A widespread coral-infecting apicomplexan with chlorophyll biosynthesis genes.

Apicomplexa is a group of obligate intracellular parasites that includes the causative agents of human diseases such as malaria and toxoplasmosis. Apicomplexans evolved from free-living phototrophic ancestors, but how this transition to parasitism occurred remains unknown. One potential clue lies in coral reefs, of which environmental DNA surveys have uncovered several lineages of uncharacterized basally branching apicomplexans1,2. Reef-building corals have a well-studied symbiotic relationship with photosynthetic Symbiodiniaceae dinoflagellates (for example, Symbiodinium3), but the identification of other key microbial symbionts of corals has proven to be challenging4,5. Here we use community surveys, genomics and microscopy analyses to identify an apicomplexan lineage-which we informally name 'corallicolids'-that was found at a high prevalence (over 80% of samples, 70% of genera) across all major groups of corals. Corallicolids were the second most abundant coral-associated microeukaryotes after the Symbiodiniaceae, and are therefore core members of the coral microbiome. In situ fluorescence and electron microscopy confirmed that corallicolids live intracellularly within the tissues of the coral gastric cavity, and that they possess apicomplexan ultrastructural features. We sequenced the genome of the corallicolid plastid, which lacked all genes for photosystem proteins; this indicates that corallicolids probably contain a non-photosynthetic plastid (an apicoplast6). However, the corallicolid plastid differs from all other known apicoplasts because it retains the four ancestral genes that are involved in chlorophyll biosynthesis. Corallicolids thus share characteristics with both their parasitic and their free-living relatives, which suggests that they are evolutionary intermediates and implies the existence of a unique biochemistry during the transition from phototrophy to parasitism.

The crown-of-thorns starfish genome as a guide for biocontrol of this coral reef pest.

The crown-of-thorns starfish (COTS, the Acanthaster planci species group) is a highly fecund predator of reef-building corals throughout the Indo-Pacific region. COTS population outbreaks cause substantial loss of coral cover, diminishing the integrity and resilience of reef ecosystems. Here we sequenced genomes of COTS from the Great Barrier Reef, Australia and Okinawa, Japan to identify gene products that underlie species-specific communication and could potentially be used in biocontrol strategies. We focused on water-borne chemical plumes released from aggregating COTS, which make the normally sedentary starfish become highly active. Peptide sequences detected in these plumes by mass spectrometry are encoded in the COTS genome and expressed in external tissues. The exoproteome released by aggregating COTS consists largely of signalling factors and hydrolytic enzymes, and includes an expanded and rapidly evolving set of starfish-specific ependymin-related proteins. These secreted proteins may be detected by members of a large family of olfactory-receptor-like G-protein-coupled receptors that are expressed externally, sometimes in a sex-specific manner. This study provides insights into COTS-specific communication that may guide the generation of peptide mimetics for use on reefs with COTS outbreaks.

A multiomic analysis of in situ coral-turf algal interactions.

Viruses, microbes, and host macroorganisms form ecological units called holobionts. Here, a combination of metagenomic sequencing, metabolomic profiling, and epifluorescence microscopy was used to investigate how the different components of the holobiont including bacteria, viruses, and their associated metabolites mediate ecological interactions between corals and turf algae. The data demonstrate that there was a microbial assemblage unique to the coral-turf algae interface displaying higher microbial abundances and larger microbial cells. This was consistent with previous studies showing that turf algae exudates feed interface and coral-associated microbial communities, often at the detriment of the coral. Further supporting this hypothesis, when the metabolites were assigned a nominal oxidation state of carbon (NOSC), we found that the turf algal metabolites were significantly more reduced (i.e., have higher potential energy) compared to the corals and interfaces. The algae feeding hypothesis was further supported when the ecological outcomes of interactions (e.g., whether coral was winning or losing) were considered. For example, coral holobionts losing the competition with turf algae had higher Bacteroidetes-to-Firmicutes ratios and an elevated abundance of genes involved in bacterial growth and division. These changes were similar to trends observed in the obese human gut microbiome, where overfeeding of the microbiome creates a dysbiosis detrimental to the long-term health of the metazoan host. Together these results show that there are specific biogeochemical changes at coral-turf algal interfaces that predict the competitive outcomes between holobionts and are consistent with algal exudates feeding coral-associated microbes.

Neuronal Modulators from the Coral-Associated Fungi Aspergillus candidus.

Three new p-terphenyl derivatives, named 4″-O-methyl-prenylterphenyllin B (1) and phenylcandilide A and B (17 and 18), and three new indole-diterpene alkaloids, asperindoles E-G (22-24), were isolated together with eighteen known analogues from the fungi Aspergillus candidus associated with the South China Sea gorgonian Junceela fragillis. The structures and absolute configurations of the new compounds were elucidated on the basis of spectroscopic analysis, and DFT/NMR and TDDFT/ECD calculations. In a primary cultured cortical neuronal network, the compounds 6, 9, 14, 17, 18 and 24 modulated spontaneous Ca2+ oscillations and 4-aminopyridine hyperexcited neuronal activity. A preliminary structure-activity relationship was discussed.

Uncovering the role of Symbiodiniaceae assemblage composition and abundance in coral bleaching response by minimizing sampling and evolutionary biases.

BACKGROUND: Biodiversity and productivity of coral-reef ecosystems depend upon reef-building corals and their associations with endosymbiotic Symbiodiniaceae, which offer diverse functional capabilities to their hosts. The number of unique symbiotic partners (richness) and relative abundances (evenness) have been hypothesized to affect host response to climate change induced thermal stress. Symbiodiniaceae assemblages with many unique phylotypes may provide greater physiological flexibility or form less stable symbioses; assemblages with low abundance phylotypes may allow corals to retain thermotolerant symbionts or represent associations with less-suitable symbionts. RESULTS: Here we demonstrate that true richness of Symbiodiniaceae phylotype assemblages is generally not discoverable from direct enumeration of unique phylotypes in association records and that cross host-species comparisons are biased by sampling and evolutionary patterns among species. These biases can be minimized through rarefaction of richness (rarefied-richness) and evenness (Probability of Interspecific Encounter, PIE), and analyses that account for phylogenetic patterns. These standardized metrics were calculated for individual Symbiodiniaceae assemblages composed of 377 unique ITS2 phylotypes associated with 123 coral species. Rarefied-richness minimized correlations with sampling effort, while maintaining important underlying characteristics across host bathymetry and geography. Phylogenetic comparative methods reveal significant increases in coral bleaching and mortality associated with increasing Symbiodiniaceae assemblage richness and evenness at the level of host species. CONCLUSIONS: These results indicate that the potential flexibility afforded by assemblages characterized by many phylotypes present at similar relative abundances does not result in decreased bleaching risk and point to the need to characterize the overall functional and genetic diversity of Symbiodiniaceae assemblages to quantify their effect on host fitness under climate change.

Diseases and pathogens of marine invertebrate corals in Indian reefs.

Diseases in marine invertebrate corals have been reported worldwide and have been associated with infection by various microbial pathogens that cause massive mortality. Several bacterial species, especially Vibrio species but also members of the cyanobacteria, fungi, viruses, and protists, are described as important pathogens associated with coral disease and mortality. The present work provides an updated overview of main diseases and implicated microbial species affecting corals in Indian reefs. Further study on pathogen diversity, classification, spread and environmental factors on pathogen-host interactions may contribute a better understanding of the coral diseases.

Condition-specific RNA editing in the coral symbiont Symbiodinium microadriaticum.

RNA editing is a rare post-transcriptional event that provides cells with an additional level of gene expression regulation. It has been implicated in various processes including adaptation, viral defence and RNA interference; however, its potential role as a mechanism in acclimatization has just recently been recognised. Here, we show that RNA editing occurs in 1.6% of all nuclear-encoded genes of Symbiodinium microadriaticum, a dinoflagellate symbiont of reef-building corals. All base-substitution edit types were present, and statistically significant motifs were associated with three edit types. Strikingly, a subset of genes exhibited condition-specific editing patterns in response to different stressors that resulted in significant increases of non-synonymous changes. We posit that this previously unrecognised mechanism extends this organism's capability to respond to stress beyond what is encoded by the genome. This in turn may provide further acclimatization capacity to these organisms, and by extension, their coral hosts.

Re-analysis of the coral Acropora digitifera transcriptome reveals a complex lncRNAs-mRNAs interaction network implicated in Symbiodinium infection.

