ADH enzyme activity and Adh gene expression in Drosophila melanogaster lines differentially selected for increased alcohol tolerance
Louis van de Zande, Evolutionary Genetics, Centre for Ecological and Evolutionary Studies, Biological Centre, University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands.
Tel.: +31 50 363 21 26; fax: +31 50 363 23 48; e-mail: firstname.lastname@example.org
In Drosophila melanogaster, alcohol dehydrogenase (ADH) activity is essential for ethanol tolerance, but its role may not be restricted to alcohol metabolism alone. Here we describe ADH activity and Adh expression level upon selection for increased alcohol tolerance in different life-stages of D. melanogaster lines with two distinct Adh genotypes: AdhFF and AdhSS. We demonstrate a positive within genotype response for increased alcohol tolerance. Life-stage dependent selection was observed in larvae only. A slight constitutive increase in adult ADH activity for all selection regimes and genotypes was observed, that was not paralleled by Adh expression. Larval Adh expression showed a constitutive increase, that was not reflected in ADH activity. Upon exposure to environmental ethanol, sex, selection regime life stage and genotype appear to have differential effects. Increased ADH activity accompanies increased ethanol tolerance in D. melanogaster but this increase is not paralleled by expression of the Adh gene.
Environmental stress is an important factor in the determination of genetic structure and evolution of populations (Hoffmann & Parsons, 1991; Bijlsma & Loeschcke, 1997; Jenkins et al., 1997; Van Delden & Kamping, 1997; Hoffmann et al., 2003). However, neither the selective force exerted by environmental stress nor the adaptive potential of the population for the prevailing stress are constant during the lifetime of individuals. Therefore the life-stage at which the environmental stress is operational is a crucial factor in determining its impact and consequences.
Insects, and especially holometabolous insects, have a complex life cycle with several stages. Larvae and adults generally have a different shape, may live in different environments and may even have a different alimentary regime. Similar environmental stresses can affect each life stage differently, and the adaptive potential to a particular stress may be dependent on or even specific to a particular life stage (Loeschcke & Krebs, 1996).
Resistance to environmental ethanol in D. melanogaster is well documented (Van Delden & Kamping, 1997), and provides a very good model to study the link between environmental stress and life stage dependent adaptation. This is because the natural breeding substrates of D. melanogaster are fermenting fruits, which can contain quite high and even toxic levels of alcohol (Gibson et al., 1981; McKechnie & Morgan, 1982). David et al. (1976) showed that alcohol dehydrogenase (ADH: EC 184.108.40.206) is a key enzyme involved in the detoxification of alcohol. Two common allozyme variants have been described for the Adh locus: AdhS and AdhF, that differ in one nucleotide (causing Lys in position 192 of the ADHS protein to change in Thr in ADHF) that always show a large difference in enzyme activity. AdhFF flies exhibit an in vitro ADH activity generally about three times higher than AdhSS flies, while heterozygotes display intermediate activity (Van Delden, 1982; Chambers, 1988, 1991; Heinstra, 1993). This higher ADH activity for the AdhFF flies is generally associated with a higher alcohol resistance. However, ADH activities show considerable variation in natural populations, even within the same Adh genotype (Barnes & Birley, 1975, 1978; Laurie-Ahlberg et al., 1980; Anderson & Gibson, 1985). The role of ADH in conferring tolerance to environmental alcohols in Drosophila has been the object of research for many years. In most Drosophila species ADH activity is positively correlated with alcohol tolerance, but the exact role of ADH in providing alcohol tolerance is not clear and its function may not be restricted to alcohol metabolism alone.
