Determining Inheritance of Sepia Eye Color and Vestigial Wing Traits in Drosophila Melanogaster

The Drosophila melanogaster, commonly referred to as the fruit fly, is an ideal model organism for use in the study of inheritance patterns of particular traits. Its suitability for these studies is due to its various peculiar characteristics. Fruit flies are quite small and have a shortened life span that allows them to increasingly mate and produce large numbers of offspring at a time. Their appendages, which allow for mutation without being lethal, are ideal for putting isolation mutation markers in studies seeking to comprehend their traits (Beckingham et al., 2005). Their growth mechanism is unique in that it allows them to bypass cytokinesis. Yet, they still contain multiple chromosomes within their cells that present a vast array of chances to detect mutations.

According to Cooper (2000), traits that are passed down from the parent to the offspring are referred to as hereditary traits. For this study, diploid flies with two genome sets were used. Offspring inherit alleles, or simply put, two copies of the same gene. The alleles code for different phenotypes which allows for genetic variations within a particular population ( Griffiths et al., 2000) . All alleles are inheritable, but their physical expression in an organism is dependent on the type of inheritance pattern. When an organism inherits dominant alleles, the alleles become physically expressed. However, recessive traits become suppressed and do not show physically. The only time a recessive trait is expressed physically in an offspring is when it is inherited from both parents. For dominant traits, only a single allele, from either parent, is needed for it to be physically expressed in the offspring. A cross involving the dominant and the recessive (heterozygous) traits will result in a phenotypic ratio of 3:1. In other words, three dominant traits and one recessive trait.

There are numerous inheritance patterns that modify how traits are expressed. Incomplete dominance results in partial expression of the dominant trait together with the recessive trait giving rise to a third new offspring. Codominance results in the distinctive appearance of both traits, dominant and recessive, in the offspring. On the other hand, complete dominance results in suppression of the recessive phenotype by the dominant trait. For example, complete dominance is exemplified when a fly with a wild type eye color is crossed with sepia eye color. All offspring will be wild type indicating suppression of the recessive sepia gene by the dominant wild type gene (Simon et al., 2016). The same inheritance pattern is exhibited in vestigial wings that have recessive genes and wild type wings which contain dominant genes.

Sepia eye color (ss) was selected as the first trait. Sepia eye color trait is a mutated trait found on the third chromosome of fruit flies. It is a recessive trait, meaning it will only be noticeable in an offspring if both parents possess the gene. Sepia is described as the brown-purple pigment, which is caused by the visual pigmentation referred to as ommochrome. The sepia color is illustrated in Figure. 1. When a wild type mates with a sepia the offspring produced is a wild type, indicating that the trait is not sex-linked, it is only autosomal recessive. A study conducted at Indiana University confirmed this inheritance pattern through crossing red-eyed flies and brown-eyed flies. Crossing a wild type and brown eyes produced wild type flies regardless of the trait possessed by the male and the female (Indiana University, 2020). The study conducted at Indiana University paralleled our study and affirmed that the inheritance pattern is recessive and not sex-linked. According to Kim et al. (2016), in a research done on the specifics of sepia eye color gene, the gene is defective for PDA synthase, which plays a crucial role in flies' biosynthesis. Their study found out that the PDA synthase enzyme occurs in limited amounts in flies of other colors but was completely absent in sepia flies. Possessing this defect strained the flies' metabolism. Thus, this defect is regarded as one of the disadvantages of the sepia eye color trait. According to Connolly et al. (1969), the inability of sepia flies to mate in darkness is the other disadvantage of the mutant eye color. Their inability to mate is due to poor adaptation of their vision to these conditions.

Figure 1 . Sepia eye color female

The wild type trait for eye color is expressed in red color, as illustrated in Figure 2. The red color is due to the drosopterin compound (w+w+). This trait is classified as dominant because it is commonly expressed in heterozygous crosses. Compared to the sepia trait, the normal wild type eye color gene is unmutated on chromosome three. The gene is dominant; hence both parents do not have to possess the wild type gene for the offspring to inherit it. Besides, it is not sex-linked since either the mother or the father can be the wild type for the offspring to inherit the trait to express the red eyes. Possessing the wild type trait for the eye color is advantageous to Drosophila melanogaster because they are regarded to be more fit than the mutants (Connolly et al., 1969). Flies with red-eye color were found to have normally functioning biosynthesis, unlike those with mutant eye colors. The wild type flies are also superior in their mating abilities due to their capability to mate in the dark as they can see in dark environments. The wild type flies have the overall advantage of possessing greater fitness in survival and mating patterns. Consequently, they tend to outnumber the mutant flies.

