Shaping of Drosophila Alcohol Dehydrogenase Through Evolution: Relationship with Enzyme Functionality

Drosophilidae is a large, widely distributed family of Diptera including 61 genera, of which Drosophila is the most representative. Drosophila feeding is part of the saprophytic trophic chain, because of its dependence upon decomposing organic matter. Many species have adapted to fermenting fruit feeding or to artificial (man-made) fermentation habitats, such as cellars and breweries. Actually, the efficient exploitation of niches with alcohols is considered one of the reasons for the worldwide success of this genus. Drosophila alcohol dehydrogenase (ADH), a member of the short-chain dehydrogenase/reductase family (SDR), is responsible for the oxidation of alcohols, but its direct involvement in fitness, including alcohol tolerance and utilization, gives rise to much controversy. Thus, it remains unclear whether ADH differentiation through evolution is somehow associated with natural adaptation to new feeding niches, and thus maybe to Drosophila speciation, or if it is a simple reflection of neutral divergence correlated with time separation between species. To build a hypothesis which could shed light on this dilemma, we analyzed the amino acid variability found in the 57 protein ADH sequences reported up to now, identified the taxon-specific residues, and localized them in a three-dimensional ADH model. Our results define three regions whose shaping has been crucial for ADH differentiation and would be compatible with a contribution of ADH to Drosophila speciation. Received: 11 August 1997 / Accepted: 30 December 1997     

Functional and biochemical features of alcohol dehydrogenase in four species of the obscura group of Drosophila

The biochemical features of ADH of four Drosophila species of the obscura group have been studied. The relationship between ethanol tolerance and ADH activity has been investigated. Propan-2-ol and acetone concentrations have been determined in propan-2-ol treated flies and ADH activity has been followed during 96 h of propan-2-ol treatment. Data on the ADH system confirm constructed phylogenies based on electrophoretic variation and chromosome homologies.     

Selection and Methionine Accumulation in the Fat Body Protein 2 Gene (FBP2), a Duplicate of the Drosophila Alcohol Dehydrogenase (ADH) Gene

The Drosophila fat body protein 2 gene (Fbp2) is an ancient duplication of the alcohol dehydrogenase gene (Adh) which encodes a protein that differs substantially from ADH in its methionine content. In D. melanogaster, there is one methionine in ADH, while there are 51 (20% of all amino acids) in FBP2. Methionine is involved in 46% of amino acid replacements when Fbp2 DNA sequences are compared between D. melanogaster and D. pseudoobscura. Methionine accumulation does not affect conserved residues of the ADH-ADH^r-FBP2 multigene family. The multigene family has evolved by replacement of mildly hydrophobic amino acids by methionine with no apparent reversion. Its short-term evolution was compared between two Drosophila species, while its long-term evolution was compared between two genera belonging respectively to acalyptrate and calyptrate Diptera, Drosophila and Sarcophaga. The pattern of nucleotide substitution was consistent with an independent accumulation of methionines at the Fbp2 locus in each lineage. Under a steady-state model, the rate of methionine accumulation was constant in the lineage leading to Drosophila, and was twice as fast as that in the calyptrate lineage. Substitution rates were consistent with a slight positive selective advantage for each methionine change in about one-half of amino acid sites in Drosophila. This shows that selection can potentially account for a large proportion of amino acid replacements in the molecular evolution of proteins. Received: 12 December 1994 / Accepted: 15 April 1996     

Genetic control of alcohol dehydrogenase levels in Drosophila

Among the progeny of Drosophila flies heterozygous for two noncomplementing Adh-negative alleles, two individuals were found that had recovered appreciable alcohol dehydrogenase activity, thereby surviving the ethanol medium used as a screen. The most likely explanation is that these Adh-positive flies are the product of intracistronic recombination within the Adh locus. Judging by the distribution of outside markers, one of the crossovers would have been a conventional reciprocal exchange while the other appears to have been an instance of nonreciprocal recombination. The enzymes produced in strains derived from the original survivors can be easily distinguished from wild-type enzymes ADH-S and ADH-F on the basis of their sensitivity to denaturing agents. None of various physical and catalytic properties tested revealed differences between the enzymes of the survivor strains except that in one of them the level of activity is 55–65% of the other. Quantitative immunological determinations of ADH gave estimates of enzyme protein which are proportional to the measured activity levels. These results are interpreted to indicate that different amounts of ADH protein are being accumulated in the two strains.This work was supported in part by NSF Grant PCM 76-19563.     