The Adh gene of D. melanogaster shows a complex pattern of regulation (Savakis et al., 1986; Lockett & Ashburner, 1989; Wu et al., 1998). The gene is temporally regulated by two distinct promoters (distal and proximal, separated by 654 bp) yielding two mRNAs that differ only in the length of their 5′ untranslated end (Benyajati et al., 1983). Transcription from the proximal promoter predominates in the early larval stages, whereas the distal promoter is active in late third instar larvae and in adults (Savakis & Ashburner, 1985). This differential regulation of Adh in the larval and adult stages may have consequences for adaptation to ethanol stress. Although Mercot et al. (1994) found a parallel between larval and adult tolerance the correlation between adult tolerance to alcohol and larval habitat is not always obvious (David & van Herrewege, 1983). On the one hand, the selection pressure exerted on D. melanogaster by ethanol produced by alcoholic fermentation occurs mainly during the juvenile stages as larvae feed and grow in the substrate and cannot escape the environment. On the other hand, adult feeding behaviour and oviposition of females does not necessarily involve a long exposure to ethanol stress, so that the selection pressure may be lower for the adults. Boulétreau & David (1981) concluded that the stay of adults on the fermenting fruits is too short to need a special metabolic adaptation, and they concluded that the selective pressure of ethanol occurs mainly during the juvenile stages. In D. melanogaster, therefore, it seems highly probable that the mechanisms involved in alcohol tolerance are different for larvae and adults. In order to investigate this hypothesis, we have applied three different procedures of selection for increased resistance to environmental ethanol. In order to discriminate between the effects of the selection pressure in the various life stages we have applied selection in the larval stage only, selection in the adult stage only and whole life selection in which the flies were continuously in contact with ethanol. In order to investigate the involvement of the Adh gene in the development of increased tolerance to environmental ethanol, we have monitored changes in ADH activity and levels of Adh mRNA in both larvae and adults of both Adh genotypes in the presence and absence of ethanol. This was done for all three regimes of selection as well as for the unselected control strain.
Two lines, homozygous for AdhS or homozygous for AdhF, were derived from a polymorphic population founded in 1983 with 403 females from a fruit market in Groningen, The Netherlands. Both lines were homozygous for the α-glycerophosphate dehydrogenase fast allele (αGpdhF) to avoid epistatic interactions with Adh (Van Delden, 1984; McKechnie & Geer, 1988). The two lines were kept on standard food (18 g agar, 54 g sucrose, 32 g dead yeast and 100 mg ampiciline per 1000 mL water) and constitute the control lines (CON-SS and CON-FF).
All lines were kept at 25 °C (50% RH and 24 h light regime), in two replicates of five bottles each (30 mL of food per bottle) with approximately 300 eggs transferred per generation per bottle. Flies emerging from the five bottle replicates were mixed before starting a new generation and the two replicate sets of five bottles were kept independently.
Procedures of selection
All selection procedures were applied for 40 generations.
Adult selection procedure (ADU)
Flies were allowed to mate and lay eggs in five bottles with 30 mL of standard food for 24 h. The density was maintained at about 300 eggs per bottle to avoid crowding conditions. The emerging adults were transferred to new bottles and kept on standard food for 7 days. To avoid egg retention in females and to keep the flies in perfect condition, the food was refreshed 24 h before the flies were transferred to bottles with 30 mL food supplemented with ethanol; 12% for AdhSS flies (ADU-SS) and 18% for AdhFF flies (ADU-FF). When approximately 25% of the flies had died, the surviving flies were transferred to bottles with standard food for a new egg-laying period of 24 h to start a new generation.
Larval selection procedure (LAR)
Female flies were transferred to vials (55 mm high, 50 mm diameter) provided with a lid containing a thin layer of normal food and a little drop of live yeast to stimulate egg laying. Females were allowed to lay eggs for a period of four hours. Subsequently, 300 eggs were transferred to bottles containing 30 mL ethanol supplemented food; 10% for AdhSS flies (LAR-SS) and 12%AdhFF flies (LAR-FF). Eggs were transferred within 8 hours after egg laying because egg-to-adult survival on ethanol medium depends on the age at which the eggs are transferred (Bijlsma-Meeles, 1979; Kerver & Rotman, 1987). The emerging adults were transferred daily to new bottles with fresh standard food, and kept for one week before starting the next generation. Twenty-four hours before starting a new generation, food was refreshed to allow females to lay eggs and avoid egg retention.
Whole life cycle selection procedure (WHO)
Flies were kept continuously on food supplemented with ethanol; 10% for AdhSS flies (WHO-SS) and 12% for AdhFF flies (WHO-FF). Each new generation, flies were transferred to fresh bottles with ethanol-supplemented food to allow egg laying (approximately 300 eggs per bottle) to initiate a new generation.
Adult survival assay
In order to avoid phenotypic effects based on physiologic processes, flies were first cultured for one generation on standard food, under identical uncrowded conditions. Then, virgin flies were collected and sexed. Groups of 10 females or males were kept separately in vials with standard food for 1 week. These 1-week-old flies were transferred to test vials (75 mm high, 25 mm diameter) containing ethanol food (30%) and the number of dead flies was recorded daily during 1 week. The number of replicate vials was 10 per selection regime, genotype and sex.
