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.

Grazing effects of sea urchin Diadema savignyi on algal abundance and coral recruitment processes.

Herbivores control algae and promote coral dominance along coral reefs. However, the majority of previous studies have focused on herbivorous fish. Here we investigated grazing effects of the sea urchin Diadema savignyi on algal abundance and coral recruitment processes. We conducted an in situ cage experiment with three density conditions of D. savignyi (0, 8, 16 indiv. m-2) for three months during the main coral recruitment season in Taiwan. Results demonstrated a strong algal control by D. savignyi. At the end of the experiment, average algal cover was 95% for 0 indiv. m-2, compared to 47% for 8 indiv. m-2 and 16% for 16 indiv. m-2. Average algal biomass at 8 indiv. m-2 declined by one third compared to 0 indiv. m-2 and almost zero at 16 indiv. m-2. On the other hand, a negative grazing effect of D. savignyi was observed on coral recruitment processes. Notably, at 16 indiv. m-2, the density of coral recruits declined and mortality of small coral fragments (proxy of coral juveniles) increased. Our results confirm findings of previous studies and indicate the need to balance both positive (strong algal control) and negative (physical damage) influences of Diadema grazing to facilitate the coral recruitment process.

Shifting in the Dominant Bacterial Group Endozoicomonas Is Independent of the Dissociation With Coral Symbiont Algae.

The coral-associated Endozoicomonas are dominant bacteria in the coral holobiont. Their relative abundance usually decreases with heat-induced coral bleaching and is proposed to be positively correlated with Symbiodiniaceae abundance. It remains unclear whether this phenomenon of decreased Endozoicomonas abundance is caused by temperature stress or a decreased abundance of Symbiodiniaceae. This study induced bleaching in the coral Euphyllia glabrescens using a dark treatment over 15 weeks. We examined shifts in Endozoicomonas abundance and experimentally reduced Symbiodiniaceae density. 16S rRNA gene amplicon sequencing was used to characterize the changes in bacterial community (incl. Endozoicomonas) over time, and the 16S rRNA gene copy number of Endozoicomonas was quantified by qPCR. We detected a high abundance of Endozoicomonas in E. glabrescens that underwent dark-induced bleaching. The results reveal that changes in the relative abundance of Endozoicomonas are unrelated to Symbiodiniaceae abundance, indicating that Endozoicomonas can be independent of Symbiodiniaceae in the coral holobiont.

A Newly Designed Primer Revealed High Phylogenetic Diversity of Endozoicomonas in Coral Reefs.

Endozoicomonas bacteria are commonly regarded as having a potentially symbiotic relationship with their coral hosts. However, their diversity and phylogeny in samples collected from various sources remain unclear. Therefore, we designed an Endozoicomonas-specific primer paired with a bacterial universal primer to detect the 16S ribosomal RNA (rRNA) genes of this taxon and conducted an in-depth investigation of the Endozoicomonas community structure in reef-building corals. The primer had high specificity in the V3-V4 region (95.6%) and its sensitivity was high, particularly when Endozoicomonas was rare in samples (e.g., in seawater, which had a higher alpha diversity of Endozoicomonas than corals). In coral samples, predominant V3-V4 ribotypes had greater divergence than predominant V1-V2 ribotypes, and were grouped into at least 9 novel clades in a phylogenetic tree, indicating Endozoicomonas had high phylogenetic diversity. Divergence within this genus was potentially higher than that among 7 outgroup genera based on the phylogenetic distances of partial 16S rDNA sequences, suggesting that the taxonomy of this genus needs to be revised. In conclusion, dominant Endozoicomonas populations had variable phylogenies; furthermore, the newly designed primers may be useful molecular tools for the reliable detection of the Endozoicomonas community in marine environments.

Dynamics of coral-associated bacterial communities acclimated to temperature stress based on recent thermal history.

Seasonal variation in temperature fluctuations may provide corals and their algal symbionts varying abilities to acclimate to changing temperatures. We hypothesized that different temperature ranges between seasons may promote temperature-tolerance of corals, which would increase stability of a bacterial community following thermal stress. Acropora muricata coral colonies were collected in summer and winter (water temperatures were 23.4-30.2 and 12.1-23.1 °C, respectively) from the Penghu Archipelago in Taiwan, then exposed to 6 temperature treatments (10-33 °C). Changes in coral-associated bacteria were determined after 12, 24, and 48 h. Based on 16S rRNA gene amplicons and Illumina sequencing, bacterial communities differed between seasons and treatments altered the dominant bacteria. Cold stress caused slower shifts in the bacterial community in winter than in summer, whereas a more rapid shift occurred under heat stress in both seasons. Results supported our hypothesis that bacterial community composition of corals in winter are more stable in cold temperatures but changed rapidly in hot temperatures, with opposite results for the bacterial communities in summer. We infer that the thermal tolerance ranges of coral-associated bacteria, with a stable community composition, are associated with their short-term (3 mo) seawater thermal history. Therefore, seasonal acclimation may increase tolerance of coral-associated bacteria to temperature fluctuations.

Long-Term Survey Is Necessary to Reveal Various Shifts of Microbial Composition in Corals.

The coral holobiont is the assemblage of coral host and its microbial symbionts, which functions as a unit and is responsive to host species and environmental factors. Although monitoring surveys have been done to determine bacteria associated with coral, none have persisted for >1 year. Therefore, potential variations in minor or dominant community members that occur over extended intervals have not been characterized. In this study, 16S rRNA gene amplicon pyrosequencing was used to investigate the relationship between bacterial communities in healthy Stylophora pistillata in tropical and subtropical Taiwan over 2 years, apparently one of the longest surveys of coral-associated microbes. Dominant bacterial genera in S. pistillata had disparate changes in different geographical setups, whereas the constitution of minor bacteria fluctuated in abundance over time. We concluded that dominant bacteria (Acinetobacter, Propionibacterium, and Pseudomonas) were stable in composition, regardless of seasonal and geographical variations, whereas Endozoicomonas had a geographical preference. In addition, by combining current data with previous studies, we concluded that a minor bacteria symbiont, Ralstonia, was a keystone species in coral. Finally, we concluded that long-term surveys for coral microbial communities were necessary to detect compositional shifts, especially for minor bacterial members in corals.

Genomic Insight into the Host-Endosymbiont Relationship of Endozoicomonas montiporae CL-33(T) with its Coral Host.

The bacterial genus Endozoicomonas was commonly detected in healthy corals in many coral-associated bacteria studies in the past decade. Although, it is likely to be a core member of coral microbiota, little is known about its ecological roles. To decipher potential interactions between bacteria and their coral hosts, we sequenced and investigated the first culturable endozoicomonal bacterium from coral, the E. montiporae CL-33(T). Its genome had potential sign of ongoing genome erosion and gene exchange with its host. Testosterone degradation and type III secretion system are commonly present in Endozoicomonas and may have roles to recognize and deliver effectors to their hosts. Moreover, genes of eukaryotic ephrin ligand B2 are present in its genome; presumably, this bacterium could move into coral cells via endocytosis after binding to coral's Eph receptors. In addition, 7,8-dihydro-8-oxoguanine triphosphatase and isocitrate lyase are possible type III secretion effectors that might help coral to prevent mitochondrial dysfunction and promote gluconeogenesis, especially under stress conditions. Based on all these findings, we inferred that E. montiporae was a facultative endosymbiont that can recognize, translocate, communicate and modulate its coral host.