I had push in cages on two of my frames with multiple queen cells. Yesterday I herded those newly emerged queens into regular queen cages. I pulled the one year old queen out of my largest hive and made up a Nuc for sale. Then I misted the bees with a bit of vanilla scented syrup and put the two caged queens in the hive. One went in to the second brood box, and the other in the honey super above a queen excluder. Today I went back in to make up a mating nuc for one of the queens. Sadly the queen in the top box was curled up and dead. I thought: they balled her because I did not wait 24 hours to introduce and the other will be dead too. But no, when I dug down to the other queen she was feisty in her cage.
So my thought: the bees clustered for the night and the queen in the super was left out and froze. Any other possibilities?