Egg to adult survival
In order to avoid phenotypic effects, flies were first cultured for one generation on standard food, under identical uncrowded conditions. Initially, 1 week old flies were allowed to lay eggs for 24 h on fresh food to avoid any egg retention. Then, females were transferred to egg laying vials (see above). Females were allowed to lay eggs during a period of 4 h. The eggs were transferred to test vials containing food supplemented with 21% ethanol, within eight hours after egg laying. The number of eggs was 50 per vial, and five replicates per selection regime and genotype. The number of emerging adults was recorded for each vial.
ADH activity assays and protein content
Samples of ten 1-week-old nonmated adults (males and females) or ten three day old larvae were frozen at −20 °C. The samples were homogenized in 0.5 mL (adults) or in 0.3 mL (larvae) of cold buffer (50 mm glycine-NaOH, 1-mm EDTA, pH 9.5). After centrifugation (5 min at 11 000 g) the supernatant was kept on ice for immediate assay.
The ADH enzyme activity was measured following a modification of the procedure described by Oudman et al. (1991). Shortly, 170 μL buffer at 30 °C, 10 μL homogenate and 20 μL reagent buffer (glycine-NaOH buffer containing 5 mm NAD+ and 200 mm 2-propanol) were mixed. The reaction rate was measured after 30 s, during 90 s at 30 °C and 340 nm (extinction of NAD) using a multi samples spectrophotometer (Spectra Max Plus; Molecular Devices, Sunnyvale, CA, USA). For each selection regime, Adh genotype and sex, five replicates were measured twice. ADH enzyme activity was expressed as nmol NADH min−1 μg prot−1.
Protein content was measured according to Bradford (1976), using the Biorad Protein Assay kit. To 0.1 mL defrosted homogenate, 5 mL reagent was added. After 15 min, the absorbance was measured at 595 nm. Total protein content was calculated in micrograms using bovine serum albumin as a standard.
RNA extraction and northern hybridization
Total RNA was isolated from third instar larvae and 1-week-old adult flies using the RNeasy kit from Qiagen. Approximately 10 μg RNA (as determined by spectrophotometry) was vacuum-dried and dissolved in 20 μL sample buffer. From each sample 5 μL was loaded onto a 1% agarose gel for later calibration. The remaining 15 μL (7.5 μg) was separated through a 1% agarose gel containing 2.2 m formaldehyde and transferred onto a nylon membrane (Hybond-N; Amersham, Little Chalfont, UK) in 20x SSC. Membranes were hybridized overnight at 42 °C to a [α32P]dCTP labelled Adh specific probe (Oppentocht et al., 2002) The filters were washed at medium to high stringency. Autoradiography was carried out for both 16 and 48 h at −80 °C using intensifying screens. Filters were stripped by pouring a boiling solution of 0.1% (w/v) SDS onto the blot and allowing it to cool to room temperature. Hybridization was repeated with a tubuline specific probe (S. Brogna, personal communication) for calibration.
Groups of ten 1-week-old virgin adults were transferred to test vials containing standard medium for 1 day or ethanol-supplemented medium (10% for AdhSS and 12% for AdhFF) for either 1 or 3 days. Additionally, 1-week-old females were allowed to mate and to lay eggs on standard medium. Eggs were then transferred to test vials containing standard or ethanol-supplemented medium (10% for AdhSS and 12% for AdhFF) for 3 days to develop into larvae. Adult flies and larvae were kept at −80 °C for later RNA extraction and ADH activity measurements.
A probit transformation was used to calculate the lethal time 50 (LT50). anovas were performed to test the effects of Adh genotype, selection procedure and sex (for the adults). Tukey's tests for multiple comparisons of means were performed for comparison of the main factors of the anovas. For the egg-to-adult survival, an arcsine transformation was realized before performed the anovas and the Tukey's tests for multiple comparisons of means. For Adh mRNA levels, two replicates per sex, and line were performed. Statistics provided for these measures should be considered with caution.
The results from the analysis of variance showed that there were no significant differences between the replicated lines, so for the analysis the data were combined for replicates within genotype and selection regime.
