All stress-resistance traits showed clear-cut direct responses to selection, which suggests a high level of genetic variation for stress resistance in the base population. In three of five stress assays (cold shock, heat shock, starvation) increased resistance was observed as correlated responses. Also, the resistance ranks averaged over all tests were higher in the selected lines compared to the control lines. These results are in agreement with numerous physiological-genetic studies reported that selection for resistance to one stress can confer resistance to other stresses. However, variation of correlated responses across stress assays and line sets indicates no single general resistance mechanism. Three sets of thermal-selected lines and the DS lines were markedly different with regard to their resistance profiles from the SS lines. Selection for starvation resistance did not result in enhanced resistance to other stresses. At the same time, each set of selection lines except HS showed a well-pronounced increase in survival rate for the starvation test. This suggests that starvation resistance easily evolves as correlated response to selection for resistance to desiccation, cold shock and knockdown by heat but the physiological mechanism of starvation resistance alone cannot explain for resistance to the above-mentioned stresses.
Survival rates under starvation and desiccation conditions in Drosophila are two most extensively studied resistance traits, for which a large number of correlations with other traits have been documented (for a review, see Hoffmann & Harshman, 1999). Particularly, a positive correlation of starvation resistance with lipid level in adult flies was consistently reported (e.g. Chippindale et al., 1996; Djawdan et al., 1998; Harshman et al., 1999a) suggesting that the accumulation of metabolic energy reserves may underlie this kind of stress resistance. There is also evidence on association of starvation resistance with increased level of glycogen (Djawdan et al., 1998). The latter was shown to contribute to enhanced desiccation resistance (e.g. Graves et al., 1992; Chippindale et al., 1998; Bradley et al., 1999) that can provide a common physiological basis for two resistance traits. The key components underlying desiccation resistance are thought to be reducing rates of water loss and increasing water storage (Hoffmann & Harshman, 1999; Gibbs & Matzkin, 2001; Folk et al., 2001). Chippindale et al. (1998) hypothesized that glycogen may function as a sponge, binding some of the extra water stored in desiccation-resistant flies. Increased starvation resistance has been repeatedly shown in Drosophila strains selected for resistance to desiccation (e.g. Hoffmann & Parsons, 1989a, 1993a; Rose et al., 1992).
In contrast to desiccation resistance, survival after cold shock has not been reported to correlate with starvation resistance in laboratory selection experiments on Drosophila. However, these traits may not be independent because cold resistance in adults is associated with the amount of lipid and carbohydrate (Ohtsu et al., 1992; Chen & Walker, 1994) and qualitative changes in the lipid composition (Ohtsu et al., 1993,1998; J. Overgaard, J.G. Sørensen, S.O. Petersen, V. Loeschcke & M. Holmstrup, unpublished). Particularly, Overgaard and coauthors revealed such changes analyzing three replicate lines from our CS regime after 10 selection generations. Accumulation of glycogen might also promote the increased cold resistance of the DS lines. Resistance to cold and desiccation should work together to prevent cellular injury and death caused by ice crystal formation. A connection between cold hardiness and desiccation has been found in a few species of arthropods (Ring & Danks, 1994). In most examples, the insect dehydrated to avoid body freezing and thus the role of glycogen as metabolic water sponge might be important.
There are also no physiological data, which could explain the increased survival of the KS lines in our starvation test. It could be suggested that the accumulation of metabolic energy reserves such as lipid and/or carbohydrate was a correlated response in lines selected for heat-knockdown resistance. However, this factor is unlikely to contribute to increased knockdown resistance: neither SS nor DS lines differ in their knockdown time from the unselected control. Some indirect evidence indicates that individuals resistant to knockdown by heat might have an increased lipid level. Bubli et al. (1998) and Sørensen & Loeschcke (2001) found enhanced heat-knockdown resistance in D. melanogaster reared at high larval densities, which has been shown to result in increased fat content (Zwaan et al., 1991; Borash & Ho, 2001).
