Materials Included In Kit

Additional Materials Required

Safety Precautions

Lull-A-Fly™ is a flammable liquid and is corrosive to eyes and skin. It is also moderately toxic by ingestion and inhalation; use only in a well-ventilated area. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Lull-A-Fly may be disposed of according to Flinn Suggested Disposal Method #5. The used vials may be cleaned and stored for future use or discarded according to Flinn Suggested Disposal Method #26a.

Lab Hints

• Enough materials are provided in this kit for 24 students working in groups of three. This laboratory experiment is very time consuming. Students will need to be allowed to check their flies every 6–8 hours. This will either entail students bringing the experiment home or the instructor counting the flies after hours.
• Flies are shipped in small containers or vials. It is not uncommon for adult fruit flies to die during shipment. However, larvae remain hardy and will be the major source of experimental flies. Remove all the adult flies and place the culture bottles in a proper storage location. Adult flies will emerge in approximately one week.
• The media of the stock cultures should be changed every three to four weeks depending upon the temperature of the room. At least two cultures of each stock should be kept in the event the media molds or grows bacteria or one of the cultures develops mites and becomes contaminated.
• It may be helpful to pass around a vial of sepia flies before beginning this activity. This will help students become familiar with and easily identify the sepia phenotype.
• If mold becomes a problem, sprinkle a small amount of methyl paraben into the media. Do not add more than 1 g per liter of media.
• The fly morgues can be made from various materials (e.g., beakers, cleaned jelly jars). Simply fill with mineral oil.
• Magnifying lenses may be used if a stereoscope is not available.

Teacher Tips

• Before beginning this activity it is crucial that students have a strong understanding of the monohybrid cross.
• Stress the importance of using virgin flies in this activity. If the virgins are not rigorously collected the result will be unpredictable and it will become frustrating for the students.

Answers to Prelab Questions

Complete the following table.

1. Why is it essential that virgin females be used in genetic crosses?

   If virgin flies are not collected the results will be inaccurate. For example, offspring will be produced which violate the predicted phenotypic and genotypic ratios.

2. Why is the black band on the ventral side of male flies not an accurate means of sex identification? What is a more accurate means of identification?

   This band appears only in older flies. It is more accurate to look for the presence or absence of sex combs on the forelegs.

Sample Data

Table 1. Phenotypic Count

Table 2. Expected Phenotypic and Genotypic Ratios

Answers to Questions

1. In this experiment a monohybrid cross was conducted. Using a biology textbook if necessary, draw two Punnett squares, one square representing the predicted genotypic results for crossing the P generation, the second displaying the results for crossing the F₁ generation.
   {10964_Answers_Figure_6}
2. Refer to the Punnett squares created in Question 1. Predict the expected ratios for the genotypes and phenotypes of both the F₁ and F₂ generations in the experiment. Record the results in Table 2.

   See Table 2.

3. Calculate the observed phenotypic ratio from Table 1 and enter the result in Table 1. Do the observed results agree with the predictions? Explain.

   The results should not deviate from what was expected. The group will most likely not get exactly 150 wild-type and 50 sepia but there should be a close 3:1 ratio.

4. While collecting flies from the F₁ generation, a student found that a small number of the 200 flies collected had a sepia phenotype.
   1. What might have caused this result?

      Sepia flies would appear in the F₁ generation if there was a female sepia fly that was already inseminated by a male sepia fly before it was removed from its original stock culture.

   2. How could this have been avoided?

      By stringently checking the flies every 6–8 hours to ensure that the virgin flies are not allowed to mate.

References

Flinn Drosophila Guide; Flinn Scientific, Batavia, IL; 2000; pp 1–11.

Introduction

Drosophila melanogaster—the common fruit fly—serves as an ideal organism to study the basic laws of heredity. Conduct a monohybrid cross to observe the recessive inheritance pattern of the sepia body color throughout the F₁ and F₂ generations.

Concepts

• Genetics
• Monohybrid cross
• Drosophila melanogaster

Background

Drosophila pass through four distinct phases during development—egg, larva, pupa and adult. When cultured at 21 °C a fresh culture of D. melanogaster will produce new adults in two weeks. The day after the egg is laid, the larva hatches. The larva molts twice, shedding the cuticle, mouth hooks and spiracles.

