Unlocking the Complex Cell Biology of Coral-Dinoflagellate Symbiosis: A Model Systems Approach.

Symbiotic interactions occur in all domains of life, providing organisms with resources to adapt to new habitats. A prime example is the endosymbiosis between corals and photosynthetic dinoflagellates. Eukaryotic dinoflagellate symbionts reside inside coral cells and transfer essential nutrients to their hosts, driving the productivity of the most biodiverse marine ecosystem. Recent advances in molecular and genomic characterization have revealed symbiosis-specific genes and mechanisms shared among symbiotic cnidarians. In this review, we focus on the cellular and molecular processes that underpin the interaction between symbiont and host. We discuss symbiont acquisition via phagocytosis, modulation of host innate immunity, symbiont integration into host cell metabolism, and nutrient exchange as a fundamental aspect of stable symbiotic associations. We emphasize the importance of using model systems to dissect the cellular complexity of endosymbiosis, which ultimately serves as the basis for understanding its ecology and capacity to adapt in the face of climate change.

Structures and organizations of PSI-AcpPCI supercomplexes from red tidal and coral symbiotic photosynthetic dinoflagellates.

Marine photosynthetic dinoflagellates are a group of successful phytoplankton that can form red tides in the ocean and also symbiosis with corals. These features are closely related to the photosynthetic properties of dinoflagellates. We report here three structures of photosystem I (PSI)-chlorophylls (Chls) a/c-peridinin protein complex (PSI-AcpPCI) from two species of dinoflagellates by single-particle cryoelectron microscopy. The crucial PsaA/B subunits of a red tidal dinoflagellate Amphidinium carterae are remarkably smaller and hence losing over 20 pigment-binding sites, whereas its PsaD/F/I/J/L/M/R subunits are larger and coordinate some additional pigment sites compared to other eukaryotic photosynthetic organisms, which may compensate for the smaller PsaA/B subunits. Similar modifications are observed in a coral symbiotic dinoflagellate Symbiodinium species, where two additional core proteins and fewer AcpPCIs are identified in the PSI-AcpPCI supercomplex. The antenna proteins AcpPCIs in dinoflagellates developed some loops and pigment sites as a result to accommodate the changed PSI core, therefore the structures of PSI-AcpPCI supercomplex of dinoflagellates reveal an unusual protein assembly pattern. A huge pigment network comprising Chls a and c and various carotenoids is revealed from the structural analysis, which provides the basis for our deeper understanding of the energy transfer and dissipation within the PSI-AcpPCI supercomplex, as well as the evolution of photosynthetic organisms.

Microtubule reorganization during mitotic cell division in the dinoflagellate Ostreospis cf. ovata.

Dinoflagellates are marine organisms that undergo seasonal proliferation events known as algal blooms. Vegetative cell proliferation is a main contributing factor in these events. However, mechanistical understanding of mitosis and cytokinesis in dinoflagellates remains rudimentary. Using an optimized immunofluorescence protocol, we analysed changes in microtubule organization occurring during the mitotic cycle of the toxic dinoflagellate Ostreopsis cf. ovata. We find that the flagella and the cortical microtubule array persist throughout the mitotic cycle. Two cytoplasmic microtubule bundles originate from the ventral area, where the basal bodies are located - a cortical bundle and a cytoplasmic bundle. The latter associates with the nucleus in the cell centre before mitosis and with the acentrosomal extranuclear spindle during mitosis. Analysis of tubulin post-translational modifications identifies two populations of spindle microtubules - polar acetylated microtubules, whose length is constant, and central tyrosinated microtubules, which elongate during chromosome segregation. During cell division a microtubule-rich structure forms along the dorsal-ventral axis, associated with the site of cytokinesis, consistent with a cytokinetic mechanism that is independent of the actomyosin ring typical of animal and yeast cells.

New plastids, old proteins: repeated endosymbiotic acquisitions in kareniacean dinoflagellates.

