Neuropeptide-like protein diversity in phylum Nematoda Paul McVeigh a, * , Susan Alexander-Bowman b , Emma Veal a , Angela Mousley a , Nikki J. Marks a , Aaron G. Maule a a Biomolecular Processes: Parasitology, School of Biological Sciences, Medical Biology Centre, 97 Lisburn Road, Queen’s University Belfast, Belfast BT9 7BL, UK b Pfizer, 7923-25-410, 7000 Portage Road, Kalamazoo, MI 49001-0199, USA Received 6 March 2008; received in revised form 24 April 2008; accepted 6 May 2008 Abstract This study reports the identification of nematode neuropeptide-like protein (nlp) sequelogs from the GenBank expressed sequence tag (EST) database, using BLAST (Basic Local Alignment Search Tool) search methodology. Search strings derived from peptides encoded by the 45 known Caenorhabditis elegans nlp genes were used to identify more than 1000 ESTs encoding a total of 26 multi-species nlp sequelogs. The remaining 18 nlps(nlp-4, -16, -24 through -36, -39, -41 and -45) were identified only in C. elegans, while the sole EST representative of nlp-23 was from Caenorhabditis remanei. Several ESTs encoding putative antibacterial peptides similar to those encoded by the C. elegans genes nlp-24–33 were observed in several parasite species. A novel gene (nlp-46) was identified, encoding a single, ami- dated dodecapeptide (NIA[I/T]GR[G/A]DG[F/L]RPG) in eight species. Secretory signal peptides were identified in at least one species representing each nlp sequelog, confirming that all 46 nematode nlp genes encode secretory peptides. A random sub-set of C. elegans NLPs was tested physiologically in Ascaris suum ovijector and body wall muscle bioassays. None of the peptides tested were able to mod- ulate ovijector activity, while only three displayed measurable myoactivity on somatic body wall muscle. AFAAGWNRamide (from nlp- 23) and AVNPFLDSIamide (nlp-3) both produced a relaxation of body wall muscle, while AIPFNGGMYamide (nlp-10) induced a tran- sient contraction. Numerical analyses of nlp-encoding ESTs demonstrate that nlp-3,-13,-14,-15 and -18 are amongst the most highly represented transcripts in the dataset. Using available bioinformatics resources, this study delineates the nlp complement of phylum Nematoda, providing a rich source of neuropeptide ligands for deorphanisation of nematode neuropeptide receptors. Ó 2008 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Bioactivity; Bioinformatics; Expressed sequence tag; EST; nlp; Peptidome; Physiology; Transcriptome 1. Introduction Neuropeptides are short chains of amino acids which are secreted by neurones, allowing communication with other cells. Peptidergic neurotransmitters are structurally and functionally diverse, performing a myriad of neuromodula- tory and neurohormonal roles throughout the eumetazoa. The neuropeptide complement of phylum Nematoda seems to be uniquely high amongst invertebrates. The model nematode Caenorhabditis elegans possesses three groups of neuropeptide genes designated as two families, the FMRFamide-like peptide (flp) and insulin-like (ins) genes, as well as one repository for all the remaining families (des- ignated neuropeptide-like protein or nlp genes), which together encode 215 distinct neuropeptides. This neuropep- tide complement exists in addition to an array of classical neurotransmitters (Brownlee et al., 2000; Li, 2005; Husson et al., 2007; Marks and Maule, 2007). Li et al. (1999) and Nathoo et al.’s (2001) described the first nlp genes from C. elegans, the latter authors reporting a total of 32 nlp genes encoding 11 putative neuropeptide families. Expressed sequence tags (ESTs) encoding similar peptides in other species were reported for all 11 families. Further studies since then have employed a combination 0020-7519/$34.00 Ó 2008 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2008.05.006 * Corresponding author. Tel.: +44 (0)28 9097 2311; fax: +44 (0)28 9097 5877. E-mail address: paul.mcveigh@qub.ac.uk (P. McVeigh). www.elsevier.com/locate/ijpara Available online at www.sciencedirect.com International Journal for Parasitology 38 (2008) 1493–1503