery at 25 °C before being frozen at −80 °C. For each temperature three replicates of 10 flies per sex and population were measured. At 38 °C all seven populations were assayed, but only six at 36.5 °C (all except Quilmes). Subsequently, flies were homogenized and the level of Hsp70 expression was assayed by Enzyme-Linked ImmunoSorbent Assay (ELISA) using the monoclonal antibody 7.FB (Velazquez et al., 1980, 1983), which is specific for the inducible Hsp70 in D. buzzatii (Sørensen et al., 1999). Each replicate was run on a 96 microwell ELISA plate and consisted of a sample from each population, sex and induction temperature (total 26 samples). All samples were measured in triplicates. The grand mean of each replicate was standardized to the grand mean of replicate one. The ELISA procedure is described in detail in Sørensen et al. (1999).

Chill coma recovery

Chill coma recovery (David et al., 1998) was measured following the protocol of Hoffmann et al. (2002). Vials with single flies of age between 4 and 5 days were cooled to 0 °C in a water/ice mixture. After 12 h at 0 °C the vials were moved back to 25 °C and chill coma recovery time was scored. Flies were considered as recovered when standing up. For each of the seven populations around 10 flies of each sex were scored.

Statistical analyses

For all resistance traits we performed anova's with population and sex as fixed factors (except knock-down where only population was analysed). Subsequently, we performed linear regression of resistance on each sex separately to evaluate the effect of altitude. The desiccation, starvation and heat-shock survival data was all arcsin-square-root transformed to improve normality and homogeneity of variances, as resistance was calculated as survival proportion. Knock-down data was log10 transformed to improve homogeneity of variances and the remaining data was untransformed. All statistical analyses were performed using the SPSS software package (SPSS, 2001).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

For desiccation resistance the anova revealed a significant effect of population and sex with no significant interaction between these factors (Table 1). For both sexes a significant negative linear regression was found between altitude and desiccation resistance [Fig. 1, linear regression, males (n = 30): r2 = 0.20, P < 0.05, b = −1.6E-04 ± 0.6E-04 (SE); females (n = 30): r2 = 0.30, P < 0.01, b = −2.1E-04 ± 0.6E-04 (SE)].

Table 1. anova results (mean squares) for tests of resistance and Hsp70 expression in D. buzzatii from Argentina. See Materials and methods for details of treatments.
SourceDesiccationStarvationHeat-shock survivalKnock-downHsp70 expressionChill coma recovery
HardeningNo hardening36.5 °C38 °C
  1. Degrees of freedom in parentheses.

  2. *P < 0.05, **P < 0.01, ***P < 0.001.

Population0.483*** (5)1.023*** (5)0.047 (5)0.110** (5)1.858*** (6)0.005 (5)0.023*** (6)234.3*** (6)
Sex1.086*** (1)0.722*** (1)0.496*** (1)0.017 (1) 0.247*** (1)0.286*** (1)19.3 (1)
Population × sex4.98E-02 (5)8.34E-02 (5)0.014 (5)0.030 (5) 0.004 (5)0.003 (6)89.4 (6)
Error3.58E-02 (48)5.79E-02 (48)0.030 (48)0.024 (48)0.175 (175)0.004 (24)0.004 (28)42.5 (152)
image

Figure 1. Desiccation resistance measured as survival (±SE) of flies without access to water (for 39 h) vs. altitude. Regressions are significant for both sexes (P = 0.014 for males and P = 0.002 for females). Females (filled symbols) and males (open symbols).

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anova of starvation data revealed a significant effect of both population and sex, with no significant interaction between these factors (Table 1). For both sexes a significant positive linear regression was found for the regression of starvation resistance on altitude [Fig. 2, linear regression, males (n = 30): r2 = 0.33, P < 0.001, b = 3.3E-04 ± 0.9E-04 (SE); females (n = 30): r2 = 0.44, P < 0.001, b = 2.8E-04 ± 0.6E-04 (SE)].

image

Figure 2. Starvation resistance measured as survival (±SE) of flies without access to food (for 120 h) vs. altitude. Regressions are significant (P < 0.001) for both sexes. Females (filled symbols) and males (open symbols).

