key: cord-0010104-retg4q82 authors: Holmes, Roger S.; Moxon, Leith N.; Parsons, Peter A. title: Genetic variability of alcohol dehydrogenase among Australian Drosophila species: Correlation of ADH biochemical phenotype with ethanol resource utilization date: 2005-05-12 journal: J Exp Zool DOI: 10.1002/jez.1402140211 sha: 79b38244855ad20528348caa34a6a943e5c079b6 doc_id: 10104 cord_uid: retg4q82 Alcohol dehydrogenase (ADH) activities, electrophoretic phenotypes, and the extent of ethanol resource utilization are compared for three groups of species distinguishable on ecological criteria: 1) the cosmopolitan species D. melanogaster, a frequent inhabitant of wineries; 2) fruit‐baited species of the typically Australian subgenus Scaptodrosophila: D. lativittata, D. nitidithorax and D. howensis; and 3) Scaptodrosophila species not attracted to fermented‐fruit baits being collected by sweeping in temperate rain forests (D. inornata, D. collessi) or from Hibiscus flowers (D. hibisci). D. melanogaster showed the highest levels of ADH activity and an electrophoretic polymorphism with two active allelic forms, while group 2) species showed intermediate ADH activities and polymorphisms, which were consistent with “high activity” and “low activity” allelic forms in natural populations of these species, and group 3) species showed only “low activity” forms. Ethanol resource utilization follows the same sequence, being 1 > 2 > 3(D. howensis and D. collessi were not tested). Therefore the species considered shown an association of ADH biochemical phenotype, laboratory ethanol utilization, and resources utilized. Alcohol dehydrogenase (ADH; E.C.l.1.1.1.) in Drosophila melanogaster has been extensively investigated in recent years, particularly for allelic and activity variation, and for the biological processes maintaining this variability in natural populations (Clarke, '75; David, '77) . ADH in D . melanogaster is encoded by a single structural locus (Adh) at position 50.1 on the second chromosome (Grell et al., '65) . Natural populations of D. melanogaster are usually polymorphic for two electrophoretic variants, designated ADHF and ADH" (O'Brien and MacIntyre, '69) . In polymorphic populations kept on food supplemented with different alcohols, a considerable rise in F allele frequency has been often found (Gibson, '70; '74; Van Delden et al., '75, '78) ; this has been interpreted as selection for the higher specific activity ADH' allozyme (Day et al., '74) . The relationship between ADH activity and mortality has been further confirmed by Thompson and Kaiser ('77) and Kamping and Van Delden ('78) , who reported a negative correlation between ADH activity and mortality among strains of identical A d h genotype, which differed in ADH activity. Several studies have shown, however, that the level of ADH activity can be modulated by other loci (Ward and Hebert, '72; McDonald e t al., '77) , some of which are on the third chromosome and regulate activity by altering the number of enzyme molecules in natural populations (McDonald and Ayala, '78 from flies collected in the cellar were more resistant than those collected outside the cellar. Apparently there is direct selection for ethanol resistance by the presence of environmental ethanol. Starmer et al. ('77 )developed a method for assessing the extent of ethanol utilization by examining Drosophila longevity when exposed to atmospheric ethanol in the absence of other food; a n increase in longevity over the control implies ethanol utilization and a decrease means a stress. Their studies demonstrated that the extent of ethanol-mediated longevity increase for adults of the cactusbreeding D. mojavensis depends upon the population, and suggested that these differences are controlled by the A d h locus and other loci including the octanol dehydrogenase locus and regulatory genes. Parsons et al. ('80) Species attracted to fermented-fruit baits use gaseous ethanol as a resource, whereas the remaining species either have a very low ethanol threshold or apparently do not utilize ethanol over the range of ethanol concentrations tested. In this communication, several Australian endemic Drosophila species are examined and compared with D. melanogaster for 1) electrophoretic phenotype, and activity variation of ADH as determined by electrophoretic techniques; 2) the specific activity of ADH using spectrophotometric methods; and 3) the utilization of ethanol as a resource by observing the longevity with ethanol vapor as the only available food resource. The results suggest that ADH activity variations at the interspecific level provide one of the major determining factors in the response of a variety of Drosophilia species to environmental ethanol. Mass bred populations of D. melanogaster (Townsville), D. latiuittata (Fairfield, Melbourne), D. nitidithorax (Perth), and D. howensis (Lord Howe Island) were obtained from fermented-fruit baits set out in the localities indicated in brackets. D. inornata and D. collessi were collected by sweeping foliage in Kinglake National Park, Victoria, and D. hibisci was aspirated from flowers of endemic Hibiscus species, especially H . heterophyllus, at the University Farm, Camden, NSW. (See Cook et al., '77; Bock and Parsons, '78; Parsons and Bock, '79 for basic biological