Juvenile hormone modulates 20-hydroxyecdysone inducible ecdysone receptor and ultraspiracle gene expression in the tobacco hornworm, Manduca sexta

Insect molting and metamorphosis are orchestrated by ecdysteroids with juve nile hormone (JH) preventing the actions of ecdysteroids necessary for meta morphosis. During the molt and metamorphosis of the dorsal abdominal epider mis of the tobacco hornworm, Manduca sexta, the isoforms involved in the ec dysone receptor (EcR)/Ultraspiracle (USP) complex change with the most dram atic switch being the loss of USP-1 and the appearance of USP-2 during the larval and pupal molts. We show here that this switch in USP isoforms is me diated by high 20-hydroxyecdysone (20E) and that the presence of JH is nece ssary for the down-regulation of USP-1 mRNA. The decrease of USP-1 mRNA in day 2 fourth instar larval epidermis in vitro required exposure to a high c oncentration (10(-5) M) of 20E equivalent to the peak ecdysteroid concentra tion in vivo, whereas the increase of USP-2 mRNA occurred at lower concentr ations (effective concentrations, EC50=6.3x10(-7) M). During the pupal molt of allatectomized larvae which lack JH, USP-2 mRNA increased normally with the increasing ecdysteroid titer, whereas USP-1 mRNA remained high until p upation, When day 2 fifth instar larval epidermis was exposed to 500 ng/ml 20E in the absence of JH to cause pupal commitment of the cells by 24 h, US P-I RNA remained at its high preculture level for 12 h, then increased two- to threefold by 24 h. The increase was prevented by the presence of 1 mu g /ml JH I which also prevents the pupal commitment of the cells. By contrast , USP-2 mRNA increased steadily with the same EC,, as in fourth stage epide rmis, irrespective of the presence or absence of JH. Under the same conditi ons, mRNAs for both EcR-B1 and EcR-A isoforms were up-regulated by 20E, eac h in its own time-dependent manner, similar to that seen in vivo. These ini tial mRNA increases were unaffected by the presence of JH I, but those seen after 12 h exposure to 20E were prevented by JH, indicating a difference i n response between larvally and pupally committed cells. The presence of JH which maintained larval commitment of the cells also prolonged the half-li fe of the EcR proteins in these cells. These results indicate that both EcR and USP RNAs are regulated by 20E and can be modulated by JH in a complex manner with only that of USP-2 apparently unaffected.