ADH and phylogenetic relationships ofDrosophila lebanonensis (Scaptodrosophila)

Summary Increasing data onDrosophila alcohol dehydrogenase (ADH) sequences have made it possible to calculate the rate of amino acid replacement per year, which is 1.7×10^–9. This value makes this protein suitable for reconstructing phylogenetic relationships within the genus for those species for which no molecular data are available such asScaptodrosophila. The amino acid sequence ofDrosophila lebanonensis is compared to all of the already knownDrosophila ADHs, stressing the unique characteristic features of this protein such as the conservation of an initiating methionine at the N-terminus, the unique replacement of a glycine by an alanine at a very conserved position in the NAD domain of all dehydrogenases, the lack of a slowmigrating peptide, and the total conservation of the maximally hydrophilic peptide. The functional significance of these features is discussed.Although the percent amino acid identity of the ADH molecule inDrosophila decreases as the number of sequences compared increases, the conservation of residue type in terms of size and hydrophobocity for the ADH molecule is shown to be very high throughout the genusDrosophila. The distance matrix and parsimony methods used to establish the phylogenetic relationships ofD. lebanonensis show that the three subgenera,Scaptodrosophila, Drosophila, andSophophora separated at approximately the same time.     

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.     

Dietary Ethanol Mediates Selection on Aldehyde Dehydrogenase Activity in Drosophila melanogaster

Ethanol is an important environmental variable for fruit-breedingDrosophila species, serving as a resource at low levels anda toxin at high levels. The first step of ethanol metabolism,the conversion of ethanol to acetaldehyde, is catalyzed primarilyby the enzyme alcohol dehydrogenase (ADH). The second step,the oxidation of acetaldehyde to acetate, has been a sourceof controversy, with some authors arguing that it is carriedout primarily by ADH itself, rather than a separate aldehydedehydrogenase (ALDH) as in mammals. We review recent evidencethat ALDH plays an important role in ethanol metabolism in Drosophila.In support of this view, we report that D. melanogaster populationsmaintained on ethanol-supplemented media evolved higher activityof ALDH, as well as of ADH. We have also tentatively identifiedthe structural gene responsible for the majority of ALDH activityin D. melanogaster. We hypothesize that variation in ALDH activitymay make an important contribution to the observed wide variationin ethanol tolerance within and among Drosophila species.     

A genomic investigation of ecological differentiation between free‐living and Drosophila‐associated bacteria

Various bacterial taxa have been identified both in association with animals and in the external environment, but the extent to which related bacteria from the two habitat types are ecologically and evolutionarily distinct is largely unknown. This study investigated the scale and pattern of genetic differentiation between bacteria of the family Acetobacteraceae isolated from the guts of Drosophila fruit flies, plant material and industrial fermentations. Genome‐scale analysis of the phylogenetic relationships and predicted functions was conducted on 44 Acetobacteraceae isolates, including newly sequenced genomes from 18 isolates from wild and laboratory Drosophila. Isolates from the external environment and Drosophila could not be assigned to distinct phylogenetic groups, nor are their genomes enriched for any different sets of genes or category of predicted gene functions. In contrast, analysis of bacteria from laboratory Drosophila showed they were genetically distinct in their universal capacity to degrade uric acid (a major nitrogenous waste product of Drosophila) and absence of flagellar motility, while these traits vary among wild Drosophila isolates. Analysis of the competitive fitness of Acetobacter discordant for these traits revealed a significant fitness deficit for bacteria that cannot degrade uric acid in culture with Drosophila. We propose that, for wild populations, frequent cycling of Acetobacter between Drosophila and the external environment prevents genetic differentiation by maintaining selection for traits adaptive in both the gut and external habitats. However, laboratory isolates bear the signs of adaptation to persistent association with the Drosophila host under tightly defined environmental conditions.     

