Genetic Experiments with Drosophila Modified from CBSC: Carolina Drosophila Manual Basic genetic mechanisms arose early enough in primitive organisms (or were so superior to alternatives) that most organisms share them. It is, therefore, possible to study the principles of genetics in one organism and gain an understanding of the modes of inheritance in many. The animal most widely used for genetic studies is the common fruit fly, Drosophila melanogaster. This model organism possesses many attributes which have contributed to its popularity. The fruit fly is easily cultured and its generation time is only two weeks at 21°C. A single female may lay as many as 500 eggs in 10 days. The small size of Drosophila cultures allows the organism to be studied in laboratories and classrooms, even those with limited space; however, each individual fly is large enough for rapid notation of mutant phenotypes. The fruit fly has been the subject of genetic studies since about 1909. Myriad spontaneous mutations have been found and many others have been induced with radiation. D. melanogaster has a tremendous number of genes for study, but practically dictates the selection of a few readily identifiable phenotypes for use in instruction.

AP Essential Understandings Completed during Lab Essential Knowledge 3.A.1

DNA, and in some cases RNA, is the primary source of heritable information. Essential Knowledge 3.A.2

In eukaryotes, heritable information is passed to the next generation via processes that include the cell cycle and mitosis or meiosis plus fertilization.

Essential Knowledge 3.A.3 The chromosomal basis of inheritance provides an understanding of the pattern of passage (transmission) of genes from parent to offspring.

Essential Knowledge 3.A.4 The inheritance pattern of many traits cannot be explained by simple Mendelian genetics.

Objectives You will be issued different stock cultures of Drosophila with “unknown” genetic makeups for the purpose of performing controlled matings. The objective of the lab are to:

1. determine the inheritance of the traits found in the stock cultures.

2. determine the inheritance of the different forms of each trait (dominant or recessive, X-linked or autosomal)

3. Determine the linkage relationship between the mutant genes

Fly Experiment Timeline/Outline and Goals for Each Step This lab will take several weeks of periodic lab sessions. Due to the length of time and space between observations, it may be difficult to remember what is being done and why. This following timeline briefs each step and the goals for each week.

Week One Make 2 vials of media Anesthetize parental flies Observe phenotypes and sexes of flies Place in each vial parental flies for one cross (you will be assigned two of the six)

Female virgin wild type X Male white eye Female virgin white eye X Male wild type Female virgin wild type X Male apterous Female virgin apterous X Male wild type Female virgin white eye X Male apterous Female virgin apterous X Male white eye

Goals Learn basic fly techniques (making media, fly manipulation, and fly husbandry) Distinguish between male and female flies Set up parental crosses to produce parental offspring (F1 generation)

Week Two Anesthetize parents Double check phenotypes and sexes Place in morgue

Goals Ensure flies placed in vials were what you wanted Remove parental flies so they are not mistaken for offspring when F1 flies emerge, nor will they

mate with F1 flies

Week Three Make 2 new vials of media Anesthetize F1 flies Sort, count, and analyze phenotypes of F1 flies Transfer F1 flies to a new vial with fresh media Save old vials in case of error

Goals Determine inheritance patters of mutants, if possible F1 flies transferred to parent F2 flies

Week Four Anesthetize and remove all flies from the second set of vials

Goals Remove adult flies so they are not mistaken for F2 flies

Week Five Anesthetize, sort, and count phenotypes of F2 flies Analyze data for inheritance patterns

Goals Use the data from the F2 flies to determine/confirm inheritance patterns

The Drosophila Life Cycle Drosophila melanogaster is a typical insect. It goes through four stages in its life cycle: egg, larva, pupa, and adult. At 21°C, the complete reproductive cycle from egg to hatching adults is about two weeks: eight days in the egg and larval stages, and six days in the pupal stage. Adult fruit flies may live as many as eight weeks under optimal conditions.