Dinoflagellates are a diverse group of ecologically significant micro-eukaryotes that can serve as a model system for plastid symbiogenesis due to their susceptibility to plastid loss and replacement via serial endosymbiosis. Kareniaceae harbor fucoxanthin-pigmented plastids instead of the ancestral peridinin-pigmented ones and support them with a diverse range of nucleus-encoded plastid-targeted proteins originating from the haptophyte endosymbiont, dinoflagellate host, and/or lateral gene transfers (LGT). Here, we present predicted plastid proteomes from seven distantly related kareniaceans in three genera (Karenia, Karlodinium, and Takayama) and analyze their evolutionary patterns using automated tree building and sorting. We project a relatively limited ( ~ 10%) haptophyte signal pointing towards a shared origin in the family Chrysochromulinaceae. Our data establish significant variations in the functional distributions of these signals, emphasizing the importance of micro-evolutionary processes in shaping the chimeric proteomes. Analysis of plastid genome sequences recontextualizes these results by a striking finding the extant kareniacean plastids are in fact not all of the same origin, as two of the studied species (Karlodinium armiger, Takayama helix) possess plastids from different haptophyte orders than the rest.

Lineage dynamics of the endosymbiotic cell type in the soft coral Xenia.

Many corals harbour symbiotic dinoflagellate algae. The algae live inside coral cells in a specialized membrane compartment known as the symbiosome, which shares the photosynthetically fixed carbon with coral host cells while host cells provide inorganic carbon to the algae for photosynthesis1. This endosymbiosis-which is critical for the maintenance of coral reef ecosystems-is increasingly threatened by environmental stressors that lead to coral bleaching (that is, the disruption of endosymbiosis), which in turn leads to coral death and the degradation of marine ecosystems2. The molecular pathways that orchestrate the recognition, uptake and maintenance of algae in coral cells remain poorly understood. Here we report the chromosome-level genome assembly of a Xenia species of fast-growing soft coral3, and use this species as a model to investigate coral-alga endosymbiosis. Single-cell RNA sequencing identified 16 cell clusters, including gastrodermal cells and cnidocytes, in Xenia sp. We identified the endosymbiotic cell type, which expresses a distinct set of genes that are implicated in the recognition, phagocytosis and/or endocytosis, and maintenance of algae, as well as in the immune modulation of host coral cells. By coupling Xenia sp. regeneration and single-cell RNA sequencing, we observed a dynamic lineage progression of the endosymbiotic cells. The conserved genes associated with endosymbiosis that are reported here may help to reveal common principles by which different corals take up or lose their endosymbionts.

Dinoflagellate vertical migration fuels an intense red tide.

Harmful algal blooms (HABs) are increasing globally, causing economic, human health, and ecosystem harm. In spite of the frequent occurrence of HABs, the mechanisms responsible for their exceptionally high biomass remain imperfectly understood. A 50-y-old hypothesis posits that some dense blooms derive from dinoflagellate motility: organisms swim upward during the day to photosynthesize and downward at night to access deep nutrients. This allows dinoflagellates to outgrow their nonmotile competitors. We tested this hypothesis with in situ data from an autonomous, ocean-wave-powered vertical profiling system. We showed that the dinoflagellate Lingulodinium polyedra's vertical migration led to depletion of deep nitrate during a 2020 red tide HAB event. Downward migration began at dusk, with the maximum migration depth determined by local nitrate concentrations. Losses of nitrate at depth were balanced by proportional increases in phytoplankton chlorophyll concentrations and suspended particle load, conclusively linking vertical migration to the access and assimilation of deep nitrate in the ocean environment. Vertical migration during the red tide created anomalous biogeochemical conditions compared to 70 y of climatological data, demonstrating the capacity of these events to temporarily reshape the coastal ocean's ecosystem and biogeochemistry. Advances in the understanding of the physiological, behavioral, and metabolic dynamics of HAB-forming organisms from cutting-edge observational techniques will improve our ability to forecast HABs and mitigate their consequences in the future.

Choanoflagellates alongside diverse uncultured predatory protists consume the abundant open-ocean cyanobacterium Prochlorococcus.

