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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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