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The algal endosymbiont Durusdinium trenchii enhances the resilience of coral reefs under thermal stress. D. trenchii can live freely or in endosymbiosis, and the analysis of genetic markers suggests that this species has undergone whole-genome duplication (WGD). However, the evolutionary mechanisms that underpin the thermotolerance of this species are largely unknown. Here, we present genome assemblies for two D. trenchii isolates, confirm WGD in these taxa, and examine how selection has shaped the duplicated genome regions using gene expression data. We assess how the free-living versus endosymbiotic lifestyles have contributed to the retention and divergence of duplicated genes, and how these processes have enhanced the thermotolerance of D. trenchii . Our combined results suggest that lifestyle is the driver of post-WGD evolution in D. trenchii , with the free-living phase being the most important, followed by endosymbiosis. Adaptations to both lifestyles likely enabled D. trenchii to provide enhanced thermal stress protection to the host coral.

The intracellular coral-dinoflagellate symbiosis is the engine that underpins the success of coral reefs, one of the most diverse ecosystems on the planet. However, the breakdown of the symbiosis and the loss of the microalgal symbiont (i.e. coral bleaching) due to environmental changes are resulting in the rapid degradation of coral reefs globally. There is an urgent need to understand the cellular physiology of coral bleaching at the mechanistic level to help develop solutions to mitigate the coral reef crisis. Here, at an unprecedented scope, we present novel models that integrate putative mechanisms of coral bleaching within a common framework according to the triggers (initiators of bleaching, e.g. heat, cold, light stress, hypoxia, hyposalinity), cascades (cellular pathways, e.g. photoinhibition, unfolded protein response, nitric oxide), and endpoints (mechanisms of symbiont loss, e.g. apoptosis, necrosis, exocytosis/vomocytosis). The models are supported by direct evidence from cnidarian systems, and indirectly through comparative evolutionary analyses from non-cnidarian systems. With this approach, new putative mechanisms have been established within and between cascades initiated by different bleaching triggers. In particular, the models provide new insights into the poorly understood connections between bleaching cascades and endpoints and highlight the role of a new mechanism of symbiont loss, i.e. 'symbiolysosomal digestion', which is different from symbiophagy. This review also increases the approachability of bleaching physiology for specialists and non-specialists by mapping the vast landscape of bleaching mechanisms in an atlas of comprehensible and detailed mechanistic models. We then discuss major knowledge gaps and how future research may improve the understanding of the connections between the diverse cascade of cellular pathways and the mechanisms of symbiont loss (endpoints)., (© 2024 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.)

Dinoflagellates in the family Symbiodiniaceae are taxonomically diverse, predominantly symbiotic lineages that are well-known for their association with corals. The ancestor of these taxa is believed to have been free-living. The establishment of symbiosis (i.e. symbiogenesis) is hypothesized to have occurred multiple times during Symbiodiniaceae evolution, but its impact on genome evolution of these taxa is largely unknown. Among Symbiodiniaceae, the genus Effrenium is a free-living lineage that is phylogenetically positioned between two robustly supported groups of genera within which symbiotic taxa have emerged. The apparent lack of symbiogenesis in Effrenium suggests that the ancestral features of Symbiodiniaceae may have been retained in this lineage. Here, we present de novo assembled genomes (1.2-1.9 Gbp in size) and transcriptome data from three isolates of Effrenium voratum and conduct a comparative analysis that includes 16 Symbiodiniaceae taxa and the other dinoflagellates. Surprisingly, we find that genome reduction, which is often associated with a symbiotic lifestyle, predates the origin of Symbiodiniaceae. The free-living lifestyle distinguishes Effrenium from symbiotic Symbiodiniaceae vis-à-vis their longer introns, more-extensive mRNA editing, fewer (~30%) lineage-specific gene sets, and lower (~10%) level of pseudogenization. These results demonstrate how genome reduction and the adaptation to distinct lifestyles intersect to drive diversification and genome evolution of Symbiodiniaceae., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Within microeukaryotes, genetic variation and functional variation sometimes accumulate more quickly than morphological differences. To understand the evolutionary history and ecology of such lineages, it is key to examine diversity at multiple levels of organization. In the dinoflagellate family Symbiodiniaceae, which can form endosymbioses with cnidarians ( e.g ., corals, octocorals, sea anemones, jellyfish), other marine invertebrates ( e.g. , sponges, molluscs, flatworms), and protists ( e.g ., foraminifera), molecular data have been used extensively over the past three decades to describe phenotypes and to make evolutionary and ecological inferences. Despite advances in Symbiodiniaceae genomics, a lack of consensus among researchers with respect to interpreting genetic data has slowed progress in the field and acted as a barrier to reconciling observations. Here, we identify key challenges regarding the assessment and interpretation of Symbiodiniaceae genetic diversity across three levels: species, populations, and communities. We summarize areas of agreement and highlight techniques and approaches that are broadly accepted. In areas where debate remains, we identify unresolved issues and discuss technologies and approaches that can help to fill knowledge gaps related to genetic and phenotypic diversity. We also discuss ways to stimulate progress, in particular by fostering a more inclusive and collaborative research community. We hope that this perspective will inspire and accelerate coral reef science by serving as a resource to those designing experiments, publishing research, and applying for funding related to Symbiodiniaceae and their symbiotic partnerships., Competing Interests: Anastazia T. Banaszak and James Davis Reimer are Academic Editors for PeerJ., (© 2023 Davies et al.)

