Selection against homozygotes in the ADH polymorphic system of Drosophila melanogaster associated with the lethal factor l(2) Stm

In the natural populations ⁺Tüb, ⁺Prov, and ⁺Rov, similar Adh ^F allele frequencies occur (q _F=0.11, 0.18, and 0.08, respectively). However, there is a discrepancy in that the Adh ^F allele in ⁺Tüb is closely linked to the lethal factor 1(2)Stm, which reduces relative fitness of the F phenotype to zero. In spite of this, polymorphism is maintained also in ⁺Tüb, because the heterozygotes are superior to the homozygous S type (relative fitness=0.88). Under laboratory culture conditions, in ⁺Tüb the relative fitness of the S genotype further decreases to 0.6. After outcrossing the lethal factor, relative fitnesses for S, FS, and F become 0.6, 1, and 0.48, respectively, implying that fitness for S remains the same. Relative values for S, FS, and F in ⁺Prov, not affected by the lethal factor, are calculated by the maximum average fitness method to be 1, 1.2, and 0.2 under the assumption that heterozygous FS are similarly superior to S as in the natural ⁺Tüb population and all allele frequencies found are stable equilibrium values.     

Chemical selection of mutants that affect ADH activity in Drosophila. III. Effects of ethanol

A chemical selection scheme is presented for the isolation of rare Adh-positive Drosophila. It makes use of the fact that flies lacking detectable ADH activity die as adults or larvae on relatively low concentrations of ethanol in the medium. We have demonstrated that this procedure is a practical one by crossing two Adh-negative alleles, screening 1.5×10⁶ embryos, and isolating 14 Adh-positive survivors.Supported by Postdoctoral Fellowship GM-57 from the National Institutes of Health to C. V. and by Grant GM-18254 from the NIH.Contribution No. 841 from the Department of Biology, The Johns Hopkins University.     

Effects on ADH activity and distribution, following selection for tolerance to ethanol in Drosophila melanogaster.

Strains of Drosophila melanogaster homozygous for either the AdhF or the AdhS allele were kept on food supplemented with ethanol for 20 generations. These strains (FE and SE) were tested for tolerance to ethanol and compared with control strains (FN and SN). The E strains showed increased tolerance to ethanol both in the adult and in the juvenile life stages. In adults the increase in tolerance was not accompanied by an increase in overall ADH activity. However, there were changes in the distribution of ADH over the body parts. Flies of the FE strain possessed significantly more ADH in the abdomen, compared with FN. Another set of FN and SN populations were started both on standard food and on ethanol food with reduced yeast concentrations. After 9 months ADH activities were determined in flies from these populations which had been placed on three different media: the food the populations had been kept on, regular food and regular food supplemented with ethanol. The phenotypic effects of yeast reduction on ADH activity were considerably, but longterm genetic effects were limited.     

Perturbation of gene frequencies in a natural population of Drosophila melanogaster: evidence for selection at the Adh locus

The cellar population of Drosophila melanogaster at the Chateau Tahbilk Winery (Victoria, Australia) was perturbed for alcohol dehydrogenase (Adh) gene frequencies. Phenol oxidase (Phox) frequencies were also perturbed and monitored as a control. Subsequent gene frequency changes, together with information on population structure, indicated that selection acted on the chromosome regions of both loci. Adh gene frequencies returned to preperturbation levels in a predictable manner. A model in which the relative fitness of Adh phenotypes was determined by temperature-dependent specific activities of enzymes of Adh genotypes adequately accounts for the rate of gene frequency change at this locus. Thus temperature behaves as a selective agent in modulating Adh gene frequencies in this cellar environment.     

Selection at the alcohol dehydrogenase locus of Drosophila melanogaster: Effects of ethanol and temperature

Drosophila melanogaster larvae were subjected to 10 generations of selection on 6% ethanol at 17, 25, and 30°C. For each temperature there was a significant (P<0.01) increase in the frequency of the Adh isoallele. Controls with no ethanol showed no change in the frequency of the Adh ^F isoallele. Larvae subjected to stronger selection on 8% ethanol confirmed the results. When adults of various ages were subjected to 16 and 32°C, the ADH^F isoenzyme retained its twofold advantage in activity over ADH^S regardless of the temperature. The same result was obtained with larvae at 16 and 35°C. Although some effect of temperature was demonstrated, it was concluded that the effect was not strong enough for temperature to be a selective factor under the conditions studied. However, ethanol is a strong selective factor for laboratory populations.     

ADH enzyme activity and Adh gene expression in Drosophila melanogaster lines differentially selected for increased alcohol tolerance

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: Adh(FF) and Adh(SS). 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.     

