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ABSTRACT While in hospite Symbiodiniaceae dinoflagellates are essential for coral health, ambient free-living counterparts are crucial for coral recruitment and resilience. Comparing free-living and in hospite Symbiodiniaceae communities can potentially provide insights into endosymbiont acquisition and recurrent recruitment in bleaching recovery. In this study, we studied coral-endosymbiotic and ambient free-living Symbiodiniaceae communities in the South China Sea. We collected samples from 183 coral and ambient plankton samples and conducted metabarcoding to investigate the diversity distribution, driving factors, and assembly mechanisms of the two groups of Symbiodiniaceae. Results revealed Cladocopium C1 and Durusdinium D1 as dominant genotypes. We detected a higher genotypic diversity in free-living than in hospite symbiodiniacean communities, but with shared dominant genotypes. This indicates a genetically diverse pool of Symbiodiniaceae available for recruitment by corals. Strikingly, we found that the cooler area had more Symbiodiniaceae thermosensitive genotypes, whereas the warmer area had more Symbiodiniaceae thermotolerant genotypes. Furthermore, in hospite and free-living Symbiodiniaceae communities were similarly affected by environmental factors, but shaped by different assembly mechanisms. The in hospite communities were controlled mainly by deterministic processes, whereas the ambient communities by stochastic processes. This study sheds light on the genetic diversity of source environmental Symbiodiniaceae and differential assembly mechanisms influencing Symbiodiniaceae inside and outside corals.IMPORTANCESymbiodiniaceae dinoflagellates play a pivotal role as key primary producers within coral reef ecosystems. Coral-endosymbiotic Symbiodiniaceae communities have been extensively studied, but relatively little work has been reported on the free-living Symbiodiniaceae community. Conducting a comparative analysis between sympatric coral-endosymbiotic and free-living Symbiodiniaceae communities can potentially enhance the understanding of how endosymbiont communities change in response to changing environments and the mechanisms driving these changes. Our findings shed light on the genetic diversity of source environmental Symbiodiniaceae and differential assembly mechanisms shaping free-living and in hospite Symbiodiniaceae communities, with implications in evaluating the adaptive and resilient capacity of corals in response to future climate change.

Dinoflagellates are ecologically important and essential to corals and other cnidarians as phytosymbionts, but their photosystems had been underexplored. Recently, photosystem I (PSI) of dinoflagellate Symbiodinium sp. was structurally characterized using cryo-Electron Microscopy (cryo-EM). These analyses revealed a distinct organization of the PSI supercomplex, including two previously unidentified subunits, PsaT and PsaU, and shed light on interactions between light harvesting antenna proteins and the PSI core. These results have implications with respect to the evolution of dinoflagellates and their association with cnidarians.

ABSTRACTLineage-wise physiological activities of plankton communities in the ocean are important but challenging to characterize. Here, we conducted whole-assemblage metatranscriptomic profiling at continental shelf and slope sites in the South China Sea to investigate carbon fixation potential in different lineages. RuBisCO expression, the proxy of Calvin carbon fixation (CCF) potential, was mainly contributed by Bacillariophyta, Chlorophyta, Cyanobacteria, and Haptophyta, which was differentially affected by environmental factors among lineages. CCF potential exhibited positive or negative correlations with phagotrophy gene expression, suggesting phagotrophy possibly enhances or complements CCF. Our data also reveal significant non-Calvin carbon fixation (NCF) potential, as indicated by the active expression of genes in all five currently recognized NCF pathways, mainly contributed by Flavobacteriales, Alteromonadales, and Oceanospirillales. Furthermore, in Flavobacteriales, Alteromonadales, Pelagibacterales, and Rhodobacterales, NCF potential was positively correlated with proton-pump rhodopsin (PPR) expression, suggesting that NCF might be energetically supported by PPR. The novel insights into the lineage-differential potential of carbon fixation, widespread mixotrophy, and PPR as an energy source for NCF lay a methodological and informational foundation for further research to understand carbon fixation and the trophic landscape in the ocean.IMPORTANCEMarine plankton plays an important role in global carbon cycling and climate regulation. Phytoplankton and cyanobacteria fix CO2 to produce organic compounds using solar energy and mainly by the Calvin cycle, whereas autotrophic bacteria and archaea may fix CO2 by non-Calvin cycle carbon fixation pathways. How active individual lineages are in carbon fixation and mixotrophy, and what energy source bacteria may employ in non-Calvin carbon fixation, in a natural plankton assemblage are poorly understood and underexplored. Using metatranscriptomics, we studied carbon fixation in marine plankton with lineage resolution in tropical marginal shelf and slope areas. Based on the sequencing results, we characterized the carbon fixation potential of different lineages and assessed Calvin- and non-Calvin- carbon fixation activities and energy sources. Data revealed a high number of unigenes (4.4 million), lineage-dependent differential potentials of Calvin carbon fixation and responses to environmental conditions, major contributors of non-Calvin carbon fixation, and their potential energy source.

A new study reports synergistic inhibitory effects of ocean acidification and phosphate limitation on the nitrogen-fixing capacity of a globally important cyanobacterium species. Inspired by the report, this Comment presents the complexity of how ocean acidification and phosphate limitation affect phytoplankton physiologies and species beyond nitrogen fixation and cyanobacteria, and what future research is needed to address the remaining crucial questions.

Microbial proton-pump rhodopsin (PPR)-based phototrophy, a light-harvesting mechanism different from chlorophyll-based photosystems, may contribute significantly to solar energy entry into the marine ecosystem. PPR transforms solar energy into cellular energy that is used for various metabolic processes in the cells or flagellar movement. Although rhodopsins or their encoding genes have been documented in a wide phylogenetic range of cultured dinoflagellates, information is limited about how widespread and how spatiotemporally dynamical dinoflagellate PPR (DiPPR) are in natural marine ecosystems. In this study, we investigated DiPPR in Long Island Sound (LIS), a temperate estuary of the Atlantic Ocean between Connecticut and Long Island, New York, USA. We isolated six novel full-length dinoflagellate proton-pump rhodopsin cDNAs, expanding the DiPPR database that is crucial to PPR research. Based on these new sequences and existing sequences of DiPPR, we designed primers and conducted quantitative PCR and sequencing to determine the abundance and diversity of DiPPR genes spatially and temporally throughout a year in the water samples collected from LIS. DiPPR genes were found year-round and throughout LIS but with higher abundances in the eutrophic Western Sound and in April and July. The gene diversity data suggest that there are at least five distinct rhodopsin-harboring groups of dinoflagellates throughout the year. The abundance of DiPPR genes, measured as copy number per mL of seawater, appeared not to be influenced by water temperature or nitrogen nutrient concentration but exhibited weak negative correlations with orthophosphate concentration and salinity and a positive correlation with the abundance of DiPPR-harboring dinoflagellates. This first quantitative profiling of PPR in natural plankton reveals the prevalence and dynamics of this plastid-independent photoenergy harvesting mechanism in a temperate estuary and provides efficient DiPPR primers potentially useful for future research. Furthermore, this study shed light on the potential role of DiPPR in phosphor nutrition and dinoflagellate population, which warrants further studies.

