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DROSOPHILA​ REPRODUCTION AND MORTALITY RATES WHEN UNDERGOING

STRESS AND EXPOSURE TO ALCOHOL


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Drosophila melanogaster ​ Reproduction and Mortality Rates When Undergoing Stress and

Exposure to Alcohol

Sarah Quigley, Sara Sarcona, Claudia Schreier, Anya Shehady, Sarah Valente, and Bella

Yedman

Marine Academy of Technology and Environmental Science


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Introduction
Drosophila melanogaster can be used for research in epigenetics and conditions such as
alcoholism. Because ​Drosophila melanogaster​, or the fruit fly, has a short lifespan, they are an
ideal organism that is used to trace alcoholism through generations (Heberlein, 2000). Fruit flies
are also used in many neurological studies due to their similarity to humans in their responses to
behavioral changes (Rodan and Rothenfluh, 2010). Alcohol dehydrogenase (ADH) activity is
essential to the ethanol tolerance in the fruit fly. Similarly, there is a gene entitled ADH, which
has two forms: ADH​+ and ADH​-​. Flies with the ADH​+ ​form of the gene have a higher alcohol
tolerance; on the other hand, flies with the ADH​- form of the gene cannot metabolize alcohol,
which makes them more likely to display intoxicated behavior when exposed to the same amount
of ethanol (Malherbe Y1, Kamping A, van Delden W, van de Zande L, 2005). A study found that
stressors such as heat can be linked to alcoholism in fruit flies (Awofala, Jones, Davies, 2011).
The experiment was designed based on the premise that recurring stress can lead to alcohol
dependence. Flies will also continue to drink alcohol, “even when researchers associate alcohol
with an aversive stimulus, such as giving the flies an electric shock whenever they drink”
(Christmann, 2014). This means that even stress cannot deter the flies from consuming alcohol,
which can indicate an alcohol dependency. In addition, researchers have shown that fruit flies do
not like the taste of alcohol, although they do like the way it smells. This is comparable to
humans developing a taste for the flavor of alcohol. This experiment can be used to link the
tendency of humans to consume alcohol while under recurring stress to the same tendency in
fruit flies. To measure alcohol consumption, the reproductive and mortality rates of both the
experimental and control flies can be used.

Objective
This experiment will analyze the reproductive and mortality rates as a function of
stress-induced alcohol consumption by ​Drosophila melanogaster​.
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Methodology
Drosophila melanogaster were acquired from culture vials that consisted of pre-bred
wild-type ADH​+ and wild-type ADH​-​. In order to create specific vials for the ADH​+ specimens
and the ADH​- specimens, the fruit flies were collected and separated. To obtain the ADH​+ flies,
the culture vial containing the specimens was gently tapped against the table until all of the fruit
flies were disoriented and were no longer adjacent to the foam stopper, preventing them from
exiting the vial. Next, the foam stopper was removed. A group member swiftly aligned a transfer
vial with the opening so the flies could be collected. After obtaining a sufficient amount of flies,
two index cards were placed between the vials and two group members separated them. Both of
the vials were tapped once again and the foam stoppers were placed on each of the vials. The
process was repeated to collect the fruit flies from the ADH​- culture vial. After obtaining both the
ADH​+ and the ADH​- ​flies, the group members counted and separated them into a control vial and
an experimental vial for each type of fly. The ideal male to female ratio was around 1:1. Once
the fruit flies were separated, there was a total of four vials: ADH​- control, ADH​- ​experimental,
ADH​+​ control, ADH​+​ experimental.
Experimental flies were exposed to external stressors, and then both control and
experimental were given alcohol to monitor the ​Drosophila ​melanogaster reproductive rates and
death rates. Every other day the experimental fruit flies were exposed to a stressor. On the first
and fifth days of each week, the flies were shaken for thirty seconds. On the third day of each
week, the flies were put in the freezer for five minutes. After each test, both experimental and
control flies were introduced to separate empty vials. Each vial contained a small cotton ball with
0.5 milliliters of alcohol. The ​Drosophila ​melanogaster specimens remained in the vial
containing the alcohol for approximately five hours.
At the conclusion of the experiment, the living flies were euthanized, separated, and
counted. In addition, the number of dead fruit flies in the medium was recorded. After counting,
all flies were disposed of.
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To observe which fly group had the highest mortality rate, the percent change was
calculated for living flies between December 13, 2017 to December 22, 2017. In order to
determine the reproductive rate, the percent change was calculated for the flies from the day the
medium was changed (December 15, 2017) to the overall flies dead and alive in the vials on
January 12, 2018. The amount in the the vials on December 15 was subtracted from the overall
total to find the total flies produced in the new generation before doing the percent change.

