Sex-Linkage of White Eye Gene in Drosophila melanogaster

Michael Hudak, Ava Kennedy, Joseph Kane, Erin Geoghegan, Kyle Lopes, Lucas Monchinski

Marine Academy of Technology and Environmental Science

Biotechnology Block 3

Mr. Sprague

December 11, 2020

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Introduction: EG + AK

The species Drosophila Melanogaster, known commonly as the fruit fly, is often used for

genetic experiments due to its simple genetic makeup and easily observable characteristics. The

“Drosophila genome is 60% homologous to that of humans, less redundant, and about 75% of

the genes responsible for human diseases have homologs in flies” (Ugur et al., 2016). This makes

the fruit fly an ideal species for both learning about genetics in an educational environment as

well as in a professional setting in which research on various viruses can be conducted. It is

common for cancer research to be performed on Drosophila due to these similarities.

Additionally, Drosophila have a relatively short life cycle with a period of about 10 days from

egg fertilization to adult life (Cherry Biotech). During crosses between flies, large amounts of

offspring are produced which allows for observing a higher population of flies that yield more

accurate results. Genetic crosses can be performed quickly and errors can be corrected simply by

performing an additional cross. Since the characteristics of the Drosophila are easily observable

under a dissecting microscope, it is simple to view the changes in genetics without having to

perform additional tests. Similarly, the low number of chromosomes and small genome size

(Gsa. (n.d.)) make the fruit fly an ideal organism to study genetics.

In most species, the two different types of chromosomes are sex chromosomes and

autosomes. Drosophila have 4 pairs of chromosomes, 3 autosomes and 1 sex chromsomes,

unlike humans who have 46 pairs of chromosomes. Sex-linked genes are genes that are located

on sex chromosomes, resulting in varied traits among males and females. While in humans male

sex chromosomes appear as XY, and female sex chromosomes appear as XX, recessive

sex-linked traits appear more in males as they have to be present on only one X rather than two.

For example, colorblindness and hemophilia in humans are caused by sex-linked genes (BD
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Editors, 2019). The pattern of inheritance of sex-linked genes on the X chromsome can be seen

in Figure 5. In Drosophila, eye-color is an example of a sex-linked gene, as it appears on the X

chromosome. The wild-type (red eyes) is dominant to any other variations, such as white eyes,

which are a recessive mutation. While recessive sex-linked traits in humans must be present on

one of two sex chromosomes, “In Drosophila, the number of X chromosomes determines sex:

two X’s result in a female and one X results in a male. In mammals, the presence of the Y

determines maleness and the absence of a Y determines femaleness.” (Griffiths, 1970). As a

result, there have been sex chromosomes that do not appear in fruit flies as XY or XX, as in

humans. Drosophila sex can appear as female with the chromosome pairs XX, XXY, XXYY, and

as male with the chromosome pairs XY and XO. Although, the genetic similarity between the

two species is a result of sex-linked traits appearing on only the X chromosome in sex

chromosomes, such as white eyes in fruit flies. Originally, in 1910, when chromosomes were

first being examined in genetics, Drosophila were used to compare dominant and recessive traits

in offspring (Gleason, 2017). While fruit flies can have a range of eye colors, primarily red or

white, these genotypes portray significant information relating to their genetic makeup.

Methodology: AK

An initial vial of Drosophila melanogaster containing white eye males and females was

sorted through with the white eye females being isolated in a prepared vial. The vials contain one

part dry medium and one part water with granules of yeast sprinkled on top, as seen in Figure 1.

Flies were sorted throughout the lab using a dissecting microscope and identified by observation

of physical characteristics by members of the lab group. The main points of identification

between male and female were the shape and color of the body as well as the noticeable
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sex-cones on the anterior legs of the male flies, which can be seen in Figure 4. In order to

properly observe with ease, the flies were napped in ice water before being observed under the

microscope. Flies were transported from vials containing medium to empty vials using a funnel

so that they could be knocked out temporarily before moving to a new vial for the next cross.

The white eye females were left isolated for a period of about a week to ensure that there was no

remaining reproductive material from the males. Another vial containing wild-type, red eye

Drosophila melanogaster was sorted through with the red eye males being added to the vial

containing the white eye females. The wild-type males and white eye females were left for a

period of a few days to breed. After eggs and larva were seen in the vial, the parent flies were

released to ensure that the offspring would not breed intergenerationally with the parent flies.

The offspring of the previous cross between the white eye females and wild-type males

were projected to be white eye males and red eye females carrying the white eye trait. However,

in this trial, the offspring were not as predicted, meaning that an error was made in identifying

one or more of the wild-type males. In order to perform the next cross effectively, flies were

taken from an effective cross performed by another lab group. Each fly was individually checked

before being placed in a new vial prepared with medium. The wild-type carrier females were left

to breed with the white eye males, and removed from the vial after eggs and larvae appeared to

prevent intergenerational breeding. The offspring of this generation are predicted to have a

1:1:1:1 ratio of wild-type females, wild-type males, white eye females, and white eye males.

