Multi-Omics Revealing the Response Patterns of Symbiotic Microorganisms and Host Metabolism in Scleractinian Coral Pavona minuta to Temperature Stresses

Global climate change has resulted in large-scale coral reef decline worldwide, for which the ocean warming has paid more attention. Coral is a typical mutually beneficial symbiotic organism with diverse symbiotic microorganisms, which maintain the stability of physiological functions. This study compared the responses of symbiotic microorganisms and host metabolism in a common coral species, Pavona minuta, under indoor simulated thermal and cold temperatures. The results showed that abnormal temperature stresses had unfavorable impact on the phenotypes of corals, resulting in bleaching and color change. The compositions of symbiotic bacteria and dinoflagellate communities only presented tiny changes under temperature stresses. However, some rare symbiotic members have been showed to be significantly influenced by water temperatures. Finally, by using ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS) method, we found that different temperature stresses had very different impacts on the metabolism of coral holobiont. The thermal and cold stresses induced the decrease of anti-oxidation metabolites, several monogalactosyldiacylglycerols (MGDGs), and the increase of lipotoxic metabolite, 10-oxo-nonadecanoic acid, in the coral holobiont, respectively. Our study indicated the response patterns of symbiotic microorganisms and host metabolism in coral to the thermal and cold stresses, providing theoretical data for the adaptation and evolution of coral to a different climate in the future.

Heat stress reduces the contribution of diazotrophs to coral holobiont nitrogen cycling

Efficient nutrient cycling in the coral-algal symbiosis requires constant but limited nitrogen availability. Coral-associated diazotrophs, i.e., prokaryotes capable of fixing dinitrogen, may thus support productivity in a stable coral-algal symbiosis but could contribute to its breakdown when overstimulated. However, the effects of environmental conditions on diazotroph communities and their interaction with other members of the coral holobiont remain poorly understood. Here we assessed the effects of heat stress on diazotroph diversity and their contribution to holobiont nutrient cycling in the reef-building coral Stylophora pistillata from the central Red Sea. In a stable symbiotic state, we found that nitrogen fixation by coral-associated diazotrophs constitutes a source of nitrogen to the algal symbionts. Heat stress caused an increase in nitrogen fixation concomitant with a change in diazotroph communities. Yet, this additional fixed nitrogen was not assimilated by the coral tissue or the algal symbionts. We conclude that although diazotrophs may support coral holobiont functioning under low nitrogen availability, altered nutrient cycling during heat stress abates the dependence of the coral host and its algal symbionts on diazotroph-derived nitrogen. Consequently, the role of nitrogen fixation in the coral holobiont is strongly dependent on its nutritional status and varies dynamically with environmental conditions.

Contrasting microbiome dynamics of putative denitrifying bacteria in two octocoral species exposed to dissolved organic carbon (DOC) and warming

Mutualistic nutrient cycling in the coral-algae symbiosis depends on limited nitrogen (N) availability for algal symbionts. Denitrifying prokaryotes capable of reducing nitrate or nitrite to dinitrogen could thus support coral holobiont functioning by limiting N availability. Octocorals show some of the highest denitrification rates among reef organisms, however little is known about the community structures of associated denitrifiers and their response to environmental fluctuations. Combining 16S rRNA gene amplicon sequencing with nirS in-silico PCR and quantitative PCR, we found differences in bacterial community dynamics between two octocorals exposed to excess dissolved organic carbon (DOC) and concomitant warming. While bacterial communities of the gorgonian Pinnigorgia flava remained largely unaffected by DOC and warming, the soft coral Xenia umbellata exhibited a pronounced shift towards Alphaproteobacteria dominance under excess DOC. Likewise, the relative abundance of denitrifiers was not altered in P. flava , but decreased by one order of magnitude in X. umbellata under excess DOC likely due to decreased proportions of Ruegeria spp. Given that holobiont C:N ratios remained stable in P. flava but showed a pronounced increase with excess DOC in X. umbellata host, our results suggest that microbial community dynamics may reflect the nutritional status of the holobiont. Hence, denitrifier abundance may be directly linked to N availability. This suggests a passive regulation of N cycling microbes, which could help stabilize nutrient limitation in the coral-algal symbiosis and thereby support holobiont functioning in a changing environment. Importance Octocorals are important members of reef-associated benthic communities that can rapidly replace scleractinian corals as the dominant ecosystem engineers on degraded reefs. Considering the substantial change in the (a)biotic environment that is commonly driving reef degradation, maintaining a dynamic and metabolically diverse microbial community might contribute to octocoral acclimatization and ecological adaptation. Nitrogen (N) cycling microbes, in particular denitrifying prokaryotes, may support holobiont functioning by limiting internal N availability, but little is known about the identity and (a)biotic drivers of octocoral-associated denitrifiers. Here, we show contrasting dynamics of bacterial communities associated with two common octocoral species, the soft coral Xenia umbellata and the gorgonian Pinnigorgia flava after a six-week exposure to excess dissolved organic carbon (DOC) under concomitant warming conditions. The specific responses of denitrifier communities associated with the two octocoral species aligned with the nutritional status of holobiont members. This suggests a passive regulation of this microbial trait based on N availability in the coral holobiont.

