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Twitch and Vinegar appear in Shantae and the Pirate's Curse. The first time Shantae meets the two girls they are on vacation on Saliva Island and are upset that there is no pool. Instead they set up their picnic (a giant ham) which Shantaeuses to open the Giga Lummox's (a giant dragon-like being napping above the pool) mouth to drool spit into the pool. As Twitch and Vinegar show off their "porcelin baby skin" and "cross the beams" they are able to help Shantae progress further by giving her the Petrify spell. Later in the game, after they finish their vacation, Shantae learns that they work for the Ammo Baron, seemingly fangirl-ing on Bran-Son nearSequin Land Palace. By giving Bran-Son his enchanted blade the girls run back to Ammo Baron in Scuttle Town, intent on revealing Bran-Son's secret identity to the Baron. Later, asBolo has inadvertently sabotaged the Baron's cannon in Scuttle Town, the girls are unable to direct the cannon to face the Palace and impact a large hole into Frostbite Islandinstead, revealing a hidden dungeon.
Twitch & Vinegar's last appearances are in a cutscene during Shantae's battle with the Pirate Master's true form in which they help Shantae, under command by Risky Boots, by firing cannonballs at the Pirate Master's head to finish him off and save Sequin Land; they then appear one last time during the ending, as Shantae gives back Scuttle Town its original appearance, the Ammo Baron's army retreats from the town and the two girls are seemingly left behind.