Stony coral tissue loss disease (SCTLD) has been causing significant whole colony mortality on reefs in Florida and the Caribbean. The cause of SCTLD remains unknown, with the limited concurrence of SCTLD-associated bacteria among studies. We conducted a meta-analysis of 16S ribosomal RNA gene datasets generated by 16 field and laboratory SCTLD studies to find consistent bacteria associated with SCTLD across disease zones (vulnerable, endemic, and epidemic), coral species, coral compartments (mucus, tissue, and skeleton), and colony health states (apparently healthy colony tissue (AH), and unaffected (DU) and lesion (DL) tissue from diseased colonies). We also evaluated bacteria in seawater and sediment, which may be sources of SCTLD transmission. Although AH colonies in endemic and epidemic zones harbor bacteria associated with SCTLD lesions, and aquaria and field samples had distinct microbial compositions, there were still clear differences in the microbial composition among AH, DU, and DL in the combined dataset. Alpha-diversity between AH and DL was not different; however, DU showed increased alpha-diversity compared to AH, indicating that, prior to lesion formation, corals may undergo a disturbance to the microbiome. This disturbance may be driven by Flavobacteriales, which were especially enriched in DU. In DL, Rhodobacterales and Peptostreptococcales-Tissierellales were prominent in structuring microbial interactions. We also predict an enrichment of an alpha-toxin in DL samples which is typically found in Clostridia. We provide a consensus of SCTLD-associated bacteria prior to and during lesion formation and identify how these taxa vary across studies, coral species, coral compartments, seawater, and sediment., (© 2023. The Author(s).)

The global impacts of climate change are evident in every marine ecosystem. On coral reefs, mass coral bleaching and mortality have emerged as ubiquitous responses to ocean warming, yet one of the greatest challenges of this epiphenomenon is linking information across scientific disciplines and spatial and temporal scales. Here we review some of the seminal and recent coral-bleaching discoveries from an ecological, physiological, and molecular perspective. We also evaluate which data and processes can improve predictive models and provide a conceptual framework that integrates measurements across biological scales. Taking an integrative approach across biological and spatial scales, using for example hierarchical models to estimate major coral-reef processes, will not only rapidly advance coral-reef science but will also provide necessary information to guide decision-making and conservation efforts. To conserve reefs, we encourage implementing mesoscale sanctuaries (thousands of km 2 ) that transcend national boundaries. Such networks of protected reefs will provide reef connectivity, through larval dispersal that transverse thermal environments, and genotypic repositories that may become essential units of selection for environmentally diverse locations. Together, multinational networks may be the best chance corals have to persist through climate change, while humanity struggles to reduce emissions of greenhouse gases to net zero., (© 2022 The Authors. Global Change Biology published by John Wiley & Sons Ltd.)

Genetic signatures caused by demographic and adaptive processes during past climatic shifts can inform predictions of species' responses to anthropogenic climate change. To identify these signatures in Acropora tenuis , a reef-building coral threatened by global warming, we first assembled the genome from long reads and then used shallow whole-genome resequencing of 150 colonies from the central inshore Great Barrier Reef to inform population genomic analyses. We identify population structure in the host that reflects a Pleistocene split, whereas photosymbiont differences between reefs most likely reflect contemporary (Holocene) conditions. Signatures of selection in the host were associated with genes linked to diverse processes including osmotic regulation, skeletal development, and the establishment and maintenance of symbiosis. Our results suggest that adaptation to post-glacial climate change in A. tenuis has involved selection on many genes, while differences in symbiont specificity between reefs appear to be unrelated to host population structure., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Reef-building corals depend upon a nutritional endosymbiosis with photosynthetic dinoflagellates of the family Symbiodiniaceae for the majority of their energetic needs. While this mutualistic relationship is impacted by numerous stressors, warming oceans are a predominant threat to coral reefs, placing the future of the world's reefs in peril. Some Symbiodiniaceae species exhibit tolerance to thermal stress, but the in hospite symbiont response to thermal stress is underexplored. To describe the underpinnings of symbiosis and heat stress response, we compared in hospite and free-living transcriptomes of Durusdinium trenchii, a pan-tropical heat-tolerant Symbiodiniaceae species, under stable temperature conditions and acute hyperthermal stress. We discovered that symbiotic state was a larger driver of the transcriptional landscape than heat stress. The majority of differentially expressed transcripts between in hospite and free-living cells were downregulated, suggesting the in hospite condition is associated with the shutdown of numerous processes uniquely required for a free-living lifestyle. In the free-living state, we identified enrichment for numerous cell signalling pathways and other functions related to detecting and responding to a changing environment, as well as transcripts relating to mitosis, meiosis, and motility. In contrast, in hospite cells exhibited enhanced transcriptional activity for photosynthesis and carbohydrate transport as well as chromatin modifications and a disrupted circadian clock. Hyperthermal stress induced drastic alteration of transcriptional activity in hospite, suggesting symbiotic engagement with the host elicited an exacerbated stress response when compared to free-living D. trenchii. Altogether, the dramatic differences in gene expression between in hospite and free-living D. trenchii indicate the importance of considering symbiotic state in investigations of symbiosis and hyperthermal stress in Symbiodiniaceae., (© 2019 John Wiley & Sons Ltd.)

