Chemical mutagenesis and thermal selection of coral photosymbionts induce adaptation to heat stress with trait trade-offs.

Despite the relevance of heat-evolved microalgal endosymbionts to coral reef restoration, to date, few Symbiodiniaceae strains have been thermally enhanced via experimental evolution. Here, we investigated whether the thermal tolerance of Symbiodiniaceae can be increased through chemical mutagenesis followed by thermal selection. Strains of Durusdinium trenchii, Fugacium kawagutii and Symbiodinium pilosum were exposed to ethyl methanesulfonate to induce random mutagenesis, and then underwent thermal selection at high temperature (31/33°C). After 4.6-5 years of experimental evolution, the in vitro thermal tolerance of these strains was assessed via reciprocal transplant experiments to ambient (27°C) and elevated (31/35°C) temperatures. Growth, photosynthetic efficiency, oxidative stress and nutrient use were measured to compare thermal tolerance between strains. Heat-evolved D. trenchii, F. kawagutii and S. pilosum strains all exhibited increased photosynthetic efficiency under thermal stress. However, trade-offs in growth rates were observed for the heat-evolved D. trenchii lineage at both ambient and elevated temperatures. Reduced phosphate and nitrate uptake rates in F. kawagutii and S. pilosum heat-evolved lineages, respectively, suggest alterations in nutrition resource usage and allocation processes may have occurred. Increased phosphate uptake rates of the heat-evolved D. trenchii strain indicate that experimental evolution resulted in further trade-offs in this species. These findings deepen our understanding of the physiological responses of Symbiodiniaceae cultures to thermal selection and their capacity to adapt to elevated temperatures. The new heat-evolved Symbiodiniaceae developed here may be beneficial for coral reef restoration efforts if their enhanced thermal tolerance can be conferred in hospite.

Long-Term Heat Selection of the Coral Endosymbiont Cladocopium C1acro (Symbiodiniaceae) Stabilizes Associated Bacterial Communities.

Heat-tolerant strains of the coral endosymbiont, Cladocopium C1acro (Symbiodiniaceae), have previously been developed via experimental evolution. Here, we examine physiological responses and bacterial community composition (using 16S rRNA gene metabarcoding) in cultures of 10 heat-evolved (SS) and 9 wild-type (WT) strains, which had been exposed for 6 years to 31 °C and 27 °C, respectively. We also examine whether the associated bacterial communities were affected by a three-week reciprocal transplantation to both temperatures. The SS strains had bacterial communities with lower diversities that showed more stability and lower variability when exposed to elevated temperatures compared with the WT strains. Amplicon sequence variants (ASVs) of the bacterial genera Labrenzia, Algiphilus, Hyphobacterium and Roseitalea were significantly more associated with the SS strains compared with the WT strains. WT strains showed higher abundance of ASVs assigned to the genera Fabibacter and Tropicimonas. We hypothesize that these compositional differences in associated bacterial communities between SS and WT strains also contribute to the thermal tolerance of the microalgae. Future research should explore functional potential between bacterial communities using metagenomics to unravel specific genomic adaptations.

Effects of Ocean Warming on the Underexplored Members of the Coral Microbiome.

The climate crisis is one of the most significant threats to marine ecosystems. It is leading to severe increases in sea surface temperatures and in the frequency and magnitude of marine heatwaves. These changing conditions are directly impacting coral reef ecosystems, which are among the most biodiverse ecosystems on Earth. Coral-associated symbionts are particularly affected because summer heatwaves cause coral bleaching-the loss of endosymbiotic microalgae (Symbiodiniaceae) from coral tissues, leading to coral starvation and death. Coral-associated Symbiodiniaceae and bacteria have been extensively studied in the context of climate change, especially in terms of community diversity and dynamics. However, data on other microorganisms and their response to climate change are scarce. Here, we review current knowledge on how increasing temperatures affect understudied coral-associated microorganisms such as archaea, fungi, viruses, and protists other than Symbiodiniaceae, as well as microbe-microbe interactions. We show that the coral-microbe symbiosis equilibrium is at risk under current and predicted future climate change and argue that coral reef conservation initiatives should include microbe-focused approaches.

Evidence for de novo acquisition of microalgal symbionts by bleached adult corals.