Prochlorococcus is a key member of open-ocean primary producer communities. Despite its importance, little is known about the predators that consume this cyanobacterium and make its biomass available to higher trophic levels. We identify potential predators along a gradient wherein Prochlorococcus abundance increased from near detection limits (coastal California) to >200,000 cells mL-1 (subtropical North Pacific Gyre). A replicated RNA-Stable Isotope Probing experiment involving the in situ community, and labeled Prochlorococcus as prey, revealed choanoflagellates as the most active predators of Prochlorococcus, alongside a radiolarian, chrysophytes, dictyochophytes, and specific MAST lineages. These predators were not appropriately highlighted in multiyear conventional 18S rRNA gene amplicon surveys where dinoflagellates and other taxa had highest relative amplicon abundances across the gradient. In identifying direct consumers of Prochlorococcus, we reveal food-web linkages of individual protistan taxa and resolve routes of carbon transfer from the base of marine food webs.

A predatory gastrula leads to symbiosis-independent settlement in Aiptasia.

The planula larvae of the sea anemone Aiptasia have so far not been reported to complete their life cycle by undergoing metamorphosis into adult forms. This has been a major obstacle in their use as a model for coral-dinoflagellate endosymbiosis. Here, we show that Aiptasia larvae actively feed on crustacean nauplii, displaying a preference for live prey. This feeding behavior relies on functional stinging cells, indicative of complex neuronal control. Regular feeding leads to significant size increase, morphological changes, and efficient settlement around 14 d postfertilization. Surprisingly, the presence of dinoflagellate endosymbionts does not affect larval growth or settlement dynamics but is crucial for sexual reproduction. Our findings finally close Aiptasia's life cycle and highlight the functional nature of its larvae, as in Haeckel's Gastrea postulate, yet reveal its active carnivory, thus contributing to our understanding of early metazoan evolution.

Conspicuous chloroplast with light harvesting-photosystem I/II megacomplex in marine Prorocentrum cordatum.

Marine photosynthetic (micro)organisms drive multiple biogeochemical cycles and display a large diversity. Among them, the bloom-forming, free-living dinoflagellate Prorocentrum cordatum CCMP 1329 (formerly P. minimum) stands out with its distinct cell biological features. Here, we obtained insights into the structural properties of the chloroplast and the photosynthetic machinery of P. cordatum using microscopic and proteogenomic approaches. High-resolution FIB/SEM analysis revealed a single large chloroplast (∼40% of total cell volume) with a continuous barrel-like structure, completely lining the inner face of the cell envelope and enclosing a single reticular mitochondrium, the Golgi apparatus, as well as diverse storage inclusions. Enriched thylakoid membrane fractions of P. cordatum were comparatively analyzed with those of the well-studied model-species Arabidopsis (Arabidopsis thaliana) using 2D BN DIGE. Strikingly, P. cordatum possessed a large photosystem-light harvesting megacomplex (>1.5 MDa), which is dominated by photosystems I and II (PSI, PSII), chloroplast complex I, and chlorophyll a-b binding light harvesting complex proteins. This finding parallels the absence of grana in its chloroplast and distinguishes from the predominant separation of PSI and PSII complexes in A. thaliana, indicating a different mode of flux balancing. Except for the core elements of the ATP synthase and the cytb6f-complex, the composition of the other complexes (PSI, PSII, and pigment-binding proteins, PBPs) of P. cordatum differed markedly from those of A. thaliana. Furthermore, a high number of PBPs was detected, accounting for a large share of the total proteomic data (∼65%) and potentially providing P. cordatum with flexible adaptation to changing light regimes.

A widespread coral-infecting apicomplexan with chlorophyll biosynthesis genes.