Egg Larva Pupa Adult

Drosophila are ready for mating within about 8-12 hours after emerging from the pupa case. After a short courtship in which the male circles the female while vibrating his wings, the female spreads her wings laterally and insemination takes place. Sperm is received by the female in seminal receptacles and is used to fertilize eggs for the major portion of her life. As sperm from the first mating is always present, it is not possible to cross the same female to different males and get accurate results. Therefore, collecting virgin females is essential. The eggs are usually fertilized at the time of laying, and early embryonic development takes place within the egg case. The female typically begins laying eggs by the second day after emerging from the pupa. She will lay from 50 to 100 eggs per day and will remain fertile as long as she lives. You can sometimes see the eggs on the surface of the food and especially around the edge where the food joins the vial. They are only about 0.5 mm long and bear 2 filaments at the anterior end. These prevent eggs from sinking into the soft food. The eggs must have contact with air and will drown if immersed for long periods. Within 2 days after the eggs have been laid, very small larvae will typically hatch out and crawl in the media. They are so tiny you may have difficulty seeing them except with a stereomicroscope. This larva represents the first instar (phase of larval growth). They will eat almost all the time and double their size about every 24 hours. After its first molt (shedding of its outer skin), the larva will form the second instar. They will eat, grow, and molt again to form the third instar, which will be about 5 mm in length. The third instar will pupate about 6 or 7 days after hatching from the egg under ideal growing conditions. The larvae crawl out of the food and adhere to a relatively dry surface (the side of the vial or the netting). The soft larval skin dries and gradually develops a brown pigmentation known as a pupa case. Within this case, metamorphosis from the larval to the adult form takes place. It is easy to tell when the time for emergence is near as folded wings can be seen as two elongated dark areas within. The pigment of the eyes will also be developed and can be seen as two bright red spots (in the case of the wild type flies). Emergence is usually expected within 24 hours when such characteristics can be seen. The adult emerges by forming its way through the anterior end of the case. The newly emerged fly will be very long, relatively unpigmented (light color), and with folded wings. It will quickly darken to a more adult appearance within the first couple of hours as they become more pigmented. Within that time, the wings also unfold and the body becomes more compact.

Methods

Records The success of any experiment depends upon a complete, concise and workable system of records. All original records should be kept in your notebook. No records are to be kept on loose sheets of paper. This lab will take several weeks to finish. Skip several pages for this in your notebook in order to keep all of the fly work together. It is necessary to make direct observations only on those characteristics you are studying in your experiment. Be sure you are certain of the differences involved in your flies and that you classify all flies completely (including sketches of your phenotypes). By the end of the lab, however, data from all student crosses should be included in your notebook to provide a full picture of how the two mutations interact with one another. Consistent mating codes must be used for easy identification. Each code should include the mating date, along with the number of flies and gender for each phenotype. Information on transferring and counting should be in your notebook.

Preparing Media Instant culture media will be used. The medium, which has a white, flaky appearance, is dehydrated and already contains a mold inhibitor. It does not need cooking or sterilization. We will use the media with an additive that turns the medium blue when dissolved in water, allowing for easier observation of some of the developmental stages. To prepare a culture, obtain a clean plastic culture vial. Place equal volumes medium and cool water in the vial, along with 3-5 grains of dry yeast. Too much yeast will produce carbon dioxide and could render the Drosophila sterile or even cause death. The medium will be quite fluid at first, but within a few minutes it will solidify to the consistency of mashed potatoes and be ready to use. Add a piece of netting and a foam plug. Once the desired fly types are identified, label the vial with a mating code using permanent marker.

Collecting Virgin Females It is important to have controlled matings for your experiments, so it is necessary to obtain females before they have mated with males. Traditionally, we can take advantage of the fact that flies are not ready for mating for 8-12 hours after emerging from the pupae cases. If a vial is emptied of all flies, females that emerge in the next 8-12 hours (possibly up to 15 hours, but fewer is better) will be virgin. Females will be ready for mating somewhat earlier than males, so be sure than an older male is not left in the vial during “clean out” periods. You will find that most of the flies tend to emerge during the early part of the day, so removing flies as early as possible will help achieve large yields of virgin females later in the day. To test virginity, females can be isolated for 3-4 days before selecting them for matings. Do not be alarmed if you find eggs in a vial of virgin flies. Virgin flies can lay eggs even if they are not inseminated, especially after they have been kept for several days without mating. Should small wiggling larvae appear in a vial of supposed virgin flies, however, then the females are not all virgin (oops…).