Reef corals and sea anemones form symbioses with unicellular symbiotic dinoflagellates. The molecular circumventions that underlie the successful intracellular colonization of hosts by symbionts are still largely unknown. We conducted proteomic analyses to determine molecular differences of Exaiptasia pallida anemones colonized by physiologically different symbiont species, in comparison with symbiont-free (aposymbiotic) anemones. We compared one homologous species, Symbiodinium linucheae , that is natively associated with the clonal Exaiptasia strain (CC7) to another heterologous species, Durusdinium trenchii , a thermally tolerant species that colonizes numerous coral species. This approach allowed the discovery of a core set of host genes that are differentially regulated as a function of symbiosis regardless of symbiont species. The findings revealed that symbiont colonization at higher densities requires circumvention of the host cellular immunological response, enhancement of ammonium regulation, and suppression of phagocytosis after a host cell in colonized. Furthermore, the heterologous symbionts failed to duplicate the same level of homologous colonization within the host, evidenced by substantially lower symbiont densities. This reduced colonization of D. trenchii correlated with its inability to circumvent key host systems including autophagy-suppressing modulators, cytoskeletal alteration, and isomerase activity. The larger capability of host molecular circumvention by homologous symbionts could be the result of a longer evolutionary history of host/symbiont interactions, which translates into a more finely tuned symbiosis. These findings are of great importance within the context of the response of reef corals to climate change since it has been suggested that coral may acclimatize to ocean warming by changing their dominant symbiont species.

Accelerating anthropogenic climate change threatens to destroy coral reefs worldwide through the processes of bleaching and disease. These major contributors to coral mortality are both closely linked with thermal stress intensified by anthropogenic climate change. Disease outbreaks typically follow bleaching events, but a direct positive linkage between bleaching and disease has been debated. By tracking 152 individual coral ramets through the 2014 mass bleaching in a South Florida coral restoration nursery, we revealed a highly significant negative correlation between bleaching and disease in the Caribbean staghorn coral, Acropora cervicornis . To explain these results, we propose a mechanism for transient immunological protection through coral bleaching: removal of Symbiodinium during bleaching may also temporarily eliminate suppressive symbiont modulation of host immunological function. We contextualize this hypothesis within an ecological perspective in order to generate testable predictions for future investigation., Competing Interests: Mauricio Rodriguez-Lanetty is an Academic Editor for PeerJ.

Corals host diverse microbial communities that are involved in acclimatization, pathogen defense, and nutrient cycling. Surveys of coral-associated microbes have been particularly directed toward Symbiodinium and bacteria. However, a holistic understanding of the total microbiome has been hindered by a lack of analyses bridging taxonomically disparate groups. Using high-throughput amplicon sequencing, we simultaneously characterized the Symbiodinium , bacterial, and fungal communities associated with the Caribbean coral Siderastrea siderea collected from two depths (17 and 27 m) on Conch reef in the Florida Keys. S. siderea hosted an exceptionally diverse Symbiodinium community, structured differently between sampled depth habitats. While dominated at 27 m by a Symbiodinium belonging to clade C, at 17 m S. siderea primarily hosted a mixture of clade B types. Most fungal operational taxonomic units were distantly related to available reference sequences, indicating the presence of a high degree of fungal novelty within the S. siderea holobiont and a lack of knowledge on the diversity of fungi on coral reefs. Network analysis showed that co-occurrence patterns in the S. siderea holobiont were prevalent among bacteria, however, also detected between fungi and bacteria. Overall, our data show a drastic shift in the associated Symbiodinium community between depths on Conch Reef, which might indicate that alteration in this community is an important mechanism facilitating local physiological adaptation of the S. siderea holobiont. In contrast, bacterial and fungal communities were not structured differently between depth habitats., Competing Interests: Mauricio Rodriguez-Lanetty is an Academic Editor for PeerJ. Trevor Graff and William Todd are employees of NASA Johnson Space Center.