Apicomplexa is a group of obligate intracellular parasites that includes the causative agents of human diseases such as malaria and toxoplasmosis. Apicomplexans evolved from free-living phototrophic ancestors, but how this transition to parasitism occurred remains unknown. One potential clue lies in coral reefs, of which environmental DNA surveys have uncovered several lineages of uncharacterized basally branching apicomplexans1,2. Reef-building corals have a well-studied symbiotic relationship with photosynthetic Symbiodiniaceae dinoflagellates (for example, Symbiodinium3), but the identification of other key microbial symbionts of corals has proven to be challenging4,5. Here we use community surveys, genomics and microscopy analyses to identify an apicomplexan lineage-which we informally name 'corallicolids'-that was found at a high prevalence (over 80% of samples, 70% of genera) across all major groups of corals. Corallicolids were the second most abundant coral-associated microeukaryotes after the Symbiodiniaceae, and are therefore core members of the coral microbiome. In situ fluorescence and electron microscopy confirmed that corallicolids live intracellularly within the tissues of the coral gastric cavity, and that they possess apicomplexan ultrastructural features. We sequenced the genome of the corallicolid plastid, which lacked all genes for photosystem proteins; this indicates that corallicolids probably contain a non-photosynthetic plastid (an apicoplast6). However, the corallicolid plastid differs from all other known apicoplasts because it retains the four ancestral genes that are involved in chlorophyll biosynthesis. Corallicolids thus share characteristics with both their parasitic and their free-living relatives, which suggests that they are evolutionary intermediates and implies the existence of a unique biochemistry during the transition from phototrophy to parasitism.

Orchestrated translation specializes dinoflagellate metabolism three times per day.

Many cells specialize for different metabolic tasks at different times over their normal ZT cycle by changes in gene expression. However, in most cases, circadian gene expression has been assessed at the mRNA accumulation level, which may not faithfully reflect protein synthesis rates. Here, we use ribosome profiling in the dinoflagellate Lingulodinium polyedra to identify thousands of transcripts showing coordinated translation. All of the components in carbon fixation are concurrently regulated at ZT0, predicting the known rhythm of carbon fixation, and many enzymes involved in DNA replication are concurrently regulated at ZT12, also predicting the known rhythm in this process. Most of the enzymes in glycolysis and the TCA cycle are also regulated together, suggesting rhythms in these processes as well. Surprisingly, a third cluster of transcripts show peak translation at approximately ZT16, and these transcripts encode enzymes involved in transcription, translation, and amino acid biosynthesis. The latter has physiological consequences, as measured free amino acid levels increase at night and thus represent a previously undocumented rhythm in this model. Our results suggest that ribosome profiling may be a more accurate predictor of changed metabolic state than transcriptomics.

A decade of dinoflagellate genomics illuminating an enigmatic eukaryote cell.

Dinoflagellates are a remarkable group of protists, not only for their association with harmful algal blooms and coral reefs but also for their numerous characteristics deviating from the rules of eukaryotic biology. Genome research on dinoflagellates has lagged due to their immense genome sizes in most species (~ 1-250 Gbp). Nevertheless, the last decade marked a fruitful era of dinoflagellate genomics, with 27 genomes sequenced and many insights attained. This review aims to synthesize information from these genomes, along with other omic data, to reflect on where we are now in understanding dinoflagellates and where we are heading in the future. The most notable insights from the decade-long genomics work include: (1) dinoflagellate genomes have been expanded in multiple times independently, probably by a combination of rampant retroposition, accumulation of repetitive DNA, and genome duplication; (2) Symbiodiniacean genomes are highly divergent, but share about 3,445 core unigenes concentrated in 219 KEGG pathways; (3) Most dinoflagellate genes are encoded unidirectionally and are not intron-poor; (4) The dinoflagellate nucleus has undergone extreme evolutionary changes, including complete or nearly complete loss of nucleosome and histone H1, and acquisition of dinoflagellate viral nuclear protein (DVNP); (5) Major basic nuclear protein (MBNP), histone-like protein (HLP), and bacterial HU-like protein (HCc) belong to the same protein family, and MBNP can be the unifying name; (6) Dinoflagellate gene expression is regulated by poorly understood mechanisms, but microRNA and other epigenetic mechanisms are likely important; (7) Over 50% of dinoflagellate genes are "dark" and their functions remain to be deciphered using functional genetics; (8) Initial insights into the genomic basis of parasitism and mutualism have emerged. The review then highlights functionally unique and interesting genes. Future research needs to obtain a finished genome, tackle large genomes, characterize the unknown genes, and develop a quantitative molecular ecological model for addressing ecological questions.

The genome of a giant clam zooxanthella (Cladocopium infistulum) offers few clues to adaptation as an extracellular symbiont with high thermotolerance.

