Discovery of a novel neurophysin-associated neuropeptide that triggers cardiac stomach contraction and retraction in starfish

Abstract

SUMMARYFeeding in starfish is a remarkable process in which the cardiac stomach is everted over prey and then retracted when prey tissue has been resorbed. Previous studies have revealed that SALMFamide-type neuropeptides trigger cardiac stomach relaxation and eversion in the starfish Asterias rubens. We hypothesized, therefore, that a counteracting neuropeptide system controls cardiac stomach contraction and retraction. Members of the NG peptide family cause muscle contraction in other echinoderms (e.g. NGFFFamide in sea urchins and NGIWYamide in sea cucumbers), so we investigated NG peptides as candidate regulators of cardiac stomach retraction in starfish. Generation and analysis of neural transcriptome sequence data from A. rubens revealed a precursor protein comprising two copies of a novel NG peptide, NGFFYamide, which was confirmed by mass spectrometry. A noteworthy feature of the NGFFYamide precursor is a C-terminal neurophysin domain, indicative of a common ancestry with vasopressin/oxytocin-type neuropeptide precursors. Interestingly, in precursors of other NG peptides the neurophysin domain has been retained (e.g. NGFFFamide) or lost (e.g. NGIWYamide and human neuropeptide S) and its functional significance remains to be determined. Investigation of the pharmacological actions of NGFFYamide in starfish revealed that it is a potent stimulator of cardiac stomach contraction in vitro and that it triggers cardiac stomach retraction in vivo. Thus, discovery of NGFFYamide provides a novel insight into neural regulation of cardiac stomach retraction as well as a rationale for chemically based strategies to control starfish that feed on economically important shellfish (e.g. mussels) or protected marine fauna (e.g. coral). Supplementary material available online at

Localization of Neuropeptide Gene Expression in Larvae of an Echinoderm, the Starfish Asterias rubens

“…The sequences of cDNAs encoding six of the seven other neuropeptide precursors were determined here for the first time, with the exception of the ArGnRH precursor cDNA, which was reported recently (Tian et al, 2016). The sequences of cDNAs encoding the F-type SALMFamide (S2) precursor (Supplementary Figure 2; GenBank accession number KP330476), asterotocin precursor (Supplementary Figure 3; GenBank accession number KT601711), NGFFYamide precursor (Supplementary Figure 4; GenBank accession number KC977457), ArGnRH precursor (Supplementary Figure 6; GenBank accession number KT601712), ArCT precursor (Supplementary Figure 7; GenBank accession number KT601715), and ArCRH precursor (Supplementary Figure 8; GenBank accession number KT601710) were found to be identical to the assembled transcript sequences reported previously (Semmens et al, 2013, 2016). However, sequencing of a cDNA encoding the A. rubens TRH-type precursor (ArTRHP) revealed a difference to the predicted transcript sequence reported previously (Semmens et al, 2016).…”

“…The sequences of transcripts encoding A. rubens neuropeptide precursors have been identified previously by analysis of radial nerve cord transcriptome sequence data from this species (Semmens et al, 2013, 2016). Here, the expression of eight of these neuropeptide precursors was investigated in A. rubens larvae using whole-mount mRNA in situ hybridization.…”

“…For this reason, NGFFYamide is a good candidate as a regulator of swimming and/or feeding behavior in A. rubens larvae. Interestingly, NGFFYamide triggers contraction and retraction of the cardiac stomach in adult starfish, indicating a role in terminating the extra-oral feeding behavior of adult starfish (Semmens et al, 2013). Thus, NGFFYamide may be involved in regulation of feeding behavior throughout the starfish lifecycle, regulating ciliary beating around the mouth in larvae and triggering cardiac stomach retraction in adults.…”

“…The foundation for the present study was the recent identification of 40 neuropeptide precursor transcript sequences in the starfish A. rubens (Semmens et al, 2013, 2016), which represents the most comprehensive resource to date for echinoderm neuropeptide research. These neuropeptide precursors were identified by analysis of the transcriptome of the radial nerve cords from adult starfish.…”

“…The eight neuropeptide precursors selected for analysis in this study include: (a) the L-type and F-type SALMFamide neuropeptide precursors, which are precursors of the prototypical SALMFamides S1 and S2, respectively, and 13 related SALMFamide-type neuropeptides, (b) the precursor of a vasopressin/oxytocin-type neuropeptide (“asterotocin”), (c) the precursor of the neuropeptide NGFFYamide, which belongs to a bilaterian family of neuropeptides that includes human neuropeptide-S (NPS) and crustacean cardioactive peptide (CCAP; Semmens et al, 2013, 2015), (d) ArTRHP, a thyrotropin-releasing hormone (TRH)-type neuropeptide precursor, (e) ArGnRHP, the precursor of a gonadotropin-releasing hormone (GnRH)-type neuropeptide, (f) ArCTP, the precursor of a calcitonin-type neuropeptide, and (g) ArCRHP, the precursor of a corticotropin-releasing hormone (CRH)-type neuropeptide.…”