After data was collected from each cross, it was organized into tables to be interpreted. Pearson’s

Chi-Squared test was used to make conclusions about data.

Data: LM + MH + KL
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Table 1: Punnett square of the first generation cross of white eye females and wild-type males, resulting in
the genotypes of the second generation. The offspring should be a 1:1 ratio of wild-type appearing
females that carry the white eye trait and white eye males.
First Cross XR Y

Xr XRXr XrY

Xr XRXr XrY

Table 2: Punnett square of the second generation cross of wild-type females carrying the recessive white
eye trait and white eye males, resulting in the genotypes of the third generation. The results of the punnett
square show that, ideally, the third generation should be evenly distributed among four different
genotypes, and accordingly, four different phenotypes. The offspring should include a 1:1:1:1 ratio of
wild-type appearing females carrying the white eye trait, white eye females, wild-type males, and white
eye males.
Second Cross Xr Y

XR XRXr XRY

Xr XrXr XrY

Table 3: Numbers of each phenotype class of flies analyzed from the F2 generation. Data was collected on
1 December 2020. Data is compared to the expected number at the conclusion of the experiment.
1 2 3 4 Total

Phenotype Class wild-type ♂ white eye ♂ wild-type ♀ white eye ♀ 4

Number of Individuals 42 42 35 47 166

(actual count)

Expected Number 41.5 41.5 41.5 41.5 166

χ2 = ∑
2 2 2 2 2
(𝑂𝑖−𝐸𝑖) (𝑂1−𝐸1) (𝑂1−𝐸1) (𝑂1−𝐸1) (𝑂1−𝐸1)
𝐸𝑖
= 𝐸1
+ 𝐸1
+ 𝐸1
+ 𝐸1
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.25 .25 42.25 30.25

χ2 =
41.5
+ 41.5
+ 41.5
+ 41.5
= 1. 759

Oi is the observed value, χ2 is chi-squared, and Ei is the expected value. The calculated chi

squared value of the second generation is 1.759. By using the χ2 table with a significance level of 0.05,

the corresponding P-value was approximately 0.6239.

Figures: AK + KL

Table 4: An unintentional cross between a wild-type female and a wild-type male must have occurred
during the first cross. Punnett squares were worked out to confirm exactly where the error occurred. It
was concluded that a wild-type female was mistaken for a wild-type male. The cross between the
wild-type female and wild-type male is shown.
XR Y

XR XRXR XRY

XR XRXR XRY

Figure 1: This figure shows the vials in which the
flies were kept. They contain one part dry medium
and one part water, with granules of yeast
sprinkled on top
Credit to Ava Kennedy for the photo.
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Figure 2: This figure shows the vial after the
addition of the sample larva from a cross between
a white eye female and a wild-type male. It is
labeled with our group, block, and the gender and
traits of the flies.
Credit to Ava Kennedy for the photo.
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Figure 3: This figure shows a sample of a white
eye female fly, which is being viewed through a
dissecting microscope. Female flies have larger,
visibly striped abdomens that are lighter in color.
Credit to Ava Kennedy for the photo.
Figure 4: This figure shows a white eye male fly
being observed under a dissecting microscope.
There are noticeable, darker sex-cones on the
anterior legs of the male flies. The top of the
abdomen is darker in color than in female flies.
Credit to Ava Kennedy for the photo.

Figure 5: This figure displays how sex linked

traits are inherited along the X chromosome. In
this experiment, the white eye trait is recessive
and linked to the X chromosome.
Credit to U.S. National Library of Medicine for
image.
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Results: LM + KL

Four phenotypic classes were present within the monohybrid cross between females

carrying the white eye mutation and males with the white eye mutation. The actual numbers

gathered for the phenotypic classes are 42 individuals for the wild-type male, 42 individuals for

the white eyed male, 35 individuals for the wild-type female, and 47 individuals for the white

eyed females, and totals to 166 (Table 3). Also shown in Table 3 is the expected values, which

were 41.5 individuals for each phenotypic class, with a sum of 166. The P-value calculated was

approximately 0.6239. Compared to the level of significance of 0.05, there was no significant

difference between the data and the expected outcome. Besides the analytical aspects of this

experiment, there was also the matter of housing, crossing, and successfully raising the sample

flies. The vials, which housed the flies for the duration of this experiment, can be seen in Figures

1 and 2. The flies themselves, can be seen in Figures 3 and 4.

Discussion: JK + MH

While examining the wild-type Drosophila melanogaster at the beginning of the lab,

precautions were taken in an attempt to ensure that exclusively male specimens would be

selected. The same group member identified all of the flies in order to mitigate the risk of a

group member unfamiliar with the distinctions between male and female flies making an error.

Males and females were distinguished using a dissecting microscope. To identify males, traits

such as a darker, rounded abdomen were sought, whereas females tend to have lighter, pointed

abdomens. Also, males have a small patch of bristes called sex combs on their forelegs (Tauber

Lab 2017). In the event that the group member identifying the flies lacked complete certainty

about a specimen’s sex, a second group member would check the microscope to confirm. If the
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uncertainty was too great, the fly would be killed, even if it was likely male. Finally, every fly

was also checked over by another group member, albeit with a naked eye, in order to act as a

final safeguard against error. Unfortunately, despite these precautions, a wild-type female must

have been accidentally introduced to the vial.

