1.
Introduction
The species known as Drosophila melanogaster belongs to the
Drosophilae family and is commonly recognized as fruit flies. Typically small
and widespread, these flies are often found in the vicinity of decaying or
underripe fruit, which accounts for their name. Additionally, they can be
spotted near decomposing vegetables. Their global distribution spans every
continent, with the exception of Antarctica, as they migrated from Africa to
Europe and subsequently spread to other parts of the world, ultimately reaching
the Americas.
In the realm of biology, Drosophila fruit flies serve as one of
the most frequently employed organisms for modeling biomedical science
("Fruit Fly Genetic," 2021). This widespread usage is attributed to
their cost-effectiveness, short generation time, and utility as genetic tools.
Their compact and easily manipulable genome renders them particularly
advantageous for genetic crosses, making them distinctive and highly valuable
for research purposes(“Genetics With Drosophila”, 2018). Other reasons as to
why they are commonly used in biomedical science is because they are able to be
kept alive at high sample sizes in order for scientific experiment, they have
short life cycles between each generation for breeding, and it is simple to
neutralize them with FlyNap anesthesia in order to transfer them from vile to
vile and examine and record data under a microscope (Lindsley & Grell, 2018).
The management of fruit flies come with ease to those
experimenting with them and as previously stated, Drosophila melanogaster are
popular among scientists because they only have 4 chromosome pairs, making it
easier to make crosses and predict future phenotypes for resulting Drosophila
generations(“Model Organisms For Biomedical Research”, n.d.). While on the
topic of predicting phenotypes, there are many studies that depict that
Mendelian autosomal inheritance is what is the driving factor for the
expression of different Drosophila phenotypes from generation to generation but
there is also evidence showing that the inheritance is caused by sex-linked
inheritance which is not a type of Mendelian inheritance (“Sex-Linked Traits”,
n.d.). In a 2017 study by Arizona State University, it was found that when
breeding F1 males that were sepia and black-eyed with F2 wild type females
that, almost ¾ of the offspring across all trials were black-eyed sepia males
and only about every 25% of female offspring were black-eyed sepia (Chen et al.,
2017). This shows that the presence of 2 X chromosomes is a crucial factor in
limiting Mendelian inheritance for eye color and eye color was instead
controlled by genetic factors (“Gender Reveal”, n.d.). Two X chromosomes means
having a higher inheritance rate of the phenotype of black-eyed sepia in males
which limits the Mendelian inheritance model that calls for equal inheritance
in males and females (Arnold et al., 2017). Contrasting this, in a study
conducted at the Berg Lab at the prestigious University of Washington, when
observing wing shape, the phenotypes appeared wingless. They concluded that it
looked like the gene alleles had formed linkage groups that showed an
inheritance pattern causing high wingless phenotype frequency in both genders
across over 100 generations (Mackay et al., 2017).This indicates the presence
of Mendelian inheritance in the wing types of fruit flies, signifying that the
transmission of genes follows a predictable pattern, with one allele copy
inherited from each parent.Thus, the dominant allele being wingless in this
case would overrule the recessive one, hence a greater prominence of the
wingless phenotype (Wertheim et al., 2023).
In this lab, we will be taking these experiments into
consideration by investigating the inheritance patterns in Drosophila
melanogaster fruit flies based on eye color and wing shape.
2.
Research Question
What are the
inheritance patterns for sepia eyes and apterous wings in Drosophila
melanogaster fruit flies?
3.
Hypotheses
Null Hypothesis
(H0):There is an
absence of a statistically significant difference anticipated in the number of
individuals displaying apterous and sepia phenotypes compared to the expected
quantities during both generations (F1 and F2) in Drosophila melanogaster. This
suggests that the inheritance pattern for the apterous and sepia mutations is
autosomal recessive.
Alternate
Hypothesis (H1):A
statistically significant distinction is anticipated in the number of
individuals exhibiting apterous and sepia phenotypes compared to the expected
quantities during both generations (F1 and F2) in Drosophila melanogaster. This
suggests a sex-linked inheritance pattern for sepia and/or apterous traits.
