Neuropeptidomics of the grey flesh fly, Neobellieria bullata Peter Verleyen, * Jurgen Huybrechts, Filip Sas, Elke Clynen, Geert Baggerman, Arnold De Loof, and Liliane Schoofs Laboratory of Developmental Physiology, Genomics and Proteomics, K.U.Leuven, Naamsestraat 59, Louvain B-3000, Belgium Received 3 February 2004 Abstract A peptidomics approach was applied to determine the peptides in the larval central nervous system of the grey flesh fly, Neo- bellieria bullata. Fractions obtained by high performance liquid chromatography were analysed by MALDI-TOF and ESI-Q-TOF mass spectrometry. This provided biochemical evidence for the presence of 18 neuropeptides, 11 of which were novel Neobellieria peptides. Most prominently present were the FMRFamide-related peptides: 7 FMRFamides, 1 FIRFamide, and Neb-myosup- pressin. The three putative capa-gene products Neb-pyrokinin and the periviscerokinins Neb-PVK-1 and -2 were detected, as well as another pyrokinin. This Neb-PK-2 was also present in the ring gland along with corazonin, Neb-myosuppressin, and Neb-AKH- GK, an intermediate processing product of the adipokinetic hormone. Furthermore, the central nervous system contained Neb- LFamide, proctolin, and FDFHTVamide, designated as Neb-TVamide. With this study, we considerably increased our knowledge of the neuropeptidome of the pest fly N. bullata, which is an important insect model for physiological research. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Neobellieria bullata; Peptidomics; Mass spectrometry; FMRFamide-related peptides; Pyrokinins; Periviscerokinins The recent publications of the genomes from the fruit fly Drosophila melanogaster [1] and the malaria mos- quito Anopheles gambiae [2] have stirred a major revolution in insect research. They enabled the straightforward identification of proteins and peptides that play a key role in the endocrinology and physiology of insects. We are interested in neuropeptides and their receptors since they occupy a high hierarchical position in the physiology of insects and regulate most (if not all) biological processes, such as reproduction, metamor- phosis, feeding, homeostasis, and behaviour. Recently, peptide profiling of the Drosophila central nervous sys- tem (CNS) has led to the isolation and sequencing of 28 neuropeptides in a single peptidomic experiment [3]. These results were confirmed by an immunocytochemi- cal approach, demonstrating the cellular localisation of the newly identified Drosophila peptide IPNamide in the larval CNS [4]. In addition, about 20 neuropeptide G- protein coupled receptors have been identified and characterised in Drosophila using reverse pharmacology or orphan receptor strategies [5–9]. While Drosophila is especially suited for functional genomic and proteomic studies, the related and larger-sized dipteran pest spe- cies, Neobellieria bullata (formerly called Sarcophaga bullata), remains more appropriate for conducting physiological experiments. The progress that has been made with respect to neuropeptide identification in Drosophila will be very useful for the identification of biologically active neu- ropeptides in the closely related grey flesh fly. In fact, to date, only 11 neuropeptides have been characterised in N. bullata, including Neb-LFamide [10], neomyosup- pressin [11], neosulfakinins [12], Neb-kinin [13], Neb- AKH [14], Neb-FMRFamide and Neb-FIRFamide [8], and Neb-periviscerokinins and Neb-pyrokinin [15], al- though immunocytochemical data suggest the presence of more peptides [16–18]. Advances in mass spectrom- etry have made it possible to identify a substantial number of neuropeptides in nervous tissue, even in species without genomic sequence information like Locusta migratoria and Cancer borealis [19,20]. Never- theless, determining the peptidome of a species without an available genomic database of a related species * Corresponding author. Fax: +32-16-32-39-02. E-mail address: peter.verleyen@bio.kuleuven.ac.be (P. Verleyen). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.02.115 Biochemical and Biophysical Research Communications 316 (2004) 763–770 BBRC www.elsevier.com/locate/ybbrc