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.     

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.     

Alcohol dehydrogenase and adaptation to ethanol in Drosophila

The subject of this research is activity and allozyme spectra of alcohol dehydrogenase (ADH), and survival of mutant strains of Drosophila kept in standard nutrient medium with added ethanol. In all experiments the ADH of flies revealed greater affinity to isopropanol than ethanol. The mutant strains considerably differed from one another and from the wild type of flies in the level of enzyme activity, which may be connected with genotypic properties in the mutants studied. The ADH variability in mutant strains seems to be caused by different alleles of the structural ADH gene, which was established as a result of investigation of activity, electrophoretic mobility and thermostability of corresponding allozymes. As follows from experiments on the genotypical structure of populations in the conditions of fly selection in the medium containing ethanol (10%), the adaptation of flies to exogenous ethanol takes place via mechanisms of allele control of the ADH activity. Phenotypical manifestation of the ADH locus and its effect on the resistance of Drosophila to alcohol are supposed to depend on complex gene interactions determined by the genotype as a whole.     

Variation in amount of enzyme protein in natural populations

Among strains of Drosophila melanogaster each derived from a single fertilized female taken from natural populations, there is variation in both alcohol dehydrogenase (ADH) activity and the amount of ADH protein. The correlation between ADH activity and number of molecules over all strains examined is 0.87 or 0.96 in late third instar larvae depending on whether the substrate is 2-propanol or ethanol. With respect to the two common electrophoretic allozymic forms, F and S, segregating in these populations, the FF strains on the whole have higher ADH activities and numbers of ADH molecules than the SS strains. Over all strains examined, enzyme extracts from FF strains have a mean catalytic efficiency per enzyme molecule higher than that of enzyme extracts from SS strains when ethanol is the substrate, and much higher when 2-propanol is the substrate. One FF strain had an ADH activity/ADH protein ratio characteristic of SS strains.     

Drosophila Alcohol Dehydrogenase Polymorphism and Carbon-13 Fluxes: Opportunities for Epistasis and Natural Selection

The influence of genetic variations in Drosophila alcohol dehydrogenase (ADH) on steady-state metabolic fluxes was studied by means of (13)C NMR spectroscopy. Four pathways were found to be operative during 8 hr of ethanol degradation in third instar larvae of Drosophila. Seven strains differed by 18-25% in the ratio between two major pathway fluxes, i.e., into glutamate-glutamine-proline vs. lactate-alanine-trehalose. In general, Adh genotypes with higher ADH activity exhibit a twofold difference in relative carbon flux from malate into lactate and alanine vs. α,α-trehalose compared to low ADH activity genotypes. Trehalose was degraded by the pentose-phosphate shunt. The pentose-phosphate shunt and malic enzyme could supply NADPH necessary for lipid synthesis from ethanol. Lactate and/or proline synthesis may maintain the NADH/NAD(+) balance during ethanol degradation. After 24 hr the flux into trehalose is increased, while the flux into lipids declines in Adh(F) larvae. In Adh(S) larvae the flux into lipids remains high. This co-ordinated nature of metabolism and the genotype-dependent differences in metabolic fluxes may form the basis for various epistatic interactions and ultimately for variations in organismal fitness.     

Conditioning to ethanol in the fruit fly-a study using an inhibitor of ADH

To identify processes involved in the choice of ethanol by adult Drosophila, flies homozygous Adh(F), reared in the absence of alcohol were placed in contact with: a) an ethanol-free medium, b) a medium containing ethanol, c) a medium supplemented with 4-methylpyrazole (4-MP, an inhibitor of the ADH pathway), d) a medium containing ethanol and 4-MP. The choice of ethanol over a medium without ethanol was evaluated by measuring the duration of extension of the proboscis of the flies in each of the media. A slight preference for the ethanol-supplemented medium was observed in the naive flies, which was enhanced by previous exposure to ethanol. Exposure to ethanol and 4-MP, however, led to an avoidance of ethanol. There was a reduction in ADH activity on treatment of the flies with 4-MP, and signs of malaise (reduced locomotor activity, loss of balance) were observed in the flies who ingested both ethanol and inhibitor. We concluded that the preference for ethanol stems from an associative learning related to ethanol utilization. Inhibition of enzymes of ADH pathway led to a conditioned aversion due to disturbance of ethanol metabolism giving rise to malaise.     

