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.     

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.     

The partial characterization of alcohol dehydrogenase null alleles from natural populations ofDrosophila melanogaster

Twenty-three alcohol dehydrogenase (ADH) putative null alleles extracted from four Tasmanian (Australia) populations of Drosophila melanogaster produce no ADH activity and are unable to form active heterodimers with either AdhF or AdhS. Twelve of these nulls were tested by enzyme-linked immunosorbent assay (ELISA) and did not produce any ADH cross-reacting material (CRM). The null homozygotes had similar, but slightly lower, mortalities on ethanol-supplemented media compared to an artificially induced null allele. Heterozygotes between the null alleles and standard AdhF and AdhS alleles had intermediate ADH activity and CRM levels.     

Enzyme Activities and Alcohol Tolerance in Isofemale Lines of Drosophila melanogaster Originating from Different Habitats

Enzyme activity variation was studied in a Drosophila melanogaster population from two villages (Tiszafüred and Tiszaszolos) in Hungary. Two habitats (distillery and farmyard) were sampled in both villages and 8-9 isofemale lines were established from each sample with a total of 35 lines. The activities of ADH, alphaGPDH, IDH and 6PGDH were determined on starch gel after electrophoresis in 10 F1 females of each of the 35 isofemale lines. Three sublines were established from three selected isofemale lines of all four samples (altogether 36 sublines). Alcohol tolerance of the adult flies was assayed in these sublines. The activity of ADH was similar in the two habitats; so was the sensitivity to ethanol. Accordingly, no differences in adaptation to environmental ethanol were detected between the two habitats. The deviations between the two habitats in average activities and in the total variation of enzyme activities were not consistent in the two villages. These results suggest that founder effects and genetic drift are more pronounced in distilleries than selection. The association among enzyme activities varied greatly both between the two villages and between the two habitats. The two parameters of alcohol tolerance were not significantly different between the two habitats in any of the two villages.     

Tolerance to 1-pentene-3-ol and to 1-pentene-3-one in relation to alcohol dehydrogenase (ADH) and aldo keto reductase (AKR) activities inDrosophila melanogaster

The detoxification of 1-pentene-3-ol (pentenol) and 1-pentene-3-one (pentenone) by Drosophila melanogaster adult flies has been studied in two homozygous lines for the AdhF and AdhS alleles (LRC lines), in their respective lines selected for tolerance to ethanol (LRSe lines) and in a homozygous strain for the Adhn4 null allele. For each line, the genotype and sex LDs50 of both compounds were estimated. Then, in order to explain the differences in LD50, both alcohol dehydrogenase (ADH) and aldo keto reductase (AKR) activities were assayed. In addition, the effects of pentenone on AKR activity were also studied. Our results show that ADH-positive flies exhibit a much higher sensitivity to pentenol than ADH-null flies. However, both ADH-positive and ADH-null flies show a similar tolerance to pentenone. Our results show that flies selected for improving tolerance to ethanol also have increased tolerance to pentenol (FF and SS flies) and pentenone (SS flies). However, this improved ability to tolerate pentenol and/or pentenone cannot be explained by changes in ADH or AKR activities. On the other hand, we have observed a beneficial effect of pentenol, but not of pentenone, in n4 flies. We also show that AKR activity is not modified by the administration of pentenone. These results suggest that, in the absence of ADH activity, pentenol may be transformed into a compound that is less toxic than pentenone and that pentenone itself might also be transformed into a less toxic compound.     

Genetical variation for enzyme activity in a population of Drosophila melanogaster. VI. Molecular variation in the control of alcohol dehydrogenase (ADH) activity

Four characters, ADH activity at 25 degrees, immunologically determined ADH protein level, total protein and body weight were measured upon 72 hour old adult female and male Drosophila melanogaster from 16 highly inbred lines, derived from the laboratory population, "Texas" (established 1966). The highest levels of ADH activity and ADH protein level were observed in the 2 lined homozygous for the AdhF allele. Amongst the 14 AdhS/S lines variation for ADH protein level was associated with genetical variation for ADH activity (r = 0.6). The genetical association between ADH activity or ADH protein level and either body weight or total protein in the 16 inbred lines was not statistically significant. A study of ADH activity, ADH protein and total protein in 8 lines representing all homozygous combinations of chromosomes I, II and III and derived from two inbred AdhS/S lines, chosen for their respective high and low ADH activities, showed that ADH activity was considerably modified by a post-translational event controlled from chromosome III. Total protein was controlled by different chromosomal effects from those controlling ADH activity. Michaelis constants for crude fly extracts of the two AdhF/F and the above two AdhS/S lines showed clear differences in affinity for isopropanol.     