Examination of host-microbe interactions in early diverging metazoans, such as cnidarians, is of great interest from an evolutionary perspective to understand how host-microbial consortia have evolved. To address this problem, we analyzed whether the bacterial community associated with the cosmopolitan and model sea anemone Exaiptasia pallida shows specific patterns across worldwide populations ranging from the Caribbean Sea, and the Atlantic and Pacific oceans. By comparing sequences of the V1-V3 hypervariable regions of the bacterial 16S rRNA gene, we revealed that anemones host a complex and diverse microbial community. When examined at the phylum level, bacterial diversity and abundance associated with E. pallida are broadly conserved across geographic space with samples, containing largely Proteobacteria and Bacteroides. However, the species-level makeup within these phyla differs drastically across space suggesting a high-level core microbiome with local adaptation of the constituents. Indeed, no bacterial OTU was ubiquitously found in all anemones samples. We also revealed changes in the microbial community structure after rearing anemone specimens in captivity within a period of four months. Furthermore, the variation in bacterial community assemblages across geographical locations did not correlate with the composition of microalgal Symbiodinium symbionts. Our findings contrast with the postulation that cnidarian hosts might actively select and maintain species-specific microbial communities that could have resulted from an intimate co-evolution process. The fact that E. pallida is likely an introduced species in most sampled localities suggests that this microbial turnover is a relatively rapid process. Our findings suggest that environmental settings, not host specificity, seem to dictate bacterial community structure associated with this sea anemone. More than maintaining a specific composition of bacterial species some cnidarians associate with a wide range of bacterial species as long as they provide the same physiological benefits towards the maintenance of a healthy host. The examination of the previously uncharacterized bacterial community associated with the cnidarian sea anemone model E. pallida is the first global-scale study of its kind., Competing Interests: Mauricio Rodriguez-Lanetty is an Academic Editor for PeerJ.

Cnidarians, in general, are long-lived organisms and hence may repeatedly encounter common pathogens during their lifespans. It remains unknown whether these early diverging animals possess some type of immunological reaction that strengthens the defense response upon repeated infections, such as that described in more evolutionary derived organisms. Here we show results that sea anemones that had previously encountered a pathogen under sub-lethal conditions had a higher survivorship during a subsequently lethal challenge than naïve anemones that encountered the pathogen for the first time. Anemones subjected to the lethal challenge two and four weeks after the sub-lethal exposure presented seven- and five-fold increases in survival, respectively, compared to the naïve anemones. However, anemones challenged six weeks after the sub-lethal exposure showed no increase in survivorship. We argue that this short-lasting priming of the defense response could be ecologically relevant if pathogen encounters are restricted to short seasons characterized by high stress. Furthermore, we discovered significant changes in proteomic profiles between naïve sea anemones and those primed after pathogen exposure suggesting a clear molecular signature associated with immunological priming in cnidarians. Our findings reveal that immunological priming may have evolved much earlier in the tree of life than previously thought.

The functional role of the bacterial organisms in the reef ecosystem and their contribution to the coral well-being remain largely unclear. The first step in addressing this gap of knowledge relies on in-depth characterization of the coral microbial community and its changes in diversity across coral species, space and time. In this study, we focused on the exploration of microbial community assemblages associated with an ecologically important Caribbean scleractinian coral, Porites astreoides, using Illumina high-throughput sequencing of the V5 fragment of 16S rRNA gene. We collected data from a large set of biological replicates, allowing us to detect patterns of geographical structure and resolve co-occurrence patterns using network analyses. The taxonomic analysis of the resolved diversity showed consistent and dominant presence of two OTUs affiliated with the order Oceanospirillales, which corroborates a specific pattern of bacterial association emerging for this coral species and for many other corals within the genus Porites. We argue that this specific association might indicate a symbiotic association with the adult coral partner. Furthermore, we identified a highly diverse rare bacterial 'biosphere' (725 OTUs) also living along with the dominant bacterial symbionts, but the assemblage of this biosphere is significantly structured along the geographical scale. We further discuss that some of these rare bacterial members show significant association with other members of the community reflecting the complexity of the networked consortia within the coral holobiont., (© 2013 John Wiley & Sons Ltd.)