Dietary ethanol and lipid synthesis in Drosophila melanogaster

When cultured on a defined diet, ethanol was an efficient substrate for lipid synthesis in wild-type Drosophila melanogaster larvae. At certain dietary levels both ethanol and sucrose could displace the other as a lipid substrate. In wild-type larvae more than 90% of the flux from ethanol to lipid was metabolized via the alcohol dehydrogenase (ADH) system. The ADH and aldehyde dehydrogenase activities of ADH were modulated in tandem by dietary ethanol, suggesting that ADH provided substrate for lipogenesis by degrading ethanol to acetaldehyde and then to acetic acid. The tissue activity of catalase was suppressed by dietary ethanol, implying that catalase was not a major factor in ethanol metabolism in larvae. The activities of lipogenic enzymes, sn-glycerol-3-phosphate dehydrogenase, fatty acid synthetase (FAS), and ADH, together with the triacylglycerol (TG) content of wild-type larvae increased in proportion to the dietary ethanol concentration to 4.5% (v/v). Dietary ethanol inhibited FAS and repressed the accumulation of TG in ADH-deficient larvae, suggesting that the levels of these factors may be subject to a complex feedback control.This research was supported by National Institutes of Health Grant GM-28779 to B.W.G. and a Monash University Research Grant to S.W.M.     

Association and the Adh locus with a lethal factor (l(2) Stm) in Drosophila melanogaster

Keeping Drosophila cultures at 28 C results in elimination of all minor multiple ADH bands, thought to be due to conformational change. Thus in diploid and triploid adults heterozygous for the Adh ^F and Adh ^Salleles, relative staining intensities are found for the three bands which were in conformity with the assumption that both alleles are equally expressed. Among all polymorphic strains derived from natural Central European and Mediterranean populations, the strain ⁺Tüb is unique in that its Adh ^Fallele is closely linked to a new recessive lethal factor, named 1(2)Stm. All Adh ^F 1/Adh^F 1 pupae are unable to emerge, and die. The lethal effect is obvious 50 hr earlier by retarded eye, bristle, and body wall pigmentation. Although all pupae of the phenotype F die, Adh ^F allele frequency scarcely seems to be lowered in this natural population.     

Alcohol metabolism in Drosophila melanogaster: Uselessness of the most active aldehyde oxidase produced by the Aldox locus

Alcohol dehydrogenase is necessary for ethanol detoxification and metabolic utilization. It has been generally assumed that aldehyde oxidase (AO) produced by the Aldox locus (3–56.7) is necessary for a further transformation of acetaldehyde into acetate. We find that various mutant strains (ma-l or Aldox ⁿ) which do not produce an active enzyme show about the same tolerance to alcohol as do wild strains. This physiological paradox is probably to be explained by the discovery of another locus (not localized) which produced a small amount of AO in all tested strains. The adaptive significance of the genetically polymorphic Aldox locus is probably to be looked for in physiological pathways other than ethanol metabolism.     

Drosophila melanogaster alcohol dehydrogenase: An electrophoretic study of the AdhS,AdhF, and AdhUF alleloenzymes

The nature and the interconversion of the three multiple forms Adh-5, Adh-4, and Adh-3 of the purified alleloenzymes Adh^S, Adh^F, and Adh^UF from the fruitflyDrosophila melanogaster have been examined. The experiments show that these multiple forms differ from those in crude extracts of flies homozygous at the Adh locus. On electrophoresis in a starch gel containing NAD or NADH, of purified Adh^S which consists of the three Adh forms S-5, S-4, and S-3, five enzymatically active zones appear. This contrasts with the single active zone that arises with crude extracts. Of the five zones that appear with purified enzyme, S-5 gives rise to one, while the other four zones come from the two minor forms S-4 and S-3. The occurrence of the three multiple forms Adh-5, Adh-4, and Adh-3 for each of the purified alleloenzymes is considered due to Adh-5 and, in the case of Adh-4 and Adh-3, deamidation of Adh-5, with the Adh-3 fraction also containing some reversible modified Adh-5. Of the labile amides, at least one must be located in the coenzyme binding region with deamidation preventing coenzyme binding. Pure NAD does not convert Adh-5 to Adh-3 and Adh-1. To produce conversion, the presence of either acetone or butanone along with NAD is necessary. Increased amounts of either acetone or butanone result in increased conversion. In contrast to this, none of the carbonyl compounds cyclohexanone, (+)- and (−)-verbenone, acetaldehyde, acrolein, or crotonaldehyde produces conversion. The ketone group binds to the alcohol binding site in the enzyme-NAD complex. Conversion is considered due to the ketone group binding to a nucleophilic amino acid residue and forming a bridge to the C-4 of the nicotinamide moiety of NAD.     

