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Neuropeptides play important roles in the regulation of physiological processes such as growth, metabolism and reproduction. In sea cucumbers (Phylum
Echinodermata), numerous neuropeptides have been identified and some are attributed to reproductive processes. In this study, our goal was to gain a better understanding of the neuropeptide repertoire for the black sea cucumber Holothuria leucospilota, a species that has been severely overfished from the wild due to human consumption. We applied in silico transcriptome analysis of the adult H. leucospilota radial nerve cord, gonad and body wall to elucidate 35 neuropeptides that are conserved throughout the Bilateria. Then, liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of radial nerve cord was employed and showed an additional 8 putative novel neuropeptide precursors, whose predicative cleaved peptides do not share sequence similarity with any reported neuropeptides. These data provide an important basis for experimental approaches to manipulate H. leucospilota broodstock reproduction and growth in culture, which will hopefully reestablish population numbers.