Evolution of wing pigmentation in Drosophila: Diversity,physiological regulation,and cis-regulatory evolution

Fruit flies (Drosophila and its close relatives, or “drosophilids”) are a group that includes an important model organism, Drosophila melanogaster, and also very diverse species distributed worldwide. Many of these species have black or brown pigmentation patterns on their wings, and have been used as material for evo-devo research. Pigmentation patterns are thought to have evolved rapidly compared with body plans or body shapes; hence they are advantageous model systems for studying evolutionary gains of traits and parallel evolution. Various groups of drosophilids, including genus Idiomyia (Hawaiian Drosophila), have a variety of pigmentations, ranging from simple black pigmentations around crossveins to a single antero-distal spot and a more complex mottled pattern. Pigmentation patterns are sometimes obviously used for sexual displays; however, in some cases they may have other functions. The process of wing formation in Drosophila, the general mechanism of pigmentation formation, and the transport of substances necessary for pigmentation, including melanin precursors, through wing veins are summarized here. Lastly, the evolution of the expression of genes regulating pigmentation patterns, the role of cis-regulatory regions, and the conditions required for the evolutionary emergence of pigmentation patterns are discussed. Future prospects for research on the evolution of wing pigmentation pattern formation in drosophilids are presented, particularly from the point of view of how they compare with other studies of the evolution of new traits.     

Separate DNA sequences are required for normal female and ecdysone-induced male expression of Drosophila melanogaster yolk protein 1

Summary Drosophila melanogaster flies were transformed with a yp1-Adh fusion gene with 890 bp of yp1 5 flanking sequence. In an Adh ^- background these flies show a stage, tissue and sex-specific pattern of alcohol dehydrogenase (ADH) activity characteristic of yolk protein genes. ADH activity is not present in dsx ^D/dsx pseudomales indicating that this fragment contains sites where the dsx gene product exerts its effect. Transformed male flies do not exhibit ADH activity when injected with 20-hydroxyecdysone while synthesis of native yolk proteins is induced. Thus the hormone inducibility and sex regulation have been separated in this construct.     

Sex determination in<Emphasis Type="Italic"> Drosophila melanogaster</Emphasis> and<Emphasis Type="Italic"> Musca domestica</Emphasis> converges at the level of the terminal regulator<Emphasis Type="Italic"> doublesex</Emphasis>

Sex-determining cascades are supposed to have evolved in a retrograde manner from bottom to top. Wilkins 1995 hypothesis finds support from our comparative studies in Drosophila melanogaster and Musca domestica, two dipteran species that separated some 120 million years ago. The sex-determining cascades in these flies differ at the level of the primary sex-determining signal and their targets, Sxl in Drosophila and F in Musca. Here we present evidence that they converge at the level of the terminal regulator, doublesex (dsx), which conveys the selected sexual fate to the differentiation genes. The dsx homologue in Musca, Md-dsx, encodes male-specific (MdDSX^M) and female-specific (MdDSX^F) protein variants which correspond in structure to those in Drosophila. Sex-specific regulation of Md-dsx is controlled by the switch gene F via a splicing mechanism that is similar but in some relevant aspects different from that in Drosophila. MdDSX^F expression can activate the vitellogenin genes in Drosophila and Musca males, and MdDSX^M expression in Drosophila females can cause male-like pigmentation of posterior tergites, suggesting that these Musca dsx variants are conserved not only in structure but also in function. Furthermore, downregulation of Md-dsx activity in Musca by injecting dsRNA into embryos leads to intersexual differentiation of the gonads. These results strongly support a role of Md-dsx as the final regulatory gene in the sex-determining hierarchy of the housefly.Edited by D. Tautz     

Isolation of Protease-Free Alcohol Dehydrogenase (ADH) from Drosophila simulans and Several Homozygous and Heterozygous Drosophila melanogaster Variants

