The Detoxification Of And Behavioral Responses To Methanol In Drosophila Melanogaster

Plants produce a large amount of methanol during growth and development, and insect feeding further stimulated the release of methanol. Moreover, fermenting plant tissues also generate a great quantity of methanol. Therefore, the insects feeding on these plant tissues may be intoxicated by methanol. These insect species should have evolved some detoxification pathways and behavioral avoidance responses. Up to now, however, only one publication has been documented the detoxification to methanol in an insect species, the Asian corn borer, Ostrinia furnacalis.In the present study, we investigated the detoxification of and behavioral reseponses to methanol in Drosophila melanogaster. Our findings will provide a useful example to understand insect-environment co-evolution, and to develop the potential pest management strategy.1. The involvement of CYPs in methanol detoxification in larvaeTo investigate the detoxification pathways in Drosophila larvae, we tested the combined toxicity effects of dietary methanol (formaldehyde) and five enzyme inhibitors, namely piperonyl butoxide (PBO),3-amino-1,2,4-triazole (3-AT),4-methylpyrazole (4-MP), diethylmeleate (DEM) and triphenyl phosphate (TPP), an inhibitor of cytochrome P450monooxygenases (CYPs), catalases, alcohol dehydrogenases (ADHs), glutathione S-transferases and esterases, respectively. For the2nd-instar larvae, the combination indices of TPP, DEM,3-AT and4-MP plus methanol were1.02,0.98,0.98and1.00, of them plus formaldehyde were1.15,1.05,1.08and1.14respectively. These mixtures were indicated to be additive effects at the ratios tested. In contrast, the combination indices of PBO plus methanol and PBO plus formaldehyde were0.28and0.18respectively, significantly below1, suggesting synergistism. These results demonstrated that CYPs were involved in methanol metabolism. Moreover, methanol exposure dramatically increased CYP activity. The ratios of the CYP activities in treated larvae to that of control reached up to3.0-,3.9-and2.7-fold, at methanol concentrations of22.6,27.9and34.5mg/g diet, respectively. In addition, methanol exposure greatly up-regulated cyp304a1, cyp9f2, cyp28a5, cyp4d2and cyp4e2genes. The proteins encoded by these genes were suggested as the candidate enzymes for methanol metabolism in D. melanogaster larvae.2. The detoxification of methanol in adultThe combination indices (CI) of PBO, TPP,3-AT,4-MP and DEM plus methanol were tested in female and male respectively. When methanol mixed with PBO or3-AT, it showed significantly synergistism. When methanol mixed with TPP or4-MP, it showed additive effects after the adults were exposed for24h, and then exhibited significantly synergistism after48h and72h of exposure. In contrast, when methanol mixed with DEM, it showed additive effects. Moreover, methanol exposure dramatically increased CYP activity and up-regulated mRNA expression levels of several cyp genes. In addition, the knockout mutation in gene encoding ADH or a-Est7resulted in decreased methanol tolerance, in contrast to the knockout mutation in gene encoding cyp4e2, cyp9f2or cyp318a1. Knocked out the gene encoding aldehyde dehydrogenase/octanol dehydrogenase led to increased methanol tolerance in the mutant after24h of methanol exposure, and then the tolerant level declined as the exposure period delayed. The results suggest that CYPs, catalases, ADHs and esterases play important roles in methanol detoxification in adults. Glutathione S-transferases were not responsible for methanol elimination. The involvement of aldehyde dehydrogenase/octanol dehydrogenase in methanol metabolism remains further study to confirm.3. The response of long-term methanol exposure in Drosophila melanogasterThe flies were maintained on methanol-supplemented diet to select for methanol tolerant strain. The survival rates varied little on methanol-supplemented diet throughout the selection process, although the LC50value increased from27.93to40.71mg/g diet after40generations of selection. The variations of realized heritability (h2), in response to selection (R) and selection differential (S) in different generations indicated that the allelic variation in resistance was eroded by methanol selection. After selection for0,2,10and40generations, the egg-to-adult developmental periods lengthened significantly from8.33,9.78,12.01and12.51days on