BACKGROUND: Cladocopium infistulum (Symbiodiniaceae) is a dinoflagellate specialized to live in symbiosis with western Pacific giant clams (Tridacnidae). Unlike coral-associated symbionts, which reside within the host cells, C. infistulum inhabits the extracellular spaces of the clam's digestive diverticula. It is phylogenetically basal to a large species complex of stress-tolerant Cladocopium, many of which are associated with important reef-building corals in the genus Porites. This close phylogenetic relationship may explain why C. infistulum exhibits high thermotolerance relative to other tridacnid symbionts. Moreover, past analyses of microsatellite loci indicated that Cladocopium underwent whole-genome duplication prior to the adaptive radiations that led to its present diversity. RESULTS: A draft genome assembly of C. infistulum was produced using long- and short-read sequences to explore the genomic basis for adaptations underlying thermotolerance and extracellular symbiosis among dinoflagellates and to look for evidence of genome duplication. Comparison to three other Cladocopium genomes revealed no obvious over-representation of gene groups or families whose functions would be important for maintaining C. infistulum's unique physiological and ecological properties. Preliminary analyses support the existence of partial or whole-genome duplication among Cladocopium, but additional high-quality genomes are required to substantiate these findings. CONCLUSION: Although this investigation of Cladocopium infistulum revealed no patterns diagnostic of heat tolerance or extracellular symbiosis in terms of overrepresentation of gene functions or genes under selection, it provided a valuable genomic resource for comparative analyses. It also indicates that ecological divergence among Cladocopium species, and potentially among other dinoflagellates, is partially governed by mechanisms other than gene content. Thus, additional high-quality, multiomic data are needed to explore the molecular basis of key phenotypes among symbiotic microalgae.

Flexibility in coral-algal symbiosis is positively correlated with the host geographic range.

Generalists are thought to adapt to broader ecological conditions compared to less flexible specialists. However, few studies have systematically tested what ecological or life-history traits are associated with organisms' ecological flexibility. Here, we used stony corals to test the relative effects of host traits and ecological factors on corals' flexibility to form photosymbioses with algae. We analysed data from 211 stony coral species to test if coral's geographic distribution, depth range, symbiont transmission mode or colony morphology predict coral-algal flexibility. We report a novel positive correlation between coral-algal flexibility and coral species' geographic range. Symbiont transmission mode was also a predictor of flexibility, albeit the result is less robust against sampling bias. Coral depth range and morphology did not show significant effects. We highlight that host-symbiont dispersal abilities, interactions and evolutionary histories likely contribute to the observed patterns. We urge conservation efforts to consider the ecological implications of coral-algal flexibility.

Interpreting the complexities of the plastid genome in dinoflagellates: a mini-review of recent advances.

Photosynthetic dinoflagellates play crucial roles in global primary production and carbon fixation. Despite their success in filling various ecological niches, numerous mysteries about their plastid evolution and plastid genomes remain unsolved. The plastid genome of dinoflagellates presents one of the most complex lineages in the biological realm, mainly due to multiple endosymbiotic plastid events in their evolutionary history. Peridinin-containing dinoflagellates possess the most reduced and fragmented genome, with only a few genes located on multiple "minicircles", whereas replacement plastids in dinoflagellate lineages have undergone different degrees of endosymbiotic gene transfer. Recent advancements in high-throughput sequencing have improved our understanding of plastid genomes and plastid-encoded gene expression in many dinoflagellate species. Plastid transcripts of dinoflagellates exhibit two unconventional processing pathways: the addition of a 3' poly(U) tail and substitutional RNA editing. These pathways are widely employed across dinoflagellate lineages, which are possibly retained from the ancestral peridinin plastid. This mini-review summarizes the developments in the plastid genomes of dinoflagellates and pinpoints the research areas that necessitate further exploration, aiming to provide valuable insights into plastid evolution in these fascinating and important organisms.

Ribosome profiling in the Symbiodiniacean dinoflagellate Fugacium kawagutii shows coordinated protein synthesis of enzymes in different pathways at different times of day.

