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Ear Worm: The little flute-sounding song that some ants are performing for the circus bugs.
Foe Yay Shipping: Hopper and Flik. Some people couldn't explain why they kept watching their No-Holds-Barred Beatdown scene repeatedly when they were children, they sure can as they got older.
Genius Bonus: Those familiar with black widow spider mating habits might view some of Rosie's lines in a different light. See also Getting Crap Past the Radar.
Harsher In Hindsight: The scene where Hopper almost feeds Dot to Tumpher is even more difficult to watch now knowing Keven Spacey's Situation.
Moral Event Horizon: He had been extorting food from the ants for years, but Hopper crosses this when he decides he's going to murder the queen.
In the documentary The Pixar Story John Lasseter, and many of the other animators give off a sense that this movie is an Old Shame. One of the animators explained that they spent more time trying to make the movie better than Toy Story, instead of simply trying to make the film good in its own right.