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They make their final appearance in Stage 6-4: Shannon's Pet, where there is a puzzle involving tossing bones in order to lead the Poison Chihuahua near the Fatty resting below a leaver, where once it reaches that location, it will bark, startling the Fatty awake and causing him to bump his head on the leaver, pushing it up and causing the bridge to extend. After this stage, they are not seen again until Stage 3-5: Puppy Kingdom, where multiple specimens of them are located, however, they do not pose any threat to Gene. The first Poison Chihuahua is seen in Stage 1-3: A Poison Chihuahua!?, where it is involved in a cutscene where a trio lead by a Fatty and his two subordinates, a Standard Henchman and a Bomb-Man are seen torturing an innocent villager with the creature, the Fatty then taunts the Villager with the serum for the poison, and tosses it into the air where it is caught by the Bomb-Man. The Poison Chihuahuas seem to behave much like real world dogs, and can be trained to attack people, and they don't seem to be inherently aggressive, as the ones in Stage 3-5 are not hostile towards Gene, and will simply behave neutrally towards him aside from harmlessly following him around if he gets close. They also all have green spots on their backs, and their muzzles, eyebrow markings underbellies, legs, and the tips of their tails are all white. The Poison Chihuahua's greatly resemble real life Chihuahua's, however, they come in a multiple variety of colors that are impossible for a real Chihuahua to have, such as purple, red, green, light blue, and orange.