Sulfakinin (SK)/cholecystokinin (CCK)-type neuropeptides regulate feeding and digestion in protostomes (e.g. insects) and chordates. Here, we characterised SK/CCK-type signalling for the first time in a non-chordate deuterostome – the starfish Asterias rubens (phylum Echinodermata). In this species, two neuropeptides (ArSK/CCK1, ArSK/CCK2) derived from the precursor protein ArSK/CCKP act as ligands for an SK/CCK-type receptor (ArSK/CCKR) and these peptides/proteins are expressed in the nervous system, digestive system, tube feet, and body wall. Furthermore, ArSK/CCK1 and ArSK/CCK2 cause dose-dependent contraction of cardiac stomach, tube foot, and apical muscle preparations in vitro, and injection of these neuropeptides in vivo triggers cardiac stomach retraction and inhibition of the onset of feeding in A. rubens. Thus, an evolutionarily ancient role of SK/CCK-type neuropeptides as inhibitory regulators of feeding-related processes in the Bilateria has been conserved in the unusual and unique context of the extra-oral feeding behaviour and pentaradial body plan of an echinoderm.

Somatostatin (SS) and allatostatin-C (ASTC) are structurally and evolutionarily related neuropeptides that act as inhibitory regulators of physiological processes in mammals and insects, respectively. Here, we report the first molecular and functional characterization of SS/ASTC-type signalling in a deuterostome invertebrate—the starfish Asterias rubens (phylum Echinodermata). Two SS/ASTC-type precursors were identified in A. rubens (ArSSP1 and ArSSP2) and the structures of neuropeptides derived from these proteins (ArSS1 and ArSS2) were analysed using mass spectrometry. Pharmacological characterization of three cloned A. rubens SS/ASTC-type receptors (ArSSR1–3) revealed that ArSS2, but not ArSS1, acts as a ligand for all three receptors. Analysis of ArSS2 expression in A. rubens using mRNA in situ hybridization and immunohistochemistry revealed stained cells/fibres in the central nervous system, the digestive system (e.g. cardiac stomach) and the body wall and its appendages (e.g. tube feet). Furthermore, in vitro pharmacological tests revealed that ArSS2 causes dose-dependent relaxation of tube foot and cardiac stomach preparations, while injection of ArSS2 in vivo causes partial eversion of the cardiac stomach. Our findings provide new insights into the molecular evolution of SS/ASTC-type signalling in the animal kingdom and reveal an ancient role of SS-type neuropeptides as inhibitory regulators of muscle contractility.

Somatostatin (SS) and allatostatin-C (ASTC) are inhibitory neuropeptides in chordates and protostomes, respectively, which hitherto were identified as orthologs. However, echinoderms have two SS/ASTC-type neuropeptides (SS1 and SS2), and here, our analysis of sequence data indicates that SS1 is an ortholog of ASTC and SS2 is an ortholog of SS. The occurrence of both SS-type and ASTC-type neuropeptides in echinoderms provides a unique context to compare their physiological roles. Investigation of the expression and actions of the ASTC-type neuropeptide ArSS1 in the starfish Asterias rubens revealed that it causes muscle contraction (myoexcitation), contrasting with myoinhibitory effects of the SS-type neuropeptide ArSS2. Our findings suggest that SS-type and ASTC-type neuropeptides are paralogous and originated by gene duplication in a common ancestor of the Bilateria, with only one type being retained in chordates (SS) and protostomes (ASTC) but with both types being retained in echinoderms. Loss of ASTC-type and SS-type neuropeptides in chordates and protostomes, respectively, may have been due to their functional redundancy as inhibitory regulators of physiological processes. Conversely, the retention of both neuropeptide types in echinoderms may be a consequence of the evolution of a myoexcitatory role for ASTC-type neuropeptides mediated by as yet unknown signaling mechanisms. Significance Somatostatin (SS) and allatostatin-C (ASTC) are related neuropeptide hormones that act as inhibitory regulators of physiological processes in chordates (e.g., humans) and protostome invertebrates (e.g., insects), respectively. We have discovered that echinoderms (e.g., starfish) uniquely have both SS-type and ASTC-type neuropeptides, which act as inhibitory and excitatory regulators of muscle activity, respectively. Our findings suggest that SS-type and ASTC-type neuropeptides evolved by duplication of a common ancestral encoding gene. Then, one of the neuropeptides was lost in protostomes and chordates, probably because of their functional redundancy as inhibitory regulators. Conversely, the unique retention of both neuropeptide types in echinoderms may be explained by evolution of an excitatory role for ASTC-type neuropeptides mediated by yet-to-be-determined signaling mechanisms.

Nerve growth factor (NGF) is a neurotrophin central to sensory neuron biology and pain signaling. NGF and NTRK1 are gnathostome paralogs within the older neurotrophin and Trk families, respectively, whereas NGFR encodes p75NTR, a member of the tumor necrosis factor receptor superfamily. Orthology assessment of NGF, NTRK1, and NGFR across vertebrates is challenging because mature neurotrophins are short, cysteine-rich ligands, NTRK1 has closely related paralogs, and NGFR lies outside the Trk receptor family. We assessed species-level joint genomic recovery of the three genes and compared evolutionary constraint among predefined ligand and receptor regions. The analysis covered 94 chordates and combined graded recovery evidence with family phylogenies, locus and domain evidence, codon models, amino acid conservation, and structural mapping. Sixteen non-gnathostome species were included to provide evolutionary context and were not used to calculate recovery rates for the three focal genes. Among 78 sampled gnathostomes, 52 had high-confidence joint recovery, eight had supported provisional joint recovery, four had component-level evidence consistent with possible co-recovery, and 14 remained incomplete. Mature NGF had a numerically lower estimate of the ratio of nonsynonymous to synonymous substitution rates (dN/dS, ω) than the pro-domain (0.067 versus 0.352). All six scaffold cysteines were invariant; in the residue-class comparison, the cysteine scaffold was the only structural class that differed from the coordinate-resolved background after correction. The dimer-interface, TrkA-contact, and p75NTR-contact classes did not differ significantly from that background after correction. Receptor analyses yielded low ω estimates in Trk kinase domains and p75NTR intracellular regions. Together, these results provide a confidence-graded map of focal-gene recovery and regional purifying constraint while placing non-gnathostome homologs within the older neurotrophin system.