Timeline
December 4th: Acquire wild-type ADH​+​ and wild-type ADH​-​ ​Drosophila
melanogaster.
December 8th: Acquire wild-type ADH​+​ and wild-type ADH​-​ ​Drosophila
melanogaster.​ Fruit flies will be separated at this stage by both sex and
ADH gene.
December 12th: Separate the specimens equally into 4 vials.
December 13th: Freeze the experimental flies for five minutes. Expose all flies to
alcohol.
December 15th: Shake each of the vials containing the fruit flies for thirty seconds and
expose them all to alcohol. Change medium within each vial as well.
December 18th: Shake the vials containing the experimental flies violently for thirty
seconds. Expose the specimens to alcohol.
December 20th: Freeze all of the experimental flies for five minutes and then expose
them to alcohol.
December 22nd: Shake the fruit flies in the experimental vials for thirty seconds and then
expose them to alcohol. Allow for all four of the vials to remain
untouched throughout the duration of the week-long break so that new
adult specimens may emerge.
January 3rd: Freeze the ​Drosophila melanogaster​ specimens within the experimental
vials for five minutes and expose them to alcohol.
January 12th: Count the total number of fruit flies flies (living and dead) that are
DROSOPHILA​ REPRODUCTION AND MORTALITY RATES WHEN UNDERGOING
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contained within each vial.

Data
Table 1: This is a record of the dead and alive ​Drosophila ​melanogaster a​ ccording to the date for
ADH​+ Experimental, ADH​+ Control, ADH​- Experimental, and ADH​- Control. The green and red
highlighted portion under 1/12/18 is the total number of flies left in the vials that were both alive
and dead in the medium.

Flies Alive
Total
12/13/17 12/15/17 12/18/17 12/20/17 12/22/17 1/3/18 1/12/18
ADH​+
Experimental 5 4 4 4 2 14 2
ADH​+
Control 8 3 3 2 1 23 16
ADH​-
Experimental 7 7 7 6 5 2 15
ADH​-​ Control 9 4 2 2 2 3 42
Flies Dead
12/13/17 12/15/17 12/18/17 12/20/17 12/22/17 1/3/18 1/12/18
ADH​+
Experimental N/A 1 0 0 2 -12 33
ADH​+
Control N/A 5 0 1 1 -22 46
ADH​-
Experimental N/A 0 0 1 1 3 23
ADH​-​ Control N/A 5 2 0 0 -1 67
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Table 2: Total amount of ​Drosophila melanogaster​ produced from December 15th,


2017 to January 12th, 2018. Values include flies both living and dead at time of count.

ADH​+ ADH​-
Experimental ADH​+ ​Control Experimental ADH​-​ Control
Alive 2 16 15 42
Dead 33 46 23 67
Subtract 12/15 4 3 7 4
Total Flies
Produced 31 59 31 105

Table 3: Percent change in ​Drosophila melanogaster p​ opulations between December 15, 2017
and January 18, 2017. Values are mortality rates only.

Percent Change Flies Living over time

ADH​+​ Experimental -60.0%


ADH​+​ Control -87.5%
ADH​-​ Experimental -28.6%
ADH​-​ Control -77.8%

Table 4: The percent change in ​Drosophila​ ​melanogaster​ produced from December 15, 2017 to
the overall amount of flies both dead and alive on January 12, 2018. It demonstrates the amount
of flies produced.

