Odekunle EA , Semmens DC , Martynyuk N , Tinoco AB , Garewal AK , Patel RR , Blowes LM , Zandawala M , Delroisse J , Slade SE , Scrivens JH , Egertová M , Elphick MR .

BB/M001644/1 Biotechnology and Biological Sciences Research Council , BB/M001032/1 Biotechnology and Biological Sciences Research Council

	Fig. 1. Asterotocin: a vasopressin/oxytocin (VP/OT)-type neuropeptide in the starfish Asterias rubens. a Amino acid sequence of the A. rubens asterotocin precursor with the predicted signal peptide shown in blue, a predicted cleavage site shown in green, the asterotocin peptide shown in red with a C-terminal glycine that is a putative substrate for amidation shown in orange and the neurophysin domain shown in purple. b Structure of the mature asterotocin peptide as determined by mass spectrometry (Additional file 1), with a disulphide bridge between the two cysteine residues and with C-terminal amidation. c Clustal-X alignment of asterotocin (A_rub) with VP/OT-type peptides from other animals reveals that the pair of cysteine residues (asterisks) is conserved in all of the peptides, whereas other residues are conserved in a subset of the peptides. Full species names within each taxonomic group (Ambulacraria, Chordata and Protostomia) and corresponding accession numbers for the peptide sequences are listed in Table S1 of Additional file 3
	Fig. 2. Phylogenetic identification and deorphanisation of an A. rubens VP/OT-type receptor. a Maximum likelihood tree showing that a VP/OT-type receptor identified by BLAST analysis of A. rubens transcriptome sequence data (boxed) is positioned within a clade comprising VP/OT-type receptors from other taxa. NPS/NG peptide/CCAP-type receptors are paralogs of VP/OT-type receptors [25]. Phylogenetic analyses of bilaterian neuropeptide receptors have shown that GnRH/AKH/ACP/CRZ-type receptors are closely related to VP/OT-type receptors and NPS/NG peptide/CCAP-type receptors [6]. Therefore, GnRH/AKH/ACP/CRZ-type receptors were included as an outgroup in the phylogenetic tree. The scale bar indicates amino acid substitutions per site, and bootstrap values are shown at nodes. Species where activation of the VP/OT-type receptor by a cognate VP/OT-type neuropeptide has been demonstrated experimentally (including A. rubens, see b) are labelled with an asterisk. Full species names and accession numbers for the receptor sequences are listed in Table S2 of Additional file 3. b Asterotocin (black circles) causes concentration-dependent activation of the A. rubens VP/OT-type receptor, demonstrated by measuring Ca2+-induced luminescence in CHO-K1 cells expressing aequorin, and transfected with the promiscuous G-protein Gα16 and the A. rubens VP/OT-type receptor (CHO-K1/G5A-ArVPOTR cells); EC50 = 5.7 × 10−8 M. CHO-K1 cells transfected with empty pcDNA vector (black square) were used as a negative control and do not exhibit luminescence when exposed to asterotocin. c Comparison of the relative luminescence responses of CHO-K1/G5A-ArVPOTR cells when exposed to asterotocin or the related peptides NGFFYamide (a paralog of asterotocin in A. rubens), human vasopressin or human oxytocin, all at a concentration of 10−4 M. Luminescence responses in the presence of vasopressin, oxytocin and NGFFYamide were not significantly higher than the basal media control (P = 0.5, P = 0.25, P = 0.25, respectively; Wilcoxon signed-rank test; n = 9)
	Fig. 3. Starfish anatomy. a Schematic vertical section of the central disc and the proximal region of an arm. b Schematic transverse section of an arm. c Schematic transverse section of a radial nerve cord. Abbreviations: a, anus; am, apical muscle; amp, ampulla; conr, circumoral nerve ring; cs, cardiac stomach; cut, cuticle; ec, ectoneural region; g, gonad; gcc, general coelomic cavity; hy, hyponeural region; m, mouth; md, madreporite; mn, marginal nerve; o, ossicle; p, papula; pc, pyloric caecum; pd., pyloric duct; ped, pedicellaria; pm, peristomial membrane; ps, pyloric stomach; rc, rectal caecum; rca, ring canal; rn, radial nerve; rw, radial water vascular canal; sp., spine; sc, stone canal; tb, Tiedemann’s body; tf, tube foot
