Crown-of-thorns starfish spines secrete defence proteins

Abstract

Background: The crown-of-thorns starfish (COTS; Acanthaster species) is a slow-moving corallivore protected by an extensive array of long, sharp toxic spines. Envenomation can result in nausea, numbness, vomiting, joint aches and sometimes paralysis. Small molecule saponins and the plancitoxin proteins have been implicated in COTS toxicity.

Methods: Brine shrimp lethality assays were used to confirm the secretion of spine toxin biomolecules. Histological analysis, followed by spine-derived proteomics helped to explain the source and identity of proteins, while quantitative RNA-sequencing and phylogeny confirmed target gene expression and relative conservation, respectively.

Results: We demonstrate the lethality of COTS spine secreted biomolecules on brine shrimp, including significant toxicity using aboral spine semi-purifications of >10 kDa (p > 0.05, 9.82 µg/ml), supporting the presence of secreted proteins as toxins. Ultrastructure observations of the COTS aboral spine showed the presence of pores that could facilitate the distribution of secreted proteins. Subsequent purification and mass spectrometry analysis of spine-derived proteins identified numerous secretory proteins, including plancitoxins, as well as those with relatively high gene expression in spines, including phospholipase A2, protease inhibitor 16-like protein, ependymin-related proteins and those uncharacterized. Some secretory proteins (e.g., vitellogenin and deleted in malignant brain tumor protein 1) were not highly expressed in spine tissue, yet the spine may serve as a storage or release site. This study contributes to our understanding of the COTS through functional, ultrastructural and proteomic analysis of aboral spines.

Keywords: Crown-of-thorns starfish; Defence; Echinoderm; Phospholipase A2; Plancitoxin; Spine; Venom.

Figure 1. Toxicity activity of crown-of-thorns starfish (COTS) venom extracts based on a brine shrimp (Artemia salina) lethality bioassay.

(A) Anatomical photo of COTS showing aboral spines. Image in the inset shows a morphology of spines at higher magnification. (B) Percent mortality was determined 48 h post-exposure to COTS extract protein concentrations (µg/mL), and compared to controls (filtered seawater; FSW). 5 biological replicates/treatment. Error bars show standard deviation (SD) of data. Asterisks indicate significant difference to the control group (p ≤ 0.05). SpE, spine extract; StWE, stress water extract.

Figure 2. Scanning electron microscopy (SEM) and histological analysis of crown-of-thorns starfish (COTS) aboral spine.

(A) SEM images showing COTS aboral spine outer surface, and at (B) higher magnification. Pores located on the spine surface are indicated. (C) SEM image of longitudinally sectioned aboral spine showing the core structure of spine, including endoskeletons (En) and outer epithelial layer (OEL). (D) Hematoxylin and eosin (H&E) stain of COTS aboral spine longitudinal section.

Figure 3. General biological functions of total proteins and gene expression of putative secreted proteins identified in the proteomic analysis of crown-of-thorns starfish (COTS) spines.

(A) WordCloud displaying the top 40 most abundant Gene Ontology (GO) terms (limited to the biological process category) for total proteins identified in the COTS spine extracts. The front size indicates the node score of each GO term. (B) Heatmap showing relative expression of genes encoding putative secreted proteins in various tissues. Two distinct clusters of genes are observed (arrows). Gene description of each gene ID is provided in Table 2.

Figure 4. Plancitoxins in the crown-of-thorns starfish (COTS).

(A) Schematic representation of deduced COTS plancitoxins (oki.27.33, oki.27.34, and oki.27.35). Sequence logo shows the conservation of amino acid sequences among all forms of COTS plancitoxins. Coloured bars at the top of sequence logo display the conserved domains and signal peptide region, and black asterisks show the position of conserved cysteine residues. (B) Graph showing relative plancitoxin gene expression (transcripts per million, TPM) in COTS tissues. An asterisk (*) indicates significantly higher (P < 0.05) gene expression in spine compared to other tissues. Red asterisks denote those identified in this study by aboral spine protein analysis. (C) Phylogenetic tree of COTS (Acanthaster cf. solaris) plancitoxin proteins with plancitoxin/plancitoxin-like proteins, deoxyribonuclease-2-alpha (DNase-II-alpha) and DNase II-alpha homologs from other species (model, Whelan and Goldman [WAG]; 1,000 bootstraps). Purple circles on the clades indicate level of bootstrap confidence (≥50%).

Figure 5. Secreted phospholipase A2 (sPLA2) proteins in the crown-of-thorns starfish (COTS).

(A) Schematic representation of deduced sPLA2 proteins identified in the COTS genome (gene IDs included). Sequence logo shows an amino acid sequence conservation among all forms. Coloured bars above a sequence logo shows the conserved domains and signal peptide region. An N-terminal insertion for oki.36.73 is shown from positions 6 to 19. Asterisks indicate highly conserved cysteine residues among COTS sPLA2, as well as sPLA2 in other animals. (B) Histogram showing relative COTS sPLA2 gene expression in TPM (transcripts per million) in COTS tissues. An asterisk (*) indicates significantly higher (P < 0.05) gene expression in spine compared to other tissues. Red asterisks denote those identified in this study by spine protein analysis. (C) Phylogenetic tree of COTS (Acanthaster cf. solaris) and other sPLA2 proteins. sPLA2 from different groups/types are included. The tree was constructed based on maximum likelihood method (model, WAG; frequency =5; 1,000 bootstraps). Purple circles on the clades indicate level of bootstrap confidence (≥50%).

Figure 6. A peptidase inbibitor 16-like protein (PI16) in the crown-of-thorns starfish (COTS).

(A) Schematic representation of COTS PI16 (oki.208.17). Coloured bars show the conserved domains and signal peptide region. Cysteine residues (‘C’) and their amino acid positions are indicated. (B) Histogram showing relative gene expression (transcripts per million; TPM) of oki.208.17 in COTS tissues. An asterisk (*) indicates significantly higher (P < 0.05) gene expression in spine compared to other tissues. (C) Phylogenetic tree of PI16s and CRISPs. Tree was constructed based on maximum likelihood method (model, WAG; frequency =5; 1,000 bootstraps). Purple circles on the clades indicate level of bootstrap confidence (≥45%).