Macro- and micro-scale adaptations allow distinct Stylophora pistillata-symbiodiniaceae holobionts to optimize performance across a broad light habitat.

In sessile organisms, phenotypic plasticity represents an important strategy for dealing with environmental variability. Here we test if phenotypic plasticity enables the common coral Stylophora pistillata to occupy a broad niche. We find clear differences in the photo-physiology of four putative species of photosynthetic dinoflagellate symbionts associated with the coral S. pistillata, namely, Cladocopium 'C35a', 'C79', 'C78a' and 'C8a'. Coral phenotypic responses were also tightly linked to symbiont identity. Corals with Cladocopium 'C8a' have more "open" macro-morphology compared to colonies associating with depth-restricted Cladocopium 'C35a' or 'C78a' in the same shallow water habitat. Corals with Cladocopium 'C8a' had 40 to 60% lower symbiont cell densities compared to other holobionts but were more efficient at acclimating over a range of light levels, with clear mechanisms to dissipate excess light energy. This holobiont contains host-based green fluorescent pigments, increased concentrations of symbiont-based mycosporine amino acids, and xanthophyll cycling in high light habitats. Photosynthetic efficiency was also adjusted over the light habitat. In contrast, limited micro-scale responses were observed between three depth-restricted symbionts: Cladocopium 'C79', 'C35a', and 'C78a'. To optimize light levels reaching the photosynthetic unit, these colonies rely on a more closed macro-morphology under high light levels, which reduces incident light levels by up to 43%, and higher symbiont densities . Our results show that distinct macro- and micro-scale adaptations lead to functional differences between four distinct S. pistillata holobionts, allowing them to co-exist by filling specific niches on a small, but environmentally diverse, spatial scale. Key index words: Light, Symbiodiniaceae, coral, pigments, Stylophora pistillata, ITS2, phenotypic plasticity, niche diversification.

Impact of catchment-derived nutrients and sediments on marine water quality on the Great Barrier Reef: An application of the eReefs marine modelling system.

Water quality of the Great Barrier Reef (GBR) is determined by a range of natural and anthropogenic drivers that are resolved in the eReefs coupled hydrodynamic - biogeochemical marine model forced by a process-based catchment model, GBR Dynamic SedNet. Model simulations presented here quantify the impact of anthropogenic catchment loads of sediments and nutrients on a range of marine water quality variables. Simulations of 2011-2018 show that reduction of anthropogenic catchment loads results in improved water quality, especially within river plumes. Within the 16 resolved river plumes, anthropogenic loads increased chlorophyll concentration by 0.10 (0.02-0.25) mg Chl m-3. Reductions of anthropogenic loads following proposed Reef 2050 Water Quality Improvement Plan targets reduced chlorophyll concentration in the plumes by 0.04 (0.01-0.10) mg Chl m-3. Our simulations demonstrate the impact of anthropogenic loads on GBR water quality and quantify the benefits of improved catchment management.

A differential k-mer analysis pipeline for comparing RNA-Seq transcriptome and meta-transcriptome datasets without a reference.

Next-generation DNA sequencing technologies, such as RNA-Seq, currently dominate genome-wide gene expression studies. A standard approach to analyse this data requires mapping sequence reads to a reference and counting the number of reads which map to each gene. However, for many transcriptome studies, a suitable reference genome is unavailable, especially for meta-transcriptome studies which assay gene expression from mixed populations of organisms. Where a reference is unavailable, it is possible to generate a reference by the de novo assembly of the sequence reads. However, the high cost of generating high-coverage data for de novo assembly hinders this approach and more importantly the accurate assembly of such data is challenging, especially for meta-transcriptome data, and resulting assemblies frequently suffer from collapsed regions or chimeric sequences. As an alternative to the standard reference mapping approach, we have developed a k-mer-based analysis pipeline (DiffKAP) to identify differentially expressed reads between RNA-Seq datasets without the requirement for a reference. We compared the DiffKAP approach with the traditional Tophat/Cuffdiff method using RNA-Seq data from soybean, which has a suitable reference genome. We subsequently examined differential gene expression for a coral meta-transcriptome where no reference is available, and validated the results using qRT-PCR. We conclude that DiffKAP is an accurate method to study differential gene expression in complex meta-transcriptomes without the requirement of a reference genome.

A-to-I RNA Editing in the Earliest-Diverging Eumetazoan Phyla.

