Prevalence, complete genome, and metabolic potentials of a phylogenetically novel cyanobacterial symbiont in the coral-killing sponge, Terpios hoshinota.

Terpios hoshinota is an aggressive, space-competing sponge that kills various stony corals. Outbreaks of this species have led to intense damage to coral reefs in many locations. Here, the first large-scale 16S rRNA gene survey across three oceans revealed that bacteria related to the taxa Prochloron, Endozoicomonas, SAR116, Ruegeria, and unclassified Proteobacteria were prevalent in T. hoshinota. A Prochloron-related bacterium was the most dominant and prevalent cyanobacterium in T. hoshinota. The complete genome of this uncultivated cyanobacterium and pigment analysis demonstrated that it has phycobiliproteins and lacks chlorophyll b, which is inconsistent with the definition of Prochloron. Furthermore, the cyanobacterium was phylogenetically distinct from Prochloron, strongly suggesting that it should be a sister taxon to Prochloron. Therefore, we proposed this symbiotic cyanobacterium as a novel species under the new genus Candidatus Paraprochloron terpiosi. Comparative genomic analyses revealed that 'Paraprochloron' and Prochloron exhibit distinct genomic features and DNA replication machinery. We also characterized the metabolic potentials of 'Paraprochloron terpiosi' in carbon and nitrogen cycling and propose a model for interactions between it and T. hoshinota. This study builds a foundation for the study of the T. hoshinota microbiome and paves the way for better understanding of ecosystems involving this coral-killing sponge.

Spatial and cross-seasonal patterns of coral diseases in reefs of Taiwan: high prevalence and regional variation.

Although research on coral diseases is increasing worldwide, it remains limited in Taiwan. Taiwan is located at the Tropic of Cancer and contains both tropical and subtropical reefs. We conducted spatial and cross-seasonal surveys in Taiwan in 2018 and identified 7 types of disease and nondisease lesions and 6 potential factors influencing coral health. The overall mean prevalence of disease and nondisease lesions varied considerably across the reef regions, and host susceptibility differed among the coral taxa. The overall mean prevalence of disease and nondisease lesions was highest in Kenting (mean ± SEM: 8.58 ± 1.81%) and lowest on the Southern Islands (2.12 ± 0.73%). Although the prevalence of diseases did not differ significantly between the seasons, cyanobacteria-related diseases-including black band disease (BBD), BBD-like syndrome, and other cyanobacterial syndromes-were slightly more prevalent in autumn than in spring. Furthermore, 3 of the potential factors influencing coral health (i.e. turf algae, bioeroding sponges, and coral bleaching) were strong predictors of disease and nondisease lesion prevalence. These results advance our understanding of coral disease ecology in Taiwan and highlight the need for further research on the correlations between diseases, hosts, and environment.

Appearance of an anomalous black band disease at upper mesophotic depths after coral bleaching.

In the summer of 2016, extensive coral bleaching occurred on the coral reefs of Okinawa, Japan, which was often lethal in shallow waters. In November 2016, after the coral bleaching event, an anomalous black band disease (ABBD) was noticed during regular monitoring of the Pachyseris-dominated upper mesophotic Ryugu Reef in Okinawa. We subsequently conducted 10 × 1 m belt transects in deeper (30-35 m depth, n = 5) and shallower areas (27-29 m, n = 7) of the reef and obtained data on coral percentage coverage, Pachyseris percentage coverage, occurrence (numbers of lesions) of ABBD, and ABBD percentage coverage on Pachyseris. Both depths showed high live coral coverage (>90%), indicating little mortality from the summer 2016 bleaching event, and Pachyseris percentage coverage was significantly higher in the deeper area (mean ± SD = 48.6 ± 45.0%) compared to the shallower area (5.1 ± 5.0%). Additionally, although numbers of ABBD occurrences (= lesions) were significantly higher in the deeper area (81.0 ± 52.8 as opposed to 8.3 ± 6.7 at shallower depths), total ABBD percentage coverage on Pachyseris was not significantly different between shallow and deep areas (7.0 ± 3.2% versus 4.7 ± 3.6%). ABBD was observed to be Pachyseris-specific at Ryugu Reef. These results indicate that similar to shallower reefs, mesophotic reefs can experience increased disease occurrences subsequent to non-lethal coral bleaching events.

Transcriptome analysis reveals limited toxic effects of the UV-filter benzophenone-3 (BP-3) on the hermatypic coral Acropora tenuis and its symbiotic dinoflagellates.