Adult ethanol resistance
On control medium, no significant differences in survival were observed between any genotype or selection line. On 30% ethanol, however, a clear difference in survival became apparent. anova for adult survival on ethanol (LT50) for both control and selected lines showed significant effects for three main factors: genotype (F1,144 =307.72, P < 0.001), sex (F1,144 = 10.84, P < 0.01) and selection procedure (F3,144 = 30.89, P < 0.001). In addition, there were significant interactions between these three factors with respect to adult resistance (genotype × sex, F1,144 = 56.28; genotype × selection procedure, F3,144 = 26.26; sex × selection procedure, F3,144 = 9.8, P < 0.001 in all cases). AdhFF flies showed significantly higher resistance to alcohol than AdhSS flies. Although AdhSS females showed a markedly low resistance to ethanol, overall (genotypes and selection procedure combined) females had significantly higher resistance than males. Furthermore, selected lines showed higher resistance than nonselected control lines. Tukey tests showed that, with the exception of ADU-SS males, flies selected at the adult stage showed a significant increase in adult resistance. Similarly, flies selected during all life stages showed a significant increase in adult resistance, with the exception of female WHO-SS (Fig. 1, Table 1).
Table 1. Effects of selection for adult resistance to ethanol for AdhSS and AdhFF genotypes.
| CON||16.58a (1.80)||2.61a (0.17)|
| ADU||22.66a,b (1.67)||8.54b (1.20)|
| LAR||29.17b (2.89)||5.89a,b (1.52)|
| WHO||26.17b (2.79)||5.61a,b (0.61)|
| CON||38.39a (3.61)||41.79a (4.89)|
| ADU||90.73b (14.78)||189.04b (22.03)|
| LAR||64.53a,b (8.61)||63.83a (4.48)|
| WHO||73.37b (4.30)||156.43b (17.06)|
Larval ethanol resistance
Both genotype (F1,32 = 85.74, P < 0.001) and selection procedure (F3,32 = 27.34, P < 0.001) had significant effects on egg-to-adult survival, without significant interaction between these two factors (genotype × selection procedure F3,32 = 2.71, P = 0.0618). As for adults, AdhFF larvae showed a higher resistance than AdhSS larvae and a significant increase in egg-to-adult survival compared to control was found for all the selection procedures, except for the ADU procedure (Fig. 1, Table 2).
Table 2. Effects of selection for egg-to-adult survival rate on 21% ethanol.
| CON||0.176a (0.035)|
| ADU||0.168a (0.010)|
| LAR||0.392b (0.030)|
| WHO||0.328b (0.039)|
| CON||0.312a (0.030)|
| ADU||0.424a (0.045)|
| LAR||0.576b (0.041)|
| WHO||0.644b (0.031)|
Adult ADH activity
As expected, there was a considerable difference in ADH activity between AdhSS and AdhFF genotypes. For AdhFF males ADH activity was 3.8 times higher than for AdhSS males, whereas AdhFF females had 3.1 times higher ADH activity than AdhSS females (F1,144 = 1448.33, P < 0.001). Exposure to ethanol for 1 day, did not result in an increase of ADH activity for either genotype, but differences in activity were still significant between genotypes (F1,144 = 1486.51, P < 0.001). However, after 3 days on ethanol, ADH activity for both male and female AdhFF genotypes had decreased significantly, whereas ADH activity for AdhSS genotypes remained constant (Table 3).
Table 3. Adult ADH enzyme activity for AdhSS and AdhFF genotypes.
