Proteomes of native and non-native symbionts reveal responses underpinning host-symbiont specificity in the cnidarian-dinoflagellate symbiosis.

Cellular mechanisms responsible for the regulation of nutrient exchange, immune responses, and symbiont population growth in the cnidarian-dinoflagellate symbiosis are poorly resolved, particularly with respect to the dinoflagellate symbiont. Here, we characterized proteomic changes in the native symbiont Breviolum minutum during colonization of its host sea anemone Exaiptasia diaphana ("Aiptasia"). We also compared the proteome of this native symbiont in the established symbiotic state with that of a non-native symbiont, Durusdinium trenchii. The onset of symbiosis between Aiptasia and Breviolum minutum increased the accumulation of symbiont proteins associated with the acquisition of inorganic carbon and photosynthesis, nitrogen metabolism, micro- and macronutrient starvation, suppression of host immune responses, tolerance to low pH, and management of oxidative stress. Such responses are consistent with a functional, persistent symbiosis. In contrast, D. trenchii predominantly showed elevated levels of immunosuppressive proteins, consistent with the view that this symbiont is an opportunist that forms a less beneficial, less well-integrated symbiosis with this model anemone. By adding symbiont analysis to the already known responses of the host proteome, our results provide a more holistic view of cellular processes that determine host-symbiont specificity and how differences in symbiont partners (i.e. native versus non-native symbionts) may impact the fitness of the cnidarian-dinoflagellate symbiosis.

Increased dominance of heat-tolerant symbionts creates resilient coral reefs in near-term ocean warming.

Climate change is radically altering coral reef ecosystems, mainly through increasingly frequent and severe bleaching events. Yet, some reefs have exhibited higher thermal tolerance after bleaching severely the first time. To understand changes in thermal tolerance in the eastern tropical Pacific (ETP), we compiled four decades of temperature, coral cover, coral bleaching, and mortality data, including three mass bleaching events during the 1982 to 1983, 1997 to 1998 and 2015 to 2016 El Niño heatwaves. Higher heat resistance in later bleaching events was detected in the dominant framework-building genus, Pocillopora, while other coral taxa exhibited similar susceptibility across events. Genetic analyses of Pocillopora spp. colonies and their algal symbionts (2014 to 2016) revealed that one of two Pocillopora lineages present in the region (Pocillopora "type 1") increased its association with thermotolerant algal symbionts (Durusdinium glynnii) during the 2015 to 2016 heat stress event. This lineage experienced lower bleaching and mortality compared with Pocillopora "type 3", which did not acquire D. glynnii. Under projected thermal stress, ETP reefs may be able to preserve high coral cover through the 2060s or later, mainly composed of Pocillopora colonies that associate with D. glynnii. However, although the low-diversity, high-cover reefs of the ETP could illustrate a potential functional state for some future reefs, this state may only be temporary unless global greenhouse gas emissions and resultant global warming are curtailed.

The seasonal investigation of Symbiodiniaceae in broadcast spawning, Acropora humilis and brooding, Pocillopora cf. damicornis corals.

The density and diversity of Symbiodiniaceae associated with corals can be influenced by seasonal changes . This study provided the first annual investigation of Symbiodiniaceae density and diversity associated with Acropora humilis and Pocillopora cf. damicornis corals in the Gulf of Thailand using both zooxanthellae cell count and next-generation sequencing (ITS-1, ITS-2 regions) techniques, respectively. The results from this study indicated that zooxanthellae cell densities in both coral species differ significantly. The number of zooxanthellae was negatively correlated with the physical environment variable (light intensity). The diversity within A. humilis consisted of two genera, Cladocopium (Cspc_C3: 56.39%, C3w: 33.62%, C93type1: 4.42% and Cspf: 3.59%) and a small amount of Durusdinium (D1: 1.03%) whereas P. cf. damicornis was found to be 100% associated with Durusdinium (D1: 95.58%, D6: 1.01% and D10: 2.7%) suggesting that each coral species may select their appropriate genus/species of Symbiodiniaceae in response to local environmental stressors. The results of this study provided some information on the coral-Symbiodiniaceae relationship between seasons, which may be applied to predict the potential adaptation of corals in localized reef environments.

Metatranscriptomic Analysis of Corals Inoculated With Tolerant and Non-Tolerant Symbiont Exposed to High Temperature and Light Stress.