Highly Variable and Non-complex Diazotroph Communities in Corals From Ambient and High CO2 Environments

The ecological success of corals depends on their association with microalgae and a diverse bacterial assemblage. Ocean acidification (OA), among other stressors, threatens to impair host-microbial metabolic interactions that underlie coral holobiont functioning. Volcanic CO2 seeps offer a unique opportunity to study the effects of OA in natural reef settings and provide insight into the long-term adaptations under a low pH environment. Here we compared nitrogen-fixing bacteria (diazotrophs) associated with four coral species (Pocillopora damicornis, Galaxea fascicularis, Acropora secale, and Porites rus) collected from CO2 seeps at Tutum Bay (Papua New Guinea) with those from a nearby ambient CO2 site using nifH amplicon sequencing to characterize the effects of seawater pH on bacterial communities and nitrogen cycling. Diazotroph communities were of generally low diversity across all coral species and for both sampling sites. Out of a total of 25 identified diazotroph taxa, 14 were associated with P. damicornis, of which 9 were shared across coral species. None of the diazotroph taxa, however, were consistently found across all coral species or across all samples within a species pointing to a high degree of diazotroph community variability. Rather, the majority of sampled colonies were dominated by one or two diazotroph taxa of high relative abundance. Pocillopora damicornis and Galaxea fascicularis that were sampled in both environments showed contrasting community assemblages between sites. In P. damicornis, Gammaproteobacteria and Cyanobacteria were prevalent under ambient pCO2, while a single member of the family Rhodobacteraceae was present at high relative abundance at the high pCO2 site. Conversely, in G. fascicularis diazotroph communities were indifferent between both sites. Diazotroph community changes in response to OA seem thus variable within as well as between host species, potentially arguing for haphazard diazotroph community assembly. This warrants further research into the underlying factors structuring diazotroph community assemblages and their functional role in the coral holobiont.

Elucidating gene expression adaptation of phylogenetically divergent coral holobionts under heat stress

As coral reefs struggle to survive under climate change, it is crucial to know whether they have the capacity to withstand changing conditions, particularly increasing seawater temperatures. Thermal tolerance requires the integrative response of the different components of the coral holobiont (coral host, algal photosymbiont, and associated microbiome). Here, using a controlled thermal stress experiment across three divergent Caribbean coral species, we attempt to dissect holobiont member metatranscriptome responses from coral taxa with different sensitivities to heat stress and use phylogenetic ANOVA to study the evolution of gene expression adaptation. We show that coral response to heat stress is a complex trait derived from multiple interactions among holobiont members. We identify host and photosymbiont genes that exhibit lineage-specific expression level adaptation and uncover potential roles for bacterial associates in supplementing the metabolic needs of the coral-photosymbiont duo during heat stress. Our results stress the importance of integrative and comparative approaches across a wide range of species to better understand coral survival under the predicted rise in sea surface temperatures.

Consensus Guidelines for Advancing Coral Holobiont Genome and Specimen Voucher Deposition

Coral research is being ushered into the genomic era. To fully capitalize on the potential discoveries from this genomic revolution, the rapidly increasing number of high-quality genomes requires effective pairing with rigorous taxonomic characterizations of specimens and the contextualization of their ecological relevance. However, to date there is no formal framework that genomicists, taxonomists, and coral scientists can collectively use to systematically acquire and link these data. Spurred by the recently announced “Coral symbiosis sensitivity to environmental change hub” under the “Aquatic Symbiosis Genomics Project” - a collaboration between the Wellcome Sanger Institute and the Gordon and Betty Moore Foundation to generate gold-standard genome sequences for coral animal hosts and their associated Symbiodiniaceae microalgae (among the sequencing of many other symbiotic aquatic species) - we outline consensus guidelines to reconcile different types of data. The metaorganism nature of the coral holobiont provides a particular challenge in this context and is a key factor to consider for developing a framework to consolidate genomic, taxonomic, and ecological (meta)data. Ideally, genomic data should be accompanied by taxonomic references, i.e., skeletal vouchers as formal morphological references for corals and strain specimens in the case of microalgal and bacterial symbionts (cultured isolates). However, exhaustive taxonomic characterization of all coral holobiont member species is currently not feasible simply because we do not have a comprehensive understanding of all the organisms that constitute the coral holobiont. Nevertheless, guidelines on minimal, recommended, and ideal-case descriptions for the major coral holobiont constituents (coral animal, Symbiodiniaceae microalgae, and prokaryotes) will undoubtedly help in future referencing and will facilitate comparative studies. We hope that the guidelines outlined here, which we will adhere to as part of the Aquatic Symbiosis Genomics Project sub-hub focused on coral symbioses, will be useful to a broader community and their implementation will facilitate cross- and meta-data comparisons and analyses.