methanol-free diet to12.59,14.08,17.37and18.10days on methanol-contained diet respectively. Correlation analyses revealed that the variations of egg-to-adult developmental durations on methanol-free diet were contributed by both the embryonic and larval developmental delays and those on methanol-contained diet resulted from the embryonic developmental delay. Methanol selection did not slow down pupal development process. Moreover, methanol selection constitutively up-regulated several genes encoding CYPs and Ests, and also increased CYP and Est activities. These results showed a fitness disadvantage in the methanol-selected population.4. Seditive effect induced by methanol exposure on adultsWhen exposed to methanol vapour slowly, the fruit flies showed sequential behavioral changes. In the initial several minutes, they became hyperactive, walked fast. And then, the flies lost motor control (infrequent movements and frequent falls during walking), and then were sedated (lying on their back). Methanol vapour could induce tolerance to mathanol seditive effect in males, but not in females. Pretreatment with enzyme inhibitor TPP, DEM,3-AT,4-MP or PBO reduced the duration of mean sedation time, but not change the variation pattern of mean sedition time after recurring methanol exposure. It seems that metabolism enzymes only tune the tolerance amplitude, they are not the tolerance mechanism to methanol sedative effect. Ingesting enzyme inhibitors increased the mortalities of the flies that treated with methanol vapour each day. Mortality for the males increased from2%in group treated only with methanol vapour to over10%in groups treated with enzyme inhibitors and methanol vapour, and for females it increased from5%to10%respectively. This suggestes that methanol metabolism enzymes may exist near and even in the central nervous system, in which these enzymes remove the methanol to protect the neurons from neurotoxicity.5. Intermale courtship induced by alcoholsCanton-S males recurring exposured to methanol or ethanol resulted in the disinhibition of sexual behavior. Treated males showed active intermale courtship behaviour. We selected eight mutants, each contains a p-element insertion at a known gene encoding protein involving in neurotrophin signaling pathway. The knockout mutants in gene Shc, drk, csw, Pi3k21B and RhoGDI, when induced by methanol, did not show intermale courtship. However, the knockout mutant in Rhol still showed active intermale courtship. These results indicate shc, drk, csw, Pi3k21B and RhoGDI play essential roles for methanol-induced intermale courtship behavior. It was suggested that recurring methanol exposure remodeled the neural circuit through neurothrophin signaling pathway, which subsequently changed the male sexual behaviour.6. The behavioral avoidance to methanol, in contrast to ammoniaDuring fruit fermentation, carbohydrates decompose to produce some alcohol volatiles and proteins generate some volatile nitrogenous compounds. We determined the olfactory responses and movement of D. melanogaster flies to ethanol, methanol and ammonia sources using a glass Y-tube olfactometer, and examined the influence of mating experience and food deprivation on orientation of the adult flies in the laboratory. We found that mating experience and food deprivation didn’t affect the response to methanol and ethanol, but significantly increased preference to ammonium bicarbonate. Mating experience and food deprivation also increased dispersal behavior. In a two-way choice experiment to test oviposition preference, the CS females refused to lay eggs on food containing methanol and ethanol, but preferred to deposit their eggs on food containing ammonium bicarbonate and potassium sulfide. Knockout genes encoding Or83b and Nc73EF influenced the oviposition preference. After knocked out of lush gene, the resulting flies lost the behavioral sensitivity to alcohols, but exhibited the preferrence to amonia and hydrogen sulphide. Moreover, Knocked out the gene encoding CaM resulted in the oviposition preferrence to ethanol. It suggested that the fruit flies in nature may rely on alcohol and nitrogenous compound volatiles to direct food and egg-laying substrates at a distance. Upon arrival, they avoided depositing their eggs on methanol-rich environment to pretect the embryoes from methanol intoxication. At the same time, they preferred protein-rich substrates for their children.