Tinoco AB , Semmens DC , Patching EC , Gunner EF , Egertová M , Elphick MR .

	Figure 1. Feeding behavior of the starfish A. rubens in a laboratory setting. (A) Here a specimen of A. rubens can be seen making contact with a mussel (M. edulis) in an aquarium tank. In experiments reported in this paper, the time elapsed from when the starfish was placed at the opposite end of the tank to the mussel to when the starfish made contact with the mussel (“time to touch”) was measured and recorded. (B,C) Here a specimen of A. rubens can be seen enclosing a mussel at the onset of a feeding bout, with photographs taken from a side view (B) and from above (C). In experiments reported in this paper, the time elapsed from when the starfish was placed at the opposite end of the tank to the mussel to when the starfish enclosed the mussel (“time to enclose”) was measured and recorded. Scale bars: (A): 2 cm; (B): 2.5 cm; (C): 1.5 cm.
	Figure 2. Recording locomotor activity of the starfish A. rubens in a laboratory setting. (A) Photograph of the experimental set-up, with a camera positioned above a Plexiglas aquarium (37 × 19.8 × 25 cm) containing 7 l of seawater, with all the walls covered with white plastic and situated on top of a lightbox to enhance video contrast, which facilitated detection of the experimental animal by the video-tracking software Ethovision XT1. (B) Photograph showing the software arena settings, with the area defined as the floor in yellow (length of 33 cm) and a starfish positioned in the center of the aquarium at the beginning of an experiment. (C) Photograph showing tracking of a starfish using Ethovision XT1, with the color scale bar representing the time spent in one position (minimum in blue to maximum in red). Scale bars: (A): 13.1 cm; (B): 4.7 cm; (C): 4.7 cm.
	Figure 3. Identification of an NPS/CCAP-type receptor in the starfish A. rubens that is activated by the neuropeptide NGFFYamide. (A) Phylogenetic tree showing that the A. rubens NPS/CCAP-type receptor (arrow) is positioned in a clade comprising NPS/CCAP-type receptors that is distinct from a clade comprising paralogous VP/OT-type receptors. Thyrotropin-releasing hormone (TRH)-type receptors were included here as an outgroup. Bootstrap support (1,000 replicates) for each clade is represented with colored stars as denoted in the key. Species in which the ligands for NPS/CCAP-type receptors and/or VP/OT-type receptors have been identified experimentally are shown with red lettering. Species: A. gam (Anopheles gambiae); A. jap (Apostichopus japonicus); A. med (Antedon mediterranea); A. rub (Asterias rubens); B. flo (Branchiostoma floridae); C. tel (Capitella teleta) D. mel (Drosophila melanogaster); E. fet (Eisenia fetida); H. sap (Homo sapiens); L. gig (Lottia gigantea); L. sta (Lymnaea stagnalis); S. kow (Saccoglossus kowalevskii); S. pur (Strongylocentrotus purpuratus); T. cas (Tribolium castaneum). The accession numbers of sequences included in the phylogenetic tree are listed in Supplementary Table 1. (B) Concentration-response curve showing that synthetic NGFFYamide (shown in red) activates the A. rubens NPS/CCAP-type receptor in CHO cells co-expressing the promiscuous G-protein Gα16 and over-expressing the GFP-aequorin fusion protein G5A. Control experiments where CHO cells were transfected with an empty pcDNA 3.1(+) vector are shown in black. Each point (± s.e.m.) represents mean values from at least two independent experiments, with each experiment performed in triplicate. Concentration-response data are shown relative (%) to the maximal response (100% activation) observed in each experiment. The EC50 value for activation of the NPS/CCAP-type receptor with NGFFYamide is 2.1 × 10−13 M.
	Figure 4. Localization of NGFFYamide precursor mRNA expression in the nervous system of A. rubens. (A) Transverse section of a radial nerve cord showing stained cells in the epithelium of the ectoneural region, largely concentrated laterally. Note the absence of stained cells in the hyponeural region. The boxed region is shown at a higher magnification in (B), where the elongate, bipolar shape of stained cells (arrowheads) can be seen. (C) Longitudinal section of a radial nerve cord showing stained cells (arrowheads) along the length of the epithelium in the ectoneural region (arrowheads). (D) Stained cells (arrowheads) in the ectoneural epithelium of the circumoral nerve ring. (E) Transverse section of an arm showing stained cells (arrowhead) in the marginal nerve, which is located lateral to the outer row of tube feet on each side of the arm. ec, ectoneural; hy, hyponeural; MN, marginal nerve; TF, tube foot. Scale bars: (A): 33 μm; (B): 16 μm; (C): 16 μm; (D): 33 μm; (E): 16 μm.
