Identification and developmental expression of mRNAs encoding putative insect cuticle hardening hormone, bursicon in the green shore crab <em>Carcinus maenas</em>

Bursicon is the ultimate hormone in insect ecdysis, which is involved in cuticle hardening. Here we show that mRNAs encoding the heterodimeric cystine knot protein bursicon (Burs alpha, beta), are present in crustaceans, suggesting ubiquity of this hormone in arthropods. We firstly report the cloning, sequencing of mRNAs encoding subunits from the water flea, Daphnia arenata and the CNS of the crab, Carcinus maenas, in comparison with insect bursicon subunits. Expression patterns of alpha and beta burs mRNAs were examined by in-situ hybridisation (ISH) and quantitative RT-PCR. In the thoracic ganglion, burs alpha and beta mRNAs were completely colocalised in neurones expressing crustacean cardioactive peptide (CCAP). However, in the brain and eyestalk, bursicon transcripts were never observed, despite a complex expression pattern of CCAP interneurones. Patterns of expression of burs alpha and beta mRNAs were constitutive during the moult cycle of adult crabs, in stark contrast to the situation in insects. Whilst copy numbers of burs beta transcripts closely matched those of CCAP, those of burs alpha mRNA were around 3-fold higher than burs beta. This pattern was apparent during embryogenesis, where bursicon transcripts were first observed at around 50% development-the same time as first expression of CCAP mRNA. Transcript ratios (burs alpha: beta) increased during development. Our studies have shown, for the first time, that bursicon mRNAs are expressed in identified neurones in the nervous system of crustaceans. These findings will now promote further investigation into the functions of bursicon during the moult cycle and development of crustaceans