N-Acetyl-d-Glucosamine-Binding Lectin in Acropora tenuis Attracts Specific Symbiodiniaceae Cell Culture Strains.

Many corals establish symbiosis with Symbiodiniaceae cells from surrounding environments, but very few Symbiodiniaceae cells exist in the water column. Given that the N-acetyl-d-glucosamine-binding lectin ActL attracts Symbiodiniaceae cells, we hypothesized that corals must attract Symbiodiniaceae cells using ActL to acquire them. Anti-ActL antibody inhibited acquisition of Symbiodiniaceae cells, and rearing seawater for juvenile Acropora tenuis contained ActL, suggesting that juvenile A. tenuis discharge ActL to attract these cells. Among eight Symbiodiniaceae cultured strains, ActL attracted NBRC102920 (Symbiodinium tridacnidorum) most strongly followed by CS-161 (Symbiodinium tridacnidorum), CCMP2556 (Durusdinium trenchii), and CCMP1633 (Breviolum sp.); however, it did not attract GTP-A6-Sy (Symbiodinium natans), CCMP421 (Effrenium voratum), FKM0207 (Fugacium sp.), and CS-156 (Fugacium sp.). Juvenile polyps of A. tenuis acquired limited Symbiodiniaceae cell strains, and the number of acquired Symbiodiniaceae cells in a polyp also differed from each other. The number of Symbiodiniaceae cells acquired by juvenile polyps of A. tenuis was correlated with the ActL chemotactic activity. Thus, ActL could be used to attract select Symbiodiniaceae cells and help Symbiodiniaceae cell acquisition in juvenile polyps of A. tenuis, facilitating establishment of symbiosis between A. tenuis and Symbiodiniaceae cells.

Skeletal structure and progression of growth anomalies in Porites australiensis in Okinawa, Japan.

Growth anomalies (GAs), one of the diseases recently reported for scleractinian corals, are characterized by an abnormal skeletal structure and reduced zooxanthella density. The pathological characteristics of GAs were studied in colonies of Porites australiensis on a reef in Kayo, Okinawa, Japan. Corallites in the GA region lost the skeletal architecture characteristic of P. australiensis, and polyp density had decreased in the GAs due to enlargement of both calices and the coenosteum. The gross productivity of isolated GA samples was lower than in healthy samples and decreased to almost 0 within 11 d after isolation. However, when GA samples were brought into contact with healthy-looking samples from the same colony, they fused and both the GA and healthy regions grew. Healthy samples fused with GA samples grew more slowly than those fused with healthy samples. For in situ GAs surrounded by healthy tissue, tissue death usually started at the center of the GA, probably due to a deficiency in the translocated energy supply from the surrounding tissue. The total area of the GA region and the dead area increased at a rate of 5.3 ± 2.9 cm2 yr-1. These results suggest that GA regions are maintained by energy supplies from surrounding healthy tissues and that GAs may have a negative impact on host corals.

The Effect of Co-Culture of Two Coral Species on Their Bacterial Composition Under Captive Environments.

Coral symbionts are important members of the coral holobiont, and coral bacterial flora are essential in host health maintenance and coral conservation. Coral symbionts are affected by various environmental factors, such as seawater temperature, pH, and salinity. Although physicochemical and chemical factors have been highlighted as possible causes of these effects, the effects of water flow and the co-culture of different species corals have not been elucidated. In this study, we designed an artificial rearing environment to examine the impact of environmental and biological factors on Acropora tenuis, one of the major coral species in Okinawa, and Montipora digitata, during their co-culture. We intervened with the water flow to reveal that the movement of the rearing environment alters the bacterial flora of A. tenuis. During the rearing under captive environment, the alpha diversity of the coral microbiota increased, suggesting the establishment of rare bacteria from the ocean. No differences in the bacterial composition between the control and water flow groups were observed under the rearing conditions. However, the structure of the bacterial flora was significantly different in the co-culture group. Comparison of bacterial community succession strongly suggested that the differences observed were due to the suppressed transmission of bacteria from the ocean in the co-culture group. These results enhance our understanding of interactions between corals and shed light on the importance of regional differences and bacterial composition of coral flora.