Alcohol dehydrogenase and ethanol tolerance at the cellular level in Drosophila melanogaster

Exposure of early third instar larvae of Drosophila melanogaster to a nonlethal dose of ethanol was detrimental to larvae lacking alcohol dehydrogenase (ADH) but beneficial to wild-type larvae in terms of surviving a later ethanol tolerance test, indicating that one of the important functions of the ADH system is to supply derivatives of ethanol to larvae that in turn promote ethanol tolerance. High intracellular concentrations of ethanol in ADH-deficient (Adhn2) larvae fed ethanol were accompanied by a decrease in the cell membrane infoldings of fat body cells, suggesting that the capacities to absorb and release molecules were reduced. Marked effects of ethanol on the endoplasmic reticulum and mitochondria of ADH-deficient larvae were also evident. The absence of similar changes in wild-type larvae that were fed moderate levels of ethanol showed that the ADH system kept the intracellular level of ethanol at a concentration low enough to avoid cell damage. A cytometric analysis of electron micrographs showed that there were ethanol-induced reductions in glycogen, lipid, and protein stores in the fat body cells of ADH-deficient larvae fed 1.25% ethanol (v/v) compared with null larvae fed an ethanol-free diet. This finding implied that the capacities to synthesize or store these compounds may be limited by high intracellular concentrations of ethanol. The cytometric analysis also revealed that the consumption of diets containing 2.5% and 4.5% ethanol by Canton-S wild-type larvae for 3 days after 4 days of feeding on an ethanol-free diet resulted in decreases in glycogen and protein deposits in fat body cells, but increased the amount of lipid deposits compared to larvae fed an ethanol-free diet. This observation, coupled with the greater weight of wild-type adults that were fed a growth-limiting concentration of ethanol compared with control adults, suggested that a metabolic defense mechanism in larvae is to convert toxic ethanol to nontoxic storage products. Dietary ethanol alone and in combination with isopropanol stimulated an increase in the size of the NAD-pool in larvae, a condition that may favor the activity of ADH. A low dietary level of isopropanol (1%) completely blocked glycogen deposition in wild-type larvae, whereas ethanol did not. Thus ethanol and isopropanol exert some different toxic effects on larval fat bodies.     

Sensitivity differences displayed by Drosophila melanogaster larvae of different ages to the toxic effects of growth on media containing aflatoxin B1.

Using Drosophila melanogaster, the relative sensitivities of various larval stages to the toxic effects of growth on media supplemented with either 0.44 or 0.88 ppm aflatoxin B1 (AFB1) were determined. Two strains of fruit flies were tested: strain A-11 which is relatively resistant to AFB1 induced toxicity, and strain A-9 which is quite sensitive. Eggs, mid-first, mid-second and early-, mid- and late-third instar larvae were transferred onto AFB1 media and allowed to complete larval and pupal development and eclose as adults. At the 0.44 ppm concentration, strain A-11 showed no effect, while only first instar larvae of strain A-9 showed significant mortality rates for first instar larvae, but the A-9 larvae die at higher rates than the A-11 larvae. In addition, second and third instar larvae of strain A-9 show significant mortality rates when grown at 0.88 ppm AFB1, while these stages are not affected in strain A-11.     

A MULTILEVEL APPROACH TO THE SIGNIFICANCE OF GENETIC VARIATION IN ALCOHOL DEHYDROGENASE OF DROSOPHILA

Prior studies showed that differences in alcohol dehydrogenase (ADH) activity across genotypes of Drosophila are decisive for the outcome of selection by ethanol. In the present paper, the effect on ADH activity and egg-to-adult survival of combinations of ethanol, propan-2-ol, and acetone in naturally occurring concentrations is examined. Propan-2-ol is converted into acetone by ADH in vitro. Acetone is considered a competitive inhibitor of ethanol for the ADH enzymes. The melanogaster-ADH-S allozyme is two times more sensitive towards inhibition by acetone than either simulans-ADH or melanogaster-ADH-F. The physiological implications of these in vitro differences for larvae were studied in short-term in vivo and long-term exposure experiments. No major differences in acetone accumulation or fitness parameters were found between the strains in response to ecologically relevant concentrations of acetone or propan-2-ol. Ethanol, however, strongly decreased egg-to-pupal survival in both Drosophila simulans strains and increased developmental time in four out of the five strains tested. Therefore, under physiological conditions only ethanol was shown to act as a selective agent on the ADH polymorphism during egg-to-pupa development in Drosophila.     