The HS lines were the only set of lines, in which starvation resistance was not affected by selection. This suggests that selection for higher survival after heat shock was associated with a resistance mechanism different from the lipid/carbohydrate accumulation. Since individuals were heat-hardened before being selected, this mechanism could involve inducible heat-shock proteins. In some studies, expression of Hsp70 was reported to increase in hardened heat-resistant flies, although this usually did not explain all variation in heat resistance (e.g. Dahlgaard et al., 1998; Lansing et al., 2000; Norry & Loeschcke, 2003). However, the enhanced survival of the KS lines in the heat-shock test and similar responses of the KS and HS lines to other stresses could not be explained by genes controlling the Hsp70 expression. We selected for knockdown resistance without prior heat hardening and thus such genes were not a primary target of selection. More likely, other Hsp genes with constitutive expression selected under both regimes were responsible for the observed parallelism in the KS and HS resistance values. The possibility of association of constitutively expressed heat-shock genes with knockdown resistance to heat has been demonstrated by studies of hsr-omega in D. melanogaster (McKechnie et al., 1998; McColl & McKechnie, 1999). However, hsp-omega does not seem to be a part of any general resistance mechanism. The selection lines used by McKechnie et al. (1998) revealed no correlation between heat-knockdown resistance and other resistance estimates, including survival after heat shock, cold shock and desiccation (Hoffmann et al., 1997).
Induced HSPS might be involved in the increased cold resistance of the HS lines in the cold-shock test. It has been reported that Drosophila larvae and adults exposed to cold express Hsp70 during recovery (Burton et al., 1988; Yiangou et al., 1997; Goto & Kimura, 1998; Sejerkilde et al., 2003) and survival after heat shock and cold shock can be improved by cold and heat acclimation/hardening, respectively (Burton et al., 1988; Sejerkilde et al., 2003). The fact that the CS lines did not show an increase in survival following heat shock indicates that the expression of inducible HSPS might be not affected by selection for cold resistance. This is in agreement with results by J. Overgaard, J.G. Sørensen, S.O. Petersen, V. Loeschcke & M. Holmstrup (unpublished), who found no changes in cold-induced Hsp70 expression for the CS lines after 10 selection generations.
The increased resistance of the DS lines in the heat-shock test suggests that HSPS could also be involved in resistance to desiccation. There is no data on Hsp expression under desiccation stress in Drosophila but some evidence has been obtained for the flesh fly Sarcophaga crassipalpis by Tammariello et al. (1999). These authors revealed that two heat-shock protein transcripts, hsp23 and hsp70, were upregulated in pupae of S. crassipalpis following a nonlethal desiccation stress. Hoffmann (1990,1991) provided indirect evidence on the possible association between induced HSPS and desiccation resistance in Drosophila. He found that flies become more resistant to desiccation after prior exposure to a nonlethal desiccation stress and after heat hardening, although the latter had a relatively small effect. In the context of these data and data on the Hsp expression in cold-stressed flies (see previous paragraph), the increased cold resistance in the DS lines might also be explained by selection on Hsp-related genes.
In the above discussion we considered two possible explanations for the observed multiple-stress resistance, one of which is accumulated metabolic energy reserves and the other the nonspecific cellular defense system based on HSPS. Apparently these physiological responses are not alternative and supplement each other in providing resistance to some stresses. For instance, it may be suggested that the increased cold resistance of our DS lines is due to a correlated response involving both carbohydrate biosynthesis genes and Hsp genes. However, HSPS and/or other factors not related to the lipid/carbohydrate accumulation appear to be more important for resistance to most of the stresses considered here. This is indicated by the fact that the lines selected for increased thermal and desiccation resistance had generally higher survival rates over stress assays than the starvation-selected lines.