Targeted single-cell genomics reveals novel host adaptation strategies of the symbiotic bacteria Endozoicomonas in Acropora tenuis coral.

BACKGROUND: Endozoicomonas bacteria symbiosis with various marine organisms is hypothesized as a potential indicator of health in corals. Although many amplicon analyses using 16S rRNA gene have suggested the diversity of Endozoicomonas species, genome analysis has been limited due to contamination of host-derived sequences and difficulties in culture and metagenomic analysis. Therefore, the evolutionary and functional potential of individual Endozoicomonas species symbiotic with the same coral species remains unresolved. RESULTS: In this study, we applied a novel single-cell genomics technique using droplet microfluidics to obtain single-cell amplified genomes (SAGs) for uncultured coral-associated Endozoicomonas spp. We obtained seven novel Endozoicomonas genomes and quantitative bacterial composition from Acropora tenuis corals at four sites in Japan. Our quantitative 16S rRNA gene and comparative genomic analysis revealed that these Endozoicomonas spp. belong to different lineages (Clade A and Clade B), with widely varying abundance among individual corals. Furthermore, each Endozoicomonas species possessed various eukaryotic-like genes in clade-specific genes. It was suggested that these eukaryotic-like genes might have a potential ability of different functions in each clade, such as infection of the host coral or suppression of host immune pathways. These Endozoicomonas species may have adopted different host adaptation strategies despite living symbiotically on the same coral. CONCLUSIONS: This study suggests that coral-associated Endozoicomonas spp. on the same species of coral have different evolutional strategies and functional potentials in each species and emphasizes the need to analyze the genome of each uncultured strain in future coral-Endozoicomonas relationships studies. Video Abstract.

Incongruence between morphotypes and genetically delimited species in the coral genus Stylophora: phenotypic plasticity, morphological convergence, morphological stasis or interspecific hybridization?

BACKGROUND: Morphological data suggest that, unlike most other groups of marine organisms, scleractinian corals of the genus Stylophora are more diverse in the western Indian Ocean and in the Red Sea than in the central Indo-Pacific. However, the morphology of corals is often a poor predictor of their actual biodiversity: hence, we conducted a genetic survey of Stylophora corals collected in Madagascar, Okinawa, the Philippines and New Caledonia in an attempt to find out the true number of species in these various locations. RESULTS: A molecular phylogenetic analysis of the mitochondrial ORF and putative control region concurs with a haploweb analysis of nuclear ITS2 sequences in delimiting three species among our dataset: species A and B are found in Madagascar whereas species C occurs in Okinawa, the Philippines and New Caledonia. Comparison of ITS1 sequences from these three species with data available online suggests that species C is also found on the Great Barrier Reef, in Malaysia, in the South China Sea and in Taiwan, and that a distinct species D occurs in the Red Sea. Shallow-water morphs of species A correspond to the morphological description of Stylophora madagascarensis, species B presents the morphology of Stylophora mordax, whereas species C comprises various morphotypes including Stylophora pistillata and Stylophora mordax. CONCLUSIONS: Genetic analysis of the coral genus Stylophora reveals species boundaries that are not congruent with morphological traits. Of the four hypotheses that may explain such discrepancy (phenotypic plasticity, morphological stasis, morphological convergence, and interspecific hybridization), the first two appear likely to play a role but the fourth one is rejected since mitochondrial and nuclear markers yield congruent species delimitations. The position of the root in our molecular phylogenies suggests that the center of origin of Stylophora is located in the western Indian Ocean, which probably explains why this genus presents a higher biodiversity in the westernmost part of its area of distribution than in the "Coral Triangle".

High-Quality Draft Genome Sequence of a Rickettsiales Bacterium Found in Acropora tenuis Coral from Okinawa, Japan.

Rickettsiales-like organisms are important for the survival and functioning of corals, prompting an investigation of their complete genomes. Earlier reports of the genomes of these organisms remain incomplete. Here, we report a novel draft genome of Rickettsiales bacterial strain SESOKO1, found in Acropora tenuis coral, using single-cell genome technology.

Luminaolide, a novel metamorphosis-enhancing macrodiolide for scleractinian coral larvae from crustose coralline algae.