Global change differentially modulates coral physiology and suggests future shifts in Caribbean reef assemblages

Global change driven by anthropogenic carbon emissions is altering ecosystems at unprecedented rates, especially coral reefs, whose symbiosis with algal endosymbionts ise particularly vulnerable to increasing ocean temperatures and altered carbonate chemistry. Here, we assess the physiological responses of the coral holobiont (animal host + algal symbiont) of three Caribbean coral species from two reef environments after exposure to simulated ocean warming (28, 31 °C), acidification (300 - 3290 μatm), and the combination of stressors for 93 days. We used multidimensional analyses to assess how multiple coral holobiont physiological parameters respond to ocean acidification and warming. Our results demonstrate significantly diminishing holobiont physiology in S. siderea and P. astreoides in response to projected ocean acidification, while future warming elicited severe declines in P. strigosa. Offshore S. siderea fragments exhibited higher physiological plasticity than inshore counterparts, suggesting that this offshore population has the capacity to modulate their physiology in response to changing conditions, but at a cost to the holobiont. Plasticity of P. strigosa and P. astreoides was not clearly different between natal reef environments, however, temperature evoked a greater plastic response in both species. Interestingly, while these species exhibit unique physiological responses to ocean acidification and warming, when data from all three species are modeled together, convergent stress responses to these conditions are observed, highlighting the overall sensitivities of tropical corals to these stressors. Our results demonstrate that while ocean warming is a severe acute stressor that will have dire consequences for coral reefs globally, chronic exposure to acidification may also impact coral physiology to a greater extent than previously assumed. The variety of responses to global change we observe across species will likely manifest in altered Caribbean reef assemblages in the future.

Relative abundance of nitrogen cycling microbes in coral holobionts reflects environmental nitrate availability

Recent research suggests that nitrogen (N) cycling microbes are important for coral holobiont functioning. In particular, coral holobionts may acquire bioavailable N via prokaryotic dinitrogen (N 2 ) fixation or remove excess N via denitrification activity. However, our understanding of environmental drivers on these processes in hospite remains limited. Employing the strong seasonality of the central Red Sea, this study assessed the effects of environmental parameters on the proportional abundances of N cycling microbes associated with the hard corals Acropora hemprichii and Stylophora pistillata. Specifically, we quantified changes in the relative ratio between nirS and nifH gene copy numbers, as a proxy for seasonal shifts in denitrification and N 2 fixation potential in corals, respectively. In addition, we assessed coral tissue-associated Symbiodiniaceae cell densities and monitored environmental parameters to provide a holobiont and environmental context, respectively. While ratios of nirS to nifH gene copy numbers varied between seasons, they revealed similar seasonal patterns in both coral species, with ratios closely following patterns in environmental nitrate availability. Symbiodiniaceae cell densities aligned with environmental nitrate availability, suggesting that the seasonal shifts in nirS to nifH gene abundance ratios were probably driven by nitrate availability in the coral holobiont. Thereby, our results suggest that N cycling in coral holobionts probably adjusts to environmental conditions by increasing and/or decreasing denitrification and N 2 fixation potential according to environmental nitrate availability. Microbial N cycling may, thus, extenuate the effects of changes in environmental nitrate availability on coral holobionts to support the maintenance of the coral–Symbiodiniaceae symbiosis.

Selecting Heat-Tolerant Corals for Proactive Reef Restoration

Coral reef restoration is an attractive tool for the management of degraded reefs; however, conventional restoration approaches will not be effective under climate change. More proactive restoration approaches must integrate future environmental conditions into project design to ensure long-term viability of restored corals during worsening bleaching events. Corals exist along a continuum of stress-tolerant phenotypes that can be leveraged to enhance the thermal resilience of reefs through selective propagation of heat-tolerant colonies. Several strategies for selecting thermally tolerant stock are currently available and range broadly in scalability, cost, reproducibility, and specificity. Different components of the coral holobiont have different utility to practitioners as diagnostics and drivers of long-term phenotypes, so selection strategies can be tailored to the resources and goals of individual projects. There are numerous unknowns and potential trade-offs to consider, but we argue that a focus on thermal tolerance is critical because corals that do not survive bleaching cannot contribute to future reef communities at all. Selective propagation uses extant corals and can be practically incorporated into existing restoration frameworks, putting researchers in a position to perform empirical tests and field trials now while there is still a window to act.