Determination of aldehyde tolerance in Drosophila

melanogaster

Andrew Guarnaccia

Instructor: James Fry

IND 395: Aldehyde Tolerance in Flies

Department of Biology, University of Rochester, Rochester, NY, 14627 USA

May 11, 2015

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Abstract

Drosophila melanogaster are known to reside in fermenting fruits, which tend to contain large

amounts of ethanol. The temperate strains of this species have been shown to have a higher

resistance to ethanol than their tropical counterparts. Ethanol breakdown involves transforming

the alcohol into acetaldehyde, a toxic substance, via ADH (alcohol dehydrogenase) then breaking

the acetaldehyde down into acetate through ALDH (acetaldehyde dehydrogenase) and ADH.

Due to this breakdown pathway, one of the questions we asked is whether a higher resistance to

ethanol stems from a higher resistance to its metabolic intermediates? This experiment looked at

the breakdown of acetaldehyde by mutating the Aldh gene in a Vienna strain (K3) and a

Cameroon strain (MD05). The flies were separated by both strain and gender and given a

specific volume of 20% ethanol, with females receiving more ethanol than males. The flies were

observed over three days and the number of living and dead flies was counted each day. In an

Aldh-null background, we found that both the male and female Vienna strains showed a higher

resistance to in vivo acetaldehyde.

Introduction

Drosophila melanogaster live and feed in fermenting fruits, which have been shown to contain

ethanol concentrations up to 6% (Gibson et al., 1981). Ethanol breaks down by alcohol

dehydrogenase (ADH) into acetaldehyde, a compound that can be toxic in animals in large

amounts. The acetaldehyde must then be broken down into acetate using aldehyde

dehydrogenase (ALDH) and a small part ADH (Leal et al., 1992). Temperate flies have been

proven to withstand ethanol better than tropical flies. We placed temperate and tropical flies in

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an Aldh-null background to test if temperate flies have a higher resistance to acetaldehyde made

in vivo in addition to a higher tolerance of ethanol.

Since it is believed that ethanol resistance is actually a resistance to the intermediates of ethanol

breakdown (Fry, 2014), we decided to compare the tolerance of in vivo acetaldehyde in a

temperate European line to a tropical African line by mutating the Aldh (aldehyde

dehydrogenase) gene and then introducing the flies to ethanol. Further information about the

mutation can be found in the Materials and Methods Section under Aldh mutation. Because

temperate flies can withstand ethanol better than tropical flies, we hypothesized that temperate

flies would withstand acetaldehyde better as well. Two separate experiments were performed:

the first one in which both strains had to be bred with an outside strain (17E) because the

Cameroon third chromosome (MD05) was lethal in the homozygous form; and the second one in

which the Cameroon strain was hybridized with another Cameroon strain (MD21, creating

MD05/21), thereby eliminating the need for 17E flies. In both tests, there was a clear difference

between the tolerances of Vienna and Cameroon flies in both males and females, with the Vienna

flies displaying a higher in vivo acetaldehyde tolerance.

Materials and Methods

Fly stocks

Two different strains of Drosophila melanogaster were used for this experiment: one from

Vienna (b AldhD17; K3), and one from Cameroon (b AldhD17; TM6C, Sb/MD05). The flies were

maintained in shell vials, containing an agar medium with cornmeal and molasses, and were

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anesthetized with minimum CO2. They were placed in 25˚C incubation with continuous lighting

and 60-70% humidity.

Aldh mutation

The mutation involved deleting a large portion of the Aldh gene, which created an Aldh mutant

#17, Aldh∆17 (Fry and Saweikis, 2006). This deletion eliminated any acetaldehyde resistance

conferred by the second chromosome, allowing a more accurate comparison between the

acetaldehyde resistances of Vienna third chromosome (K3) and of the Cameroon third

chromosome (MD05). In the first few experiments, due to a lethality of MD05 in the

homozygous form, both Vienna and Cameroon flies were individually bred to a separate

unmarked 17E line (b AldhD17), also containing an Aldh mutant #17. Additionally, the Cameroon

flies contained a third-chromosome balancer (tm6c) as well as a third-chromosome molecular

marker (Sb, short bristles). Tm6c permitted the maintenance of a heterozygous MD05/TM6C

stock since both genes are recessive lethal, and prevented crossing over on the third chromosome

since the balancer is inverted. Sb allowed us to determine which flies contained both MD05 and

17E in their third chromosomes, since those with short bristles carried the tm6c and Sb genes and

therefore lacked either MD05 or 17E. In the last few experiments, a new Cameroon strain

(MD05/21) was acquired through hybridization between the MD05 strain and a Cameroon

MD21 strain (created by Dr. Fry). This new strain did not experience lethality in the

homozygous third chromosome form, so the Vienna and Cameroon strains could be compared

directly with no 17E needed.