information on these species). Ethanol resource utilization and tolerances were assessed by exposing adults to vapor over various concentrations of ethanol in water, following the procedure of Starmer et al. ('77) as modified by Parsons et al. ('80) . D. inornata and D. hibisci were tested 1 day after collecting flies from the field. For these two species, five replicates of ten flies per sex were tested for each of the points plotted in Figure 2 . The data are expressed as LT,,,'s calculated by linear interpolation at the various ethanol concentrations used. Individual Droaophila of various species were extracted in it multiple sample homogenizer (Roberts, '71) in 50 pl of 50 mM Tris-0.1% Triton X-100-HC1 buffer, pH 8.0 (extraction buffer), and centrifuged a t 15,000 g x 15 minutes prior to electrophoretic examination. Extracts for spectrophotometric analysis of Drosophila ADH activity were made by homogenizing 20 flies in 2mls of extraction buffer using a n Ultra-Turrax homogenizer and subsequently centrifuging at 15,000 g x 15 minutes. Model 634 spectrophotometer a t 25". The absorbance at 340 nm was recorded after addition of 100 pl of extract to 3 ml of reaction mixture containing 100 mM ethanol, 0.4 mM NAD+, and 50 mM Tris-IIC1 buffer, pH 8.0. Units of specific activity of ADH are expressed in terms of International Units (pmolesiminute) per gm wet weight of Drosophila. Cellulose acetate electrophoresis and ADH staining Homogenate supernatants were subjected to zone electrophoresis on Titan 111 cellulose acetate plates (60 by 75 mm) (Helena Labs., Texas) with tris-glycine buffer (25 mM tris, 192 mM glycine, pH 8.5 a t 25 V/cm for 20 minutes. Three 0.5 p1 applications ofextract were placed a t the origin, situated approximately 3 cm from the cathodal edge of the plate, prior to electrophoresis. The plates were then stained for activity, washed, dried, and photographed. ADH was stained by a n agar overlay technique described in detail elsewhere (Holmes, '78) . Control stains containing 100 mM tris-HC1 (pH 8.01, 0.9 mM MTT, and 0.3 mM PMS were also used to reveal aldehyde oxidase activity in the absence of coenzyme (100 mM ethanol incorporated) and dehydrogenase activity in the absence of substrate (0.4 mM NAD' incorporated). Figure 1 illustrates the phenotypic variation of ADH extracted from individual flies of the species above. The results for D. melanogaster conform with many previous studies showing a singleAdh locus with two major allelic forms in natural populations: Adh" (A; or ADH-S isozyme) andAdh" (A? or ADH-F isozyme), giving a 3 allelic isozyme phenotype in hybrid animals (A:, A;'A", A!) (O'Brien and MacIntyre, '69; David, '77) . Additional multiple forms are also observed for each allelic isozyme of melanogaster; however, these represent epigenetic products arising from the differential binding of the coenzyme, NAD', to ADH (Schwartz e t al., '79). In contrast to this electrophoretic heterogeneity, variation in activity of a single form of ADH was observed in nitidithorax and lativittata individuals. Three distinct phenotypes were observed: "high" activity, "intermediate" activity and "null" activity for ADH, with t h e genotypes being designated Adh"Adh", Adh"Adh" and AdhbAdhh respectively. This proposal assumes that the observed activity is regulated by two codominant alleles at a single locus, which is supported by a close fit to a Hardy-Weinberg distribution in each case (Table 1) . It is of course not known whether this is the structural or "regulator" locus for ADH. D. collessi individuals exhibited a uniformly single "low" form of ADH activity. The remaining species, D. inornata and D. hibisci, gave "null" patterns of ADH activity when examined electrophoretically (Fig. 1) . However, staining of the "null" variants of D. lativittata, D. nitidithorax andD. howensis, and the high frequency "null" variants of D. inornata and D. hibisci, using 100mM ethanol (compared with 50mM for Fig. I) , revealed some ADH activity migrating in the same position as the "higher activity" variants. Apparently the ADH "null" variants in these species are actually "low activity-higher Km" variants, which appear as light bands a t lower concentrations of substrate. Table 1 gives ADH specific activities and allelic frequencies, from which the species can be placed into three groups for both categories: 1) D. melarwgaster, 2) the fruit-baited Scaptodrosophila species, and 3) the nonfruit-baited Scaptodrosophila species. Similarly, ethanolLx;ource utilization thresholds and the values (Fig. 2) are in the sequence, D. melanogaster >> D. lativittata - showing that the D. melamgaster population has a n extremely high ethanol utilization threshold compared with the two other fruitbaited species, which in turn are able to use ethanol more effectively than the nonfruitbaited species, D. inornata. D. hibisci presents a special case since 1% ethanol is a stress and 0.5?0 ethanol does not increase longevity, although the possibility of ethanol utilization a t extremely low concentrations has not of course been eliminated. As documented in the introduction, selection for ethanol resistance in D. melanogaster may occur in the presence of environmental ethanol. The molecular basis of this phenomenon is under extensive investigation. In