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Heat-shock survival was measured with and without prior hardening. anova showed a significant effect of population only for the non-hardening treatment, whereas only sex was significant in the hardened samples (Table 1). No significant linear regressions were found between heat-shock resistance and altitude in any of the resistance tests (Fig. 3a, with hardening, linear regression, males (n = 30): r2 = 0.05, n.s., b = 0.5E-04 ± 0.4E-04 (SE); females (n = 30): r2 = 0.06, n.s., b = 0.6E-04 ± 0.4E-04 (SE); Fig. 3b, without hardening, linear regression, males (n = 30): r2 = 0.01, n.s., b = 0.1E-04 ± 0.4E-04 (SE); females (n = 30): r2 = 0.06, n.s. 84, b = 0.6E-04 ± 0.4E-04 (SE)].

image

Figure 3. Heat-shock resistance measured as survival to heat (±SE) vs. altitude. Regressions are non significant. Females (filled symbols) and males (open symbols). (a) Flies tested for resistance after a 1 h heat hardening treatment. (b) Flies were tested without prior heat hardening.

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For knock-down time anova showed a significant effect of population (only males were studied here, Table 1). A significant negative linear regression was found by regressing heat knock-down resistance on altitude [Fig. 4, linear regression, males (n = 182): r2 = 0.26, P < 0.001, b = −0.12 ± 0.02 (SE)].

image

Figure 4. Knock-down resistance of males measured as time to heat induced coma (±SE) vs. altitude. Regression is significant (P < 0.001).

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The heat induced Hsp70 expression data was analysed with two-way anova using population and sex as fixed factors for each heat hardening treatment. Only sex was found to be significant when using an induction temperature of 36.5 °C. In contrast, both population and sex showed significant effects when using a higher induction temperature of 38 °C (Table 1). After the milder heat treatment of 36.5 °C no relation between altitude and expression level was found (Fig. 5a, 36.5 °C, linear regression, males (n = 18): r2 = 0.04, n.s., b = −0.2E-04 ± 0.3E-04 (SE); females (n = 18): r2 = 0.03, n.s., b = 0.1E-04 ± 0.2E-04 (SE)]. Significant positive linear regressions were found between Hsp70 expression and altitude in both sexes after exposure to 38 °C [Fig. 5b, 38 °C, linear regression, males (n = 21): r2 = 0.38, P < 0.01, b = 0.6E-04 ± 0.2E-04 (SE); females (n = 21): r2 = 0.38, P < 0.01, b = 0.8E-04 ± 0.2E-04 (SE)].

image

Figure 5. Hsp70 expression (measured as absorbance) after exposure to heat (±SE) vs. altitude. Females (filled symbols) and males (open symbols). (a) Hsp70 expression induced by 1 h at 36.5 °C. (b) Hsp70 expression induced by 1 h at 38 °C. Regressions are significant (P = 0.003) for both sexes at 38 °C but not at 36.5 °C.

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Chill-coma recovery was analysed with anova using population and sex as fixed factors. anova showed a significant effect of population and no effect of sex or sex-by-population interaction (Table 1). In both sexes there was no significant regression of chill coma recovery time on altitude [Fig. 6, linear regression, males (n = 81): r2 = 0.01, n.s., b = 1E-05 ± 0.001 (SE); females (n = 85): r2 = 0.01, n.s., b = −0.001 ± 0.001(SE)].

image

Figure 6. Cold resistance measured as chill coma recovery time at 25 °C (±SE) of flies kept at 0 °C (for 12 h) vs. altitude. Regressions are not significant for both sexes. Females (filled symbols) and males (open symbols).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

A negative relationship was found between desiccation resistance and altitude. Given that high temperature is a cause of desiccation in the wild, the positive altitudinal cline we found for desiccation resistance is consistent with our climatic gradient where temperatures differ markedly, especially for maximal temperature during summer. During the winter, the geographical area where the populations were sampled is characterized by very limited rainfall. Therefore, a generally high desiccation resistance could be expected to be selected even in winter.