Enzymatic responses of Drosophila melanogaster to long- and short-term exposures to ethanol

The effects of environmental ethanol on larva-to-pupa survival and on the activities of four enzymes were investigated in three Drosophila melanogaster strains. The strains had different allelic combinations at the Odh and Aldox loci on their third chromosomes, but they all carried the Adh ^S -Gpdh ^F allelic combination on the second chromosome. Replicates of each of the strains were exposed to three different ethanol treatments: (i) no ethanol in the medium (control); (ii) 5% ethanol for a single generation (short-term exposure); (iii) 5% ethanol for 20 generations (long-term exposure). In all experiments, the activities of four enzymes (ADH, ODH, GPDH and AOX) were measured in larvae, pupae and adults. The results showed that (i) the larval and adult metabolic responses to environmental ethanol were different; (ii) enzyme activity changes under short-term exposure differed from those measured under long-term exposure; (iii) the activities of the allozymes common to all strains (ADH-S and GPDH-F), differed depending on the genetic background. Changes in larva-to-pupa survival were seen when the larvae of control and exposed lines of the three strains were confronted with various concentrations of ethanol. In all three strains, the exposed lines had significantly higher initial survival rate and ethanol tolerance than the control lines. Strain-specific differences were observed in the ethanol tolerance of both types of line. Received: 26 November 1996 / Accepted: 14 February 1997     

Enzymatic responses of Drosophila melanogaster to long- and short-term exposures to ethanol

The effects of environmental ethanol on larva-to-pupa survival and on the activities of four enzymes were investigated in three Drosophila melanogaster strains. The strains had different allelic combinations at the Odh and Aldox loci on their third chromosomes, but they all carried the Adh ^S -Gpdh ^F allelic combination on the second chromosome. Replicates of each of the strains were exposed to three different ethanol treatments: (i) no ethanol in the medium (control); (ii) 5% ethanol for a single generation (short-term exposure); (iii) 5% ethanol for 20 generations (long-term exposure). In all experiments, the activities of four enzymes (ADH, ODH, GPDH and AOX) were measured in larvae, pupae and adults. The results showed that (i) the larval and adult metabolic responses to environmental ethanol were different; (ii) enzyme activity changes under short-term exposure differed from those measured under long-term exposure; (iii) the activities of the allozymes common to all strains (ADH-S and GPDH-F), differed depending on the genetic background. Changes in larva-to-pupa survival were seen when the larvae of control and exposed lines of the three strains were confronted with various concentrations of ethanol. In all three strains, the exposed lines had significantly higher initial survival rate and ethanol tolerance than the control lines. Strain-specific differences were observed in the ethanol tolerance of both types of line.     

Acetaldehyde detoxification mechanisms in Drosophila melanogaster adults involving aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) enzymes

The mechanism of acetaldehyde detoxification in Drosophila melanogaster adults has been studied by comparing physiological in vitro and in vivo data. ADH⁺ and ADH⁻ flies, both lacking aldehyde dehydrogenase activity from ADH (ALDH^ADH, ALDH (ALDH) or both enzymes were exposed to acetaldehyde or ethanol, and the toxicity and internal accumulation of both compounds were determined. Acetaldehyde was extremely lethal for flies whose ALDH activity had been inhibited by cyanamide, though acetaldehyde was effectively detoxified by flies whose ALDH^ADH activity had been inhibited by acetone. After exposure to acetaldehyde, both acetaldehyde and ethanol rapidly accumulated in flies lacking ALDH activity, but not in flies lacking ALDH^ADH activity. However, ethanol but not acetaldehyde quickly accumulated in flies lacking ALDH activity after exposure to ethanol. Our results provide in vivo evidence that, as opposed to larvae, in D. melanogaster adults acetaldehyde is mainly oxidized into acetate by means of ALDH enzymes. However, the reducing activity of the ADH enzyme, which transforms acetaldehyde into ethanol, also plays an essential role in the detoxification of acetaldehyde. Differences in ALDH activity might be important to explain the differences in ethanol tolerance found in natural populations.     

The Effect of Oxygen on the Frequency of Somatic Recombination in DROSOPHILA MELANOGASTER

The influence of oxygen on the frequency of somatic recombination in the yellow singed system on the X chromosome of Drosophila melanogaster was studied under a variety of experimental conditions. Flies raised from egg to adult in atmospheres containing 70-90% oxygen were found to have significantly more mosaic spots on their abdominal tergites than were observed in flies which developed in air. First instar larvae X-rayed in from 0 to 100% oxygen demonstrated the existence of an oxygen effect for somatic recombination in the cells which form the abdominal hypoderm. The mosaic spot counts, beginning with the lowest numbers which were found in flies X-rayed in nitrogen, increased rapidly with rising oxygen tensions until the percentage in air was reached, then leveled off at the higher concentrations. Post-treatment with nitrogen of larvae X-rayed in air or oxygen created a substantially higher number of mosaic spots than were found when larvae, after being similarly irradiated, were instead placed into air or oxygen.     