A new metamorphosis-enhancing macrodiolide, luminaolide (1), was isolated from the crustose coralline algae (CCA) Hydrolithon reinboldii. Its structure was determined by spectroscopic analysis. A fraction (1.30 mug/mL) eluted with 80% aqueous MeOH by ODS gel column chromatography of the same CCA extract induced larval metamorphosis (25.9 +/- 7.4%) against Leptastrea purpurea, and its metamorphosis-inducing activity was further enhanced to 92.6 +/- 2.9% with the addition of 1 (25.6 ng/mL).

Continuous-flow complete-mixing system for assessing the effects of environmental factors on colony-level coral metabolism.

A small-scale chamber experimental system was designed to study the effects of temperature on colony-level coral metabolism. The system continuously supplies fresh seawater to the chamber, where it is mixed immediately and completely with the seawater already present. This continuous-flow complete-mixing system (CFCM system), in conjunction with theoretical equations, allows quantitative determination of chemical uptake and release rates by coral under controlled environmental conditions. We used the massive hermatypic coral Goniastrea aspera to examine variations in pH, total alkalinity, and total inorganic carbon for 16 days at 27 degrees C under controlled light intensities (300 and 0 micromol m(-2) s(-1)). We confirmed the stability of the CFCM system with respect to coral photosynthetic and calcification fluxes. In addition, we obtained daily photosynthetic and calcification rates at different temperatures (27 degrees C, 29 degrees C, 31 degrees C, and 33 degrees C). When seawater temperature was raised from 31 degrees C to 33 degrees C, the gross primary production rate (Pgross) decreased 29.5%, and the calcification rate (G) decreased 85.7% within 2 days. The CFCM system allows quantitative evaluation of coral colony chemical release and uptake rates, and metabolism.

Microplastics disturb the anthozoan-algae symbiotic relationship.

World production of plastic has dramatically increased from the 1950's and now it reaches approximately 311 million tons per year. The resulting accumulation of small plastic detritus less than 5 mm in size, termed "microplastics", has started threatening the life cycles of marine organisms. Here we show the first evidence that microplastics disturb the initiation of symbiotic relationships in anthozoan-algae symbiosis. We found in both the aposymbiotic sea-anemone Aiptasia sp. and the coral Favites chinensis that the infectivity of symbiotic algae into the host is severely suppressed by microspheres fed either directly or indirectly through microsphere-fed Artemia sp. Similar trends were seen when microplastics collected from commercial facewash were used instead of microspheres. Therefore, ongoing accumulation of microplastics in the ocean might disturb the healthy anthozoan-algae symbiotic relationships, which are cornerstones of the biologically enriched coral reef ecosystem.

Coral individuality - confluence of change physical splitting and developmental ability of embryos.

Previous studies have suggested that blastomeres from the 2-, 4-, or 8-cell stage of corals have the ability to develop into normal primary polyps. However, it is still not known which developmental stage's blastomere produces which juvenile. In this study, we demonstrated that only the blastomeres with animal hemispheres have the capacity to develop into normal primary polyps. Individuality was evaluated using blastomeres isolated from the corals Acropora digitifera, A. intermedia, Dipsastraea lizardensis, and Favites chinensis. On commencement of embryo cleavage, the animal pole was marked using Neutral red staining, and at the 2-, 4-, and 8-cell stages, embryos were divided into individual blastomeres using glass needles. We found that the survival rate and percentage metamorphosis were higher in the larger-sized blastomeres with animal hemispheres. The vegetal hemisphere alone is incapable of developing into a normal primary polyp; however, a ball-shaped embryo with incomplete mesenteries and no pharynx developed in some cases. These results indicate that the animal hemisphere is needed for corals to develop into normal primary polyps, and that the individuality of corals is possibly determined by a combination of the chance physical splitting of embryos by waves and their innate developmental ability.

Molecular analysis of a major soluble egg protein in the scleractinian coral Favites chinensis.