The highly conserved ADAR enzymes, found in all multicellular metazoans, catalyze the editing of mRNA transcripts by the deamination of adenosines to inosines. This type of editing has two general outcomes: site specific editing, which frequently leads to recoding, and clustered editing, which is usually found in transcribed genomic repeats. Here, for the first time, we looked for both editing of isolated sites and clustered, non-specific sites in a basal metazoan, the coral Acropora millepora during spawning event, in order to reveal its editing pattern. We found that the coral editome resembles the mammalian one: it contains more than 500,000 sites, virtually all of which are clustered in non-coding regions that are enriched for predicted dsRNA structures. RNA editing levels were increased during spawning and increased further still in newly released gametes. This may suggest that editing plays a role in introducing variability in coral gametes.

Signaling cascades and the importance of moonlight in coral broadcast mass spawning.

Many reef-building corals participate in a mass-spawning event that occurs yearly on the Great Barrier Reef. This coral reproductive event is one of earth's most prominent examples of synchronised behavior, and coral reproductive success is vital to the persistence of coral reef ecosystems. Although several environmental cues have been implicated in the timing of mass spawning, the specific sensory cues that function together with endogenous clock mechanisms to ensure accurate timing of gamete release are largely unknown. Here, we show that moonlight is an important external stimulus for mass spawning synchrony and describe the potential mechanisms underlying the ability of corals to detect environmental triggers for the signaling cascades that ultimately result in gamete release. Our study increases the understanding of reproductive chronobiology in corals and strongly supports the hypothesis that coral gamete release is achieved by a complex array of potential neurohormones and light-sensing molecules.

Transcriptomic Changes in Coral Holobionts Provide Insights into Physiological Challenges of Future Climate and Ocean Change.

Tropical reef-building coral stress levels will intensify with the predicted rising atmospheric CO2 resulting in ocean temperature and acidification increase. Most studies to date have focused on the destabilization of coral-dinoflagellate symbioses due to warming oceans, or declining calcification due to ocean acidification. In our study, pH and temperature conditions consistent with the end-of-century scenarios of the Intergovernmental Panel on Climate Change (IPCC) caused major changes in photosynthesis and respiration, in addition to decreased calcification rates in the coral Acropora millepora. Population density of symbiotic dinoflagellates (Symbiodinium) under high levels of ocean acidification and temperature (Representative Concentration Pathway, RCP8.5) decreased to half of that found under present day conditions, with photosynthetic and respiratory rates also being reduced by 40%. These physiological changes were accompanied by evidence for gene regulation of calcium and bicarbonate transporters along with components of the organic matrix. Metatranscriptomic RNA-Seq data analyses showed an overall down regulation of metabolic transcripts, and an increased abundance of transcripts involved in circadian clock control, controlling the damage of oxidative stress, calcium signaling/homeostasis, cytoskeletal interactions, transcription regulation, DNA repair, Wnt signaling and apoptosis/immunity/ toxins. We suggest that increased maintenance costs under ocean acidification and warming, and diversion of cellular ATP to pH homeostasis, oxidative stress response, UPR and DNA repair, along with metabolic suppression, may underpin why Acroporid species tend not to thrive under future environmental stress. Our study highlights the potential increased energy demand when the coral holobiont is exposed to high levels of ocean warming and acidification.

Unfolding the secrets of coral-algal symbiosis.

Dinoflagellates from the genus Symbiodinium form a mutualistic symbiotic relationship with reef-building corals. Here we applied massively parallel Illumina sequencing to assess genetic similarity and diversity among four phylogenetically diverse dinoflagellate clades (A, B, C and D) that are commonly associated with corals. We obtained more than 30,000 predicted genes for each Symbiodinium clade, with a majority of the aligned transcripts corresponding to sequence data sets of symbiotic dinoflagellates and <2% of sequences having bacterial or other foreign origin. We report 1053 genes, orthologous among four Symbiodinium clades, that share a high level of sequence identity to known proteins from the SwissProt (SP) database. Approximately 80% of the transcripts aligning to the 1053 SP genes were unique to Symbiodinium species and did not align to other dinoflagellates and unrelated eukaryotic transcriptomes/genomes. Six pathways were common to all four Symbiodinium clades including the phosphatidylinositol signaling system and inositol phosphate metabolism pathways. The list of Symbiodinium transcripts common to all four clades included conserved genes such as heat shock proteins (Hsp70 and Hsp90), calmodulin, actin and tubulin, several ribosomal, photosynthetic and cytochrome genes and chloroplast-based heme-containing cytochrome P450, involved in the biosynthesis of xanthophylls. Antioxidant genes, which are important in stress responses, were also preserved, as were a number of calcium-dependent and calcium/calmodulin-dependent protein kinases that may play a role in the establishment of symbiosis. Our findings disclose new knowledge about the genetic uniqueness of symbiotic dinoflagellates and provide a list of homologous genes important for the foundation of coral-algal symbiosis.

