APPENDIX
K
Culture of Drosophila
A. LIFE CYCLE OF DROSOPHILA There are four distinct stages in the life cycle of the fruit fly (Fig. K-1): egg, larva, pupa, and adult. At 25°C, a fresh culture of Drosophila will produce new adults in 9 or 10 days: about 5 days in the egg and larval stages and 4 days in the pupal stage. The adult flies may live for several weeks. Drosophila cultures should not be exposed to high temperatures (e.g., 30 °C), because such exposure results in sterilization or death of the flies, nor to low temperatures (e.g., 10 °C), because it prolongs the life cycle (perhaps 57 days) and reduces viability.
1. Egg The adult Drosophila female starts to deposit eggs on the second day after emergence from the pupa. Each egg is about 0.5 mm long and is ovoid in shape and white in color. Extending from its anterior end are two thin stalks that expand into flat-
tened, spoonlike terminal parts. Embryonic development of the egg takes about one day at 25°C.
2. Larva The larva is white, segmented, and wormlike. It has black mouth parts (jaw hooks) in a narrowed head region, but no eyes and so is completely blind. The larva also lacks appendages and must literally eat and push its way through its environment. It breathes by trachea and has a pair of conspicuous spiracles (air pores) both at the anterior end and at the posterior end of the body. The larval stage in the Drosophila life cycle is one of rapid eating and growing. It consists of three subdivisions called instars. The first and second instars terminate in molts. Each molt consists of a complete shedding of the skin and mouth parts of the larva and is the mechanism by which the animal grows. The third instar terminates in pupation. Just before pupation the animal ceases to feed, crawls to some relatively dry surface, and
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APPENDIX K
FIG. K-1. Life cycle of Drosophila melanogaster .
CULTURE OF DROSOPHILA
everts its anterior spiracles. The larval stage takes about 4 days at 25°C for completion, at which time the third instar is about 4.5 mm long.
3. Pupa The pupal stage is considered a reorganizational stage of the fly's life cycle in which most larval structures are destroyed and adult structures are developed from embryonic tissues called anlagen (imaginal discs). These embryonic tissues have been lying dormant in the animal since their differentiation in the egg stage. The animal pupates within the last larval skin, which is at first soft and white but slowly hardens and darkens in color. The transformation that takes place in the pupa results in the development of the body form and structures of the adult ( imago). The pupal stage takes about 4 days at 25 °C to be complete, at which time the adult fly emerges from the puparium (pupal case).
4. Adult The adult stage is considered the reproductive stage of the life cycle. The fly emerges from the puparium by forcing its way through the anterior end. At first the adult fly is greatly elongated, with its wings unexpanded. Within an hour, however, the wings expand and the body gradually attains the more rounded form typical of an adult. The adult is also relatively light in color when it first emerges, but within the first few hours it darkens to its characteristic adult colors. Adult Drosophila mate about 6 hours after emerging from the puparium. The sperm are then stored in the spermathecae and ventral receptacles of the female and are released gradually into the oviduct as eggs are produced and passed through the oviduct into the vagina. As stated earlier, the female begins to deposit eggs about 2 days after it has emerged. It may deposit as many as 50 to 75 eggs per day for the first few days. Thereafter, its egg production decreases with time. The average lifespan of adult flies is 37 days at 25 °C.
B. CULTURE MEDIUM A number of media have been developed for the culture of Drosophila. However, the easiest to use are the “instant media” available from many bio-
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logical supply houses. A concentrated medium requires only the addition of water to be immediately usable. No cooking is necessary. Pour the medium into chemically clean bottles. These bottles may be of any size, but 4-ounce (120 ml), wide-mouthed bottles or half-pint milk bottles are satisfactory. (It is best to transfer the mixture to a beaker for pouring. None of the medium should come into contact with the neck of the bottle.) Fill the bottles with medium to a depth of about 1 inch (2.5 cm). Then place a piece of nonabsorbent paper such as brown wrapping paper in each bottle so that it extends down into the medium (a double piece of paper is more satisfactory). The paper should be about an inch wide and should extend upward to a point about onehalf inch (12 mm) below the neck of the bottle. This paper provides a dry place on which the larvae can pupate. Stopper the bottles with cotton plugs (such as those used in bacteriology) or with disposable foam plugs, which are more convenient. Sterilize the bottles in an autoclave at 20 pounds pressure for 20 minutes. At the same time, it is wise to sterilize other materials that you may wish to use in handling flies. Before placing the flies in the cooled bottles of medium, shake a small amount of dry yeast on the medium. The yeast will grow and serve as food for the developing fly larvae.
