The Effect of Caffeine on Drosophila Melanogaster

Alec, Avery, Chiara, TeAndre

March - April 2017

Purpose:
The purpose of this experiment was to test and observe the effects of caffeine and
caffeine addiction on fruit flies and their offspring. It is important to see how an addiction
to any substance can change both individuals and populations.

Prediction:
We predicted that Drosophila exposed to caffeine would be more inclined to prefer
caffeinated food sources. And by extension, the next generation that grew up with
caffeine in their food, would prefer it as well.

Introduction:
Drosophila Melanogaster are what we are using to run our experiment, theyre also
known as fruit flies. They are a model organisms because they have a short life span
and a fast reproduction rate, allowing us to see the affects on their offspring. You can
get large number of them at a time giving you a larger sample size, giving more
accurate results across the board. Animal models are important for biological research
because they allow scientists to reproduce human diseases or abnormal behavior
without the ethical concerns inherent in human studies. Although there are obvious
differences between humans and other animals, there are many molecular and cellular
processes that are shared among all species through evolution. For this experiment, we
will be using artificial selection to see the effects of caffeine preference on the offspring
of fruit flies that have only been exposed to caffeine, and those who have only been
exposed to normal food, and if theres a difference. Artificial selection is the breeding of
plants and or animals to produce desirable traits. Humans share about 60% of their
DNA with fruit flies, and over half of the adults in america are addicted to caffeine.
 Materials:

Three plastic tubes: one for the caffeine group, one for the non caffeine group
and one for both options
Caffeine Pills
Fly food ( teaspoon food, 4 mL water)
Mortar and Pestle
Divider between food options (cardboard)
Camera (For time lapse)
CO2

Independent Variable: Food (Caffeinated or normal)

Dependant Variable: Which food the flies choose to go to (pre-disposed to caffeine
and non).
Confounding Variables: Death of flies, reproduction rates

Procedure #1

1. Serial Dilution
a. Test caffeine pills on flies using serial dilution to determine a safe but
effective quantity of caffeine for our experiment.
b. Crush up caffeine pill and mix with water. Add parts of caffeine water
solution to regular water, reducing the previous caffeine content by 9/10.
c. Setup 4 test tubes with fly food mix and put each different caffeine level of
the caffeine water solution in each tube.
d. Test on flies to see which caffeine level will affect the flies, but not kill
them.

2. Setup habitats
a. Take three test tubes and place a 1 inch cardboard divider in one of the
tubes to split the caffeine and non caffeine food sources.
b. Put cotton balls on the top to allow the flies to breath and to allow us to
anesthetize the flies easier.
3. Make the food
a. 1/2 teaspoon dry fly food (leveled off) and 4ml H2O. Tap and swirl vial
immediately after addition of H2O. Let sit for 10 min.
b. Make the caffeine food source. add an amount (depending on serial
dilution, .1 for our group) of a crushed up caffeine pill to the H2O before
mixing with fly food powder. Repeat above steps.

4. Add Food to Habitats

a. Add caffeinated food source to the one habitat and half of the surface of
another habitat.
b. add non-caffeinated food source to the third habitat (our control group)
and to the other half of the surface of the second habitat. *It is important
to have a control group so you know if your findings are significant
or not.

5. Put Flies in Habitat

a. Use CO2 to anesthetize flies.
b. Separate equally by number and sex into each experiment habitat.
c. Make sure theres enough females and males together to reproduce in the
separate groups.
d. Place the one fly group into the tube with the caffeinated food source, and
the other group into the non-caffeinated food source.

6. Reproduction
a. Wait for 2-3 weeks for flies to reproduce. This will form a new population,
selectively bred with exposure to caffeine.

7. Test Offspring
a. Using CO2, anesthetize offspring from caffeine and non-caffeine tubes
separately at different times.
b. Move offspring into the both options tube at separate times.
c. Record with slow-mo camera knocking them to the bottom of the tube (10
times).
d. See how many go to the caffeine and non-caffeine food sources. Record
results.

8. Compare data from both groups

 Procedure #2:

1. Create Habitats
a. After running serial dilution and determining a safe amount of caffeine for
the flies to consume, put a caffeinated food source in one tube.
b. Put a non-caffeinated food source into another tube.