	Fig. 4. Localisation of asterotocin in A. rubens using in situ hybridisation (ISH) and immunohistochemistry (IHC). a Asterotocin precursor transcript-expressing cells (AstPtc) in a radial nerve cord (black arrowheads) and tube feet (white arrowheads). Inset shows the absence of staining with sense probes. b Asterotocin-immunoreactive (Ast-ir) cells (arrowheads in inset) and fibres (white asterisks) in a radial nerve cord. c AstPtc in the circumoral nerve ring (arrowheads and inset). d Ast-ir cells (arrowheads) and fibres (white asterisks) in the circumoral nerve ring. e AstPtc in marginal nerve. f Ast-ir fibres in marginal nerve. g AstPtc in tube foot disc. h Ast-ir in the basal nerve ring (black arrow) and longitudinal nerve tract (grey arrow) of a tube foot. i AstPtc in the peristomial membrane. j AstPtc in the oesophagus. k Ast-ir in the peristomial membrane (square bracket) and oesophagus. l AstPtc in the cardiac stomach. m Ast-ir in the cardiac stomach. n AstPtc in the pyloric stomach. o AstPtc in a pyloric duct. p Ast-ir in the pyloric stomach. q AstPtc in the coelomic epithelium of the body wall. r Ast-ir in the coelomic basiepithelial nerve plexus of body wall. s Ast-ir in the basiepithelial nerve plexus of the apical muscle. t AstPtc in a papula. u Ast-ir in a papula. v AstPtc in the body wall. w Ast-ir in the body wall. x AstPtc in a pedicellaria. y Ast-ir in a pedicellaria. z AstPtc in a spine. z’ Ast-ir in an ambulacral spine. Abbreviations: AM, apical muscle; BNR, basal nerve ring; BNP, basiepithelial nerve plexus; CMLNP, circular muscle layer nerve plexus; CBNP, coelomic basiepithelial nerve plexus; CE, coelomic epithelium; CT, collagenous tissue; Di, disc; Ec, ectoneural region; EE, external epithelium; Hy, hyponeural region; LNT, longitudinal nerve tract; Lu, lumen; MN, marginal nerve; ML, mucosal layer; MuL, muscle layer; Oes, oesophagus; Pa, papula; RHS, radial hemal sinus; TF, tube foot; VML, visceral muscle layer. Scale bars: a, a inset, h, l, m, n, o, p, u = 60 μm; b, d, g, z’ = 40 μm; i, j, l inset, p inset, t, w, x = 30 μm; B inset, c, d inset, e, k, n inset, o inset, s, v, y, z = 20 μm; c inset, f, m inset, q, r = 10 μm
	Fig. 5. Localisation of asterotocin receptor in A. rubens using in situ hybridisation (ISH) and immunohistochemistry (IHC). a Asterotocin receptor transcript-expressing cells (AstRtc) in a radial nerve cord (black arrowheads) and tube feet (white arrowheads). Inset shows the absence of staining with sense probes. b AstR-immunoreactive (AstR-ir) cells (black arrowhead) and processes (arrows and in inset) in a radial nerve cord. Stained fibres in the adjacent tube foot (white arrow). c AstRtc in the circumoral nerve ring. d AstR-ir cells (arrowheads) and fibres (arrow) in the circumoral nerve ring. e AstRtc in a marginal nerve. f AstR-ir cell (arrowhead) and stained process (black arrow) in a marginal nerve. Stained fibres in the adjacent tube foot (white arrow). g AstRtc in a tube foot (black arrowhead). h Ast-ir in a tube foot. i AstRtc in the cardiac stomach. j AstR-ir in the mucosal layer (white arrowhead), basiepithelial nerve plexus (black arrowhead) and visceral muscle layer (black arrow; inset) of the cardiac stomach. k AstRtc in the coelomic epithelium of the body wall. l AstR-ir cell in the coelomic epithelium of the body wall. m AstRtc in the external epithelium of the body wall. n AstR-ir cell in the external epithelium of the body wall. o AstRtc in a papula. p AstR-ir cells in an ambulacral spine. q AstR-ir cells in a pedicellaria. Abbreviations: BNR, basal nerve ring; BNP, basiepithelial nerve plexus; CMLNP, circular muscle layer nerve plexus; Ce, coelom; CBNP, coelomic basiepithelial nerve plexus; CT, collagenous tissue; Di, disc; Ec, ectoneural region; EE, external epithelium; Hy, hyponeural region; Lu, lumen; MN, marginal nerve; ML, mucosal layer; Pa, papula; RHS, radial hemal sinus; TF, tube foot; VML, visceral muscle layer. Scale bars: a, a inset, b, c, g, h, o, p, q = 60 μm; d, e, j = 30 μm; c inset, f, g inset, n = 20 μm; b inset, e inset, f inset, i, i inset, j inset, k, l, m, o = 10 μm