ABSTRACT Dissolved organic phosphorus (DOP) is a potential source of aquatic eutrophication and pollution because it can potentially stimulate growth in some species and inhibit growth in other species of algae, the foundation of the marine ecosystem. Inositol hexaphosphate (also named phytic acid or PA), an abundant organophosphate, is presumably ubiquitous in the marine environment, but how it affects marine primary producers is poorly understood. Here, we investigated the bioavailability of this DOP to the cosmopolitan coccolithophore Emiliania huxleyi. Our results showed that E. huxleyi cells can take up PA and dissolved inorganic phosphorus (DIP) simultaneously. Absorbed PA can efficiently support algal growth, producing cell yield between DIP and phosphorus (P)-depleted conditions. Accordingly, PA supply as the sole P source highly influences cellular metabolism and nutrient stoichiometry. Particularly, PA-grown cultures exhibited enhanced carbon fixation, increased lipid content, activated energy metabolism, and induced nitrogen assimilation. However, our data suggest that PA may also exert some levels of toxic effects on E. huxleyi. This study provides novel insights into the variable effects of a DOP on marine phytoplankton, which will inform new inquiries about how the complex DOP constituencies in the ocean will shape phytoplankton community structure and function. IMPORTANCE The dissolved organic phosphorus (DOP) utilization in phytoplankton plays vital roles in cellular P homeostasis, P-nutrient niche, and the dynamics of community structure in marine ecosystems, but its mechanisms, potentially varying with species, are far from clear. In this study, we investigated the utilization of a widespread DOP species, which is commonly produced by plants (land plants and marine macrophytes) and released into coastal areas, in a globally distributed bloom-forming coccolithophore species in various phosphorus environments. Using a combination of physiological and transcriptomic measurements and analyses, our experimental results revealed the complex mechanism and two-sided effects of DOP (major algal growth-supporting and minor toxic effects) in this species, providing a novel perspective on phytoplankton nutrient regulation.

Abstract The mixoplankton green Noctiluca scintillans (gNoctiluca) is known to form extensive green tides in tropical coastal ecosystems prone to eutrophication. In the Arabian Sea, their recent appearance and annual recurrence have upended an ecosystem that was once exclusively dominated by diatoms. Despite evidence of strong links to eutrophication, hypoxia and warming, the mechanisms underlying outbreaks of this mixoplanktonic dinoflagellate remain uncertain. Here we have used eco-physiological measurements and transcriptomic profiling to ascribe gNoctiluca’s explosive growth during bloom formation to the form of sexual reproduction that produces numerous gametes. Rapid growth of gNoctiluca coincided with active ammonium and phosphate release from gNoctiluca cells, which exhibited high transcriptional activity of phagocytosis and metabolism generating ammonium. This grazing-driven nutrient flow ostensibly promotes the growth of phytoplankton as prey and offers positive support successively for bloom formation and maintenance. We also provide the first evidence that the host gNoctiluca cell could be manipulating growth of its endosymbiont population in order to exploit their photosynthetic products and meet critical energy needs. These findings illuminate gNoctiluca’s little known nutritional and reproductive strategies that facilitate its ability to form intense and expansive gNoctiluca blooms to the detriment of regional water, food and the socio-economic security in several tropical countries.

ABSTRACT The development and demise of a harmful algal bloom (HAB) are generally regulated by multiple processes; identifying specific critical drivers for a specific bloom is important yet challenging. Here, we conducted a whole-assemblage molecular ecological study on a dinoflagellate bloom to address the hypothesis that energy and nutrient acquisition, defense against grazing and microbial attacks, and sexual reproduction are critical to the rise and demise of the bloom. Microscopic and molecular analyses identified the bloom-causing species as Karenia longicanalis and showed that the ciliate Strombidinopsis sp. was dominant in a nonbloom plankton community, whereas the diatom Chaetoceros sp. dominated the after-bloom community, along with remarkable shifts in the community structure for both eukaryotes and prokaryotes. Metatranscriptomic analysis indicated that heightened energy and nutrient acquisition in K. longicanalis significantly contributed to bloom development. In contrast, active grazing by the ciliate Strombidinopsis sp. and attacks by algicidal bacteria (Rhodobacteracea, Cryomorphaceae, and Rhodobacteracea) and viruses prevented (at nonbloom stage) or collapsed the bloom (in after-bloom stage). Additionally, nutrition competition by the Chaetoceros diatoms plausibly contributed to bloom demise. The findings suggest the importance of energy and nutrients in promoting this K. longicanalis bloom and the failure of antimicrobial defense and competition of diatoms as the major bloom suppressor and terminator. This study provides novel insights into bloom-regulating mechanisms and the first transcriptomic data set of K. longicanalis, which will be a valuable resource and essential foundation for further elucidation of bloom regulators of this and related species of Kareniaceae in the future. IMPORTANCE HABs have increasingly occurred and impacted human health, aquatic ecosystems, and coastal economies. Despite great efforts, the factors that drive the development and termination of a bloom are poorly understood, largely due to inadequate in situ data about the physiology and metabolism of the causal species and the community. Using an integrative molecular ecological approach, we determined that heightened energy and nutrient acquisition promoted the bloom, while resource allocation in defense and failure to defend against grazing and microbial attacks likely prevented or terminated the bloom. Our findings reveal the differential roles of multiple abiotic and biotic environmental factors in driving the formation or demise of a toxic dinoflagellate bloom, suggesting the importance of a balanced biodiverse ecosystem in preventing a dinoflagellate bloom. The study also demonstrates the power of whole-assemblage metatranscriptomics coupled to DNA barcoding in illuminating plankton ecological processes and the underlying species and functional diversities.

Phosphoesters are a dominant component of marine dissolved organic phosphorus (DOP) and an important source of the phosphorus nutrient for phytoplankton, but the molecular mechanisms of their utilization by phytoplankton are divergent and poorly understood. In this study, we used the model diatom Phaeodactylum tricornutum to investigate and compare the utilization mechanisms of two different phosphoesters, adenosine triphosphate (ATP) and glucose-6-phosphate (G6P). We found that ATP and G6P can both be efficiently used to support the growth of P. tricornutum. Cells grown on ATP or G6P showed lower pigment contents and photosynthetic rates but higher cellular lipids relative to those grown on NaH2PO4 (DIP). Surprisingly, in neither the ATP nor the G6P group were significant increases in whole-cell alkaline phosphatase (AP) activity detected, suggesting that utilization of both DOPs was not reliant on extracellular AP. Yet, ATP-grown cultures released DIP into the medium (i.e., ATP hydrolyzed extracellularly) whereas G6P-grown cultures did not. Furthermore, transcriptomic and RT-qPCR results showed that the gene encoding 5’ nucleotidase (5NT) in the ATP group and PhoD in the G6P group was upregulated. These results indicated that different pathways are involved in the use of these two DOPs, with ATP being hydrolyzed extracellularly likely by 5NT (PHATRDRAFT_44177) to release DIP for uptake, and G6P being directly absorbed and hydrolyzed intracellularly likely by PhoD (PHATRDRAFT_45757). Nevertheless, P. tricornutum under ATP and G6P conditions showed more similar transcriptomic profiles to each other than either compared to DIP-grown cultures, indicating similar metabolic functions of these two DOPs. These findings demonstrate that despite the high similarity in transcriptomic response to ATP and G6P conditions, the utilization mechanisms of these phosphoesters in the same species can be totally different, and the lack of AP activity does not necessarily signal the absence of DIP deficiency or the absence of DOP utilization.