Figure 2 . Wild type eye color male

Vestigial wings were (vv) were considered as the second trait that would be investigated in this study. Vestigial wings are a mutated trait found on fruit flies' second chromosome (Williams et al., 1991). The chromosome possesses a deletion of intro 2 that functions to control flight appendages and wing disc formation in wild type flies. The gene is only present during the larval stage. Hence, if fruit flies have this mutation, their full wings do not develop since the gene affects the flies embryologically, causing the cells to die. Deletion mutations are regarded as the most severe since they are quite disadvantageous to the organisms. The fruit flies' wings become small and deformed, thus making them be less mobile and have weaker flying ability. The small and deformed wings phenomenon is illustrated in Figure 3 . On the other hand, the wildtype wing trait flies have larger wings that allow them to fly fast and more adequately. The larger wings are illustrated in Figure 4 . Another difference between vestigial and wild type flies is exhibited by the ability of vestigial flies to store more glycogen in their bodies than the wild type flies. The higher glycogen storage capability in vestigial flies has been described as a compensatory mechanism for the short and deformed wings that inhibits their flying. A study found out that vestigial flies use less glycogen when flying. In the study, the seven-day-old vestigial flies had stored about 10 micrograms more glycogen than the wild type flies (Barnes, 1994). This lab study was conducted with an aim to comprehend the various patterns of inheritance through visualizing key traits of the Drosophila melanogaster .

Figure 3 . Vestigial winged female

Figure 4 . Wildtype winged male

METHODS

Handling Methods and Materials — On the first day, we were given tubes of sepia eye color flies, vestigial eye color flies, and wild type flies. To make tubes for the crosses, we mixed yeast and nutrients with water to make the base for flies to eat and lay eggs in. For every tube made, we first labeled which traits were going to be crossed so there would be no confusion later. Finally, we covered the tubes with a sponge so the flies couldn't escape. Each day we began by initially observing the flies and noting down any changes (larvae, dead flies). In order to microscopically observe the flies, the flies must be first put to sleep via FlyNap®. FlyNap® is a chemical substance used to temporarily anesthetize the flies so that they don't fly away and disrupt the results. We dipped a small brush in FlyNap® and put it in the tube but also kept the sponge on. We then waited five minutes until all the flies were asleep. To observe the traits and sex of the flies, we used a dissection microscope. After recording all the flies, we killed the adult flies by putting them in a soap-water solution but kept the larvae. We killed the adult flies after the first and second crosses. We made sure we recorded all the information as we progressed through the lab to ensure that we don't leave out any essential points. After the entire lab was completed, we disposed of all the flies, removed the labels, and cleaned out all the tubes with soap water.

Sex Determination — When sexing a fly, certain characteristics signify male versus female. Males predominantly have sex combs on their tarsus of the leg and a darkly pigmented tail. Females have striped tails (Ferris and Poulson, 1950). When separating our flies before and after crosses, we used these methods to distinguish male versus female to keep track of how prevalent the traits were in each gender.

Monohybrid Crosses — For this cross, we separated all the males and females from the tubes given by Dr. Gustafson. Then, we grouped them into wildtype and sepia eyes, and then into male and female within the trait groups. In tube 3, three male wildtypes and three female sepias were put into the tube. In tube 4, two sepia female and two male wildtypes were put into the tube. We initially hypothesized that sepia would be a sex-linked dominant trait. Therefore, we expected sepia offspring from this cross.

For the next cross, we separated them the same way as the first. Our group took the offspring from tube 3 and selected five wildtype males and five wildtype females and put them into a new tube labeled 5. Since the results from generation F1 did not match our initial hypothesis, we changed it to predict that the sepia trait was autosomal recessive, and the wildtype was dominant.