Anesthetizing Procedure Investigators have used many means to immobilize fruit flies for examination. They have crowded them into constricted glass tubes, chilled them, knocked them out with ether and methoxyflurane, and kept them in a continuous flow of carbon dioxide. Using these techniques, the novice kills of loses many flies. To avoid issues of loss, death, or explosion, we will use an easy-to-use anesthetic called FlyNap. Exposure based on directions should anesthetize flies for about 50 minutes, but they may remain knocked out for several hours (especially young flies). Dip an anesthetic wand into the FlyNap bottle, making sure to remove excess liquid by running the wand across the edge of the bottle. To get flies away from the plug, invert the culture vial. Since flies are negatively geotropic, they tend to crawl up and away from the plug. Flies that remain near the plug can be shaken away by turning the vial upright and tapping the vial down gently, but with enough force to dislodge them from the foam. Place the wand into the vial by using a finger to push the plug slightly to one side. The FlyNap will evaporate into the vial. Follow the directions for the product to determine a proper length of time to anesthetize. Once all flies are anesthetized, remove the plug, and pour the flies onto a white note card. If necessary, place under a stereomicroscope for examination. Do not overanesthetize your flies – they will die!

Handling Drosophila As you observe the Drosophila of your vials, you may fear that handling such tiny creatures will be difficult. With a little practice in the proper techniques, however, you should find it relatively simple. All flies should be placed on a white note card for ease of observation. Use of a fine brush, teasing needle, or any other suitable tool is necessary to move flies about. Closer examination should be conducted with a stereomicroscope, if possible. Discarded flies should always be dropped into a morgue – a jar of alcohol. Sorting may be done in any variety of ways, but often works best when the flies are placed in a row on the note card, and moved to different groups based on phenotype. This allows for easy movement under the microscope to examine the different flies in the row. To manipulate the flies while viewing them, use the brush. Practice moving the flies by turning them over and becoming familiar with every part of the body. Specifically note the colors of the eyes and the presence of wings. If any flies in the original stock cultures appear to have a much lighter body shade than the other flies, it probably means they are newly emerged. These flies may also have wings that are still folded tightly. This is normal and within a couple of hours the wings will be expanded. If a fly has wings folded together over the back and the abdomen curved downward, it has been overanesthetized and is dead. These flies may be discarded in the morgue.

Sexing In selecting flies for genetic mating, it is absolutely essential the sex of each fly be properly identified. The sex of Drosophila is noticeable through many phenotypic characteristics, with the most reliably distinguished through examination of the genital organs with magnification. If a male and female are compared side by side, note the distinctions in the:

Presence of heavy dark bristles on the male genitalia, which are completely lacking in females

Pigmentation and banding at the posterior of the abdomen when viewed from the dorsal surface

Shape of the posterior tip of the abdomen (Pigmentation and Shape may not be distinct in newly emerged adults)

Presence of a pair of dark, bristle-like sex combs, on the front legs of males

Differences in size between sexes of the same vial

Experimental Crosses Mating and Counting of Drosophila Cultures

1. Every vial should be clearly labeled with the number and characteristics of each parent (mating code). In addition to the vital Drosophila information, the mating date, period, and lab group information should be present.

2. Virgin flies (usually 3-6) selected for a cross should be tapped into a labeled vial containing

approximately the same number of males of another strain. For reciprocal crosses, set up additional cultures but reverse the sex of each strain.

