Endocrine scaffolds and peptide networks: How is the molt cycle and ecdysis programme controlled in crustaceans?

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In contrast to insects, our understanding of the neuropeptide networks involved in crustacean ecdysis is rudimentary. However, our recent research has identified novel potential control mechanisms that promise to revolutionise our understanding of crustacean molt control. The overarching objective of this work is to develop a much more detailed model of the neuroendocrine control of crustacean molting, by functional analysis of neuroendocrine scaffolds and networks, comparing with insects. Three interrelated themes will be investigated: 1) The control of ecdysteroid synthesis, and entry into premolt, 2) The initiation of the "ecdysis cassette" via a proposed ecdysis-triggering hormone/eclosion hormone (ETH/EH) signalling network. 3) The interaction of these peptides in regulating sequential release of the hormones critical in integrating successful ecdysis in or crab model, Carcinus maenas.
PacBio Iso-Seq transcriptomes will be produced (specific-YO and epidermis and whole crab), and analysed to identify full-length transcripts for receptors (MIH, CHH, ETH and EH) and peptides (ETH, EH). The receptors will be functionally deorphaned by transient expression in cell-based assays (GPCRs- aequorin Ca2+ reporting, mGCs, cGMP measurement). The cells (neurones and peripheral non-neuronal ETH producing cells) expressing the receptor and peptide transcripts will be identified using RNAScope, quantitative PCR (dd-PCR) and IHC by developing antisera for ETH and EH. Functional analysis of neuropeptide networks with be done by using RNAi, peptide injections and measurement of perturbations of neuropeptide cascades in the ecdysis cassette and ultrasensitive TR-FIA for ETH, EH and CRZ. CRZ abrogation of MIH and CHH signalling in the YO will be investigated using ecdysteroid and cGMP RIA. By using these multifaceted approaches, we aim to answer a big question in arthropod endocrinology-How does neuropeptide signalling integrate molting processes in crustaceans?

The research questions posed in this proposal are firstly of interest to academic groupings, in Biological Sciences and particularly to invertebrate endocrinologists and neuroscientists. We are attempting to answer outstanding, fundamental questions in arthropod neuroendocrinology and physiology, by taking a comparative approach; the work is unifying, in that we are using the considerable body of knowledge that is available for insect model systems, to investigate these in a well-established crustacean non-model. It is now becoming clear that arthropods possess a common "toolbox" of neuropeptides and receptors, as befits their shared ancestry. Intuitively, for the most pervading phenomena in the life history of arthropods- growth and ecdysis, we should expect that common neuroendocrine control mechanisms are ubiquitous. Counterintuitively, some are very different (for example in the control of molting hormone synthesis in the two subphyla), yet others are instantly recognisable (the hormonal control of exuviation- the "ecdysis cassette"). By identifying core neuropeptide signalling pathways involved in crustacean ecdysis, we are trying to answer a big question that impacts upon every aspect of arthropod physiology and development. Thus, we have every confidence that the results from our studies will, in the near future, appear in biology textbooks. Publishing primary papers and reviews in high impact journals, presenting our work at national and international meetings, will disseminate our findings. We anticipate that the proposed work will lead to up to eight high quality, primary research papers. Crustaceans and insects are immensely charismatic animals. Crustacean ecdysis and insect metamorphosis and eclosion are fascinating behaviours that capture interest, as evidenced by our BBC coverage of crab ecdysis, (The One Show). Thus, we plan to maximise public exposure to our research (see Pathways to Impact statement). Both DCW and SGW are passionate about public engagement and have had extensive media coverage. To summarise; we will engage public interest by taking advantage of, and creating available opportunities. Relevance and impact to industry. The proposed work has a direct impact on crustacean aquaculture. Firstly, the current shrimp (penaeid) industry, presently worth 39 billion USD pa, is being transformed by development of environmentally sustainable indoor recirculating aquaculture systems (RAS) as opposed to extensive culture, which has many negative environmental and disease issues. Furthermore, the by-product of molting- the old exuviae are used to extract chitosan for pharmaceutical processes- a market worth 1.5 billion USD, which is set to grow to over 17 billion USD in the next 5 years. At the moment, we know very little regarding the hormonal control of molting in penaeid shrimp, yet we have a huge body of research in crabs, which is translational and can be directly applied to shrimp aquaculture, and quite fundamental issues which dramatically decrease productivity, such as molt-related mortality. Further potential impacts relate firstly to animal welfare issues, particularly during transportation of live crustaceans, the responses due to stress, indicated by our research implicates a common set of neuropeptides that are involved in the control of molting, and the stress response, and are probably involved in the considerable mortality of crustaceans during transport to market. Finally, a future, but possibly critical impact relates to issues involving the use of stable pseudopeptide mimetics in insect pest control. There is a renewed interest in developing these to target groups or single species of insects, and those associated with disruption of growth and ecdysis are ones that are currently demanding attention. Thus, knowledge of possible off target effects on crustaceans, with regard to disruption of molting will be vital in assessing the potential impact of these on non-target organisms.