Neuropeptide function in a decentralised nervous system

Neuropeptides are "messenger molecules" that enable nervous systems to co-ordinate physiological processes and behaviour in humans and other animals. Research on neuropeptides has provided important insights on the molecular basis of the physiological mechanisms that underlie how we feel and behave. For example, we know that morphine exerts its pain-relieving effect in humans because it mimics the action of endogenous neuropeptides - the "endorphins". Whilst discovery of the actions of the neuropeptide oxytocin in neural mechanisms of social behaviour in humans and other animals have led to it being labeled as the "hormone for love and trust". But when did these key molecular regulators of behaviour originate in the evolutionary history of animal life on earth? Answering this question has proven to be difficult because neuropeptides are comprised of only short strings of amino acids. Thus, when comparing neuropeptides in humans with those present in, for example, a distantly related "model" invertebrate such as the fruit fly Drosophila, identifying of relationships between neuropeptides is challenging because of amino acid sequence divergence. However, with the falling cost of DNA sequencing it has become feasible to identify neuropeptide-encoding genes in an increasingly wide range of animals, and this has provided important new insights on the evolutionary history of neuropeptide signaling systems. In particular, it has been the availability of DNA sequence data from a variety of marine invertebrates that has provided critical "missing pieces" in the "jigsaw puzzle" of neuropeptide evolution. For example, the hormone thyrotropin-releasing hormone (TRH), which has a key role in regulating growth in humans, was until recently thought to occur only in vertebrates. But our research has revealed that TRH-type neuropeptides also occur in echinoderms (e.g. sea urchins, starfish), demonstrating that TRH has a much more ancient evolutionary history than hitherto thought.

The aim of this project is to investigate and determine the physiological/behavioural roles of neuropeptides in the common European starfish Asterias rubens. Why this species and why now? Firstly, as an echinoderm, this species belongs to the same branch of the animal kingdom (deuterostomes) as vertebrates and therefore it provides a "missing link" between the well-characterised neuropeptide systems of vertebrates and protostomian invertebrates such as Drosophila. Secondly, starfish and other echinoderms have a pentaradial body plan without a brain (a "decentralized" nervous system), providing a unique context in which to determine how neuropeptide systems are used to control physiological/behavioural processes. Thirdly, Asterias rubens is a very abundant species in European waters, which makes it easy to obtain for experimental studies. Fourthly, using new DNA sequencing technology, we have recently determined the sequences of ~16,000 genes that are expressed in the nervous system of this species, including many neuropeptide genes. Thus, we are now for the first time able to comprehensively investigate neuropeptide function in this echinoderm species. By investigating neuropeptide expression and action in Asterias rubens using a range of techniques (see Technical Summary), we will i). discover the physiological roles of neuropeptides in starfish, providing for the first time comprehensive insights on how neuropeptide systems are used to regulate physiological processes and behaviour in echinoderms, pentaradially symmetrical animals that have a decentralized nervous system without a brain ii). determine to what extent there has been conservation/diversification of neuropeptide function in echinoderms, by comparison of our findings with knowledge of neuropeptide function in other animals (vertebrates and other invertebrates).

This will be the first multi-gene analysis of neuropeptide expression and function in the nervous system of an echinoderm. We have used Illumina HiSeq to sequence the neural transcriptome of the starfish Asterias rubens, employing SOAP de novo to assemble ~16,000 contigs (>1000 bp). We have identified over 30 neuropeptide precursors and 8 of these have been selected for analysis in this project based on the following criteria: a). they contain a peptide that is a member of an evolutionarily conserved bilaterian neuropeptide family b). they give rise to only one or two putative neuropeptides These are: 1. a vasopressin/oxytocin-type neuropeptide; 2). a NPS/CCAP-type neuropeptide (NGFFYamide); 3. a GnRH-type neuropeptide; 4. a TRH-type neuropeptide; 5. two CCK/gastrin-type neuropeptides; 6. a luqin-type neuropeptide; 7. an orexin-type neuropeptide and 8. a calcitonin-type neuropeptide.