Early transcriptional changes in the reef-building coral Acropora aspera in response to thermal and nutrient stress.

BACKGROUND: Changes to the environment as a result of human activities can result in a range of impacts on reef building corals that include coral bleaching (reduced concentrations of algal symbionts), decreased coral growth and calcification, and increased incidence of diseases and mortality. Understanding how elevated temperatures and nutrient concentration affect early transcriptional changes in corals and their algal endosymbionts is critically important for evaluating the responses of coral reefs to global changes happening in the environment. Here, we investigated the expression of genes in colonies of the reef-building coral Acropora aspera exposed to short-term sub-lethal levels of thermal (+6°C) and nutrient stress (ammonium-enrichment: 20 µM). RESULTS: The RNA-Seq data provided hundreds of differentially expressed genes (DEGs) corresponding to various stress regimes, with 115 up- and 78 down-regulated genes common to all stress regimes. A list of DEGs included up-regulated coral genes like cytochrome c oxidase and NADH-ubiquinone oxidoreductase and up-regulated photosynthetic genes of algal origin, whereas coral GFP-like fluorescent chromoprotein and sodium/potassium-transporting ATPase showed reduced transcript levels. Taxonomic analyses of the coral holobiont disclosed the dominant presence of transcripts from coral (~70%) and Symbiodinium (~10-12%), as well as ~15-20% of unknown sequences which lacked sequence identity to known genes. Gene ontology analyses revealed enriched pathways, which led to changes in the dynamics of protein networks affecting growth, cellular processes, and energy requirement. CONCLUSIONS: In corals with preserved symbiont physiological performance (based on Fv/Fm, photo-pigment and symbiont density), transcriptomic changes and DEGs provided important insight into early stages of the stress response in the coral holobiont. Although there were no signs of coral bleaching after exposure to short-term thermal and nutrient stress conditions, we managed to detect oxidative stress and apoptotic changes on a molecular level and provide a list of prospective stress biomarkers for both partners in symbiosis. Consequently, our findings are important for understanding and anticipating impacts of anthropogenic global climate change on coral reefs.

New-old hemoglobin-like proteins of symbiotic dinoflagellates.

Symbiotic dinoflagellates are unicellular photosynthetic algae that live in mutualistic symbioses with many marine organisms. Within the transcriptome of coral endosymbionts Symbiodinium sp. (type C3), we discovered the sequences of two novel and highly polymorphic hemoglobin-like genes and proposed their 3D protein structures. At the protein level, four isoforms shared between 87 and 97% sequence identity for Hb-1 and 78-99% for Hb-2, whereas between Hb-1 and Hb-2 proteins, only 15-21% sequence homology has been preserved. Phylogenetic analyses of the dinoflagellate encoding Hb sequences have revealed a separate evolutionary origin of the discovered globin genes and indicated the possibility of horizontal gene transfer. Transcriptional regulation of the Hb-like genes was studied in the reef-building coral Acropora aspera exposed to elevated temperatures (6-7°C above average sea temperature) over a 24-h period and a 72-h period, as well as to nutrient stress. Exposure to elevated temperatures resulted in an increased Hb-1 gene expression of 31% after 72 h only, whereas transcript abundance of the Hb-2 gene was enhanced by up to 59% by both 1-day and 3-day thermal stress conditions. Nutrient stress also increased gene expression of Hb-2 gene by 70%. Our findings describe the differential expression patterns of two novel Hb genes from symbiotic dinoflagellates and their polymorphic nature. Furthermore, the inducible nature of Hb-2 gene by both thermal and nutrient stressors indicates a prospective role of this form of hemoglobin in the initial coral-algal responses to changes in environmental conditions. This novel hemoglobin has potential use as a stress biomarker.

Major cellular and physiological impacts of ocean acidification on a reef building coral.