Algal symbionts of corals can influence host stress resistance; for example, in the Pacific Ocean, whereas Cladocopium (C-type) is generally dominant in corals, Durusdinium (D-type) is found in more heat-resistant corals. Thus, the presence of D-type symbiont likely increases coral heat tolerance, and this symbiotic relationship potentially provides a hint to increase the stress tolerance of coral-algal symbioses. In this study, transcriptome profiles of Cladocopium- and Durusdinium-harboring Acropora solitaryensis (C-coral and D-coral, respectively) and algal photosystem functioning (F v /F m ) under bleaching conditions (high temperature and light stress) were compared. Stress treatment caused algal photoinhibition that the F v /F m value of Symbiodiniaceae was immediately reduced. The transcriptome analysis of corals revealed that genes involved in the following processes were detected: endoplasmic reticulum (ER) stress, mitophagy, apoptosis, endocytosis, metabolic processes (acetyl-CoA, chitin metabolic processes, etc.), and the PI3K-AKT pathway were upregulated, while DNA replication and the calcium signaling pathway were downregulated in both C- and D-corals. These results suggest that unrepaired DNA and protein damages were accumulated in corals under high temperature and light stress. Additionally, some differentially expressed genes (DEGs) were specific to C- or D-corals, which includes genes involved in transient receptor potential (TRP) channels and vitamin B metabolic processes. Algal transcriptome analysis showed the increased expression of gene encoding photosystem and molecular chaperone especially in D-type symbiont. The transcriptome data imply a possible difference in the stress reactions on C-type and D-type symbionts. The results reveal the basic process of coral heat/light stress response and symbiont-type-specific coral transcriptional responses, which provides a perspective on the mechanisms that cause differences in coral stress tolerance.

Identification of coral endosymbionts of Veedhalai and Mandapam coasts of Palk Bay, India using small subunit rDNA.

Coral endosymbionts act as a bio-indicator of coral ecosystem under extreme environmental conditions. The health of the coral ecosystem depends on the endosymbiont cell density of the coral hosts. Therefore, it is of interest to analyze ten coral fragments found to be under the genera Acropora, Favites, Favia, and Porites collected at various locations from Veedhalai to Mandapam, southeast coast of India during January 2019 to March 2019. The zooxanthellae cell count ranged between 4.08 (Porites sp.9) and 13.75x105 cells cm2 -1 (Favites sp.3). This indicates the health of the corals in the region. The genus (clade) level identification of endosymbionts was detected using the host excluding primers of small subunit DNA (nssrDNA). Bidirectional sequencing of 18S nrDNA gene (SSU) of all ten coral fragments show that the Veedhalai corals is associated with the genus Durusdinium (Clade D) but the corals of Mandapam is associated with the genera, Cladocopium (Clade C) and Durusdinium (Clade D). It is known that the thermal stress has negative impact on coral reef ecosystem of the world. The dominance of the genus Durusdinium in the scleractinian corals of Palk Bay may be due to frequent exposure to thermal stress. This thermotolerant endosymbionts is opportunistic. Thus, the corals of Veedhalai and Mandapam coasts, Palk Bay, India are necessarily packed with thermotolerant endosymbionts enabling conservation.

Gene clusters for biosynthesis of mycosporine-like amino acids in dinoflagellate nuclear genomes: Possible recent horizontal gene transfer between species of Symbiodiniaceae (Dinophyceae).

Global warming increases the temperature of the ocean surface, which can disrupt dinoflagellate-coral symbioses and result in coral bleaching. Photosynthetic dinoflagellates of the family Symbiodiniaceae include bleaching-tolerant and bleaching-sensitive coral symbionts. Therefore, understanding the molecular mechanisms for changing symbiont diversity is potentially useful to assist recovery of coral holobionts (corals and their associated microbes, including multiple species of Symbiodiniaceae), although sexual reproduction has not been observed in the Symbiodiniaceae. Recent molecular phylogenetic analyses estimate that the Symbiodiniaceae appeared 160 million years ago and diversified into 15 groups, five genera of which now have available draft genomes (i.e., Symbiodinium, Durusdinium, Breviolum, Fugacium, and Cladocopium). Comparative genomic analyses have suggested that crown groups have fewer gene families than early-diverging groups, although many genes that were probably acquired via gene duplications and horizontal gene transfers (HGTs) have been found in each decoded genome. Because UV stress is likely a contributor to coral bleaching, and because the highly conserved gene cluster for mycosporine-like amino acid (MAA) biosynthesis has been found in thermal-tolerant symbiont genomes, I reviewed genomic features of the Symbiodiniaceae, focusing on possible acquisition of a biosynthetic gene cluster for MAAs, which absorb UV radiation. On the basis of highly conserved noncoding sequences, I hypothesized that HGTs have occurred among members of the Symbiodiniaceae and have contributed to the diversification of Symbiodiniaceae-host relationships. Finally, I proposed that bleaching tolerance may be strengthened by multiple MAAs from both symbiotic dinoflagellates and corals.