Reduced metabolic rate, which has also been hypothesized as a general stress-resistance mechanism (Hoffmann & Parsons, 1989a,b,1991), may explain resistance to a variety of, but not to all stresses. One exception seems to be cold resistance: at low temperature a reduced metabolism would decrease rather than increase survival, leading to an early stop of vitally important physiological processes (see e.g. Wieser, 1973). Hoffmann & Parsons (1989b) reported no change in cold resistance for the lines of D. melanogaster, which were selected for enhanced desiccation resistance and had reduced mass-specific metabolic rate. However, in our experiment a similar selection resulted in increased resistance to cold shock. Also, the HS lines had improved survival in the cold-shock assay. These results are not consistent with the above-mentioned hypothesis indicating that reduced metabolic rate should not be a key factor of the observed cross-resistance.
Unlike stress-resistance traits, the direct response to selection on longevity was not statistically significant. This fitness parameter was measured after a lower number of selection generations, which together with the fact of a generally low lifespan heritability may explain the present results. Whereas estimates of heritability for desiccation and starvation resistance reached 60 and 100%, respectively (Hoffmann & Harshman, 1999), for longevity they ranged from 3 to 20% being typically around 3–4% (Baret et al., 1995). Selection on age of reproduction often resulted in a significant longevity increase only under stressful or suboptimal conditions such as high larval density (e.g. Clare & Luckinbill, 1985; Luckinbill & Clare, 1986; Service et al., 1988). We also found no correlated response for longevity in any set of the resistance-selected lines. Selection for stress resistance has been reported to extend lifespan in some Drosophila studies (Rose et al., 1992; Hoffmann & Parsons, 1993b; Norry & Loeschcke, 2003) but not in others (Harshman et al., 1999b; Norry & Loeschcke, 2002a). The lack of evidence on longevity changes in experiments with stress-resistant lines including present work does not agree with the hypothesis known as the stress theory of ageing (Parsons, 1995). The latter suggests that a generalized stress resistance is the major mechanism underlying extended longevity and that a more direct procedure for increasing longevity is to select for stress resistance as the primary trait.
However, our starvation test showed that the lines selected for longevity were more resistant than the unselected control. In addition, the values of resistance ranks across all tests for the LS lines were similar to that of the SS lines. An increase in starvation resistance has been reported for different long-lived Drosophila strains (e.g. Service et al., 1985; Zwaan et al., 1995; Rose & Archer, 1996) and a recent QTL analysis in D. melanogaster discovered a linkage group on the second chromosome that conferred both starvation resistance and longer lifespan (Wang et al., 2004). There is also evidence on increased lipid content in strains selected for extended longevity (Rose, 1984; Service, 1987; Djawdan et al., 1998). The fact that the LS lines showed a clear-cut correlated response for starvation resistance, which is known to be consistently associated with the lipid accumulation (Hoffmann & Harshman, 1999), suggests that the latter might be a factor contributing to prolonged lifespan. On the other hand, there was no indication that the LS lines were more resistant in other stress assays and this was again not in agreement with the stress theory of ageing.
It should be noted that data on association between lifespan and resistance traits are frequently inconsistent, suggesting that different long-lived strains can utilize different combinations of physiological mechanisms to alter their longevity and stress resistance. For instance, one set of long-lived lines sometimes called ‘the UC Irvine lines’ (Rose, 1984) were reported to have increased starvation, desiccation and oxidative stress resistance (Service et al., 1985; Rose et al., 1992; Harshman & Haberer, 2000) as well as decreased metabolic rate and increased lipid and glycogen content (Service, 1987; Graves et al., 1992). In contrast, another set of longevity-selected lines known as ‘the Wayne State University lines’ (Luckinbill et al., 1984) showed increased resistance to oxidative stress but no change in metabolic rate, starvation resistance and lipid content, and any correlation with desiccation resistance and glycogen content was marginal (Arking et al., 1988; Dudas & Arking, 1995;Force et al., 1995). Apparently, such a difference in correlated responses could be due to initial genetic differences between the base populations, which is typical for laboratory selection experiments on Drosophila (Harshman & Hoffmann, 2000). On the other hand, the evident increase in starvation resistance observed in the LS lines as well as in most of the stress-resistance selection lines seems to reflect another feature of laboratory selection studies. As has been pointed out by Harshman & Hoffmann (2000), the selection response may tend to be based on the accumulation of energy storage compounds when food resources are abundant in every generation during the course of the experiment. In contrast, adaptation by energy conservation via reduced metabolic rate could be a common response to stress in natural populations where food supplies are limited (Hoffmann & Parsons, 1991).