This study aimed to investigate the toxic and transcriptomic effects of the ultraviolet filter benzophenone-3 (BP-3) on Acropora tenuis and its symbiotic dinoflagellates while using acetone as a solvent. Seven-day exposure to 50 and 500 µg/L, which is higher than most BP-3 records from coastal waters, did not affect coral colour or dinoflagellate photosynthesis. Differentially expressed genes (DEGs) between seawater and solvent controls were <20 in both corals and dinoflagellates. Eleven coral DEGs were detected after treatment with 50 µg/L BP-3. Fourteen coral DEGs, including several fluorescent protein genes, were detected after treatment with 500 µg/L BP-3. In contrast, no dinoflagellate DEGs were detected in the BP-3 treatment group. These results suggest that the effects of 50-500 µg/L BP-3 on adult A. tenuis and its dinoflagellates are limited. Our experimental methods with lower acetone toxicity provide a basis for establishing standard ecotoxicity tests for corals.

Mutualistic Interactions between Dinoflagellates and Pigmented Bacteria Mitigate Environmental Stress.

Scleractinian corals form symbiotic relationships with a variety of microorganisms, including endosymbiotic dinoflagellates of the family Symbiodiniaceae, and with bacteria, which are collectively termed coral holobionts. Interactions between hosts and their symbionts are critical to the physiological status of corals. Coral-microorganism interactions have been studied extensively, but dinoflagellate-bacterial interactions remain largely unexplored. Here, we developed a microbiome manipulation method employing KAS-antibiotic treatment (kanamycin, ampicillin, and streptomycin) to favor pigmented bacteria residing on cultured Cladocopium and Durusdinium, major endosymbionts of corals, and isolated several carotenoid-producing bacteria from cell surfaces of the microalgae. Following KAS-antibiotic treatment of Cladocopium sp. strain NIES-4077, pigmented bacteria increased 8-fold based on colony-forming assays from the parental strain, and 100% of bacterial sequences retrieved through 16S rRNA amplicon sequencing were affiliated with the genus Maribacter. Microbiome manipulation enabled host microalgae to maintain higher maximum quantum yield of photosystem II (variable fluorescence divided by maximum fluorescence [Fv/Fm]) under light-stress conditions, compared to the parental strain. Furthermore, by combining culture-dependent and -independent techniques, we demonstrated that species of the family Symbiodiniaceae and pigmented bacteria form strong interactions. Dinoflagellates protected bacteria from antibiotics, while pigmented bacteria protected microalgal cells from light stress via carotenoid production. Here, we describe for the first time a symbiotic relationship in which dinoflagellates and bacteria mutually reduce environmental stress. Investigations of microalgal-bacterial interactions further document bacterial contributions to coral holobionts and may facilitate development of novel techniques for microbiome-mediated coral reef conservation. IMPORTANCE Coral reefs cover less than 0.1% of the ocean floor, but about 25% of all marine species depend on coral reefs at some point in their life cycles. However, rising ocean temperatures associated with global climate change are a serious threat to coral reefs, causing dysfunction of the photosynthetic apparatus of endosymbiotic microalgae of corals, and overproducing reactive oxygen species harmful to corals. We manipulated the microbiome using an antibiotic treatment to favor pigmented bacteria, enabling their symbiotic microalgal partners to maintain higher photosynthetic function under insolation stress. Furthermore, we investigated mechanisms underlying microalgal-bacterial interactions, describing for the first time a symbiotic relationship in which the two symbionts mutually reduce environmental stress. Our findings extend current insights about microalgal-bacterial interactions, enabling better understanding of bacterial contributions to coral holobionts under stressful conditions and offering hope of reducing the adverse impacts of global warming on coral reefs.

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.

High-resolution spatial and genomic characterization of coral-associated microbial aggregates in the coral Stylophora pistillata.

Bacteria commonly form aggregates in a range of coral species [termed coral-associated microbial aggregates (CAMAs)], although these structures remain poorly characterized despite extensive efforts studying the coral microbiome. Here, we comprehensively characterize CAMAs associated with Stylophora pistillata and quantify their cell abundance. Our analysis reveals that multiple Endozoicomonas phylotypes coexist inside a single CAMA. Nanoscale secondary ion mass spectrometry imaging revealed that the Endozoicomonas cells were enriched with phosphorus, with the elemental compositions of CAMAs different from coral tissues and endosymbiotic Symbiodiniaceae, highlighting a role in sequestering and cycling phosphate between coral holobiont partners. Consensus metagenome-assembled genomes of the two dominant Endozoicomonas phylotypes confirmed their metabolic potential for polyphosphate accumulation along with genomic signatures including type VI secretion systems allowing host association. Our findings provide unprecedented insights into Endozoicomonas-dominated CAMAs and the first direct physiological and genomic linked evidence of their biological role in the coral holobiont.

