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Is there any chance that wild flies could have gotten to the cultures? If all of your cultures have developed the trait at once, then that's the best bet. The flight trait is dominant, so any fly that mates with flying flies will have flying offspring. I remember back in biology class about how Fransesco Redi proved spontaneous generation was false. He took a piece of meat and put it in a jar, then covered the jar's mouth with a cloth. The flies laid eggs on the cloth. I'm betting that's what happened to your cultures, except that the maggots hatched out and fell in the medium. It is also possible that they may have spontaniously mutated back to flying ability, but since this just happened suddenly and to all but one of your cultures, this isn't likely.
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