Justification For
Hypothesis: Our
hypothesis is testing the inheritance pattern of sepia and apterous mutations
based on the actual number of flies expressing the phenotypes for sepia and
apterous mutations versus the number of flies that we expected over the course
of multiple generations. If the observed results that we find do not have a
statistically significant difference to the ratios predicted in the autosomal
recessive pattern, we will accept the null hypothesis meaning that both
mutations use the autosomal recessive pattern of inheritance. If there is a
statistically significant difference in the ratios for the autosomal recessive
pattern, we can reject the null hypothesis meaning that one or both mutations
would likely be sex-linked. By using our hypotheses, we will be able to
accurately determine the mode of inheritance for sepia eyes and apterous wings
based on how significant the differences between our observed and expected
results are.
4.
Variables
Independent
Variable: Mutation
Dependent
Variable:
Inheritance pattern
5.
Experimental Design
In this lab, we
investigated the patterns of inheritance for the Drosophila melanogaster fruit
flies containing the mutations, sepia eyes and apterous wings. Doing so will
allow us to deeply understand the impacts of alterations in one’s genetic
compositions, therefore affecting the characteristics of future generations in
comparison to the species’ wild type organisms (Chen et al., 2018). Students
will understand the importance of Drosophila melanogaster being used in the
experiment to be used as the “model organism” and predicting genotypes through
Punnett Squares, a mechanism used to illustrate the genotypes of a breeding or
crossing experiment (Reue et al., 2018). To outline the laboratory procedure,
we will engage in an extended experiment spanning several weeks. Throughout
this period, we will systematically cross male and female flies with different mutations,
concurrently tallying the exact number of each gender and scrutinizing their
physical phenotypic characteristics, including eye color and wing shape. (Singh
et al, 2023). To sex the individuals, we observed not only the genitalia of
both sexes, where adult males hold dark, multifaceted components on the lower
abdomen including the lateral plate, genital arch, and penis, and females hold
lightly colored, protruding genitalia with few visible features, but also the
presence or absence of sex combs (Chen &Reue, 2019). Males obtain combs
located on the front legs utilized to grasp onto the female when looking to
reproduce. Understanding these differences were crucial to the isolation of
each sex, later leading to the production of future generations.
In addition, before
going into depth on how we plan on accomplishing our goal of understanding
inheritance patterns of Drosophila melanogaster flies, obtaining knowledge on
the phenotypes of the parental flies will aid with reader visuals. The Wild
Type fly obtains bright red colored eyes with short antennae and typical, round
wings while the Recessive Autosomal Sepia fly obtains large, brown (sepia)
colored eyes and apterous wings, meaning no wings (Chen at al., 2018). Also,
the Recessive Sex-Linked Flies obtain large, white eyes. Thus, this leads us to
our experimental plan. Initially, we will acquire the parent flies,
representing the first generation, from both the wild-type (A) and sepia-type
(B) stock containers. Allowing them to reproduce for approximately one week or
more, we will then proceed to cross the male flies of the parent generations
with new virgin females exhibiting different mutations of our selection (e.g.,
a cross between A and B flies as illustrated in Table 5), resulting in the production
of our F1 generation (Green et al., 2018). Finally, we will cross the F1 males
with new virgin females to generate the F2 generation. Through this systematic
approach, we aim to unravel the inheritance patterns of traits, identifying
which traits are passed on to subsequent generations and gaining insights into
predicting phenotypes using Punnett Squares.
Materials
• FlyNap Anesthetic
• FlyNap Applicator (Wand)
• Stereo Microscope
• Four relatively wide vials with foam caps
• Yeast
• Carolina’s Formula 4-24® Instant Drosophila
Medium
• Deionized Water
• Small Tipped Brush
• Two samples of vestigial Drosophila
melanogaster with unknown mutations
• Notecards or Blank Paper
• Timer (phone or watch)
• Optional/Recommended: Note Taking Device
• Phone or Camera
6.
Procedure
Part 1 – Making
the P Vials
1. Acquire 2 pristine
vials.
2. Place one portion
of media into each vial designated for use.
3. Pour about 17 mL
of water into each vial.
4. Sprinkle between 2
and 4 yeast grains onto the media surface within each vial.
5. Obtain a specified
FlyNap vial and one vial housing Drosophila melanogaster.
6. Invert the D.
Melanogaster container directly over the FlyNap vial, aligning and joining
their openings.
7. Tap the original
vial gently to allow some flies to descend without spilling any media.
8. Recap the
respective vials securely.