Contact Reactions between Individuals of the Coral-killing sponge, Terpios hoshinota.

Outbreaks of the coral-killing sponge Terpios hoshinota Rützler and Muzik, 1993 have become a threat to corals and result in coral reef deterioration. This species has an increasing distribution in the Indo-Pacific Ocean and thrives in patches on some reefs in Okinawa, Japan. However, the dispersal process and mechanisms involved remain unknown. We observed the self and non-self recognition capabilities of T. hoshinota by performing contact assays in aquarium and in the field. In the contact assays (indirect and direct contact), allogeneic sets did not fuse and showed a rejection reaction as they formed boundaries (approx. 0.2 mm width) between their tissues. Although the initial reaction between individuals involved adhesion in allogeneic sets, the two individuals remained distant from each other. Histological observations showed that soft tissues (such as collagen) were not present in the boundary zones. These boundaries were maintained for more than 2 weeks. Boundary formations were also confirmed at three field sites in Okinawa, Japan. Our results suggest that T. hoshinota can distinguish self and non-self individuals. Contact assays are a useful method for evaluating the spatial distribution and local population structures of T. hoshinota in coral reefs.

Snails associated with the coral-killing sponge Terpios hoshinota in Okinawa Island, Japan.

Terpios hoshinota is a thin encrusting sponge that overgrows live scleractinian corals and it is linked to coral loss in many reefs. However, our knowledge of the species associated with this sponge species is poor. During a periodical survey of T. hoshinota in 2020, we found tiny snails crawling on the sponge in the subtropical waters around Okinawa Island, Japan. We observed egg capsules inside the sponge tissue and veliger larvae released from the egg capsules. Molecular analyses of both the snails and veliger larvae (cytochrome oxidase I, COI) showed that they were identical and belonged to Joculator sp. (family Cerithiopsidae). There was no direct observation of predation on the sponge by this snail; however, to the best of our knowledge, this is the first report on a close association between a snail and the sponge T. hoshinota.

Specific Detection of Coral-Associated Ruegeria, a Potential Probiotic Bacterium, in Corals and Subtropical Seawater.

Coral microbial flora has been attracting attention because of their potential to protect corals from environmental stresses or pathogens. Although coral-associated bacteria are considered to be acquired from seawater, little is known about the relationships between microbial composition in corals and its surrounding seawater. Here, we tested several methods to identify coral-associated bacteria in coral and its surrounding seawater to detect specific types of Ruegeria species, some of which exhibit growth inhibition activities against the coral pathogen Vibrio coralliilyticus. We first isolated coral-associated bacteria from the reef-building coral Galaxea fascicularis collected at Sesoko Island, Okinawa, Japan, via random colony picking, which showed the existence of varieties of bacteria including Ruegeria species. Using newly constructed primers for colony PCR, several Ruegeria species were successfully isolated from G. fascicularis and seawater. We further investigated the seawater microbiome in association with the distance from coral reefs. By seasonal sampling, it was suggested that the seawater microbiome is more affected by seasonality than the distance from coral reefs. These methods and results may contribute to investigating and understanding the relationships between the presence of corals and microbial diversity in seawater, in addition to the efficient isolation of specific bacterial species from coral or its surrounding seawater.

Usage of UV-curable oligomer-based adhesive agent in hermatypic coral experimental research.

The global decline of the tropical and subtropical coral reefs requires urgent completion of various experiments that will reveal the factors influencing coral health. We describe the procedure of a new inexpensive, easy, and fast method for attaching fragments of the hermatypic coral Acropora spp. to small polycarbonate hexagon head bolts using Bondic®, an ultraviolet (UV)-curable oligomer-based adhesive agent made by Laser Bonding Tech, Inc. (Aurora, ON, Canada). The attachment was hardened within 10 s after applying the adhesive to the cut surface of the coral fragment. The corals attached to polycarbonate bolt were tolerant to long-distance aerial transport 3 days after the attachment. In addition to its implementation in various experiments using hermatypic corals, this method will contribute to aquaculture of hermatypic corals, exhibition of corals in aquariums, and coral reef restoration. The advantages of this new method are summarized below: •A new UV-curable oligomer-based adhesive agent is used as an artificial substrate for coral.•This method is inexpensive, easy to use, and coral attaches quickly to the artificial substrate.•Corals attached to the artificial substrate can withstand long periods of transportation.

