Neurohormone cascades during crustacean ecdysis

All arthropods (such as insects and crabs) must periodically shed their exoskeletons in order to grow and matamorphose. This process, called ecdysis, is under complex hormonal control. Just before ecdysis a precisely timed series of hormonal 'cascades' begin, which set off a series of behavioural and morphological changes allowing the animal to emerge, swell to its final (larger size) and eventually harden the exoskeleton. For insects, and particularly for the fruit fly, Drosophila much is known about the hormones involved in these processes, and in particular, the hormones called crustacean cardioactive peptide and bursicon are particularly well studied. For crustaceans hardly anything is known, despite the fact that crustacean cardioactive peptide was, as its name suggests firstly found in crabs! With regard to bursicon, absolutely nothing was known until very recently, when I discovered genes encoding similar 'bursicon-like' hormones in crabs. However, we do not know much about how these hormones work, or their functions in crustaceans, and since aquatic crustaceans (i.e. crabs) and insects (i.e.flies) have very different lifestyles, it would be fascinating to determine how these hormones work and interact during ecdysis. This will be done by determining the hormones' distribution and levels in the nerve cells in the central nervous system of the crab, and how they are released during ecdysis by measuring patterns of release using highly sensitive assay techniques to determine the vanishingly small amounts of hormones released at precisely timed stages of emergence from the old exoskeleton, and as the new one develops and hardens. I will also use a powerful technique called 'RNA interference' (the founders of this technique have just been awarded a Nobel prize), to reduce the amount of hormones and/or the levels of gene expression (bursicon and crustacean cardioactive peptide) present in the crabs nervous system during moulting, or reduce the numbers of bursicon receptors in the crabs skin (the target tissue for bursicon in insects), to see if I can disrupt the normal moulting pattern and behaviour of the animal, thus shedding light on the functions of the hormones. Finally, I will try to 'rescue' these abnormal moulting patterns by injecting the hormones, or pharmacological agents involved in transmitting the hormonal signal to the cellular machinery involved in producing the new exoskeleton, and observing subsequent events during and after moulting. In this way, a much more detailed understanding of an exquisitely complex process- ecdysis- can be obtained. This will not only allow us to compare differences in the way these hormones act between the two largest arthropod groups, but also may in the future, allow manipulation of moulting in economically important crustaceans, to maximise yield in an economically sustainable way.

This project seeks to address topical issues concerning the distribution, transcription and release patterns of the insect cuticle tanning hormone (bursicon) in crustaceans, and relate these hormone cascades such as that involving crustacean cardioactive peptide (CCAP) in crustaceans, with particular emphasis on the functions of bursicon. This approach is particularly timely, since I have recently found bursicon transcripts encoding a putative heterodimer in the crab Carcinus maenas, which are extraordinarily similar to those identified just last year in the model insect, Drosophila. Thus this project seeks to capitalise upon entirely novel, exciting findings. To determine transcripts of bursicon (burs), partner of bursicon (pburs) and CCAP in the CNS as well as their cognate peptides, in-situ hybridisation, ICC Q-PCR will be used on precisely staged crabs. Hormone titres will likewise be measured to obtain fine temporal scale hormone cascade patterns. Functional issues will be addressed by using bioassay with native (or recombinant) peptides in ecdysing crabs and transcript knockdown and hence phenotype will be determined by developing RNAi techniques using long dsRNA. Disrupted phenotypes (behavioural, morphological) will be described, together with detailed quantitative information on transcript, hormone and second messenger profiles in these animals during the ecdysis programme. Sequencing the cognate bursicon receptor (the crab homologue of the insect rickets (rk) receptor, and using the sequence information in RNAi experiments will extend studies. Once knockdown phenotypes have been identified, I will attempt to rescue these by hormone injection or pharmacological intervention (stimulation of the second messenger pathway). Using these approaches, which span from gene, to whole organism, novel and exciting results will be obtained which will impact significantly on our understanding of hormone cascades involved in ecdysis in arthropods.