Early life stages of most coral species acquire microalgal endosymbionts (Symbiodiniaceae) from the environment, but whether exogenous symbiont uptake is possible in the adult life stage is unclear. Deep sequencing of the Symbiodiniaceae ITS2 genetic marker has revealed novel symbionts in adult corals following bleaching; however these strains may have already been present at densities below detection limits. To test whether acquisition of symbionts from the environment occurs, we subjected adult fragments of corals (six species in four families) to a chemical bleaching treatment (menthol and DCMU). The treatment reduced the native microalgal symbiont abundance to below 2% of their starting densities. The bleached corals were then inoculated with a cultured Cladocopium C1acro strain. Genotyping of the Symbiodiniaceae communities before bleaching and after reinoculation showed that fragments of all six coral species acquired the Cladocopium C1acro strain used for inoculation. Our results provide strong evidence for the uptake of Symbiodiniaceae from the environment by adult corals. We also demonstrate the feasibility of chemical bleaching followed by reinoculation to manipulate the Symbiodiniaceae communities of adult corals, providing an innovative approach to establish new symbioses between adult corals and heat-evolved microalgal symbionts, which could prove highly relevant to coral reef restoration efforts.

Colonization and metabolite profiles of homologous, heterologous and experimentally evolved algal symbionts in the sea anemone Exaiptasia diaphana.

The sea anemone, Exaiptasia diaphana, is a model of coral-dinoflagellate (Symbiodiniaceae) symbiosis. However, little is known of its potential to form symbiosis with Cladocopium-a key Indo-Pacific algal symbiont of scleractinian corals, nor the host nutritional consequences of such an association. Aposymbiotic anemones were inoculated with homologous algal symbionts, Breviolum minutum, and seven heterologous strains of Cladocopium C1acro (wild-type and heat-evolved) under ambient conditions. Despite lower initial algal cell density, Cladocopium C1acro-anemeones achieved similar cell densities as B. minutum-anemones by week 77. Wild-type and heat-evolved Cladocopium C1acro showed similar colonization patterns. Targeted LC-MS-based metabolomics revealed that almost all significantly different metabolites in the host and Symbiodiniaceae fractions were due to differences between Cladocopium C1acro and B. minutum, with little difference between heat-evolved and wild-type Cladocopium C1acro at week 9. The algal fraction of Cladocopium C1acro-anemones was enriched in metabolites related to nitrogen storage, while the host fraction of B. minutum-anemones was enriched in sugar-related metabolites. Compared to B. minutum, Cladocopium C1acro is likely slightly less nutritionally beneficial to the host under ambient conditions, but more capable of maintaining its own growth when host nitrogen supply is limited. Our findings demonstrate the value of E. diaphana to study experimentally evolved Cladocopium.

Adaptive responses of free-living and symbiotic microalgae to simulated future ocean conditions.

Marine microalgae are a diverse group of microscopic eukaryotic and prokaryotic organisms capable of photosynthesis. They are important primary producers and carbon sinks but their physiology and persistence are severely affected by global climate change. Powerful experimental evolution technologies are being used to examine the potential of microalgae to respond adaptively to current and predicted future conditions, as well as to develop resources to facilitate species conservation and restoration of ecosystem functions. This review synthesizes findings and insights from experimental evolution studies of marine microalgae in response to elevated temperature and/or pCO2 . Adaptation to these environmental conditions has been observed in many studies of marine dinoflagellates, diatoms and coccolithophores. An enhancement in traits such as growth and photo-physiological performance and an increase in upper thermal limit have been shown to be possible, although the extent and rate of change differ between microalgal taxa. Studies employing multiple monoclonal replicates showed variation in responses among replicates and revealed the stochasticity of mutations. The work to date is already providing valuable information on species' climate sensitivity or resilience to managers and policymakers but extrapolating these insights to ecosystem- and community-level impacts continues to be a challenge. We recommend future work should include in situ experiments, diurnal and seasonal fluctuations, multiple drivers and multiple starting genotypes. Fitness trade-offs, stable versus plastic responses and the genetic bases of the changes also need investigating, and the incorporation of genome resequencing into experimental designs will be invaluable.

Gene regulation underpinning increased thermal tolerance in a laboratory-evolved coral photosymbiont.

