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.     

Analysis of microdifferentiation in a Spanish cellar population of Drosophila melanogaster

Variation in Adh and Gpdh-1 gene frequencies has been used to check for microdifferentiation in Spanish samples of Drosophila melanogaster inside and outside a wine cellar. Flies were collected after vintage and after overwintering respectively; within each period samples were taken on up to five consecutive days each month. Variation of gene frequencies of Adh and Gpdh-1 can be considered random when samples collected each month are taken into account. When mean monthly frequencies are considered, Gpdh-1 does not show any significant variation all over the year; yet, variation of the frequency of Adh ^S shows a cyclical pattern, its frequency being maximum at the end of the summer and minimum after overwintering. Due to the parallel change of the frequency of the inversion In(2L)t and the Adh ^S allele, no decision can be made whether the Adh locus itself or the inversion are responsible for the changes.     

An attempt to measure selection coefficients affecting the alcohol dehydrogenase polymorphism in Drosophila melanogaster populations maintained on ethanol media

Eleven populations polymorphic for Adh F and S alleles were established from wild-caught D. melanogaster. Seven of the populations were founded from mass collections each of several hundred flies and the other four were isofemale lines. Subcultures from all eleven were maintained on one of four different types of medium—standard laboratory medium, standard media supplemented to 3% or 9% (v/v) ethanol, and simulated wine seepages. These subcultures were rescored for F and S gene frequencies after 10, 20 and 30 generations. Maximum-likelihood methods were then used to estimate selection coefficients among the Adh genotypes and analyses of deviance were carried out to test the coefficients against hypotheses of neutrality and various modes of selection. No significant selective changes in Adh frequencies were observed in the seven mass cultures on any of the four different media types. However, highly significant selective effects were detected in the four isofemale lines; these effects were homogeneous across the four lines and the four media types and the underlying fitness set was estimated as 1.00:1.04:1.09 for S/S:F/S:F/F.     

Allozyme variation in Greek wild populations of Drosophila melanogaster and D. simulans along a North-South gradient

Allozyme frequency data from five Greek wild sympatric populations of Drosophila melanogaster and D. simulans along a North-South gradient were analyzed for genotype-environment relationships. The regression coefficient of genetic distance on geographic distance indicates that there is a significant relationship between these parameters for D. melanogaster only. Highly significant differences in specific alleles at certain loci were found between the various local populations studied. The changes in Gpdh ^F of D. melanogaster and Est-6 ^F of D. simulans exhibited clinal patterns in allele frequencies. In addition, analysis of D. melanogaster Gpdh ^Fand Adh ^F allele frequencies shows that the Greek data do not have regression coefficients (regressing allele frequency on degrees North of latitude) of the same sign as East-and West-Coast United-States populations. These contradictory data are discussed in relation to what is known about the maintenance of the Adh and Gpdh polymorphisms.     

Adaptive significance of the action of the Drosophila melanogaster alcohol dehydrogenase locus through the heat shock protein system

Four Drosophila melanogaster strains, each homozygous for one of the two major ADH aliozymes, Fast and Slow (Adh ^F1, Adh ^S , Adh ^F2 and Adh ^S2) were used to study the interaction of the Adh locus with ethanol and temperature. The separate and especially the combined effects of these two parameters allow the conclusion that the Adh locus of D. melanogaster intervenes in the adaptation process through the heat shock protein system.     

Annual variation of enzyme polymorphism in four natural populations ofDrosophila melanogaster occupying different niches

Gene and gametic frequencies of theAdh, Aox, α-Gpdh andEst-6 loci (map position: II-50.I, III-56.6, II-20.5 and III-36.0 respectively) have been studied in two wine cellar populations at 40 km distance from each other, and two field populations, one near each of the wine cellars. Only one locus,Aox was polymorphic for more than two electromorphs. TheAdh locus seems to follow a different mode of change in genic frequencies in wine cellars, than in the field, while the other loci exhibit a similar pattern of polymorphism in the field and in the wine cellar. Linkage disequilibrium in the gameteAdh/α-Gpdh is affected by different phenomena in the field than in the wine cellar. A stronger linkage disequilibrium in theAdh/gametes observed in the field, may be a consequence of a mixture of subpopulations subjected to differential selection at theAdh locus. TheAdh locus is the only one in which selection is detected in one environment. It is neutral in other environments. Theα-Gpdh, Aox andEst-6 polymorphisms, however, behave as though they were neutral both in the wine cellar and in the field.     

Temporal variation of Drosophila melanogaster Adh allele frequencies,inversion freqencies,and population sizes

The frequencies of cosmopolitan inversions and of Adh alleles in D. melanogaster, and population sizes of D. melanogaster and D. simulans have been monitored for two years in seven south-eastern Queensland sites. D. melanogaster population sizes showed greater summer increases and winter decreases than those of D. simulans. Adh allele frequencies showed no seasonal or yearly variation, and no significant correlations with environmental variables after accounting for linkage disequilibrium with In(2L)t. Some inversion frequencies showed regular summer to winter declines, yearly variation, and significant correlations with population size. Thus, the temporal variation of some inversion frequencies, unlike that for Adh allele frequencies, paralleled previously reported frequency variation over latitude.     