3. Seven to ten days after a cross is started, the parents will be discarded from the vial to avoid

breeding between generations and confusion during counting. Once the F1 flies have emerged, they must be sorted and counted. From these F1 flies, choose approximately six pairs, which need not be virgin, to parent the next generation. The rest may be returned to the vial in case of error.

4. F1 parental flies must be removed as outlined above. Counting of progeny for the F2 generation

should begin within a couple days of the first flies emerging. Cultures generally produce more females earlier in this process. On successive days, the proportion of males tends to increase until the sex ratio balances.

5. Anesthetize and count progeny every other day for 10 days. Counts made for fewer than 10 days

may omit individuals with slow developmental rates due to their sex or a mutation. Counts beyond 10 days risk including flies of the next generation. Do not return counted flies to the same vial.

Concepts to Consider

The F1 Generation If the genes are autosomal what do you expect?

If the genes are X-linked what do you expect?

How can you tell which traits are dominant and which are recessive?

Sib Matings What are your expected results from this cross if the genes are linked? What if the genes are not

linked?

The F2 Generation At least 100 flies from each cross, preferably over 200, are desired to do the calculations for this project. Larger values will provide more certainty in data and less random results. Sort and count each fly based upon phenotypes. Data from all groups may be combined to ensure large values, where needed.

If the genes are autosomal what do you expect?

If the genes are X-linked what do you expect?

How can you tell which traits are dominant and which are recessive?

Are the two mutations linked?

Cleanup You are responsible for cleaning up your mess in the lab room and the area you are assigned for keeping your flies. Follow your instructor’s directions on clean up.

Data analysis 1. Propose a hypothesis for the different mutations

(linked or unlinked genes, autosomal or x-linked, dominant or recessive, Mendelian)

Do this after the F1 generation

Use Punnett squares from the predicted parental strains (including expected ratios) to demonstrate how you came up with this hypothesis

2. Include Punnett squares for the F1 crosses (including expected ratios) to further support your hypothesis

3. Perform Chi-Square analyses for both the F1 and F2 data 4. Determine whether your hypotheses are supported or rejected and describe why using data

(linkage, X-linkage, extensions to Mendelian inheritance, or non-Mendelian inheritance)

5. If your genes are linked, you need to map the genes on the chromosome Directions for completing this are located at the end of the packet. If genes are not linked, you may skip this step.

Conclusion A full set of conclusion expectations may be provided near the end of the lab if additional content or effort is warranted

Chi-Square Analysis – Example of what should be in notebook This should be used for EACH of the genetic crosses for which you have data

Phenotype # Observed

(o) # Expected

(e) (o-e) (o-e)2 (o-e)2/e

Χ2 =

p-value =

Drosophila Record Sheet – Example of what should be in notebook

_______ X

_______

Drosophila melanogaster Data P female

phenotype P male

phenotype P generation pairing date

P generation removal date

F1 female phenotype

F1 male phenotype

F1 generation pairing date(s)

F1 generation removal date(s)

F2 generation phenotype data*

vial # date

counted female male female male

Total

Inferred Ratio**

* It is essential to list out the phenotypes for both females and males - you never know if inheritance

patterns will show sex linkage (It’s always possible to combine values later…)

** This will be compared to your Punnett square hypothesis

Gene Linkage – Determining a Gene Map A. Figure out phenotypic numbers for the cross:

a is the number of flies with both dominant phenotypes

b is the number of flies with one homozygous recessive phenotype

c is the number of flies with the other homozygous recessive phenotype d is the number of flies with both homozygous recessive phenotypes

B. Figure out, do we have a coupling cross or a repulsion cross?

A coupling cross is: P X P double dominant true breeding phenotypes x double recessive true breeding phenotypes

A repulsion cross is: P X P one recessive true breeding phenotype X the other recessive true breeding phenotype

C. Use this equation for a coupling cross

b * c = Z a * d

Use this equation for a repulsion cross

a * d = Z value b * c

D. Use table on the following page to find the approximate map units for the cross. Look in the appropriate column and then look over to the left to get the Crossover Value

E. Multiply the Crossover Value by 100 to get the map units