ABSTRACT In the Paleozoic era, more than 400 million years ago, insects continued molting after forming functional wings. Today, however, all flying insects stop molting after metamorphosis when they become fully winged. The only exception is the mayflies (Ephemeroptera), which molt in the subimago, a flying intermediate stage between the nymph and the adult. However, the identity and homology of the subimago remains underexplored. Debate remains regarding whether this stage represents a modified nymph, an adult, or a pupa like that of butterflies. Another relevant question is why do mayflies maintain the subimago stage despite the risk of molting fragile membranous wings. These questions have intrigued numerous authors but nonetheless, clear answers have not yet been found. However, by combining morphological studies, hormonal treatments, and molecular analysis in the mayfly species Cloeon dipterum , we found new answers to these old questions. We observed that treatment with a juvenile hormone analog in the last nymphal instar stimulated the expression of Kr-h1 gene and reduced that of E93 , which suppress and trigger metamorphosis, respectively. Consequently, the subimago is not formed in these treated mayflies. This indicates that metamorphosis is determined prior to the formation of the subimago, which must therefore be considered an instar of the adult stage. We also observed that the forelegs dramatically grow between the last nymphal instar, the subimago, and the adult. This necessary growth is spread over the last two stages, which could explain, at least in part, the adaptive sense of the subimago.

The mayfly subimago explained. The regulation of metamorphosis in Ephemeroptera