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

Initially discovered over 35 years ago in the bacterium Escherichia coli as a defense system against invasion of viral (or other exogenous) DNA into the genome, CRISPR/Cas has ushered in a new era of functional genetics and served as a versatile genetic tool in all branches of life science. CRISPR/Cas has revolutionized the methodology of gene knockout with simplicity and rapidity, but it is also powerful for gene knock-in and gene modification. In the field of marine biology and ecology, this tool has been instrumental in the functional characterization of ‘dark’ genes and the documentation of the functional differentiation of gene paralogs. Powerful as it is, challenges exist that have hindered the advances in functional genetics in some important lineages. This review examines the status of applications of CRISPR/Cas in marine research and assesses the prospect of quickly expanding the deployment of this powerful tool to address the myriad fundamental marine biology and biological oceanography questions.

Using CRISPR-Cas9 mediated-knockout and overexpression analyses, this study shows that a trypsin in the diatom Phaeodactylum tricornutum promotes phosphorus uptake and inhibits nitrogen uptake but its expression is downregulated under nitrogen stress and upregulated under phosphorus stress. Together, the findings suggest this trypsin is a coordinate regulator of nutrient homeostasis.

As a well-known pseudo-persistent environmental pollutant, oxybenzone (BP-3) and its related organic ultraviolet (UV) filters have been verified to directly contribute to the increasing mortality rate of coral reefs. Previous studies have revealed the potential role of symbiotic Symbiodiniaceae in protecting corals from the toxic effects of UV filters. However, the detailed protection mechanism(s) have not been explained. Here, the impacts of BP-3 on the symbiotic Symbiodiniaceae Cladocopium goreaui were explored. C. goreaui cells exhibited distinct cell growth at different BP-3 doses, with increasing growth at the lower concentration (2 mg L–1) and rapid death at a higher concentration (20 mg L–1). Furthermore, C. goreaui cells showed a significant BP-3 uptake at the lower BP-3 concentration. BP-3 absorbing cells exhibited elevated photosynthetic efficiency, and decreased cellular carbon and nitrogen contents. Besides, the derivatives of BP-3 and aromatic amino acid metabolism highly responded to BP-3 absorption and biodegradation. Our physiological and metabolic results reveal that the symbiotic Symbiodiniaceae could resist the toxicity of a range of BP-3 through promoting cell division, photosynthesis, and reprogramming amino acid metabolism. This study provides novel insights into the influences of organic UV filters to coral reef ecosystems, which urgently needs increasing attention and management.

With rapid growth and high lipid contents, microalgae have become promising environmentally friendly candidates for renewable biodiesel and health supplements in our era of global warming and energy depletion. Various pathways have been explored to enhance algal lipid production, especially gene editing. Previously, we found that the functional loss of PhoD-type alkaline phosphatase (AP), a phosphorus-stress indicator in phytoplankton, could lead to increased lipid contents in the model diatom Phaeodactylum tricornutum, but how the AP mutation may change lipid composition remains unexplored. This study addresses the gap in the research and investigates the effects of PhoD-type AP mutation on the lipid composition and metabolic regulation in P. tricornutum using transcriptomic and lipidomic analyses. We observed significantly modified lipid composition and elevated production of fatty acids, lysophosphatidylcholine, lysophosphatidylethanolamine, ceramide, phosphatidylinositol bisphosphate, and monogalactosylmonoacylglycerol after PhoD_45757 mutation. Meanwhile, genes involved in fatty acid biosynthesis were upregulated in mutant cells. Moreover, the mutant exhibited increased contents of ω-3 long-chain polyunsaturated fatty acid (LC-PUFA)-bound phospholipids, indicating that PhoD_45757 mutation could improve the potential bioavailability of PUFAs. Our findings indicate that AP mutation could influence cellular lipid synthesis and probably redirect carbon toward lipid production and further demonstrate that AP mutation is a promising approach for the development of high-value microalgal strains for biomedical and other applications.

Coastal eutrophication and hypoxia remain a persistent environmental crisis despite the great efforts to reduce nutrient loading and mitigate associated environmental damages. Symptoms of this crisis have appeared to spread rapidly, reaching developing countries in Asia with emergences in Southern America and Africa. The pace of changes and the underlying drivers remain not so clear. To address the gap, we review the up-to-date status and mechanisms of eutrophication and hypoxia in global coastal oceans, upon which we examine the trajectories of changes over the 40 years or longer in six model coastal systems with varying socio-economic development statuses and different levels and histories of eutrophication. Although these coastal systems share common features of eutrophication, site-specific characteristics are also substantial, depending on the regional environmental setting and level of social-economic development along with policy implementation and management. Nevertheless, ecosystem recovery generally needs greater reduction in pressures compared to that initiated degradation and becomes less feasible to achieve past norms with a longer time anthropogenic pressures on the ecosystems. While the qualitative causality between drivers and consequences is well established, quantitative attribution of these drivers to eutrophication and hypoxia remains difficult especially when we consider the social economic drivers because the changes in coastal ecosystems are subject to multiple influences and the cause–effect relationship is often non-linear. Such relationships are further complicated by climate changes that have been accelerating over the past few decades. The knowledge gaps that limit our quantitative and mechanistic understanding of the human-coastal ocean nexus are identified, which is essential for science-based policy making. Recognizing lessons from past management practices, we advocate for a better, more efficient indexing system of coastal eutrophication and an advanced regional earth system modeling framework with optimal modules of human dimensions to facilitate the development and evaluation of effective policy and restoration actions.

Knowledge of in situ diet of widespread rotifers is crucial for accurately understanding the trophic position, ecological function, and adaptability to environmental changes in aquatic ecosystems. However, it is challenging to achieve the in situ diet information due to the lack of efficient and comprehensive methods. Here, we investigated the diet composition of Polyarthra in a subtropical lake using high-throughput sequencing (HTS) of a rRNA metabarcode for Polyarthra and ambient water samples. After eliminating Polyarthra sequences, a total of 159 operational taxonomic units (OTUs) from taxa in 15 phyla were detected from Polyarthra gut content samples. Most of the OTUs belong to Chlorophyta, followed by unclassified Fungi, Chrysophyta, Dinoflagellata, Ciliophora, Bacillariophyta, Cryptophyta, Arthropoda, Cercozoa, Mollusca, Apicomplexa, Haptophyta, Amoebozoa, Chordata and other eukaryotes. Our results showed that Polyarthra mainly grazed on Chlorophyta, which may result from the high relative abundance of Chlorophyta in ambient waters. In contrast, Chrysophyceae and Synurophyceae were enriched in Polyarthra’s gut, indicating that this rotifer prefers these taxa as food. Moreover, correlation analysis showed that total nitrogen, transparency, depth, Chlorophyll-a and total phosphorus were key factors for the variation of the eukaryotic community in the Polyarthra gut contents. When the concentration of nutrients in the water environment decreased, Polyarthra shifted from herbivorous feeding to more carnivorous feeding. Thus, Polyarthra is generally omnivorous but preference for Chrysophytes and Synurophytes, and it responds to the environmental changes by adopting a flexible feeding strategy. This could partly explain why the widespread rotifers have apparently wide tolerance toward spatial and environmental changes.

How coral–Symbiodiniaceae mutualistic symbiosis is established, maintained, and disrupted is arguably the most fundamental and central area of coral research. The breakdown of this symbiosis, and consequent coral bleaching, have been frequently attributed to thermal stress, although microbial attack and pollution have also been blamed. Despite the perceived intense and broad research, it is unclear whether all the potential causes have been given adequate attention and whether some important areas have been overlooked. This work aims to comprehensively review the literature on coral and Symbiodiniaceae research and provide a portrait of the current coral research landscape, hence identifying areas that require more research effort. Data of publication output were extracted from the Web of Science (WoS) from 1986 to 2022 by using the keywords “coral” and “Symbiodiniaceae.” A total of 43,089 and 3,191 papers in the coral and Symbiodiniaceae were identified, mostly published after 2002. The journal Coral Reefs was ranked first regarding the total number of publications on coral or Symbiodiniaceae. The USA, Australia, and China were the top three countries in the number of publications. The network co-occurrence analysis of all keywords in coral and Symbiodiniaceae using VOSviewer showed that biodiversity, climate change, nutrient, and survival were the central research areas in coral and Symbiodiniaceae. Among them, climate change was the most invested research field, as revealed by the high proportion of published literature, while nutrient was the most understudied area. Thematic evolution analysis revealed that nutrient enrichment combined with elevated temperature was an emerging research field about coral and Symbiodiniaceae. Besides, nitrogen is currently the most studied nutrient. The findings from this study shed light on the trends of coral and Symbiodiniaceae research in the past 36 years, current research hotspots in the field, and areas that need more research investment going forward.

Kaidian Zhang et al. identify SPX-containing genes in the phytoplankton, Phaeodactylum triconutum, and examine their contribution toward phosphorous regulation. Their results suggest that SPX is a negative regulator of phosphorous uptake, providing a better understanding of how phytoplankton adapt to phosphorous availability in the oceans.

Abstract Genetic differentiations and phylogeographical patterns of small organisms may be shaped by spatial isolation, environmental gradients, and gene flow. However, knowledge about genetic differentiation of rotifers at the intercontinental scale is still limited. Polyarthra dolichoptera and P. vulgaris are cosmopolitan rotifers that are tolerant to environmental changes, offering an excellent model to address the research gap. Here, we investigated the populations in Southeastern China and eastern North America and evaluated the phylogeographical patterns from their geographical range sizes, geographic–genetic distance relationships and their responses to spatial‐environmental factors. Using the mitochondrial cytochrome c oxidase subunit I gene as the DNA marker, we analyzed a total of 170 individuals. Our results showed that some putative cryptic species, also known as entities were widely distributed, but most of them were limited to single areas. The divergence of P. dolichoptera and P. vulgaris indicated that gene flow between continents was limited while that within each continent was stronger. Oceanographic barriers do affect the phylogeographic pattern of rotifers in continental waters and serve to maintain genetic diversity in nature. The genetic distance of P. dolichoptera and P. vulgaris populations showed significant positive correlation with geographic distance. This might be due to the combined effects of habitat heterogeneity, long‐distance colonization, and oceanographic barriers. Furthermore, at the intercontinental scale, spatial distance had a stronger influence than environmental variables on the genetic differentiations of both populations. Wind‐ and animal‐mediated transport and even historical events of continental plate tectonics are potential factors for phylogeography of cosmopolitan rotifers.

Global warming is expected to cause decreases in nutrient availability, photosynthesis, and potentially carbon export in the ocean. But how, and by what molecular mechanisms, nutrient limitation affects biological pump (BP) efficiency of phytoplankton are poorly understood. Here, using transcriptomics, miRNAomics, and physiological measurements, we report that phosphorus (P)-limitation increased cellular carbon and calcium contents and sinking rate of the cosmopolitan phytoplankton Emiliania huxleyi. Under P-limitation, when photosynthesis was depressed, there were substantial increases in cellular organic (3.4-fold) and inorganic (fivefold) carbon contents due to cell division arrest and, as our transcriptomic data suggest, CO2 incorporation into C4 compounds. Furthermore, calcification was increased by 46% through transcriptional and epigenetic regulations. An increase in sinking rate by 37-44% was detected. Although calcification releases equivalent amounts of CO2, the considerable increase in cellular carbon content and sinking rate far outweighed the CO2 release, leading to an elevated efficiency of carbon export by E. huxleyi, which would partially offset the decrease in BP capacity resulting from lower growth rate under P deficiency. However, how the observed sinking rate and its increase under P limitation on the laboratory cultures will translate into BP efficiency still requires further examination using in situ or mesocosm experiments.

Dinoflagellates, including harmful algal bloom species, are known to co-exist with and rely upon bacteria but how the microbiome changes with the physiologies of the cognate dinoflagellates is poorly understood. Here, we used 16S rRNA gene meta-barcoding to characterize the bacterial community in the cultures of Gambierdiscus balechii, a ciguatoxin-producing benthic dinoflagellate, under different nitrogen (N)-nutrient conditions and at different ciguatoxin-producing growth. The high-throughput sequencing of a total of 12 libraries generated 926,438 reads which were classified into 16 phyla. We observed a shift of the G. balechii-associated microbiome from N-replete to low-N conditions and from the early (low toxin) to the late exponential (high toxin) growth stage. Common across these conditions were species from families Rhodobacteraceae and Flavobacteriaceae. Species abundant in the low-N condition mainly included Planctomyces, Ekhidna, and Lactobacillus. Dominant or highly abundant microbial taxa in the high toxin-producing stage (N-replete, late exponential stage) were Oceanococcus and Marinoscillum. Under this condition, one Rhizobiales bacterium, Oricola, also increased in relative abundance. Our study documents the high diversity and dynamics of the G. balechii-associated microbiome, and identifies condition-specific sub-communities: the core (constitutive) microbiome that stably co-exists with G. balechii, the bacterial lineages that are responsive to N-nutrient variations, and species whose abundances are correlated with toxin content of the dinoflagellate. These findings demonstrate that particular bacterial groups are responsive to N-nutrient or toxicity changes of G. balechii and thus will be useful for further investigations on the associated microbiome’s interactions with benthic dinoflagellates and functions in the course of benthic harmful algae blooms.

