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Unravelling the ecdysis cascade in crustaceans: Can we unify neuropeptide and receptor identities and functions in arthropods?

ReferenceBB/L021242/1
Principal Investigator / Supervisor Dr David Wilcockson
Co-Investigators /
Co-Supervisors		 Dr Martin Swain
Institution Aberystwyth University
DepartmentIBERS
Funding typeResearch
Value (£) 254,896
StatusCompleted
TypeResearch Grant
Start date 01/11/2014
End date 31/10/2017
Duration36 months

Abstract

The hormone cascades leading to execution of stepwise orchestrated behaviours culminating in ecdysis in insects have been the subject of intense and fruitful research in the past few years, not least for genetically tractable models. However, for crustaceans, much less is known. Our research programme will address this issue, uncovering both common and unique mechanisms involved in ecdysis in our crab model, Carcinus maenas. We will firstly discover the wide array of peptide hormone and GPCR receptor transcripts involved by using RNAseq technologies to produce neurotranscriptomes at several stages of the molt cycle. Thus we will discover and measure expression levels of many candidates in a global context. Functional identification of these, will build upon what is known for homologous systems in insects. To identify, and deorphanize receptor ligand pairs, we will use well-proven aequorin reporter assays in which cloned candidate GPCRs signal through a promiscuous Galpha subunit. Peptidergic neurones and networks that are organisers of the endocrine cascade will be identified, together with the receptors for the command peptides that initiate ecdysis using immunochemistry and in-situ hybridization. We will couple these results with precise measurement of peptide hormone cascades at a very fine temporal scale. These findings will lead to novel functional studies on selected neuropeptides and receptors, performed via systemic RNAi to determine disrupted phenotypes (transcript, hormone and behavioural/phenotype). Finally we will attempt to contrast conserved neuropeptide/receptor hierarchies involved in ecdysis in athropods with that of a regulatory system unique to crustacaceans, the molt-inhibiting hormone (MIH) and will answer a long-standing question in crustacean endocrinology by identifying and deorphanizing the cognate receptor for the first time, using a combination of NGS, bioinformatics and functional receptor screening in the aequorin assay.

Summary

Arthropods are the most successful multicellular organisms on earth in terms of diversity, species, and habitat utilisation. Insects are our major competitors for food resources, vectors of disease, but yet are vital as pollinators. Crustaceans are immensely important high value food resources. The success of arthropods is due in part to their amazing plasticity in growth. This involves periodic shedding (ecdysis) of the cuticle, controlled by a complex interplay of hormones. Whilst we know much about the roles of many of the key peptide hormones and their signalling pathways (receptors) involved in ecdysis in insects, much less is known about these in crustaceans. Since insects and crustaceans evolved from a common ancestor over 500 million years ago, their molting endocrinology involves some of the most highly evolved integrative processes, yet show commonality despite their divergence and contrasting life histories! This project will seek to find the common (and unique) endocrine mechanisms involved in ecdysis in crustaceans by pioneering recent fundamental advances in molecular techniques. Using a variety of state-of-the-art technologies, we will unravel the complexities of the hormonal control of crustacean molting (in a crab model). We will identify novel hormones and their putative receptors, using next generation sequencing technologies, and bioinformatics. In this way we can find the crustacean equivalents of insect peptide hormones and their receptors and identify changes in their expression during the molt cycle. We will identify neuropeptide receptors using novel technologies in which we transfer the genes that express the receptors into genetically engineered cells that, when exposed to hormone, produce bioluminescent light. Thus we can functionally identify the correct hormone with its receptor. We will identify the neurones in the crab nervous system, which express peptide receptors, and link this to the anatomy peptide producing neurones. This work will allow us to piece together the "neural networks" involved in the various behavioural events involved in molting. We will measure hormone levels during ecdysis using ultrasensitive assays.This will give us a unique insight into the various "hormone cascades", each lasting a few minutes, that are vital in allowing progression and behavioural repertoires of the various stages of ecdysis. We will manipulate the hormonal cascade, silencing genes by RNA interference (RNAi). We can thus target each process, given the information on the identity of the hormones and receptors we have identified, and answer the critical question: Can we change specific behaviours such as emergence from the old shell, water uptake during ecdysis, cuticle hardening by using these techniques? What then happens to the expression of others? One of the key hormones involved in crustacean molting is one that inhibits this process (moult-inhibiting hormone, MIH). The receptor for MIH is unknown, and this endocrine system is unique to crustaceans. Thus, understanding the signalling system is of great importance. We will approach this problem in a novel way, using a strategy to identify the receptor by using next generation sequencing and bioinformatics, and then proving functionality in the bioluminescent cell assay. The research described here will answer one of the outstanding questions in arthropod endocrinology. How do the precisely timed series of hormonal cascades orchestrate successful ecdysis?- a process that has "zero tolerance" to variability, but is nevertheless inordinately successful! The research has impact in aquaculture. We must improve yields of farmed crustaceans (shrimp, crab) to ensure global food security, yet mortality during molting results in tremendous losses (10% per molt) If we can understand the hormonal basis of the ecdysis cascade, caused by inadequate husbandry, culture and transport, we have a first step to wards redressing these problems.

