Till Bayer

Symbiodinium Transcriptomes: Genome Insights into the Dinoflagellate Symbionts of Reef-Building Corals

Dinoflagellates are unicellular algae that are ubiquitously abundant in aquatic environments. Species of the genus Symbiodinium form symbiotic relationships with reef-building corals and other marine invertebrates. Despite their ecologic importance, little is known about the genetics of dinoflagellates in general and Symbiodinium in particular. Here, we used 454 sequencing to generate transcriptome data from two Symbiodinium species from different clades (clade A and clade B). With more than 56,000 assembled sequences per species, these data represent the largest transcriptomic resource for dinoflagellates to date. Our results corroborate previous observations that dinoflagellates possess the complete nucleosome machinery. We found a complete set of core histones as well as several H3 variants and H2A.Z in one species. Furthermore, transcriptome analysis points toward a low number of transcription factors in Symbiodinium spp. that also differ in the distribution of DNA-binding domains relative to other eukaryotes. In particular the cold shock domain was predominant among transcription factors. Additionally, we found a high number of antioxidative genes in comparison to non-symbiotic but evolutionary related organisms. These findings might be of relevance in the context of the role that Symbiodinium spp. play as coral symbionts. Our data represent the most comprehensive dinoflagellate EST data set to date. This study provides a comprehensive resource to further analyze the genetic makeup, metabolic capacities, and gene repertoire of Symbiodinium and dinoflagellates. Overall, our findings indicate that Symbiodinium possesses some unique characteristics, in particular the transcriptional regulation in Symbiodinium may differ from the currently known mechanisms of eukaryotic gene regulation.

The Microbiome of the Red Sea Coral Stylophora pistillata Is Dominated by Tissue-Associated Endozoicomonas Bacteria

ABSTRACT Endozoicomonas bacteria were found highly associated with the coral Stylophora pistillata, and these bacteria are also ubiquitously associated with diverse corals worldwide. Novel Endozoicomonas-specific probes revealed that Endozoicomonas bacteria were abundant in the endodermal tissues of S. pistillata and appear to have an intimate relationship with the coral.

Assessing Symbiodinium diversity in scleractinian corals via next-generation sequencing-based genotyping of the ITS2 rDNA region

The persistence of coral reef ecosystems relies on the symbiotic relationship between scleractinian corals and intracellular, photosynthetic dinoflagellates in the genus Symbiodinium. Genetic evidence indicates that these symbionts are biologically diverse and exhibit discrete patterns of environmental and host distribution. This makes the assessment of Symbiodinium diversity critical to understanding the symbiosis ecology of corals. Here, we applied pyrosequencing to the elucidation of Symbiodinium diversity via analysis of the internal transcribed spacer 2 (ITS2) region, a multicopy genetic marker commonly used to analyse Symbiodinium diversity. Replicated data generated from isoclonal Symbiodinium cultures showed that all genomes contained numerous, yet mostly rare, ITS2 sequence variants. Pyrosequencing data were consistent with more traditional denaturing gradient gel electrophoresis (DGGE) approaches to the screening of ITS2 PCR amplifications, where the most common sequences appeared as the most intense bands. Further, we developed an operational taxonomic unit (OTU)‐based pipeline for Symbiodinium ITS2 diversity typing to provisionally resolve ecologically discrete entities from intragenomic variation. A genetic distance cut‐off of 0.03 collapsed intragenomic ITS2 variants of isoclonal cultures into single OTUs. When applied to the analysis of field‐collected coral samples, our analyses confirm that much of the commonly observed Symbiodinium ITS2 diversity can be attributed to intragenomic variation. We conclude that by analysing Symbiodinium populations in an OTU‐based framework, we can improve objectivity, comparability and simplicity when assessing ITS2 diversity in field‐based studies.

Genomes of coral dinoflagellate symbionts highlight evolutionary adaptations conducive to a symbiotic lifestyle