Genetical variation for enzyme activity in a population of Drosophila melanogaster. VII. Genotype-environment interaction for alcohol dehydrogenase (ADH) activity

Genotype-environment interaction was detected for ADH activity amongst a set of 18 highly inbred lines of Drosophila melanogaster which had been extracted from the laboratory population, "Texas". The genotype-environment interaction for ADH activity was not wholly associated with genotype-environment interaction for body weight or total protein level. Detailed analyses of the responses of the individual inbred lines in ADH activity in relation to the environmental index, ej and following the procedure of Jinks and Pooni (1979), showed substantial diversity in the form of response. Lines homozygous for the AdhF allele were more environmentally sensitive than AdhS/S lines. Amongst the 16 AdhS/S lines, models of linear, quadratic or two intersecting-straight-lines were used to illustrate the varied responses of genotypes to the environment. The heterogeneity in the response characteristics of the inbred lines was attributed to variations in the conditions of culture media normally present within populations and laboratories. Moreover the non-linear responses shown by some lines to the environment are consistent with a model of genotype-environment interaction for ADH activity mediated by varied genotype-specific sensitivities to different environmental factors.     

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

Strains of Drosophila melanogaster homozygous for either the Adh ^F or the Adh ^S 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.     

Physiological Significance of the Alcohol Dehydrogenase Polymorphism in Larvae of Drosophila

This study deals with biochemical and metabolic-physiological aspects of the relationship between variation in in vivo alcohol dehydrogenase activity and fitness in larvae homozygous for the alleles Adh71k, AdhF, AdhS, of Drosophila melanogaster, and for the common Adh allele of Drosophila simulans. The Adh genotypes differ in the maximum oxidation rates of propan-2-ol into acetone in vivo. There are smaller differences between the Adh genotypes in rates of ethanol elimination. Rates of accumulation of ethanol in vivo are negatively associated with larval-to-adult survival of the Adh genotypes. The rank order of the maximum rates of the ADHs in elimination of propan-2-ol, as well as ethanol, is ADH-71k greater than ADH-F greater than ADH-S greater than simulans-ADH. The ratio of this maximum rate to ADH quantity reveals the rank order of ADH-S greater than ADH-F greater than ADH-71k greater than simulans-ADH, suggesting a compensation for allozymic efficiency by the ADH quantity in D. melanogaster.Our findings show that natural selection may act on the Adh polymorphism in larvae via differences in rates of alcohol metabolism.     

Genetic Divergence under Uniform Selection. II. Different Responses to Selection for Knockdown Resistance to Ethanol among DROSOPHILA MELANOGASTER Populations and Their Replicate Lines

We have tested the hypothesis that genetic differences among conspecific populations may result in diverse responses to selection, using natural populations of Drosophila melanogaster. Selection for ethanol tolerance in a tube measuring knockdown resistance was imposed on five West Coast populations. In 24 generations the selected lines increased their mean knockdown times, on average, by a factor of 2.7. An initially weak latitudinal cline was steepened by selection. The two southernmost populations showed the same increases in the selected character, but differed consistently in their correlated responses in characters related to ethanol tolerance. This result indicates that the populations responded to selection by different genetic changes. Selection decreased female body weight and increased resistance to acetone, suggesting components of the response unrelated to ethanol metabolism. The Adhs allele was favored by selection in all populations at the onset, but increased in frequency only in the selected lines of the southernmost population. There was a correlation between latitude and Adh frequency changes, suggesting that fitnesses of the Adh alleles were dependent on the genetic background. Genetic background also had a large effect on the loss of fitness due to selection. Genetic drift between replicate lines caused more variation in selection response than initial genetic differences between populations. This result demonstrates the importance of genetic drift in divergence among natural populations undergoing uniform selection, since the effective population sizes approached those of small natural populations. Drift caused greater divergence between selected replicates than control replicates. Implications of this result for the genetic model of selection response are discussed.     

Kinetics and thermodynamics of ethanol oxidation catalyzed by genetic variants of the alcohol dehydrogenase from Drosophila melanogaster and D. simulans

Four naturally occurring variants of the alcohol dehydrogenase enzyme (ADH; EC 1.1.1.1) from Drosophila melanogaster and D. simulans, with different primary structures, have been subjected to kinetic studies of ethanol oxidation at five temperatures. Two amino acid replacements in the N-terminal region which distinguish the ADH of D. simulans from the three ADH allozymes of D. melanogaster generate a significantly different activation enthalpy and entropy, and Gibbs free energy change. The one or two amino acid replacements in the C-terminal region between the ADH allozymes of D. melanogaster do not have such clear-cut effects. All four ADH variants show highly negative activation entropies. Sarcosine oxidation by the ADH-71k variant of D. melanogaster has an activation energy barrier similar to that of ethanol oxidation. Three amino acid differences between the ADH of D. simulans and the ADH-F variant of D. melanogaster influence the kappa cat and kappa cat/Kethm constant by a maximum factor of about 2 and 2.5, respectively, over the whole temperature range. Product inhibition patterns suggest a 'rapid equilibrium random' mechanism of ethanol oxidation by the ADH-71k, and the ADH of D. simulans.     

