Acetaldehyde utilization and toxicity in Drosophila adults lacking alcohol dehydrogenase or aldehyde oxidase

Metabolic utilization and toxicity of acetaldehyde were studied in flies lacking alcohol dehydrogenase (ADH), aldehyde oxidase (AO), or both functions. Prior to the experiments, mutant alleles Adhn4 and mal were transferred to the same genetic background by 10 successive backcrosses. By comparison with wild-type flies, various deleterious, pleiotropic effects could be attributed to the mal allele but not to Adhn4. Of the four genotypes studied (mal, Adhn4, mal Adhn4, and wild), all were able to use acetaldehyde as a resource in a similar way. In spite of its high toxicity, acetaldehyde appeared a better resource than ethanol. Flies treated with intermediate acetaldehyde concentrations (around 0.5%) exhibited a very high interindividual heterogeneity which could reflect a physiological adaptation occurring as a consequence of the aldehyde treatment. Toxicity tests showed that ADH-negative flies were more sensitive to acetaldehyde than wild type, but this is most likely explained by the transformation of the aldehyde into alcohol. Our results show that the aldehyde metabolizing enzyme (AME) system in Drosophila is neither ADH nor AO. The existence of an aldehyde dehydrogenase is plausible.     

Role of peroxisomal fatty acid beta-oxidation in ethanol metabolism

The contribution of peroxisomal fatty acid beta-oxidation to ethanol metabolism was examined in deermice hepatocytes. Addition of 1 mM oleate to hepatocytes isolated from fasted alcohol dehydrogenase (ADH)-positive deermice in the presence of 4-methylpyrazole or to hepatocytes from fasted or fed ADH-negative deermice produced only a slight and statistically not significant increase in ethanol oxidation. Lactate (10 mM), which is not a peroxisomal substrate, showed a greater effect on ethanol oxidation. There was also a lack of oleate effect on the oxidation of ethanol by hepatocytes of ADH-positive deermice. Furthermore, in ADH-negative deermice, the catalase inhibitor azide (0.1 mM) did not inhibit the increase in ethanol oxidation by oleate and lactate. The rate of oleate oxidation by hepatocytes from fasted ADH-negative deermice was much lower than that of ethanol. These results indicate that in deermice hepatocytes, peroxisomal fatty acid oxidation does not play major role in ethanol metabolism.     

Circadian modulation of acute alcohol sensitivity but not acute tolerance in Drosophila

An increased understanding of the factors affecting behavioral and neurological responses to alcohol and alcohol physiology is necessary given the tremendous toll alcohol abuse and alcoholism exert on individuals and society. At the behavioral and molecular levels, the response to alcohol appears remarkably conserved from Drosophila to humans, suggesting that investigations across model species can provide insight into the identification of common modulatory factors. We investigated the interaction between the circadian clock and alcohol sensitivity, alcohol tolerance, and alcohol absorbance in Drosophila melanogaster. Using a loss-of-righting reflex (LoRR) assay, we found that flies exhibit a circadian rhythm in the LoRR, with the greatest sensitivity to alcohol occurring from mid to late night, corresponding to the flies' inactive phase. As predicted, a circadian rhythm in the LoRR was absent in circadian mutant flies and under conditions in which the circadian clock was nonfunctional. Circadian modulation of the response to alcohol was not due to circadian regulation of alcohol absorbance. Similar to other animals, Drosophila develop acute and chronic tolerance to alcohol upon repeat exposures. We found that the circadian clock did not modulate the development of acute alcohol tolerance measured as the difference in sensitivity to alcohol between na?ve and pre-exposed flies. Thus, the circadian clock modulates some, but not all, of the behavioral responses to alcohol exposure, suggesting that specific mechanisms underlie the observed circadian modulation of LoRR rather than global cellular circadian regulation. This study provides valuable new insights in our understanding of the circadian modulation of alcohol-induced behaviors that ultimately could facilitate preventative measures in combating alcohol abuse and alcoholism.     

The use of promoter fusions in Drosophila genetics: isolation of mutations affecting the heat shock response

We have constructed a gene fusion using the promoter of Drosophila hsp70 and the structural gene for Drosophila alcohol dehydrogenase (Adh) and used this construct to transform Adh-deficient flies. In these transformants, Adh is expressed only after heat shock. Like hsp70 itself, this heat-shock-inducible Adh (Adhhs) is induced in a wide variety of tissues. It fails to be induced in primary spermatocytes. Although the tissue distribution of Adh activity is very different from wild type, this does not appear to be deleterious. Indeed, the induction of Adhhs allows flies to survive exposure to ethanol. We have used this latter characteristic to select dominant, trans-acting mutations that alter the response of flies to heat shock.     