Despite half a century of research, the biology of dinoflagellates remains enigmatic: they defy many functional and genetic traits attributed to typical eukaryotic cells. Genomic approaches to study dinoflagellates are often stymied due to their large, multi-gigabase genomes. Members of the genus Symbiodinium are photosynthetic endosymbionts of stony corals that provide the foundation of coral reef ecosystems. Their smaller genome sizes provide an opportunity to interrogate evolution and functionality of dinoflagellate genomes and endosymbiosis. We sequenced the genome of the ancestral Symbiodinium microadriaticum and compared it to the genomes of the more derived Symbiodinium minutum and Symbiodinium kawagutii and eukaryote model systems as well as transcriptomes from other dinoflagellates. Comparative analyses of genome and transcriptome protein sets show that all dinoflagellates, not only Symbiodinium, possess significantly more transmembrane transporters involved in the exchange of amino acids, lipids, and glycerol than other eukaryotes. Importantly, we find that only Symbiodinium harbor an extensive transporter repertoire associated with the provisioning of carbon and nitrogen. Analyses of these transporters show species-specific expansions, which provides a genomic basis to explain differential compatibilities to an array of hosts and environments, and highlights the putative importance of gene duplications as an evolutionary mechanism in dinoflagellates and Symbiodinium.

Bacterial profiling of White Plague Disease in a comparative coral species framework

Coral reefs are threatened throughout the world. A major factor contributing to their decline is outbreaks and propagation of coral diseases. Due to the complexity of coral-associated microbe communities, little is understood in terms of disease agents, hosts and vectors. It is known that compromised health in corals is correlated with shifts in bacterial assemblages colonizing coral mucus and tissue. However, general disease patterns remain, to a large extent, ambiguous as comparative studies over species, regions, or diseases are scarce. Here, we compare bacterial assemblages of samples from healthy (HH) colonies and such displaying signs of White Plague Disease (WPD) of two different coral species (Pavona duerdeni and Porites lutea) from the same reef in Koh Tao, Thailand, using 16S rRNA gene microarrays. In line with other studies, we found an increase of bacterial diversity in diseased (DD) corals, and a higher abundance of taxa from the families that include known coral pathogens (Alteromonadaceae, Rhodobacteraceae, Vibrionaceae). In our comparative framework analysis, we found differences in microbial assemblages between coral species and coral health states. Notably, patterns of bacterial community structures from HH and DD corals were maintained over species boundaries. Moreover, microbes that differentiated the two coral species did not overlap with microbes that were indicative of HH and DD corals. This suggests that while corals harbor distinct species-specific microbial assemblages, disease-specific bacterial abundance patterns exist that are maintained over coral species boundaries.

In-situ Effects of Eutrophication and Overfishing on Physiology and Bacterial Diversity of the Red Sea Coral Acropora hemprichii

Coral reefs of the Central Red Sea display a high degree of endemism, and are increasingly threatened by anthropogenic effects due to intense local coastal development measures. Overfishing and eutrophication are among the most significant local pressures on these reefs, but there is no information available about their potential effects on the associated microbial community. Therefore, we compared holobiont physiology and 16S-based bacterial communities of tissue and mucus of the hard coral Acropora hemprichii after 1 and 16 weeks of in-situ inorganic nutrient enrichment (via fertilizer diffusion) and/or herbivore exclusion (via caging) in an offshore reef of the Central Red Sea. Simulated eutrophication and/or overfishing treatments did not affect coral physiology with respect to coral respiration rates, chlorophyll a content, zooxanthellae abundance, or δ 15N isotopic signatures. The bacterial community of A. hemprichii was rich and uneven, and diversity increased over time in all treatments. While distinct bacterial species were identified as a consequence of eutrophication, overfishing, or both, two bacterial species that could be classified to the genus Endozoicomonas were consistently abundant and constituted two thirds of bacteria in the coral. Several nitrogen-fixing and denitrifying bacteria were found in the coral specimens that were exposed to experimentally increased nutrients. However, no particular bacterial species was consistently associated with the coral under a given treatment and the single effects of manipulated eutrophication and overfishing could not predict the combined effect. Our data underlines the importance of conducting field studies in a holobiont framework, taking both, physiological and molecular measures into account.

Microbiome structure of the fungid coral Ctenactis echinata aligns with environmental differences

The significance of bacteria for eukaryotic functioning is increasingly recognized. Coral reef ecosystems critically rely on the relationship between coral hosts and their intracellular photosynthetic dinoflagellates, but the role of the associated bacteria remains largely theoretical. Here, we set out to relate coral‐associated bacterial communities of the fungid host species Ctenactis echinata to environmental settings (geographic location, substrate cover, summer/winter, nutrient and suspended matter concentrations) and coral host abundance. We show that bacterial diversity of C. echinata aligns with ecological differences between sites and that coral colonies sampled at the species’ preferred habitats are primarily structured by one bacterial taxon (genus Endozoicomonas) representing more than 60% of all bacteria. In contrast, host microbiomes from lower populated coral habitats are less structured and more diverse. Our study demonstrates that the content and structure of the coral microbiome aligns with environmental differences and denotes habitat adequacy. Availability of a range of coral host habitats might be important for the conservation of distinct microbiome structures and diversity.

