In the present day, archaeologist and puzzle master Professor Layton and his young apprentice Luke Triton solve the mystery of the theft of Big Ben, deducing the culprit to be Layton's perennial adversary, Don Paolo. Following this resolution, the pair return to Layton's office, where they listen to "The Eternal Diva", a record by an old student of the professor's named Jenis Quatlane, prompting them to recall one of their earliest adventures together from three years ago...
Layton is invited by Jenis to attend an opera in which she is performing, centered around the legendary lost land of Ambrosia and the secret of eternal life it claims to hold, which it is claimed will be rediscovered when its queen returns. Jenis believes recent strange occurrences are somehow connected to the opera: in addition to girls disappearing from London, the opera's composer Oswald Whistler has recently adopted a young girl who has claimed to be Whistler's deceased daughter, and Jenis' friend, Melina, restored to life.
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