Circadian Modulation of Acute Alcohol Sensitivity But Not Acute Tolerance in Drosophila

An increased understanding of the factors affecting behavioral and neurological responses to alcohol and alcohol physiology is necessary given the tremendous toll alcohol abuse and alcoholism exert on individuals and society. At the behavioral and molecular levels, the response to alcohol appears remarkably conserved from Drosophila to humans, suggesting that investigations across model species can provide insight into the identification of common modulatory factors. We investigated the interaction between the circadian clock and alcohol sensitivity, alcohol tolerance, and alcohol absorbance in Drosophila melanogaster. Using a loss-of-righting reflex (LoRR) assay, we found that flies exhibit a circadian rhythm in the LoRR, with the greatest sensitivity to alcohol occurring from mid to late night, corresponding to the flies' inactive phase. As predicted, a circadian rhythm in the LoRR was absent in circadian mutant flies and under conditions in which the circadian clock was nonfunctional. Circadian modulation of the response to alcohol was not due to circadian regulation of alcohol absorbance. Similar to other animals, Drosophila develop acute and chronic tolerance to alcohol upon repeat exposures. We found that the circadian clock did not modulate the development of acute alcohol tolerance measured as the difference in sensitivity to alcohol between naïve and pre-exposed flies. Thus, the circadian clock modulates some, but not all, of the behavioral responses to alcohol exposure, suggesting that specific mechanisms underlie the observed circadian modulation of LoRR rather than global cellular circadian regulation. This study provides valuable new insights in our understanding of the circadian modulation of alcohol-induced behaviors that ultimately could facilitate preventative measures in combating alcohol abuse and alcoholism. (Author correspondence: lyons@bio.fsu.edu)     

Genetic tolerance to ethanol in drosophila melanogaster: Increase by selection and analysis of correlated responses

After 28 generations of selection, the alcohol tolerance of a French strain was increased from its initial value of 18% (lethal concentration 50 for adults after two days of treatment) to about 26%. A decrease in the between-individual variation was observed in the selected line. Comparison of the LC 50 after 1, 2 or 3 days of treatment showed that resistant flies had a better capacity to withstand a prolonged exposure to alcohol.Study of various biometrical and physiological traits in the selected and unselected line showed only minor differences between them. The considerable size difference observed between European and tropical African strains cannot be considered to be the cause of the higher alcohol tolerance found in French strains.     

Peromyscus alcohol dehydrogenase: Lack of cross-reacting material in enzyme-negative animals

Two forms of alcohol dehydrogenase (ADH), coded by allelic genes, have been purified to homogeneity from Peromyscus. Monospecific antisera to the purified enzymes have been raised in rabbits. These antisera fail to detect cross-reacting material in the liver of ADH-negative animals on Ouchterlony plates. Immuno-titration of anti-ADH antiserum with ADH in liver extracts from AdhS/AdhS and AdhS/AdhN animals results in identical equivalence points, again suggesting the absence of cross-reacting material coded by the AdhN allele. Over a wide range of anti-ADH antiserum dilutions, radiolabeled protein was not immunoprecipitable from liver extracts of AdhN/AdhN animals. These immunochemical tests, in conjunction with previous studies, suggest that the AdhN allele in Peromyscus does not produce inactive polypeptide in normal levels that bears immunological determinants similar to those of the fast and slow ADH isozymes.     

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.     

Transcriptional response to alcohol exposure in Drosophila melanogaster

Background  

Alcoholism presents widespread social and human health problems. Alcohol sensitivity, the development of tolerance to alcohol and susceptibility to addiction vary in the population. Genetic factors that predispose to alcoholism remain largely unknown due to extensive genetic and environmental variation in human populations. Drosophila, however, allows studies on genetically identical individuals in controlled environments. Although addiction to alcohol has not been demonstrated in Drosophila, flies show responses to alcohol exposure that resemble human intoxication, including hyperactivity, loss of postural control, sedation, and exposure-dependent development of tolerance.     