The symbiosis of coral-Symbiodiniaceae is the quintessential basis of the coral reef ecosystem, and its breakdown results in coral bleaching, one of the most severe ecological catastrophes in the ocean. Critical to the establishment of the symbiosis is the host’s specific recognition of the symbionts through the binding of the coral host’s pattern recognition receptors (PRRs) to the symbiont cell surface’s glycoconjugates. However, the molecular basis for this recognition process is poorly understood. The present study investigated the binding affinities of the coral galectin PdGLT-1 to different symbiodiniacean species under different temperatures. At 25°C, the PdGLT-1 recombinant protein (rPdGLT-1) exhibited different binding affinities to different symbiodiniacean species from five genera, with a significantly higher binding affinity (p < 0.05) to Fugacium kawagutii (2.6-fold) and Cladocopium goreaui (1.9-fold) than Symbiodinium microadriaticum. The binding topology of rPdGLT-1 differed among the five symbiodiniacean species; for S. microadriaticum, Breviolum minutum, and Durusdinium trenchii, the binding was on some specific sites on the cell surface, whereas for C. goreaui and F. kawagutii, the binding signals were detected over the whole cell surface. Interestingly, PdGLT-1 binding induced agglutination of F. kawagutii cells but not of C. goreaui, explaining why C. goreaui was the most dominant symbiodiniacean symbionts in corals. Moreover, the affinity of rPdGLT-1 to Symbiodiniaceae was affected by temperature, and the highest binding affinities were observed at 30, 20, 30, 35, and 30°C for S. microadriaticum, B. minutum, C. goreaui, D. trenchii, and F. kawagutii, respectively. The optimal binding temperatures were consistent with the current understanding that D. trenchii was the most thermal resistant among these species. These results suggest that the binding affinity of the PRR PdGLT-1 may determine the specificity of host-symbiont pairing and explain why Cladocopium is the dominant symbionts of coral P. damicornis at normal temperature, and corals with Durusdinium symbionts may survive better at high temperature.

Dinoflagellates is one dominant group in coastal marine phytoplankton communities and, on occasion, form blooms in estuaries and coastal ecosystems. While relationships between dinoflagellate bloom dynamics and nutrients are well-studied, information regarding bloom dissipation in estuaries is limited. We studied the dissipation of dinoflagellate Polykrikos geminatum blooms in the Pearl River Estuary, South China Sea, during August of 2011 using ecological, molecular, and satellite remote sensing data. We found that the dinoflagellate bloom was associated with water temperatures of 29.2–31 °C, salinities ranging 16.4–20, and ambient water nutrient concentrations that were not limited. The abundance of the ciliate Euplotes rariseta, which feeds on P. geminatum cell debris and bacteria, functions as an indicator species of P. geminatum bloom dissipation. In situ and satellite data indicate that bloom water masses were transferred from the central to inner estuary near Shenzhen Bay, driven by continuous, strong southerly winds; at which point in time, P. geminatum blooms dissipated to a high-salinity area near the estuary mouth driven by northerly winds and freshwater discharge, whereupon the blooms rapidly vanished. A low tolerance to low or high salinities resulted in P. geminatum bloom demise in the Pearl River Estuary. We propose that interactions among salinity, wind, and freshwater incursion result in P. geminatum bloom dissipation in the Pearl River Estuary.

Symbiodiniaceae community structure in corals is crucial for understanding the plasticity of different holobionts under environmental stress. While this relies on molecular analyses, accuracy of molecular quantification, as influenced by DNA extraction efficiency and rDNA copy number variations in particular, has rarely been systematically investigated. Here, we report the development of a set of genus-specific qPCR assays. First, a protocol for efficient DNA isolation and accurate measurements of genome size and rDNA copy number was established. Second, seven newly designed genus-specific ITS2 primer sets were validated using computational and empirical analyses and qPCR assays were developed. We find that while the genome size ranges between 1.75 ± 0.21 and 4.5 ± 0.96 Gbp, rDNA copy number shows over 10-fold variation among Symbiodiniaceae species. Our protocol produced standard curves with high efficiencies (89.8–99.3%; R2 ≥ 0.999) and tight Cq values over different PCR conditions, illustrating high specificity and sensitivity of the qPCR assays. Tested on mock communities of mixed culture species, our qPCR results agreed well with microscopic counts and facilitated calibration of metabarcoding data. To test the applicability of our protocol for field samples, we analyzed three different Hong Kong coral samples. Six Symbiodiniaceae genera were detected in Acropora valida, Oulastrea crispata, and Platygyra acuta, with Breviolum, Effrenium, Fugacium, and Gerakladium sp. being reported for the first time. Our results suggest that aggressively disrupting cells to ensure thorough cell lysis, estimating cell loss and DNA loss, and validating qPCR assays are critical for success. The number of species examined here is limited, but the primers are potentially applicable to most species in respective genera, and the protocol and the approach to develop it provide a base and template toward a standardized procedure for quantitatively characterizing Symbiodiniaceae communities in corals.

Cytosine methyltransferases (DNMTs) often silence transposons in eukaryotic genomes. Here the authors describe the recurrent acquisition of DNMTs by transposons from two distantly-related eukaryotes and suggest that methylation of CG dinucleotides by transposon DNMTs could modify the host epigenome in dinoflagellates.

Free-living (FL) and particle-associated (PA) bacterioplankton communities play critical roles in biogeochemical cycles in the ocean. However, their community composition, assembly process and functions in the continental shelf and slope regions are poorly understood. Based on 16S rRNA gene amplicon sequencing, we investigated bacterial communities’ driving factors, assembly processes and functional potentials at a subtropical marginal sea. The bacterioplankton community showed specific distribution patterns with respect to lifestyle (free living vs. particle associated), habitat (slope vs. shelf) and depth (surface vs. DCM and Bottom). Salinity and water temperature were the key factors modulating turnover in the FL community, whereas nitrite, silicate and phosphate were the key factors for the PA community. Model analyses revealed that stochastic processes outweighed deterministic processes and had stronger influences on PA than FL. Homogeneous selection (Hos) was more responsible for the assembly and turnover of FL, while drift and dispersal limitation contributed more to the assembly of PA. Importantly, the primary contributor to Hos in PA was Gammaproteobacteria:Others, whereas that in FL was Cyanobacteria:Bin6. Finally, the PICRUSt2 analysis indicated that the potential metabolisms of carbohydrates, cofactors, amino acids, terpenoids, polyketides, lipids and antibiotic resistance were markedly enriched in PA than FL.

