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Vanshika Singh (2023), A Study on Genetic Inheritance of Mutations in Drosophila Melanogaster, Spectrum of Emerging Sciences, 3(02) 2023, pp 37-46, 10.55878/SES2023-3-2-6

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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

Chart, bar chart, waterfall chart

Description automatically generated

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

Chart, waterfall chart

Description automatically generated

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

Chart, bar chart

Description automatically generated

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

Chart, bar chart, waterfall chart

Description automatically generated

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.


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