9. Dip the wand into
the FlyNap container (not the vial) and position it alongside the foam cap and
vial edge, ensuring it rests just below the cap for the flies to access the
FlyNap.
10. Start a timer for
50 seconds and monitor the fly movement during FlyNap anesthesia. Avoid leaving
them in the vial beyond 50 seconds to prevent increased mortality or prolonged
sedation.
11. Open the FlyNap
vial and transfer the flies onto an index card for observation beneath a stereo
microscope.
12. Assess fly gender
by examining abdomen color, size, and the presence of sex characteristics.
13. Segregate five
males and five females, moving them to the vial with prepared medium, returning
the rest to their original stock vial.
14. Repeat steps 5
through 13 for each desired Drosophila melanogaster sample.
15. Label the vials
with the stock vial's designated letter, the date, and the quantity of flies
(male and female) contained within.
Part 2 – Making
the F1 Vials:
1. Check the P vials
daily to observe any larvae development.
2. Upon reaching a
substantial quantity of visible larvae, remove the parent flies and proceed
with euthanization:
a. Transfer the parent flies into a
dedicated FlyNap vial.
b. Implement the FlyNap method as previously
demonstrated, allowing 60-90 seconds for effective euthanization.
c. Place the deceased flies into the
designated alcohol morgue.
3. Obtain two fresh,
uncontaminated vials and repeat steps 2 through 4 from Part 1.
4. Regularly monitor
the parent generation flies and eliminate any newly emerged individuals.
5. Apply FlyNap to
the emerging flies and subsequently ascertain their genders.
6. House the emerging
flies in an F1 vial until obtaining five virgin females and five males.
7. Label the vials
indicating their respective stock origin, the date, and the quantity of flies
(male and female) contained within.
Part 3 – Making
the F2 Vials:
1. Monitor the F1
vials daily for developments.
2. Upon the emergence
of a significant number of visible larvae, remove the P flies and carry out
euthanization by repeating steps 2a to 2c.
3. Await the
emergence of the larvae.
4. Apply FlyNap to
the new F1 flies and utilize a stereo microscope to determine their genders and
phenotype based on their traits.
5. Place
approximately 5 male and 5 female flies from the same vial into a new vial.
6. Replicate these
steps for each individual vial.
Part 4 –
Collecting Data from F2 Flies
1. Regularly observe
the F2 vials for any developments.
2. Once a noticeable
quantity of visible larvae is present, remove the F1 flies and carry out
euthanization by repeating steps 2a to 2c.
3. Await the
emergence of the larvae.
4. Apply FlyNap to
the new F2 flies and employ a stereo microscope to ascertain their genders and
phenotype based on their traits.
5. Euthanize the
remaining F2 flies and deposit them in the designated fly morgue.
7.
Data
A - Winged, Sepia
B - Winged,
Sex-Linked White Eyes
C - Apterous Wings,
Wild Type Eyes
Table 1
Number
of parental Drosophila melanogaster in F1 vial (AB cross)
Table 2
Number
of parentalDrosophilamelanogaster in F1 vial (BC cross)
Table 3
Number
of parentalDrosophila melanogaster in F1 vial (AB x AB cross)
Table 4
Number
of parental Drosophila melanogaster in F1 vial (BC x BC cross)
Table 5
Number
of F1 Drosophila melanogaster after the A and B cross.
Graph
1

Number of individuals with
different trait types in A Female x B Male population.
Table 6
Number
of F1 Drosophila melanogaster after the B and C cross
Graph
2

Number of individuals with
different trait types in B Female x C Male population.
Table 7

Number of F2 Drosophila
melanogaster after the AB x AB cross
Graph
3

Number of individuals with
different trait types in AB Female x AB Male population.
Table 8
Number
of F2 Drosophila melanogaster after the BC and BC cross
Graph
4

Number of individuals with
different trait types in BC Female x BC Male population.
8.
Analysis of Results
We studied the
mutations of sepia eyes and apterous wings in Drosophila melanogaster.
