Breeding Experiments in Drosophila Melanogaster

Life cycle of Drosophila melanogaster
Drosophila melanogaster is a common fruit fly used as a test system and has contributed to the establishment of the basic principles of heredity. It is also called the “Cinderella of Genetics”. Drosophila melanogaster is a dipterous, holometabolous insect. It has a characteristic larval stage preceded by the egg and succeeded by the pupalstage.

Egg
Egg is about 0.5 mm in length, ovoid in shape, and white. Extending from the anterior dorsal surface, there is a pair of egg filaments. The terminal portion of these filaments are flattened into spoonlike floats. This floats keep the egg from sinking into the semi-liquid medium.

Larva
The larva hatches out from the egg. It is white, segmented, and wormlike. The head is narrow and has black mouth parts (jaw hooks). The larva undergoes 2 moults, so that the larval phase consists of 3 instars. After this stage, the larva crawls out of the medium and finally attaches to the inner drier surface of the bottle. This culminates in pupation.

Pupa
Soon after the formation of the “pupal horn” from the anterior spiracle, the larval body is shortened and the skin becomes hardened and pigmented. The pupa is considered a reorganization stage. During this process, most of the
adult structures are developed from the imaginal disc. A fully transformed adult fly emerges out through the anterior end of the pupal case. At the times of eclosion, the fly is greatly elongated and light in color, with wings yet to be unfolded. Immediately after this, the wings unfold and the body gradually turns dark and brown. After 6 hours of emergence, the adult fly attains the ability to participate in reproduction.

Adult
The body is divided into head, thorax, and abdomen. The head has a pair of compound eyes and a pair of antennae. The thorax is divided into 3 segments— prothorax, mesothorax, and metathorax, each with a pair of legs. The mesothorax has a pair of wings and the metathorax has a pair of halters. The abdomen is segmented in 4 or 5 sections in males and 6 or 7 in females. The abdominal tip in males is darkly pigmented.

Morphology of Drosophila Melanogaster
The body of an adult Drosophila melanogaster is divided into 3 parts, namely the head, thorax, and abdomen.

1. Head. The head is composed of 6 fused segments, a pair of antennae with
   plumose aristae, and a licking proboscis without mandibles. On the dorsal
   side of the head between the compound eyes are 3 simple eyes called
   ocelli. Bristles are found on the head.
2. Thorax. It is composed of 3 fused segments, namely the prothorax,
   mesothorax, and metathorax. All 3 segments have a pair of wings. The
   metathorax has halters (reduced wings).
3. Abdomen. The abdomen consists of 7 or 8 visible segments in the female
   and 5 or 6 segments in male.

Differentiation between Male and Female Drosophila

Media Preparation
Drosophila melanogaster, like other animals, requires an optimum temperature for its survival, growth, and breeding. The optimum temperature for the maintenance of Drosophila melanogaster is between 20°C to 25°C. The temperature around and above 31°C makes the flies sterile and reduces the oviposition, and may result in death. At any lower temperature, the life cycle is prolonged and the viability may be impaired.

The routinely used food media for the maintenance of Drosophila melanogaster is cream of white agar medium. The ingredients of this media are:
1000 mL of distilled water
100 gm of wheat flour (sooji)
100 gm of jaggery
10 gm of agar agar
7.5 mL of propionic acid, and
Yeast granule.

Heat-sterilized bottles should be used for preparing culture. Similarly, sterilized cotton has to be used to plug the bottles. As the condition of the medium deteriorates with time, the flies have to be transferred from old to new bottles, with fresh culture medium at least once every 3 weeks.

Procedure
Take a clean vessel and boil 1000 mL of water. Then add 10 gm of jaggery and stir well. To this add 10 gm of agar agar, which acts as a solidifying agent. Once it boils, add 100 gm of sooji. Then add 7.5 mL of propionic acid, which
acts as an antimicrobial agent. By constant stirring, the medium becomes a viscous fluid. The hot mixture is transferred into the culture bottle. The bottles are left for cooling, yeast is added, and they are plugged with cotton. Bottles are ready to use only after adding the yeast solution.

Etherization
When flies have to be analyzed, either for routine observation or for experiments, they are anaesthetized to make them inactive. The procedure is to transfer flies from the media bottle to another empty wide-mouthed bottle, referred to as an etherizer. The mouth of this bottle is to be covered with a stopper sprayed with ether. It takes a minute or so to anaesthetize flies. After this, if etherized flies revive before completion of observation, they have to be re-etherized by using re-etherizer. The re-etherizer is an ether-soaked filter paper fitted in a petri plate, which has to be placed over the flies on the glass plate.

Sexing
Adult flies are 2–3 mm long, while females are slightly larger than males. The males carry a sex comb on the first tarsal segment of the first leg. Males can also be identified by the presence of black pigmentation at the tip of the rounded abdomen. The tip of the female’s abdomen is pointed and nonpigmented. After the separation of the 2 sexes, the unwanted flies should be discarded immediately into the morgue (a bowl of mineral oil or detergent in water).

Isolation of Virgins
In many experiments with Drosophila, it is essential that the sperm of a particular genotype (male) is used for fertilization of a particular female. To ensure this, it is often essential to isolate virgin females. The females can store and utilize the sperms from 1 insemination for a large part of the reproductive life. Females that have any chance of being nonvirgins should not be used for crosses. For ensuring virginity, females are removed before 6 hrs of their emergence and males are removed from the culture bottle before 12 hrs of emergence.
Making and Conducting Crosses To make crosses between different strains 1–10 virgins from the first strain are mated in a culture bottle with the corresponding number of males from other strains. The reciprocal mating of the 2 strains is also done. As soon as the cross is made, the bottle is marked with the nature of the cross and the date of crossing. If the larva does not appear after 5–7 days, then the culture is discarded. If the culture is successful, then the parents are discarded and the already-laid eggs are allowed to develop into adults.

Analysis of the Progeny
The aim is to understand the pattern of inheritance of a character from parents to offspring and to subsequent generations. Therefore, each progeny (F1, F2, and test cross) has to be carefully analyzed and classified according to the phenotype and sex of each individual. Utmost care must be taken to record the number of flies in each category. From each experiment bottle, the counting must be restricted to the first 7 days from the third day of eclosion. A minimum of 200 flies must be analyzed from each of the F1, F2, and test cross progenies.
Statistical Test and Confirmation of Results The data obtained from the analysis of the progeny have to be tested with an established hypothesis. This has to be done to ascertain whether the observed data work with the hypothesis or not. The routinely used statistical test for such an experiment is the Chi square test:

x2 = ε (0 – E)2
E________

The chi square test obtained from this test and the degree of freedom (df ) df = n – 1 are checked for the level of significance in the chi square probability table.

If the calculated value of the chi square is less than the table value at a particular level of significance, the difference between the observed and expected frequency is not significant. Then we have to accept the hypothesis.

On the other hand, when the calculated value is more than the table value, the difference between the observed and expected value is significant, then we have to reject the hypothesis.