Barlotta, Pereira, Putnam, Walkowicz, and Weiner 1

Examining Sex-Linked Traits Across Multiple Generations of

Drosophila melanogaster
13 January 2019
Alessandro Barlotta, Lexi Pereira, Jack Putnam, Devyn Walkowicz, and
Blake Weiner
Marine Academy of Technology and Environmental Science
Block 1 Biotechnology
 Barlotta, Pereira, Putnam, Walkowicz, and Weiner 2

Introduction:
Drosophila melanogaster, otherwise known as the fruit fly, is a frequently used organism
in genetic research that has been used to study heredity since the early twentieth century. (Rodan
& Rothenfluh, 2012). The fruit fly has been an optimal organism in research due to its many
favorable characteristics, including: a short life cycle of around 10-15 days (allowing several
generations to be observed in a single sample study), its fast reproduction rates (bearing
hundreds of offspring in a single mating), its easily indistinguishable sexes, and its ability to be
cultured very easily (“Sex Linked Inheritance”) (Rodan & Rothenfluh, 2012). Drosophila also
has a relatively simple genome, bearing only 4 pairs of chromosomes, with the first pair being a
sex chromosome (♀-XX, ♂-XY) and the other 3 pairs being autosomes. Additionally, the
genome of Drosophila melanogaster has been found to be around 60% similar to that of humans,
allowing connections to be drawn between the simple fruit fly organism and the complex human
body (Echalier, 2018).
The most common eye color exhibited in Drosophila is red-brown, caused by two
different types of pigments: pteridines, which are red, and ommochromes, which are brown (“An
Eye on Trafficking Genes”). In some cases, fruit flies show the absence of both of these
pigments, resulting in the mutation that is the white-colored eye (“An Eye on Trafficking
Genes”). Because the white eyed gene is found due to a recessive gene found in sex
chromosomes, the allele can be considered sex-linked. Also, the white-eye phenotype is present
in females so therefore, the determination of the eye color of Drosophila is seen on the X
chromosome, which is sex-determining (“Sex Chromosomes and Sex-Linked Inheritance”). The
white-eye phenotype can thus be considered X-linked, because it is found on the X chromosome
(“Drosophila as a Model System”). For example, a male with the white-eye mutation would
display the white-eye phenotype because it has only one X chromosome. However, in order for a
female to exhibit the same phenotype, it must have the trait on both X chromosomes (Easter).
The males of Drosophila can be seen as hemizygous because they exhibit the sex-linked trait
regardless of being dominant or recessive (“X-Linked Inheritance”).
When performing specific test crosses amongst wild-type and white-eye fruit flies it is
extremely crucial to have said fruit flies correctly identified. There are several key characteristics
to look for when identifying male vs. female Drosophila flies. Male fruit flies have a darker,
rounder abdomen, while females are larger, lighter in color, and have a pointed abdomen.
Additionally, the males bear a set of sex combs on the fourth set of their front legs (Lembke).
Identifying these traits allow for confirmation on the success of a genetic cross.
The common fruit fly allows for an easier method of analysis which can be mirrored to
humans. This project, while observing the different generations and how the sex-linked trait was
prevalent, along with comparing the results with what was expected, one will be able to draw a
line between the sex-linkage in Drosophila melanogaster and Homeo sapiens.
Blake Weiner
 Barlotta, Pereira, Putnam, Walkowicz, and Weiner 3

