Smith MK et al. (2023), Structure and proteomic analysis of the crown-o...

ECB-ART-51573

Sci Rep 2023 Feb 27;131:3349. doi: 10.1038/s41598-023-30425-1.

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

Smith MK , Rotgans BA , Lang T , Johnston R , Wang T , Suwansa-Ard S , Bose U , Satoh N , Egertova M , Hall MR , Byrne M , Elphick MR , Motti CA , Cummins SF .

Abstract
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.

PubMed ID: 36849815
PMC ID: PMC9971248
Article link: Sci Rep
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Article Images: [+] show captions

	Figure 1. Images of crown-of-thorns species. (A) Crown-of-thorns starfish, Acanthaster sp., and (B) Short-spined crown-of-thorns starfish, Acanthaster brevispinus.
	Figure 2. Scanning electron microscope (SEM) and histology (trichrome stain) of the radial nerve cord (RNC). (A–C) Adult COTS: (A) SEM of the RNC and adjacent ambulacral ossicles. The tissue bordering the neural bulbs is a cut away section of epithelium; (B) SEM of the RNC, showing neural bulbs (arrows); (C) Histology, showing neural bulbs (arrow) protruding from the ectoneural surface of the RNC. (D,E) Juvenile COTS: Histology, showing the smooth surface of the RNC and absence of the bulbous structures seen in adult. (F–H) A. brevispinus: (F,G) SEM of the RNC, showing its smooth ectoneural surface; (H) Histology, showing the absence of neural bulbs on the ectoneural surface of the RNC. In histology, blue represents connective tissue, dark red/purple indicates nuclei and red/pink is cytoplasm. TF, Tube foot; Ec, ectoneural layer; Hy, hyponeural layer; hs, haemal sinus; hc, hyponeural coelom. Scale bars (A,F) = 500 μm; (B,G) = 200 μm; (C,H) = 50 μm; (D,E) = 40 µm.
	Figure 3. TEM of the adult COTS radial nerve cord (RNC). (A) Transverse section showing the hyponeural region (h), connective tissue layer (c), and ectoneural region (e). (B) Hyponeural region, connective tissue layer, and uppermost part of the ectoneural neuropile. (C) High-magnification view of a large cell in the hyponeural region with a portion of the neuropile and myofilaments visible. (D) Ectoneural neuropile showing, dense-cored vesicles and the fibre bundle of the radial-glia-like process. (E) Lower border of the ectoneural neuropile, with dense protein vesicle-like cell granules surrounded by endoplasmic reticulum. (F) Neuroepithelium containing large vesiculated cells and the extracellular cuticle-like cover through which microvilli of the epithelial cells traverse to the oral surface; this is the lowermost portion of the ectoneural tissue and is the surface exposed to the seawater environment. (G) Goblet-like cells from the epithelial portion of the ectoneural tissue. CF collagen fibres, Cil cilium, DCV dense-cored vesicles, ER endoplasmic reticulum, FB fibre bundles, GA Golgi apparatus, GC goblet-like cell, hc hyponeural coelom, Mf myofilament, Mt mitochondria, Mv microvilli, N nucleus, Nl nucleolus, NP neural plexus, RCG releasing-like cell granule, SM smooth muscle cell-like, V vacuole.
	Figure 4. Histology and TEM of the neural bulbs of COTS radial nerve cord (RNC). (A) Histology of the ectoneural surface (H&E stain) (B) TEM of bulb edge showing what may be secretory vesicles, containing electron dense diffuse material at various stages of activity leading to exocytosis of contents into the external space. (C) Secretory vesicle located at ~ 10 μm from the microvilli border. (D) Secretory vesicle fusing with the outer surface of the bulb, note the liberated granular contents visible at the base of the microvilli layer. (E) Secretory vesicle fused with an adjacent microvilli layer, forming a small pore, and releasing its granular contents to the exterior environment. (F) Cilium with rootlet projecting through the microvilli of the epithelial surface. (G) High magnification view of ciliary rootlet and a small corner of the endoplasmic reticulum cisternae, from which light cored vesicles can be seen emerging. (H) Epithelial region of ectoneural tissue populated with multiple cilia projecting past the microvilli layer. Cil cilium, Cis cisternae of endoplasmic reticulum, CR ciliary rootlet, FB fibre bundles, Mv microvilli, N nucleus, NB neural bulb, NE neuroepithelium, NP neural plexus, P pore, SV secretory Vesicle, V vacuole.
	Figure 5. Analysis of putative secreted proteins identified in adult COTS radial nerve cord (RNC). (A) An area-proportional Venn diagram showing distribution of total RNC proteins identified from soluble (RP-HPLC) and insoluble (SDS-PAGE) RNC preparations and neural bulbs. In parenthesis is the number of predicted secreted proteins. (B) BLAST2GO analysis with presentation of molecular function and biological process. (C) Heatmap showing relative tissue gene expression of each identified protein, with scale bar representing the z-score values calculated based on log2-transformed expression (Transcripts Per Million) across all tissues. Along the sides of the heatmap, (right) groups of genes are categorized based on general functions, and (left) gene representatives from each group are shown. Full list of gene names and gene expression are provided in Table S4.
	Figure 6. Summary of COTS neuropeptide precursors and those with mass spectral support (grey shading) in adult radial nerve cord. ‘NB’ indicates neuropeptides found in the neural bulbs; ‘aa’, amino acids.
	Figure 7. Precursor schematics of deleted in Malignant Brain Tumor 1 (or DMBT1)-like proteins identified from the adult COTS radial nerve cord.

References [+] :

Babcock, Assessing Different Causes of Crown-of-Thorns Starfish Outbreaks and Appropriate Responses for Management on the Great Barrier Reef. 2016, Pubmed, Echinobase