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

节肢动物是地球上最成功的多细胞生物，在多样性、物种和栖息地利用方面。昆虫是我们食物资源的主要竞争对手，是疾病的传播媒介，但也是至关重要的传粉者。甲壳类动物是非常重要的高价值食物资源。节肢动物的成功部分是由于它们惊人的生长可塑性。这包括角质层的周期性脱落（蜕皮），由激素的复杂相互作用控制。虽然我们对昆虫蜕皮过程中许多关键肽激素及其信号通路（受体）的作用了解得很多，但对甲壳类动物的这些作用知之甚少。由于昆虫和甲壳类动物在5亿多年前从一个共同的祖先进化而来，它们的蜕皮内分泌涉及一些最高度进化的综合过程，尽管它们的差异和不同的生活史，但仍显示出共同点！该项目将通过开拓分子技术的最新基本进展，寻求找到甲壳类动物中涉及蜕皮的共同（和独特）内分泌机制。使用各种最先进的技术，我们将解开甲壳类动物蜕皮的激素控制的复杂性（在螃蟹模型中）。我们将利用下一代测序技术和生物信息学来鉴定新的激素及其可能的受体。通过这种方法，我们可以找到昆虫肽激素及其受体的甲壳类等价物，并确定它们在蜕皮周期中的表达变化。我们将使用新技术识别神经肽受体，我们将表达受体的基因转移到基因工程细胞中，当暴露于激素时，产生生物发光。因此，我们可以从功能上识别出正确的激素及其受体。我们将识别螃蟹神经系统中表达肽受体的神经元，并将其与解剖学上产生肽的神经元联系起来。这项工作将使我们能够拼凑出与换羽有关的各种行为事件有关的“神经网络”。我们将测量激素水平在电解过程中使用超灵敏的测定。这将使我们对各种“激素级联反应”有一个独特的见解，这些“激素级联反应”每次持续几分钟，对于允许蜕化的各个阶段的进展和行为反应至关重要。我们将操纵激素级联，通过RNA干扰（RNAi）沉默基因。因此，我们可以针对每个过程，给出我们已经确定的激素和受体的身份信息，并回答关键问题：我们能否通过使用这些技术改变特定的行为，如从旧壳中出现，在蜕皮过程中吸收水分，角质层硬化？那么其他人的表达会发生什么变化呢？在甲壳类动物的蜕皮过程中，有一种关键的激素可以抑制蜕皮过程（MIH）。MIH的受体是未知的，这种内分泌系统是甲壳类动物所特有的。因此，了解信号系统是非常重要的。我们将以一种新颖的方式解决这个问题，使用一种策略，通过使用下一代测序和生物信息学来识别受体，然后在生物发光细胞试验中证明其功能。这里描述的研究将回答节肢动物内分泌学中一个突出的问题。精确定时的一系列激素级联是如何协调成功的蜕皮的？-一个对变化“零容忍”的过程，但仍然非常成功！该研究对水产养殖产生了影响。我们必须提高养殖甲壳类动物（虾、蟹）的产量，以确保全球粮食安全，但蜕皮过程中的死亡率造成巨大损失（每次蜕皮10%）。如果我们能够了解蜕皮级联的激素基础，这是由于饲养、养殖和运输不足造成的，我们就迈出了解决这些问题的第一步。

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.