Drosophila: Introduction and Genetics

Author: Betty Ann Wonderly Abstract: Introduction to Drosophila including life cycle, sexing, and identifying mutations. Used as the introductory lab to Drosophila Genetics . DROSOPHILA GENETICS LAB I PURPOSE: To become familiar with the stages of the life cycle, the sex differences and mutations of Drosophila. To perfect techniques for anesthetizing, identifying, and counting Drosophila. DROSOPHILA GENETICS LAB 2 PURPOSE: To discover how certain traits are inherited in Drosophila.

DROSOPHILA GENETICS LAB I PURPOSE: To become familiar with the stages of the life cycle, the sex differences and mutations of Drosophila. To perfect techniques for anesthetizing, identifying, and counting Drosophila. BACKGROUND MATERIAL: LIFE CYCLE There are four distinct stages in the life cycle of the fruit fly: egg, larva, pupa, and adult. At 25 C a fresh culture of Drosophila will produce new adults in 9-10 days. Five days in the egg and larval stages and four days in the pupal stage. The adult flies may live for several weeks. Drosophila cultures should not be exposed to high temperatures (e.g.. above 30 C) which result in sterilization or death of the flies no to low temperatures (e.g. below 10 C) which result in a prolonged life cycle (perhaps 57 days) and reduced viability. The adult Drosophila female starts to deposit eggs on the second day after emergence. Embryonic development of the egg takes about one day at 25 C. The larva is white, segmented and worm like. The larval stage is a feeding stage and consists of three subdivisions called instars. The first and second instars stages end in molting which allows the larva to grow. The third instar ends with pupation. Prior to pupation, the animal stops feeding and crawls to some relatively dry surface and the cuticle hardens and darkens to form the puparium. Metamorphosis occurs in the puparium and takes about four days. The pupa begins to darken just prior to the emergence of the adult fly. Most flies eclose (emerge) from the

pupa in the early morning hours. CULTURE MEDIUM We will use instant Drosophila Medium. It is essential that you use clean vials and plugs as mites and fungus infections can wipe out your Drosophila and lead to agony as you have to start your whole experiment over. C are at the beginning will save you grief in the long run. Mix equal amount of medium and tap water in the bottom of a vial. Try to keep the sides relatively clean. you do not need more than an inch of medium in the bottom. Too much can be as bad as not enough. Sprinkle a few grains of dry yeast on the medium. After about 1 minute flies can be introduced into the vial. Always place anesthetized flies on the side of the vial and leave the vial on its side until they awaken. Anesthetized flies can become stuck in the medium and die. Be sure to label the vial with the type of cross, the date, and your name. ANESTHETIZING FLIES We will use carbon dioxide and ice to anesthetize our flies. This is the safest, cleanest, and least obnoxious way of anesthetizing the flies. We will have several carbon dioxide generators. These are side arm flasks with rubber tubing attached to the side arm. The other end of the tube will have a Pasteur pipette attached. Drop 2 Alka Seltzers into about 2 inches of fresh water in the bottom of the flask. Replace the cork. Place the vial of flies on its side and insert the Pasteur pipette between the vial and the plug. Watch until all flies have stopped moving. Remove the plug and empty the flies on a piece of filter paper in a plastic petri dish. Place the dish on a container of ice that you can get from the freezer. Keep the lid on and the flies on ice while you are observing them. You may remove the top for separating and moving but it is a good idea to keep the lid on most of the time while you are just observing. If you are placing the flies back into a culture vial be sure you keep the vial on its side until the flies awake. If you will not need the flies for further use place them in the morgue. Be sure you are through with them before you send them to meet their maker. You do not have the ability to resurrect fruit flies! Please return all ice containers to freezer when not in use. SEXING FLIES It is essential that you learn to distinguish the males from the females. With practice this becomes relatively easy and you will probably be able to sex the flies without magnification. At first it will be best to use the dissecting scopes. There are several ways of distinguishing the males from the females. I think the most reliable way is to look for the sex comb on the front legs of the male. They do not appear on the front legs of the female. This characteristic is present even in the pupa. In older flies the posterior part of the abdomen is quite dark in males and considerably lighter in females. The tip of the abdomen in more rounded in males than in females. In general the male is smaller than the female. Use a camel hair brush for moving flies around. It is easy to damage the flies if you use pencils or other objects.