Symbiont shuffling induces differential DNA methylation responses to thermal stress in the coral Montastraea cavernosa.

Algal symbiont shuffling in favour of more thermotolerant species has been shown to enhance coral resistance to heat-stress. Yet, the mechanistic underpinnings and long-term implications of these changes are poorly understood. This work studied the modifications in coral DNA methylation, an epigenetic mechanism involved in coral acclimatization, in response to symbiont manipulation and subsequent heat stress exposure. Symbiont composition was manipulated in the great star coral Montastraea cavernosa through controlled thermal bleaching and recovery, producing paired ramets of three genets dominated by either their native symbionts (genus Cladocopium) or the thermotolerant species (Durusdinium trenchi). Single-base genome-wide analyses showed significant modifications in DNA methylation concentrated in intergenic regions, introns and transposable elements. Remarkably, DNA methylation changes in response to heat stress were dependent on the dominant symbiont, with twice as many differentially methylated regions found in heat-stressed corals hosting different symbionts (Cladocopium vs. D. trenchii) compared to all other comparisons. Interestingly, while differential gene body methylation was not correlated with gene expression, an enrichment in differentially methylated regions was evident in repetitive genome regions. Overall, these results suggest that changes in algal symbionts favouring heat tolerant associations are accompanied by changes in DNA methylation in the coral host. The implications of these results for coral adaptation, along with future avenues of research based on current knowledge gaps, are discussed in the present work.

A New Dinoflagellate Genome Illuminates a Conserved Gene Cluster Involved in Sunscreen Biosynthesis.

Photosynthetic dinoflagellates of the Family Symbiodiniaceae live symbiotically with many organisms that inhabit coral reefs and are currently classified into fifteen groups, including seven genera. Draft genomes from four genera, Symbiodinium, Breviolum, Fugacium, and Cladocopium, which have been isolated from corals, have been reported. However, no genome is available from the genus Durusdinium, which occupies an intermediate phylogenetic position in the Family Symbiodiniaceae and is well known for thermal tolerance (resistance to bleaching). We sequenced, assembled, and annotated the genome of Durusdinium trenchii, isolated from the coral, Favia speciosa, in Okinawa, Japan. Assembled short reads amounted to 670 Mb with ∼47% GC content. This GC content was intermediate among taxa belonging to the Symbiodiniaceae. Approximately 30,000 protein-coding genes were predicted in the D. trenchii genome, fewer than in other genomes from the Symbiodiniaceae. However, annotations revealed that the D. trenchii genome encodes a cluster of genes for synthesis of mycosporine-like amino acids, which absorb UV radiation. Interestingly, a neighboring gene in the cluster encodes a glucose-methanol-choline oxidoreductase with a flavin adenine dinucleotide domain that is also found in Symbiodinium tridacnidorum. This conservation seems to partially clarify an ancestral genomic structure in the Symbiodiniaceae and its loss in late-branching lineages, including Breviolum and Cladocopium, after splitting from the Durusdinium lineage. Our analysis suggests that approximately half of the taxa in the Symbiodiniaceae may maintain the ability to synthesize mycosporine-like amino acids. Thus, this work provides a significant genomic resource for understanding the genomic diversity of Symbiodiniaceae in corals.

Lipidomes of phylogenetically different symbiotic dinoflagellates of corals.