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Breeding flies

17E flies were bred to the Vienna and Cameroon flies separately for the first few experiments

due to the lethality of Cameroon third chromosome (MD05) in the homozygous form. 17E

female virgins were collected and transferred to collecting vials, which contain a small amount

of food. After a few days, they were bred to the two separate strains and transferred to new

feeding vials, which contain more food than the collecting vials. 4 female 17E flies were bred

with 4 males from the respective strains.

In the second set of experiments, a new Cameroon strain (MD05/21) was used that could be

maintained in the homozygous form, so no 17E flies were needed. At first, 6 females of one

strain were bred to 5 males of the same strain. However, the K3 flies were not very healthy, so

instead 8-9 female Vienna flies were bred with 4-5 male Vienna flies, and 6-7 female Cameroon

flies were bred with 4-5 male Cameroon flies, with each vial receiving roughly 10-20g of yeast.

Collecting flies

Once the flies were put in feeding vials to breed, they were put in incubation. After 1 week (4-5

days with the MD05/21 experiments), flies present in the vials were turned over to a new set of

feeding vials. After another week, the flies in the new vials were disposed while flies in the old

vials were turned over to a set of collecting vials. These collecting vials sat in incubation for 2-3

days, after which they were put into ethanol vials. 1 week after the transfer from old vials to

collecting vials, flies from the new vials were turned over into collecting vials, where they also

sat for 2-3 days in incubation before being turned over to ethanol vials.

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Feeding ethanol

Flies were divided up into 15 flies per sex per strain in each ethanol vial making sure to acquire

an equal number of vials of Vienna females, Vienna males, Cameroon females, and Cameroon

males. With the 17E experiments, Cameroon male and female flies with long bristles (lacking the

tm6c marker) were collected, since they contained the MD05 and 17E third chromosomes.

Ethanol vials were set up by tamping down a 0.5g ball of cotton into an empty vial then pipetting

1.0mL of 5% sugar water into the cotton. Flies were anaesthetized and transferred to these vials,

with another 0.5g ball of cotton placed into the middle of vial to seal the flies in. A cork was then

placed in the vial, which would be important later for trapping ethanol vapors.

After 1 day in ethanol vials, the corks were removed and 20% ethanol was pipetted into

individual vials based on the sex and strain of the flies inside. For the 17E experiments, 125uL

was used with females while 75uL was used with males. With the K3 and MD05/21

experiments, 110uL was used with females and 90uL was used with males. All of the ethanol

volumes are shown in Table 1. Upon pipetting ethanol into the vials, the cork was placed back in

their respective vials. As ethanol evaporated, the cork would keep the vapors from escaping.

After putting ethanol into the vials, the flies were put back into incubation, where they stayed for

three days. During that time, the vials would be briefly removed once a day to count how many

flies had died. On the third day the vials were discarded.

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Analyzing the data

For the 17E experiments, we analyzed 11 vials of Vienna females, 10 vials of Cameroon

females, 9 vials of Vienna males, and 8 vials of Cameroon males, with each vial containing 15

flies In testing for significance, the Mann-Whitney-U Test was used to analyze the survival over

all three days between strains for both genders. Error bars are one standard error of the mean in

both the positive and negative directions.

Working with the MD05/21 strains, 30 vials each were used for Vienna females, Cameroon

females, Vienna males, and Cameroon males, with each vial containing 15 flies. A T-Test was

done to test the average difference in survival over all three days between Vienna and Cameroon

in both genders. Error bars are one standard error of the mean in both the positive and negative

directions.

Results

17E crosses

The results from the crosses with 17E flies are shown in Figures 1 and 2. In both males and

females, Vienna flies display a higher tolerance for ethanol than their Cameroon counterparts

when the ALDH enzyme was impaired. When exposed to 125uL 20% ethanol for three days,

female Vienna flies displayed a higher survival rate than the Cameroon flies (P < 0.01). The

same results can be found when male Vienna flies were compared to male Cameroon flies under

75uL 20% ethanol (P < 0.005).

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Working with MD05/21

Figures 3 and 4 show the survival rates between the Vienna flies and the new Cameroon flies.

