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 Shantae uses 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 near Sequin 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, as Bolo 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 Island instead, 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.
Twitch & Vinegar return in Shantae: Half-Genie Hero. Still working for the Ammo Baron. They give Shantae a quest to get them some Gator Steaks in exchange for a race ticket. Shantae later finds out they have been stealing magic carpets under orders of the Ammo Baron and Twitch & Vinegar fight Shantae in their air assualt vehicles as a mid boss. They are later seen again carrying away Ammo Baron after his defeat.
The creation of the characters actually dates back to the 1990s, as Matt Bozon revealed in an interview that Twitch's name and design were actually those of Sky. When was developped, he decided to modify the character a bit. Years later, he chose to bring back Twitch as a character for , although he altered her so that she would be split into two characters, Twitch and Vinegar. The Ammo Baron was supposed to introduce them to Shantae by name in the game, but due to a programming error the line took the shaft, and they therefore remained unnamed. In a small promotional comic on Matt Bozon's twitter, Matt later joked about this fact; but the release of cards and wallpapers for the Steam version established Twitch's & Vinegar's official names. The quote "Why am I staring at this totally buff gorgeous loser?" attributed to Twitch on the wallpaper allows to ascertain that the girl with the white top is Twitch as she is the one to say this in-game; making the girl with the orange and blue outfit Vinegar.
Steam Wallpaper that revealed their names: