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Page 4 Embryonic development (following fertilization) takes place within the egg membrane. The egg hatches and produces a larva which feeds by burrowing through the medium. As the larva grows, it undergoes two molts so that the larval period consists of three stages (instars), the first instars being the newly hatched larva. The final larval stage or third instar may attain a length of 4.5 mm. The third instar stage, toward the end of the larval period, will crawl up the sides of the culture jar, attach itself to a dry surface (the jar, the filter paper, etc.) and form the pupa. After a period of time the adult or imago will emerge. The duration of the above stages will vary with the temperature; at 20°C (68°F) the average length of the egg-larval period is eight days, while at 25°C (77°F) it is reduced to five days. Thus at 25°C (77°F) the life cycle may be completed in about ten days, while 20°C (68°F), fifteen days are required. The sperm received by a female fly during mating is retained, serving to fertilize a number of eggs. Therefore, in an experimental cross between two different strains, virgin females must be used. This can be done quite simply by taking advantage of the fact that females do not mate before twelve hours from the time of emergence. If all the adult flies are removed from a culture with many pupae, all of the females collected within the next twelve hours will probably be virgin. III. Experimental Crosses Perhaps the best way to explain the technique used in making experimental crosses would be to trace the step-by-step procedure, using an example. Careful records should be kept of all experiments, giving dates of crosses, results of crosses, comments about technique, and results of the experiments. For our experiment, we will use the wild type (+) strain and the vestigial wing (vg) strain. 1. Select virgin vestigial (vg) females and place six in each of three culture bottles. Into each bottle place six wild type (+) males. Mark the date and the type of mating on each bottle. (vg O ) + (+ O ) 2/14 2. After seven days remove the parent flies from the mating bottle and discard. There should be many larvae in the bottle. 3. When the flies begin to emerge, examine them and record the characteristics. This is the F1 generation. In this case, since the (vg) is recessive, all of the flies should exhibit (+) characteristics. (Note: If any (vg) flies appear in a bottle, one of the parent females was NOT a virgin and the culture should not be used in the rest of the experiment.) 4. Place six males and six females of the F1 generation in each of three culture bottles. Mark each bottle with the date, the type of original mating, and the generation. (vg O) + (+ O ) F1 2/28 5. Remove the F1 flies from the culture bottles after seven days and discard them.