Dinoflagellates respond to daily changes in light and dark by changes in cellular metabolism, yet the mechanisms used are still unclear. For example, Fugacium (previously Symbiodinium) kawagutii shows little difference in the transcriptome between day and night suggesting little transcriptional control over gene expression. Here, we have performed ribosome profiling at 2 h intervals over a daily light-dark cycle to assess the degree to which protein synthesis rates might change over the daily cycle. The number of F. kawagutii coding sequences with significant differences in the number of ribosome-protected fragments (RPF) over the 24-h cycle was 2923 using JTK_Cycle and 3655 using ECHO. The majority of the regulated transcripts showed peak translation at the onset of the dark period. The regulated sequences were assigned to different KEGG pathways and transcripts that were translated at roughly the same time were termed concurrently regulated. Both analyses revealed concurrent regulation of many transcripts whose gene products were involved in spliceosome or lysosome biogenesis with peak translation rates around the onset of the dark period, while others, involved in nitrate metabolism and ribosomal proteins, were preferentially translated around the onset of the day phase or the end of the night phase, respectively. In addition, some sequences involved in DNA synthesis were preferentially translated at the end of the day. We conclude that light-dark cycles seem able to synchronize translation of some transcripts encoding proteins involved in a range of different cellular processes, and propose that these changes may help the cells adapt and alter their metabolism as a function of the time of day.

Starvation differentially affects gene expression, immunity and pathogen susceptibility across symbiotic states in a model cnidarian.

Mutualistic symbioses between cnidarians and photosynthetic algae are modulated by complex interactions between host immunity and environmental conditions. Here, we investigate how symbiosis interacts with food limitation to influence gene expression and stress response programming in the sea anemone Exaiptasia pallida (Aiptasia). Transcriptomic responses to starvation were similar between symbiotic and aposymbiotic Aiptasia; however, aposymbiotic anemone responses were stronger. Starved Aiptasia of both symbiotic states exhibited increased protein levels of immune-related transcription factor NF-κB, its associated gene pathways, and putative target genes. However, this starvation-induced increase in NF-κB correlated with increased immunity only in symbiotic anemones. Furthermore, starvation had opposite effects on Aiptasia susceptibility to pathogen and oxidative stress challenges, suggesting distinct energetic priorities under food scarce conditions. Finally, when we compared starvation responses in Aiptasia to those of a facultative coral and non-symbiotic anemone, 'defence' responses were similarly regulated in Aiptasia and the facultative coral, but not in the non-symbiotic anemone. This pattern suggests that capacity for symbiosis influences immune responses in cnidarians. In summary, expression of certain immune pathways-including NF-κB-does not necessarily predict susceptibility to pathogens, highlighting the complexities of cnidarian immunity and the influence of symbiosis under varying energetic demands.

Trade-off between photosymbiosis and innate immunity influences cnidarian's response to pathogenic bacteria.

Mutualistic relationships with photosynthetic organisms are common in cnidarians, which form an intracellular symbiosis with dinoflagellates in the family Symbiodiniaceae. The establishment and maintenance of these symbionts are associated with the suppression of key host immune factors. Because of this, there are potential trade-offs between the nutrition that cnidarian hosts gain from their symbionts and their ability to successfully defend themselves from pathogens. To investigate these potential trade-offs, we utilized the facultatively symbiotic polyps of the upside-down jellyfish Cassiopea xamachana and exposed aposymbiotic and symbiotic polyps to the pathogen Serratia marcescens. Symbiotic polyps had a lower probability of survival following S. marcescens exposure. Gene expression analyses 24 hours following pathogen exposure indicate that symbiotic animals mounted a more damaging immune response, with higher levels of inflammation and oxidative stress likely resulting in more severe disruptions to cellular homeostasis. Underlying this more damaging immune response may be differences in constitutive and pathogen-induced expression of immune transcription factors between aposymbiotic and symbiotic polyps rather than broadscale immune suppression during symbiosis. Our findings indicate that in facultatively symbiotic polyps, hosting symbionts limits C. xamachana's ability to survive pathogen exposure, indicating a trade-off between symbiosis and immunity that has potential implications for coral disease research.

PACMan: A software package for automated single-cell chlorophyll fluorometry.