Small increases in ocean temperature can disrupt the obligate symbiosis between corals and dinoflagellate microalgae, resulting in coral bleaching. Little is known about the genes that drive the physiological and bleaching response of algal symbionts to elevated temperature. Moreover, many studies to-date have compared highly divergent strains, making it challenging to accredit specific genes to contrasting traits. Here, we compare transcriptional responses at ambient (27°C) and bleaching-relevant (31°C) temperatures in a monoclonal, wild-type (WT) strain of Symbiodiniaceae to those of a selected-strain (SS), derived from the same monoclonal culture and experimentally evolved to elevated temperature over 80 generations (2.5 years). Thousands of genes were differentially expressed at a log fold-change of >8 between the WT and SS over a 35 days temperature treatment period. At 31°C, WT cells exhibited a temporally unstable transcriptomic response upregulating genes involved in the universal stress response such as molecular chaperoning, protein repair, protein degradation and DNA repair. Comparatively, SS cells exhibited a temporally stable transcriptomic response and downregulated many stress response genes that were upregulated by the WT. Among the most highly upregulated genes in the SS at 31°C were algal transcription factors and a gene probably of bacterial origin that encodes a type II secretion system protein, suggesting interactions with bacteria may contribute to the increased thermal tolerance of the SS. Genes and functional pathways conferring thermal tolerance in the SS could be targeted in future genetic engineering experiments designed to develop thermally resilient algal symbionts for use in coral restoration and conservation.

A PCR-Based Assay Targeting the Major Capsid Protein Gene of a Dinorna-Like ssRNA Virus That Infects Coral Photosymbionts.

The coral-Symbiodinium association is a critical component of coral reefs as it is the main primary producer and builds the reef's 3-dimensional structure. A breakdown of this endosymbiosis causes a loss of the dinoflagellate photosymbiont, Symbiodinium, and/or its photosynthetic pigments from the coral tissues (i.e., coral bleaching), and can lead to coral mortality. Coral bleaching has mostly been attributed to environmental stressors, and in some cases to bacterial infection. Viral lysis of Symbiodinium has been proposed as another possible cause of some instances of coral bleaching, but this hypothesis has not yet been experimentally confirmed. In this study, we used coral virome data to develop a novel PCR-based assay for examining the presence and diversity of a single-stranded RNA (ssRNA) virus by targeting its major capsid protein (MCP) gene. Illumina sequence analysis of amplicons obtained with novel primers showed 99.8% of the reads had the closest taxonomic affinity with the MCP gene of the virus, Heterocapsa circularisquama RNA virus (HcRNAV) known to infect dinoflagellates, indicating that dinorna-like viruses are commonly associated with corals on the Great Barrier Reef. A phylogenetic analysis of MCP gene sequences revealed strong coral species specificity of viral operational taxon units (OTUs). This assay allows a relatively easy and rapid evaluation of the presence and diversity of this particular viral group and will assist in enhancing our understanding of the role of viral lysis in coral bleaching.

Coral larvae for restoration and research: a large-scale method for rearing Acropora millepora larvae, inducing settlement, and establishing symbiosis.

Here we describe an efficient and effective technique for rearing sexually-derived coral propagules from spawning through larval settlement and symbiont uptake with minimal impact on natural coral populations. We sought to maximize larval survival while minimizing expense and daily husbandry maintenance by experimentally determining optimized conditions and protocols for gamete fertilization, larval cultivation, induction of larval settlement by crustose coralline algae, and inoculation of newly settled juveniles with their dinoflagellate symbiont Symbiodinium. Larval rearing densities at or below 0.2 larvae mL-1 were found to maximize larval survival and settlement success in culture tanks while minimizing maintenance effort. Induction of larval settlement via the addition of a ground mixture of diverse crustose coralline algae (CCA) is recommended, given the challenging nature of in situ CCA identification and our finding that non settlement-inducing CCA assemblages do not inhibit larval settlement if suitable assemblages are present. Although order of magnitude differences in infectivity were found between common Great Barrier Reef Symbiodinium clades C and D, no significant differences in Symbiodinium uptake were observed between laboratory-cultured and wild-harvested symbionts in each case. The technique presented here for Acropora millepora can be adapted for research and restoration efforts in a wide range of broadcast spawning coral species.

Genetic, morphological and growth characterisation of a new Roseofilum strain (Oscillatoriales, Cyanobacteria) associated with coral black band disease.

Black band disease (BBD) is a common disease of reef-building corals with a worldwide distribution that causes tissue loss at a rate of up to 3 cm/day. Critical for a mechanistic understanding of the disease's aetiology is the cultivation of its proposed pathogen, filamentous cyanobacteria (genus Roseofilum). Here, we optimise existing protocols for the isolation and cultivation of Roseofilum cyanobacteria using a new strain from the central Great Barrier Reef. We demonstrate that the isolation of this bacterium via inoculation onto agar plates was highly effective with a low percentage agar of 0.6% and that growth monitoring was most sensitive with fluorescence measurements of chlorophyll-a (440/685 nm). Cell growth curves in liquid and solid media were generated for the first time for this cyanobacterium and showed best growth rates for the previously untested L1-medium (growth rate k = 0.214 biomass/day; doubling time t gen = 4.67 days). Our results suggest that the trace metals contained in L1-medium maximise biomass increase over time for this cyanobacterium. Since the newly isolated Roseofilum strain is genetically closest to Pseudoscillatoria coralii, but in terms of pigmentation and cell size closer to Roseofilum reptotaenium, we formally merge the two species into a single taxon by providing an emended species description, Roseofilum reptotaenium (Rasoulouniriana) Casamatta emend. Following this optimized protocol is recommended for fast isolation and cultivation of Roseofilum cyanobacteria, for growth curve generation in strain comparisons and for maximisation of biomass in genetic studies.

CRISPR-Cas Defense System and Potential Prophages in Cyanobacteria Associated with the Coral Black Band Disease.

Understanding how pathogens maintain their virulence is critical to developing tools to mitigate disease in animal populations. We sequenced and assembled the first draft genome of Roseofilum reptotaenium AO1, the dominant cyanobacterium underlying pathogenicity of the virulent coral black band disease (BBD), and analyzed parts of the BBD-associated Geitlerinema sp. BBD_1991 genome in silico. Both cyanobacteria are equipped with an adaptive, heritable clustered regularly interspaced short palindromic repeats (CRISPR)-Cas defense system type I-D and have potential virulence genes located within several prophage regions. The defense system helps to prevent infection by viruses and mobile genetic elements via identification of short fingerprints of the intruding DNA, which are stored as templates in the bacterial genome, in so-called "CRISPRs." Analysis of CRISPR target sequences (protospacers) revealed an unusually high number of self-targeting spacers in R. reptotaenium AO1 and extraordinary long CRIPSR arrays of up to 260 spacers in Geitlerinema sp. BBD_1991. The self-targeting spacers are unlikely to be a form of autoimmunity; instead these target an incomplete lysogenic bacteriophage. Lysogenic virus induction experiments with mitomycin C and UV light did not reveal an actively replicating virus population in R. reptotaenium AO1 cultures, suggesting that phage functionality is compromised or excision could be blocked by the CRISPR-Cas system. Potential prophages were identified in three regions of R. reptotaenium AO1 and five regions of Geitlerinema sp. BBD_1991, containing putative BBD relevant virulence genes, such as an NAD-dependent epimerase/dehydratase (a homolog in terms of functionality to the third and fourth most expressed gene in BBD), lysozyme/metalloendopeptidases and other lipopolysaccharide modification genes. To date, viruses have not been considered to be a component of the BBD consortium or a contributor to the virulence of R. reptotaenium AO1 and Geitlerinema sp. BBD_1991. We suggest that the presence of virulence genes in potential prophage regions, and the CRISPR-Cas defense systems are evidence of an arms race between the respective cyanobacteria and their bacteriophage predators. The presence of such a defense system likely reduces the number of successful bacteriophage infections and mortality in the cyanobacteria, facilitating the progress of BBD.

From cholera to corals: Viruses as drivers of virulence in a major coral bacterial pathogen.

Disease is an increasing threat to reef-building corals. One of the few identified pathogens of coral disease is the bacterium Vibrio coralliilyticus. In Vibrio cholerae, infection by a bacterial virus (bacteriophage) results in the conversion of non-pathogenic strains to pathogenic strains and this can lead to cholera pandemics. Pathogenicity islands encoded in the V. cholerae genome play an important role in pathogenesis. Here we analyse five whole genome sequences of V. coralliilyticus to examine whether virulence is similarly driven by horizontally acquired elements. We demonstrate that bacteriophage genomes encoding toxin genes with homology to those found in pathogenic V. cholerae are integrated in V. coralliilyticus genomes. Virulence factors located on chromosomal pathogenicity islands also exist in some strains of V. coralliilyticus. The presence of these genetic signatures indicates virulence in V. coralliilyticus is driven by prophages and other horizontally acquired elements. Screening for pathogens of coral disease should target conserved regions in these elements.