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Mario and Luigi are first shown in the episode Mr. Fugu and Chowder and Chowder is assigned to go with Mr. Fugu and take him to food places.
After they go to the taco stand and the thrice cream stand, they go into the rib place then comes mario and luigi. He takes them to a rib hat place and if you see closely you can see mario and luigi.
Mario and Luigi share a resemblance to the video game characters.