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A novel pathway of nutrient absorption in crustaceans: branchial amino acid uptake in the green shore crab (Carcinus maenas).

ABSTRACT: Estuaries are environments enriched with dissolved nutrients such as amino acids. To date, marine arthropods are the only invertebrate group that have not been demonstrated to access this potentially important nutrient resource. Using in vitro gill perfusion techniques, we sought to investigate the ability of the green shore crab (Carcinus maenas) to take up the amino acid l-leucine directly from the water. Investigation of the concentration-dependent transport kinetics of radiolabelled l-leucine showed that there are two specific transport pathways across Carcinus gills, one with high affinity and low capacity, and the other with high capacity and low affinity. Using putative competitive substrates and reduced sodium preparations, we were able to identify the putative amino acid transport system associated with high-affinity uptake. This is the first study to demonstrate the absorption of dissolved organic nutrients across the gill epithelium of a marine arthropod.

SUBMITTER: Blewett TA

PROVIDER: S-EPMC5740269 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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A novel pathway of nutrient absorption in crustaceans: branchial amino acid uptake in the green shore crab (Carcinus maenas).

Blewett Tamzin A TA Goss Greg G GG

Proceedings. Biological sciences 20171201 1868

Estuaries are environments enriched with dissolved nutrients such as amino acids. To date, marine arthropods are the only invertebrate group that have not been demonstrated to access this potentially important nutrient resource. Using in vitro gill perfusion techniques, we sought to investigate the ability of the green shore crab (Carcinus maenas) to take up the amino acid l-leucine directly from the water. Investigation of the concentration-dependent transport kinetics of radiolab ...[more]

PMID: 29212719

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Project description: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.

Project description:Neuropeptides play a central role as neurotransmitters, neuromodulators and hormones in orchestrating arthropod physiology. The post-genomic surge in identified neuropeptides and their putative receptors has not been matched by functional characterization of ligand-receptor pairs. Indeed, until very recently no G protein-coupled receptors (GPCRs) had been functionally defined in any crustacean. Here we explore the structurally-related, functionally-diverse gonadotropin-releasing hormone paralogs, corazonin (CRZ) and red-pigment concentrating hormone (RPCH) and their G-protein coupled receptors (GPCRs) in the crab, Carcinus maenas. Using aequorin luminescence to measure in vitro Ca2+ mobilization we demonstrated receptor-ligand pairings of CRZ and RPCH. CRZR-activated cell signaling in a dose-dependent manner (EC50 0.75 nM) and comparative studies with insect CRZ peptides suggest that the C-terminus of this peptide is important in receptor-ligand interaction. RPCH interacted with RPCHR with extremely high sensitivity (EC50 20 pM). Neither receptor bound GnRH, nor the AKH/CRZ-related peptide. Transcript distributions of both receptors indicate that CRZR expression was, unexpectedly, restricted to the Y-organs (YO). Application of CRZ peptide to YO had no effect on ecdysteroid biosynthesis, excepting a modest stimulation in early post-molt. CRZ had no effect on heart activity, blood glucose levels, lipid mobilization or pigment distribution in chromatophores, a scenario that reflected the distribution of its mRNA. Apart from the well-known activity of RPCH as a chromatophorotropin, it also indirectly elicited hyperglycemia (which was eyestalk-dependent). RPCHR mRNA was also expressed in the ovary, indicating possible roles in reproduction. The anatomy of CRZ and RPCH neurons in the nervous system is described in detail by immunohistochemistry and in situ hybridization. Each peptide has extensive but non-overlapping distribution in the CNS, and neuroanatomy suggests that both are possibly released from the post-commissural organs. This study is one of the first to deorphanize a GPCR in a crustacean and to provide evidence for hitherto unknown and diverse functions of these evolutionarily-related neuropeptides.

Project description:There is an increasing interest to restore the ecosystem services that eelgrass provides, after their continuous worldwide decline. Most attempts to restore eelgrass using seeds are challenged by very high seed losses and the reasons for these losses are not all clear. We assess the impact of predation on seed loss and eelgrass establishment, and explore methods to decrease seed loss during restoration in the Swedish northwest coast. In a laboratory study we identified three previously undescribed seed predators, the shore crab Carcinus maenas, the hermit crab Pagurus bernhardus and the sea urchin Strongylocentrotus droebachiensis, of which shore crabs consumed 2-7 times more seeds than the other two species. The importance of shore crabs as seed predators was supported in field cage experiments where one enclosed crab caused 73% loss of seeds over a 1-week period on average (~ 21 seeds per day). Seedling establishment was significantly higher (14%) in cages that excluded predators over an 8-month period than in uncaged plots and cages that allowed predators but prevented seed-transport (0.5%), suggesting that seed predation constitutes a major source of seed loss in the study area. Burying the seeds 2 cm below the sediment surface prevented seed predation in the laboratory and decreased predation in the field, constituting a way to decrease seed loss during restoration. Shore crabs may act as a key feedback mechanism that prevent the return of eelgrass both by direct consumption of eelgrass seeds and as a predator of algal mesograzers, allowing algal mats to overgrow eelgrass beds. This shore crab feedback mechanism could become self-generating by promoting the growth of its own nursery habitat (algal mats) and by decreasing the nursery habitat (seagrass meadow) of its dominant predator (cod). This double feedback-loop is supported by a strong increase of shore crab abundance in the last decades and may partly explain the regime shift in vegetation observed along the Swedish west coast.

Project description: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.

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A novel pathway of nutrient absorption in crustaceans: branchial amino acid uptake in the green shore crab (Carcinus maenas).

Publications

A novel pathway of nutrient absorption in crustaceans: branchial amino acid uptake in the green shore crab (<i>Carcinus maenas</i>).

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