Transcriptomic analysis of crustacean neuropeptide signaling during the moult cycle in the green shore crab, Carcinus maenas

Andrew Oliphant, Jodi L. Alexander, Martin T. Swain, Simon G. Webster, David C. Wilcockson

Ecdysis is an innate behaviour programme by which all arthropods moult their exoskeletons. The complex suite of interacting neuropeptides that orchestrate ecdysis is well studied in insects, but details of the crustacean ecdysis cassette are fragmented and our understanding of this process is comparatively crude, preventing a meaningful evolutionary comparison. To begin to address this issue we identified transcripts coding for neuropeptides and their putative receptors in the central nervous system (CNS) and Y-organs (YO) within the crab, Carcinus maenas, and mapped their expression profiles across accurately defined stages of the moult cycle using RNA-sequencing. We also studied gene expression within the epidermally-derived YO, the only defined role for which is the synthesis of ecdysteroid moulting hormones, to elucidate peptides and G protein-coupled receptors (GPCRs) that might have a function in ecdysis. Transcriptome mining of the CNS transcriptome yielded neuropeptide transcripts representing 47 neuropeptide families and 66 putative GPCRs. Neuropeptide transcripts that were differentially expressed across the moult cycle included carcikinin, crustacean hyperglycemic hormone-2, and crustacean cardioactive peptide, whilst a single putative neuropeptide receptor, proctolin R1, was differentially expressed. Carcikinin mRNA in particular exhibited dramatic increases in expression pre-moult, suggesting a role in ecdysis regulation. Crustacean hyperglycemic hormone-2 mRNA expression was elevated post- and pre-moult whilst that for crustacean cardioactive peptide, which regulates insect ecdysis and plays a role in stereotyped motor activity during crustacean ecdysis, was elevated in pre-moult. In the YO, several putative neuropeptide receptor transcripts were differentially expressed across the moult cycle, as was the mRNA for the neuropeptide, neuroparsin-1. Whilst differential gene expression of putative neuropeptide receptors was expected, the discovery and differential expression of neuropeptide transcripts was surprising. Analysis of GPCR transcript expression between YO and epidermis revealed 11 to be upregulated in the YO and thus are now candidates for peptide control of ecdysis. The data presented represent a comprehensive survey of the deduced C. maenas neuropeptidome and putative GPCRs. Importantly, we have described the differential expression profiles of these transcripts across accurately staged moult cycles in tissues key to the ecdysis programme. This study provides important avenues for the future exploration of functionality of receptor-ligand pairs in crustaceans.