Similar to the crosses with 17E, the Vienna flies demonstrated a greater tolerance for ethanol

than the new Cameroon flies when ALDH was compromised. After exposure to 110uL 20%

ethanol, the female Vienna flies had a higher survival rate than the female Cameroon flies (P <

0.05). Additionally, male Vienna flies also appeared to have fared better than Cameroon male

flies under 90uL 20% ethanol (P < 0.05).

Discussion

As predicted, we found Vienna flies had a higher resistance to in vivo acetaldehyde upon having

Aldh mutated. Since ethanol breakdown involves the conversion to lethal acetaldehyde before

converting it to acetate, the question we asked in determining the causal difference in ethanol

tolerance between temperate and tropic flies was whether this tolerance derives from a tolerance

to one or both of its breakdown intermediates. Drosophila melanogaster third chromosome is the

main contributor for ethanol resistance, so the comparison of acetaldehyde resistance had to be

made by mutating the Aldh gene on the second chromosome, thereby giving both strains the

same genetically null background and allowing a proper comparison of the third chromosomes.

The finding that Vienna flies had a higher resistance to the ethanol, even when unable to

properly dispose of acetaldehyde, is consistent with numerous other findings that illuminate a

tendency in temperate flies to have a higher ethanol resistance than tropical flies. A high

tolerance of ethanol would likely stem from a high tolerance to ethanol metabolites, especially

considering how toxic the metabolite acetaldehyde is. Higher tolerance of acetate, the other

ethanol breakdown intermediate, has already been proven (Fry, 2014).

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In conclusion, we have shown that there is a higher resistance in Vienna flies than there is in

Cameroon flies when dealing with in vivo acetaldehyde. These results are consistent with the

hypothesis that temperate flies are better resistant to ethanol than tropical flies due to a better

resistance to ethanol metabolites.

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References

Fry J (2014) Mechanisms of naturally evolved ethanol resistance in Drosophila melanogaster.

Journal of Experimental Biology, 217, 3996-4003.

Fry J & Saweikis M (2006) Aldehyde Dehydrogenase is essential for both adult and larval

ethanol resistance in Drosophila melanogaster. Cambridge University Press, 87, 87-92

Gibson JB, May TW, & Wilkis AV (1981) Genetic variation at the alcohol dehydrogenase locus

in Drosophila melanogaster in relation to environmental variation: Ethanol levels in

breeding sites and allozyme frequencies. Oecologia, 51, 191-198

Leal JFM & Barbancho M (1992) Acetaldehyde detoxification mechanisms in Drosophila

melanogaster adults involving aldehyde dehydrogenase (ALDH) and alcohol

dehydrogenase (ADH) enzymes. Insect Biochemistry and Molecular Biology, Volume

22, Issue 8, 885-892

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Figure Legends

Table 1. Volume 20% ethanol used per sex per strain for the two experiments. When bred with

17E, females received 125uL and males received 75uL. When not bred with 17E, females

received 110uL and males received 90uL.

Figure 1. Survival rates in females between K3 and MD05 flies when bred with the 17E flies.

K3 flies showed a statistically significantly higher tolerance for ethanol than MD05 (P < 0.05).

All 3 days favored K3 in terms of survival (Day 1 and Day 2 P < 0.005; Day 3 P < 0.05). Error

bars are one standard error of the mean in both directions.

Figure 2. Survival rates in males between K3 and MD05 flies when bred with the 17E flies. K3

flies showed a statistically significantly higher tolerance for ethanol than MD05 (P < 0.05). All 3

days favored K3 in terms of survival (Day 1 P < 0.005; Day 2 P < 0.0005; Day 3 P < 0.005).

Error bars are one standard error of the mean in both directions.

Figure 3. Survival rates in females between K3 and MD05/21 flies. K3 flies showed a

statistically significantly higher tolerance for ethanol than MD05/21 (P < 0.05). All 3 days

favored K3 survival (Day 1, Day 2, and Day 3 P < 0.05). Error bars are one standard error of the

mean in both directions.

Figure 4. Survival rates in males between K3 and MD05/21 flies. K3 flies showed a statistically

significantly higher tolerance for ethanol than MD05/21 (P < 0.05). All 3 days favored K3

survival (Day 1, Day 2, and Day 3 P < 0.05). Error bars are one standard error of the mean in

both directions.

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Figures and Tables Table 1

17E x K3 17E x MD05 K3 MD05/21 Female 125uL 125uL 110uL 110uL Male 75uL 75uL 90uL 90uL

Figure 1

Figure 2

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Figure 3

Figure 4