Introduction

Drosophilia melanogaster, the fruit fly, is an excellent organism for genetics studies because it has simple food requirements, occupies little space, is hardy, completes its life cycle in about 12 days at room temperature, produces large numbers of offspring, can be immobilized readily for examination and sorting, and has many types of heredity variations that can be observed with low power magnification. Drosophila has a small number of chromosomes ( four pairs). These chromosomes are easily located in the large salivary gland cells.

The Life Cycle of Drosophila

• The eggs. The eggs are small, oval shaped, and have two filaments at one end. They are usually laid on the surface of the culture medium and, with practice, can be seen with the naked eye. The eggs hatch into larvae after about a day.
• The larval stage. The worm like larvae eats most continuously, and its black mouth parts can be seen moving back and forth even when the larvae is less distinct. Larvae tunnel through the culture medium when eating; thus channels are a good indication of a successful growth culture. The larvae molt twice as it increases in size. In the last of the three larval stages, the cells of the salivary glands contain giant chromosomes, which may be seen readily under low-power magnification after proper staining.
• The pupal stage. When a mature larvae in a laboratory culture is about to become a pupa, it usually climbs up the side of the container or on to a paper strip provided in the culture bottle. The last larval covering becomes harder and darker, forming a pupal case.
• The adult stage. When metamorphosis is complete, the adult flies emerge from the pupal case. They are fragile and light in color and their wings are not full expanded. These flies darken in a few hours and take on the appearance of an adult fly. They live a month or more and then die. A female does not mate for 10-12 hours after emerging from the pupa. Once she has mated, she stores a considerable quantity of sperm in receptacles and fertilized her eggs as she lays them. To ensure a controlled mating, it is necessary to use females that have not been mated before(virgins).

Figure 7.1 The Life Cycle of Drosophila melanogaster

 

Design of the Exercise

This genetics experiment will be carried on for several weeks. Drosophila with well-defined mutant traits will be assigned to you by your teacher. You are responsible for making observations and keeping records concerning what happens as mutant traits are passed from generation to the next.

You will be assigned to study a certain mode of inheritance using particular genetic crosses of flies having one or two mutations. The modes of inheritance most commonly used are:

• Monohybrid. In these experiments, the mode of inheritance is determined when a single contrasting pair of traits is involved.
• Dihybrid. In these experiments, the mode of inheritance is determined when two pairs of contrasting traits are considered at the same time.
• Sex-linked. In these experiments, the mode of inheritance is determined when the mutant characteristic is associated with the X chromosome.

Procedure

1. Obtain a vial of wild- type flies. Practice immobilizing and sexing these flies. Examine these flies and note the characteristics of their eyes, wings, bristles, and antennae.

2. To make handling easier, immobilize the flies with fly-nap, or by twirling the vial in ice for several minutes. Place the immobilized flies on a piece of filter paper inside a petri dish. Place this under a dissecting microscope to view the flies.

3. Distinguish male flies from female flies by looking for the following characteristics:

a). Males are usually smaller than females.

b). Males have dark, blunt abdomens, and females have lighter, pointed abdomens.

c). Only males have sex combs. which are groups of black bristles on the upper most joint of the forelegs.

Figure 7.2: Female and Male Drosophilia

Female

 

Male

 

Male with vestigial wings

4. Obtain a vial containing pairs of experimental flies. Record the cross number of the vial. This number will serve as a record as to which cross you obtained. These flies are the parental generation(P₁) and have already mated. The female should have already laid eggs on the surface of the culture medium. The eggs represent the first filial, F₁ generation and will be emerging from their pupal cases in about a week.

5. First week (today): Immobilize and remove the adult flies. Observe them carefully under the dissecting microscope. Separate the males from the females and look for the mutation(s). Note whether the mutation(s) is/are associated with the males or females. Identify the mutation(s) and give them a made up name and symbol. Record the phenotype and symbol in Table 7.1. The findings should be confirmed by your teacher.

6. Place the parents in the morgue(jar containing alcohol). Label the vial containing the eggs or larvae with the symbols for the mating. Also label the vial with your name and date. Place the vial in a warm location.

7. Second week: Begin by observing the F₁ flies. Immobilize and examine all the flies. Record their sex and characteristics. Consider the conclusion s that can be drawn from these data. Place five or six pairs of F₁ flies in a fresh culture bottle and the rest of the flies in the morgue. For this cross the females need not be virgins. Label the vial with the symbols , name, and date.

8. Third week: Remove the F₁ flies from the vials and place them into the morgue. The F₂ generation are the eggs and /or larvae in the vial. Place the vial in a warm place.

9. Fourth week: Begin removing the F₂ flies. Record their sex and the presence or absence of mutation(s). The more F₂ flies collected, the more reliable the data will be. You may have to collect flies over a three-or four day period. Try to collect at least 200 flies.

10. To analyze your data, you will need to learn how to use the Chi-Square Test. Go to the Statistical Analysis Section to review the technique.

Table 7.1: F₁ Generation Data

Date _________________

Table 7.1: F₂Generation Data

Date _________________

Analysis of Results

1. Describe and name the observed mutation(s).

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2. Write a hypothesis which describes the mode of inheritance of the trait(s) you studied. This is your null hypothesis ( as described in the statistical analysis section).

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3. Refer to a textbook and review Punnett squares. In the space below, construct two Punnett squares to predict the expected results of both the parental and F₁ crosses from your null hypothesis.

4. Refer to the Punnett squares above. Record the expected ratios for the genotypes and phenotypes of the F₁ and F₂ in the experiment below.

5. Do the actual results deviate from what was expected? If so explain how.

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6. From the results describe your cross: is it

Sex-linked or autosomal?fffffffffffffffffffffffffffffffffffffffffffffffffffff_________________________

A dominant mutation or a recessive mutation? fffffffffffffffffffffffffffff_________________________

Monohybrid or dihybrid? ffffffffffffffffffffffffffffffffffffffffffffffffffff _________________________

7. Are the deviations for the phenotypic ratio of the F2 generation within the limits expected by chance? To answer this question, statistically analyze the data using the Chi-square analysis. Calculate the Chi-square statistic for the F2 generation in the chart below.

a). Calculate the Chi-square value for these data.

sssss1. How many degrees of freedom are there? ___________________

sssss2. Chi-square (X²) = __________________

sssss3. Reffering to the critical values chart, what is the probability value for these data? _____________

b). According to the probability value, can you accept or reject your null hypothesis? Explain why.

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Topics for Discussion

1. Why was it necessary for the females of the parental generation to be virgin?

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2. Why was it not necessary to isolate virgin females for the F₁ cross?

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3. Why were the adult flies removed from the vials at weeks 2 and 4?

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For those individuals that have access to the internet, this entire lab can be completed by using the Virtual Fly Lab web site. This unique site will allow students to pick and choose among various mutations without telling them the type of trait transmission. Chi-square is also an option to prove the null hypothesis. I have used this site with my students for the past two years with great success. In the Lab section of this web site, there is an alternate lab that may be used in place of this lab or as enrichment.