Microalgae, small photosynthetic unicells, are of great interest to ecology, ecotoxicology and biotechnology and there is a growing need to investigate the ability of cells to photosynthesize under variable conditions. Current strategies involve hand-operated pulse-amplitude-modulated (PAM) chlorophyll fluorimeters, which can provide detailed insights into the photophysiology of entire populations- or individual cells of microalgae but are typically limited in their throughput. To increase the throughput of a commercially available MICROSCOPY-PAM system, we present the PAM Automation Control Manager ('PACMan'), an open-source Python software package that automates image acquisition, microscopy stage control and the triggering of external hardware components. PACMan comes with a user-friendly graphical user interface and is released together with a stand-alone tool (PAMalysis) for the automated calculation of per-cell maximum quantum efficiencies (= Fv /Fm ). Using these two software packages, we successfully tracked the photophysiology of >1000 individual cells of green algae (Chlamydomonas reinhardtii) and dinoflagellates (genus Symbiodiniaceae) within custom-made microfluidic devices. Compared to the manual operation of MICROSCOPY-PAM systems, this represents a 10-fold increase in throughput. During experiments, PACMan coordinated the movement of the microscope stage and triggered the MICROSCOPY-PAM system to repeatedly capture high-quality image data across multiple positions. Finally, we analyzed single-cell Fv /Fm with the manufacturer-supplied software and PAMalysis, demonstrating a median difference <0.5% between both methods. We foresee that PACMan, and its auxiliary software package will help increase the experimental throughput in a range of microalgae studies currently relying on hand-operated MICROSCOPY-PAM technologies.

The microbiome dynamics and interaction of endosymbiotic Symbiodiniaceae and fungi are associated with thermal bleaching susceptibility of coral holobionts.

The thermal bleaching percentage of coral holobionts shows interspecific differences under heat-stress conditions, which are closely related to the coral-associated microbiome. However, the ecological effects of community dynamics and interactions between Symbiodiniaceae and fungi on coral thermal bleaching susceptibility remain unclear. In this study, we analyzed the diversity, community structure, functions, and potential interaction of Symbiodiniaceae and fungi among 18 coral species from a high thermal bleaching risk atoll using next-generation sequencing. The results showed that heat-tolerant C3u sub-clade and Durusdinium dominated the Symbiodiniaceae community of corals and that there were no core amplicon sequence variants in the coral-associated fungal community. Fungal richness and the abundance of confirmed functional animal-plant pathogens were significantly positively correlated with the coral thermal bleaching percentage. Fungal indicators, including Didymellaceae, Chaetomiaceae, Schizophyllum, and Colletotrichum, were identified in corals. Each coral species had a complex Symbiodiniaceae-fungi interaction network (SFIN), which was driven by the dominant Symbiodiniaceae sub-clades. The SFINs of coral holobionts with low thermal bleaching susceptibility exhibited low complexity and high betweenness centrality. These results indicate that the extra heat tolerance of coral in Huangyan Island may be linked to the high abundance of heat-tolerant Symbiodiniaceae. Fungal communities have high interspecific flexibility, and the increase of fungal diversity and pathogen abundance was correlated with higher thermal bleaching susceptibility of corals. Moreover, fungal indicators were associated with the degrees of coral thermal bleaching susceptibility, including both high and intermediate levels. The topological properties of SFINs suggest that heat-tolerant coral have limited fungal parasitism and strong microbial network resilience.IMPORTANCEGlobal warming and enhanced marine heatwaves have led to a rapid decline in coral reef ecosystems worldwide. Several studies have focused on the impact of coral-associated microbiomes on thermal bleaching susceptibility in corals; however, the ecological functions and interactions between Symbiodiniaceae and fungi remain unclear. We investigated the microbiome dynamics and potential interactions of Symbiodiniaceae and fungi among 18 coral species in Huangyan Island. Our study found that the Symbiodiniaceae community of corals was mainly composed of heat-tolerant C3u sub-clade and Durusdinium. The increase in fungal diversity and pathogen abundance has close associations with higher coral thermal bleaching susceptibility. We first constructed an interaction network between Symbiodiniaceae and fungi in corals, which indicated that restricting fungal parasitism and strong interaction network resilience would promote heat acclimatization of corals. Accordingly, this study provides insights into the role of microorganisms and their interaction as drivers of interspecific differences in coral thermal bleaching.