Materials Included In Kit

Additional Materials Required

Prelab Preparation

Morgue

1. Label 4 specimen jars “morgue.”
2. Place 20 mL of isopropyl alcohol into each specimen jar.

Culture Media

1. Shake the capped bottle of culture media until thoroughly mixed.
2. Prepare media on an as-needed basis. Media can mold or become contaminated with mites and bacteria after being prepared and left open to the environment.
3. For each vial, place 10 mL of culture media in a clean beaker.
4. Add 10 mL of distilled water to the beaker.
5. Using a clean glass stirring rod, mix the components in the beaker until the water has been completely absorbed. The culture media will be “slushy” but will solidify over time.
6. Add the media to a clean culture vial.
7. Cap the culture vial with a foam plug, tip the vials at a 45° angle, and allow the media to solidify.
8. Insert one piece of vial netting into the culture tube and recap.

Breeding Drosophila
Flies are shipped in small vials. It is not uncommon for adult flies to die during shipment. Larvae, however, are very hardy during shipping and are the major source of experimental flies. Remove all dead adult flies from the vial. New adults should emerge in about one week.

When performing a genetic cross, it is extremely important to remove and sort the newly emerged fruit flies into the proper culture tubes. A female Drosophila can store and use sperm from a single insemination for the major portion of her reproductive life. For genetic crosses, it is important to control the characteristics of the mating flies. Thus, it is necessary to use virgin females while conducting genetic cross experiments. Virgin females are able to lay eggs. However, there will only be a few of these sterile eggs laid whereas an inseminated female can lay up to 100 eggs per day. In order to ensure virginity, females should be selected before they are 8 hours old from a vial that has not contained adults for 8–12 hours prior to the eggs hatching. As an extra precaution, the supposed virgin females can be isolated for three days before crossing with a male in order to ensure virginity.

1. Prepare two culture vials for each type of fruit fly, one for males and one for females.
2. As the adults emerge, and before they are 8 hours old, anesthetize the flies using the information included in the Anesthetizing Drosophila Handout.
3. Determine the sex of each fly and separate the sexes into the proper culture vials.
4. Perform a cross by putting three males into a new culture tube with three virgin females.
5. After 6–8 days, purge the vial of adult flies by placing them all in the morgue. This should leave only larvae and pupae.

Safety Precautions

Lull-A-Fly™ is extremely flammable. It is toxic by ingestion and inhalation and corrosive to eyes and skin. Handle carefully, do not inhale fumes or allow Lull-A-Fly to come in contact with 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 consult current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for specific procedures and general guidelines, 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. Drosophila Culture Media and dead flies may be disposed of according to Flinn Biological Disposal Method VI, common garbage waste.

Lab Hints

• Enough materials are provided in this kit for eight groups of students. This laboratory will take four weeks to complete. The first day requires a full lab period. The remaining days will take partial lab periods with more time required when the adult flies are being analyzed. Advanced preparation requires two weeks before the flies are ready to be distributed to the students.
• Ensure that students label all materials with their group number and the activity number.
• Ensure that fly food remains moist, but not wet, at all times. Dry food will inhibit larval growth and eclosion (hatching of adult flies).
• Do not expose fly stocks to extremes in temperature for extended periods of time, that is less than 18 °C or greater than 25 °C.
• Always transfer stocks every three weeks to prevent and minimize the chance of mite infestation. Mites are transparent with eight legs, and can be seen crawling on the sides of the vial when viewed with a stereoscope.
• Clean used culture vials in warm, soapy water and then autoclave them. As an alternative to autoclaving, the freshly washed vials can be soaked in a 3% bleach solution for 30 minutes and rinsed with distilled water.
• Encourage students to count every fly using a stereoscope. Counting hundreds of flies is very tedious and students are often tempted to estimate answers or to “eyeball” the flies instead of using the stereoscope. Both shortcuts will result in inaccurate results.
• White eyes and dumpy wings are visible without the use of a stereoscope.

Teacher Tips

• Pooling data from similar crosses will create a larger sample size. Larger sample size, in turn, will lend validity to the calculated ratios.

