Drosophila Introduction

Experimental Genetics Biol 334

An Introduction to Drosophila melanogaster

The fruit fly, Drosophila melanogaster, has been used extensively for basic genetic research purposes since the time of T. H. Morgan, who along with his colleagues made many important discoveries using this small dipteran in the early 1900's. Drosophila is a good organism for genetic research purposes because it 1) has a short life cycle (less than 2 weeks), 2) is very prolific (a single female will lay hundreds of eggs), 3) has a large number of morphological and biochemical mutants to be studied, and 4) has unusual chromosomes (polytene chromosomes) that help in cytological investigations.

Drosophila has a typical dipteran life cycle, passing through the following four stages "egg," larva, pupa, and imago (adult). The minimum duration of the pre-adult stages at 24ºC is: egg = 1 day; larva = 4 days; pupa = 4 day.

Life Cycle: The female receives a packet of sperm from the male during copulation and fertilization in Drosophila is internal. The "egg" that the female lays has already begun development and is actually an early embryo. The embryo will remain in the egg case until the larva is ready to hatch. The white eggs are small but visible with the naked eye and are seen to have two antenna-like projections sticking out from one end. The whitish larva that hatches from the egg case about a day after being laid and then spends its time eating its way through the food. There are three stages, or instars, in the larval stage. At the end of the first and second instar the larva molts, that is it sheds its exoskeleton to allow for growth. Therefore, each instar is larger that the previous one. Late third instar larvae are often seen crawling on the side of the vial. These larvae find a dry place, stop moving, and the cuticle hardens into the puparium. These three larval instars take a minimum of four days. Feeding third instar larvae (not those preparing for pupation) are the best for observing polytene chromosomes. Metamorphosis occurs during this pupa stage as the adult fly, or imago, develops inside. The pupal stage lasts at least 4 days and at the end of that period the imago ecloses (or emerges) from the pupal case. The newly emerged fly's wings are folded and its body is elongated and have little color. Adult males can produce sperm almost immediately, but adult females cannot be inseminated for at least 8 hours. Therefore, females can lay eggs on the day they eclose from the pupal case. So, the minimum length of the Drosophila life cycle is 9 days (egg=1, larva=4, pupa=4, adults produce eggs the day they eclose). In actuality, at 24ºC, the next generation of flies are not usually seen until the 10th day after a cross has been made. A knowledge of this life cycle is necessary in order to be able to use this organism in genetic experiments.

Culturing Flies: Flies will be cultured in plastic vials at room temperature and fed Carolina Biological Supply's Instant Drosophila Medium or Nutri-Fly prepared fly food. To set up a cross, flies must first be anesthetized. Ether or Carolina's Fly Nap may be used. However, we will be using cold to anesthetize flies. Knock flies into a clean, empty vial and plug with cotton. Get a frozen, sealed petri dish (water inside) from the freezer and let it thaw for about 5 min. Place the vial in ice until flies are completely motionless. Immediately dump the flies onto the painted side of the petri dish. Flies are best sorted and manipulated under a dissecting microscope with a fine artist's brush. Be careful not to turn on the bottom light of the dissecting microscope as this may generate enough heat to kill the flies.

Fly Food: To set up a cross, an equal volume of instant food and water should be put into a clean vial. For Carolina medium, put about 3/4 of a "cup" (small plastic cup supplied) of the dry food mix in a vial then add about 3/4 "cup" water. However, use one cup for the dry food (keep that cup dry) and a separate one for the water. After the mixture sets and the water has all been absorbed, add a very small quantity of live yeast. Two or three grains is enough. Much more than that will grow and compete with (kill) developing larvae. Plug the vials with cotton (or spong plug). If you make more food vials than you need, be sure to plug them. Never leave food vials open for more than a few seconds. Always clean up your mess when you are finished: place the dry cup in the food bag and close it then wipe down the counter with a wet paper towel. Nutri-Fly bottles will be made up beforehand.

Transferring and Manipulating Flies: If the flies you are placing in the vial do not need to be anesthetized, you can, with a little practice, get them in the new vial without anesthetizing them. For example, you may with to transfer some adults from one vial to one with fresh food. To do so, tap the vial with flies in it on the table top a couple of times to knock the flies to the bottom (not too much or they will stick in the wet food), then quickly remove the plug and invert a vial with fresh food over this vial. Holding the two vials together, turn them over and tap until all flies fall into the new vial. Watch the food in the old vial as you do this so you do not dislodge it and dump it into the new vial. Then, quickly plug the new vial. If you are adding anesthetized flies to the new vial, be sure there is no water on the inside of the vial, lay it on its side, and brush the anesthetized flies into the vial so they lie on the side of the vial. Plug the vial and wait for them to recover before standing the vial upright. This prevents the anesthetized flies from becoming stuck in the food.

Crosses: The day a cross is set up will be counted as DAY 0. To make a cross, place 3-10 females and 3-10 males in vial with fresh food. On DAY 3 to 7 these parents can be discarded (or transferred to fresh food if you need a lot of progeny). Beginning on about DAY 10 you will begin to see the next generation (F1) appearing. If you are going to rear an F2 generation, you must collect F1 flies before DAY 19 and place some of the males and females in a new vial. The reason this must be done before DAY 19 is that beginning on that day, the flies you think are F1 flies may themselves be F2 flies, since the minimum life cycle is 9 days (9+9=18). Flies are discarded by dumping anesthetized flies into the "morgue," which is a mixture of water and alcohol (or water and detergent). Always replace the cork in the morgue to keep the alcohol for evaporating. For an example of setting up a cross, click here (.pdf) and to download a printable blank calendar.

Distinguishing Males from Females: In order to set up crosses, it will of course be necessary to be able to distinguish males from females. In mature adults, this is no problem. The slightly smaller male has a broad black band on the dorsal side of the posterior abdomen. This band is posterior to three black bands. The female's abdomen is slight more pointed at the posterior tip, but lacks the broad black band. Instead, the stripes continue to the tip. This characteristic will be good enough to distinguish males from females in most cases. However, newly emerged flies present a problem since the shape of the abdomen is distorted and there is little pigment present for several minutes. For newly emerged adults (they look pale and may still have their wings folded up), the best differentiating characteristic is the appearance of the genitalia. The male genitalia with its surrounding bristles on the ventral tip of the abdomen are absent in females. Also, sex combs can be seen on the front legs of males under high magnification.

Collecting Virgins: When beginning a cross, there is one more problem. Drosophila females can store sperm for a long time, so if a female has previous copulated with another fly and you use that female in a cross, the progeny you get from a cross you set up may not actually be the progeny from the male you added, but rather those of a previous acquaintance. The only way to get around this problem is to be sure you are beginning with virgin females. Again, a knowledge of the Drosophila life cycle makes it possible to collect virgins. Since the female cannot be inseminated within 8 hours after it ecloses from the pupa case, any fly 8 hours old or younger is a virgin. So, if you are setting up a cross, the females must first be collected from a bottle in which flies are emerging. For example, you might come into lab at 8:00 AM and discard any flies in such a vial (you don't know how old these females are). If you then return to lab that afternoon at 3:30 PM and collect any females (and keep them separate from males on fresh food), they will be virgins since they are less than 8 hours old. You can then use these females for your cross. You may have to repeat this procedure several times to collect enough virgin females. This "8-hour rule" at room temperature and become a "14-hour rule" at 17°C, which makes virgins collecting easier.