As atmospheric levels of CO(2) increase, reef-building corals are under greater stress from both increased sea surface temperatures and declining sea water pH. To date, most studies have focused on either coral bleaching due to warming oceans or declining calcification due to decreasing oceanic carbonate ion concentrations. Here, through the use of physiology measurements and cDNA microarrays, we show that changes in pH and ocean chemistry consistent with two scenarios put forward by the Intergovernmental Panel on Climate Change (IPCC) drive major changes in gene expression, respiration, photosynthesis and symbiosis of the coral, Acropora millepora, before affects on biomineralisation are apparent at the phenotype level. Under high CO(2) conditions corals at the phenotype level lost over half their Symbiodinium populations, and had a decrease in both photosynthesis and respiration. Changes in gene expression were consistent with metabolic suppression, an increase in oxidative stress, apoptosis and symbiont loss. Other expression patterns demonstrate upregulation of membrane transporters, as well as the regulation of genes involved in membrane cytoskeletal interactions and cytoskeletal remodeling. These widespread changes in gene expression emphasize the need to expand future studies of ocean acidification to include a wider spectrum of cellular processes, many of which may occur before impacts on calcification.

IMPORTANCE OF MACRO- VERSUS MICROSTRUCTURE IN MODULATING LIGHT LEVELS INSIDE CORAL COLONIES(1).

Adjusting the light exposure and capture of their symbiotic photosynthetic dinoflagellates (genus Symbiodinium Freud.) is central to the success of reef-building corals (order Scleractinia) across high spatio-temporal variation in the light environment of coral reefs. We tested the hypothesis that optical properties of tissues in some coral species can provide light management at the tissue scale comparable to light modulation by colony architecture in other species. We compared within-tissue scalar irradiance in two coral species from the same light habitat but with contrasting colony growth forms: branching Stylophora pistillata and massive Lobophyllia corymbosa. Scalar irradiance at the level of the symbionts (2 mm into the coral tissues) were <10% of ambient irradiance and nearly identical for the two species, despite substantially different light environments at the tissue surface. In S. pistillata, light attenuation (90% relative to ambient) was observed predominantly at the colony level as a result of branch-to-branch self-shading, while in L. corymbosa, near-complete light attenuation (97% relative to ambient) was occurring due to tissue optical properties. The latter could be explained partly by differences in photosynthetic pigment content in the symbiont cells and pigmentation in the coral host tissue. Our results demonstrate that different strategies of light modulation at colony, polyp, and cellular levels by contrasting morphologies are equally effective in achieving favorable irradiances at the level of coral photosymbionts.

Coral skeletons defend against ultraviolet radiation.

BACKGROUND: Many coral reef organisms are photosynthetic or have evolved in tight symbiosis with photosynthetic symbionts. As such, the tissues of reef organisms are often exposed to intense solar radiation in clear tropical waters and have adapted to trap and harness photosynthetically active radiation (PAR). High levels of ultraviolet radiation (UVR) associated with sunlight, however, represent a potential problem in terms of tissue damage. METHODOLOGY/PRINCIPAL FINDINGS: By measuring UVR and PAR reflectance from intact and ground bare coral skeletons we show that the property of calcium carbonate skeletons to absorb downwelling UVR to a significant extent, while reflecting PAR back to the overlying tissue, has biological advantages. We placed cnidarians on top of bare skeletons and a UVR reflective substrate and showed that under ambient UVR levels, UVR transmitted through the tissues of cnidarians placed on top of bare skeletons were four times lower compared to their counterparts placed on a UVR reflective white substrate. In accordance with the lower levels of UVR measured in cnidarians on top of coral skeletons, a similar drop in UVR damage to their DNA was detected. The skeletons emitted absorbed UVR as yellow fluorescence, which allows for safe dissipation of the otherwise harmful radiation. CONCLUSIONS/SIGNIFICANCE: Our study presents a novel defensive role for coral skeletons and reveals that the strong UVR absorbance by the skeleton can contribute to the ability of corals, and potentially other calcifiers, to thrive under UVR levels that are detrimental to most marine life.

Phototropic growth in a reef flat acroporid branching coral species.

Many terrestrial plants form complex morphological structures and will alter these growth patterns in response to light direction. Similarly reef building corals have high morphological variation across coral families, with many species also displaying phenotypic plasticity across environmental gradients. In particular, the colony geometry in branching corals is altered by the frequency, location and direction of branch initiation and growth. This study demonstrates that for the branching species Acropora pulchra, light plays a key role in axial polyp differentiation and therefore axial corallite development--the basis for new branch formation. A. pulchra branches exhibited a directional growth response, with axial corallites only developing when light was available, and towards the incident light. Field experimentation revealed that there was a light intensity threshold of 45 micromol m(-2) s(-1), below which axial corallites would not develop and this response was blue light (408-508 nm) dependent. There was a twofold increase in axial corallite growth above this light intensity threshold and a fourfold increase in axial corallite growth under the blue light treatment. These features of coral branch growth are highly reminiscent of the initiation of phototropic branch growth in terrestrial plants, which is directed by the blue light component of sunlight.