Vibrio coralliilyticus is known to cause coral diseases, especially under environmental perturbation, but its impact on coral physiology and underpinning mechanism is poorly understood. In the present study, we investigated cytological, immunological, and metatranscriptomic responses of the scleractinian coral Pocillopora damicornis to V. coralliilyticus infection. The density and chlorophyll content of symbiotic zooxanthellae decreased significantly at 12 and 24 h after Vibrio challenge. The activities of antioxidant enzymes such as superoxide dismutase and catalase, nitric oxide synthase, phenoloxidase (PO), and the activation level of caspase3 all rose significantly in P. damicornis after Vibrio challenge. In the metatranscriptomic analysis, we found 10 significantly upregulated genes in the symbionts at 24 h after the challenge, which were mostly involved in the metabolism of nucleic acid and polysaccharide, and 133 significantly down-regulated symbiont genes, which were mainly related to amino acid catabolism and transport. Meanwhile, 1432 significantly upregulated coral genes were revealed, highly overrepresented in GO terms that are mostly related to the regulation of immune response, the regulation of cytokine production, and innate immune response. Furthermore, at 24 h after Vibrio challenge, 890 coral genes were significantly downregulated, highly overrepresented in four GO terms implicated in defense response. These results in concert suggest that V. coralliilyticus infection triggered the innate immune response including the redox, PO, and apoptosis systems, but repressed the response of the complement system in the scleractinian coral P. damicornis, accompanied by symbiont density decrease and symbiosis collapse through disordering the metabolism of the symbionts. These findings shed light on the molecular regulatory processes underlying bleaching and degradation of P. damicornis resulting from the infection of V. coralliilyticus.

Diatoms and dinoflagellates dominate coastal marine phytoplankton communities as major players of marine biogeochemical cycles and their seasonal succession often leads to harmful algal blooms (HABs). What regulates their respective dominances and the development of the HABs remains elusive. Here we conducted time-sequential metatranscriptomic profiling on a natural assemblage that evolved from diatom dominance to a dinoflagellate bloom to interrogate the underlying major metabolic and ecological drivers. Data reveals similarity between diatoms and dinoflagellates in exhibiting high capacities of energy production, nutrient acquisition, and stress protection in their respective dominance stages. The diatom-to-dinoflagellate succession coincided with an increase in turbidity and sharp declines in silicate and phosphate availability, concomitant with the transcriptomic shift from expression of silicate uptake and urea utilization genes in diatoms to that of genes for light harvesting, diversified phosphorus acquisition and autophagy-based internal nutrient recycling in dinoflagellates. Furthermore, the diatom-dominant community featured strong potential to carbohydrate metabolism and a strikingly high expression of trypsin potentially promoting frustule building. In contrast, the dinoflagellate bloom featured elevated expression of xanthorhodopsin, and antimicrobial defensin genes, indicating potential importance of energy harnessing and microbial defense in bloom development. This study sheds light on mechanisms potentially governing diatom- and dinoflagellate-dominance and regulating bloom development in the natural environment and raises new questions to be addressed in future studies.

Pigmented bacteria cells, including aerobic anoxygenic phototrophic (AAP) bacteria, contribute significantly to secondary production and aquatic carbon cycling but their distribution in the deep sea is still not well understood, especially in the South China Sea. In this study, microscopic, flow cytometric, and molecular analyses were carried out to investigate the abundance and diversity of AAP bacteria at seven stations in the South China Sea. The results revealed the existence of bacteriochlorophyll-containing bacteria below 500 m from two of seven stations. Flow cytometric analysis detected red and infra-red fluorescence under blue (488 nm) light excitation from fluorescent cells. Blue light-excited red fluorescence of these cells from the 1000 m depth at station E403 were verified using epifluorescence microscopy. Based on fluorescence and side scatter features, fluorescent cells were sorted and subjected to molecular analysis. DNA was extracted from these sorted cells from both stations for PCR amplification using 16S rDNA primers. Sequencing of the PCR products showed that the sorted cells from the 1000 m depth at station E403 belonged to the genus Porphyrobacter. The cell population sorted from 500 m at station E703 contained Sphingomonas and a Methylobacterium-like taxon. All these three taxa belong to aerobic anoxygenic phototrophic alpha-proteobacteria. Using flow cytometric analysis, we found that the abundance of Porphyrobacter sp. at 1000 m was 2.71-2.95×104 cells mL-1 whereas cell counts of Sphingomonas sp. and Methylobacterium at 500 m were about 3.75-4.12×105 cells mL-1. These results indicate that albeit not ubiquitous in deep water, bacteriochlorophyll-containing bacteria can be abundant in the deep-sea aphotic zone.

The lack of a robust gene transformation tool that allows proper expression of foreign genes and functional testing for the vast number of nuclear genes in dinoflagellates has greatly hampered our understanding of the fundamental biology in this ecologically important and evolutionarily unique lineage of microeukaryotes. Here, we report the development of a dinoflagellate expression vector containing various DNA elements from phylogenetically separate dinoflagellate lineages, an electroporation protocol, and successful expression of introduced genes in an early branching dinoflagellate, Oxyrrhis marina. This protocol, involving the use of Lonza’s Nucleofector and a codon-optimized antibiotic resistance gene, has been successfully used to produce consistent results in several independent experiments for O. marina. It is anticipated that this protocol will be adaptable for other dinoflagellates and will allow characterization of many novel dinoflagellate genes.

Cataloging an accurate functional gene set for the Symbiodiniaceae species is crucial for addressing biological questions of dinoflagellate symbiosis with corals and other invertebrates. To improve the gene models of Fugacium kawagutii, we conducted high-throughput chromosome conformation capture (Hi-C) for the genome and Illumina combined with PacBio sequencing for the transcriptome to achieve a new genome assembly and gene prediction. A 0.937-Gbp assembly of F. kawagutii were obtained, with a N50 > 13 Mbp and the longest scaffold of 121 Mbp capped with telomere motif at both ends. Gene annotation produced 45,192 protein-coding genes, among which, 11,984 are new compared to previous versions of the genome. The newly identified genes are mainly enriched in 38 KEGG pathways including N-Glycan biosynthesis, mRNA surveillance pathway, cell cycle, autophagy, mitophagy, and fatty acid synthesis, which are important for symbiosis, nutrition, and reproduction. The newly identified genes also included those encoding O-methyltransferase (O-MT), 3-dehydroquinate synthase, homologous-pairing protein 2-like (HOP2) and meiosis protein 2 (MEI2), which function in mycosporine-like amino acids (MAAs) biosynthesis and sexual reproduction, respectively. The improved version of the gene set (Fugka_Geneset _V3) raised transcriptomic read mapping rate from 33% to 54% and BUSCO match from 29% to 55%. Further differential gene expression analysis yielded a set of stably expressed genes under variable trace metal conditions, of which 115 with annotated functions have recently been found to be stably expressed under three other conditions, thus further developing the “core gene set” of F. kawagutii. This improved genome will prove useful for future Symbiodiniaceae transcriptomic, gene structure, and gene expression studies, and the refined “core gene set” will be a valuable resource from which to develop reference genes for gene expression studies.