| CON-SS||0.156a (0.004)||0.185a (0.015)||0.137a (0.004)|
| ADU-SS||0.198b (0.010)||0.126a (0.011)||0.142a (0.004)|
| LAR-SS||0.194b (0.009)||0.158a (0.014)||0.161b (0.005)|
| WHO-SS||0.203b (0.010)||0.182a (0.021)||0.140a (0.004)|
| CON-SS||0.111a (0.002)||0.113a (0.005)||0.099a,b (0.009)|
| ADU-SS||0.152b (0.016)||0.130a (0.027)||0.079a (0.007)|
| LAR-SS||0.129a,b (0.006)||0.147a (0.006)||0.117b (0.006)|
| WHO-SS||0.120a,b (0.004)||0.144a (0.014)||0.094a,b (0.005)|
| CON-FF||0.587a (0.022)||0.579a (0.041)||0.296a (0.013)|
| ADU-FF||0.788b (0.029)||0.903c (0.054)||0.467b (0.030)|
| LAR-FF||0.760b (0.041)||0.721a,b (0.016)||0.465b (0.015)|
| WHO-FF||0.855b (0.037)||0.749b (0.032)||0.456b (0.019)|
| CON-FF||0.339a (0.022)||0.375a (0.020)||0.206a (0.010)|
| ADU-FF||0.457b (0.029)||0.653b (0.038)||0.260b (0.008)|
| LAR-FF||0.398a,b (0.041)||0.609b (0.019)||0.329c (0.013)|
| WHO-FF||0.389a,b (0.037)||0.565b (0.024)||0.384d (0.015)|
All selected lines showed a constitutive increase in ADH activity, that was apparent in both sexes, but for females significant in the ADU selection regime only (Table 3). This is in concordance with the finding that there is a positive correlation between ethanol tolerance and amount of ADH protein in adult flies (Geer et al., 1993). The same pattern of decreasing ADH activity after 3-day exposure to ethanol was still observed in the selected lines (Table 3). However, compared with control values, selection for ethanol tolerance led to increased adult ADH activity upon 1- and 3-day exposure to ethanol. This increase was most evident in ADHFF genotypes and most prominent in females (Fig. 2; Table 3).
Larval ADH activity
For the larval stage, similar results as for the adult stage were obtained: ADH activity was not or only very slightly increased upon exposure to ethanol (Table 4), but there were evident differences between the AdhSS and AdhFF genotypes both on standard medium (F1,72 = 746.00, P < 0.001) and on ethanol medium (F1,72 = 493.00, P < 0.001).
Table 4. Larval ADH enzyme activity for AdhSS and AdhFF genotypes.
|CON-SS||0.154a (0.014)||0.211a (0.020)|
|ADU-SS||0.151a (0.006)||0.328b (0.033)|
|LAR-SS||0.165a (0.012)||0.222a (0.026)|
|WHO-SS||0.175a (0.012)||0.216a (0.022)|
|CON-FF||0.725a (0.021)||0.866a (0.041)|
|ADU-FF||0.923a (0.060)||1.106a,b (0.060)|
|LAR-FF||0.915a (0.069)||1.198b (0.115)|
|WHO-FF||1.350b (0.071)||1.167b (0.041)|
Selection for increased ethanol tolerance, irrespective of selection regime, did not lead to constitutive increase of ADH activity in the larval stage, with the exception of the WHO-FF line (Table 4). Again, this result is in line with the results of Geer et al. (1993), who found no relation between the amount of ADH protein and ethanol tolerance of larvae. Exposure to ethanol, however, seems to induce ADH activity, most prominently in AdhFF genotypes (Fig. 2; Table 4).
Adult Adh expression
Control and selected lines
There was no apparent difference in expression level between AdhSS and AdhFF genotypes on normal food (F3,4 = 0.5, n.s.; Table 5). These results are in accordance with the fact that the difference of activity is not primarily due to differences in mRNA levels between AdhSS and AdhFF genotypes (Parsch et al., 2000). Compared with controls, selection for ethanol tolerance had no significant effect on adult Adh expression for either genotype or selection regime, with the exception of WHO-FF males (Table 5). For all lines and both sexes, Adh expression increased upon 1-day exposure to ethanol, whereas 3-day exposure caused the Adh expression level to return to default values (Table 5). However, an increase above control values was observed for the AdhFF genotype only, although not significantly (Table 5, Fig. 3). This expression profile of AdhFF genotypes was not completely reflected in the activity pattern, although the decrease in expression after 3 days of ethanol exposure for AdhFF genotypes was also observed in the activity pattern. However, the increased ADH activity in AdhFF females after 1-day exposure to ethanol was reflected in the Adh expression level (Table 5).