Percent Change Total Flies Produced


ADH​+​ Experimental 675.0%
ADH​+​ Control 1866.7%
ADH​-​ ​ ​Experimental 342.9%

ADH​-​ Control 2525.0%


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Results
From December 13 to December 22, the first generation of flies collected were
experimented on. The percent change for living flies was calculated for that time. The groups
tested were the ADH​+ Experimental, ADH​- Experimental, ADH​+ Control, and the ADH​- Control.
The percent change for ADH​+ Experimental was a 60.0% decrease, from 5 to 2 flies. The percent
change for the ADH​- Experimental was a 28.6% decrease, from 7 to 5. The percent change for
the ADH​+ Control was a 87.5% decrease, from 8 to 1 flies. The percent change for the ADH​-
Control was a 77.8% decrease, from 9 to 2 flies. Overall, the ADH​+ Control group of flies had
the greatest decrease in living flies. The ADH​- Experimental group had the least decrease of flies.
Overall, the ADH​+ flies decreased the most by comparing the experimental vials and the control
vials.
In order to determine which fly group had the best reproductive rate, the percent change
was calculated for the date the medium was changed on December 15 to the overall flies
produced by the end of the experiment on January 12. The percent change for ADH​+
Experimental was a 675.0% increase, from 4 to 31 flies. The percent change for ADH​+ Control
was a 1866.7% increase, from 3 to 59 flies. The percent change for ADH​- Experimental was a
342.9% increase, from 7 to 31 flies. The percent change for ADH​- Control was a 2525.0%
increase, from 4 to 105 flies. The ADH​- Control had the greatest increase in flies. The ADH​-
Experimental increased the least out of the four groups. The controls of both ADH​+ and ADH​-
had the greatest increase in the amount of flies, which means they reproduced the most.
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Figure 1: The ADH​+​ Experimental ​Drosophila​ ​melanogaster g​ roup was exposed to stress and
alcohol on every other day from December 13, 2017 to December 22, 2017. This graph shows
the decrease in the amount of living flies overtime. The ADH​+​ Experimental group had a 60.0%
decrease from December 13 to December 22.

Figure 2: The ADH​+​ Control ​Drosophila​ ​melanogaster g​ roup was exposed to alcohol on every
other day from December 13, 2017 to December 22, 2017. This graph shows the decrease in the
DROSOPHILA​ REPRODUCTION AND MORTALITY RATES WHEN UNDERGOING
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amount of living flies overtime. The ADH​+​ Control group had a 87.5% decrease from December
13 to December 22.

Figure 3: The ADH​-​ Experimental ​Drosophila​ ​melanogaster ​group was exposed to stress
and alcohol on every other day from December 13, 2017 to December 22, 2017. This graph
shows the decrease in the amount of living flies overtime. The ADH​-​ Experimental group had a
28.6% decrease from December 13 to December 22.

Figure 4: The ADH​-​ Control ​Drosophila​ ​melanogaster g​ roup was exposed to stress and alcohol
on every other day from December 13, 2017 to December 22, 2017. This graph shows the
DROSOPHILA​ REPRODUCTION AND MORTALITY RATES WHEN UNDERGOING
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decrease in the amount of living flies overtime. The ADH​-​ Control group had a 77.8% decrease
from December 13 to December 22.

Discussion

The effects of alcohol on both human and ​Drosophila melanogaster reproductive
systems are severe. Through the possession of the ADH gene, some ​Drosophila melanogaster
are less sensitive to the depressant nature of alcohol, meaning that their vital functions are less
affected (Malherbe Y1, Kamping A, van Delden W, van de Zande L, 2005). In the experiment,
Drosophila melanogaster ​that expressed the ADH​- gene were not able to metabolize alcohol. For
this reason, the ADH​- flies became intoxicated, leading to temporary incapacitation. They were
not able to reproduce while in this state, but they also did not consume more alcohol than they
could handle (Edenberg, 2007). In addition, ADH​- flies needed time to recover before
reproducing, leading to less alcohol in their systems. This caused lower reproduction rates for
ADH​- flies and a lower mortality rate as well. However, flies with the ADH​+ gene were able to
metabolize alcohol, so they had the ability to consume alcohol without reaching a tolerance
threshold (Edenberg, 2007). These flies were still able to reproduce, but their body toxicities
were higher than those of the ADH​- flies. The high body toxicity resulted in more deaths and
developmental issues in eggs and offspring (Ranganathan, Davis, Hood, 1987) . Because both
males and females were exposed to the same amount of alcohol, female flies consumed alcohol
as much as males did. Because reproductive females consumed alcohol, their offspring were
likely to have developmental problems (Ledig et. al, 1998). In addition, studies have shown that
alcohol may be a direct toxicant to sperm, causing males to compromise the development of their
offspring as well (Cicero, 1994). Since both parents consumed excessive amounts of alcohol,
their offspring were at a greater risk for developmental issues. The factors of parental
consumption of alcohol due to stress and the ability of ADH​+ ​flies to metabolize alcohol supports
the fact that the experimental ADH​+ ​flies had both high reproduction and mortality rates.
The behavior of the Drosophila melanogaster, or the common fruit fly, can be greatly
influenced by the introduction of stress into their environments. For example, if a female fruit
fly refuses to mate with a certain male, then the male fruit fly will begin to consume alcohol in
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an attempt to reduce the disquietude of having been rejected, similar to humans (Rodan and
Rothenfluh, 2010). The control group vials containing the ADH​+ and ADH​- ​Drosophila
melanogaster s​ pecimens did not experience any increased levels of stress while they were
exposed to alcohol and therefore were not as desperately trying to consume alcohol in order to
reduce their levels of stress (Dunn, 2012). Because of this, their reproductive rates were higher
than those of the vials containing the flies that were introduced the high-stress and low-stress
environments (The Tech Museum of Innovation, 2008). Less alcohol in their systems means that
there were less negative effects of that alcohol. The ADH​+ and ADH​- experimental groups of
Drosophila melanogaster were more inclined to consume alcohol due to the amount of pressure
that resulted from their exposure to high and low-stress environments (Clark et al, 1998). While
the ADH​+ flies are able to metabolize the alcohol, their death rates were higher. This is because,
when stressed, the flies are also likely to drink more of the food, which was alcohol in this
experiment (Wong et al, 2009). Even though the ADH​+ were not feeling the effects of the
alcohol, the effects of the toxicity were still impacting their health, resulting in death. So, while
the fruit flies were not directly feeling the damage they were doing, they drank an excessive
amount of whatever was available to them, which is what led to more ADH​+ dying than ADH-
(Christmann, 2014). The ADH​- fruit flies were also put through high-stress and low-stress
environments; however, they were able to feel the effects of the alcohol and became so
intoxicated that they could not drink enough alcohol to result in the same rate of death as the flies
with the ADH​+ gene (Christmann, 2014). For these reasons, ADH​+ ​experimental flies had higher
reproductive rates than ADH​- experimental flies, but higher body toxicities in ADH​+ led to
greater deaths.

Conclusion
Overall, the ADH​+ and ADH​- experimental groups’ reproductive and mortality rates were
the most affected by stress compared to the ADH​+ and ADH​- control groups. The control groups
had the greatest percent change in reproduction, and as they were not introduced into stressful
environments it can be inferred that ​Drosophila melanogaster specimens do not consume larger
quantities of alcohol unless they are provoked to do so. When the ADH​+ flies within the
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experimental vial were exposed to stress they drank the alcohol but, because they are able to
metabolize it, they were able to reproduce immediately after consumption; therefore, they had
higher reproductive rates. On the other hand, the ADH​- fruit flies that were contained within the
experimental vial responded differently because their bodies were temporarily crippled after
ingesting the alcohol. Since the specimens were unable to move, they could not reproduce while
they were intoxicated and, therefore, they had a lower reproductive rate. Regarding the mortality
rates, more ADH​+ specimens perished than the ADH​- specimens because, although they were
able to tolerate the alcohol, they continued to ingest it excessively and, as a result, their organs
failed because there was simply too much alcohol in their systems. Overall, the data collected
throughout the duration of this experiment supports the null hypothesis which stated that humans
and ​Drosophila melanogaster specimens consume greater quantities of alcohol when they are
stressed. Moreover, depending on the genetics of the fruit fly or the person that is ingesting the
alcohol, they will either be physically capable or incapable of tolerating the copious amounts of
alcohol in their systems.

Acknowledgements
Thank you to Mr. Sprague for giving the Biotechnology class the opportunity to perform
this lab and for helping with the fruit flies and alcohol needed to compare and correlate the
characteristics of the ADH gene. Also, thanks to MATES for the student access to any lab
equipment used in the experiments.
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Figure 5: The original vial from which


all flies were obtained.

Figure 6: FlyNap is an anesthetic that was used on the flies while


transferring them from one vial to another, or used while counting the
flies.
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