	Fig. 6. Comparison of asterotocin and asterotocin receptor expression in A. rubens using double-labelling fluorescence immunohistochemistry. Comparison of the distribution of asterotocin immunoreactivity (Ast-ir; green) and asterotocin receptor immunoreactivity (AstR-ir; red) in A. rubens reveals ‘salt and pepper’ patterns of labelling consistent with expression largely in different, but often adjacent, populations of cells/processes. In the few instances where labelling appears yellow/orange, this could be due to the co-localisation of asterotocin and the asterotocin receptor in processes or alternatively it may simply reflect where asterotocin-containing fibres happen to be positioned directly above asterotocin receptor containing fibres. a Radial nerve cord showing Ast-ir cells (yellow arrowheads) and AstR-ir cells (white arrowheads) in the ectoneural epithelium and Ast-ir processes (yellow arrow) and AstR processes (white arrow) in the ectoneural neuropile. b Marginal nerve; AstR-ir cells (white arrowhead) in the epithelial layer and both Ast-ir processes (yellow arrow) and AstR-ir processes (white arrow) in the underlying neuropile. c Disc region of a tube foot; Ast-ir processes (yellow arrow) and AstR-ir processes (white arrow) in the basal nerve ring. d Cardiac stomach; Ast-ir processes (white arrowhead) and AstR-ir processes (blue arrow) in the basiepithelial nerve plexus and mucosa, while in the visceral muscle layer, only AstR-ir is present (white arrow). Abbreviations: BNR, basal nerve ring; BNP, basiepithelial nerve plexus; CS, cardiac stomach; CT, collagenous tissue; Ec, ectoneural; Lu, lumen; ML, mucosal layer; TF, tube foot; VML, visceral muscle layer. Scale bars: d = 30 μm; c = 20 μm; a, b = 10 μm
	Fig. 7. Asterotocin causes relaxation of in vitro cardiac stomach and apical muscle preparations from A. rubens. a Representative recording showing that asterotocin causes relaxation of a cardiac stomach preparation. Seawater supplemented with KCl (3 × 10−2 M) was used to induce contraction of the cardiac stomach prior to the application of asterotocin. The relaxing effect of asterotocin is reversed when the preparation is washed with KCl-supplemented seawater. b Graph showing the concentration-dependent relaxing effect of asterotocin on cardiac stomach preparations at concentrations ranging from 3 × 10−11 M to 10−6 M. The responses are expressed as the mean relative percentage (± SEM; n = 16) of the maximal relaxing effect of asterotocin in each preparation. c Representative recording from a cardiac stomach preparation that compares the relaxing effects of asterotocin and the SALMFamide-type neuropeptide S2, both at 10−7 M. As in a, the cardiac stomach preparation was pre-contracted with KCl-supplemented seawater prior to the application of the neuropeptides. Inset compares the effects of asterotocin and S2 on cardiac stomach preparations at a concentration of 10−7 M, expressed as mean percentages (± SEM; n = 5) with the relaxing effect of S2 defined as 100%. The relaxing effect of asterotocin is significantly larger than the effect S2 (Mann-Whitney U test; P = 0.0079; n = 5). d Representative recording showing that asterotocin (10−6 M) causes relaxation of an apical muscle preparation. 10−6 M acetylcholine (ACh) was used to induce contraction of the apical muscle preparation prior to the application of asterotocin. Following washing of the preparation with artificial seawater, it returns to its basal relaxed state. Inset shows the mean percentage (± SEM; n = 4) reversal of 10−6 M ACh-induced contraction of apical muscle preparations caused by 10−6 M asterotocin over a 50-s period after peptide administration
	Fig. 8. In vivo injection of asterotocin triggers cardiac stomach eversion in A. rubens. a Dose-dependent effect of asterotocin in inducing cardiac stomach eversion. The graph shows the percentage of animals (n = 5 per group) that exhibit cardiac stomach eversion when injected with 10 μl asterotocin at concentrations ranging from 10−6 M to 10−3 M, by comparison with 10 μl of the starfish neuropeptide S2 (10−3 M) or 10 μl of water. Stomach eversion occurred in 100%, 100% and 80% of the animals injected with 10−5 M, 10−4 M and 10−3 M asterotocin, respectively, but stomach eversion was not observed in any of the animals injected with 10−6 M asterotocin, 10−3 M S2 or water. b Temporal dynamics of asterotocin-induced (10 μl of 10−3 M) cardiac stomach eversion. The graph shows mean area (± SEM; n = 13) of the cardiac stomach everted expressed as the percentage of the area of the central disc region at 30-s intervals over a 10-min period following injection of asterotocin. c Images from video recordings of the experiment in b showing a representative water-injected (control) starfish (i–iii) and a representative asterotocin-injected starfish (iv–vi) at 0 min (immediately after injection), after 5 min and after 10 min. The area of the cardiac stomach everted is shown with a dashed line in v and vi. The representative video recordings used to generate the images in c are in Additional file 7

