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

Abstract

BackgroundEcdysis 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.ResultsTranscriptome 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5057-3) contains supplementary material, which is available to authorized users.

The Crustacean Hyperglycemic Hormone Superfamily: Progress Made in the Past Decade

“…Similarly, only two mih genes have been identified in several species ( 111 , 118 ). Some are clearly derived from recent duplications and show little divergence ( 119 ), while others differ substantially from the most represented isoforms and clearly demonstrate that diversity has been underestimated ( 45 ). It is very likely that additional transcriptomic analyses will uncover new isoforms, even in species that have already been well studied ( 119 ).…”

“…Some are clearly derived from recent duplications and show little divergence ( 119 ), while others differ substantially from the most represented isoforms and clearly demonstrate that diversity has been underestimated ( 45 ). It is very likely that additional transcriptomic analyses will uncover new isoforms, even in species that have already been well studied ( 119 ). This diversity supports the importance of this molecular family at the physiological level and could also explain the apparent pleiotropy of the CHHs.…”

Comparative and Evolutionary Physiology of Vasopressin/ Oxytocin-Type Neuropeptide Signaling in Invertebrates

“…Expression of a VP/OT-type neuropeptide precursor in the shore crab Carcinus maenas has been examined in the context of ecdysis, which is regulated by release of ecdysteroid molting hormones from the endocrine Y-organs. Changes in Y-organ expression of the VP/OT-type neuropeptide precursor transcripts were detected over the molt cycle, but no changes in VP/OT-type receptor expression were observed (76).…”

Identification and characterisation of crustacean hyperglycaemic hormone (CHH) from Mediterranean shore crab Carcinusaestuarii

“…The synthesis of both 5' and 3' RACE cDNA was carried out using the SMART cDNA synthesis kit (BD Biosciences), with specific CHH primers drawn with Primer3Plus v. 4.0.0 (Untergasser, 2012) and OligoCalc v. 3.27 (Kibbe, 2007) to exclude possible dimers and hairpin formations. A CHH from C. maenas was used as template (GenBank: X17596) and the resulting primers (shown in Table 1) were further compared with additional CHH XO-type (namely, AF286078, AF286079, AF286080 and AF286091) together with CHH-XO characterized in the transcriptomic study by Oliphant et al (2018) to verify correspondence with the sequences. Green SuperMix (Quantabio), 0.3 µM each primer and 1 µL of cDNA.…”

“…Oliphant et al (2018) from C. maenas, where the majority of the CHHs have a Gln30.However, the variant 2 and the XO-CHH(Oliphant et al, 2018) from C. maenas have also a Pro20, within the signal peptide, where other CHHs, including the one from C. aestuarii, have an Ala20.…”