Variation in alcohol dehydrogenase activity in vitro in flies from natural populations ofDrosophila melanogaster

Alcohol dehydrogenase (ADH) activity variation in male flies taken directly from seven natural populations ofDrosophila melanogaster is largely accounted for by segregation of alleles at theAdh structural gene locus. There was little overlap in the ADH activities ofAdh ^F andAdh ^s homozygotes. Body weights varied only slightly betweenAdh genotypes and contributed little to ADH variation. Between and within population variation in ADH activity and ADH protein in flies in the wild is mainly due to the relative frequencies ofAdh ^F andAdh ^s.     

Extension of life duration by dietary ethanol in Drosophila melanogaster: response to selection in two strains of different origins

Lines homozygous for the Adh ^S and Adh ^F alleles were extracted from a French and an African natural population of D. melanogaster. The lines from each geographic region were then crossed and the Mendelian F₂ constituted the first generation subjected to selection for an increase in adult survival in the presence of 2% ethanol.The responses to selection were similar in the two strains, in spite of their different ethanol tolerance. In less than 10 generations, life span with ethanol increased from about 7 to more than 12 days, with realized heritabilities of 0.14 and 0.18. This extension of longevity was also observed with other concentrations of ethanol but not with water alone. The increase in life span in presence of alcohol appears to result from unknown metabolic processes, which are not obligatorily related to the capacity of the flies to tolerate starvation, nor to their size, their lipid content of their ethanol tolerance. In the two lines, however, the Adh ^S allele was quickly eliminated, suggesting a selective advantage for the F allele.     

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.     

Complexity in evolved regulatory variation for alcohol dehydrogenase genes in HawaiianDrosophila

Summary The alcohol dehydrogenase gene (Adh gene) ofDrosophila affinidisjuncta is expressed at a higher level in the larval midgut and Malpighian tubules than the homologous gene fromDrosophila hawaiiensis. This study analyzed thecis-acting sequences responsible for these regulatory differences in larval tissues ofDrosophila melanogaster transformants. A series of 10 chimeric and deletedAdh genes was introduced into the germ line ofD. melanogaster, and tissue-specific expression levels were quantified by gel electrophoresis of tissue extracts. Sequences in the upstream region of the two genes had the strongest influence on enzyme production in the midgut and Malpighian tubules. Other sequence elements also showed effects, some of which were tissue specific. Most gene fragments displayed context-dependent effects, thus supporting the proposed model of polygenic regulation ofAdh gene expression.     

Micro-evolution in a wine cellar population: An historical perspective

The population ofDrosophila melanogaster inside the wine cellar of Chateau Tahbilk of Victoria, Australia was found by McKenzie and Parsons (1974) to have undergone microevolution for greater alcohol tolerance when compared to the neighboring population outside the cellar. This triggered additional studies at Tahbilk, and at other wine cellars throughout the world. The contributions and interactions of researchers and the development of ideas on the ecology and genetics of this unique experimental system are traced. Although the ADH-F/ADH-S polymorphism was found to be maintained by selection in the Tahbilk populations, the selection is not significantly associated with alcohol tolerance. The environment inside the Tahbilk wine cellar is not as rich in ethanol as was originally anticipated, and selection that affects the alcohol dehydrogenase polymorphism may be more concerned with the relative efficiency with which ethanol is used as a nutrient byD. melanogaster. The synthesis and modification of lipids, particularly in membranes, appears to be important to alcohol tolerance. The studies of the Tahbilk population are at a crossroad. New experimental approaches promise to provide the keys to the selection that maintains the alcohol dehydrogenase polymorphism, and to factors that are important to alcohol tolerance and stress adaptation. From these research foundations at Tahbilk very significant contributions to our future understanding of the genetic processes of evolution can be made.     

Adh n5: A temperature-sensitive mutant at the Adh locus in Drosophila

Individuals of an alcohol dehydrogenase-negative strain of Drosophila melanogaster designated Adh ⁿ⁵ are resistant to ethanol poisoning at low but not at high temperatures. The basis for this behavior is that Adh ⁿ⁵ flies contain a mutant form of alcohol dehydrogenase which is less heat stable than that of wild-type flies. The mutation in Adh ⁿ⁵ maps at, or very close to, the presumptive structural locus and is not complemented by any of 11 other alcohol dehydrogenase-null mutants.This research was supported by Grant No. GM 18254 from the National Institutes of Health and Grant No. M55.2217 from the National Cancer Institute.Publication No. 768 from the Department of Biology, Johns Hopkins University.     