Metamorphosis into an adult fly occurs within the puparium. As the pupa matures, it becomes dark in color. About one day after emergence, the folded wings appear as two dark elliptical bodies and the pigment of the large eyes is visible through the puparium. When metamorphosis is complete, the adult emerges by forcing its way through the anterior end of the puparium. Within 8 to 12 hours of emergence, a fruit fly is capable of mating.

A female Drosophila can store and use sperm from a single insemination for the major portion of her reproductive life. This fact underscores the necessity of using only virgin females for genetic crosses. Older males will mate with newly emerge females. Therefore, it is essential all adult flies be removed from a culture 8 to 12 hours before it is used for virgin female selection. To ensure virginity, females should be selected before they are eight hours old.

Physical Characterization
Like all insects, Drosophila have three main body parts—the head, the thorax and the abdomen (see Figure 1). The major structures on the head of a wild-type fruit fly are the large red compound eyes. On top of the head are two antennae the fly uses for smelling. The mouth is a proboscis—the fly lowers it to suck up food like a vacuum cleaner. The thorax has six legs, two wings and, on the dorsal (top) side, a number of long dark bristles.

Culturing Drosophila

Media Preparation
Drosophila can be raised on a variety of media. In nature, fermenting fruits make up the standard diet of a fruit fly. In the lab it is more practical to use dry, prepared media. Culture media are typically blue in color so that developing flies will be more visible. Dry media inhibit mold growth and will not liquefy or give off unpleasant odors as more traditional food sources would.

1. Place 10 mL of culture media in a clean culture tube.
2. Using a graduated cylinder, add 10 mL of water to the 10 mL of media. Note: It is best to add slightly less water until some of the water has been absorbed by the media. Avoid adding too much water or the media will become soupy.
3. Gently agitate the tube from side to side to mix the water and media.

Transferring Flies Between Vials
Sometimes it becomes necessary to transfer flies from one vial to another. Reasons can vary from moldy media to simply wanting to increase stock. To transfer flies from one vial to another, first tap the vial containing the flies down so that the adults are gathered near the bottom. Quickly remove the foam stopper and invert the tube on top of the second vial containing fresh media. Note: A funnel may be placed in the receiving vial to reduce chances of the flies escaping. Tap the vials on the table so that the flies fall into the new vial and then quickly stopper the vial (see Figure 2).

Anesthetizing Flies
In order for students to have enough time to thoroughly examine and sex their Drosophila, it is necessary to anesthetize flies. When anesthetized with Lull-A-Fly™, Drosophila can be expected to remain “asleep” for 45 minutes to an hour.

1. Follow standard Drosophila transfer technique to knock the flies to be anesthetized to the bottom of the vial, or transfer them to an empty vial used for anesthetization (see Figure 2).
2. Dip the tip of the wand into the Lull-A-Fly solution. Note: Do not submerse the entire wand! Only the very tip of the wand should enter the solution
3. Replace the cap on the Lull-A-Fly bottle.
4. Quickly place the end of the wand into the vial, just below the vial plug (see Figure 3).
   {10964_Background_Figure_3_Anesthetizing Drosophila}
5. Observe the flies as they become anesthetized. When the last fly has been anesthetized, remove the wand.
6. Remove the plug from the vial and dump the flies onto a white study surface. Note: A note card provides an excellent surface for observation and a camel hair brush is an ideal manipulation tool.
7. Replace the plug in the culture vial while studying the anesthetized flies. Note: Do not “store” the anesthetizing wand in the culture tube.
8. Use a stereoscope to examine flies if necessary.

Sexing Flies
When selecting flies for genetic mating, it is critical that the sex of each fly be properly identified. Identification of sex is most reliably done by examination of the genital organs with the aid of magnification. The external reproductive organs of both the male and female are located on the ventral-posterior part of the abdomen. The male genetalia are surrounded by heavy, dark bristles which are not present on the female (see Figure 4, Part A). This characteristic is quite distinct even in a fly that has just emerged from the puparium.

As flies age, the posterior portion of the abdomen becomes very dark in males and considerably lighter in females. The tip of the abdomen is rounded in males and more pointed in females. Generally, male fruit flies tend to be smaller than females but this is not a reliable characteristic to sort the sexes. Females have stripes on every segment of their abdomen. Males have shorter abdomens, and the last few segments of the abdomen are solid black. Males also have a set of brown anal plates on the ventral (bottom) side of the abdomen.

The front legs can also be used to distinguish sexes. There are sex combs on the front legs of the male fly (see Figure 4).

