18.12.

2022
UZAY CAN DUMAN
21702852
SECTION WED
BILKENT UNIVERSITY FALL 2022
MBG210 – GENETICS

DROSOPHILA MELANOGASTER LAB REPORT

AIM
Aim of the first lab of the Drosophila Melanogaster is:

To learn how to work with this experiment’s model organism Drosophila Melanogaster in a
laboratory environment.
To learn and observe the life cycle of the model organism as well as being able to designate
the physical differences between male and female organisms also, phenotypical traits of
different mutants of Drosophila Melanogaster.
To learn how to prepare stocks for mutant organisms and how to use corresponding
laboratory equipment.

Aim of the first Drosophila cross (parental cross) is:

To cross mutant strain males with mutant strain females-one type of mutant strain- and obtain
the F1 generation.

Aim of the second Drosophila cross (F1 cross) is:

To cross virgin mutant females that were obtained from parental cross and wildtype males,
thus, obtain F2 generation.

Aim of the third Drosophila cross (F2 cross) is:

To cross F2 generation with each other by selecting mutant strain and wild type strain, thus
obtaining F3 generation and make a Chi Square test.

The general aim of Drosophila laboratory sessions is to work with an easy to work with
model, which is Drosophila Melanogaster to make experiments and observation about
crosses, alleles and generations. Finally to see how well the results are match up with the
statistical numbers.

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INTRODUCTION
Drosophila Melanogaster which is known as fruit fly or vinegar fly. Drosophila is commonly
used in biological research thanks to its rapid life cycle which is around 2 weeks, its size that
is not too small and too large granting easy handling and good observability, it is easy to
breed, there are different strains that can be used, after breeding large number of offspring
can be obtained and it is simple to anesthetize.
Life cycle:
Egg: Approximately 0.5mm. Adult female produces 50 to 75 eggs daily.

Larva: This stage takes 3 instar stages. Every instar stage is 24 hours long and after the third
instar, pupa rises.

Pupa: At first, pupa comes out from larva stage as soft and white then, it becomes harder and
its color darkens. Metamorphosis takes place in this stage and it lasts about 4 to 5 days.
Adult: After metamorphosis is complete, adult Drosophila lives around 30 days depending
on the temperature. Female becomes receptive and starts to produce 50 to 75 eggs daily. It
stores sperms inside and use it [1].

Mutations:
Ebony: Recessive mutation that carried on autosomal chromosomes. Physical trait is to have
brown-blackish body color

Vestigial: Recessive mutation that carried on autosomal chromosomes. Physical trait is to have
short wings that restricts flying capability.
White eye: Sex linked gene that carried with X-chromosome. Causes normally red eyes to be
white.

Yellow body: Yellow body gene is also sex linked and it is carried with X-chromosome. In
this mutation, body of the fly is colored yellow.

Monohybrid cross is not sex dependent which is autosomal. It is crossing only one pair of
alleles. Dihybrid cross is also not sex linked however, cross is performed in two different
alleles together. In addition, sex linked cross is performed between male and female sex with
their sex linked gene.
For the first, Monohybrid cross was performed between ebony male and ebony female which
is ee x ee. Then, for the Parental cross, because ebony reproduction was failed, vestigial and
wildtype was crossed which is VV X vv (wildtype was taken from TA’s). Finally when F1
generation emerged, mixed cross was made with both vestigial males and females and
wildtype males and females.
For the chi square analysis 1 degree of freedom will be used since there are two traits which
are wiltype and vestigial (V and v, 2-1=1). 0.05 threshold should be used because higher
deviations lowers the significance of the experiment.

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MATERIALS AND METHODS
First we need to have 70% ethanol for the sterilization purposes. For the preparation of the
stocks: Falcon tube, Drosophila medium, distilled water and yeast is needed. For the falcon
tubes to be able breath, opening of the tube should be closed with cotton. To be able to gently
transfer the flies, a brush is needed. Also, a stereo microscope is needed to observe the flies.
Finally in order to anesthetize the flies, ether is needed.
Anesthetization was performed dipping a cotton bud to ether and putting the cotton bud in the
falcon tube with flies. After about 5 minutes flies are anesthetized. This procedure should be
done carefully since if flies are exposed to ether too long they will die and if they expose too
short they will not be anesthetized.