The impact of disease outbreaks on coral physiology represents an increasing concern for the fitness and resilience of reef ecosystems. Predicting the tolerance of corals to disease relies on an understanding of the coral immune response to pathogenic interactions. This study explored the transcriptional response of two putative immune genes (c3 and c-type lectin) and one stress response gene (hsp70) in the reef building coral, Acropora millepora challenged for 48 hours with bacterial strains, Vibrio coralliilyticus and Alteromonas sp. at concentrations of 10(6) cells ml(-1). Coral fragments challenged with V. coralliilyticus appeared healthy while fragments challenged with Alteromonas sp. showed signs of tissue lesions after 48 hr. Coral-associated bacterial community profiles assessed using denaturing gradient gel electrophoresis changed after challenge by both bacterial strains with the Alteromonas sp. treatment demonstrating the greatest community shift. Transcriptional profiles of c3 and hsp70 increased at 24 hours and correlated with disease signs in the Alteromonas sp. treatment. The expression of hsp70 also showed a significant increase in V. coralliilyticus inoculated corals at 24 h suggesting that even in the absence of disease signs, the microbial inoculum activated a stress response in the coral. C-type lectin did not show a response to any of the bacterial treatments. Increase in gene expression of c3 and hsp70 in corals showing signs of disease indicates their potential involvement in immune and stress response to microbial challenges.

Background: Ecosystems worldwide are suffering the consequences of anthropogenic impact. The diverse ecosystem of coral reefs, for example, are globally threatened by increases in sea surface temperatures due to global warming. Studies to date have focused on determining genetic diversity, the sequence variability of genes in a species, as a proxy to estimate and predict the potential adaptive response of coral populations to environmental changes linked to climate changes. However, the examination of natural gene expression variation has received less attention. This variation has been implicated as an important factor in evolutionary processes, upon which natural selection can act., Results: We acclimatized coral nubbins from six colonies of the reef-building coral Acropora millepora to a common garden in Heron Island (Great Barrier Reef, GBR) for a period of four weeks to remove any site-specific environmental effects on the physiology of the coral nubbins. By using a cDNA microarray platform, we detected a high level of gene expression variation, with 17% (488) of the unigenes differentially expressed across coral nubbins of the six colonies (jsFDR-corrected, p < 0.01). Among the main categories of biological processes found differentially expressed were transport, translation, response to stimulus, oxidation-reduction processes, and apoptosis. We found that the transcriptional profiles did not correspond to the genotype of the colony characterized using either an intron of the carbonic anhydrase gene or microsatellite loci markers., Conclusion: Our results provide evidence of the high inter-colony variation in A. millepora at the transcriptomic level grown under a common garden and without a correspondence with genotypic identity. This finding brings to our attention the importance of taking into account natural variation between reef corals when assessing experimental gene expression differences. The high transcriptional variation detected in this study is interpreted and discussed within the context of adaptive potential and phenotypic plasticity of reef corals. Whether this variation will allow coral reefs to survive to current challenges remains unknown.

Discovering how corals can adjust their thermal sensitivity in the context of global climate change is important in understanding the long-term persistence of coral reefs. In this study, we showed that short-term preconditioning to higher temperatures, 3°C below the experimentally determined bleaching threshold, for a period of 10 days provides thermal tolerance for the symbiosis stability between the scleractinian coral, Acropora millepora and Symbiodinium. Based on genotypic analysis, our results indicate that the acclimatization of this coral species to thermal stress does not come down to simple changes in Symbiodinium and/or the bacterial communities that associate with reef-building corals. This suggests that the physiological plasticity of the host and/or symbiotic components appears to play an important role in responding to ocean warming. The further study of host and symbiont physiology, both of Symbiodinium and prokaryotes, is of paramount importance in the context of global climate change, as mechanisms for rapid holobiont acclimatization will become increasingly important to the long-standing persistence of coral reefs.

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.

The acclimatization capacity of corals is a critical consideration in the persistence of coral reefs under stresses imposed by global climate change. The stress history of corals plays a role in subsequent response to heat stress, but the transcriptomic changes associated with these plastic changes have not been previously explored. In order to identify host transcriptomic changes associated with acquired thermal tolerance in the scleractinian coral Acropora millepora, corals preconditioned to a sub-lethal temperature of 3°C below bleaching threshold temperature were compared to both non-preconditioned corals and untreated controls using a cDNA microarray platform. After eight days of hyperthermal challenge, conditions under which non-preconditioned corals bleached and preconditioned corals (thermal-tolerant) maintained Symbiodinium density, a clear differentiation in the transcriptional profiles was revealed among the condition examined. Among these changes, nine differentially expressed genes separated preconditioned corals from non-preconditioned corals, with 42 genes differentially expressed between control and preconditioned treatments, and 70 genes between non-preconditioned corals and controls. Differentially expressed genes included components of an apoptotic signaling cascade, which suggest the inhibition of apoptosis in preconditioned corals. Additionally, lectins and genes involved in response to oxidative stress were also detected. One dominant pattern was the apparent tuning of gene expression observed between preconditioned and non-preconditioned treatments; that is, differences in expression magnitude were more apparent than differences in the identity of genes differentially expressed. Our work revealed a transcriptomic signature underlying the tolerance associated with coral thermal history, and suggests that understanding the molecular mechanisms behind physiological acclimatization would be critical for the modeling of reefs in impending climate change scenarios.