The enzyme alcohol dehydrogenase (ADH) fromseveral naturally occurring ADH variants ofDrosophila melanogaster and Drosophilasimulans was isolated. Affinity chromatography withthe ligand Cibacron Blue and elution with NAD⁺ showed similarbehavior for D. melanogaster ADH-FF, ADH-71k,and D. simulans ADH. Introduction of a secondCibacron Blue affinity chromatography step, withgradient elution with NAD⁺, resulted in pure and stable enzymes. D.melanogaster ADH-SS cannot be eluted from theaffinity chromatography column at a high concentrationof NAD⁺ and required a pH gradient for itspurification, preceded by a wash step with a high concentration ofNAD⁺. Hybrid Drosophila melanogasteralcohol dehydrogenase FS has been isolated fromheterozygous flies, using affinity chromatography withfirst elution at a high concentration NAD⁺, directlyfollowed by affinity chromatography elution with a pHgradient. Incubation of equal amounts of pure homodimersof Drosophila melanogaster ADH-FF and ADH-SS,in the presence of 3 M urea at pH 8.6, for 30 min at roomtemperature, followed by reassociation yielded activeDrosophila melanogaster ADH-FS heterodimers. Noproteolytic degradation was found after incubation ofpurified enzyme preparations in the absence or presenceof SDS, except for some degradation of ADH-SS after verylong incubation times. The thermostabilities of D.melanogaster ADH-71k and ADH-SS were almostidentical and were higher than those of D.melanogaster ADH-FF and D. simulans ADH. Thethermostability of D. melanogaster ADH-FS waslower than those of D. melanogaster ADH-FF andADH-SS. D. melanogaster ADH-FF and ADH-71k have identical inhibition constantswith the ligand Cibacron Blue at pH 8.6, which are twotimes higher at pH 9.5. The K_i values forD. simulans ADH are three times lower at bothpH values. D. melanogaster ADH-SS and ADH-FS havesimilar K_i values, which are lower than thosefor D. melanogaster ADH-FF at pH 8.6. But at pH9.5 the K_i value for ADH-FS is the same as atpH 8.6, while that of ADH-SS is seven times higher. Kinetic parameters ofDrosophila melanogaster ADH-FF, ADH-SS, andADH-71k and Drosophila simulans ADH, at pH 8.6and 9.5, showed little or no variation inK_m ^eth values. TheK_m ^NAD values measured at pH 9.5for Drosophila alcohol dehydrogenases are alllower than those measured at pH 8.6. The rate constants(k_cat) determined for all fourDrosophila alcohol dehydrogenases are higher at pH 9.5 than at pH 8.6. D.melanogaster ADH-FS showed nonlinear kinetics.     

Visual system of the stalk-eyed fly,Cyrtodiopsis quinqueguttata (Diopsidae,Diptera): an anatomical investigation of unusual eyes

Diopsid flies have eye stalks up to a centimeter in length, displacing the retina laterally from the rest of the head. This bizarre condition, called hypercephaly, is rare, but has evolved independently among several insect orders and is most common in flies (Diptera). Earlier studies of geometrical optics and behavior have led to various hypotheses about possible adaptive advantages of eye stalks, such as enhanced stereoscopic vision while other hypothesis suggest that eye stalks are an outcome of sexual selection. Here, we focus on how these curious distortions of head/eye morphology are accompanied by changes in the neural organization of the visual system of Cyrtodiopsis quinqueguttata. Histological examinations reveal that the optic lobes, lamina (La), medulla (Me), lobula (Lo), and lobula plate (LP) are contained entirely within the fly's eye bulbs, which are located at the distal ends of the eye stalks. We report that the organization of the peripheral visual system (La and Me) is similar to that of other Diptera (e.g., Musca and Drosophila), but deeper visual areas (Lo and LP) have been more strongly modified. For example, in both the lobula and lobula plate, fewer but larger giant collector neurons are found. The most pronounced difference is the reduction in the number of wide-field vertical cells of the lobula plate, where there are only four relatively large fibers, as opposed to 11 in Musca. The “fewer but larger” neural organization may enhance the conduction velocities of these cells, but may result in a loss of spatial resolution. At the base of the eye bulb, axon bundles collect and form a long optic nerve that extends the length of the eye stalk. We suggest that this organization of the diopsid visual system provides evidence for the costs of possessing long eye stalks. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 449–468, 1998     