	Figure 5. Localization of NGFFYamide precursor mRNA expression in the digestive system of A. rubens. (A) Low magnification image showing stained cells (arrowheads) widely distributed in a section of a highly folded aboral region of the cardiac stomach. (B) Stained cells in the cardiac stomach, with the boxed region shown at a higher magnification in (C), where stained cells (arrowheads) can be seen to be located in the mucosal layer or just beneath the basiepithelial nerve plexus. (D) Transverse section of the central disk region showing the cardiac stomach, pyloric stomach, rectal caeca and apical muscle. Stained cells can be seen in the cardiac stomach, pyloric stomach and apical muscle (arrowheads) but not in the rectal caeca. A higher magnification image of expression in the apical muscle is shown in Figure 6E and the boxed region of the pyloric stomach is shown at higher magnification in (E). (F) Stained cells (arrowheads) in a transverse section of a pyloric duct. AM, apical muscle; BNP, basiepithelial nerve plexus; CT, collagenous tissue layer; CS, cardiac stomach; ML, mucosal layer; PS, pyloric stomach; RC, rectal caeca; VML, visceral muscle layer. Scale bars: (A): 33 μm; (B): 16 μm; (C): 6 μm; (D): 33 μm; (E): 16 μm; (F): 16 μm.
	Figure 6. Localization of NGFFYamide precursor mRNA expression in the tube feet, apical muscle and body wall of A. rubens. (A) Longitudinal sections of two tube feet, showing stained cells (arrowheads) in the sucker region. (B) Longitudinal sections of three tube feet, with the boxed region shown at higher magnification in (C); here stained cells (arrowhead) can be seen near to the basal nerve ring (*) and in the adhesive region of the tube foot sucker (arrow). (D) Transverse section of an arm showing stained cells (arrowheads) in the coelomic epithelium. (E) Transverse section at the junction between an arm and the central disk showing stained cells (arrowheads) in the coelomic epithelial lining the apical muscle. (F) Transverse section of an arm showing stained cells (arrowheads) in the external epithelium of the body wall. AM, apical muscle; CE, coelomic epithelium; PS, pyloric stomach. Scale bars: (A): 33 μm; (B): 33 μm; (C): 6 μm; (D): 16 μm; (E): 16 μm; (F): 16 μm.
	Figure 7. NGFFYamide causes relaxation of in vitro acetylcholine-contracted apical muscle preparations from A. rubens. A representative recording is shown, with 10−6 M NGFFYamide causing transient partial reversal of the contracting action 10−6 M acetylcholine (ACh). Application of ACh and NGFFYamide are labeled with upward pointing arrowheads and washing of the preparation is labeled with a downward pointing arrowhead. The graph shows the mean (± s.e.m.) relaxant effect of NGFFYamide when tested at a concentration of 10−6 M (n = 4).
	Figure 8. Application of NGFFYamide to in vitro tube foot preparations from A. rubens triggers phasic contractions that are superimposed upon an increase in basal tone. Representative recordings from a single tube foot preparation are shown, comparing the effect of (A) acetylcholine (ACh; 10−6 M) and the effect of (B) NGFFYamide (10−6 M). Application of ACh and NGFFYamide are labeled with upward pointing arrowheads and washing of the preparation is labeled with a downward pointing arrowhead.
	Figure 9. The effects of NGFFYamide in causing both tonic and phasic contraction of tube foot preparations from A. rubens are concentration dependent. (A) Concentration-dependence of the effect of NGFFYamide in causing tonic contraction of tube feet. (B) Concentration-dependence of the effect of NGFFYamide on the peak amplitude of NGFFYamide-induced phasic contractions. (C) Concentration-dependence of the effect of NGFFYamide on the peak frequency of NGFFYamide-induced phasic contractions. The effects of NGFFYamide shown in (A,C) are normalized to the contractile effect observed with 10−6 M Acetylcholine (ACh). Results are expressed as mean ± s.e.m. (n = 6). Different letters indicate statistically significant differences (p < 0.05) by one-way ANOVA (A) or Kruskal-Wallis test (B,C).

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