Unicellular photosynthetic algae (dinoflagellate) from the genus Symbiodinium live in mutualistic symbiosis with reef-building corals. Cultured Symbiodinium sp. (clade C) were exposed to a range of environmental stresses that included elevated temperatures (29°C and 32°C) under high (100 μmol quanta m(-2) s(-1) Photosynthetic Active Radiation) and low (10 μmol quanta m(-2) s(-1)) irradiances. Using real-time RT-PCR the stability of expression for the nine selected putative housekeeping genes (HKGs) was tested. The most stable expression pattern was identified for cyclophilin and S-adenosyl methionine synthetase (SAM) followed by S4 ribosomal protein (Rp-S4), Calmodulin (Cal), and Cytochrome oxidase subunit 1 (Cox), respectively. Thermal stress alone resulted in the highest expression stability for Rp-S4 and SAM, with a minimum of two reference genes required for data normalization. For Symbiodinium exposed to both, light and thermal stresses, at least five reference genes were recommended by geNorm analysis. In parallel, the expression of Hsp90 for Symbiodinium in culture and in symbiosis within coral host (Acropora millepora) was evaluated using the most stable HKGs. Our results revealed a drop in Hsp90 expression after an 18 h-period and a 24 h-period of exposure to elevated temperatures indicating the similar Hsp90 expression profile in symbiotic and non-symbiotic environments. This study provides the first list of the HKGs and will provide a useful reference in future gene expression studies in symbiotic dinoflagellates.

High-resolution melting (HRM) analysis is a closed-tube, rapid and sensitive technique able to detect DNA variations. It relies on the fluorescence melting curves that are obtained from the transition of double-stranded DNA (dsDNA) to single-stranded DNA (ssDNA) as a result of temperature increase. In this study, we evaluated the effectiveness of HRM as a tool to rapidly and precisely genotype monotypic Symbiodinium populations using the internal transcribed spacer, region 2, ribosomal DNA (ITS2 rDNA). For this, Symbiodinium denaturing gradient gel electrophoresis (DGGE) profiles, where gel bands were excised and sequenced, were compared to HRM genotypes. Results showed that twenty cultures were correctly genotyped in <2 h using HRM analysis with a percentage of confidence >90%. Limitations of the technique were also investigated. Unlike other techniques used for genotyping Symbiodinium, such as DGGE and other fingerprint profiles, HRM is a technique of great advantage for field coral reef ecologists and physiologists as no expertise in advanced molecular methods is required., (© 2010 Blackwell Publishing Ltd.)

Circadian regulation of plant-animal endosymbioses is complicated by a diversity of internal and external cues. Here, we show that stress-related genes in corals are coupled to the circadian clock, anticipating major changes in the intracellular milieu. In this regard, numerous chaperones are "hard-wired" to the clock, effectively preparing the coral for the consequences of oxidative protein damage imposed by symbiont photosynthesis (when O(2) > 250% saturation), including synexpression of antioxidant genes being light-gated. Conversely, central metabolism appears to be regulated by the hypoxia-inducible factor system in coral. These results reveal the complexity of endosymbiosis as well as the plasticity regulation downstream of the circadian clock.

Most of the work on the impact of elevated temperature and light on Symbiodinium-invertebrate symbioses have focused primarily on how the photosynthetic (algal) partner is impacted. Understanding how the same stresses affect the invertebrate host, however, is in its infancy. In this study, we re-examined the direct effect of elevated temperatures on the invertebrate host exploring the early transcriptional response of aposymbiotic (without algal symbionts) coral larvae. The temperatures tested in the experimental design were 24 degrees C (ambient seawater temperature), 28 degrees C and 31 degrees C; and the sampling points were 3 and 10 h after temperature exposure. We explored relative changes in transcription using a cDNA microarray constructed for the scleractinian coral, Acropora millepora, and containing 18 142 expressed sequence tag (EST) clones/8386 unigenes. Our study identified 29 genes that were significantly up- and down-regulated when A. millepora coral larvae were exposed to elevated temperatures. Down-regulation of several key components of DNA/RNA metabolism was detected implying inhibition of general cellular processes. The down-regulation of protein synthesis, however, was not simple and random, which suggested that the stress response was a more complicated adjustment of cellular metabolism. We identified four significant outcomes during the very early hours of the transcriptional response to hyperthermal stress in coral larvae. First, the expression of heat-shock proteins increased rapidly (within 3 h) in response to hyperthermal stress. Second, a fluorescent protein homologue, DsRed-type FP, decreased its expression in response to elevated temperature reinforcing a potential role as a molecular marker for monitoring hyperthermal stress in nature. Third, the down-regulation of a coral mannose-binding C-type lectin under elevated temperature suggests that heat stress might compromise some components of the coral immune defence and therefore might bring about susceptibility to pathogenic diseases. And last, genes involved in protecting cells against oxidative stress showed little response at the early hours to heat stress, supporting the proposal that up-regulation of cnidarian host oxidative stress genes may require reactive oxygen species generated by stressed algal symbionts.

