Adaptation to ethanol, and absence of evidence for trade-offs
I observed adaptation to ethanol in four large laboratory populations that were maintained wholly (E1 and E2) or partly (M1 and M2) on ethanol-supplemented food for ca. 100 generations: when tested on ethanol-supplemented food, these populations had higher egg-to-adult survival and faster development rate than populations (R1 and R2) which had not previously been exposed to ethanol. In addition, the E populations had higher survival on ethanol food than the M populations, showing that rearing 50% of the flies on regular food each generation significantly slowed the selection response of the latter populations.
In spite of the clear superiority of the E and M populations on ethanol-supplemented food, I found no evidence that these populations were inferior to the R populations in survival or development rate on regular food. In contrast, when Oakeshott et al. (1985) selected eight different base populations for tolerance of 9% ethanol food for 30 generations, the selected lines took an average of a day longer to develop on regular food than the unselected lines. Oakeshott et al. used a different method for measuring development time, and it is possible that this, as well as the different base populations used, caused the difference between their results and mine. A possibility that needs to be seriously considered, however, is that the declines in development rate on regular food that Oakeshott et al. observed were the result of inbreeding depression. Each generation of each selection line was initiated with approximately 40 adults of each sex; even if one generously assumes that effective population size was 75% of actual population size (Frankham, 1995), the inbreeding coefficient after 30 generations would have been 0.22, about the same as for a full-sib mating. Furthermore, inbreeding is likely to have been more severe in the ethanol-selected lines because strong selection reduces effective population size (Robertson, 1961). Although development rate has received relatively little attention in studies of inbreeding depression in Drosophila, Roper et al. (1993) showed that inbreeding can depress development rate in selected populations. Inbreeding depression does not appear to have had a substantial effect on my results; when I crossed the replicate E and R populations, the hybrids had similar development rate on regular food as the pure populations (J. D. Fry, unpublished data).
An alternative explanation as to why I did not observe trade-offs is that modifiers were selected for in the E and M populations that mitigated initial negative effects of alleles conferring higher ethanol tolerance. Such modifiers would have to have been in high frequency by generation 50, by which time the E and M populations were similar to the R populations in development time on regular food (indeed, the slight tendency at this time was for slower development in the R populations; Fig. 3). There is some reason to doubt the ubiquity of modifiers that can ameliorate negative effects of genes favoured under stressful regimes, however. In spite of the clear example of a modifier of the fitness effects of an organophosphate resistance gene in the Australian sheep blowfly (McKenzie & Game, 1987; Davies et al., 1996), similar resistance modifiers in other insect species do not appear to be common (Roush & McKenzie, 1987). Perhaps more relevant to the current experiment, in at least four experiments with Drosophila, apparent negative pleiotropic effects of adaptation to stressful regimes have persisted in experiments lasting about the same number of generations as the current experiment. Selecting for accelerated development for 125 generations resulted in a 10% reduction of larval viability (Chippindale et al., 1997); maintaining populations at 16.5 °C for 5 years resulted in lower survival at 25 °C (Partridge et al., 1994), selecting for large body size for 49 generations reduced larval competitive ability (Partridge & Fowler, 1993); maintaining populations under severe crowding for 50 generations resulted in lower survival to pupation under food-limited conditions (Joshi & Mueller, 1996). In all of these cases, inbreeding depression was either ruled out or does not appear to have been likely as a cause of the declines.
Like those of Oakeshott et al. (1985), my ethanol-selected populations did not have lower survival on regular food under uncrowded conditions than the nonselected populations. This is not surprising because survival was uniformly high (ca. 80%) in both studies, indicating that uncrowded regular food is a benign environment. Because trade-offs are sometimes easier to detect under harsh conditions than under benign conditions (e.g. Partridge & Fowler, 1993), I also tested the E and R populations for survival and development rate on dilute regular food under competitive conditions. Although the differences were not quite significant, the E populations appeared to do better than the R populations by both measures when the competitor strain was related to the R populations. In contrast, when the competitor strain was related to the E populations there was no difference in development rate, but the R populations may have had slightly higher survival than the E populations, although this difference was also nonsignificant. These results therefore suggest the possibility that each population did worse in competition with the marker stock more closely related to itself than in competition with the less closely related stock. Numerous studies have documented such negative frequency-dependent selection for viability in Drosophila (reviewed in Ayala & Campbell, 1974; Antonovics & Kareiva, 1988). The most important conclusion from the competitive assays, however, is that adaptation to ethanol did not lower the general competitive ability of the E populations.
Induction of ethanol tolerance by exposure of eggs to ethanol
My results confirm that ethanol tolerance in D. melanogaster larvae is inducible: larvae that hatch from eggs that develop in contact with ethanol have higher survival on ethanol-supplemented food than those hatching from eggs not exposed to ethanol (Bijlsma-Meeles, 1979; Kerver & Rotman, 1987; Bijlsma & Bijlsma-Meeles, 1991). The inducibility of ethanol tolerance provides a potential mechanism by which ethanol-tolerant genotypes could avoid having low fitness in the absence of ethanol: even if the mechanisms responsible for high ethanol tolerance are costly in the absence of ethanol, the costs will be realized only to the extent that the mechanisms are activated constitutively. If I had observed no difference between the populations in ethanol tolerance of larvae hatching from eggs not exposed to ethanol, there would have been little reason to expect larvae from the E and M populations to have had lower survival or slower development in the absence of ethanol than larvae from the R populations. Such differences were observed, however, both for survival (Fig. 4) and development rate (Figs 5 & 6), indicating that ethanol tolerance in the E and M populations was to some extent constitutive.