Correlated responses to selection for wing length in allozyme systems of Drosophila melanogaster

Summary Significant changes of genotypic structure in 20 lines selected for wing length are detected by analysis of the allelic frequencies of several enzyme loci (XDH, LAP-D, EST-6, 1-APH, ADH, -GPDH). These changes are not haphazard but a consequence of the effects of selection on the genetic structure of the population, since replicate lines always behave in a parallel way. The changes are larger in the lines selected for short wings, in which the genetic variability decreases considerably. This decrease is the result of selection for homozygosity, detected at the allozyme loci, but most probably reflects homozygosity of more or less extended chromosomal segments. Selection for wing length, especially for short wings, favoured recombinants of the initial founder chromosomes. Only in the 1-APH and the EST-6 loci, separated by 11.7 centimorgans on the genetic map, do the alleles linked in the founder lines change in parallel in the control and long wing lines. The correlated response in the allozyme allele frequencies cannot be accounted for by a direct influence of the allozymes on the variability in wing length. The changes in the EST-6, 1-APH and perhaps in the LAP-D, can be explained by a direct effect of natural selection on the allozyme loci, probably in interaction with the effect of selection for wing length on linked loci. This last effect seems to be the main factor contributing to the change detected in the XDH locus.     

Directional selection in lines founded from different parts of the phenotypic distribution of sternopleural chaetae number in Drosophila melanogaster

Summary Divergent directional selection lines were initiated from base populations founded from parents taken from different parts of the sternopleural chaetae distribution in a cage population of Drosophila melanogaster. Lines founded from parents taken from the central part of the distribution showed greater response and higher realised heritability than lines derived from parents with extreme high or extreme low chaetae number. The results suggest that centrally derived phenotypes have higher heterozygosity for chaetae factors than extreme phenotypes and that these factors have a large effect on the character.     

Enzyme polymorphism in Drosophila melanogaster populations in Iraq

Electrophoretic studies of the degree and pattern of polymorphism at two third-chromosome loci, esterase-6 (Est-6) and phosphoglucomutase (PGM), were carried out in three Drosophila melanogaster populations collected from different localities in Iraq: Mosul, Tuwaitha, and Basrah. The results show that only the Tuwaitha population was polymorphic for both loci; the other two populations were polymorphic for Est-6 and monomorphic for PGM. The allele frequency changes at both loci were followed for 20 generations in an experimental cage derived from the Tuwaitha population; it was found that there is a deviation from Hardy-Weinberg equilibrium at both loci toward the homozygote.     

The effect of dietary ethanol on the composition of lipids of Drosophila melanogaster larvae

At a moderate concentration (2.5%, v/v) dietary ethanol reduced the chain length of total fatty acids (FA) and increased the desaturation of short-chain FA in Drosophila melanogaster larvae with a functional alcohol dehydrogenase (ADH). The changes in length in total FA were postulated to be due to the modulation of the termination specificity of fatty acid synthetase. Because the ethanol-stimulated reduction in the length of unsaturated FA was blocked by linoleic acid, it was thought to reflect the properties of FA 9-desaturase. Although the ethanol-stimulated reduction in chain length of unsaturated FA was also observed in ADH-null larvae, ethanol promoted an increase in the length of total FA of the mutant larvae. Thus, the ethanolstimulated change in FA length was ADH dependent but the ethanol effect on FA desaturation was not. Ethanol also stimulated a decrease in the relative amount of phosphatidylcholine and an increase in phosphatidylethanolamine. Because similar ethanol-induced changes have been found in membrane lipids of other animals, ethanol may alter the properties of membranes in larvae. It is proposed that ethanol tolerance in D. melanogaster may be dependent on genes that specify lipids that are resistant to the detrimental effects of ethanol.This research was supported by National Institutes of Health Grant GM-28779 to B.W.G. and a Monash University Research Grant to S.W.M.     

Reproductive fitness and artificial selection in animal breeding: culling on fitness prevents a decline in reproductive fitness in lines of Drosophila melanogaster selected for increased inebriation time

Summary The maintenance of reproductive fitness in lines subjected to artificial selection is one of the major problems in animal breeding. The decline in reproductive performance has neither been predictable from heritabilities and genetic correlations, nor have conventional selection indices been adequate to avoid the problem. Gowe (1983) has suggested that the heritabilities of reproductive traits are non-linear, with heritabilities being higher on the lower fitness side. Consequently, he has predicted that culling on reproductive fitness in artificial selection lines will be effective in preventing the usual declines in fitness. An experimental evaluation of Gowe's prediction has been carried out by comparing fitnesses of replicated lines of three treatments: selection for increased inebriation time without culling on fitness (HO), selection for inebriation time with culling of 20% (4/20) of selected females on reproductive fitness (HS), and unselected controls (C). Response to selection for inebriation time in the two selection treatments was similar. After 25 generations, the competitive index, a measure of reproductive fitness, was significantly lower in the HO treatment than the HS treatment, while the HS treatment did not differ from the control lines or the base population. These results demonstrate for the first time that culling on reproductive fitness in selection lines can be used to prevent the usual decline in reproductive performance.     