Developmental toxicity of ethanol in Drosophila melanogaster

Ethanol was tested for teratogenicity in Drosophila melanogaster. Treatment consisted of rearing the fly larvae in media containing initial ethanol concentrations of 0%, 4%, 8%, or 14% by weight. Emerging flies were inspected for gross malformations. A low frequency of malformations was seen among controls (0.82%), increasing to 10.36% of emerging adults at the highest ethanol dose. The most common malformation involved the legs (segments missing or distorted or complete absence) and wings (uninflated, distorted, or absent). Less frequent defects included fused or missing mouth parts and missing halteres. Also, by exposing staged larvae to ethanol and examining the emerging flies, developmental stage sensitivity of Drosophila was investigated in terms of timing of treatment initiation. The results suggested that the incidence of defects increased with length of exposure. These results support the assumption that ethanol itself is the causative agent in ethanol-induced developmental toxicity and further support the use of Drosophila for developmental toxicity screening.     

Larval ethanol exposure alters adult circadian free-running locomotor activity rhythm in Drosophila melanogaster

Alcohol consumption causes disruptions in a variety of daily rhythms, including the sleep-wake cycle. Few studies have explored the effect of alcohol exposure only during developmental stages preceding maturation of the adult circadian clock, and none have examined the effects of alcohol on clock function in Drosophila. This study investigates developmental and behavioral correlates between larval ethanol exposure and the adult circadian clock in Drosophila melanogaster, a well-established model for studying circadian rhythms and effects of ethanol exposure. We reared Drosophila larvae on 0%, 10%, or 20% ethanol-supplemented food and assessed effects upon eclosion and the free-running period of the circadian rhythm of locomotor activity. We observed a dose-dependent effect of ethanol on period, with higher doses resulting in shorter periods. We also identified the third larval instar stage as a critical time for the developmental effects of 10% ethanol on circadian period. These results demonstrate that developmental ethanol exposure causes sustainable shortening of the adult free-running period in Drosophila melanogaster, even after adult exposure to ethanol is terminated, and suggests that the third instar is a sensitive time for this effect.     

Genetic sexing in Drosophila melanogaster using the alcohol dehydrogenase locus and a Y-linked translocation

Summary By incorporating ethanol (4% v/v) into the larval rearing medium of a specially constructed Drosophila melanogaster strain it was possible to produce only male adults; the female larvae died.In this strain, the male determining chromosome was linked with a positive Alcohol dehydrogenase (ADH) allele by a translocation. The females were homozygous for the null allele and hence sensitive to ethanol.This genetic sexing method is discussed in relation to its use in the genetic control of insects.     

Correct developmental expression of a cloned alcohol dehydrogenase gene transduced into the Drosophila germ line

We have used P-element-mediated transformation to introduce a cloned Drosophila alcohol dehydrogenase (Adh) gene into the germ line of ADH null flies. Six independent transformants expressing ADH were identified by their acquired resistance to ethanol. Each transformant carries a single copy of the cloned Adh gene in a different chromosomal location. Four of the six transformant lines exhibit normal Adh expression by the following criteria: quantitative levels of ADH enzyme activity in larvae and adults; qualitative tissue specificity; the size of stable Adh mRNA; and the characteristic developmental switch in utilization of two different Adh promoters. The remaining two transformants express ADH enzyme activity with the correct tissue specificity, but at a lower level than wild type. These results demonstrate that an 11.8 kb chromosomal fragment containing the Adh gene includes the cis-acting sequences necessary for its correct developmental expression, and that a variety of chromosomal sites permit proper Adh gene function.     

Aldehyde oxidase activity in the tumorous-head strain of Drosophila melanogaster

Gross aldehyde oxidase activity from the egg-stage through 10-day-old adults and distribution of the enzyme in eye-antennal imaginal discs in third instar larvae were determined for the tumorous-head strain of Drosophila melanogaster. Aldehyde oxidase activity of several laboratory strains was measured for comparative purposes. Aldehyde oxidase activity was 100% higher during embryogenesis in tuh(ASU) eggs than in Oregon-R-C eggs. A second period of elevated aldehyde oxidase activity was observed during metamorphosis where tuh(ASU) pupae averaged 65% more enzyme activity than Oregon-R-C. Therefore, during determination and differentiation of the eye-antennal imaginal disc, the tuh(ASU) strain possesses a high aldehyde oxidase activity. Wild-type Drosophila melanogaster antennal imaginal discs are aldehyde oxidase positive, whereas attached eye imaginal discs are apparently aldehyde oxidase negative. A sample of eye-antennal imaginal discs from tuh(ASU) third instar larvae revealed that either one or both eye discs of 64% of the larvae were aldehyde oxidase positive. Aldehyde oxidase activity may be correlated with the homoeotic transformation in parts of the eye disc.