After the first cross had been completed, the resulting offspring were counted. The first

twenty flies checked included eleven white eyed males and nine red eyed females. This was

expected, as the ratio between the two was very close to the expected one-to-one, as shown in

Table 1. Only white eyed males and wild-type females were expected. This expectation was

based on the fact that “the genetic factor controlling eye color in the flies was on the same

chromosome that determined sex” (Gleason 2017). Thus, all the males would carry one X

chromosome with the linked white eye trait. However, the twenty-first fly removed from the vial

was a red eyed male. If exclusively wild-type males had bred with the white eyed females as had

been intended, then such a result would have been impossible. Each male offspring would have

received the gene for white eyes from the female parent, as is shown in Table 1. The only

possible way that a red eyed male could have been produced as an offspring, since it is known

that there were no white eyed males, would be if a wild-type female had bred with a wild-type

male, as shown in Table 4. It is likely that only a single wild-type female had been introduced to

the vial. This is evidenced by the fact that only a single fly out of the twenty one flies counted

was a wild-type female.

Despite the fact that wild-type males could be eliminated from breeding in the second

cross, the fact that at least one wild-type female had bred in the first cross introduced the

possibility of there being wild-type females in the offspring of our first cross that did not carry

the gene for white eyes. Because the differentiation in genotype could not be recognized visually,
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the females had to be thrown out, lest the second cross be potentially contaminated. As long as

the males had white eyes, they could be included in the second cross. Female flies were donated

by another group that had a successful first cross.

The second cross turned out as predicted. A roughly equal number of flies of each

possible phenotype were counted, as would be expected given the crossed flies. A Pearson’s

chi-squared test was calculated to determine whether or not the data collected were significantly

different than what would be expected of a ratio of one-one-one-one. The calculated P-value of

0.6239 indicates that there was no significant difference between the collected and expected data,

which in turn indicates an accurate second cross. To have a reliable chi-squared test, a great

enough sample is necessary. As a general rule, the expected values must be greater than 5

(Gleason 2017). Given that all expected values were 41.5, the chi-squared text is both reliable

and accurate to the expected result.

Conclusion: KL + EG

To summarize, the data collected throughout the experiment has no significant difference

in relation to the expected values. Thus, the data do not indicate enough information to disprove

the null hypothesis. The experiment began by mapping out an estimated timeline of events,

which was used to keep track of how the experiment would be conducted. First, the vials were

created, which served as the temporary habitat of the sample flies, Drosophila melanogaster.

Then, the necessary crosses were conducted and the offspring of each new cross was examined.

However, the results of the breeding did not meet our expectations. These results came about due

to a mistake involving the males’ genotypes within the vial. As a result, the second generation

did not produce the desired results in order to complete the next experiment. In order to keep
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performing the experiment, another group allowed for the use of their flies to create a new vial

from a successful cross for the second generation of flies. These flies were used to cross

wild-type females and white eyed males and achieved the desired results. These results consisted

of 166 individuals and a P-value of 0.6239. In order to improve the fly testing portion of the

experiment, each individual fly should have been checked more carefully as they were placed in

the vial to secure accurate results. Further, a greater sample size would have achieved even more

significant results. The outcome of this experiment correctly lined up with our data and

hypothesis based on scientific evidence.

Acknowledgments: LM

This research would not have been possible without the help of Michael Hudak, Ava

Kennedy, Joseph Kane, Erin Geoghegan, Kyle Lopes, and Lucas Monchinski. Thank you again

to Ava Kennedy for photographing this experiment. A special thanks goes out to Mr. Adam

Sprague for his guidance throughout the research process. Finally, our gratitude is given to the

Marine Academy of Technology and Environmental Science for the providing use of its science

laboratories and apparatus.

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References: KL

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https://www.cherrybiotech.com/scientific-note/drosophila-life-cycle-and-fly-anatomy

Editors, B. (2019, October 05). Sex Linked Genes. Retrieved from

https://biologydictionary.net/sex-linked-genes/

Gleason, K. (2017, May 22). The Embryo Project Encyclopedia. Retrieved from

https://embryo.asu.edu/pages/sex-limited-inheritance-drosophila-1910-thomas-hunt-

morgan

Griffiths, A. J. (2000, January 01). Sex chromosomes and sex-linked inheritance. Retrieved

from https://www.ncbi.nlm.nih.gov/books/NBK22079/

Gsa. (n.d.). Drosophila as a Model Organism. Retrieved from

http://modencode.sciencemag.org/drosophila/introduction

Libretexts. (2020, August 15). 12.2E: Sex-Linked Traits. Retrieved from

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Mirzoyan, Z., Sollazzo, M., Allocca, M., Valenza, A., Grifoni, D., & Bellosta, P. (2019,

January 21). Drosophila melanogaster: A Model Organism to Study Cancer.

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