Methodology:
To begin, two master vials containing homozygous wild-type and homozygous
white-eyed flies were split among the class. Each group received one vial of male and female
homozygous wild-type flies and one vial of male and female homozygous white-eyed flies.
These flies received would act as the parent generation to perform the F1 cross. The type of cross
that would be performed was determined: wild-type females and white-eyed males.
The parent generation of wild-type and white-eyed flies was sorted by gender. Four vials
with a stopper were labeled using a marker and tape as follows: wild-type female, wild-type
male, white-eyed female, and white-eyed male. A dissecting microscope was used to determine
the sex of the flies. The fruit flies were put to sleep by placing the two original vials into a beaker
of ice for approximately 5 minutes or until visibly sedated. To sort the wild-type flies and
white-eyed flies 5 flies were poured out under the microscope at a time and a paintbrush was
used to move the flies under the focus. Sex was identified based upon the following
characteristics: male fruit flies are smaller, have sex combs on their forelegs, and have a dark
abdomen, while female flies are large, have a striped/light colored abdomen, and their abdomen
is pointed. Flies were sorted until 10 wild-type females and 10 white-eyed males were collected.
In a new vial, food was made for the cross of the parent generation: about one-fifth of
the vial was filled with medium, a pinch of yeast was added to the medium, and an equal amount
of water was poured into the mixture. The medium was left to dry until slightly damp. The flies
were transferred from the wild-type vial into the medium-containing vial. The
medium-containing vial was held upright, a funnel was placed into the medium-containing vial,
and the original vial was placed on top. The stopper was removed from the original vial allowing
for the flies to transfer vials. Once the 10 flies left the wild-type vial a stopper was placed on the
medium containing vial. The same process was repeated to transfer the white-eyed flies into the
medium-containing vial. This medium-containing vial was labeled F1 ♀ wild-type v. ♂
white-eyed. After 8 days the parent flies were released from the vial.
The F1 cross was performed. Females from the F1 generation were crossed with wild-type
males. The same procedure was used at this point as that in paragraph 2 to sort the flies by
gender, however, four vials with a stopper were labeled using a marker and tape as follows: F1
female, F1 male, white-eyed female, and white-eyed male. The F1 generation and white-eyed
flies were sorted by gender so there were 16 F1 females and 16 white-eyed males. A new vial
with medium was made and the sorted flies were transferred into the medium-containing vial.
This vial was labeled F2 and placed into the incubator for 10 days. After 10 days the parent flies
were released from the vial leaving the F2 generation.
The F2 cross was performed. White-eyed males were crossed with unspecified wild-type
females. The same procedure was used at this point as in paragraph 2 to sort the flies by gender.
Two vials were labeled as follows: white-eyed males and wild-type females. The F2 generation
was sorted by gender so there were 8 males and 8 females. A new vial with medium was made
and the sorted files were transferred into the medium-containing vial. This vial was labeled F3
 Barlotta, Pereira, Putnam, Walkowicz, and Weiner 4

and placed into the incubator until taken home over winter break. The F2 generation was released
leaving larvae to hatch as the final F3 generation.
The final F3 generation was sorted by gender and whether the flies were wild-type or
white-eyed. Two vials were labeled: ♀ wild-type, ♂ wild-type, ♀ white-eyed, and ♂ white-eyed.
While sorting the flies, the amount of each type was recorded. A Chi-square test was used to
analyze our results. Based off the Chi-squared test a P-value was found to determine whether our
data failed to reject or reject the null hypothesis.

Timeline:
31 October 2019: Parents were crossed (♀ wild-type + ♂ white-eyed)
9 November 2019: Parent flies of F1 generation were released
19 November 2019: F1 generation was crossed (♀ wild-type + ♂ wild-type)
27 November 2019: Parent flies of F2 generation were released
3 December 2019: F2 generation was crossed (♀ wild-type + ♂ white-eyed)
17 December 2019: Parent flies of F3 generation were released
8 January 2019: F3 generation was sorted
Lexi Pereira

Data:

Table 1: A display of the actual numbers (top) and expected numbers based on ratios obtained
from Punnett squares (bottom) of each type of Drosophila melanogaster obtained in the final
generation of the experiment (wild-type/red-eyed males and females and white-eyed males and
females).
Wild-Type White-Eyed

Gender Male Female Male Female Total

Actual 41 52 7 13 113

Expected 42 42 14 14 112
 Barlotta, Pereira, Putnam, Walkowicz, and Weiner 5

X’ Y

X XX’ XY

X XX’ XY
Figure 1: Punnett square for the expected ratios of the first generation of flies. Red-eyed females
are indicated by light pink, and red-eyed males are indicated by dark pink.

X Y

X XX XY

X’ XX’ X’Y
Figure 2: Punnett square for the expected ratios of the second generation of flies. Red-eyed
females are indicated by light pink, red-eyed males are indicated by dark pink, and white-eyed
males are indicated by dark grey.

Figure 3: Punnett squares displaying the expected ratios of the final generation (male flies are
labeled on the top and female flies are labeled on the side). From a white-eyed male and red-eyed
female (ambiguous genotype) cross, ⅜ of the flies will be red-eyed female (as indicated by the
light red color), ⅜ will be red-eyed male (as indicated by the dark red color), ⅛ will be
white-eyed female (as indicated by the light grey color), and ⅛ will be white-eyed male (as
indicated by the dark grey color). These probabilities were multiplied by the total number of flies
(113 flies, rounded down to 112 to avoid quarter-flies) to obtain the expected values for 42
red-eyed male, 42 red-eyed female, 14 white-eyed male, and 14 white-eyed female.
 Barlotta, Pereira, Putnam, Walkowicz, and Weiner 6

Figure 4: A bar graph displaying the numbers of each type of Drosophila melanogaster based on
gender and eye color. The expected values are shown in blue, and the actual values are shown in
Alessandro Barlotta and Jack Putnam