PROCEDURE You and your partner will be given a vial of flies. Label this with your name and the date. Guard it with your life! Anesthetize the flies in your culture and observe. Practice sexing the flies. When you are familiar with your flies look at the other types of flies in the lab. You will be expected to recognize all the different mutants we have. Make a new vial of culture medium and return your flies to the new medium. Be sure to label the new culture. This will serve as a back up in case anything happens to your original culture. Bring a shoe box and create a lovely home for your new fly culture. Clean up and prepare for a quiz over the material in this lab. Record the making of your new culture in you log book. Each person must keep his own log book. Write in this book only what you do and observe. Log book checks will not be announced and are vital to your grade. Keep you book up to date and bring it to class every day. If you do not have it when I check you will receive a 0. No exceptions! Be sure to date each entry and to record each observation and every cross you make. Be sure to put your flies away and to keep your supplies with your flies. If you leave your supplies out you will have to buy them back and if you lose your flies WOE BE UNTO YOU! Please be careful. DROSOPHILA LAB II I. PURPOSE: To discover how certain traits are inherited in Drosophila. II. MATERIALS: Drosophila medium, Drosophila cultures, Anesthetizer, brush, dissecting scopes, infinite patience. III. PROCEDURE: A. ISOLATING VIRGINS: A female Drosophila can store and use sperm from a single insemination for the major portion of her reproductive life. Thus, it is necessary to select virgin females for genetic crosses. The old double standard holds for Drosophila also, therefore it is not necessary that the males be virgins. Older males will mate with newly hatched females. Therefore it is essential that all adult flies be cleared from the vial that is being used to hatch virgin females. It is a good idea to keep these flies as part of your backup culture rather than killing them. When pupae are ready for hatching they will darken. Clear all adult flies from the vial as late in the evening as feasible. Flies tend to hatch in the early morning in the greatest numbers. Check the vial within 10 hours of clearing and remove and sex any flies present. Begin your 10 hour timing again and continue to remove and sex the flies that

hatch within ten hours. If you will devote yourself to this time schedule for one or two days you should be able to get all the virgins you need. Remember that insuring virginity and correct sexing of these flies is crucial to the success of your experiment. B. MAKING CROSSES: The strains of flies we are using are all true breeding so are assumed to be homozygous. You will be concerned only with the traits listed on the vial for your flies. + is used to represent the wild type flies. Using procedures studied for isolating and identifying flies, select at least 3 virgin females of the type needed fo r the cross assigned to you. Place these virgin flies and at least three males of the type needed for the cross assigned to you in a vial with fresh culture medium. You may add these flies one or two at a time if necessary. You do not have to add them all at once. For instance, if you get only one virgin female you may place her in the vial alone. However, try to limit the number of times you must anesthetize your flies as this sometimes decreases viability. Be sure you label the vial as your P 1 cross and record all your data in your log book. Locate a group that is making the reciprocal of your cross. For example, if your cross a wild female with a white-eyed male the reciprocal would be a white-eyed female with a wild male. Observe both crosses daily and record your observations in your log book. After about 7 or 8 days remove the parent flies. Anesthetize them and place them in the morgue. We will have a wake and mass burial later. Flies of the F1 generation will begin to appear about 10 days after mating. After several flies have appeared, anesthetize them, identify them as to characteristic and sex, record your results and place them in a new vial with fresh medium. This should be labeled as your F1 cross. Since all members of the F1 are the same it is not necessary to obtain virgins. You should cross at least 6 females and 6 males. The numbers are not critical, however, the more flies you cross the more flies you will get in your F2 and the better your results will be. Continue to identify, count and record the flies that hatch out in your F1 vial until no more flies emerge. Observe the F1 vial daily and record your results. As these flies hatch, anesthetize, identify, and count them. Remember to note the characteristics and sex of each fly. Once the flies are counted they may be placed in the morgue. Check with the group that did your reciprocal cross and record their total counts for each generation. Determine the F2 phenotyp ic ratios for both crosses and form an hypothesis as to the manner of inheritance. Calculate the X2 values for each of the crosses. IV. LAB REPORT: This will be a formal lab report and will contain the following: Page 1 Cover page- Name, date, Title of Lab

Page 2 Abstract- Brief summary of the rationale and the experiment. Page 3 Hypothesis- Even though you have included this in your abstract, please place it on a separate sheet and label it your hypothesis. Page 4 Methods and materials- a brief summary of the procedures used. You may assume that your reader is familiar with basic techniques and need not give details of making medium, identifying flies, etc. Page 5 Data charts for F1 and F2 for your cross and for the reciprocal cross. Page 6 Conclusion- either support or refute your hypothesis using your X2 data. If necessary, offer a new hypothesis or cite specific errors in your procedures to explain your data deviations.