BACKGROUND: Being critically important to the ecosystem, the stability of coral reefs is directly related to the marine and surrounding terrestrial environments. However, coral reefs are now undergoing massive and accelerating devastation due to bleaching. The fact that the breakdown of symbiosis between coral and the dinoflagellate, zooxanthellae, has been well elucidated as the main cause of bleaching, implying the establishment of symbiosis with zooxanthellae plays a crucial role in maintaining coral survival. However, the relevant molecular and cellular mechanisms have not been well studied yet. In this study, based on the deep RNA-sequencing data derived from Mohamed, A. R. et al., an integrated transcriptome analysis was performed to deeply investigate global transcriptome changes of the coral Acropora digitifera in response to infection by dinoflagellate of the genus Symbiodinium. RESULTS: The results revealed that compared to RefTranscriptome_v1.0 (A. digitifera transcriptome assembly v1.0), numerous novel transcripts and isoforms were identified, the Symbiodinium-infected coral larvae at 4 h generated the highest proportion of specific isoforms. Alternative splicing analysis showed that intron retention predominated in all alternative transcripts among six statuses. Additionally, 8117 lncRNAs were predicted via a stringent stepwise filtering pipeline. A complex lncRNAs-mRNAs network including 815 lncRNAs and 6395 mRNAs were established, in which 21 lncRNAs were differentially expressed at 4 h post infection. Functional clustering analysis for those differentially lncRNAs-coexpressed mRNAs demonstrated that several biological processes and pathways related to protein kinase activity, metabolic pathways, mitochondrion, ribosome, etc. were enriched. CONCLUSIONS: Our study not only refined A. digitifera transcriptome via identification of novel transcripts and isoforms, but also predicted a high-confidence dataset of lncRNAs. Functional study based on the construction of lncRNAs-mRNAs co-expression network has disclosed a complex lncRNA-mediated regulation in response to Symbiodinium infection exhibited in A. digitifera. Once validated, these lncRNAs could be good potential targets for treatment and prevention of bleaching in coral.

Diazotrophic community and associated dinitrogen fixation within the temperate coral Oculina patagonica.

Dinitrogen (N2 ) fixing bacteria (diazotrophs) are an important source of new nitrogen in oligotrophic environments and represent stable members of the microbiome in tropical corals, while information on corals from temperate oligotrophic regions is lacking. Therefore, this study provides new insights into the diversity and activity of diazotrophs associated with the temperate coral Oculina patagonica from the Mediterranean Sea by combining metabarcoding sequencing of amplicons of both the 16S rRNA and nifH genes and 15 N2 stable isotope tracer analysis to assess diazotroph-derived nitrogen (DDN) assimilation by the coral. Results show that the diazotrophic community of O. patagonica is dominated by autotrophic bacteria (i.e. Cyanobacteria and Chlorobia). The majority of DDN was assimilated into the tissue and skeletal matrix, and DDN assimilation significantly increased in bleached corals. Thus, diazotrophs may constitute an additional nitrogen source for the coral host, when nutrient exchange with Symbiodinium is disrupted (e.g. bleaching) and external food supply is limited (e.g. oligotrophic summer season). Furthermore, we hypothesize that DDN can facilitate the fast proliferation of endolithic algae, which provide an alternative carbon source for bleached O. patagonica. Overall, O. patagonica could serve as a good model for investigating the importance of diazotrophs in coral recovery from bleaching.

Role of host genetics and heat-tolerant algal symbionts in sustaining populations of the endangered coral Orbicella faveolata in the Florida Keys with ocean warming.

Identifying which factors lead to coral bleaching resistance is a priority given the global decline of coral reefs with ocean warming. During the second year of back-to-back bleaching events in the Florida Keys in 2014 and 2015, we characterized key environmental and biological factors associated with bleaching resilience in the threatened reef-building coral Orbicella faveolata. Ten reefs (five inshore, five offshore, 179 corals total) were sampled during bleaching (September 2015) and recovery (May 2016). Corals were genotyped with 2bRAD and profiled for algal symbiont abundance and type. O. faveolata at the inshore sites, despite higher temperatures, demonstrated significantly higher bleaching resistance and better recovery compared to offshore. The thermotolerant Durusdinium trenchii (formerly Symbiondinium trenchii) was the dominant endosymbiont type region-wide during initial (78.0% of corals sampled) and final (77.2%) sampling; >90% of the nonbleached corals were dominated by D. trenchii. 2bRAD host genotyping found no genetic structure among reefs, but inshore sites showed a high level of clonality. While none of the measured environmental parameters were correlated with bleaching, 71% of variation in bleaching resistance and 73% of variation in the proportion of D. trenchii was attributable to differences between genets, highlighting the leading role of genetics in shaping natural bleaching patterns. Notably, D. trenchii was rarely dominant in O. faveolata from the Florida Keys in previous studies, even during bleaching. The region-wide high abundance of D. trenchii was likely driven by repeated bleaching associated with the two warmest years on record for the Florida Keys (2014 and 2015). On inshore reefs in the Upper Florida Keys, O. faveolata was most abundant, had the highest bleaching resistance, and contained the most corals dominated by D. trenchii, illustrating a causal link between heat tolerance and ecosystem resilience with global change.

New Species of Closely Related Endosymbiotic Dinoflagellates in the Greater Caribbean have Niches Corresponding to Host Coral Phylogeny.

Symbiotic dinoflagellates in the genus Breviolum (formerly Symbiodinium Clade B) dominate coral communities in shallow waters across the Greater Caribbean. While some formally described species exist, mounting genetic, and ecological evidence indicate that numerous more comprise this genus, many of which are closely related. To test this, colonies of common reef-building corals were sampled across a large geographical range. Phylogenetic and population genetic markers then used to examine evolutionary divergence and delineate boundaries of genetic recombination. Three new candidate species were distinguished by fixed differences in nucleotide sequences from nuclear and chloroplast DNA. Population connectivity was evident within each lineage over thousands of kilometers, however, substantial genetic structure persisted between lineages co-occurring within sampling locations, signifying reproductive isolation. While geographically widespread with overlapping distributions, each species is ecologically distinct, exhibiting specific mutualisms with phylogenetically distinct coral hosts. Moreover, significant differences in mean cell sizes provide some morphological evidence substantiating formal species distinctions. In providing evidence that satisfies the biological, phylogenetic, ecological, and morphological species concepts, we classify and formally name Breviolum faviinorum n. sp., primarily associated with Caribbean corals belonging to the Caribbean subfamily Faviinae; B. meandrinium n. sp., associated with corals belonging to the family Meandrinidae; and B. dendrogyrum n. sp., a symbiont harbored exclusively by the threatened coral Dendrogyra cylindrus. These findings support the primary importance of niche diversification (i.e. host habitat) in the speciation of symbiotic dinoflagellates.

Potent Cytotoxic Analogs of Amphidinolides from the Atlantic Octocoral Stragulum bicolor.

Amphidinolides are cytotoxic macrolides produced by symbiotic unicellular microalgae of the genus Amphidinium. Here we describe the identification of four related molecules belonging to this macrolide family isolated from the invertebrate Stragulum bicolor. The new molecules, named amphidinolide PX1-PX3 and stragulin A (1⁻4), show an unprecedented carbon skeleton whose complete stereochemistry has been determined by spectroscopic and computational methods. Differences in the structures of these molecules modulate their biological activity in a panel of tumor cell lines, but the opened derivative stragulin (4) shows a very potent and specific cytotoxic activity (IC50 0.18 µM) against the aggressive human melanoma cell A2058.

Global diversity and distribution of close relatives of apicomplexan parasites.

Apicomplexans are a group of obligate intracellular parasites, but their retention of a relict non-photosynthetic plastid reveals that they evolved from free-living photosynthetic ancestors. The closest relatives of apicomplexans include photosynthetic chromerid algae (e.g., Chromera and Vitrella), non-photosynthetic colpodellid predators (e.g., Colpodella) and several environmental clades collectively called Apicomplexan-Related Lineages (ARLs). Here we investigate the global distribution and inferred ecology of the ARLs by expansively searching for apicomplexan-related plastid small ribosomal subunit (SSU) genes in large-scale high-throughput bacterial amplicon surveys. Searching more than 220 million sequences from 224 geographical sites worldwide revealed 94 324 ARL plastid SSU sequences. Meta-analyses confirm that all ARLs are coral reef associated and not to marine environments generally, but only a subset is actually associated with coral itself. Most unexpectedly, Chromera was found exclusively in coral biogenous sediments, and not within coral tissue, indicating that it is not a coral symbiont, as typically thought. In contrast, ARL-V is the most diverse, geographically widespread and abundant of all ARL clades and is strictly associated with coral tissue and mucus. ARL-V was found in 19 coral species in reefs, including azooxanthellate corals at depths greater than 500 m. We suggest this is indicative of a parasitic or commensal relationship, and not of photosynthetic symbiosis, further underscoring the importance of isolating ARL-V and determining its relationship with the coral host.