My results give some information, albeit highly indirect, on whether activation of mechanisms necessary for high ethanol tolerance would reduce fitness when ethanol is absent. Exposing eggs to ethanol did not lower the subsequent survival of larvae on regular food, giving no evidence for such a cost. However, ethanol treatment of eggs slowed egg-to-adult development on both regular and ethanol-supplemented food, partly by increasing the amount of time it took eggs to hatch. It is possible that the delays in development rate caused by treating eggs with ethanol reflect a cost of induction; alternatively, the delays may simply have resulted from toxic effects of ethanol. Some evidence argues against the former explanation. If the slowing of development rate reflected a cost of induction, then those populations showing greater induction of tolerance would be expected to show greater slowing of development rate when eggs are treated with ethanol. Contrary to this prediction, ethanol slowed the embryonic development period of the E and R populations to a similar extent (Fig. 7) but ethanol tolerance appeared to be more strongly inducible in the E populations than in the R populations. This can be seen from the following comparison (Fig. 4): on 16% ethanol, ethanol treatment of eggs increased survival of the E populations from 0.06 to 0.64, whereas on 12% ethanol, ethanol treatment of eggs increased survival of the R populations from 0.14 to only 0.50. The degree of induction of tolerance in the different populations can also be visualized in a scatter plot of survival of ethanol-treated eggs on the ordinate vs. survival of control eggs on the abscissa (Fig. 8); for a given survival of control eggs, the distance of a point from the diagonal line reflects the degree to which survival is improved by treating eggs with ethanol. For most of the plot, the points from the R populations lie below those from the other populations (Fig. 8).
Figure . 8. Proportion surviving on 16% (six leftmost points), 12% (six middle points) and 8% ethanol (six rightmost points) in the E, M and R populations in the main experiment. The diagonal line denotes equal survival of ethanol-treated and control eggs; the height of a point above the line therefore reflects the degree of induction of ethanol tolerance. The probit transformation was used because it produces approximately linear relationships within each of the populations.
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One might have predicted that the M populations, in which only half the individuals encounter ethanol each generation, would show a greater degree of inducibility of ethanol tolerance than the E populations, in which every individual encounters ethanol. In the E populations, constitutive tolerance should serve as well as inducible tolerance; in fact, if there is a cost to maintaining mechanisms for responding to ethanol concentration (a.k.a., a cost of phenotypic plasticity; Van Tienderen, 1991), constitutive tolerance might be favoured over inducible tolerance in the E populations. Figure 8 shows, however, that inducibility of tolerance in the M populations was not greater than in the E populations. It is possible that not enough time had elapsed for such a difference to emerge; I plan to retest the populations after another ca. 100 generations of selection to check this possibility. It is also possible, of course, that no cost of phenotypic plasticity exists in this system.
On regular food, the development delay of the R populations caused by treating eggs with ethanol was greater than in the E and R populations, and much greater than can be accounted for by the effect of ethanol exposure on egg development. The simplest explanation for this is that the R population larvae were affected by residual ethanol in the agar pieces that were used to transfer eggs to the regular food vials; although the agar was rinsed with distilled water after being soaked in ethanol, some ethanol undoubtedly remained.
In addition to causing high larval mortality, ethanol in the food delays egg-to-adult development considerably. The fact that exposing eggs to ethanol alleviates the first effect but not the second suggests that the lowering of survival and slowing of development may arise from different causes. In support of this view, it appeared that the majority of mortality on ethanol-supplemented food took place within 24 h after the eggs had hatched (J.D. Fry, unpublished observations). The slowing of development, in contrast, seems more likely to have resulted from the effects of feeding on ethanol-supplemented food throughout larval development, rather than being caused only by exposure to ethanol within the first 24 h. Late in larval development, it is likely that most of the ethanol in the medium had been converted to acetic acid by microbial activity (Hageman et al., 1990); therefore it is possible that the slowing of development was partly caused by acetic acid buildup.
The mechanism of the induction of ethanol tolerance is not fully understood. Exposing eggs to ethanol causes an increase in alcohol dehydrogenase activity (Bijlsma-Meeles, 1979; Kerver & Rotman, 1987) but increases the ethanol tolerance of Adh null strains as well (Bijlsma & Bijlsma-Meeles, 1991). It is possible that other enzymes involved in ethanol degradation (Geer et al., 1993) are induced by exposing eggs to ethanol. In addition, ethanol induces production of stress (heat-shock) proteins in a variety of organisms (Li & Hahn, 1978; Michel & Starka, 1987; Feder et al., 1995), and this response has been associated with increased ethanol resistance in a bacterium (Michel & Starka, 1987) and yeast (Feder et al., 1995).
The inducibility of ethanol tolerance explains some initially puzzling observations I made. Flies from the R populations produce large numbers of adult progeny when allowed to lay eggs in vials containing 10–12% ethanol (J.D. Fry, unpublished data). For example, in the initial development rate comparisons on 10% ethanol, in which five females were allowed to lay eggs for 5 h, the mean number of progeny was about 60, and there were no significant differences among the selection regimes in progeny number. At about the same time, however, preliminary comparisons of larval survival were made by transferring larvae to vials with 10% ethanol food. Survival of larvae from the R populations was extremely low, only about 15%, compared with ca. 50% survival of larvae from the E populations (J.D. Fry, unpublished data). This difference in results makes sense in light of the induction effect: the eggs laid in ethanol food developed in contact with ethanol whereas the larvae transferred in the survival experiment had not been previously exposed to ethanol. The induction response appears critical for allowing laboratory populations to thrive on medium with 10% or more ethanol. It is notable that even after nearly 100 generations of selection, survival of noninduced eggs from the E populations on 12 and 16% ethanol was lower than that of induced eggs from the R populations (Fig. 4).