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

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.

"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."