Lab 5- Drosophila Genetics 

Introduction 

The common fruit fly Drosophila melanogaster is an ideal model organism for genetic studies. It is small, easy to raise, and relatively easy to observe and manipulate. In addition, it has a short generation time and is developmentally complex. This developmental complexity is reflected in the results of the recent analysis of the human genome: the human genome contains only twice as many genes as the fruit fly and many genes are identical.  

As fruit flies develop, they pass through several well-defined life stages, which are illustrated below. From a fertilized egg, which may be difficult to see in the vials that serve as the lab homes of fruit flies, a larva hatches after 1-2 days. This larva, called the first instar, is the first of several larval forms that eat, molt, and develop. Molting is the process of shedding the cuticle, or external covering, and mouth hooks. When the developing fly molts, the new larva is called a second instar larva. After a second molt, the fly enters its third and final larval phase, the third instar.  

Together these larval phases last about 6 days, and you will be able to see the larvae eating channels through the medium in the vials. When the cuticle of the third instar darkens, the developing fly is then called a pupa. Metamorphosis to the adult form takes place during the pupal phase, which lasts another five or six days. About one day before emergence, the folded wings and eye pigments will be visible through the cuticle (which is now called a puparium). After a total of about 12-14 days, an adult fly emerges from the puparium. Females can start laying eggs two days after emergence.  

 

 

In this experiment, you will perform a genetic cross between a wild-type fruit fly and a mutant fly. The wild type has red eyes and normal wings; the mutant you will use has sepia (grayish-brown) eyes and is wingless (called apterous). Both strains are true-breeding strains (i.e., ++/++ and apap/sese, respectively), and the genes are autosomal. Drosophila has four chromosomes.

Procedures:  

Day 1 

1.         Observe the wild-type flies in your vial. Look for differences between the sexes. You should be able to distinguish males from females by their size, abdomen shape, and abdomen color. The males are shorter and have a rounded posterior abdomen that is dark in color. When you observe the flies under the stereomicroscope, you will see other differences between the sexes. The drawings below   illustrate some of the sex differences.

 

 

 

2.         Insert the anesthetizing wand into a bottle of FlyNap. As you withdraw it, pass the wand bristles gently against the plastic cap at the mouth of the bottle to remove excess liquid. 

3.         The following steps must be done quickly to prevent flies from escaping. Watch the demonstration and practice with an empty fly vial before trying it with a vial of live flies. 

            a.         Gently tap the bottom of the plastic vial on the table or your thigh. The flies near the top of  the vial should fall to the bottom. 

            b.         In one smooth motion, use one finger to compress the plastic plug to one side of the vial and insert the wand through the opening. Try not to get any anesthetic on the plastic plug  or on the sides of the vial. 

            c.         Release the plug, allowing it to hold the wand in place with the bristles protruding beneath the plug. (You will find that a slight kink in the wand will prevent the bristles from contacting  the side of the vial.) 

            d.         Lay the vial on its side. 

4.         Allow the FlyNap to work for 4-5 minutes, or slightly longer if some flies continue to move. The flies should begin falling to the bottom of the vial after 2-3 minutes. Remove the wand using the same technique you used to insert it. (Flies should remain anesthetized for 40 minutes.) 

5.         Tap the vial to loosen any flies that may be stuck to the sides, and then dump them onto a white viewing card.  

6.         Observe the flies under the stereomicroscope and develop some skill distinguishing males from  females. Note the eye and wing phenotypes. Sketch examples of each sex.  

7.         Repeat the anesthesia treatment with a vial of mutant flies. Note the differences between the  phenotypes of wild-type and mutant flies and sketch examples of the differences. (After making  crosses, you will need to be able to recognize and separate quickly the progeny flies by sex as well  as wing and eye phenotypes.) It is also interesting to look for eggs and to observe the various larval  and pupal stages. 

8.         With your partner, isolate 3 to 5 wild-type male flies and 3 to 5 mutant females. (Alternatively, you   may reverse the sexes; just keep track of your cross.) Place them together in one empty vial and label with the genotype cross, date, and your initials. This is your parental cross.

 9.         Place the vial at 25°C. Predict the F₁ phenotypes and genotypes. 

10.        Obtain a vial of F₁ flies, the result of your parental cross. Anesthetize these flies according to Steps 2-5 above and score for sex, wing, and eye-color phenotypes.  

11.        Isolate 4 or 5 of the F₁ male flies and 4 or 5 F₁ females. Place them together in one vial (F₂) with growth medium and label the vial with the genotypes of the crossed flies, date, and your initials.  Place the vial at 25°C. 

12.        Based on the results thus far, develop a hypothesis that predicts the outcome of the F₁ cross. 

Day 7 or 8 

13.        Anesthetize the flies in your F₂ vial. Put these (F₁) adults in the morgue. (It is important to remove    adult flies before new flies begin to emerge from the pupae; adult males will mate immediately with newly emerged females.) The F₂ flies should begin to emerge on Day 11. 

Day 12 

14.        All the flies in the vial are now F₂ flies. Anesthetize the F₂ flies and score them.  

            a.         Label four vials with the appropriate phenotypes. (What are these?) 

            b.         Count and sort the flies into the appropriate vials. 

Days 12-15 (or longer;teammates should trade off days.) 

15.        Anesthetize the new F₂ flies as they emerge each day and score them, saving them in the same vials used previously. (If you are careful, you can quickly add the anesthetized flies to the vial of active flies by thumping the vial, quickly retracting the plug, and dumping the new flies into the vial using a slightly creased sheet of paper. Otherwise, anesthetize the old flies as well.)

 

Assignment 

Type a two-page summary that includes the following: 

            a.         the hypothesis that you were testing in this lab (Step 12), stated clearly and precisely;

            b.         a neat and easy-to-interpret table summarizing your results;

            c.         a Chi-square analysis of those results (and class results if we have them); and

            d.         a brief conclusion that comments on the soundness of the original hypothesis and any potential sources of error.

Materials 

various vials of Drosophila                                               paint brush

5 empty vials and 1 vial with medium                               white card

stereomicroscope                                                          labels

FlyNap                                                                          morgue

anesthetizing wand                                                        notepad

  

References: