To identify the tissue that produces 8K-BLP, gene expression of 8K-BLP in various tissues was analyzed by real-time quantitative RT-PCR (qRT-PCR). The 8K-BLP gene was predominantly expressed in the fat body after pupal ecdysis ( Fig. 4A ). Gene expression was very low during the larval stage but increased slightly at the pharate pupal stage, and this was followed by a steep increase after pupal ecydsis. Thereafter, the expression further increased towards adult eclosion ( Fig. 4B ). The 8K-BLP peptide became detectable in the fat body after pupal ecdysis ( Fig. 4C ), in agreement with the fluctuation pattern of 8K-BLP levels in hemolymph ( Fig. 1 ). Production of 8K-BLP by the fat body was also confirmed by immunohistochemistry using a novel monoclonal antibody D7H3, specific for the C-domain of 8K-BLP ( Fig. 4D ). Although the staining intensity of the cells increased during adult development, the number of fat body cells decreased dramatically from mid-adult development onwards, due to their disruption. These results indicate that the fat body is the main source of the hemolymph 8K-BLP.
AB - We previously demonstrated that two ecdysteroid-regulated genes, Mblk-1/E93 and E74, are expressed selectively in Kenyon cell subtypes in the mushroom bodies of the honeybee (Apis mellifera L.) brain. To further examine the possible involvement of ecdysteroid-regulated genes in brain function as well as in oogenesis in the honeybee, we isolated cDNAs for two other ecdysteroid-regulated genes, Broad-Complex (BR-C) and E75, and analyzed their expression in the worker brain as well as in the queen abdomen. In situ hybridization revealed that BR-C, like Mblk-1/E93, is expressed selectively in the large-type Kenyon cells of the mushroom bodies in the worker brain, whereas E75 is expressed in all mushroom body neuron subtypes, suggesting a difference in the mode of response to ecdysteroid among Kenyon cell subtypes. In the queen ovary, both BR-C and £75 are expressed preferentially in the follicle cells that surround egg cells at the late stage, suggesting their role in oogenesis. These results suggest that BR-C and E75 are involved in the regulation of brain function as well as in reproductive physiology in the adult honeybee.
AB - Investigations of ecdysteroid-regulated gene cascades in Drosophila have shown that characteristics of downstream genes in such cascades include their repression by high ecdysteroid levels, their expression at low hormone levels, and the dependence of their expression on protein synthesis. In an earlier study, we identified a gene, esr20, which is expressed in the tracheae of the tobacco hornworm, Manduca sexta, prior to larval and pupal ecdyses. Initial characterization of the expression of esr20 suggested that it had the above characteristics of a downstream gene in an ecdysteroid-regulated cascade. The present study shows that, unlike the downstream genes in Drosophila, the expression of esr20 in tracheae cannot be induced by changes in the ecdysteroid levels alone. We present evidence which suggests that a decline in ecdysteroid is necessary but not sufficient for expression. Soon after pupal ecdysis the level of the esr20 transcript drops fourfold, and by 24 h after ecdysis the transcript is undetectable. Evidence is presented which suggests that this decline in transcript levels requires protein synthesis and appears to result from a decline in the stability of the transcript.