Differential sensitivity of coral larvae to natural levels of ultraviolet radiation during the onset of larval competence.

Scleractinian corals are the major builders of the complex structural framework of coral reefs. They live in tropical waters around the globe where they are frequently exposed to potentially harmful ultraviolet radiation (UVR). The eggs and early embryonic stages of some coral species are highly buoyant and remain near the sea surface for prolonged periods of time and may therefore be the most sensitive life stages with respect to UVR. Here, we analysed gene expression changes in five developmental stages of the Caribbean coral Montastraea faveolata to natural levels of UVR using high‐density cDNA microarrays (10 930 clones). We found that larvae exhibit low sensitivity to natural levels of UVR during early development as reflected by comparatively few transcriptomic changes in response to UVR. However, we identified a time window of high UVR sensitivity that coincides with the motile planula stage and the onset of larval competence. These processes have been shown to be affected by UVR exposure, and the transcriptional changes we identified explain these observations well. Our analysis of differentially expressed genes indicates that UVR alters the expression of genes associated with stress response, the endoplasmic reticulum, Ca2+ homoeostasis, development and apoptosis during the motile planula stage and affects the expression of neurogenesis‐related genes that are linked to swimming and settlement behaviour at later stages. Taken together, our study provides further data on the impact of natural levels of UVR on coral larvae. Furthermore, our results might allow a better prediction of settlement and recruitment rates after coral spawning events if UVR climate data are taken into account.

The role of the segmentation gene hairy in Tribolium

Hairy stripes in Tribolium are generated during blastoderm and germ band extension, but a direct role for Tc-h in trunk segmentation was not found. We have studied here several aspects of hairy function and expression in Tribolium, to further elucidate its role. First, we show that there is no functional redundancy with other hairy paralogues in Tribolium. Second, we cloned the hairy orthologue from Tribolium confusum and show that its expression mimics that of Tribolium castaneum, implying that stripe expression should be functional in some way. Third, we show that the dynamics of stripe formation in the growth zone is not compatible with an oscillatory mechanism comparable to the one driving the expression of hairy homologues in vertebrates. Fourth, we use parental RNAi experiments to study Tc-h function and we find that mandible and labium are particularly sensitive to loss of Tc-h, reminiscent of a pair-rule function in the head region. In addition, lack of Tc-h leads to cell death in the gnathal region at later embryonic stages, resulting in a detachment of the head. Cell death patterns are also altered in the midline. Finally, we have analysed the effect of Tc-h knockdown on two of the target genes of hairy in Drosophila, namely fushi tarazu and paired. We find that the trunk expression of Tc-h is required to regulate Tc-ftz, although Tc-ftz is itself also not required for trunk segmentation in Tribolium. Our results imply that there is considerable divergence in hairy function between Tribolium and Drosophila.

Metatranscriptome analysis of the reef-building coral Orbicella faveolata indicates holobiont response to coral disease

White Plague Disease (WPD) is implicated in coral reef decline in the Caribbean and is characterized by microbial community shifts in coral mucus and tissue. Studies thus far have focused on assessing microbial communities or the identification of specific pathogens, yet few have addressed holobiont response across metaorganism compartments in coral disease. Here, we report on the first metatranscriptomic assessment of the coral host, algal symbiont, and microbial compartment in order to survey holobiont structure and function in healthy and diseased samples from Orbicella faveolata collected at reef sites off Puerto Rico. Our data indicate metaorganism-wide as well as compartment-specific responses to WPD. Gene expression changes in the diseased coral host involved proteins playing a role in innate immunity, cytoskeletal integrity, cell adhesion, oxidative stress, chemical defense, and retroelements. In contrast, the algal symbiont showed comparatively few expression changes, but of large magnitude, of genes related to stress, photosynthesis, and metal transport. Concordant with the coral host response, the bacterial compartment showed increased abundance of heat shock proteins, genes related to oxidative stress, DNA repair, and potential retroelement activity. Importantly, analysis of the expressed bacterial gene functions establishes the participation of multiple bacterial families in WPD pathogenesis and also suggests a possible involvement of viruses and/or phages in structuring the bacterial assemblage. In this study, we implement an experimental approach to partition the coral holobiont and resolve compartment- and taxa-specific responses in order to understand metaorganism function in coral disease.