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

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?

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.