Using Ebony protein either expressed in Escherichia coli or in Schneider S2 cells, we provide evidence for its substrate specificity and reaction mechanism. Ebony activates ␤-alanine to aminoacyladenylate by an adenylation domain and covalently attaches it as a thioester to a thiolation domain in a nonribosomal peptide synthetase (NRPS) related mechanism. In a second reaction, biogenic amines act as external nucleophiles on ␤-alanyl-S-pantetheine-Ebony, thereby releasing in a fast reaction the dipeptide (peptidoamine) in a process that is novel in higher eucaryotes. Therefore, we define Ebony as a ␤-alanyl-biogenic amine synthetase. Insight into the reaction mechanism stems from mutational analysis of an invariant serine that disclosed Ebony as a multienzyme with functional analogy to the starting modules of NRPSs. In light of a putative biogenic amine-deactivating capacity, Ebony function in the nervous system must be reconsidered. We propose that in the Drosophila eye Ebony is involved in the transmission process by inactivation of histamine through ␤-alanyl conjugation.

The Drosophila ebony mutation (Bridges and Morgan, [1923] Publs Carnegie Inst Wash 327:50) reveals a pleiotropic phenotype with cuticular and behavioral defects. To understand Ebony function in the nervous system, particularly in transmission of the visual signal, it is essential to know the cell type and temporal characteristics of its expression throughout development. Therefore, we raised an antiserum against an Ebony peptide to detect the protein in whole-mount and slice preparations of Drosophila. Attention was focused on ebony expression in the adult optic neuropiles of the fly. Colocalization of Ebony with neuronal or glial cell markers in frozen sections showed non-neuronal expression of ebony in the lamina and medulla neuropiles. Furthermore, colocalization with glial cell markers demonstrated glial expression of ebony in epithelial glia of the lamina and neuropile glia of the distal medulla. This finding was confirmed for the lamina epithelial glia by electron microscopic examination of immunolabeling by using the diaminobenzidine method. These glia have in common that they match the two sites of histamine release from the compound eye's photoreceptors. Possible ways in which the biochemical activity of Ebony might function with respect to histamine release are considered.

Olfactory stimulation induces an odor-guided crawling behavior of Drosophila melanogaster larvae characterized by either an attractive or a repellent reaction. In order to understand the underlying processes leading to these orientations we stimulated single olfactory receptor neurons (ORNs) through photo-activation within an intact neuronal network. Using the Gal4-UAS system two light inducible proteins, the light-sensitive cation channel channelrhodopsin-2 (ChR-2) or the light-sensitive adenylyl cyclase (Pacα) were expressed in all or in individual ORNs of the larval olfactory system. Blue light stimulation caused an activation of these neurons, ultimately producing the illusion of an odor stimulus. Larvae were tested in a phototaxis assay for their orientation toward or away from the light source. Here we show that activation of Pacα expressing ORNs bearing the receptors Or33b or Or45a in blind norpA mutant larvae induces a repellent behavior away from the light. Conversely, photo-activation of the majority of ORNs induces attraction towards the light. Interestingly, in wild type larvae two ligands of Or33b and Or45a, octyl acetate and propionic ethylester, respectively, have been found to cause an escape reaction. Therefore, we combined light and odor stimulation to analyze the function of Or33b and Or45a expressing ORNs. We show that the larval olfactory system contains a designated neuronal pathway for repellent odorants and that activation of a specific class of ORNs already determines olfactory avoidance behavior.