The positive relationship we found between starvation resistance and altitude would be expected if feeding resources for D. buzzatii are much less abundant in highland than in lowland populations (Hasson et al., 1995). However, other explanations are possible. In particular, starvation resistance might not be coupled with altitude itself but with size, with larger size allowing storage of higher amounts of lipid energy. Size was not measured in this study, but is expected to increase with increasing latitude and altitude. In D. buzzatii, latitudinal clines in size or inversion frequencies (affecting size) have been found in Australia and Argentina (Knibb & Barker, 1988; Norry et al., 1995; Loeschcke et al., 2000). In addition, a study with four populations of D. buzzatii also found wing size to increase with altitude (Norry et al., 2001).

No relationship was detected between heat-shock survival and altitude. Although the climate varies predictably with altitude and populations are expected to be adapted to this climate, only weak selection is expected to take place for this resistance trait in the wild. This resistance trait might not be very relevant ecologically as temperatures are not expected to change from 25 °C to 40 °C without warning. Even a gradual warming (or cooling) over a few hours would give the flies time to prepare countermeasures (i.e. to induce the stress response) which would be a different resistance trait. No clear pattern was found in the assay of survival with no hardening, underlining the complexity of the trait and the local selection that might occur. The hardening treatment took place at 36.5 °C which was one of the temperatures used to induce Hsp70 expression. In both of these assays relatively little difference was observed among populations. Compared with the results of the non-hardened heat-shock survival assay, the survival frequencies seem much more consistent and stable after hardening at 36.5 °C. This could be because of the strong effect of Hsp70 levels (that did not vary much among populations) induced by the hardening. A strong effect of Hsp70 expression on survival has been observed in many studies (e.g. 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 due to the effect of expression levels of Hsp70 at that temperature (Sørensen et al., 2001).

For knock-down resistance a negative relationship with altitude was found. This confirms the hypothesis that this trait is ecologically relevant for heat adaptation (Sørensen et al., 2001). Other studies also found this trait to be ecologically relevant exhibiting adaptative variation (for review see Hoffmann et al., 2003a). The mechanisms and genes involved in heat knock-down resistance are only partially known (McColl et al., 1996; McKechnie et al., 1998), and have been further explored using a QTL approach (Norry et al., 2004). The trait responds to a hardening treatment, but with a smaller benefit to resistance than is observed for heat-shock survival (Sørensen et al., 2001). Possibly molecular chaperone genes other than hsp70 are the primary heat-shock genes involved in resistance for this trait. hsp68 and hsr-omega respond to selection for heat knock-down resistance indicating that these genes have a role in resistance (McColl et al., 1996; McKechnie et al., 1998). Interestingly, a marked adaptive change in this trait controlled by the circadian rhythm has been observed in a heat adapted population of D. buzzatii (Sørensen & Loeschcke, 2002). The change occurred fast just prior to the expected activity period for the heat adapted population. The response was absent in another population collected from a less heat exposed environment and was not related to expression of Hsp70 (Sørensen & Loeschcke, 2002). Thus, much is still to be learned about the mechanisms behind this ecologically important trait.