Gene-enzyme system of alcohol dehydrogenase and adaptation of Drosophila to increased temperature]

Properties and allelic ADH (alcohol dehydrogenase) control in Drosophila melanogaster were studied upon flies' cultivation under conditions of hypotherm of different intensity and duration. Lines homozygotic for F allele (vg) and S allele (cn) of the Adh gene as well as genetically enriched experimental cn' and vg' populations containing a small number of AdhF/AdhS heterozygotes at the initial stage were used. It was found out that physiological adaptation of the species to momentary influence of elevated temperature is accompanied by modification of physical properties of ADH-F according to ADH-S. Constant influence (during the life span of 25 generations) of elevated temperature on the population changes its genetic structure, due to selective advantages of the S allele of Adh under these conditions.     

Dynamics of the genetic structure in experimental Drosophila populations for the Adh locus as affected by ethanol

Electrophoresis method was used to analyze the genetic structure of four experimental Drosophila populations (the control one and those with 5, 10 and 15% of ethanol in the nutrient medium) with initial frequency of AdhF = 0.5 for 50 monitoring generations. It was established that F and S alleles had different selective values. Natural selection favoured the F-allele. Addition of ethanol to the nutrient medium increased the selection intensity in the first 15 generations but did not change the relative adaptability of three genotypes. This caused a similarity of genetic structure in experimental populations during 50 generations.     

Biochemical differences between alcohol dehydrogenases of Drosophila melanogaster.

This paper describes substrate specificities, developmental changes in activity, pH profiles, and heat stabilities of isozymes produced by four Adh genotypes in D. melanogaster. No differences are found in the substrate specificities of isozymes from the different genotypes but studies of the other three properties reveal significant differences between the isozymes. Thus relatively low activities are found among extracts of AdhF Adhn2 larvae and among extracts of AdhF AdhF adults aged 44 days. Also AdhF AdhS and AdhS AdhS extracts have relatively high activities at pH 6-5, and AdhF Adhn2 extracts have relatively low activities at pH values above 10-0. Finally, extracts of AdhF AdhF and AdhF AdhS are more stable at 40 degrees C than are those of AdhS AdhS and AdhF Adhn2.     

Latitudinal Adh allozymic variation and ethanol tolerance in Indian populations of Drosophila melanogaster

Ten Indian geographical populations of D. melanogaster were assayed electrophoretically for Adh genic variation. The Indian geographical populations of D. melanogaster revealed significant clinal variation (3 % for 1 d? latitude) at Adh locus and Adh^F allelic frequency correlated significantly with increase in latitude. It was suggested that the abundance of secondary alcohols in the southern Indian tropical and humid environment might exert selective pressure favouring higher frequency of Adh^S allele. Patterns of ethanol utilization as well as ethanol tolerance were analyzed in larval and adult individuals of six geographical populations of D. melanogaster. Latitudinal variation in ethanol tolerance was observed in D. melanogaster populations from India. The parallel occurrence of latitudinal variation it Adh locus as well as ethanol tolerance in Indian geographical populations of D. melanogaster could be maintained by balancing natural selection varying spatially along the north-south axis of the Indian sub-continent.     

Experimental reduction of codon bias in the Drosophila alcohol dehydrogenase gene results in decreased ethanol tolerance of adult flies

The ethanol tolerance of adult transgenic flies of Drosophila containing between zero and ten unpreferred synonymous mutations that reduced codon bias in the alcohol dehydrogenase (Adh) gene was assayed. As the amino acid sequences of the ADH protein were identical in the four genotypes assayed, differences in ethanol tolerance were due to differences in the abundance of ADH protein, presumably driven by the effects of codon bias on translational efficiency. The ethanol tolerance of genotypes decreased with the number of unpreferred synonymous mutations, and a positive correlation between ADH protein abundance and ethanol tolerance was observed. This work confirms that the fitness effects of unpreferred synonymous mutations that reduce codon bias in a highly expressed gene are experimentally measurable in Drosophila melanogaster.     

