In insects ecdysone causes molting and metamorphosis and juvenile hormone (JH) allows molting but prevents the metamorphic action of ecdysone. Ongoing immunocytochemical studies of how JH modulates the ecdysone-induced cascade of transcription factors in the tobacco hornworm, Manduca sexta, pupal wing at the outset of adult development will be completed. The technique of RNA interference to eliminate specific mRNAs in this pathway will be used to determine the roles of key transcription factors of this cascade in pattern formation and cuticle deposition. This same transcription factor cascade will be analyzed immunocytochemically during pupal commitment of the wing imaginal disc and of the eye primordium during the molt to the final larval stage and at the initiation of pupal development. The role of JH in the maintenance of the larval state of these two adult precursors will be assessed by experimental manipulations, both in the animal and in tissue culture Moling, an epidermal protein that appears only in the last larval stage of Manduca, belongs to the Takeout family of proteins that includes some JH-binding proteins. Recombinant Moling will be produced by a baculovirus system, then used to test whether it binds JH and, if so, the binding will be characterized. Also, the pattern of its disappearance in the epidermis relative to the time of pupal commitment and the appearance of Broad expression will be studied immunocytochemically. Putative homologs of Moling in Drosophila that are regulated by JH will be analyzed for their presence and possible role in larval epidermal development and metamorphosis. The role of USP as a possible JH receptor in Drosophila is being studied in collaboration with Dr. Grace Jones. Bioassays of JH in the prevention of abdominal metamorphosis will be done with animals bearing mutated forms of USP hypothesized to have little or no JH binding activity.
