Bursicon and neuropeptide cascades during the ecdysis program of the shore crab, Carcinus maenas

Very little is known regarding the release patterns of neuropeptides involved in ecdysis of crustaceans compared to insects. In particular, the dynamics of release of the insect cuticle hardening hormone bursicon, which has only recently been discovered in crustaceans, is unknown. Bursicon has not previously been identified as a circulating neurohormone in these animals. Since patterns of release were likely to be ephemeral, bursicon, as well as two other neurohormones involved in the ecdysis program in crustaceans, crustacean cardioactive peptide (CCAP) and crustacean hyperglycaemic hormone (CHH) were measured in single haemolymph samples in Carcinus maenas. For bursicon, an ultrasensitive time resolved-fluoroimmunoassay (TR-FIA) was developed, which firstly involved its characterisation by HPLC, bioassay and immunoassay. Simultaneous measurement of three neurohormones was performed at unparalleled levels of resolution, which has not previously been reported in any invertebrate. Additionally, expression patterns and architecture of neurones expressing both bursicon and CCAP were determined in the CNS during the moult cycle. Bursicon and CCAP are released in a massive surge, likely a single global exocytotic event on emergence, just after release of CHH. Despite co-localisation of CCAP and bursicon in neurones of the CNS, observations suggest that differential packaging of CCAP can occur in the pericardial organs in a small population of secretory boutons, thus accounting for observations showing release of some CCAP during the penultimate stages of the ecdysis program. The results obtained vividly illustrate the dynamism of neuropeptide cascades occurring during crustacean ecdysis, and also allow proposal of a hypothesis of its endocrine control.