Alcohol dehydrogenase genes: restriction fragment length polymorphisms for ADH4 (π-ADH) and ADH5 (χ-ADH) and construction of haplotypes among different ADH classes

Of the five human alcohol dehydrogenase (ADH) genes located in the region q21–25 of chromosome 4, genetic markers have been reported previously only for class I enzymes, ADH1-3. Here, new restriction fragment length polymorphisms (RFLPs) are described for the genes of two other classes, ADH4 () and ADH5 ( or formaldehyde dehydrogenase, FDH). The frequencies and modes of inheritance of these RFLPs were determined with DNA both from unrelated individuals and from families. A polymorphic PstI site is assigned to the fourth intron of the ADH4 gene. Pairwise linkage disequilibrium calculations for these new RFLPs and already known RFLPs at the ADH2 and ADH3 loci establish strong linkage disequilibria between polymorphic MspI and BstXI sites in the ADH5 gene as well as between XbaI and MspI sites in the ADH3 gene. Furthermore, linkage disequilibria were detected between RFLPs of the ADH2 and ADH3 genes as well as between those of the ADH4 and ADH5 genes. The latter disequilibrium implies a hitherto unknown physical proximity of two genes belonging to different ADH classes. The RFLPs were used to construct chromosomal haplotypes that include three ADH classes. Of the 16 possible haplotypes for four RFLP markers used here, 10 were experimentally detected. The potential application of the ADH RFLPs and haplotypes in linkage or association studies of inherited diseases such as familial alcoholism is discussed.     

Telomere-Specialized Retroelements in Drosophila: Adaptive Symbionts of the Genome,Neutral, or in Conflict?

Linear chromosomes shorten in every round of replication. In Drosophila, telomere-specialized long interspersed retrotransposable elements (LINEs) belonging to the jockey clade offset this shortening by forming head-to-tail arrays at Drosophila telomere ends. As such, these telomeric LINEs have been considered adaptive symbionts of the genome, protecting it from premature decay, particularly as Drosophila lacks a conventional telomerase holoenzyme. However, as reviewed here, recent work reveals a high degree of variation and turnover in the telomere-specialized LINE lineages across Drosophila. There appears to be no absolute requirement for LINE activity to maintain telomeres in flies, hence the suggestion that the telomere-specialized LINEs may instead be neutral or in conflict with the host, rather than adaptive.     

Abamectin resistance in Drosophila is related to increased expression of P-glycoprotein via the dEGFR and dAkt pathways

Many insects have evolved resistance to abamectin but the mechanisms involved in this resistance have not been well characterized. P-glycoprotein (P-gp), an ATP-dependent drug-efflux pump transmembrane protein, may be involved in abamectin resistance. We investigated the role of P-gp in abamectin (ABM) resistance in Drosophila using an ABM-resistant strain developed in the laboratory. A toxicity assay, Western blotting analysis and a vanadate-sensitive ATPase activity assay all demonstrated the existence of a direct relationship between P-gp expression and ABM resistance in these flies. Our observations indicate that P-gp levels in flies' heads were higher than in their thorax and abdomen, and that both P-gp levels and LC₅₀ values were higher in resistant than in susceptible and P-gp-deficient strains. In addition, P-gp levels in the blood–brain barrier (BBB) of resistant flies were higher than in susceptible and P-gp-deficient flies, which is further evidence that a high level of P-gp in the BBB is related to ABM resistance. Furthermore, we found greater expression of Drosophila EGFR (dEGFR) in the resistant strain than in the susceptible strain, and that the level of Drosophila Akt (dAkt) was much higher in resistant than in susceptible flies, whereas that in P-gp-deficient flies was very low. Compared to susceptible flies, P-gp levels in the resistant strain were markedly suppressed by the dEGFR and dAkt inhibitors lapatinib and wortmannin. These results suggest that the increased P-gp in resistant flies was regulated by the dEGFR and dAkt pathways and that increased expression of P-gp is an important component of ABM resistance in insects.     