Hsp70 expression was measured after induction at two temperatures. No differences were observed at the lower temperature of 36.5 °C. This suggests that the first part of the reaction norm for Hsp70 expression level with temperature is alike among populations. Induction temperature seems thus not to be a major target of selection. At the higher temperature of 38 °C a positive relationship was observed with altitude. The climatic data and the results of the knock-down resistance assay support the hypothesis that these populations were exposed and responded adaptively to high temperatures in the field. However, we observed a decreased Hsp70 expression with increased temperature. High levels of Hsp70 expression seem not to be a prerequisite for thermal adaptation, on the contrary, heat-induced Hsp70 expression has been found to be inversely related to high temperature adaptation in several studies both on laboratory natural evolution (Bettencourt et al., 1999; Sørensen et al., 1999) and in the wild (Sørensen et al., 2001, 2003). The positive cline of Hsp70 expression with altitude thus confirms a previous hypothesis that selection for high-temperature resistance leads to a decreased expression of Hsp70 under both natural (Sørensen et al., 2001) and simulated natural conditions (Sørensen et al., 1999). The hypothesis states that a given sub-lethal heat exposure is less stressful for heat-adapted populations thus leading to less cellular damage and less induction of stress proteins. Although the role of Hsp70 in natural adaptation is not very clear (see Sørensen et al., 2003 for review), the apparent involvement and importance of this Hsp is strongly underlined by consistent experimental evolution in expression levels (Bettencourt et al., 1999), allele frequency variation (Bettencourt et al., 2002) and copy number (Bettencourt & Feder, 2001; Evgenev et al., 2004) of the hsp70 genes in Drosophila.

Chill-coma recovery has been used by several authors and seems to be a trait causally related to cold adaptation both within and between species (Gibert et al., 2001; Hallas et al., 2002; Hoffmann et al., 2002). However, no altitudinal cline was found for this trait, suggesting that adaptation to cold is not of major importance for D. buzzatii in north-western Argentina. This species is considered a more warm-adapted species than e.g. D. melanogaster, and occurs in generally warmer habitats, yet it is likely that D. buzzatii are exposed to low temperatures occasionally – especially populations originating from higher altitudes and latitudes during cold seasons. The high elevation populations in this study probably represent the most extreme distribution with regard to low temperature exposure available for this species, thus the most likely to be cold exposed. It is possible that cold adaptation is achieved through seasonal plastic responses. Shortening day length might induce adaptive changes in resistance, primarily through diapause in the adult or larval stages as known from other Drosophila species (Goto & Kimura, 1998). Not much is known about D. buzzatii diapause, overwintering or cold adaptation. Probably, overwintering can take place in old rots in the plant stems that extend down to some cm underground, so that flies are protected from overnight frost. Adult flies and larvae have been observed in these old rots during winter, but the main life stages and overwintering mechanisms involved are yet to be determined (J.S.F. Barker, personal communication).

Although climate and especially temperature in these populations generally varies with altitude, the 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. Only some of these characteristics are predictably related to altitude. Lack of correlation between candidate resistance traits and climate variables can occur even if resistance is selected for by the climate. Possibly the resistance trait measured is not the most relevant for the climate trait. Furthermore, plastic reponses such as diapause, stress responses, maternal effects etc. are common in Drosophila, changing properties over seasons (Goto & Kimura, 1998) and during days (Sørensen & Loeschcke, 2002). These plastic responses often make adaptations invisible under standard conditions and thus make the study of adaptation more difficult.

Clear adaptive patterns were observed for desiccation and knock-down resistance and Hsp70 expression. No pattern was obvious for chill coma recovery and heat-shock survival. The pattern observed for starvation resistance was also clear-cut. To a large degree these results are in concordance with latitudinal patterns of tolerance to climatic stress (Karan et al., 1998; Hoffmann et al., 2002, 2003a) supporting the hypothesis of the importance of climatic adaptation. Overall, these results provide further insight into the ecological importance of environmental stress and adaptation, and the traits that are responding to environmental selection pressures.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We are grateful to Stuart Barker and two anonymous reviewers for helpful comments, to Doth Andersen and Trine Gammelgaard for collecting the flies, to Susan Lindquist and Michael Evgenev for providing the 7.FB antibody and to Doth Andersen, Iben Skov Jensen and Pablo Sambucetti for excellent technical assistance. The work was supported by the Danish Natural Sciences Research Council by Centre and Frame grants. Support from UBACyT-x139, ANPCyT and CONICET (Argentina) to FN is also acknowledged.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References