Structure and proteomic analysis of the crown-of-thorns starfish (Acanthaster sp.) radial nerve cord

The nervous system of the Asteroidea (starfish or seastar) consists of radial nerve cords (RNCs) that interconnect with a ring nerve. Despite its relative simplicity, it facilitates the movement of multiple arms and numerous tube feet, as well as regeneration of damaged limbs. Here, we investigated the RNC ultrastructure and its molecular components within the of Pacific crown-of-thorns starfish (COTS; Acanthaster sp.), a well-known coral predator that in high-density outbreaks has major ecological impacts on coral reefs. We describe the presence of an array of unique small bulbous bulbs (40–100 μm diameter) that project from the ectoneural region of the adult RNC. Each comprise large secretory-like cells and prominent cilia. In contrast, juvenile COTS and its congener Acanthaster brevispinus lack these features, both of which are non-corallivorous. Proteomic analysis of the RNC (and isolated neural bulbs) provides the first comprehensive echinoderm protein database for neural tissue, including numerous secreted proteins associated with signalling, transport and defence. The neural bulbs contained several neuropeptides (e.g., bombyxin-type, starfish myorelaxant peptide, secretogranin 7B2-like, Ap15a-like, and ApNp35) and Deleted in Malignant Brain Tumor 1-like proteins. In summary, this study provides a new insight into the novel traits of COTS, a major pest on coral reefs, and a proteomics resource that can be used to develop (bio)control strategies and understand molecular mechanisms of regeneration.journal articl

Characterisation of <i>Biomphalaria glabrata</i> IRs and iGluRs.

<p>(A) Molecular phylogeny for IR and iGluRs from <i>B</i>. <i>blabrata</i> (<i>Bgla</i>), <i>A</i>. <i>californica</i> (Acal), <i>S</i>. <i>gregaria</i> (Sgre), <i>D</i>. <i>ponderosae</i> (Dpon), <i>P</i>. <i>argus</i> (Parg) and <i>D</i>. <i>melanogaster</i> (Dmel). Bootstrap supports two IR subfamilies. The 7 newly identified <i>Biomphalaria</i> IRs are highlighted with red diamonds. Phylogenetic tree of nonIR8a/25a IRs is shown for 5 <i>B</i>. <i>glabrata</i>, 2 from <i>P</i>. <i>argus</i> and 9 from <i>A</i>. <i>californica</i>. Clades are indicated by different colours. All gene accession numbers can be found in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156380#pone.0156380.s004" target="_blank">S2 Table</a>.</b> (B) Alignment of predicted amino acid sequences of 5 candidate <i>Biomphalaria</i> IRs (BglaIR1-5), including regions encoding putative ligand-binding domains; S1 and S2 domains are shown by black asterisks below the sequences. Three key ligand-binding residues (R, T and D/E) are marked with red asterisks. Blue shading indicates identical or similar amino acids. Sequence logo conservation is presented above the sequence.</p

Analysis of ligand-binding domains in <i>Biomphalaria glabrata</i> IRs and iGluRs.

<p>(A) Left: Protein domain structure of conventional iGluRs/IRs in schematic form [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156380#pone.0156380.ref008" target="_blank">8</a>]. Right: Illustration of the three Pfam domains present in iGluRs and IRs. Both IR8a and IR25a possess the Pfam domain corresponding to the iGluR ATD. All other IRs lack the same homology to the ATD. (B) Alignment of S1 and S2 ligand-binding domains from putative <i>B</i>. <i>glabrata</i> iGluRs and IRs with <i>A</i>.<i>californica</i> iGluRs. <i>Biomphalaria</i> and <i>Aplysia</i> S1 and S2 ligand-binding domains were manually aligned. Blue shading indicates identical or similar amino acids. Three key ligand-binding residues (R, T and D/E) are boxed. S1 and S2 domains are marked with coloured lines at the bottom. (C) Schematic representation of <i>Biomphalaria</i> iGluRs, showing conserved and invariable amino acids. Predicted ATD site is highlighted in red and the region of key ligand-binding residues is magnified and shown in yellow and green.</p