The FlyBar: Administering Alcohol to Flies

Fruit flies (Drosophila melanogaster) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian clock modulates alcohol sensitivity, but not the formation of tolerance. Here, we describe our protocol in detail. Alcohol is administered to the flies using the FlyBar. In this setup, saturated alcohol vapor is mixed with humidified air in set proportions, and administered to the flies in four tubes simultaneously. Flies are reared under standardized conditions in order to minimize variation between the replicates. Three-day old flies of different genotypes or treatments are used for the experiments, preferably by matching flies of two different time points (e.g., CT 5 and CT 17) making direct comparisons possible. During the experiment, flies are exposed for 1 hr to the pre-determined percentage of alcohol vapor and the number of flies that exhibit the Loss of Righting reflex (LoRR) or sedation are counted every 5 min. The data can be analyzed using three different statistical approaches. The first is to determine the time at which 50% of the flies have lost their righting reflex and use an Analysis of the Variance (ANOVA) to determine whether significant differences exist between time points. The second is to determine the percentage flies that show LoRR after a specified number of minutes, followed by an ANOVA analysis. The last method is to analyze the whole times series using multivariate statistics. The protocol can also be used for non-circadian experiments or comparisons between genotypes.     

Repellent effectiveness of seven plant essential oils,sunflower oil and natural insecticides against horn flies on pastured dairy cows and heifers

Plant essential oils (basil, geranium, balsam fir, lavender, lemongrass, peppermint, pine and tea tree), mixed with either sunflower oil or ethyl alcohol, were applied at 5% concentrations to the sides of Holstein cattle. Pastured cattle treated with essential oils diluted in sunflower oil had less flies than the untreated control for a 24‐h period. However, the essential oil treatments were not significantly different than the carrier oil alone. Barn‐held heifers treated with essential oils and sunflower oil alone had significantly less flies than the untreated control for up to 8 h after treatment. Basil, geranium, lavender, lemongrass and peppermint repelled more flies than sunflower oil alone for a period ranging from 1.5 to 4 h after treatments applied to heifers. All essential oils repelled > 75% of the flies on the treated area for 6 and 8 h on pastured cows and indoor heifers, respectively. Geranium, lemongrass and peppermint stayed effective for a longer duration. Essential oils mixed with ethyl alcohol demonstrated less repellence than when mixed with the carrier oil. Safer's soap, natural pyrethrins without piperonyl butoxide and ethyl alcohol alone were not efficient at repelling flies. Essential oils could be formulated for use as fly repellents in livestock production.     

Chemical Selection of Mutants That Affect Alcohol Dehydrogenase in Drosophila II. Use of 1–PENTYNE–3–Ol

We describe a procedure for the selection of alcohol dehydrogenase negative mutants in Drosophila. The method consists of exposing eggs and larvae to low concentrations of 1-pentyne-3-ol dissolved in the culture medium. Only those flies with greatly reduced levels of alcohol dehydrogenase activity survive. In addition, genotypically negative flies die if their mothers are alcohol dehydrogenase positive. Using this procedure and formaldehyde to generate mutants, we were able to detect seven alcohol dehydrogenase negative mutants out of 350,000 individuals subjected to selection. At least five of the mutants contain small deletions that include the alcohol dehydrogenase locus.     

Exposure to stable flies reduces spatial learning in mice: involvement of endogenous opioid systems

Abstract. Biting flies influence both the physiology and behaviour of domestic and wild animals. This study demonstrates that relatively brief (60min) exposure to stable flies, Stomoxys calcitrans (L.), affects the spatial abilities of male mice. Stable fly exposure resulted in poorer subsequent performance in a water maze task in which individual mice had to learn the spatial location of a submerged hidden platform using extramaze visual cues. Determinations of spatial acquisition and retention were made with mice that had been previously exposed for 60min to either stable flies or house flies, Musca domestica (L.). Mice exposed to stable flies displayed over one day of testing (six blocks or sets of four trials) significantly poorer acquisition and retention of the water maze task than either mice that had been exposed to house flies or fly-naive mice. This attenuation of spatial learning occurred in the absence of any evident sensorimotor or motivational impairments. The reduction in spatial abilites involved endogenous opioid systems, as the decreased performance resulting from stable fly exposure was blocked by pre-treatment with the prototypic opiate antagonist, naltrexone. These results indicate that relatively brief exposure to biting flies can lead to a decrease in spatial abilities which is associated with enhanced endogenous opioid activity. These results support the involvement of endogenous opioid systems in the mediation of the behavioural and physiological effects of biting fly exposure. They further suggest that decreases in spatial abilities and performance may be part of the behavioural consequences of biting fly exposure in domestic and wild animals.     