Punnett Squares:
Winged - W
Apterous wings - w
Wild Type eyes - E
Sepia eyes - e
Cross Figure 1
P1
Cross - (A) WWee x (B) WWEE
Expected Genotypic Ratio - 100% WWEe
Expected Phenotypic Ratio - 100% Winged and Wild-Type Eyes
Cross
Figure 2
F1
Cross - (AB) WWEe x (AB) WWEe
Expected Genotypic Ratio - 8 WWEe : 4 WWEE, 4 WWee
Expected Phenotypic Ratio – 12 Winged and Wild-Type Eyes :
4 Winged and Sepia Eyes
Cross Figure 3
P1
Cross - (B) WWEE x (C) wwEE
Expected Genotypic Ratio - 100% WwEE
Expected Phenotypic Ratio - 100% Winged and Wild-type eyes.
Cross
Figure 4
F1
Cross - (BC) WwEE x (BC) WwEE
Expected Genotypic Ratio - 8 WwEE : 4 WWEE : 4 wwEE
Expected Phenotypic Ratio – 12 Winged and Wild-type eyes :
4 Apterous Wings and Wild-type eyes
Our observed results do not
substantially deviate from our expected values and appear to align with the
autosomal recessive pattern of inheritance since they did not have a
statistically significant difference from our predictions with the Punnett
Squares. This means that we cannot reject our null hypothesis and that both the
apterous and sepia mutations have an autosomal recessive pattern.
Furthermore, the
deviations for the phenotypic ratios of the F2 generations are not
statistically significant and very well could have occurred by chance. After
completing a Chi-Square Test, we can conclude that on a statistical level our
observed results for the wing and eye phenotypes align with our expected
results from the Punnett square because the chi-square values did not exceed
the 0.05 critical value. As a result, it is likely that the differences between
the expected and observed values resulted from random chance. Our genetic
conclusion from our testing is that the sepia and apterous wings are inherited
through an autosomal recessive pattern since we are failing to reject the null
hypothesis based on our chi-square value.
9.
Chi-Square Test
Table 9
Chi-square
results of wing phenotype in Winged Male/Female flies and Apterous Male/Female
flies.
In Table 9, we fail
to reject the null hypothesis. Our p-value is 7.815 and the p-value is not less
than or equal to our significance level which we found as 6.157. This indicates
that there is no notable statistical distinction between the anticipated number
of flies in each phenotype and the observed count. This lack of significance
could be attributed to the limited number of observed flies or a relatively
even distribution among the observed phenotypes. Nevertheless, based on this,
we uphold our null hypothesis asserting that there will be no statistically
significant difference in the quantity of individuals expressing apterous and
sepia phenotypes (focusing on wings in this table) as shown in Graph 1, Graph
2, Graph 3, and Graph 4, and the expected amounts over the course of two
generations (F1 and F2) in Drosophila melanogaster meaning the inheritance
pattern is autosomal recessive for the apterous and sepia mutations to be true
(“An Introduction to Fruit Flies”, n.d.).
Table 10
Chi-square
results of eye phenotype in Wild-Type Male/Female flies and Sepia Male/Female
flies.
In Table 10, our null hypothesis stands unchallenged. The p-value,
calculated as 7.815, is far from reaching or surpassing our established
significance level of 1.547. This outcome signifies that there is no
substantial difference between the expected and observed counts of flies in
each phenotype. The pronounced disparity in numbers and the considerably higher
p-value compared to our significance level suggest a lack of statistical
significance. If represented graphically, there would be extensive overlap in
the error bars, underscoring the absence of a statistically significant
difference. This may be attributed to the limited number of observed flies or
an overall even distribution among observed phenotypes. Despite these
considerations, we maintain our null hypothesis, affirming that there is no
statistically significant difference in the quantity of individuals expressing
apterous and sepia phenotypes (with a focus on eyes in this table) compared to
the expected counts over two generations (F1 and F2) in Drosophila
melanogaster. This reinforces the belief that the inheritance pattern for the
apterous and sepia mutations is autosomal recessive.
Conclusion
From our experiment
and the chi-square analysis, we have concluded that we have failed to reject
the null hypothesis. In our Punnett squares, which are Table 1, Table 2, Table
3, Table 4, Cross Figure 1, Cross Figure 2, Cross Figure 3, and Cross Figure 4,
we looked at each of the mutations as if they were inherited as autosomal
recessive and compared this to the results that we found during our experiment
after crossing the Drosophila melanogaster flies over multiple generations. We
also looked at Table 6, Table 7, and Table 8 in order to determine the number
of melanogaster flies after the multiple crosses allowing us to do a
comparison.