Experiment Overview

A monohybrid cross will be conducted using sepia females (se♀) and wild-type males (+♂). The sepia body color is a recessive trait.

Materials

Prelab Questions

Complete the following table.

1. Why is it essential that only virgin females be used in genetic crosses?
2. Why is the black band on the ventral side of male flies not an accurate means of sex identification? What is a more accurate means of identification?

Safety Precautions

Lull-A-Fly™ is a flammable liquid. It is also moderately toxic by ingestion and inhalation; use only in a well-ventilated area; corrosive to eyes and skin. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines.

Procedure

Part A. Obtaining a Pure Culture

1. Obtain a stock culture of sepia Drosophila.
2. Anesthetize all adults in the sepia Drosophila culture according to the procedure in the Background section.
3. Discard the anesthetized flies in the fly morgue. This step clears the vial of current adults so all the newly emerged females will be virgins.
4. Allow the vial to sit undisturbed for 6–8 hours at room temperature (21 °C).
5. Prepare a fresh culture vial as described in the Background section. Label this vial se♀ and add your initials and the date. Note: Labeling the vial this way indicates the females are virgins.
6. Anesthetize the newly emerged flies in the first vial and separate the flies according to sex. See the Background/Sexing flies section. Note: A stereoscope may be used if desired.
7. Place the females into the fresh culture vial made in step 5.
8. Repeat steps 6–7 until 15–20 virgin sepia females have been obtained. All females may be added to the same vial prepared in step 5.
9. The virgin sepia females should be kept isolated for 3–4 days to assure virginity. Observe the vial to be sure no larvae appear at the bottom of the virgin culture.
10. Obtain a stock culture of wild-type Drosophila.
11. Prepare a fresh culture vial as in step 5. Label this vial +♂ and add your initials and the date.
12. Anesthetize the wild-type culture and separate the flies according to sex. Place the males in the vial labeled +♂ prepared in step 11.
13. Repeat step 12 until approximately 15–20 males have been obtained.

Part B. Crossing the P Generation

1. Prepare three fresh culture vials. Label each vial se♀ X +♂ and add your initials and the date.
2. Anesthetize all flies in the vials labeled se♀ prepared in Part A.
3. Once all flies are anesthetized, add six se♀ to each of the three culture vials prepared in Part B step 1.
4. Repeat steps 2 and 3 using the +♂ culture prepared in Part A. Add six +♂ to each of the three vials containing the six se♀.
5. Leave the se♀ and +♂ adults in the cross-culture vials for 7–10 days. Note: Larvae should be visible in 3–4 days. If pupae are visible, remove the adult flies immediately.

Part C. Crossing the F1 Generation

1. Prepare three fresh vials and label each vial se♀ X se♂. Add your initials and the date. These vials will be used in step 6.
2. After 7–10 days, remove the P generation adults from the vials labeled se♀ X +♂. Anesthetize and dispose of the adults in the fly morgue.
3. As the F₁ adults emerge, anesthetize and count every 6–8 hours. Determine the phenotype (wild type or sepia) of each F₁ adult fly.
4. Tabulate the results and fill in Table 1 on the Drosophila Basics Worksheet. Do not discard F₁ flies!
5. Repeat step 2 every 6–8 hours until at leaset 200 flies from the F₁ generation have been sorted and counted.
6. As the F₁ adults are being counted and sorted (steps 2–4), place 6 se/+♀ and 6 se/+♂ into a fresh vial prepared in step 1. Tip: Once each vial has 6 se/+♀ and 6 se/+♂, the future F₁ flies may be discarded after counting.
7. Allow the F₁ adults to remain in the se/+ X se/+ vials for 7–10 days. After this time, remove the adults so the F₂ generation can emerge without contamination.
8. Once the F₁ adults have been removed, check the vials labeled se/+ X se/+ every 6–8 hours.
9. Anesthetize and remove the F₂ adult flies. Count the results. Identify their phenotype and discard the flies in the fly morgue as they are counted. Tally the results in Table 1.
   {10964_Procedure_Figure_5_Flowchart for the experiment}
   Figure 5 visually represents the overall process of the experiment, Parts A–C. It is important to realize that the symbols represent an overall generation, such as F₁, not a specific phenotype. It is meant to serve as a general guide to aid in understanding the overall procedure.
10. Discard dead flies in the fly morgue. Consult your instructor for appropriate disposal procedures.

Student Worksheet PDF

10964_Student1.pdf