The juvenile hormone analog pyriproxyfen affects ecdysteroid-dependent cuticle melanization and shifts the pupal ecdysteroid peak in the honey bee (Apis mellifera)

The control of the pupal melanization in the honey bee by ecdysteroids, and the modulation of these processes by a juvenile hormone analog were investigated by a combination of in vivo and in vitro experiments. Injection of 1-5 microg of 20-hydroxyecdysone (20E) into unpigmented pupae showed a dose- and stage-dependent effect. The higher the dose and the later the injection was performed, the more pronounced was the delay in cuticle pigmentation. This inhibition of cuticular melanization by artificially elevated ecdysteroid titers was corroborated by in vitro experiments, culturing integument from unpigmented, dark-eyed pupae for 1-4 days in the presence of 20E (2 or 5 microg/ml culture medium). Topical application (1 microg) of pyriproxyfen to unpigmented, white-eyed pupae had the opposite effect, leading to precocious and enhanced melanization of the pupal cuticle. In vitro incubation of integuments in the presence of this juvenile hormone analog (1 microg/ml) confirmed these results, showing that pyriproxyfen is apparently capable of triggering melanization. The in vivo mode of action of pyriproxyfen was further investigated by quantifying hemolymph ecdysteroids by radioimmunoassays. Topical application leads to a delay of the pupal ecdysteroid peak by 4 days. The pyriproxyfen-induced low ecdysteroid titers during early pupal development could account for precocious pigmentation by removing an inhibition on prophenoloxidase activation normally imposed by the elevated ecdysteroid titer during this phase.