Table 5. Adult Adh expression for AdhSS and AdhFF genotypes.
| CON-SS||0.71 (0.10)||1.33 (0.11)||0.52 (0.08)|
| ADU-SS||0.64 (0.01)||1.61 (0.34)||0.27 (0.11)|
| LAR-SS||0.37 (0.04)||1.42 (0.08)||0.33 (0.14)|
| WHO-SS||0.49 (0.36)||1.89 (0.00)||2.28* (0.15)|
| CON-SS||0.49 (0.10)||0.85 (0.09)||0.32 (0.04)|
| ADU-SS||0.44 (0.05)||0.95 (0.09)||0.18 (0.13)|
| LAR-SS||0.67 (0.26)||0.93 (0.06)||0.06|
| WHO-SS||0.66 (0.02)||1.16 (0.11)||0.49 (0.01)|
| CON-FF||0.54 (0.30)||2.00||0.69 (0.09)|
| ADU-FF||0.48 (0.03)||2.09||0.45 (0.13)|
| LAR-FF||0.45 (0.39)||2.10 (0.29)||0.17* (0.03)|
| WHO-FF||2.28 (0.54)||1.95 (0.21)||0.88 (0.01)|
| CON-FF||0.41 (0.15)||0.70 (0.14)||0.61 (0.17)|
| ADU-FF||0.15 (0.06)||1.20 (0.29)||0.23 (0.00)|
| LAR-FF||0.25 (0.03)||1.34 (0.09)||0.22 (0.09)|
| WHO-FF||0.54 (0.13)||1.61 (0.06)||0.51 (0.02)|
Larval Adh expression
Control and selected lines
As in adults, there was no significant difference in expression level between the Adh genotypes (t2 = 3.38, P = 0.077; Table 6). In contrast to adults, selection for ethanol tolerance seemed to lead to increased constitutive Adh expression in larvae irrespective of the selection regime (Table 6). This apparent increase is, however, also not statistically significant (AdhSSF3,3 = 7.98 P = 0.061; AdhFFF3,4 = 3.0; n.s.). Exposure to ethanol diminished this increased expression relative to the controls (Fig. 3). Interestingly, the increased expression of the Adh gene did not lead to increased activity of ADH in larvae. Adh expression of unselected larvae also seemed to increase upon 1-day exposure to ethanol (Table 6). Because of the high standard errors, however, this increase is not statistically significant. If selected lines are included in the analysis, however, increased Adh expression upon exposure to ethanol becomes more evident (AdhSSF7,7 =7.78 P = 0.0074; AdhFFF7,8 = 7.39 P < 0.01).
Table 6. Larval Adh expression for AdhSS and AdhFF genotypes.
|CON-SS||0.18 (0.02)||0.69 (0.32)|
|ADU-SS||0.32 (0.06)||1.08 (0.04)|
|WHO-SS||0.41 (0.01)||0.94 (0.05)|
|CON-FF||0.48 (0.08)||1.10 (0.18)|
|ADU-FF||0.67 (0.07)||1.47 (0.21)|
|LAR-FF||0.79 (0.11)||1.69 (0.27)|
|WHO-FF||0.86 (0.12)||1.30 (0.05)|
Our results clearly demonstrate that selection for increased ethanol resistance within Adh genotypes leads to a pronounced positive response for both AdhSS and AdhFF genotypes.
Selecting for increased ethanol resistance leads to a positive response in adults, irrespective of the selection regime. For egg-to-adult survival, however, a positive response was observed only when the larval stage was included in the selection regime. Apparently a different, probably additional, genetic network is involved in establishing larval tolerance to high levels of environmental ethanol. This finding partly supports the hypothesis of Boulétreau & David (1981) that larval adaptation to ethanol requires a specific metabolic adaptation. It also demonstrates, however, that selection for ethanol resistance in the larval stage extends to the adult stage as well. Therefore, the suggestion by Loeschcke & Krebs (1996) that a similar environmental stress can affect each life stage differently, and that the resistance to a particular stress may be specific to a particular life stage holds for the larval stage, but not for the adult stage. Given the fact that many developmental processes are in operation during the larval stage or will be implemented in later stages, while the metabolic pathways in larvae and adults may be quite similar, it is likely that similar adaptations of the metabolic pathway constitute ethanol resistance in adults and larvae. The additional developmental tasks of the larval stage, however, make adaptive changes in additional pathways necessary. This would explain the fact that resistance obtained in the larval stage, including metabolic and additional pathways, extends to the adult stage, whereas resistance obtained in the adult stage, including metabolism only, is insufficient to include the larval stage.