Dihybrid Crosses — For this cross, we repeated the same initial protocol from the monohybrid cross. For the first cross, we categorized them into wildtype and sepia eyes, vestigial and wildtype wings, and then into male and female. Tubes 1 and 2 had three sepia males and five vestigial females. After seeing the results from the monohybrid cross, we hypothesized that these traits were also autosomal recessive and that the wildtype trait was dominant.

For the second cross, we separated them the same way as the first. Our group took the offspring from tube 1 and selected five female wildtypes for sepia and vestigial (red eyes, normal wings) and five male wildtypes for sepia and vestigial. These ten flies were put into tube 6. Two more tubes were made, but the flies came from tube 2 and were put into tubes 7 and 8. We kept our original hypothesis as the two traits were autosomal recessive in relation to the wildtype.

Statistical Analyses — To analyze our data, we used Chi-Squared calculations to test how likely our observed values were due to chance. Chi-Squared is used to determine if the data is comparable to the "null" data of 9:3:3:1 (Ling, 2008). For the monohybrid cross, our null hypothesis was that the cross would follow the 3:1 phenotypic ratio, which supported our hypothesis that sepia was a recessive trait. A monohybrid cross for vestigial wings was not performed as we did not have enough flies with a vestigial trait. For the dihybrid cross, our null hypothesis was that the cross would follow the 9:3:3:1 pattern, which supported our hypothesis that vestigial was a recessive trait. We started by listing our observed crosses (s+v+, s+v, sv+, sv) of each type along with the expected ratio (9:3:3:1). After calculating the ratios and the degrees of freedom, we determined the p-value to be greater than 0.2, which supported the null as it was much greater than 0.05. The p-value is an indicator to show if the results are extreme, which would help support or reject the null hypothesis. The ratio also came out very closely to 9:3:3:1, therefore, supporting the null hypothesis.

RESULTS

For the F0 generation (ex. first cross), it yielded all wild types for both the monohybrid and dihybrid. In the initial tubes, our group crossed each trait and then determined the monohybrid and dihybrids. The yields of the monohybrid and dihybrid in this initial cross are summarized in Table 1. For the monohybrid cross, we chose the sepia trait. All of the crosses led to the production of only wild type offspring- for both sepia eyes (ss) and vestigial wings (vv).

The F1 generation (ex. second cross) yielded all wild type offspring. The results for the monohybrid and dihybrid cross are summarized in Table 2 . All offspring produced were heterozygous.

The F2 generation (chi-squared) for the dihybrid and monohybrid crosses indicated how our observed versus expected ratios compared as summarized in Tables 3 and 4, respectively. The statistical results for the dihybrid are as follows: Χ2 = 0.2181, df = 3, p > 0.2 . The statistical results for the monohybrid are as follows: Χ2 = 0.0036, df = 1, p > 0.2. These values, both for the dihybrid and monohybrid crosses, give p-values that support the null hypothesis.

Table 1 . Initial crosses with F0 generation for each trait, including wild types. (F= female, M= male, Monohybrid tubes- 1&2, Dihybrid tubes- 3&4)

Tubes	Initial Crosses
1	5F (vv), 3M (ss)
2 3	5F (vv), 3M (ss) 3F (ss), 3M (S+S+)
4	2F (ss), 2M (S+S+)

Table 2 . Second cross with F1 generation (Monohybrid tube-5, Dihybrid tubes 6-8)

Tubes	2nd Crosses
5	5F (S+s), 5M (S+s)
6	5F (S+v+), 5M (S+v+)
7 8	5F (S+v+), 5M (S+v+) 5F (S+v+), 5M (S+v+)

Table 3 . F2 generation chi-squared results from the dihybrid cross. The results show that both traits are recessive, not sex-linked and that the total is very similar to the expected ratio ( Χ2 = 0.2181, df = 3, p > 0.2 )

Phenotype	Expected ratio		Observed Tubes		Total
(S+v+)	9	17F,14M	10F,23M			17F,18M 99
(sv+) (S+v)	3 3	10F,12M 8F,4M	2F,1M 8F,5M		3F,5M 33 8F,3M 36
(sv)	1	3F,0M	2F,0M		5F,1M 11