ACHER, [The structure of bovine vasopressin]. 1954, Pubmed

ACHER, [The structure of bovine vasopressin]. 1954, Pubmed
Aikins, Vasopressin-like peptide and its receptor function in an indirect diuretic signaling pathway in the red flour beetle. 2008, Pubmed
Balment, Arginine vasotocin a key hormone in fish physiology and behaviour: a review with insights from mammalian models. 2006, Pubmed
Baubet, Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level. 2000, Pubmed
Beets, Ancient neuromodulation by vasopressin/oxytocin-related peptides. 2013, Pubmed
Beets, Vasopressin/oxytocin-related signaling regulates gustatory associative learning in C. elegans. 2012, Pubmed
Blowes, Body wall structure in the starfish Asterias rubens. 2017, Pubmed , Echinobase
Burke, A genomic view of the sea urchin nervous system. 2006, Pubmed , Echinobase
Cai, Biochemical, Anatomical, and Pharmacological Characterization of Calcitonin-Type Neuropeptides in Starfish: Discovery of an Ancient Role as Muscle Relaxants. 2018, Pubmed , Echinobase
Cruz, Invertebrate vasopressin/oxytocin homologs. Characterization of peptides from Conus geographus and Conus straitus venoms. 1987, Pubmed
Dale, The Action of Extracts of the Pituitary Body. 1909, Pubmed
Elphick, Correction: Evolution of neuropeptide signalling systems (doi:10.1242/jeb.151092). 2018, Pubmed
Elphick, The SALMFamides: a new family of neuropeptides isolated from an echinoderm. 1991, Pubmed , Echinobase
Elphick, NGFFFamide and echinotocin: structurally unrelated myoactive neuropeptides derived from neurophysin-containing precursors in sea urchins. 2009, Pubmed , Echinobase
Elphick, Distribution and action of SALMFamide neuropeptides in the starfish Asterias rubens. 1995, Pubmed , Echinobase
Garrison, Oxytocin/vasopressin-related peptides have an ancient role in reproductive behavior. 2012, Pubmed
Gesto, Arginine vasotocin treatment induces a stress response and exerts a potent anorexigenic effect in rainbow trout, Oncorhynchus mykiss. 2014, Pubmed
Gruber, Physiology of invertebrate oxytocin and vasopressin neuropeptides. 2014, Pubmed
Guindon, New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. 2010, Pubmed
Holmes, Vagal afferent fibres determine the oxytocin-induced modulation of gastric tone. 2013, Pubmed
Johnson, Oxytocin and vasopressin neural networks: Implications for social behavioral diversity and translational neuroscience. 2017, Pubmed
Jékely, The long and the short of it - a perspective on peptidergic regulation of circuits and behaviour. 2018, Pubmed
Jékely, Global view of the evolution and diversity of metazoan neuropeptide signaling. 2013, Pubmed
Kanda, Cloning of Octopus cephalotocin receptor, a member of the oxytocin/vasopressin superfamily. 2003, Pubmed
Kanda, Novel evolutionary lineages of the invertebrate oxytocin/vasopressin superfamily peptides and their receptors in the common octopus (Octopus vulgaris). 2005, Pubmed
Kawada, Characterization of a novel vasopressin/oxytocin superfamily peptide and its receptor from an ascidian, Ciona intestinalis. 2008, Pubmed
Kawada, Identification of a novel receptor for an invertebrate oxytocin/vasopressin superfamily peptide: molecular and functional evolution of the oxytocin/vasopressin superfamily. 2004, Pubmed
Kumar, MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. 2016, Pubmed
Lawson, The effects of oxytocin on eating behaviour and metabolism in humans. 2017, Pubmed
Lin, Cellular localization of relaxin-like gonad-stimulating peptide expression in Asterias rubens: New insights into neurohormonal control of spawning in starfish. 2017, Pubmed , Echinobase
