Neuropeptide function in a decentralised nervous system

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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 protection
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.