Elevated temperature and solar radiation, including ultraviolet radiation, are now recognized as the primary environmental stresses that lead to mass cnidarian bleaching. This study takes a functional genomics approach to identifying genes that change expression soon after exposure to these stressors in the temperate sea anemone Anthopleura elegantissima that harbors Symbiodinium, the same genus of symbionts found in reef-building corals. Symbiotic anemones were subjected to elevated temperature or UV over a 24 h period. cDNA from these animals was hybridized to a 10,000-feature cDNA microarray of A. elegantissima. Overall 2.7% of the 10,000 features were found to be differentially expressed as a function of temperature or UV stress. Of the 86 features sequenced, 45% displayed significant homology to sequences in GenBank. There are 27 features that were differentially expressed in both stress conditions. Gene ontology analysis placed the differentially expressed genes in a wide range of categories including cytoskeleton organization and biogenesis, protein biosynthesis, cell proliferation, apoptosis and transport. This suggests that the early stress response to elevated temperature and UV involves essentially all aspects of host cellular regulation and machinery and that downstream cnidarian bleaching is a complex cellular response in host tissues., (2008 Elsevier Inc. All rights reserved.)

Corals provide the foundation of important tropical reef ecosystems but are in global decline for multiple reasons, including climate change. Coral health depends on a fragile partnership with intracellular dinoflagellate symbionts. We argue here that progress in understanding coral biology requires intensive study of the cellular processes underlying this symbiosis. Such study will inform us on how the coral symbiosis will be affected by climate change, mechanisms driving coral bleaching and disease, and the coevolution of this symbiosis in the context of other host-microbe interactions. Drawing lessons from the broader history of molecular and cell biology and the study of other host-microbe interactions, we argue that a model-systems approach is essential for making effective progress in understanding coral cell biology.

Research in gene function using Quantitative Reverse Transcription PCR (q-RT-PCR) and microarray approaches are emerging and just about to explode in the field of coral and cnidarian biology. These approaches are showing the great potential to significantly advance our understanding of how corals respond to abiotic and biotic stresses, and how host cnidarians/dinoflagellates symbioses are maintained and regulated. With these genomic advances, however, new analytical challenges are also emerging, such as the normalization of gene expression data derived from q-RT-PCR. In this study, an effective analytical method is introduced to identify candidate housekeeping genes (HKG) from a sea anemone (Anthopleura elegantissima) cDNA microarray platform that can be used as internal control genes to normalize q-RT-PCR gene expression data. It is shown that the identified HKGs were stable among the experimental conditions tested in this study. The three most stables genes identified, in term of gene expression, were beta-actin, ribosomal protein L12, and a Poly(a) binding protein. The applications of these HKGs in other cnidarian systems are further discussed.

Background: Cnidarian-dinoflagellate intracellular symbioses are one of the most important mutualisms in the marine environment. They form the trophic and structural foundation of coral reef ecosystems, and have played a key role in the evolutionary radiation and biodiversity of cnidarian species. Despite the prevalence of these symbioses, we still know very little about the molecular modulators that initiate, regulate, and maintain the interaction between these two different biological entities. In this study, we conducted a comparative host anemone transcriptome analysis using a cDNA microarray platform to identify genes involved in cnidarian-algal symbiosis., Results: We detected statistically significant differences in host gene expression profiles between sea anemones (Anthopleura elegantissima) in a symbiotic and non-symbiotic state. The group of genes, whose expression is altered, is diverse, suggesting that the molecular regulation of the symbiosis is governed by changes in multiple cellular processes. In the context of cnidarian-dinoflagellate symbioses, we discuss pivotal host gene expression changes involved in lipid metabolism, cell adhesion, cell proliferation, apoptosis, and oxidative stress., Conclusion: Our data do not support the existence of symbiosis-specific genes involved in controlling and regulating the symbiosis. Instead, it appears that the symbiosis is maintained by altering expression of existing genes involved in vital cellular processes. Specifically, the finding of key genes involved in cell cycle progression and apoptosis have led us to hypothesize that a suppression of apoptosis, together with a deregulation of the host cell cycle, create a platform that might be necessary for symbiont and/or symbiont-containing host cell survival. This first comprehensive molecular examination of the cnidarian-dinoflagellate associations provides critical insights into the maintenance and regulation of the symbiosis.