Communities of microscopic dinoflagellates are omnipresent in aquatic ecosystems. Consequently, their traits drive community processes with profound effects on global biogeochemistry. Species traits are, however, not necessarily static but respond to environmental changes in order to maintain fitness and may differ with cell size that scales physiological rates. Comprehending such trait characteristics is necessary for a mechanistic understanding of plankton community dynamics and resulting biogeochemical impacts. Here, we used information theory to analyze metatranscriptomes of micro- and nano-dinoflagellate communities in three ecosystems. Measures of gene expression variations were set as a proxy to determine conserved and plastic community traits and the environmental influence on trait changes. Using metabarcoding, we further investigated if communities with a more similar taxon composition also express more similar traits. Our results indicate that plastic community traits mainly arise from membrane vesicle associated processes in all the environments we investigated. A specific environmental influence on trait plasticity was observed to arise from nitrogen availability in both size classes. Species interactions also appeared to be responsible for trait plasticity in the smaller-sized dinoflagellates. Additionally, the smaller-sized dinoflagellate communities are characterized by the expression of a large pool of habitat specific genes despite being taxonomically more similar across the habitats, in contrast to the microplanktonic assemblages that adapted to their environments by changing species composition. Our data highlight the functional diversification on the gene level as a signature of smaller sized dinoflagellates, nitrogen availability and species interactions as drivers of trait plasticity, and traits most likely linked to fitness and community performance.

Emiliania huxleyi, a cosmopolitan coccolithophore in the modern ocean, plays an important role in the carbon cycle and local climate feedback as it can form extensive blooms, calcify, and produce dimethylsulfoniopropionate (DMSP) leading to the generation of dimethyl sulfide (DMS) which affects climate when oxidized in the atmosphere. It is known to be able to utilize dissolved organic phosphorus (DOP) by expressing a specific type of alkaline phosphatase (EHAP1) under phosphorus-limited conditions. In this study, we identified a new alkaline phosphatase (EH-PhoAaty) in this species, which we found belongs to the newly classified PhoAaty family. The expression of this atypical phosphatase was up-regulated under P-depleted conditions at both the transcriptional and translational levels, suggesting that E. huxleyi is able to express this AP to cope with phosphorus limitation. Comparative analysis revealed different transcriptional expression dynamics between eh-PhoAaty and ehap1, although both genes exhibited inducible expression under phosphate deficiency. In addition, after AP activity was eliminated by using EDTA to chelate metal ions, we found that AP activity was recovered with the supplement of Ca2+ and Zn2+, indicative of the adoption of Ca2+ as the cofactor under Zn-P co-limited conditions, likely a result of adaptation to oceanic environments where Zn2+ is often limiting.

[This corrects the article DOI: 10.1371/journal.pone.0187837.].

Glyphosate is a widely used herbicide that can potentially be a phosphorus (P) source for phytoplankton and microbes when discharged into the coastal ocean. In contrast to bacteria, few eukaryotic phytoplankton species appear capable of directly utilizing glyphosate. In this study, we observed, after a long delay (>60 days), Prorocentrum donghaiense, a dinoflagellate known to cause major harmful algal blooms in the East China Sea, could grow in a medium with glyphosate as the sole P source; suggesting that P. donghaiense growth was through bacterial mediation. To understand how the bacteria community might respond to glyphosate, we analyzed the 16S rRNA genes of the microbial community present in P. donghaiense cultures when grown under lower (36 μM) and higher (360 μM) glyphosate concentrations. Based on both Sanger and Illumina high throughput sequencing, we obtained more than 55,323 good-quality sequences, which were classified into six phyla. As the concentration of glyphosate rose, our results showed a significant increase in the phyla Proteobacteria and Firmicutes and a decrease in the phylum Bacteroidetes. Further qPCR (Quantitative PCR) analysis showed higher abundances of two specific phylotypes in the higher-glyphosate P. donghaiense cultures when compared to the lower-glyphosate and no-glyphosate cultures. Correspondingly, qPCR displayed the same trend for the abundance of a gammaproteobacterial type of phnJ, a gene encoding Alpha-D-ribose 1-methylphosphonate 5-phosphate C-P lyase, which is responsible for phosphonate degradation. In addition, Tax4Fun analysis based on our 16S rRNA gene sequences results in higher predicted abundances of phosphonate metabolizing genes in glyphosate-treated cultures. This study demonstrates that glyphosate could selectively promote the growth of particular groups of bacteria within an algal culture and in glyphosate enriched coastal waters, this interaction may potentially further facilitate the growth of alga.

Dinoflagellates undergo a typical eukaryotic cell cycle consisting of G1, S, G2, and M phases and some of the typical cell cycle related genes have been computationally identified. However, very few of these genes have been experimentally linked to the cell cycle phases. Besides, although thecate dinoflagellates are known to possess theca composed of cellulose, information on cellulose synthesis and degradation associated with the cell cycle is also limited. In this study, we isolated G1/S cyclin, cellulose synthase and cellulase encoding genes in dinoflagellate Prorocentrum donghaiense. Further, using reverse transcription quantitative PCR (RT-qPCR), we characterized the expression profiles of the three genes throughout the cell cycle. All three showed clear expression dynamics throughout the cell cycle, with fold changes of 26, 2.4 and 9.3 for G1/S cyclin, cellulose synthase and cellulase gene, respectively. The transcript abundance of G1/S cyclin increased in late G1 phase and dropped in early S phase, indicating that this protein is involved in the G1/S transition. Throughout the cell cycle, the average transcript level of cellulose synthase was 4.5-fold higher than that of cellulase. Cellulose synthase and cellulase gene expressions showed peak transcript abundances at middle G1 phase and G2M phase, respectively, indicating the respective roles of these enzymes in the growth of newly divided cells and in cytokinesis. Our results suggest that G1/S cyclin, cellulase, and cellulose synthase genes associated with G1/S transition, G2M, and G1 phases of the cell cycle and are candidates of biomarkers for assessing growth status of P. donghaiense.

Urea has been shown to contribute more than half of total nitrogen (N) required by phytoplankton in some estuaries and coastal waters and to provide a substantial portion of the N demand for many harmful algal blooms (HABs) of dinoflagellates. In this study, we investigated the physiological and transcriptional responses in Prorocentrum donghaiense to changes in nitrate and urea availability. We found that this species could efficiently utilize urea as sole N source and achieve comparable growth rate and photosynthesis capability as it did under nitrate. These physiological parameters were markedly lower in cultures grown under nitrate- or urea-limited conditions. P. donghaiense N content was similarly low under nitrate- or urea-limited culture condition, but was markedly higher under urea-replete condition than under nitrate-replete condition. Carbon (C) content was consistently elevated under N-limited condition. Consequently, the C:N ratio was as high as 21:1 under nitrate- or urea-limitation, but 7:1 under urea-replete condition and 9:1 to 10:1 under nitrate-replete condition. Using quantitative reverse transcription PCR, we investigated the expression pattern for four genes involved in N transport and assimilation. The results indicated that genes encoding nitrate transport, urea hydrolysis, and nickel transporter gene were sensitive to changes in general N nutrient availability whereas the urea transporter gene responded much more strongly to changes in urea concentration. Taken together, our study shows the high bioavailability of urea, its impact on C:N stoichiometry, and the sensitivity of urea transporter gene expression to urea availability.