this paper, we extend considerations from the intraspecific level usually studied to the interspecific level with a comparative electrophoretic and spectrophotometric analysis of ADH from Australian species, and have attempted to correlate these results with ethanol resource utilization studies and ecological information. In general, ADH specific activity is associated with ethanol tolerance and resource utilization, ranging from D. melanogaster a t the high extreme, to D. hibisci, where evidence for ethanol utilization was not found. Low to undetectable use of ethanol as a food resource Fig. 1 . Cellulose acetate zymogram and diagrammatic illustration of electrophoretic and activity variants of alcohol dehydrogenase (ADH) from various endemic Austrdhan species of Drosophrla. Proposed genotypes encoding electrophoretic variants of melamgaster ADH and those determining the activity of ADH in otherDrosophila species are given. Ethanol concentration used was 50 mM. "Null" variants for nctidithorar, latiuittata, inornata, and hcbisct showed low activity on the zymogram when stained using 100 mM ethanol a s substrate. Anodal migrating zones of aldehyde oxidase activity (which stain in the absence of NAD') have not been included in these zymograms as in D . inornata and D. hibisci may well be quite common in the genus Drosophila as a whole given the number of species in the Australian fauna not attracted to fermented-fruit baits (Parsons, '77, '80) . Compared with these species,D. lativittata andD. nitidithorax show a much increased capacity to utilize ethanol, which may reflect their adaptation to the availability of fermented fruits and other ethanol-containing resources in their habitats. They are commonly found in orchardhrban habitats in the southwest and east (lativittata) and southwest (nitidithorax) of Australia respectively (Bock and Parsons, '78) and have presumably spread into such habitats following European settlement from temperate and subtropical forests where fermented-fruit resources are not common (Parsons, '77) . At the intraspecific level the three species not attracted to fermented-fruit baits did not exhibit detectable activit,y variation, and it would appear that the "low" A d h allele found in these species is either fixed or in a very high frequency. By comparison, in the three Scaptodro- LT 50 Control sophila species attracted to fermented-fruit baits, three ADH phenotypes were observed: "high," "intermediate," and "low" ADH activities. Assuming the "low" activity allelic form of ADH is representative of the ancestral phenotype, then it is possible that the " h i g h activity allele is favored in these three species in their new orchardlurban environment, so permitting the species to spread. On this argument, the ADH specific activity andAdh alleles of D. melanogaster, form a response to the almost totally domesticated array of habitats occupied by this species, which of course includes wineries. The argument that "low" activity alleles for ADH a r e ancestral gains support from Throckmorton's ('75) review of the phylogeny, ecology, and geography of Drosophila. He argues that originally the Drosophilidae were probably associated with slowly fermenting leaves and other fleshy plant parts on the forest floor, as well as sap and broken and damaged plant parts of living plants themselves. This was a relatively austere existence since resources exploited were not rich in carbohydrates. But it provided a step towards the exploitation of the fermentation mode of existence. It may be that in comparison with D . inornata and D . hibisci, such species as D. lativattata and D . nitidithorax respond to natural selection by altered genetic constitutions in relation to ADH genotypes. Thus, these species would be better able to exploit the normally richer resources of the orchardlurban environments into which they have spread." Intra-and interpopulation selection concerning the alcohol dehydrogenase locus in Drosophila melanogaster The subgenusscaptodrosophila (Diptera: Drosophilidae The contribution of ecological genetics to evolutionary theory: Detecting the direct effects of natural selection on particular polymer loci Australian endemic Drosophila 11. 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Lord Howe Island, with description of a new species of the coracine group Environmental alcohol at low concentrations: Longevity and development in the sibling species Drosophila melanogaster and D. simulans Private communication Origin of the multiple forms of alcohol dehydrogenase from Drosophila melanogaster Extension of longevity in Drosophila mojauensis by environmental ethanol: Differences between subraces Selection upon slow-migrating A d h alleles differing in enzyme activity The phylogeny, ecology and geography of Drosophila Selection a t the alcohol dehydrogenase locus in Drosophila melanogaster The alcohol dehydrogenase polymorphism in populations of Drosophila melanogaster. 1. Selection in different environments Variability of alcohol dehydrogenase activit,y in a natural population ofDrosophila melanogaster The technical assistance of Tracey Smith, Glenn Timms, and Garry Spence is gratefully acknowledged. This research was supported in part by a grant from the Australian Research Grants Committee.