Table 4 . F2 generation for the monohybrid cross. The results show that both traits are recessive, not sex-linked and that the total is very similar to the expected ratio ( Χ2 = 0.0036, df = 1, p > 0.2 )

Phenotype	Expected ratio	Observed
(ss)	1	23
(S+S+)	3	68

DISCUSSION

Both the monohybrid and the dihybrid crosses supported the null hypothesis. The null was set to observed = expected in order to test the hypothesis. In the monohybrid cross, the phenotypic ratio was 3:1, for the wildtype to sepia traits. In the dihybrid cross, the phenotypic ratio was 9:3:3:1, for all the traits. The results of the F2 monohybrid cross that were obtained were very close to the expected 3:1 ratio phenotypic ratio, as indicated in Table 4. Similarly, the results of the dihybrid cross were quite close to the expected 9:3:3:1 ratio, as shown in Table 3. The chi-squared test values were found to be x 2 = 0.0036 and 1 degree of freedom in the monohybrid and x 2 = 0.2181 and 3 degrees of freedom in the dihybrid. These values were compared to the extremes on the p-value chart, and it was quite distinct that our results supported the null hypothesis. The results validated our hypothesis. It was clear that both traits, sepia eye colors, and vestigial wing type are autosomal recessive. Working backward from these results, it can be assumed that the parents of the initial monohybrid cross had produced all heterozygotes, which became the parents for the second cross for sepia. The second cross produced textbook results for the 3:1 phenotypic ratio of inheritance, proving that the parents were heterozygous. In the dihybrid cross, the parents had one recessive vestigial trait and one wildtype (S+S+vv and ssV+V+) making their offspring be (S+sV+v), thus giving the ratio 9:3:3:1

A look at the phenotypes of the fruit flies and comparing them to the gender of the flies leads one to conclude that the traits are not sex-linked. It did not matter who had the recessive or dominant allele, the male or the female; all traits were autosomal. The results of this study closely paralleled the background literature that was found. An almost similar study conducted at Indiana University found out that both traits were autosomal recessive and that and not sex-linked. To investigate the patterns of the traits, they undertook the study using two combinations of the traits (Indiana University, 2020). The first combination involved a wildtype male and a mutant female. The second involved a mutant male and a wildtype female.

The main aim of conducting this study was to understand how the pattern of inheritance can be applied by visualizing common traits of the fruit fly. The first-hand experience of conducting a study involving crosses, using flies, has provided us with a better understanding of how to design an experiment and effectively implement it. We recommend that in future labs, more flies should be made available right from the initial crosses. This is because we lacked some vestigial mutants, and therefore, we were unable to do a monohybrid cross involving them. Although this absence did not impact our results, it would have been plausible to see how this cross would play out. Furthermore, the cross would have provided more data for the study. The PDA synthase enzyme is one of the research areas that I would like to explore in future experiments. I would like to learn more about whether it is defective or functioning in various mutant flies, such as those with the vestigial wings (Kim et al., 2006). It would also be engrossing to carry out a study involving only the variations in eye color. The study would track the fruit flies' mating patterns in the darkness since the wild types have the best vision in complete darkness when compared to the other flies. Through this study, we could determine if it is all or none of the flies, or if different eye colors have various level of sight in the dark (Connolly et al., 1969).

CONTRIBUTIONS

Our group, #4, was composed of five members, namely, Ashley McCleary, Sophia Day, Garrett Dunn, and Yesenia Eichholz, and I. As a group, we worked very well. Everyone handled the task assigned to him or her very well as well as keep the rest of the group well informed. Sophia was responsible for ensuring the group kept time. She was also responsible for recording observations or any information collected in the study. The task of observing the flies under a microscope was assigned to Ashley an I. Apart from observing the flies, were also required to count them and group them for our crosses. Garrett and Yessina were responsible for categorizing the flies and grouping them. Yesenia also helped Sophia to document our findings or the information we collected. The entire group prepared the tubes to be used in the experiment. Each tube was labeled correctly for ease of reference. We also took photos of our laboratory experiment and shared among the group members. It was a big group effort at the start with the FlyNap®, as we needed two people to be assigned to each tube to ensure the flies did not escape. Throughout the experiment, the group collaborated very well. In addition, we had a great group dynamic. We have the same dynamic going into the presentation as well.

References

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