Drosophila Melanogaster As a Research Specimen

AP Biology Lab

Drosophila melanogaster as a Research Specimen

The animal most widely used for genetic studies is Drosophila melanogaster (the common fruit fly). The fly is easily cultured, and its generation time is only two weeks at 21oC. One female may lay as many as 500 eggs in 10 days. Because Drosophila is small, cultures occupy little space; however, the fly is large enough that mutations can be observed. Drosophila only has 4 pairs of chromosomes. This may not sound like much, but as many as 10,000 genes can fit on one chromosome. By studying inheritance patterns in Drosophila, scientists can learn a lot about the way our genes work also.

When two flies are mated, they are called the Parent generation (P1). Their offspring are referred to as the F1 generation. If the F1 generation (siblings) are crossed, their offspring are referred to as the F2 generation.

There are four distinct stages in the life of the fruit fly: egg, larva, pupa and adult. At 21oC, a fresh culture of flies will produce new adults within 2 weeks; 8 days in the egg and larval stage and 6 days in the pupal stage (see diagram below). Adult flies may live for several weeks.

Fruit Fly Life Cycle

The following research project will be used to help students learn about inheritance patterns in Drosophila. Students will observe the inherited characteristics in offspring of known parental stock for three crosses. Inheritance of a sex linked trait will be compared with inheritance of an autosomal trait. A dihybrid cross will be studied as well. Students will predict the outcome of the crosses using Punnett Squares. Observed data will be compared with the predicted values using statistical analysis.

Drosophila Lab Day 1: Determining the Sex of your Flies

Females have pointed posterior ends with thin black stripes (left).

Males have blunt posterior ends with a thick, black stripe (right).

Procedure

1. The flies have already been anesthetized for you today. Remove 5 flies from vial #1. These are wildtype (normal) flies, with no mutations. We use the symbolwt to abbreviate the phenotype of these flies.

1. Observe your flies under low power of your microscope. Use a paint brush to move them around. Record the eye color, wing shape, and abbreviation in the data table. Remember, wildtype flies are completely normal-they have no mutations. The mutations in the other flies will either be in wing shape or eye color, and each fly will have only one mutation. Also record how many male and female flies you have.

1. Remove 5 flies from vial #2. Follow the procedure from steps 1 & 2. We use the symbol se to abbreviate the phenotype of these flies.

1. Remove 5 flies from vial #3. Follow the procedure from steps 1 & 2. We use the symbol vg to abbreviate the phenotype of these flies.

1. Remove 5 flies from vial #4. Follow the procedure from steps 1 & 2. We use the symbol w to abbreviate the phenotype of these flies.

1. When you are finished, place the flies back in the vial and wash your Petri dishes out.

Data Table

Questions

1. What traits do wildtype (Wt) flies have?

1. Name 2 mutations you found in the other flies.

1. How can you tell the difference between a male and a female fly?

1. How many females did you count in total? _____ How many males? _____

Is this the ratio you should realistically come up with? Explain.

1. Why are fruit flies good candidates for genetic studies?

1. What are the 4 stages in a fruit fly life cycle?

1. How long does it take Drosophila to go from egg to adult?

1. What is the purpose of placing a sponge stopper in the top of the vials instead of a plastic cap?

Drosophila Lab Day 2: Dominant and Recessive Traits

Prelab Exercise

The parent flies (P1) were crossed two weeks ago. The flies you will be observing today will be their offspring (the F1 generation). The original parent crossesare shown below. Underneath each abbreviation, color and fill in the eye color and wing shape for the phenotype indicated. Also, adjust the striping for the two sexes.