Ionotropic Receptors Identified within the Tentacle of the Freshwater Snail <i>Biomphalaria glabrata</i>, an Intermediate Host of <i>Schistosoma mansoni</i>

<div><p><i>Biomphalaria glabrata</i> (<i>B</i>. <i>glabrata</i>) is an air-breathing aquatic mollusc found in freshwater habitats across the Western Hemisphere. It is most well-known for its recognized capacity to act as a major intermediate host for <i>Schistosoma mansoni</i>, the human blood fluke parasite. Ionotropic receptors (IRs), a variant family of the ionotropic glutamate receptors (iGluR), have an evolutionary ancient function in detecting odors to initiate chemosensory signaling. In this study, we applied an array of methods towards the goal of identifying IR-like family members in <i>B</i>. <i>glabrata</i>, ultimately revealing two types, the iGluR and IR. Sequence alignment showed that three ligand-binding residues are conserved in most <i>Biomphalaria</i> iGluR sequences, while the IRs did exhibit a variable pattern, lacking some or all known glutamate-interactingresidues, supporting their distinct classification from the iGluRs. We show that <i>B</i>. <i>glabrata</i> contains 7 putative IRs, some of which are expressed within its chemosensory organs. To further investigate a role for the more ancient <i>IR25a</i> type in chemoreception, we tested its spatial distribution pattern within the snail cephalic tentacle by <i>in situ</i> hybridization. The presence of <i>IR25a</i> within presumptive sensory neurons supports a role for this receptor in olfactory processing, contributing to our understanding of the molecular pathways that are involved in <i>Biomphalaria</i> olfactory processing.</p></div

Analysis of <i>Biomphalaria glabrata</i> IR25a.

<p>(A) The protein domain organization of a typical IR25a is shown above a protein alignment of <i>Biomphalaria</i> (Bgla), <i>Aplysia</i> (Acal), <i>Panulirus</i> (Parg) and <i>Drosophila</i> (Dmel) IR25a. Conserved amino acid residues are highlighted in purple (≥80% conserved) and blue (≥50% conserved), and ligand-binding domain S1 and S2 domains are shown with red lines above the sequences. Three key ligand-binding residues (R, T and D/E) are marked with a black dot. (B) Schematic representation of <i>Biomphalaria</i> IRs, showing conserved and invariable amino acids. Predicted S1 and S2 region are highlighted in green and yellow, respectively. (C) Structure of BglaIR25a predicted by SWISS-MODEL in conjunction with MDS. Top: tertiary structure, purple-α helix, blue-3-10 helix, yellow-β sheet, cyan-turn and white-random coil. Bottom: space filling of predicted binding site, yellow-predicted ligand binding S1 region, green-predicted ligand binding S2 region, and blue-predicted TM region.</p

Candidate IRs and iGluRs identified from the <i>B</i>. <i>glabrata</i> genome.

<p>Candidate IRs and iGluRs identified from the <i>B</i>. <i>glabrata</i> genome.</p

Expression of <i>BglaIR25a</i> as detected by <i>in situ</i> hybridization in <i>Biomphalaria glabrata</i> tentacle.

<p>(A) Control whole-mount <i>in situ</i> hybridization on tentacle tissue with a DIG-labelled sense riboprobe for <i>BglaIR25a</i>. No signal is apparent. (B-D) Whole-mount tentacle probed with antisense riboprobe for <i>BglaIR25a</i>. (E-I) Cryostat sections showing cellular localization of <i>IR25a</i> within central and peripheral cells (arrows). d, distal; p, proximal.</p

Tissue expression of <i>Biomphalaria glabrata</i> IRs.

<p>Top: Schematic representation of <i>B</i>.<i>glabrata</i> showing tissues used for RT-PCR. Bottom: RT-PCR detection of 7 <i>Biomphalaria</i> IR genes in different tissues. <i>Biomphalaria</i> IRs can be detected in both olfactory and non-olfactory tissues. No expression could be detected from the lung or gonad. No amplification was detected in RNA samples in the absence of reverse transcription (data not shown) or template (-ve). Control RT-PCR products for comparative analysis of gene expression correspond to the β-actin.</p