Unexpected mixed-mode transmission and moderate genetic regulation of Symbiodinium communities in a brooding coral.

Determining the extent to which Symbiodinium communities in corals are inherited versus environmentally acquired is fundamental to understanding coral resilience and to predicting coral responses to stressors like warming oceans that disrupt this critical endosymbiosis. We examined the fidelity with which Symbiodinium communities in the brooding coral Seriatopora hystrix are vertically transmitted and the extent to which communities are genetically regulated, by genotyping the symbiont communities within 60 larvae and their parents (9 maternal and 45 paternal colonies) using high-throughput sequencing of the ITS2 locus. Unexpectedly, Symbiodinium communities associated with brooded larvae were distinct from those within parent colonies, including the presence of types not detected in adults. Bayesian heritability (h2) analysis revealed that 33% of variability in larval Symbiodinium communities was genetically controlled. Results highlight flexibility in the establishment of larval symbiont communities and demonstrate that symbiont transmission is not exclusively vertical in brooding corals. Instead, we show that Symbiodinium transmission in S. hystrix involves a mixed-mode strategy, similar to many terrestrial invertebrate symbioses. Also, variation in the abundances of common Symbiodinium types among adult corals suggests that microhabitat differences influence the structure of in hospite Symbiodinium communities. Partial genetic regulation coupled with flexibility in the environmentally acquired component of Symbiodinium communities implies that corals with vertical transmission, like S. hystrix, may be more resilient to environmental change than previously thought.

Cell Cycle Dynamics of Cultured Coral Endosymbiotic Microalgae (Symbiodinium) Across Different Types (Species) Under Alternate Light and Temperature Conditions.

Dinoflagellates of the genus Symbiodinium live in symbiosis with many invertebrates, including reef-building corals. Hosts maintain this symbiosis through continuous regulation of Symbiodinium cell density via expulsion and degradation (postmitotic) and/or constraining cell growth and division through manipulation of the symbiont cell cycle (premitotic). Importance of premitotic regulation is unknown since little data exists on cell cycles for the immense genetic diversity of Symbiodinium. We therefore examined cell cycle progression for several distinct SymbiodiniumITS2-types (B1, C1, D1a). All types exhibited typical microalgal cell cycle progression, G1 phase through to S phase during the light period, and S phase to G2 /M phase during the dark period. However, the proportion of cells in these phases differed between strains and reflected differences in growth rates. Undivided larger cells with 3n DNA content were observed especially in type D1a, which exhibited a distinct cell cycle pattern. We further compared cell cycle patterns under different growth light intensities and thermal regimes. Whilst light intensity did not affect cell cycle patterns, heat stress inhibited cell cycle progression and arrested all strains in G1 phase. We discuss the importance of understanding Symbiodinium functional diversity and how our findings apply to clarify stability of host-Symbiodinium symbioses.

Coral Symbiodinium Community Composition Across the Belize Mesoamerican Barrier Reef System is Influenced by Host Species and Thermal Variability.

