Maybe Spike was asking how she knew it was Twi's butt 
If the angle showed her cutie mark, it'd be unmistakable. I'm assuming no two are exactly the same. Plus I think she's the only Pony who comes in in that delightful shade of lavender.
How often
do people call you a cinnamon roll, dear?
Maybe Twi thought it was a science or history question, which she considers her purview, and resents having another queen bee in the hive... I mean, I don't know if we have any mothers here on this board, but if your learned your son were going to one of your friends for help on his homework rather than you, though you're fully capable of assisting him, wouldn't you feel a touch put out?