Sample Data

Observing Wild-Type Worksheet

Vial Number varies

Crossing Drosophila Worksheet

Vial Number example shown of wild–type male crossed with a vestigial female

Table 1 Table 2 Table 3 Table 4 Table 5 Statistical Analysis Worksheet

1. How many degrees of freedom are there?

df = 1 for a monohybrid cross. df = 3 for a dihybrid cross

2. What is the probability value for this data?

Varies (in example, 0.90–0.70)

Answers to Questions

Observing Wild-Type

1. Why is it necessary to use a stereoscope when examining Drosophila?

Many of the features of Drosophila are too small to be easily identified without the use of magnification.

2. Describe a male wild-type fly in detail.

Male wild-type flies have red compound eyes, two antennae, two full wings, six legs, dark bristles on the dorsal side of the thorax, a short, black, rounded abdomen, sex combs on their forelegs, and a brown anal plate on the ventral side of the abdomen.

3. Describe a female wild-type fly in detail.

Female wild-type flies have red compound eyes, two antennae, two full wings, six legs, dark bristles on the dorsal side of the thorax, a long, narrow banded abdomen with a bump at the end, and a brown anal plate on the ventral side of the abdomen.

Crossing Drosophila

1. Is this a monohybrid, dihybrid or sex-linked cross?

Ensure answer matches the type of cross that the group was assigned. In the above example vestigial is an autosomal, monohybrid cross.

2. Write a hypothesis that describes the mode of inheritance of the trait(s) you studied. This is your null hypothesis.

The observed pattern of inheritance matches that of (the assigned) type of genetic cross.

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.

Please refer to the Flinn Drosophila Guide pages 11–14 for examples. Results are shown for an autosomal monohybrid cross.

4. Refer to the Punnett squares created in Question 3. In the following table, record the expected ratios for the genotypes and phenotypes of the F₁ and F₂ crosses in the experiment.

Please refer to the Flinn Drosophila Guide pages 11–14 for examples. Results are shown for an autosomal monohybrid cross.

5. Do the actual results appear to deviate from what was expected? Explain.

The results should not deviate from what was expected.

6. Describe your cross by answering the following questions.
   1. Is the mutation sex-linked or autosomal?

      As assigned (in example, autosomal)

   2. Is the mutation dominant or recessive?

      As assigned (in example, recessive)

Is the cross a monohybrid or a dihybrid?

As assigned (in example, monohybrid)

Statistical Analysis

1. According to the probability value, can the null hypothesis be accepted? Explain why or why not.

Varies (in example, accept null hypothesis)

References

Biology: Lab Manual; College Entrance Examination Board: 2001.

Introduction

How does the physical appearance, or phenotype, of an organism relate to its genetic composition or genotype? How are phenotypes transmitted to future generations?

Objectives
After completing this laboratory, you should be able to:

• Investigate the independent assortment of two genes.
• Determine whether the genes are autosomal or sex-linked using a multigenerational experiment.
• Analyze the data from your genetic crosses using chi-square statistical analysis.

Concepts

• Genetic crosses
• Sex-linked trait
• Chi-square analysis of data
• Independent assortment
• Autosomal trait

Background

I. Genetics

Genetics is the scientific study of heredity. Scientists substitute simple organisms for humans when studying inherited diseases and disorders. About 61% of known human disease genes have a recognizable match in the genetic code of the common fruit fly (Drosophila melanogaster), and 50% of Drosophila’s protein sequences are similar to those of mammals. Fruit flies are commonly used in genetic research because these gene and protein similarities are contained in an organism with only four pairs of chromosomes—the X/Y sex chromosomes and three autosomes, numbered 2, 3 and 4. The four pairs of chromosomes contain 132 million base pairs of DNA, comprising 13,676 genes. For comparison, the human genome has 3.2 billion base pairs comprising 30,000 genes on 23 chromosomes. Other advantages to using Drosophila are that they breed and mature rapidly, are inexpensive and easy to raise, produce several hundred offspring per generation, and require very little space. The fruit fly is also an ideal candidate for genetic studies because simple mutations cause obvious phenotype differences, and its genome map has been fully sequenced (completed in 2000).

Genes are sections of a chromosome that code for individual proteins. A trait is defined as a physical characteristic that can be passed from parent to offspring. Alternate forms of a gene are called alleles. Most organisms have two copies of every gene, one inherited on the chromosome from the mother and one on the chromosome inherited from the father. Individuals carrying two identical versions or alleles of a given gene, which may be either AA or aa, are said to be homozygous for the gene. Similarly, when two different alleles are present in a gene pair, labeled Aa, the individual is said to have a heterozygous genotype. The homozygous dominant genotype (AA) and the heterozygous genotype (Aa) will both show dominant phenotypes (because A is dominant to a), whereas the homozygous recessive genotype (aa) will exhibit a recessive phenotype. These rules apply not only for a single characteristic or traits resulting from a monohybrid cross, but also for a dihybrid cross in which two genes associated with different traits with contrasting characteristics are considered. A special case exists for genes on the sex chromosomes. Since the Y chromosome contains very few genes, the only copy of a gene in a male resides on the X chromosome which may cause a recessive gene to be expressed even though there is only one copy of the gene present. Sex-linked inheritance occurs mostly in males because a female has two copies of the X chromosome and therefore her genotype will follow normal inheritance rules.

II. Drosophila

Characterization
Like all insects, Drosophila has 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.

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 (see Figure 3).

Life Cycle
Drosophila passes through four distinct phases of development: egg, larva, pupa, and adult (see Figure 2). The time spent in each phase varies with temperature because insects are poikilothermic, or “cold-blooded.” At 21–25 °C a new generation will grow from egg to adult in 12–14 days, while at 18 °C the generation time is 20–24 days. This characteristic will be helpful when completing the lab since the culture vials may be refrigerated to time the emergence of adults for weekdays.

At room temperature, a typical generation time is 12 days and it includes the following stages. The day after an egg is laid a larva hatches from the egg. The larva molts twice, shedding the cuticle, mouth hooks, and spiracles. During the periods of growth before and after molting, the larva is called an instar. The fruit fly has three instars. The first two each last about one day while the third and final instar lasts about two days. Each larva form feeds by burrowing through the media. The final instar larva crawls out of the media about 120 hours after being laid for pupariation on a dry surface. The cuticle of the larva hardens to form the puparium.

The puparium contains the fruit fly as it undergoes metamorphosis into an adult fly. As the pupa matures, it becomes dark in color. About one day before 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. The newly emerged fly is pale in color, its shape is elongated, and the wings are unexpanded. In a few hours the adult matures, developing its characteristic color and shape, and within 10 hours it is capable of mating. The adult fruit fly may live for several weeks.

Sexing Flies

In selecting flies for genetic mating, it is absolutely essential 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, using a stereoscope. The external reproductive organs of both the male and the female are located on the ventral, posterior part of the abdomen (see Figure 3). The male genitalia are surrounded by heavy, dark bristles that are not found on the female. This characteristic is quite distinct even in a fly that has just emerged from the puparium. Female genitalia are seen as a small bump on the end of the abdomen.

In older flies the posterior part of the abdomen is quite dark in males and considerably lighter in females. The tip of the abdomen is also rounded in males and more pointed in females. Male fruit flies tend to be smaller than females, but this is not a reliable characteristic to be used to sort the sexes.

With practice and care, the front legs can also be used to distinguish the sexes. There are sex combs on the front legs of the male fly (used for grasping the female).

Drosophila Mutations

The wild-type fruit fly has full wings, red eyes and black coloring, along with bristles and antennae. There are many trait mutations available for crossing. Most mutations involve a change in the eyes, wings, bristles or antennae. The changes may be the complete absence of the feature, such as no eyes, a change in shape, such as bar-shaped eyes or a change in color, such as white eyes. Each mutant type is given a name suggesting the main distinguishing feature. The name is usually a descriptive adjective, such as “black,” or a noun, such as “bar.” For convenience in listing and labeling, a representative symbol is assigned to each mutant type. By convention, if the trait is recessive it is listed as lower case letter(s), while dominant traits are listed as upper case letter(s). Wild-type is designated by a plus sign (+). See Table 1 for a list of common trait mutations in Drosophila.