Soluble proteins were extracted from eggs of the scleractinian coral Favites chinensis, and analyzed using SDS-polyacrylamide gel electrophoresis. Two major proteins, named FcEP-1 and 2, were detected, and two partial amino acid sequences of FcEP-1 were determined. A cDNA encoding FcEP-1 was identified, using reverse transcription PCR with degenerate oligonucleotide primers designed based on the amino acid sequences, and rapid amplification of cDNA ends. Upon translation of the cDNA, FcEP-1 was predicted to consist of 648 amino acids, and the protein sequence exhibited similarity to vertebrate and invertebrate vitellogenins. FcEP-1 transcripts were already present approximately 6 months before spawning, when the size of oocytes was approximately 1/60 of the mature egg, and could be detected throughout the vitellogenic period. These observations suggest that detection of FcEP-1 transcripts may be useful to monitor the vitellogenic activity in F. chinensis.

The effects of thermal and high-CO2 stresses on the metabolism and surrounding microenvironment of the coral Galaxea fascicularis.

The effects of elevated temperature and high pCO2 on the metabolism of Galaxea fascicularis were studied with oxygen and pH microsensors. Photosynthesis and respiration rates were evaluated from the oxygen fluxes from and to the coral polyps. High-temperature alone lowered both photosynthetic and respiration rates. High pCO2 alone did not significantly affect either photosynthesis or respiration rates. Under a combination of high-temperature and high-CO2, the photosynthetic rate increased to values close to those of the controls. The same pH in the diffusion boundary layer was observed under light in both (400 and 750 ppm) CO2 treatments, but decreased significantly in the dark as a result of increased CO2. The ATP contents decreased with increasing temperature. The effects of temperature on the metabolism of corals were stronger than the effects of increased CO2. The effects of acidification were minimal without combined temperature stress. However, acidification combined with higher temperature may affect coral metabolism due to the amplification of diel variations in the microenvironment surrounding the coral and the decrease in ATP contents.

Comparative embryology of eleven species of stony corals (Scleractinia).

A comprehensive understanding of coral reproduction and development is needed because corals are threatened in many ways by human activity. Major threats include the loss of their photosynthetic symbionts (Symbiodinium) caused by rising temperatures (bleaching), reduced ability to calcify caused by ocean acidification, increased storm severity associated with global climate change and an increase in predators caused by runoff from human agricultural activity. In spite of these threats, detailed descriptions of embryonic development are not available for many coral species. The current consensus is that there are two major groups of stony corals, the "complex" and the "robust". In this paper we describe the embryonic development of four "complex" species, Pseudosiderastrea tayamai, Galaxea fascicularis, Montipora hispida, and Pavona Decussata, and seven "robust" species, Oulastrea crispata, Platygyra contorta, Favites abdita, Echinophyllia aspera, Goniastrea favulus, Dipsastraea speciosa (previously Favia speciosa), and Phymastrea valenciennesi (previously Montastrea valenciennesi). Data from both histologically sectioned embryos and whole mounts are presented. One apparent difference between these two major groups is that before gastrulation the cells of the complex corals thus far described (mainly Acropora species) spread and flatten to produce the so-called prawn chip, which lacks a blastocoel. Our present broad survey of robust and complex corals reveals that prawn chip formation is not a synapomorphy of complex corals, as Pavona Decussata does not form a prawn chip and has a well-developed blastocoel. Although prawn chip formation cannot be used to separate the two clades, none of the robust corals which we surveyed has such a stage. Many robust coral embryos pass through two periods of invagination, separated by a return to a spherical shape. However, only the second of these periods is associated with endoderm formation. We have therefore termed the first invagination a pseudo-blastopore.

Large-scale mono-clonal structure in the north peripheral population of blue coral, Heliopora coerulea.

We examined the genotypic diversity of the large population of Heliopora coerulea, discovered recently in Ooura Bay, northern Okinawa Main Island Japan, together with another large population in Shiraho Reef, also in southwest Japan, using 9 polymorphic microsatellite markers. From each population, 40 samples were systematically collected along 2 transect lines with 4-m intervals. Surprisingly, all 40 samples from Ooura Bay were mono-genotypic, implying that the huge coral structure (30 m×80 m) originated from a single larva. Conversely, the 40 samples collected from the Shiraho Reef site all had different genotypes; measurements of genetic diversity, H(E) and H(O), were 0.075-0.975 and 0.064-0.655, respectively. At least four factors are considered to make such a huge H. coerulea population with a single genotype in Ooura Bay, (1) origin of single larva or fragment (2) a genetic bottleneck, (3) post settlement selection, and (4) frequent asexual propagation.