I figured it out, There is a “sRGB” setting that said “uncheck if using alpha channels individually as masks” I thought applied to me. Re-checking it made the texture match again.
I went back to tappy chicken to double check what I’d found. Sure enough, there is a grey border to the chicken’s (eyes?) that gets lost (bleached white) when you press play. My sprite is matching much better now, but I wonder why Tappy Chicken is still brightening by the smallest ammount…