Correlated responses to selection for stress resistance and longevity were observed under optimum laboratory conditions in two of three additionally considered fitness-related traits. Productivity estimated over the first 4 days after eclosion showed no statistically significant heterogeneity among the selection regimes. The trade-off between longer lifespan and decreased early age reproduction in Drosophila has been observed in numerous age-at-reproduction selection experiments (e.g. Rose, 1984; Luckinbill et al., 1984; Partridge et al., 1999) as well as in an experiment on direct selection for extended longevity (Zwaan et al., 1995). There is also some evidence on a possible trade-off between fecundity and adult stress resistance in lines selected for resistance to cold (Watson & Hoffmann, 1996) and desiccation (Hoffmann & Parsons, 1989a). It should be mentioned, that in our study differences in early productivity between lines could be expected not only due to the above-mentioned trade-offs but also because of variation in age of parents at reproduction. To start every second generation, 1.5–2.5-day-old and 4–6.5-week-old flies were used in the SS and LS lines, respectively. For other lines, this age ranged from 5–6 (control) to 10–13 (CS) days. In fact, selection for extended lifespan in the LS lines was associated with delayed reproduction as compared to other lines, whereas the SS lines were selected for the earliest reproduction. However, as it is seen from the homogeneity of productivity values, any selection on this trait was inefficient. Apparently, the low heritability of fecundity (for references, see e.g. Roff & Mousseau, 1987) and relatively short duration of selection did not allow obtaining differentiation in early productivity.
In contrast, developmental time and thorax length, which are known to have relatively high heritability (Roff & Mousseau, 1987), were affected by selection. Developmental time was higher in two sets of lines, SS and KS, as compared to the control lines. A positive correlation with developmental time in Drosophila has been previously reported for starvation resistance by Chippindale et al. (1996) and Harshman et al. (1999a). Chippindale et al. (1996) suggested a trade-off between larval developmental time and adult storage compound acquisition arguing that slower development allows the lipid accumulation for the adult stage. Our data do not contradict this hypothesis: both SS and KS line sets had an increased survival in the starvation test. Selection for slower development in the SS lines was probably quite intensive because it overbalanced selection in the opposite direction due to earlier reproduction in the SS lines as compared to the control lines. At the same time, the absence of statistically significant correlated responses for developmental time in other sets of lines with enhanced starvation resistance (LS, CS, DS) indicates that delayed development is not a necessary cost of this resistance.
Thorax length in our experiment was reduced only in the longevity-selected lines. In most Drosophila studies, body size did not reveal a correlated response to selection on lifespan (Rose et al., 1984; Luckinbill et al., 1988; Zwaan et al., 1995; Norry & Loeschcke, 2002b). However, Buck et al. (2000) reported a decrease in dry body weight for two independent sets of long-lived strains, in which developmental time was also reduced. The parallel changes in body size and developmental time in the above-mentioned study were in the direction predicted by a well-known positive genetic correlation between them (for references, see Roff, 2000). In our LS lines, developmental time was not different from that of the control lines but body size was decreased, indicating a response to selection for lifespan independent of developmental time. Such independence is in agreement with other studies that compared the effects of developmental time and adult body size on longevity (Zwaan et al., 1991; Hillesheim & Steams, 1992; Partridge et al., 1999). Small body size is known to result not only from shortened preadult period but also from decreased rate of growth. A decrease in growth rate appears to be a plausible explanation for the reduced body size in our study that may be considered as a cost of selection for extended longevity.