III. Chi-Square Analysis of Data

Experimental measurements always have some random error, which means that results or conclusions can never be drawn with complete certainty. Statistics, or mathematical analysis of data, provides a set of tools that scientists can use to accept a conclusion that has a high probability of being correct, and to reject a conclusion that has a low probability of being correct. In particular, chi-square (χ²) analysis is a statistical test geneticists often use to determine whether an observed phenotype distribution occurs by chance or not. In other words, when you see fruit flies with certain physical characteristics, are the number of flies produced because they have the expected genotype or because of chance. Analyzing the results using a statistical test is important because the results observed from the experiment are unlikely to exactly match the expected results. The chi-square test determines whether or not the deviation from the expected results is due to chance or due to what is called an invalid null hypothesis. A null hypothesis is a hypothesis that states there is no statistically significant difference between the observed results of the experiment and the expected results and the hypothesis is correct. It is expecially important to run a chi-square test when experimenting with very few numbers of test organisms, such as will be used in this laboratory.

The formula for chi-square is:

The top line of the equation, abbreviated (o – e), calculates the difference, or deviation, between the number of flies having a specific phenotype and the number expected for that phenotype. The number of fruit flies expected for an experiment is based on the Punnett square for the cross. In order to prevent the deviations from equaling a negative number, the deviations are squared and the resultant is divided by the expected number of fruit flies. The chi-square value is calculated for each trial of the experiment. In this activity, each F₁ or F₂ generation, for each group performing the same cross, could be considered a trial.

The chi-square results are then compared to a set of reference numbers contained in a statistical table called a Probability Table or a Chi-square Table. The table allows a scientist to determine if the value determined by the chi-square equation is statistically different from what was expected. The table is based upon the concept of degrees of freedom (df) where df = n – 1. Degrees of freedom are used instead of the number of variables (n) as a way of correcting for taking the statistic of a statistic. In this experiment, the variable (n) is the number of different phenotypes. For example, in a monohybrid cross there are two possible phenotypes (not genotypes), one expressing the dominant form and one expressing the recessive form. Therefore, a monohybrid cross would have one degree of freedom.

In a Probability Table, the degrees of freedom are listed in the left-hand column of the table, the probabilities are listed across the top row, and the chi-square values are listed in the body of the table (see Table 2). When there is only a small difference between the observed results and the expected results, a very low chi-square value is obtained, and the probability that the hypothesis is correct is considered very high. In order to read the table, calculate the chi-square value and determine the correct degrees of freedom. Read along the correct degree of freedom row until the chi-square value fits between two columns. Follow the column up to read the probability that the chi-square result supports the null hypothesis. Any probability greater than 0.05 supports the null hypothesis, while a probability less than 0.05 rejects the null hypothesis. Note that statistical probability in not the same as percent chance. A probability of 0.05 is not the same as saying that there is a 5% chance that the observed results are statistically the same as the expected results.

Experiment Overview

Activity 1 emphasizes the observation and characterization of wild-type Drosophila. In Activity 2, a mutant Drosophila and a wild-type will be crossed and the F₁ and F₂ generations will be observed and characterized. Activity 3 involves the statistical analysis of the data collected in Activity 2.

Materials

Safety Precautions

Lull-A-Fly™ is extremely flammable. It is toxic by ingestion and inhalation and corrosive to eyes and skin. Handle carefully, do not inhale fumes or allow Lull-A-Fly to come in contact with skin. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Activity 1. Observations of Wild-Type Drosophila

1. Read the Anesthetizing Drosophila Handout.
2. Obtain a vial of wild-type Drosophila.
3. Record the number of the vial on the Observing Wild-Type Worksheet.
4. Anesthetize the flies using the procedure described on the Anesthetizing Drosophila Handout.
5. Place the anesthetized flies on a sheet of white paper.
6. Examine the adult flies using a stereoscope and a brush. Record observations and measurements on the Observing Wild-Type Worksheet.
7. After completing the examination, carefully return the flies to their vial. Lay the vial on its side and place the flies on the side of the vial. This ensures that the flies do not drown in any moisture nor get stuck in the culture media. An alternative is to place the flies into a clean, dry vial until they awaken and then transfer them using the funnel technique shown in Figure 1 in the Anesthetizing Drosophila Handout.