Cross number 1

Sepia eye (se) femaleXWildtype (Wt) male

Cross number 2

White eye (w) femaleXWildtype (wt) male

Cross number 3

Sepia eye (se)-long wing (wt)female X Red eye (wt)-vestigial wing (vg) male

Procedure

1. Remove 8 offspring from the first vial.
2. Record eye color and wing shape for all the males and females of each cross. Record your information. Make sure you have approximately 50% males and 50% females.
3. Place the flies into a new vial and label the vial with the parent cross (se X wt), today’s date, the period, and your group number. Also label them “F1 Cross.”
4. These flies have either already mated or will begin mating right away. The females should start laying eggs by tomorrow.
5. We will release these F1 adult offspring in 1 week. Why?
6. Repeat this procedure for each of the other two crosses (w X wt & se X vg).

Data Table Cross 1 (se X wt)

Data Table Cross 2 (w X wt)

Data Table Cross 3 (se X vg)

Questions

1. In cross #1, which eye color appears to be dominant? Is “sepia” an autosomal or sex-linked trait? How can you tell?

1. How do the results of cross #2 differ from the results of cross #1? Is “white” an autosomal or sex-linked trait? How can you tell?

1. The phenotype symbols for eye color are wt, wand se. Now that you know the color that is dominant, what letters should we use to represent the alleles for eye color? What is different about the notation for the white eye gene?

1. Are vestigial wings dominant or recessive? How can you tell? Is this an autosomal or sex-linked trait? How can you tell?

1. The phenotype symbols for wing shape are wt and vg. Now that you know the wing shape that is dominant, what letters should we use to represent the alleles for wing shape?

1. How long should it be until we can observe our F2 generation?

1. Why do we need to release the F1 adults in one week?

1. Why do you think we are creating an F2 generation?

1. Is there any correlation between recessiveness and mutations? Why do you think this is beneficial?

Drosophila Lab Day 3: Prelab Predictions

Prelab Exercise

Mendel’s work gave us a valuable tool. A Punnett Square is used to predict the ratio of the possible phenotypes (traits) in the next generation. The first letter of the dominant trait is used to represent the two varieties. A capital letter represents the dominant characteristic and the same letter in lower case is used to represent the recessive characteristic. In the following example, red eyes are dominant to brown (sepia eyes). Therefore the letter “R” is used to represent the genes for eye color from the purebred red-eyed parent and “r” is used for the sepia-eyed parent’s genes for eye color. When parents mate, they each give away half of their chromosomes along with the attached genes. When the offspring are produced, they each contain a pair of genes for each trait—one from each parent.

(F1 Prediction)

Use the Punnett Square below to predict the F2 generation for these flies. The parents are the offspring of the previous Punnett Square.

(F2 prediction)

Write a hypothesis for the F2 generation of this cross (If…then…)

______

In a sex linked trait, the gene for the trait is associated with the X chromosome. Use the Punnett Squares below to predict the F1 and F2 generations for the white eye female (XrXr) and a normal red eye male (XRY)

(F1 prediction) (F2 prediction)

Write a hypothesis for the F2 generation of this cross (If…then…):

______

Prelab Questions:

1. Write the complete genotype of the F1 generation of cross # 3.

1. Write the four possible gamete combinations for these individuals.

1. Fill in the Punnett Square to predict the ratio of phenotypes in the F2 generation of cross # 3.

1. What are the four phenotypes found in the Punnett Square above?

1. What is the expected (predicted) phenotype ratio according to the Punnett Square above?

1. Write a hypothesis for this cross (If…then…):

______

Drosophila Lab Day 4: The F2 Generation

Procedure

1. Separate all flies by phenotype.
2. Count all flies and record in the data table.
3. Determine the phenotype ratio by dividing each phenotype total by the smallest one.
4. Analyze the data using a Chi-square analysis.
5. Write a formal lab report.

Cross # 1: se x wt

*First check to make sure there are males and females in both phenotypes

Cross # 2: w x wt

Cross # 3: se x vg

Data Analysis

1. Write the expected phenotype ratio for each cross (see Day 3).

1. Write the observed phenotype ratio for each cross (see tables above).

1. Fill in the tables below forthe Chi-square analysis (one for each cross).To determine the expected numbers, multiply the predicted ratio from the hypothesis by the total number of flies counted in the experiment. For example, 9/16 is the predicted ratio for the red/normal phenotype (according to the hypothesis for cross # 3). Multiply this by the total number of fruit flies counted today. The following formula is the Chi-square equation:

Chi-square = (Expected value-Observed value)2/ (Expected value)

Perform this function for each of the phenotypes and enter the value in the final column.