Reef-building corals maintain a symbiotic relationship with dinoflagellate algae of the genus Symbiodinium, and this symbiosis is vital for the survival of the coral holobiont. Symbiodinium community composition within the coral host has been shown to influence a coral's ability to resist and recover from stress. A multitude of stressors including ocean warming, ocean acidification, and eutrophication have been linked to global scale decline in coral health and cover in recent decades. Three distinct thermal regimes (highTP, modTP, and lowTP) following an inshore-offshore gradient of declining average temperatures and thermal variation were identified on the Belize Mesoamerican Barrier Reef System (MBRS). Quantitative metabarcoding of the ITS-2 locus was employed to investigate differences and similarities in Symbiodinium genetic diversity of the Caribbean corals Siderastrea siderea, S. radians, and Pseudodiploria strigosa between the three thermal regimes. A total of ten Symbiodinium lineages were identified across the three coral host species. S. siderea was associated with distinct Symbiodinium communities; however, Symbiodinium communities of its congener, S. radians and P. strigosa, were more similar to one another. Thermal regime played a role in defining Symbiodinium communities in S. siderea but not S. radians or P. strigosa. Against expectations, Symbiodinium trenchii, a symbiont known to confer thermal tolerance, was dominant only in S. siderea at one sampled offshore site and was rare inshore, suggesting that coral thermal tolerance in more thermally variable inshore habitats is achieved through alternative mechanisms. Overall, thermal parameters alone were likely not the only primary drivers of Symbiodinium community composition, suggesting that environmental variables unrelated to temperature (i.e., light availability or nutrients) may play key roles in structuring coral-algal communities in Belize and that the relative importance of these environmental variables may vary by coral host species.

Microbial invasion of the Caribbean by an Indo-Pacific coral zooxanthella.

Human-induced environmental changes have ushered in the rapid decline of coral reef ecosystems, particularly by disrupting the symbioses between reef-building corals and their photosymbionts. However, escalating stressful conditions enable some symbionts to thrive as opportunists. We present evidence that a stress-tolerant "zooxanthella" from the Indo-Pacific Ocean, Symbiodinium trenchii, has rapidly spread to coral communities across the Greater Caribbean. In marked contrast to populations from the Indo-Pacific, Atlantic populations of S. trenchii contained exceptionally low genetic diversity, including several widespread and genetically similar clones. Colonies with this symbiont tolerate temperatures 1-2 °C higher than other host-symbiont combinations; however, calcification by hosts harboring S. trenchii is reduced by nearly half, compared with those harboring natives, and suggests that these new symbioses are maladapted. Unforeseen opportunism and geographical expansion by invasive mutualistic microbes could profoundly influence the response of reef coral symbioses to major environmental perturbations but may ultimately compromise ecosystem stability and function.

Coral host cells acidify symbiotic algal microenvironment to promote photosynthesis.

Symbiotic dinoflagellate algae residing inside coral tissues supply the host with the majority of their energy requirements through the translocation of photosynthetically fixed carbon. The algae, in turn, rely on the host for the supply of inorganic carbon. Carbon must be concentrated as CO2 in order for photosynthesis to proceed, and here we show that the coral host plays an active role in this process. The host-derived symbiosome membrane surrounding the algae abundantly expresses vacuolar H(+)-ATPase (VHA), which acidifies the symbiosome space down to pH ∼ 4. Inhibition of VHA results in a significant decrease in average H(+) activity in the symbiosome of up to 75% and a significant reduction in O2 production rate, a measure of photosynthetic activity. These results suggest that host VHA is part of a previously unidentified carbon concentrating mechanism for algal photosynthesis and provide mechanistic evidence that coral host cells can actively modulate the physiology of their symbionts.

"Super-quenching" state protects Symbiodinium from thermal stress - Implications for coral bleaching.

The global rise in sea surface temperatures causes regular exposure of corals to high temperature and high light stress, leading to worldwide disastrous coral bleaching events (loss of symbiotic dinoflagellates (Symbiodinium) from reef-building corals). Our picosecond chlorophyll fluorescence experiments on cultured Symbiodinium clade C cells exposed to coral bleaching conditions uncovered the transformations of the alga's photosynthetic apparatus (PSA) that activate an extremely efficient non-photochemical "super-quenching" mechanism. The mechanism is associated with a transition from an initially heterogeneous photosystem II (PSII) pool to a homogeneous "spillover" pool, where nearly all excitation energy is transferred to photosystem I (PSI). There, the inherently higher stability of PSI and high quenching efficiency of P(700)(+) allow dumping of PSII excess excitation energy into heat, resulting in almost complete cessation of photosynthetic electron transport (PET). This potentially reversible "super-quenching" mechanism protects the PSA against destruction at the cost of a loss of photosynthetic activity. We suggest that the inhibition of PET and the consequent inhibition of organic carbon production (e.g. sugars) in the symbiotic Symbiodinium provide a trigger for the symbiont expulsion, i.e. bleaching.