The alcohol dehydrogenase alleloenzymes AdhS and AdhF from the fruitfly Drosophila melanogaster: An enzymatic rate assay to determine the active-site concentration

A rapid and reproducible enzymatic rate assay for the quantitative determination of the concentration of active sites is presented for the alleloenzymes Adh^S and Adh^F from Drosophila melanogaster. Using this procedure the turnover numbers as catalytic-center activities were found to be 12.2 sec^–1 for Adh^F and 3.4 sec^–1 for Adh^S with secondary alcohols. This showed a slower dissociation of the coenzyme from the binary enzyme-NADH complex with Adh^S and hence a stronger binding of NADH to this alleloenzyme. With ethanol, the catalytic-center activity was 1.4 sec^–1 for Adh^S and 2.8 sec^–1 for Adh^F, and hence the single amino acid mutation distinguishing the two alleloenzymes also affected hydride transfer.     

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.     

Isozyme variability in species of the genus Drosophila. VI. Frequency-property-environment relationships of allelic alcohol dehydrogenases in D. melanogaster

Adult Drosophila melanogaster from naturally occurring populations in the Eastern United States were examined by gel electrophoresis for their alcohol dehydrogenase (ADH) phenotype. The ADH enzymes were partially purified and characterized. Frequencies of the controlling alleles, Adh ⁴ and Adh ⁶, were discovered to vary in a clinal pattern. Adh ⁶ reaches a maximum frequency of about 0.90 in the South and minimum of about 0.50 in the North. Partially purified enzymes from the three Adh genotypes varied according to specific activity, substrate specificity, and heat stability. A differential influence of pH was indicated. There was little variation in K _m values for ethanol and DPN⁺ among the enzymes.This work was supported by AEC Contract No. AT-(40-1)-3980 and by PHS Research Grant No. GM 11546.Paper No. 3880 of the Journal Series of the North Carolina Experiment Station, Raleigh, North Carolina. This work incorporates, in part, the thesis research of C. L. Vigue to be submitted in partial fulfillment of the Ph.D. requirements in Genetics.     

Ethanol Tolerance and Alcohol Dehydrogenase (ADH; EC1.1.1.1) Activity in Species of the cardini Group of Drosophila

Ethanol tolerance, alcohol dehydrogenase (ADH;EC1.1.1.1) activity, and tissue-specific expression wereexamined in species of the cardini group ofDrosophila using D. melanogaster as astandard of comparison. In contrast to most fruit-breeding species, allcardini species examined, two from the cardini subgroupand five from the dunni subgroup, were ethanol sensitive(LC₅₀ 2.05%) and the mean ADH activityof males ranges from only 8 to 16% that of D.melanogaster Adh^FF. Among all sevencardini species, there were small but significantdifferences in ethanol tolerance and ADH activity.Differences in enzyme mobility were in accordance with the proposedphylogeny for the dunni-subgroup species. ADH isexpressed in the fat body and midgut. Males of D.acutilabella and of D. belladunni havesignificantly less ethanol tolerance and express less ADH activitythan females in zymograms and histologicalpreparations.     

Patterns of natural selection at the Alcohol dehydrogenase gene of Drosophila americana

Similar outcomes are often observed in species exposed to similar selective regimes, but it is unclear how often the same mechanism of adaptive evolution is followed. Here we present an analysis of selection affecting sequence variation in the Alcohol dehydrogenase (Adh) gene of Drosophila americana, a species endemic to a large climate range that has been colonized by D. melanogaster. Unlike D. melanogaster, there is no evidence of selection on allozymes of ADH across the sampled range. This indicates that if there has been a similar adaptive response to climate in D. americana, it is not within the coding region of Adh. Instead, analyses of a combined dataset containing 86 alleles of Adh reveal purifying selection on the Adh gene, especially within its intron sequences. Frequency spectra of derived unpreferred variants at synonymous sites indicate that these sites are affected by weak purifying selection, but the deviation from neutrality is less drastic than observed for derived variants in noncoding introns. This contrast further supports the notion that noncoding sites in Drosophila are often subject to stronger selection pressures than synonymous sites.     

Spatial variation of biochemical and ecological phenotypes in Drosophila: Electrophoretic and quantitative variation

Electrophoretic variation at three enzyme loci-alcohol dehydrogenase (Adh), glycerophosphate dehydrogenase (Gpdh), triosephosphate isomerase (Tpi)- is compared in Australian Drosophila melanogaster populations at three levels of spatial heterogeneity; among breeding sites, within populations, and between populations at the geographic level. Heterogeneity at the breeding site level greatly exceeds that among adults within populations, indicating greater intermixing at the mobile adult stage than at the developmentally immature and less migratory larval stage. Heterogeneity at the microspatial level is large relative to the geographic level at two of these loci. Spatial patterns of variation in ecological phenotypes are also considered. It is argued that electrophoretic variants may contribute little to an understanding of this quantitative variation, and that a more useful approach in ecological genetics is to consider ecological phenotypes as primary data.     

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.