The structural base of all membranes of symbiotic dinoflagellates (SD) is composed of glycolipids and betaine lipids, whereas triacylglycerols (TG) constitute an energy reserve and are involved in biosynthesis of glycolipids. Since data on the SD lipidome and the host's influence on symbionts' lipidome are scanty, we analyzed and compared the lipidomes of SD isolated from the zoantharian Palythoa tuberculosa and the alcyonarian Sinularia heterospiculata. A sequencing of nuclear gene regions showed that both cnidarians hosted the dinoflagellates Cladocopium sp. (subclades C1 and C3), but the zoantharian also contained the dinoflagellates Durusdinium trenchii (clade D). The presence of the thermotolerant D. trenchii resulted in a higher unsaturation of mono- and digalactosyldiacylglycerols (MGDG and DGDG), but a lower unsaturation of sulfoquinovosyldiacylglycerol (SQDG). The same features were earlier described for same SD from a reef-building coral. Hence, the profile of glycolipid molecules, which form SD thylakoid membranes, seems to be species-specific and does not depend on the host's taxonomic position. In contrast, the betaine lipid molecular species profile of diacylglyceryl-3-O-carboxyhydroxymethylcholine (DGCC), which forms SD cell membranes, can be influenced by the host. The profiles of the TG molecular species from freshly isolated SD have been determined for the first time. These molecular species can be divided on the basis of the acyl group in sn-2 position. The TG with 16:0 acyl group in sn-2 position may enrich total TG of a cnidarian colony and originate from SD cytoplasm. In contrast, TG 18:3/18:4/18:3 may be biosynthetically related with DGDG and concentrated in SD plastoglobules. Our data may be useful for further investigations of natural and technogenic variations in microalgal lipids and symbiont-host interactions in marine ecosystems.

Thermotolerant coral symbionts modulate heat stress-responsive genes in their hosts.

Some corals may become more resistant to bleaching by shuffling their Symbiodiniaceae communities toward thermally tolerant species, and manipulations to boost the abundance of these symbionts in corals may increase resilience in warming oceans. However, the thermotolerant symbiont Durusdinium trenchii may reduce growth and fecundity in Caribbean corals, and these tradeoffs need to be better understood as this symbiont spreads through the region. We sought to understand how D. trenchii modulates coral gene expression by manipulating symbiont communities in Montastraea cavernosa to produce replicate ramets containing D. trenchii together with paired ramets of these same genets (n = 3) containing Cladocopium C3 symbionts. We then examined differences in global gene expression between corals hosting Durusdinium and Cladocopium under control temperatures, and in response to short-term heat stress. We identified numerous transcriptional differences associated with symbiont identity, which explained 2%-14% of the transcriptional variance. Corals with D. trenchii upregulated genes related to translation, ribosomal structure and biogenesis, and downregulated genes related to extracellular structures, and carbohydrate and lipid transport and metabolism, relative to corals with Cladocopium. Unexpectedly, these changes were similar to those observed in Cladocopium-dominated corals in response to heat stress, suggesting that thermotolerant D. trenchii may cause corals to increase expression of heat stress-responsive genes, explaining both the increased heat tolerance and the associated energetic tradeoffs in corals containing D. trenchii. These findings provide insight into the ecological changes occurring on contemporary coral reefs in response to climate change, and the diverse ways in which different symbionts modulate emergent phenotypes of their hosts.

Dispersal, genetic variation, and symbiont interaction network of heat-tolerant endosymbiont Durusdinium trenchii: Insights into the adaptive potential of coral to climate change.

Global warming has degraded coral reef ecosystems worldwide. Some corals develop thermal tolerance by associating with heat-tolerant Symbiodiniaceae. Here, we studied the mechanisms surrounding the dispersal, genetic variation and symbionts interaction of heat-tolerant Durusdinium trenchii across 13° latitudes in the South China Sea (SCS), to explore the possible mechanisms underlying these changes. Our results showed that Durusdinium trenchii are widely distributed in the seawater from the SCS. Our analyses of microsatellite loci revealed that D. trenchii has a high genetic diversity in the SCS; STRUCTURE analysis indicated that D. trenchii can be divided into four populations within the SCS; There exist positive correlations between genetic variation and geographic isolation, average sea surface temperature (SST) and variations in SST. Network modelling inferences showed that D. trenchii is a key species in the Symbiodiniaceae communities in the tropical SCS and contributes the greatest number of co-exclusion relationships. These results indicated that D. trenchii can affect the rare Symbiodiniaceae community. The long lifespan and the monsoon-driven ocean currents have shaped the wide distribution of D. trenchii. But low SST limits the ability of D. trenchii to establish stable symbioses with coral in the subtropical habitats. Geographical isolation and SST have shaped significant genetic variation of D.trenchii around the SCS. Our data reveals the biogeography and genetic population characteristics of D. trenchii in the Indo-Pacific region, and suggests that heat-tolerance and high genetic diversity of D. trenchii aid the corals with their adaptation to climate change.

Proteomic Basis of Symbiosis: A Heterologous Partner Fails to Duplicate Homologous Colonization in a Novel Cnidarian- Symbiodiniaceae Mutualism.

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.

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.