Metagenomic, phylogenetic, and functional characterization of predominant endolithic green sulfur bacteria in the coral Isopora palifera.

BACKGROUND: Endolithic microbes in coral skeletons are known to be a nutrient source for the coral host. In addition to aerobic endolithic algae and Cyanobacteria, which are usually described in the various corals and form a green layer beneath coral tissues, the anaerobic photoautotrophic green sulfur bacteria (GSB) Prosthecochloris is dominant in the skeleton of Isopora palifera. However, due to inherent challenges in studying anaerobic microbes in coral skeleton, the reason for its niche preference and function are largely unknown. RESULTS: This study characterized a diverse and dynamic community of endolithic microbes shaped by the availability of light and oxygen. In addition, anaerobic bacteria isolated from the coral skeleton were cultured for the first time to experimentally clarify the role of these GSB. This characterization includes GSB's abundance, genetic and genomic profiles, organelle structure, and specific metabolic functions and activity. Our results explain the advantages endolithic GSB receive from living in coral skeletons, the potential metabolic role of a clade of coral-associated Prosthecochloris (CAP) in the skeleton, and the nitrogen fixation ability of CAP. CONCLUSION: We suggest that the endolithic microbial community in coral skeletons is diverse and dynamic and that light and oxygen are two crucial factors for shaping it. This study is the first to demonstrate the ability of nitrogen uptake by specific coral-associated endolithic bacteria and shed light on the role of endolithic bacteria in coral skeletons.

Continuous recordings of the coral bleaching process on Sesoko Island, Okinawa, Japan, over about 50 days using an underwater camera equipped with a lens wiper.

The colours of the hermatypic corals Porites sp. and Acropora cytherea at Sesoko Island, Okinawa, Japan, were photographed continuously, from 19 July to 6 September 2016, by an underwater camera equipped with a lens wiper. The average seawater temperature during the study period was 29.9 °C. The daily average seawater temperature (DAST) was >30.0 °C until 23 August 2016, and a maximum value of 31.2 °C was recorded on 2 August 2016. Red, green, and blue (RGB) values of these corals were analysed based on photographs taken at 14:00. The RGB values of Porites sp. were stable throughout the observation period, while those of A. cytherea gradually increased (i.e. moved toward the "white" end of the spectrum) until the beginning of September. The present study demonstrated the usefulness of RGB analysis of photographs taken by an underwater camera equipped with a lens wiper for monitoring coral beaching.

Bloom of the cyanobacterium Moorea bouillonii on the gorgonian coral Annella reticulata in Japan.

Coral populations are in decline due to environmental changes and biological attacks by predators and infectious diseases. Here, we report a localized bloom of the benthic filamentous cyanobacterium Moorea bouillonii (formerly Lyngbya bouillonii) observed exclusively on the gorgonian (sea fan) coral Annella reticulata at around 20 m depth in Japan. The degree of infection has reached 26% among different sizes of Annella colonies. Thick and continuous growth of Moorea may be sustained partly by symbiotic alpheid shrimp, which affix Moorea filaments to gorgonian corals for use as food and shelter. Most filaments get entangled on the coral colony, some penetrate into the stem of the coral with a swollen end like a root hair, which appears to function as an anchor in Annella. In addition to the cyanobacterium-shrimp interaction, the new trait of anchoring by the cyanobacterium into gorgonian coral may contribute to persistence of this bloom.

Localized outbreak of attached diatoms on the coral Montipora due to low-temperature stress.

A short-term, localized outbreak of diatoms attached to live corals was observed along the coast of Sesoko Island, Okinawa, Japan in February, 2011. Diatoms are recognized as brown patches in the initial stage, becoming fluffy encrustations and resulting in complete or partial coral death. Attached diatoms, including Licmophora, Climacosphenia, Ardissonea and others, attached and overgrew exclusively Montipora corals, which are dominant corals in some parts of Sesoko reef. Heavily-covered colonies or branches died. The rate of affected corals reached 80% in the worst-affected area. Microscopic observation showed that most diatoms settled directly with polysaccharide stalks or pads onto the partly-bared skeleton of coral branches, although some settled on coral soft tissues. Although no similar phenomenon was reported from other areas of Japan, cold-water events might have important roles in coral weakening, as a consequence, enabling diatom attachment on corals, thus leading to coral death in this area.