Award details

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

Reference	BB/T005084/1
Principal Investigator / Supervisor	Dr David Wilcockson
Co-Investigators / Co-Supervisors
Institution	Aberystwyth University
Department	IBERS
Funding type	Research
Value (£)	140,958
Status	Current
Type	Research Grant
Start date	16/03/2020
End date	15/03/2024
Duration	48 months

Abstract

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?

Summary

Arthropods are the most successful multicellular organisms on earth, in terms of diversity, species number and habitat utilisation. Arguably, they are the most important on earth; providing key ecosystem roles, as pollinators, pests and vectors of disease, yet many, for example crustaceans, are high value food resources. Their success is due to their growth format, which involves periodic shedding of the exoskeleton to allow growth, and development. Recently, there has been an explosion of information regarding the nature of insect peptide hormones, and the genetic tractability of several model insect species, together with genome and transcriptome analyses has led to dramatic, indeed fundamental advances in our knowledge of the hormonal control of insect ecdysis programmes. However, in comparison, knowledge of the equivalent processes in crustaceans is far less well developed. Whilst arthropods share a common ancestry, (having diverged over 500mya), which is beautifully reflected in the similarity of many of the peptides and receptors of insects and crustaceans, there has been a divergence in function of a number of these. This is illustrated nicely when comparing molt control in crustaceans and insects: The core components involved in stereotyped behaviours involved in emergence from the old exoskeleton (the "ecdysis cassette") are similar, yet the control mechanisms that drive increases in molting hormone synthesis are entirely different. We have recently discovered a novel neuropeptide signalling system (corazonin- CRZ) which targets the molting gland (Y-organ) that will completely reappraise models of crustacean molting with regard to the control of ecdysteroid synthesis. In between these (premolt initiation and ecdysis), we have discovered tantalising glimpses of a peptide and receptor signalling system (ecdysis triggering hormone, ETH, and eclosion hormone, EH) which appears to be homologous to those in insects, but for which we have no functional information in crustaceans. In this project, the over-arching objective is to unravel the complexities of neuropeptide/receptor signalling involved in the three phases of the molt cycle outlined here, using our well established crab (Carcinus maenas) model. Firstly, will investigate the novel role of CRZ signalling in relation to its proposed role in controlling the action of the molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone CHH that will likely be a key control in entry into premolt. Since the receptors for these hormones have not been identified, we will identify and functionally characterise these by a single transcript sequencing and bioinformatics approach, whereby full length transcript sequences of putative receptors expressed by the Y-organ will be identified, followed by functional "deorphaning" in heterologous expression systems. Secondly, we will identify the peptide signalling pathway and expression of ETH and EH and their cognate receptors, which are thus far only known in insects using the approaches alluded to here. Finally we aim to discover the roles of these hormone signalling systems in the ultimate stage of molting- the precisely integrated behavioural and hormonal cascades involved in ecdysis using a targeted approach. We will manipulate or disrupt the ecdysis "cassette" by altering expression and/or timing of release of these hormones, which will involve determining alterations in the sequential release of key hormones during ecdysis. The proposed research will firstly answer the big question in crustacean endocrinology: How is ecdysis controlled? Furthermore, it has direct potential application and benefits to aquaculture, since it is translational. We propose that the hormonal control mechanisms that we identify in a crab model will, since these are core processes, be directly translatable to shrimp aquaculture, where there is almost no knowledge of functional characterised neuroendocrine control of ecdysis.

Impact Summary

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 publicengagement 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.

Committee	Research Committee A (Animal disease, health and welfare)
Research Topics	Neuroscience and Behaviour
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/T005912/1 Endocrine scaffolds and peptide networks: How is the molt cycle and ecdysis programme controlled in crustaceans?

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