Lin, Pedal peptide/orcokinin-type neuropeptide signaling in a deuterostome: The anatomy and pharmacology of starfish myorelaxant peptide in Asterias rubens. 2017, Pubmed , Echinobase
Lin, Functional characterization of a second pedal peptide/orcokinin-type neuropeptide signaling system in the starfish Asterias rubens. 2018, Pubmed , Echinobase
Lockard, Oxytocin mediated behavior in invertebrates: An evolutionary perspective. 2017, Pubmed
Mayorova, Localization of Neuropeptide Gene Expression in Larvae of an Echinoderm, the Starfish Asterias rubens. 2016, Pubmed , Echinobase
Melarange, Comparative analysis of nitric oxide and SALMFamide neuropeptides as general muscle relaxants in starfish. 2003, Pubmed , Echinobase
Mennigen, The nonapeptide isotocin in goldfish: Evidence for serotonergic regulation and functional roles in the control of food intake and pituitary hormone release. 2017, Pubmed
Mirabeau, Molecular evolution of peptidergic signaling systems in bilaterians. 2013, Pubmed
Oliver, On the Physiological Action of Extracts of Pituitary Body and certain other Glandular Organs: Preliminary Communication. 1895, Pubmed
Oumi, Annetocin: an oxytocin-related peptide isolated from the earthworm, Eisenia foetida. 1994, Pubmed
Oumi, Annetocin, an annelid oxytocin-related peptide, induces egg-laying behavior in the earthworm, Eisenia foetida. 1996, Pubmed
Pentreath, Neurobiology of echinodermata. 1972, Pubmed , Echinobase
Priyam, Sequenceserver: A Modern Graphical User Interface for Custom BLAST Databases. 2019, Pubmed
Proux, Identification of an arginine vasopressin-like diuretic hormone from Locusta migratoria. 1987, Pubmed
Richard, Central effects of oxytocin. 1991, Pubmed
SMITH, The motor nervous system of the starfish, Astropecten irregularis (Pennant), with special reference to the innervation of the tube feet and ampullae. 1950, Pubmed , Echinobase
Sawyer, Evolution of neurohypophyseal hormones and their receptors. 1977, Pubmed
Semmens, The evolution of neuropeptide signalling: insights from echinoderms. 2017, Pubmed , Echinobase
Semmens, Discovery of sea urchin NGFFFamide receptor unites a bilaterian neuropeptide family. 2015, Pubmed , Echinobase
Semmens, Transcriptomic identification of starfish neuropeptide precursors yields new insights into neuropeptide evolution. 2016, Pubmed , Echinobase
Semmens, Discovery of a novel neurophysin-associated neuropeptide that triggers cardiac stomach contraction and retraction in starfish. 2013, Pubmed , Echinobase
Skinner, The relationship between oxytocin, dietary intake and feeding: A systematic review and meta-analysis of studies in mice and rats. 2019, Pubmed
Spetter, Current findings on the role of oxytocin in the regulation of food intake. 2017, Pubmed
Tahara, Pharmacological characterization of the human vasopressin receptor subtypes stably expressed in Chinese hamster ovary cells. 1998, Pubmed
Takuwa-Kuroda, Octopus, which owns the most advanced brain in invertebrates, has two members of vasopressin/oxytocin superfamily as in vertebrates. 2003, Pubmed
Talavera, Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. 2007, Pubmed
Tian, Functional Characterization of Paralogous Gonadotropin-Releasing Hormone-Type and Corazonin-Type Neuropeptides in an Echinoderm. 2017, Pubmed , Echinobase
Tian, Urbilaterian origin of paralogous GnRH and corazonin neuropeptide signalling pathways. 2016, Pubmed , Echinobase
Tinoco, Characterization of NGFFYamide Signaling in Starfish Reveals Roles in Regulation of Feeding Behavior and Locomotory Systems. 2018, Pubmed , Echinobase
Ukena, Unique form and osmoregulatory function of a neurohypophysial hormone in a urochordate. 2008, Pubmed
Van Kesteren, Structural and functional evolution of the vasopressin/oxytocin superfamily: vasopressin-related conopressin is the only member present in Lymnaea, and is involved in the control of sexual behavior. 1995, Pubmed
Yañez-Guerra, Discovery and functional characterisation of a luqin-type neuropeptide signalling system in a deuterostome. 2018, Pubmed , Echinobase
van Kesteren, Evolution of the vasopressin/oxytocin superfamily: characterization of a cDNA encoding a vasopressin-related precursor, preproconopressin, from the mollusc Lymnaea stagnalis. 1992, Pubmed
van den Pol, Neuropeptide transmission in brain circuits. 2012, Pubmed