Activity 2. Crossing Drosophila

1. Obtain a vial containing mating pairs of experimental flies.
2. Record the cross number of the vial on the Crossing Drosophila Worksheet. These flies represent the parental generation (P) and have already mated. The females should have already laid eggs on the surface of the culture medium. The eggs (or larvae) represent the first filial (F₁) generation—the flies should emerge from their puparia in about a week.

P Generation

1. Anesthetize the adult flies using the information included in the Anesthetizing Drosophila Handout.
2. Observe each adult fly using a stereoscope.
3. Use a camel hair brush to separate the males from the females and observe all the flies to identify any mutation(s) that may be present. Record whether the mutation(s) is/are associated with the males or the females in Table 1 on the Crossing Drosophila Worksheet.
4. Record the phenotype for each adult fly in Table 1 Common Drosophila mutations on page 3.
5. Identify the mutations using Table 1 on the Observing Drosophila Worksheet. Record the mutation’s symbol in Table 1 on the Crossing Drosophila Worksheet. Note: Recessive alleles are represented by lower-case letters while dominant alleles are represented by upper-case letters.
6. Place the adult flies into the morgue.
7. Label the vial containing the eggs (or larvae) with the symbols for the cross. An example would be “+ ♀ X ap ♂.” Also label the vial with your group’s initials and the date.
8. Place the vial in the incubation area as directed by your teacher.

F₁ Generation

1. Inspect the eggs and larvae daily and record any observations in Table 2 on the Crossing Drosophila Worksheet.
2. Once the adult flies emerge from their puparium, begin the observations of the F₁ Generation by anesthetizing the adult flies as directed on the Anesthetizing Drosophila Handout.
3. In Table 3 on the Cross Worksheet, describe each observed phenotype. Record the phenotype symbol in the second column of Table 3. Place a tally mark in the appropriate gender column for each phenotype. For example, a vestigial winged male would have a tally mark in the male, vestigial-wing row, while a full-winged male would have a tally mark in the male, full-winged row (see Figure 4).

4. Place 5 pairs of F₁ flies in a fresh vial and the remaining adult flies into the morgue.
5. Label the new vial “F₁ × F₁.” Also label the vial with your group name, the date, and the symbol of the mutation as outlined in step 7.
6. Place the vial in the incubation area as directed by your teacher.
7. Check the vial daily for eggs.
8. After the females have laid their eggs, remove the F₁ adult flies from the vial and place them in the morgue. The eggs (or larvae) in the vial are the F₂ generation.
9. Place the vial in the incubation area as directed by your teacher.

F₂ Generation

1. Inspect the eggs and larvae daily and record only observations in Table 4 on the Crossing Drosophila Worksheet.
2. Once the adult flies emerge from their puparia, begin the observations about the F₂ Generation by anesthetizing the adult flies as directed on the Anesthetizing Drosophila sheet.
3. Sort the flies into appropriate categories using the camel hair brush to move them.
4. In Table 5 on the Crossing Drosophila Worksheet, describe each phenotype observed. Record the phenotype symbol in the second column of Table 5. Place a tally mark in the appropriate gender column for each phenotype (see Figure 4). Collect at least 200 flies. This may take several days. Note: The more F₂ flies collected, the more reliable the data will be.
5. After observing the adult flies, place them into the morgue.

Activity 3. Statistical Analysis of Experimental Data

1. Record results in the Statistical Analysis Worksheet.
2. Record the observed numbers of organisms in each phenotype.
3. Transfer the expected number of organisms from question 4 on the Crossing Drosophila Worksheet.
4. For each phenotype, calculate the chi-squared value using Equation 1 in the Background section.
5. Determine the degrees of freedom.
6. Use Table 1 from the Background section to determine whether or not the χ² value is within acceptable or unacceptable probability.
7. Consult your instructor for appropriate disposal procedures.

Student Worksheet PDF

13809_Student1.pdf