Cross 1

1. The degrees of freedom for a Chi-square analysis are the number of equations minus one.
2. How many equations (phenotypes) were analyzed for cross 1?_____ How many degrees of freedom do you have? ______

Degrees of freedom ______significance P=0.05

What value should you stay below at P=0.05 for cross # 1? ______

1. Is your hypothesis supported (see hypothesis on Day 3)? ______

Cross 2

1. The degrees of freedom for a Chi-square analysis are the number of equations minus one.
2. How many equations (phenotypes) were analyzed for cross 2?_____ How many degrees of freedom do you have? ______
3. Degrees of freedom ______significance P=0.05

What value should you stay below at P=0.05 for cross # 1? ______

1. Is your hypothesis supported (see hypothesis on Day 3)? ______

Cross 3

1. The degrees of freedom for a Chi-square analysis are the number of equations minus one.
2. How many equations (phenotypes) were analyzed for cross 2?_____ How many degrees of freedom do you have? ______
3. Degrees of freedom ______significance P=0.05

What value should you stay below at P=0.05 for cross # 1? ______

1. Is your hypothesis supported (see hypothesis on Day 3)? ______

Drosophila Lab Day 5: LAB WRITE-UP

The following template should be used for this formal lab write-up. It should be typed, double spaced, and paragraph format (no bullets or quotation marks). This is a formal paper. Headings for introduction, procedure, and results & discussion should be bold.

Title:

Write a short and concise title that reflects the purpose of the experiment. (+5)

Introduction:

• The first paragraph should include at least 5 sentences of background information about Drosophila melanogaster (+5)
• The second paragraph should include at least 5 sentences discussing autosomal and sex linkage, dominant and recessive traits, and the phenotype of heterozygous versus homozygous individuals. You will do your own research to explain these terms and give examples. (+5)
• The third paragraph should consist of at least 5 sentences and should include a sentence such as: “The purpose of this experiment was to ______” as well as your hypothesis written in “If…then…” format. See Drosophila lab Day 3. (+5)

Procedure (+10)-explain what was done and how. Include the following sentences:

• Three crosses were tested: cross 1 ( ______X ______), cross 2 (______X ______), and cross 3 ( ______X ______) on (give date).
• The ______generations for each cross were observed two weeks later so that dominant and recessive traits could be determined.
• The ______generations were then crossed.
• The ______were released one week later so that ______
• The ______generations were observed and counted. The data were analyzed by a Chi-square test to determine the validity of the results.

Results and Discussion

• In first paragraph, briefly restate the purpose, hypotheses and how these were tested. (+5)
• In the second paragraph, give results: total the number of Drosophila counted for each phenotype for each cross (class data found under “observed” in Chi Square chart). (+5)
• In the third paragraph, give the Chi-square test results and explain whether or not there was a significant difference between expected and observed for each cross. What could be done to improve the results? (+5)
• Suggested sentences to use for each test in this last paragraph of paper:

“The Chi-square value for the ______cross was ______. At P= ______, with _____ degrees of freedom, the data ______the hypothesis. In order to improve the results ______. Another mutation that could be tested is ______.”

• The fourth paragraph is for you to do some outside literature research. Suggested questions are “What Drosophila mutations could be tested next time?” How did these mutations get discovered? Why do we (+5)
• Two Punnett squares for each cross (6 total) showing the predicted results of the parental cross and the F1 cross (+30)
• Graph showing the results of the F1 cross for each trait (+30)

Additionally you will be graded on:

• neatness and spelling (+5)
• punctuation and grammar (+5)

Total points for this project:

Lab handout with all 3 labs completed:30

Write-up:110

140 points

Drosophila Lab and Paper Grading Rubric

Chi Square Table

DF=Degrees of Freedom