Analysis of the Ecdysiotropic activity in larval brains of the tobacco hornworm, Manduca sexta

Abstract

The ability of Manduca sexta larval brain extracts to stimulate ecdysteroid synthesis by day-3, last-instar M. sexta prothoracic glands was examined using methods previously described for M. sexta, Bombyx mori and Lymantria dispar. Ecdysteroid synthesis was quantified by enzyme-linked immunosorbent assay (ELISA) with an antiserum that recognized both ecdysone and 3-dehydroecdysone and by radioimmunoassay (RIA) with an antiserum that recognized primarily ecdysone. Time-course analysis by RIA revealed that a basal synthesis rate of about 135 pg ecdysone equivalents/gland/h was reached after 3h of incubation in Grace's medium and that at this time, glands could be activated in a dose-dependent manner about 9-fold with brain extract, as compared to Grace's medium controls. Size-exclusion analysis of the ecdysiotropic activity in brains that had been stored frozen at − 20 °C revealed activity in size ranges not previously reported for M. sexta, 11.5 kDa and 0.65 kDa. Extracts from brains that had been stored in 90% methanol/0.1% TFA at − 50 °C, however, revealed primarily the 11.5 kDa form of ecdysiotropin, suggesting possible degradation of the larger form at − 20 °C and an artifactual production of the 0.65 kDa form. Both forms were sensitive to protease digestion.