C. ETHERIZATION OF FLIES It is necessary to etherize flies in order to keep them inactive while they are being examined and when they are being transferred into culture bottles for matings. The etherizing bottle can consist of any small container that has an opening the same size as that of the culture bottle and a tightfitting cork. Into the bottom of the cork, tack a pad of cotton. Just before employing the technique outlined below (Fig. K-2), douse the cotton with a few dropperfuls of ether. 1. Shake the flies down into the bottom of the culture bottle by tapping the bottle on a rubber pad on a desk. 2. Remove the cotton plug from the culture bottle and, in its place, quickly insert the empty etherizing bottle. Make certain you have blown any residual fumes from the etherizing bottle before you invert it over the culture bottle.
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APPENDIX K
FIG. K-2. Procedure for etherizing flies.
CULTURE OF DROSOPHILA
3. Reverse the position of the two bottles so that the etherizer is now on the bottom. 4. Hold the two bottles firmly together and shake the flies from the culture bottle into the etherizer. This is best done by tapping the sides of the culture bottle horizontally so that the flies are dislodged and fall into the etherizer. Make certain you do not shake the food loose in the culture bottle. 5. After you have shaken the flies into the etherizer, remove the culture bottle and quickly replug it. Simultaneously plug the etherizer with its cork. The cotton on the cork should feel moist with ether but not wet. 6. Observe the flies in the etherizer. Thirty seconds after they have stopped walking around, pour them out onto your counting plate. If the flies have been overetherized (killed), their wings will be extended at right angles to their bodies. 7. Normally, the flies will remain etherized from 5 to 10 minutes. If they begin to awaken on the plate, they can be re-etherized as follows: on the inner surface of a Petri dish, tape a piece of paper toweling. Moisten the paper toweling with ether, and place the Petri dish over the flies to form an etherizing chamber.
D. DISTINGUISHING THE SEX Examination of the external genitalia under magnification is the best means of distinguishing the sex of flies. Only male flies exhibit darkly colored external genitalia, which are visible on the ventral side of the tip of the abdomen. The following characteristics may also be helpful in distinguishing males from females: 1. Size: Females are usually larger than males. 2. Shape: The abdomen of the male is round and blunt, whereas that of the female is sharp and protruding. The abdomen of the male is relatively narrow and cylindrical, whereas that of the female is distended and appears spherical or ovate. 3. Color : Black pigment is more extensive on the abdomen of the male than on that of the female. On the male, the markings extend com pletely around the abdomen and meet on the ventral side. On the female, the pigment is present only in the dorsal region.
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4. Sex Combs: Only males have a small tuft of black bristles called a sex comb on the anterior margin at the basal tarsal joint of each front leg. Magnification is necessary to see the sex combs.
E. ISOLATING VIRGINS Females of D. melanogaster can store and use sperm from one insemination for a large part of their reproductive lives. As a result, only virgin females should be used in making crosses. Females of this species can mate 6 hours after they have emerged from the puparium. Therefore, a procedure must be followed that will ensure the collection of females that are no more than 6 hours old. A number of possible procedures are available to you, depending on your preference and the accessibility of the laboratory. If the laboratory is generally accessible, 1. Shake out and discard into the morgue (a bottle containing ethanol, oil. or detergent) all adult flies (10 or more days after the introduction of the parental flies). This step should be done early in the morning (8–10 A.M.) for most efficient results. 2. Return to the laboratory within 4 to 6 hours, and examine the newly hatched flies. The females in this group may be presumed to be virgin and can he used in experimental matings. If the laboratory is not generally accessible, 1. Collect darkened pupae from the paper or sides of the bottle with a fine camel-hair brush. 2. Turn each pupa so that the legs are visible through the pupal case. Examine under high power the uppermost or proximal joint of the tarsi of the front legs. The presence of sex combs indicates a male. 3. Collect the pupae that are without sex combs, hence female, and place them individually in fresh culture bottles. After the flies have hatched from their pupal cases, examine them to be certain that no male pupae have accidentally been placed in a culture. All the flies that are female can be used in experimental matings.
REFERENCE Levine, L. and N. M. Schwartz. 1973. Laboratory Exercises in Genetics. Mosby.