Symbiodinium-like dinoflagellates have been shown to be a diverse group of endosymbionts that associate mutualistically with many kinds of coral reef dwellers, including cnidarians, molluscs, and protists. A high number of genetically ITS types of symbionts have been reported to date. However, whether these recently identified Symbiodinium ITS types indeed represent independent evolutionary lineages is still unsettled. Here I tested the null hypothesis that certain group of symbionts sampled from different geographical locations are derived from a single evolutionary lineage using a nested clade analysis (NCA). I analyzed a total of 174 ITS1 sequences from GenBank and pooled them into 74 ITS1 distinct haplotypes. Using these haplotypes, the statistical parsimony criterion produced 23 independent network trees, each one corresponding to a genetically independent evolving lineage. Some of these lineages revealed certain degree of specificity with some host groups at least at the phylum level. Within the previously described 28S-rDNA phylotype A, five ITS1 lineages were resolved. Phylotypes B and C resolved each in two ITS1 lineages. The highest ITS1 symbiont diversity was observed within the phylotype F, in which 11 lineages were resolved. Moreover, most of these lineages were associated uniquely with protist hosts from the group of foraminiferans. Here it is suggested that this high genetic diversity of endosymbionts associated with foraminiferans is linked with the evolution of soritacean foraminifera, which seems to have been driven by endosymbiosis. Lastly, the absence of genetic recombination presented in this study, suggest a lack of hybridisation at least among the major 28S-rDNA phylotypes within Symbiodinium-like dinoflagellates. This supports highly the idea that these phylotypes are indeed independent evolutionary units, which should be considered at least as different species. Whether they belong to the same genus or to different higher taxa still needs to be revised.

Eleven distinct families of resistance gene analogs (RGAs) with the characteristic nucleotide-binding sequence (NBS) were identified in two wild apple species, Malus prunifolia and M. baccata, and two cultivated apple cultivars, M. domestica cv. Fuji and M. domestica cv. Hong-ok, using PCR approaches with degenerate primers based on two conserved motifs of known NBS-LRR resistance genes. These RGA families were found to be represented in all the apple species tested, including wild and cultivated species. However, their sequences are very divergent from each other. Furthermore, the low level of recombination detected within their RGA families supports the idea that the evolution of NBS-encoding sequences in apple species involves the gradual accumulation of mutations. Despite the high diversity of the RGA families found in all apple species, the apparent lack of differentiation between wild and cultivated forms suggests that other factors, such as the capacity to tolerate pathogens, might play an important role in the survival of wild-type species.

Changes in genetic variability and allele frequency can be responses from natural populations when encountering a novel contaminated environment. The genetic diversity and population structuring of natural populations of the gastropod Littorina brevicula from heavy-metal polluted and unpolluted environments along the southeast coast of Korea were examined using two mtDNA markers, cyt b and ND6. This study applied a nested clade analysis to test the existence of structuring association of haplotype distribution with environments (polluted and unpolluted). No genetic differences within cyt b mtDNA were detected between environments. On the other hand, differences in population haplotype diversity and structuring were found within ND6 mtDNA between polluted and unpolluted environments. The ND6-mtDNA haplotype (=genetic) diversity was significant lower in polluted environments. This decreased genetic diversity along with differences in the haplotype distribution within heavy-metal polluted environments compared to those unpolluted ones stand out as emergent effects from pollution at a population level. In this study, we propose the use of different approaches, such as the NCA, that takes into account the rare haplotypes, when assessing the effects of pollution on population genetic structuring.

Whereas terrestrial animal populations might show genetic connectivity within a continent, marine species, such as hermatypic corals, may have connectivity stretching to all corners of the planet. We quantified the genetic variability within and among populations of the widespread scleractinian coral, Plesiastrea versipora along the eastern Australian seaboard (4145 km) and the Ryukyu Archipelago (Japan, 681 km) using sequences of internal transcribed spacers (ITS1-2) from ribosomal DNA. Geographic patterns in genetic variability were deduced from a nested clade analysis (NCA) performed on a parsimony network haplotype. This analysis allowed the establishment of geographical associations in the distribution of haplotypes within the network cladogram, therefore allowing us to deduce phylogeographical patterns based under models of restricted gene flow, fragmentation and range expansion. No significant structure was found among Ryukyu Archipelago populations. The lack of an association between the positions of haplotypes in the cladogram with geographical location of these populations may be accounted for by a high level of gene flow of P. versipora within this region, probably due to the strong Kuroshio Current. In contrast, strong geographical associations were apparent among populations of P. versipora along the south-east coast of Australia. This pattern of restricted genetic connectivity among populations of P. versipora on the eastern seaboard of Australia seems to be associated with the present surface ocean current (the East Australian Current) on this side of the south-western Pacific Ocean.