Evolutionary ecology of learning: insights from fruit flies

Ecologically and evolutionarily oriented research on learning has traditionally been carried out on vertebrates and bees. While less sophisticated than those animals, fruit flies (Drosophila) are capable of several forms of learning, and have the advantage of a short generation time, which makes them an ideal system for experimental evolution studies. This review summarizes the insights into evolutionary questions about learning gained in the last decade from evolutionary experiments on Drosophila. These experiments demonstrate that Drosophila has the genetic potential to evolve a substantially improved learning performance in ecologically relevant learning tasks. In at least one set of selected populations, the improved learning generalized to a task other than that used to impose selection, involving a different behavior, different stimuli, and a different sensory channel for the aversive reinforcement. This improvement in learning ability was associated with reductions in other fitness-related traits, such as larval competitive ability and lifespan, pointing to evolutionary trade-offs for improved learning. These trade-offs were confirmed by other evolutionary experiments where a reduction in learning performance was observed as a correlated response to selection for tolerance to larval nutritional stress or for delayed aging. Such trade-offs could be one reason why fruit flies have not fully used up their evolutionary potential for learning. Finally, another evolutionary experiment with Drosophila provided the first direct evidence for the long-standing idea that learning can under some circumstances accelerate and in others slow down genetically based evolutionary change. These results demonstrate the usefulness of fruit flies as a model system to address evolutionary questions about learning.     

Natural alcohol exposure: is ethanol the main substrate for alcohol dehydrogenases in animals?

Alcohol dehydrogenase (ADH) activity is widely distributed in all phyla. In animals, three non-homologous NAD(P)(+)-dependent ADH protein families are reported. These arose independently throughout evolution and possess different structures and mechanisms of reaction: type I (medium-chain) ADHs are zinc-containing enzymes and comprise the most studied group in vertebrates; type II (short-chain) ADHs lack metal cofactor and have been extensively studied in Drosophila; and type III ADHs are iron-dependent/-activated enzymes that were initially identified only in microorganisms. The presence of these different ADHs in animals has been assumed to be a consequence of chronic exposure to ethanol. By far the most common natural source of ethanol is fermentation of fruit sugars by yeast, and available data support that this fruit trait evolved in concert with the characteristics of their frugivorous seed dispersers. Therefore, if the presence of ADHs in animals evolved as an adaptive response to dietary ethanol exposure, then it can be expected that the enzymogenesis of these enzymes began after the appearance of angiosperms with fleshy fruits, because substrate availability must precede enzyme selection. In this work, available evidence supporting this possibility is discussed. Phylogenetic analyses reveal that type II ADHs suffered several duplications, all of these restricted to flies (order Diptera). Induction of type II Adh by ethanol exposure, a positive correlation between ADH activity and ethanol resistance, and the fact that flies and type II Adh diversification occurred in concert with angiosperm diversification, strongly suggest that type II ADHs were recruited to allow larval flies to exploit new restricted niches with high ethanol content. In contrast, phyletic distribution of types I and III ADHs in animals showed that these appeared before angiosperms and land plants, independently of ethanol availability. Because these enzymes are not induced by ethanol exposure and possess a high affinity and/or catalytic efficiency for non-ethanol endogenous substrates, it can be concluded that the participation of types I and III ADHs in ethanol metabolism can be considered as incidental, and not adaptive.     

In vivo gap repair in Drosophila: a one-way street with many destinations

While it has long been possible to study the process of recombination in yeast and other single-celled organisms, it has been difficult to distinguish between pathways of meiotic and mitotic recombination in multicellular eukaryotes. The experimental system described here bridges the historically separated fields of Genetic Recombination and DNA Repair in Drosophila. It is now feasible to study the repair of unique double-strand breaks induced in the Drosophila genome by the excision of a P-transposable element or by cleavage at an introduced endonuclease recognition sequence. This repair can be studied in both somatic cells and mitotically dividing germ cells. The repair of these breaks occurs mainly by copying sequence from a template located anywhere in the karyoplasm, and occurs in both male and female flies. This system, which was the first of its kind in metazoan organisms, is now being used for gene targeting in Drosophila. This review summarizes results that provide new insights into the process of gap repair in Drosophila and outline some recent experiments that demonstrate the power of the gene targeting technique. BioEssays 20: 317-327, 1998.© 1998 John Wiley & Sons, Inc.