The molecular structure of the neuropeptides will first be determined by analysis of starfish nerve extracts using nanoflow liquid chromatography with electrospray ionization quadrupole time-of-flight tandem mass spectrometry. Neuropeptide precursor mRNA expression patterns in A. rubens will then be determined using in situ hybridization (ISH) methods, employing enzyme-based and/or fluorescent markers to reveal DIG-labelled probes in serial sections of starfish. Specific antibodies to the neuropeptides will be generated and used for immunocytochemical (ICC) visualization of neuropeptides in starfish. Detailed analysis of neuropeptide expression in A. rubens using ISH and ICC will provide a neuroanatomical framework for investigation of the pharmacological actions of the neuropeptides, using in vivo or in vitro assays to test effects of neuropeptides on a). stomach eversion/retraction b). righting behaviour c). muscle activity d). body wall stiffness e). arm autotomy f). gamete release.

Who might benefit from this research?

1. Academic beneficiaries: (see above)
2. UK expertise: training the next generation of comparative physiologists
3. Aquaculture industry: shellfish food security
4. Ecosystem services: protection of coral reefs

How might they benefit from this research?

UK expertise: training the next generation of comparative physiologists

The UK has a rich tradition of research in comparative physiology and we are at the beginning of a new era where the falling cost of DNA sequencing is enabling any species to have its genome/transcriptome sequenced. This is expanding the range of animal species that can be utilized to gain molecular insights on physiological processes. The Elphick lab provides a unique contribution to UK research in comparative physiology in using echinoderms (in particular starfish) as experimental systems for neuropeptide biology. Elphick established this field of research as a PhD student (1988-1991; funded by SERC, forerunner to BBSRC) and then moved on to other areas of research, focusing on NO signaling and endocannabinoid signaling (supported by BBSRC grants). Next generation transcriptome sequencing has enabled Elphick to return to a field of research that he pioneered over twenty years ago. In the proposed project Elphick will train a PDRA in echinoderm neurobiology, which will contribute toward maintaining UK expertise and research excellence in comparative neurobiology and echinoderm biology. Furthermore, Elphick's outreach project "A five-sided life: the amazing biology of starfish" is designed to inspire sixth-formers to become the next generation of comparative physiologists in the post-genomic era.

Aquaculture industry: shellfish food security
The common European starfish Asterias rubens feeds on bivalve molluscan species that are economically important as foodstuffs - e.g. mussels, clams, scallops. Starfish predation impacts on the productivity of shellfish aquaculture facilities. For example, a recent study assessing the impact of starfish on scallop aquaculture concludes: "methods for reducing scallop predation by sea stars are necessary". In the proposed project we will obtain new insights into neural mechanisms that control the feeding behaviour of starfish. By identifying and characterizing the mechanisms of action of neuropeptides that trigger stomach eversion or retraction in starfish, we will obtain data that may provide a basis for development of novel strategies for chemical control of starfish predation on shellfish. As a step towards this objective, we will investigate nitric oxide synthase inhibitors as small-molecule inhibitors of neuropeptide-mediated stomach eversion in starfish.

Ecosystem services: protection of coral reefs
The environmental impact of starfish predation is perhaps most infamously associated with the crown-of-thorns-starfish (COTS) Acanthaster plancii, which feeds on coral, causing destruction of the Great Barrier Reef in Australia. Efforts to develop novel strategies to control COTS are focusing on the ongoing sequencing of the genome/transcriptome of Acanthaster plancii, led by Dr Mike Hall at the Australian Institute for Marine Sciences. Recognising that our research on neuropeptidergic control of feeding behaviour in the European starfish Asterias rubens has potential translational relevance to control of COTS, we have established contact with Dr Hall. In this project, we will utilize our Asterias rubens sequence data to identify orthologous neuropeptides in COTS. Informed by experimental findings from this project, we will then seek to secure funding to establish a collaborative project with Dr Hall's group that would directly investigate the actions of neuropeptides on the feeding behaviour of COTS. This may then provide a basis for development of novel methods for chemical control of COTS, with concomitant potential benefits to coral reefs as ecosystem services.