VARIATION IN ALCOHOL DEHYDROGENASE ACTIVITY AND FLOOD TOLERANCE IN WHITE CLOVER,TRIFOLIUM REPENS

Flooding results in induction of anaerobic metabolism in many higher plants. As an important component of anaerobic energy production, alcohol dehydrogenase (ADH) activity increases markedly in response to flooding in white clover, Trifolium repens. Significant inter-individual variation in flood-induced ADH activity exists in natural populations of T. repens. The genetic basis of this variation was analyzed by offspring-midparent regression of data from 75 greenhouse reared families; the estimated heritability of flood-induced ADH activity was 0.55 (±0.13). Genetic variation in flood-induced ADH activity has pronounced effects on physiological response and flood tolerance in this species. ADH activity is positively correlated with the rate of ethanol production, indicating that observed in vitro activity differences are manifested in in vivo physiological function. T. repens plants with higher ADH activities during flooding have greater flood tolerance (measured as growth rate when flooded/unflooded growth rate). Variation in ADH activity during flooding accounts for more than 79% of the variance in flood tolerance. On the basis of a limited field survey of populations occupying three sites differing in exposure to flooding conditions, individuals from site C, the most frequently flooded site, expressed significantly higher average ADH activity when flooded than individuals from site A, a site with no history of flooding. Since ADH activity levels are not correlated with electrophoretic mobility variation in T. repens, this work supports previous suggestions that regulatory variation in enzyme activity may play a central role in biochemical adaptations to environmental stress.     

Hepatic aldehyde dehydrogenase activity in Peromyscus genetically deficient in alcohol dehydrogenase

1. Hepatic aldehyde dehydrogenase (ALDH) activity was measured in two strains of deer-mouse, Peromyscus maniculatus. 2. There is no difference in the subcellular distribution of ALDH activity in the two strains. Animals of AdhN/AdhN genotype, lacking liver alcohol dehydrogenase (ADH), had 90% of total ALDH activity in the mitochondrial fraction compared to 94% for the AdhF/AdhF animals with normal ADH activity. Almost all of the remaining ALDH activity was in the hepatic cytosol with less than 1% in the microsomal fraction. 3. By contrast, in mice (Mus musculus) 43% of total hepatic ALDH activity was found in the cytosolic fraction and 55% in the mitochondrial. 4. It was concluded that the subcellular distribution of hepatic ALDH activity in Peromyscus does not vary with the presence or absence of ADH and that this ALDH distribution is not similar to that reported for other rodents.     

The effect of selection for desiccation resistance on cold tolerance of Drosophila melanogaster

Abstract.  Low temperature and desiccation stress are thought to be mechanistically similar in insects, and several studies indicate that there is a degree of cross-tolerance between them, such that increased cold tolerance results in greater desiccation tolerance and vice versa . This assertion is tested at an evolutionary scale by examining basal cold tolerance, rapid cold-hardening (RCH) and chill coma recovery in replicate populations of Drosophila melanogaster selected for desiccation resistance (with controls for both selection and concomitant starvation) for over 50 generations. All of the populations display a RCH response, and there is no effect of selection regime on RCH or basal cold tolerance, although there are differences in basal cold tolerance between sampling dates, apparently related to inter-individual variation in development time. Flies selected for desiccation tolerance recover from chill coma slightly, but significantly, faster than control and starvation-control flies. These findings provide little support for cross-tolerance between survival of near-lethal cold and desiccation stress in D. melanogaster .     

Dynamics of Correlated Genetic Systems. IV. Multilocus Effects of Ethanol Stress Environments

Four replicate populations of Drosophila melanogaster, two reared on medium supplemented with ethanol and two reared on standard medium, were electrophoretically monitored for 28 generations. During the first 12 generations, allelic, genotypic and gametic frequencies were determined for eight polymorphic enzymes: GOT, alpha-GPDH, MDH, ADH, TO, E6, Ec and ODH. Samples from generation 18 and 28 were electrophoretically typed for ADH and alpha-GPDH. In addition, samples from generation 27 were analyzed for the presence of inversion heterozygotes. The experimental results showed rapid gene-frequency divergence between control and treatment populations at the Adh locus in a direction consistent with the activity hierarchy of Adh genotypes. Gene-frequency divergence between control and treatment populations also occurred at the alpha-Gpdh locus, although the agreement among replicates appeared to have broken down by generation 28. No differential gene-frequency change occurred at any of the six remaining marker loci. Furthermore, values of linkage disequilibria among all linked pairs of genes were initially small and remained small throughout the course of the experiment. Taking these facts into account, it is argued that the gene-frequency response observed at ADH is most probably caused by selection at the Adh locus. The gene frequency response at alpha-Gpdh can also be be accounted for in terms of the effect of ethanol on energy metabolism, although other explanations cannot be excluded.