The results obtained for ADH activity are in support of this scenario. For unselected lines, no increase in ADH activity was observed upon exposure to environmental ethanol, for both the larval and the adult stage. Selection for increased ethanol resistance, however, leads to constitutive increase of ADH activity in adults, irrespective of the selection regime. In larvae, no such increase was observed. This positive correlation between ADH protein abundance and ethanol tolerance for adults but not for larvae has already been reported by Geer et al. (1993). Apparently, the metabolic adaptations to environmental ethanol cause a higher ADH activity. In larvae, however, this higher activity may be in conflict with other, probably developmental, pathways. An attractive explanation is the involvement of ADH in other pathways than ethanol degradation only. Support for this may come from the fact that AdhSS flies generally have longer developmental times than AdhFF flies. Moreover, it has been shown by Oppentocht et al. (2002) that D. kunzei, an extremely ethanol sensitive species, has levels of both larval and adult Adh mRNA that are similar to that of D. melanogaster, whereas the ability to convert ethanol of the D. kunzei ADH is much lower. This finding is highly indicative of the involvement of Adh in other pathways than ethanol degradation only. The fact that larvae can be selected for increased ethanol tolerance, despite the possible ‘multitasking’ of Adh is indicative of the high flexibility of D. melanogaster in adaptation to environmental ethanol. This is also reflected in the results of Fry (2001) who found no trade-off between selection for larval ethanol tolerance and developmental time.
In unselected flies, Adh mRNA levels are increased in both adult and larval stages upon exposure to ethanol, but no increase in ADH activity was observed. Similarly, the findings on ADH activity upon selection are not reflected in the levels of Adh mRNA. On the contrary, Adh mRNA levels are constitutively increased in larvae, but not in adults, whereas the reverse was observed for ADH activity. The only clear relative increase in Adh expression was found in AdhFF females upon 1-day exposure to ethanol. Apparently, the regulation of ADH activity, or protein abundance, upon selection for increased ethanol resistance, is not at the level of Adh transcription. Only in adults, after 3 days of ethanol exposure, was a positive correlation between ADH activity and Adh mRNA found, in particular for the AdhFF genotypes. This may be the result of the fact that too high ADH activity leads to intolerable levels of toxic intermediates (acetaldehydes). As ADHFF has a higher activity than ADHSS, the more pronounced effect is to be expected for the AdhFF genotypes (Van't Land, 1997; Oppentocht, 2001).
Previous research has demonstrated that Adh mRNA levels are not necessarily associated with higher ADH activity or, indeed, alcohol tolerance (Choudhary & Laurie, 1991; Laurie et al., 1991). Moreover, the regulation of ADH activity can be effectuated at all possible levels. The two promoter system allows life time specific regulation at the transcription level, enhancer elements allow regulation at the transcription level within life stages, regulatory elements present within exons and at the 3′ region of the mRNA are involved in post-transcriptional regulation (Stam & Laurie, 1996). Recently it has been observed that codon bias plays an important role in translation efficiency of the Adh gene, pointing at the importance of tRNA availability for the efficient translation of the Adh mRNA (Carlini, 2004). In this light it may be no surprise that no clear correlation between ethanol tolerance and Adh gene expression was observed in this study. It may even be true that Adh is not crucial in adaptation to elevated levels of environmental ethanol and that the observed effects are merely a metabolic response and that other physiological systems are the basis for increased ethanol resistance (see Geer et al., 1993). Both lipid metabolism and membrane structure have been put forward in this respect (Eanes, 1999).
We observed some differences between the two genotypes in response to selection and subsequent exposure to environmental ethanol. In general, AdhFF genotypes gave a more pronounced increase in both ADH activity and Adh mRNA levels than the AdhSS genotypes. In addition, we observed clear differences between adult male and female responses. Females seem to display a more pronounced response to selection at all items investigated than males. This could be the consequence of the fact that females have to oviposit and cannot stop feeding on ethanol supplemented food, because of the fact that they need the energy to produce eggs. Males, however, can sustain longer periods of refraining from feeding, and may ingest smaller amounts of ethanol. The differences between male and female response to selection should become apparent in the examination of correlated responses to selection for increased ethanol tolerance. These responses will be discussed elsewhere.
In summary, our results indicate that selection for increased tolerance within genotypes is possible, that life-stage dependent selection leads to correlated results for the larval stage but not for the adult stage and to a constitutive increase in ADH activity in adults. Neither increased ethanol tolerance nor ADH activity, however, is simply correlated with increased Adh mRNA levels. The establishment of resistance to environmental ethanol is a complex trait, with different but overlapping compounds in the larval and adult life stages.