2. Record Weight
a. weigh both tubes to find the amount/volume of food in each.
b. Write down the weight.

3. Place Flies
a. Put an equal amount of flies into each habitat.
b. The number of flies was 10 in each.

4. Let the tubes sit for an extended period of time, preferably a week.

5. Test New Weights

a. Now that the flies are accustomed to the tubes and are eating the food, it
is time to see if the volume of food is changing at a different rate
depending on difference of the food source.
b. Anesthetize and remove all flies and larvae in both exhibits.
c. Measure the weight of the tubes.
d. Record weights.

6. Calculate Results
a. Use math to find out how the volume of food is changing over time. We
can draw conclusions from how the amount changes.
b. Write down any observations or changes.

Procedure #3:
1. Setup Habitats
a. Cut out a piece of cardboard to divide the tube into two equal halves.
b. Fill one side with caffeinated food predetermined by the serial dilution and
fill up the other side with non-caffeinated food.
c. Wait for food to dry
2. Prepare to Test Flies
a. Anesthetize flies individually and place into testing vile.

3. Run experiment.
a. When fly wakes up, knock the tube on the table.
b. Record which food source the fly goes to via negative geotaxis.

4. Record Results
a. Create a table for the first ten trials and the second ten trials.
b. Record this for each fly and repeat as many times as possible.
 Results
Figure 1: Raw Data Table & Graph
Fly 1 Fly 2 Fly 3 Fly 4

1st 10 2nd 10 1st 10 2nd 10 1st 10 2nd 10 1st 10 2nd 10

Caffeine 2 8 3 8 1 6 3 8

Normal 8 2 7 2 9 4 7 2
Flowgram
 Conclusion:

Meaning of Data
What we concluded was that caffeine has a strong effect on fruit flies. Not only
did our original experiment fail, but all of the flies in the caffeine test tube died. Even
though our original plan and method for recording the caffeine addiction in fruit flies
didnt succeed there is still more to be taken away. Something interesting that we
observed was that not only did all of the flies die, but the volume of food was much
lower than the non-caffeinated.
This can mean many things and we cant make an exact claim on why this
happened, but we came up with two possible explanations. Either the fruit flies became
addicted to caffeine and then overate, or the caffeine shortened the lifespan of the flies
and they had to consume more food. This could explain why the food volume was
different, but not how they died. We again hypothesize two possibilities. Either the flies
became addicted to the caffeine in their food and died via overeating, or the caffeine
was too strong and caused major health problems for the drosophila.

Next Steps
To salvage our experiment and project, we decided to follow up on these
outcomes. We ran two follow-up experiments, the first testing the difference in food
volume between caffeinated and non-caffeinated food sources. The second was testing
on individual flies preference to caffeine over time.
The first experiment was set up with one test tube with a caffeinated food source,
and one with a regular food source. We measured the mass of each tube before we put
in flies, and then we put the same amount of flies in each tube. This experiment is still a
work in progress, but we will measure the weight of the tubes after the flies have been
inhabiting the tube for some time. To avoid confounding variables, we will remove all
flies, eggs, and larvae before weighing the tubes. This experiment is to test whether or
not the flies are really overeating or just being affected by health factors.
The second test is about preference and to see if flies become addicted to
caffeine over time. We split a test tube into two sections, caffeinated and normal food
sources. We then put one fly in at a time and measured which side they went to. We
observe an individual fly ten times, and then test the next one. What we observed is that
there does seem to be a development of preference or even addiction beginning in the
fruit flies.
This is shown through the average of the four flies we tested. The first ten times
we tested their preferences, the flies either went to the caffeine or normal side. The first
ten times the flies chose caffeine 2.25 times on average, and the second time, 7.5
chose caffeine on average. This shows the preference grows over time from exposure
to the substance.

Future Improvement
While this data is significant, there is still room for growth in our experiment
since we have only tested four flies. To make this experiment better and improve next
time, We should run more tests and trials, and carry out the food volume theory for a
longer period of time to better show the significance. For our original idea and
procedure, there is clearly more to learn. We werent able to test any offspring due to
the extreme effects the caffeine has on fruit flies. To improve this we must give the flies
a much smaller concentration of caffeine. This would allow our flies to have offspring
that will survive and that still are affected by caffeine.