Long-term selection for a quantitative character in large replicate populations of Drosophila melanogaster

Summary Six replicate lines of Drosophila melanogaster, which had been selected for increased abdominal bristle number for more than 85 generations, were assayed by hierarchical analysis of variance and offspring on parent regression immediately after selection ceased, and by single-generation realised heritability after more than 25 generations of subsequent relaxed selection.Half-sib estimates of heritability in 5 lines were as high as in the base population and much higher than observed genetic gains would suggest, excluding lack of sufficient additive genetic variance as a cause of ineffective selection in these lines. Also, there was considerable diversity among the six lines in composition of phenotypic variability: in addition to differences in the additive genetic component, one or more of the components due to dominance, epistasis, sex-linkage or genotype-environment interaction appeared to be important in different lines.Even after relaxed selection, single-generation realised heritabilities in four lines were as high as in the base population. As a large proportion of total genetic gain must have been made by fixation of favourable alleles, the compensatory increase of genetic variability has been sought in a genetic model involving genes at low initial frequencies, enhancement of gene effects during selection and/or new mutations.     

P element transposon-induced quantitative genetic variation for inebriation time in Drosophila melanogaster

Summary Bi-directional selection was carried out in coisogenic stocks with and without mobilised P element transposons to determine whether P elements induce quantitative genetic variation for inebriation time in Drosophila. There was significant response to 11 generations of selection in both pairs of replicates of bi-directional selection from an isogenic base stock in which P elements had been mobilised. Conversely, there was no significant response to 11 generations of identical selection in the control lines derived from a relatively inbred line lacking P elements. Thus, P elements have induced quantitative genetic variation for inebriation time.     

Indirect thermal selection in Drosophila melanogaster and adaptive consequences

Summary Short-term indirect selection in Drosophila melanogaster for heat-sensitivity and heat resistance resulted in two strains, one heat sensitive and another heat resistant, and correlated responses were found for the rate of heat shock protein synthesis, behavioral patterns (asymmetrical sexual isolation) and fitness components (fecundity, fertility, viability, developmental time), as well as for several enzyme activities (MDH, G-6-PDH, ADH, ACHE). These responses associated with temperature selection may reflect the effects of differential inbreeding depression caused by homozygosity of temperature sensitive mutations with different pleiotropic effects. Selection even of a very short duration can induce significant adaptive and evolutionary changes.     

Direct and correlated responses to selection for larval ethanol tolerance in Drosophila melanogaster

Ethanol is an important larval resource and toxin for natural Drosophila melanogaster populations, and ethanol tolerance is genetically variable within and among populations. If ethanol‐tolerant genotypes have relatively low fitness in the absence of ethanol, as suggested by the results of an earlier study, genetic variation for ethanol tolerance could be maintained by variation in ethanol levels among breeding sites. I selected for ethanol tolerance in large laboratory populations by maintaining flies on ethanol‐supplemented media. After 90 generations, the populations were compared with control populations in egg‐to‐adult survival and development rate on ethanol‐supplemented and unsupplemented food. When compared on ethanol‐supplemented food, the ethanol‐selected populations had higher survival and faster development than the control populations, but on unsupplemented food, the populations did not differ in either trait. These results give no evidence for a ‘trade‐off’ between the ability to survive and develop rapidly in the presence of ethanol and the ability to do so in its absence. The effect of physiological induction of ethanol tolerance by exposing eggs to ethanol was also investigated; exposing eggs to ethanol strongly increased subsequent larval survival on ethanol‐supplemented food, but did not affect survival on regular food, and slowed development on both ethanol‐supplemented and regular food, partly by delaying egg hatch.     

X-linked mutations that give rise to overproduction of glucose-6-phosphate dehydrogenase in Drosophila melanogaster

Two X-linked mutations that give rise to overproduction of glucose-6-phosphate dehydrogenase (G6PD) were found among the progenies of isogenic strains which had been subjected to selection for high G6PD activity. Mapping of the high-activity factor in these mutants was carried out using car Zw ^B sw males of low G6PD activity. As a result, the factor mapped 0.02–0.04 unit to the left of the Zw locus. The amount of the G6PD gene was also quantitated utilizing a cloned G6PD gene as a probe, but no significant difference was found between the mutants and low-G6PD activity flies which shared the same X, second, and third chromosomes with the mutants. These findings are consistent with our notion that the mutations might be regulatory mutations, possibly resulting from the insertion of a novel class of transposable genetic elements.This research was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.