Abstract
The functional characterization of crustacean neuropeptides and their cognate receptors has not kept pace with the recent advances in sequence determination and, therefore, our understanding of the physiological roles of neuropeptides in this important arthropod sub-phylum is rather limited. We identified a candidate receptor-ligand pairing for diuretic hormone 31 (DH31) in a neural transcriptome of the crab, Carcinus maenas. In insects, DH31 plays species -specific but central roles in many facets of physiology, including fluid secretion, myoactivity, and gut peristalsis but little is known concerning its functions in crustaceans. The C. maenas DH31 transcript codes for a 147 amino acid prepropeptide, and a single receptor transcript translates to a secretin-like (Class B1) G protein-coupled receptor (GPCR). We used an in vitro aequorin luminescence Ca2+ mobilization assay to demonstrate that this candidate DH31R is activated byCarcinus and insect DH31s in a dose-dependent manner (EC50 15–30 nM). Whole mount immunohistochemical and in situ hybridization localization revealed extensive DH31 expressing neurons throughout the central nervous system, most notably in the abdominal ganglion where large, unpaired cells give rise to medial nerves, which terminate in extensive DH31 immunopositive dendritic fields intimately associated with oesophageal musculature. This system constitutes a large and hitherto undescribed neurohemal area adjacent to key muscle groups associated with the gastric system. DH31 expressing neurons were also seen in the cardiac, commissural, oesophageal, and stomatogastric ganglia and intense labeling was seen in dendrites innervating fore- and hindgut musculature but not with limb muscles. These labeling patterns, together with measurement of DH31R mRNA in the heart and hindgut, prompted us test the effects of DH31 on semi-isolated heart preparations. Cardiac superfusion with peptide evoked increased heart rates (10–100 nM). The neuroanatomical distribution of DH31 and its receptor transcripts, particularly that associated with gastric and cardiac musculature, coupled with the cardio- acceleratory effects of the peptide implicate this peptide in key myoactive roles, likely related to rhythmic coordination.
Highlights
In recent years, de novo assembly and data mining of transcriptomes and genomes of arthropods has revealed a wonderfully rich and diverse collection of neuropeptide signaling molecules, and their putative receptors (GPCRs)
Deduced amino acid sequences for DH31R and comparisons of functionally identified diuretic hormone 31 (DH31) receptors from insects are shown on Figure 1 and the prepro- DH31, with comparisons with selected crustacean and insect mature peptides are shown on Figure 2, together with a cladogram for crustacean DH31s
Our C. maenas neurotranscriptomes revealed just one DH31R transcript which is a member of the secretin (Class B1) G protein-coupled receptor (GPCR) family; as are other identified arthropod DH31 GPCRs which show high sequence identity in the transmembrane regions, and in the extracellular N-terminal region where the six conserved Cys residues are located, which are typical for the secretin receptor family
Summary
De novo assembly and data mining of transcriptomes and genomes of arthropods has revealed a wonderfully rich and diverse collection of neuropeptide signaling molecules, and their putative (mainly G protein- coupled) receptors (GPCRs). Many of these are common to both insects and crustaceans- perhaps unsurprisingly, given the consensus that all arthropod lineages arose from a monophyletic ancestor (Cook et al, 2001; Regier et al, 2010). During the passing of 400 million years of divergent arthropod evolution, commonality in structure across the phylum cannot be assumed to reflect current function
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