Impact Summary

The research questions posed in this proposal are firstly of major interest to academic groupings in Biological Sciences. For invertebrate endocrinologists and neuroscientists, our state-of-the art approaches to answering fundamental questions related to arthropod physiology, neuroendocrinology and endocrine homeostasis, will have considerable impact. We are we making a bold attempt to unify arthropod endocrinology, which will be of broad interest to evolutionary biologists. By unravelling core mechanisms involved in ecdysis, we are trying to answer a big question that impacts on quite a number of issues that involve every aspect of arthropod physiology and development. Thus, we have every confidence that our studies will, in the near future, appear in biology textbooks. We will disseminate our findings by publishing primary papers and reviews in high impact journals, presenting our work at national and international meetings. We anticipate that the proposed work will lead to up to 6 high-quality, primary research papers. Crustaceans, and insects are (in their own way) immensely charismatic animals. Crustacean ecdysis and insect eclosion are fascinating behaviours that capture the imagination of young and old alike. Our findings will be of general interest to the public, and we have planned a vigorous, far reaching and enthusiastic series of activities (Pathways to Impact) to ensure that we maximise public exposure to our research. Both DCW and SGW are passionate about public engagement, and have recently had extensive media coverage. To summarise; we will engage public interest by taking advantage of all available opportunities. Relevance and impact to industry. The proposed work has direct impact upon crustacean aquaculture. There are two major issues for which our work will be relevant. Firstly, in the broadest sense, since we know relatively little regarding endocrine mechanisms involved in growth and reproduction in crustaceans, our findings will be important in future (technologically driven) aquaculture. More topically, and of immediate relevance, our research deals with endocrine cascades during ecdysis. At this time (preparation for, and execution of ecdysis), there are substantial losses in aquaculture. Soft-shell crab fisheries in the USA (Blue crab, Callinectes sapidus) suffer quite enormous losses from stress-related mortality in captive crabs prior to marketing. Apropos this, we should highlight welfare issues in transported crustaceans. It is not generally appreciated that almost all live crustaceans undergo extensive periods of transport, often between continents. These animals invariably become tremendously stressed, and experience severe hypoxic episodes, with high mortality. Since our research exactly involves the neurohormones involved in adaptation to stressful episodes, and use of technologies developed in the research programme (for example measurement of neurohormone levels in experiments designed to reduce transport stress) our findings will be relevant to animal welfare. The research programme involves collaboration with scientists at FERA, (cell-based aequorin receptor assays). This group investigates rational design of potential peptide mimetic pesticides, hence their interest in receptor/ligand interactions on economically important insect pests. Since, the central driver of our research programme is to unify peptide signalling systems involved in arthropod ecdysis, there is a potential possibility that our respective research programmes will interact synergistically. Training opportunities. The research programme proposed here offers superb training opportunities in data driven research (bioinformatics, NGS technologies), state of the art molecular techniques, that are in our experience, in short supply in the UK. Thus, the impact of this research is direct in that it has the immediate potential of increasing UK competitiveness in Biosciences research.
Committee Research Committee A (Animal disease, health and welfare)
Research TopicsNeuroscience and Behaviour
Research PriorityX – Research Priority information not available
Research Initiative X - not in an Initiative
Funding SchemeX – not Funded via a specific Funding Scheme
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