Induction of Suppressed ADH Activity in Drosophila pachea Exposed to Different Alcohols

Laboratory-reared males of the cactophilic Drosophila pachea exhibit a spontaneous and sex-specific suppression of alcohol dehydrogenase (ADH) activity within 4 days after eclosion. A lack of ADH activity also is usually seen in wild-caught males, although relatively high activity is always seen in female flies. In the present study we examined the effectiveness of different alcohols and related compounds, including several found naturally in necroses of the host cactus, to induce suppressed ADH activity in wild males of D. pachea and to serve as enzyme substrates. The primary alcohols (methanol, ethanol, 1-propanol, 1-butanol, and 1-pentanol), and the secondary alcohols (2-propanol and 2-butanol), each induced activity after 24 h exposure, although to different degrees. 1,2-Propanediol was usually effective as an inducer, but 2,3-butanediol usually was ineffective. Little or no induction was seen with 1-octanol, 2-pentanol, 3-methyl-1-butanol, 3-hydroxy-2-butanone, or acetaldehyde. Although the compounds tested varied in their ability to function as ADH substrates, methanol was the only alcohol that showed no activity staining. Ethanol induction of ADH activity was apparent after 3-6 h exposure and induced activity decreased dramatically within 1 week of flies being placed in an alcohol-free environment. Ethanol exposure did not induce ADH in adult female D. pachea, or in adult males and females of D. acutilabella in which control males show reduced ADH activity compared to females. The implications of the loss of ADH activity in adult males of D. pachea, as they relate to feeding ecology and fitness, are discussed.     

Effects of differential alcohol dehydrogenase activity on the developmental toxicity of ethanol in Drosophila melanogaster

The role of alcohol dehydrogenase (ADH) activity in ethanol toxicity was investigated in Drosophila melanogaster. Flies from three congenic Adh strains (high, medium, and low ADH activity) were allowed to deposit eggs on medium containing 0, 4, or 8% ethanol. The resulting larvae were allowed to complete their development in the medium, and emerging flies were examined for defects. Flies with high ADH activity had malformation incidences of 0.8, 2.4, and 5.2% at 0, 4, and 8% ethanol, respectively. The comparable incidences for the low ADH strain were 1.0, 4.1, and 8.4%, while those for the medium ADH strain were intermediate in value. These results indicate that ethanol teratogenesis may be inversely related to ADH activity. When larvae were treated with ethanol for different lengths of time during development, the incidence of defects in flies from the high ADH strain was 3.9% when exposure started at the first instar and 3.09% when exposure started at the third instar. Results of the same exposures for the intermediate ADH strain were 5.2 and 3.4%, respectively, while those for the low ADH strain were 6.9 and 5.5%, respectively. Thus, length of ethanol exposure was directly related to the increased incidence of malformations in all tested Drosophila strains. For all tested strains, defect incidences appeared to be dose-related as well, regardless of length of exposure. ADH in Drosophila has a dual function and thus can catalyze oxidation of both ethanol and its toxic metabolite, acetaldehyde. This suggests that ethanol is the proximate teratogen in Drosophila.     

Change in Free Amino Acids and Phospholipids in the Head of Adult Sarcophaga peregrina with Age

Abstract. The amino acid and phospholipid contents in the head of adult Sarcophaga peregrina (flesh-fly) were measured with age. Only aspartic acid was found to increase significantly with aging. A significant difference was also detected in the fatty acid composition of the phospholipids. Phospholipids isolated from old flies contained more unsaturated fatty acid than young flies, suggesting that the properties of the membrane of head cells change with age. These changes were observed irrespective of sex or rearing conditions. Thus, it is possible that certain metabolic pathways in post-mitotic cells are selectively affected by aging.     

Alcohol consumption as self-medication against blood-borne parasites in the fruit fly

Plants and fungi often produce toxic secondary metabolites that limit their consumption, but herbivores and fungivores that evolve resistance gain access to these resources and can also gain protection against nonresistant predators and parasites. Given that Drosophila melanogaster fruit fly larvae consume yeasts growing on rotting fruit and have evolved resistance to fermentation products, we decided to test whether alcohol protects flies from one of their common natural parasites, endoparasitoid wasps. Here, we show that exposure to ethanol reduces wasp oviposition into fruit fly larvae. Furthermore, if infected, ethanol consumption by fruit fly larvae causes increased death of wasp larvae growing in the hemocoel and increased fly survival without need of the stereotypical antiwasp immune response. This multifaceted protection afforded to fly larvae by ethanol is significantly more effective against a generalist wasp than a wasp that specializes on D. melanogaster. Finally, fly larvae seek out ethanol-containing food when infected, indicating that they use alcohol as an antiwasp medicine. Although the high resistance of D. melanogaster may make it uniquely suited to exploit curative properties of alcohol, it is possible that alcohol consumption may have similar protective effects in other organisms.