RESULTS
In the first lab session, ebony and ebony was crossed according to the assigned flies to
experimenters. However next week it was observed that there were no embryos.
For the second session, because ebony cross was failed, I have carried on with friend’s cross
which was vestigial. In this session, 2 vestigial females and 2 wildtype males were crossed.
Next week significant amount of embryos were observed and parents were removed.

Finally, during the third session F1 offspring was counted and there was 8 flies with 3 of
them were vestigial, normally all wildtype was expected since both vestigial and wildtype
was homozygous but probably vestigial female was not virgin. After counting, 2 wildtype, 1
vestigial females and 2 wildtype, 1 vestigial males were selected and crossed. Next week
parents were removed and several embryos were observed. During the final week, F2
offspring was observed and there were 1 vestigial and 3 wildtype flies.

e e v v v v

e ee ee V Vv Vv V Vv Vv

e ee ee V vv vv v vv vv

First Cross Parental Cross (cross made F1 Cross

with homozygous wildtype
but results were like this)

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DISCUSSION
After first cross was performed between ebony and ebony, there were no offspring however,
flies were alive. If the cross was successful, offspring would be only ebony since ebony is
homozygous recessive. After observing no offspring, and discussed the result with TA’s, it
was told that ebony strain known to be problematic about reproduction and this result is not a
surprising result. Besides this, the reason for no offspring could be from not properly
prepared stock and/or improper ambient temperature.
For the parental cross, because ebony cross was failed, vestigial was used as a mutant strain.
First, 2 virgin female vestigial were selected from the falcon tube that consists of vestigial
cross then 2 male wildtype were selected and stock was prepared. As a result of this cross 3
vestigial and 5 wildtype flies occurred. In theory, because parents were homozygous, F1
generation should not consist vestigial flies. Reason for this result is most probably from
failing to choose virgin vestigial flies. If that is the case, vestigial female hold sperm in it and
used them. Thus, making the offspring not as planned.
Chi Square Analysis for parental cross:

2
(𝑂 − 𝐸)2
𝑥 =∑ [2]
𝐸
Above equation stands for chi square analysis. Degree of freedom was determined as 1. Both
vestigial and wildtype have the same probability of 1/2 and with 8 flies expected number:

1/2 x 8 = 4
Applying the equation:

x2 = (3 – 4)2/4 + (5-4)2/4 = 0.5

Here, according to chi square values table, 0.5 is slightly larger than 0.455 and it corresponds
to P = 0.50 with 1 degree of freedom. Obviously vestigial flies were not expected in this cross
however, considering Vv x vv cross, this result is not bad especially with this sample size. It
is far from null hypothesis criteria of 0.05 so rejection was not made.

Finally for the F1 cross flies were selected as 2 wildtype, 1 vestigial females and 2 wildtype,
1 vestigial male. 2 weeks later offspring was observed and there were only 4 flies 1 is
vestigial 3 are wildtype. Sample size here is significantly low for a chi square analysis
however an analysis will be made. Further observing, there were several dead flies inside the
tube. Flies could be died from sticking to the wall of the tube. Reason why offspring is small
is could be from poorly prepared stock and/or dead parent flies and also yeast amount could
be improper however, quality of the stock was good according to experimenter.

Both vestigial and wildtype have the same probability of 1/2 and with 4 flies expected number:
1/2 x 4 = 2

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Applying the equation:
x2 = (1 – 2)2/2 + (3-2)2/2 = 1

Here, according to chi square values table, 1 is between 0.455 and 1.642 and it corresponds to
P ≈ 0.35 with 1 degree of freedom. This result is not bad with this sample size. It is far from
null hypothesis criteria of 0.05 so rejection was not made [2].

Challenge faced in this lab was mostly handling the flies. Transporting without harming them
and flies waking from anesthetize was really challenging.

REFERENCES

[1] Mlblevins. (2010, March 12). Stages in the life cycle of a drosophila melanogaster.
Biology Wise. Retrieved December 18, 2022, from https://biologywise.com/drosophila-
melanogaster-life-cycle

[2] R. J. Brooker, Genetics: Analysis and principles. New York: McGraw Hill Education,
2018.