Results:
After the cross between white-eyed male and red-eyed carrier female fruit flies, the final
generation of flies contained 41 wild-type males, 52 wild type females, 7 white-eyed males, and
13 white eyed females. The expected values for these flies are 42, 42, 14, and 14, respectively. In
comparing these with the actual values of the flies, the closest ones were those of the wild-type
males and white eyed females, each with one less than their expected values, a small enough
difference to be attributed to probability. The wild-type females contained 10 more flies than
expected, and the white-eyed males contained 7 less than, or half of the expected number. The
expected values were obtained from the respective ratios of each type of fly from punnett
squares: ⅜, ⅜, ⅛ and ⅛.
A chi square test was conducted to evaluate the significance of the data. By using the
actual and expected values of the fruit flies, the equation gave a value of 5.976. When relating
this to the degrees of freedom value of 3, a P-value of approximately 0.1128 was obtained. This
shows that the null hypothesis has failed to be rejected; solely based on the data, it is suggested
that there is no significant difference and a correlation between the actual and expected values of
the number of Drosophila in the final generation.
Alessandro Barlotta
 Barlotta, Pereira, Putnam, Walkowicz, and Weiner 7

Discussion:
Through the use of a chi square test, it was determined that there was no significant
difference between the actual and expected amounts of Drosophila melanogaster and that there
was a correlation between these values. It is essentially suggested from the data that the punnett
square ratios relate to the actual number of offspring resulting from the cross performed. With
this knowledge, it provides a steady basis for further research to be done.
The majority of our problems can be directly linked to human error; however, since we
demonstrated relatively proper technique, the human error was kept to as minimal as possible,
given the circumstances. The largest being that the F2 parents were not separated from the F3
offspring in a timely fashion, thus causing a very high chance for inbreeding. This could provide
an explanation for the discrepancies between the actual and predicted values, as the offspring
between these two generations would have different genotypes and phenotypes when compared
to their parents. On top of this, flies escaping from the vials when moving them from one vial to
another was also an issue that could very well have been of significance, as this happened on
numerous occasions within our group. One such accidental release could have resulted in
upwards of 15 flies, all of which could have potentially caused a change in results. As far as
naturally occuring reasons as to why there may have been differences between the actual and
expected results, it has been shown that, amongst white eyed flies, there are some cases of
homosexual male courtship, preventing females from being impregnated by white-eyed male
flies (Vett). This might account for minute discrepancies however as it is a relatively rare
happening.
There were definitely ways that this experiment could have run smoother, although it
does show potential in terms of the scientific discoveries that could be made. Since fruit flies and
humans are so similar in terms of genetic makeup, and we were able to effectively run the
experiment with data close to what we expected, this makes the fruit flies able to have lots of
potential when it comes to studying a multitude of different diseases and conditions (Yamamoto).
Devyn Walkowicz

Conclusion:
In conclusion, after experimenting from October 31, 2019 to January 8, 2020, the data
collected showed that the difference between the actual and expected values of the number of
individuals of Drosophila melanogaster was not significant. Despite there being some
differences between the expected and actual values of the flies, a suggested correlation is seen
when comparing these values. These differences can be accounted for by some issues that were
encountered during the study, including those related to the possible inbreeding of fly
generations and the escaping of important flies from the final generation, thus skewing the
results. The relation of the genomes of fruit flies and humans is strikingly important to the
understanding of genotypic layout of not only humans but other organisms as well, something
only attainable from the further analysis of Drosophila. Alessandro Barlotta
 Barlotta, Pereira, Putnam, Walkowicz, and Weiner 8

Acknowledgements:
We would like to thank our instructor, Mr. Sprague, for supplying us with the proper
materials to complete the lab and for his guidance throughout the it, which allowed us to
understand all aspects of our experimentation. Also, we are thankful for the Marine Academy of
Technology and Environmental Science for permitting us to work with the fruit flies and
complete this lab.
Blake Weiner

Miscellaneous Graphics:

Figure 5: An example of a male red-eyed fly similar to the ones seen during the experiment.

Figure 6: An example of a female red-eyed fly similar to the ones seen during the experiment.

Figure 7: A dissecting microscope similar to the one used in the experiment that was used to sort
the flies based on sex and eye color.
 Barlotta, Pereira, Putnam, Walkowicz, and Weiner 9

Figure 8: The medium and yeast used for the feeding of the fruit flies in their vials, which are
also shown.

Devyn Walkowicz

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Griffiths, A. J. F. (1970, January 1). Sex chromosomes and sex-linked inheritance. Retrieved

from https://www.ncbi.nlm.nih.gov/books/NBK22079/.
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 Barlotta, Pereira, Putnam, Walkowicz, and Weiner 11

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Jack Putnam