Our chi-square values
for both the apterous wing and sepia eye mutations did not exceed the critical
value meaning that there was not a statistically significant difference between
our observed and expected results that we predicted using the Punnett Squares.
This means that they are similar enough for us to consider it a match based on
the data that we have collected. As a result, we are unable to reject the null
hypothesis ultimately meaning that the pattern of inheritance for both examined
mutations are autosomal recessive.
Leveraging our
existing understanding of genetic concepts, we successfully identified various
aspects in this laboratory experiment. Distinguishing between autosomal
inheritance and sex-linked inheritance was straightforward, thanks to our prior
knowledge and the analysis of the collected data. While we approached the
results with confidence, we noted a significant enhancement in our
comprehension of genetic inheritance and mutations through this lab.
Conducting the
experiment in person, utilizing live D. melanogaster flies over an extended
duration, provided valuable insights into how diverse phenotypes are
transmitted from parent to offspring. This hands-on approach also afforded us
an understanding of the life cycle of flies, spanning from the larval stage to
pupa and adulthood, enriching our knowledge through a first-person perspective.
Looking ahead, if our
interest in D. melanogaster flies persists, we aspire to explore crossbreeding
with other mutations beyond those selected in our current experiment (Green et
al., 2018). By scrutinizing the frequency of various phenotypes such as no
wings, small eyes, and crooked wings, we aim to ascertain potential
associations between these traits and identify any linked genes based on their
relative prevalence (Morgan, 2018).
Although we
successfully completed this lab with confidence in our results, there were still
some limitations during the lab that affected our ability to get clear data at
some points. For example, due to a smaller sample size, the data we gained
might not be as accurate since the flies that we select might not be a good
representation of the entire population. However, with this experiment being
controlled, the data could be skewed as compared to what could occur if there
is no control on the behavior of the D. melanogaster. Additionally, because
female flies remain virgin for only 8 hours, this creates great difficulty
especially when trying to encourage further reproduction of the fly. Obtaining
virgin females flies means that we can further understand our goal of
recognizing biological patterns and crossing them means that we can further understand
inheritance patterns, however, if students are not able to capture the virgins
within the necessary time frame, they will not be able to succeed. Ultimately,
the conduction of this experiment allowed us to gain a deeper understanding of
inheritance patterns and how our knowledge of genetic crosses can be applied in
such studies.
In this research, we
embarked on a comprehensive investigation into the inheritance patterns of
mutations in Drosophila melanogaster, specifically focusing on apterous wing
and sepia eye mutations. Through the utilization of Punnett squares and
chi-square analysis, we rigorously examined the inheritance dynamics over
multiple generations. The key novelty of our study lies in the meticulous
exploration of autosomal recessive inheritance for these mutations,
substantiated by statistical analyses that failed to reject the null
hypothesis. Significantly, our hands-on approach, involving the observation of
real D. melanogaster flies throughout their life cycle, provided a unique perspective
and enriched our comprehension of genetic inheritance (Bhalla et al., 2019).
While our prior knowledge facilitated the differentiation between autosomal and
sex-linked inheritance, this research acted as a transformative experience,
augmenting our understanding of intricate genetic mechanisms. Looking forward,
our proposal to extend this research to explore the cross between different
mutations unveils the potential for uncovering linked genes and furthering the
understanding of phenotypic prevalence. Despite the limitations posed by a
smaller sample size and the temporal constraints in capturing virgin female
flies, our controlled experiment stands as a pioneering effort, shedding light
on the complexities of inheritance patterns and the application of genetic
crosses in empirical studies.
Acknowledgement
I am deeply grateful for the
invaluable guidance and support provided by Dr. Ginny Berkemeier, my esteemed
research advisor, throughout the course of this research endeavor. Her
unwavering commitment to excellence, wealth of knowledge, and insightful
feedback have been instrumental in shaping the trajectory of our project. Dr.
Berkemeier's mentorship has been a beacon of inspiration, steering us through
the complexities of our research experiment. Additionally, I extend my sincere
appreciation for her generosity in providing us with the necessary materials
and resources, which proved crucial in the successful completion of our
research. This acknowledgment is a testament to Dr. Berkemeier's profound
impact on both the academic and personal dimensions of this research journey,
and I am truly fortunate to have had the privilege of working under her
guidance.