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Microbiome
2020 Aug 24;81:123. doi: 10.1186/s40168-020-00880-3.
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A ubiquitous subcuticular bacterial symbiont of a coral predator, the crown-of-thorns starfish, in the Indo-Pacific.
Wada N
,
Yuasa H
,
Kajitani R
,
Gotoh Y
,
Ogura Y
,
Yoshimura D
,
Toyoda A
,
Tang SL
,
Higashimura Y
,
Sweatman H
,
Forsman Z
,
Bronstein O
,
Eyal G
,
Thongtham N
,
Itoh T
,
Hayashi T
,
Yasuda N
.
Abstract
BACKGROUND: Population outbreaks of the crown-of-thorns starfish (Acanthaster planci sensu lato; COTS), a primary predator of reef-building corals in the Indo-Pacific Ocean, are a major threat to coral reefs. While biological and ecological knowledge of COTS has been accumulating since the 1960s, little is known about its associated bacteria. The aim of this study was to provide fundamental information on the dominant COTS-associated bacteria through a multifaceted molecular approach.
METHODS: A total of 205 COTS individuals from 17 locations throughout the Indo-Pacific Ocean were examined for the presence of COTS-associated bacteria. We conducted 16S rRNA metabarcoding of COTS to determine the bacterial profiles of different parts of the body and generated a full-length 16S rRNA gene sequence from a single dominant bacterium, which we designated COTS27. We performed phylogenetic analysis to determine the taxonomy, screening of COTS27 across the Indo-Pacific, FISH to visualize it within the COTS tissues, and reconstruction of the bacterial genome from the hologenome sequence data.
RESULTS: We discovered that a single bacterium exists at high densities in the subcuticular space in COTS forming a biofilm-like structure between the cuticle and the epidermis. COTS27 belongs to a clade that presumably represents a distinct order (so-called marine spirochetes) in the phylum Spirochaetes and is universally present in COTS throughout the Indo-Pacific Ocean. The reconstructed genome of COTS27 includes some genetic traits that are probably linked to adaptation to marine environments and evolution as an extracellular endosymbiont in subcuticular spaces.
CONCLUSIONS: COTS27 can be found in three allopatric COTS species, ranging from the northern Red Sea to the Pacific, implying that the symbiotic relationship arose before the speciation events (approximately 2 million years ago). The universal association of COTS27 with COTS and nearly mono-specific association at least with the Indo-Pacific COTS provides a useful model system for studying symbiont-host interactions in marine invertebrates and may have applications for coral reef conservation. Video Abstract.
Fig. 1. Geographic and anatomical distributions of COTS individuals and the COTS body parts analysed in this study. The seventeen locations where the COTS individuals were collected (a) and the dissected body parts of COTS for the analyses (b) are shown. The dashed yellow line (b) indicates the dissection line for the cross-sectional view. c Details of the samples used in each analysis are shown: [1] 16S rRNA metabarcoding, [2] phylogenetic analysis using the full-length 16S rRNA gene sequences, [3] PCR screening and sequencing of the 16S rRNA gene sequences of COTS27, [4] FISH analysis, and [5] hologenome sequencing analysis. *1: This analysis was performed in triplicate for each sample. *2: The same individuals were used in analyses [1, 2]
Fig. 2. The relative abundances of the 25 most abundant OTUs, including COTS27 (OTU 1; red), in the total samples analysed in this study. The bubble chart of the relative abundances was calculated from the merged replicates of each body part (spines, tube feet, and pyloric stomachs) in each COTS individual. The phylogenies of each OTU were determined based on the results (best hit) of BLAST searches against the NCBI nr/nt database. *1: The phylogenies of OTU3 and OTU4 were determined in the All-species Living Tree Project and RDP databases, respectively
Fig. 3. The phylogenetic position of the dominant OTU 1 (COTS27) in the phylum Spirochaetes. Maximum likelihood (ML) trees were constructed based on the full-length 16S rRNA gene sequences (a) and the sequences of 43 conserved marker genes identified by CheckM (b). The bootstrap values in a were calculated by resampling 1000 times. The scale bars indicate substitutions per site. *1: The gene with accession No. FQ660021.1 in a was obtained from a polycycle aromatic hydrocarbon (PAH)-contaminated soil sample in a mitigated wetland.
Fig. 4. FISH analysis of three serial sections of a COTS disc spine. Each section was hybridized with the EUB338mix (a, purple; a general probe for bacteria), COTSsymb (b, red; a COTS27-specific probe), or Non338 (c, purple; a negative control to detect non-specific binding) probes. The probes were labelled with Cy3 in all panels and coloured with purple in a and c and with red in b. The green signals are tissue-derived autofluorescence. The arrowheads in a and b indicate layer-like signals from the general probe for bacteria (a) and the COTS27- specific probe (b). N.S. and C.U. indicate regions with non-specific binding and the outer cuticle complex, respectively. The scale bars represent 20âμm (aâc)
Fig. 5. Visualization of the COTS27 cells in different body parts of COTS using FISH. COTS27 cells (red) residing in the subcuticular spaces of the body walls were detected with COTSsymb, a COTS27-specific probe, in the tips (a) and bases (b) of aboral spines on the discs and arms, respectively, dermal papula (c), pedicellariae on the aboral side (d; 3D image [left] and 3D rendering image [right]), and tube feet (e). Many non-COTS27 bacteria (purple) were detected in the pyloric stomachs (f) using the EUB338mix probe. No visible bacteria were detected in the pyloric caeca (g) and gonads (h) in this study (the images were obtained applying the COTS27-specific probe). The arrowheads indicate signals from COTS27. The green signals are tissue-derived autofluorescence. CU, outer cuticle complex; CYA, cyanobacteria-like cells. Scale bars (aâc and eâh) indicate 20âμm. The 3D image in panel d was taken with an original objective of Ãâ40
Fig. 6. Circular view of the COTS27 chromosome. From the outside to the centre, each circle indicates forward strand CDSs, reverse strand CDSs, forward strand tRNA and rRNA genes, reverse strand tRNA and rRNA genes, GC-content, and GC skew. The CDSs were coloured according to the COG functional category of each CDS. The circular maps were created using CGView Server and the designations were then superposed manually
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