Female Drosophila store sperm after mating and are capable of laying fertile eggs for many days. Therefore in order to set up a cross between males and females of known genotypes, it is essential to collect virgin females. To do this, in the morning, empty all the flies from bottles that have adult flies just emerging from pupae. Then collect flies that eclose over the next 8 hours and separate into sexes. Virgins have dark meconium in the gut, visible through the ventral abdominal wall. Virgin females do lay some eggs; however, these are unfertilized and will not hatch.
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