Ciliates from the genus Mesodinium are globally distributed in marine and freshwater ecosystems and may possess either heterotrophic or mixotrophic nutritional modes. Members of the M. major/rubrum species complex photosynthesize by sequestering and maintaining organelles from cryptophyte prey, and under certain conditions form periodic or recurrent blooms (= red tides). Here we present an analysis of the genetic diversity of Mesodinium and cryptophyte populations from 10 environmental samples (8 globally dispersed habitats including 5 Mesodinium blooms), using group-specific primers for Mesodinium partial 18S, ITS, and partial 28S rRNA genes as well as cryptophyte large subunit RuBisCO genes (rbcL). In addition, 22 new cryptophyte and 4 new M. rubrum cultures were used to extract DNA and sequence rbcL and 18S-ITS-28S genes, respectively, in order to provide a stronger phylogenetic context for our environmental sequences. Bloom samples were analyzed from coastal Brazil, Chile, two Northeastern locations in the United States, and the Pribilof Islands within the Bering Sea. Additionally, samples were also analyzed from the Baltic and Barents Seas and coastal California under non-bloom conditions. Most blooms were dominated by a single Mesodinium genotype, with coastal Brazil and Chile blooms composed of M. major and the Eastern USA blooms dominated by M. rubrum variant B. Sequences from all 4 blooms were dominated by T. amphioxeia-like cryptophytes. Non-bloom communities revealed more diverse assemblages of Mesodinium spp., including heterotrophic species and the mixotrophic M. chamaeleon. Similarly, cryptophyte diversity was also higher in non-bloom samples. Our results confirm that Mesodinium blooms may be caused by M. major, as well as multiple variants of M. rubrum, and further implicate T. amphioxeia as the key cryptophyte species linked to these phenomena in temperate and subtropical regions.

Alkaline phosphatase (AP) enables marine phytoplankton to utilize dissolved organic phosphorus (DOP) when dissolved inorganic phosphate (DIP) is depleted in the ocean. Dinoflagellate AP (Dino-AP) represents a newly classified atypical type of AP, PhoAaty. Despite While being a conventional AP, PhoAEC is known to recruit Zn2+ and Mg2+ in the active center, and the cofactors required by PhoAaty have been contended and remain unclear. In this study, we investigated the metal ion requirement of AP in five dinoflagellate species. After AP activity was eliminated by using EDTA to chelate metal ions, the enzymatic activity could be recovered by the supplementation of Ca2+, Mg2+ and Mn2+ in all cases but not by that of Zn2+. Furthermore, the same analysis conducted on the purified recombinant ACAAP (AP of Amphidinium carterae) verified that the enzyme could be activated by Ca2+, Mg2+, and Mn2+ but not Zn2+. We further developed an antiserum against ACAAP, and a western blot analysis using this antibody showed a remarkable up-regulation of ACAAP under a phosphate limitation, consistent with elevated AP activity. The unconventional metal cofactor requirement of Dino-AP may be an adaptation to trace metal limitations in the ocean, which warrants further research to understand the niche differentiation between dinoflagellates and other phytoplankton that use Zn−Mg AP in utilizing DOP.

Coral reef-associated Symbiodiniaceae live in tropical and oligotrophic environments and are prone to heat and nutrient stress. How their metabolic pathways respond to pulses of warming and phosphorus (P) depletion is underexplored. Here, we conducted RNA-seq analysis to investigate transcriptomic responses to thermal stress, phosphate deprivation, and organic phosphorus (OP) replacement in Fugacium kawagutii. Using dual-algorithm (edgeR and NOIseq) to remedy the problem of no replicates, we conservatively found 357 differentially expressed genes (DEGs) under heat stress, potentially regulating cell wall modulation and the transport of iron, oxygen, and major nutrients. About 396 DEGs were detected under P deprivation and 671 under OP utilization, both mostly up-regulated and potentially involved in photosystem and defensome, despite different KEGG pathway enrichments. Additionally, we identified 221 genes that showed relatively stable expression levels across all conditions (likely core genes), mostly catalytic and binding proteins. This study reveals a wide range of, and in many cases previously unrecognized, molecular mechanisms in F. kawagutii to cope with heat stress and phosphorus-deficiency stress. Their quantitative expression dynamics, however, requires further verification with triplicated experiments, and the data reported here only provide clues for generating testable hypotheses about molecular mechanisms underpinning responses and adaptation in F. kawagutii to temperature and nutrient stresses.

Heterotrophic dinoflagellates are prevalent protists in marine environments, which play an important role in the carbon cycling and energy flow in the marine planktonic community. Oxyrrhis marina (Dinophyceae), a widespread heterotrophic dinoflagellate, is a model species used for a broad range of ecological, biogeographic, and evolutionary studies. Despite the increasing research effort on this species, there lacks a synthesis of the existing data and a coherent picture of this organism. Here we reviewed the literature to provide an overview of what is known regarding the biology of O. marina, and identify areas where further studies are needed. As an early branch of the dinoflagellate lineage, O. marina shares similarity with typical dinoflagellates in permanent condensed chromosomes, less abundant nucleosome proteins compared to other eukaryotes, multiple gene copies, the occurrence of trans-splicing in nucleus-encoded mRNAs, highly fragmented mitochondrial genome, and disuse of ATG as a start codon for mitochondrial genes. On the other hand, O. marina also exhibits some distinct cytological features (e.g., different flagellar structure, absence of girdle and sulcus or pustules, use of intranuclear spindle in mitosis, presence of nuclear plaque, and absence of birefringent periodic banded chromosomal structure) and genetic features (e.g., a single histone-like DNA-associated protein, cob-cox3 gene fusion, 5′ oligo-U cap in the mitochondrial transcripts of protein-coding genes, the absence of mRNA editing, the presence of stop codon in the fused cob-cox3 mRNA produced by post-transcriptional oligoadenylation, and vestigial plastid genes). The best-studied biology of this dinoflagellate is probably the prey and predators types, which include a wide range of organisms. On the other hand, the abundance of this species in the natural waters and its controlling factors, genome organization and gene expression regulation that underlie the unusual cytological and ecological characteristics are among the areas that urgently need study.

The original version of this Article contained an error in the spelling of the author Hongfei Li, which was incorrectly given as Fei Hong. This has now been corrected in both the PDF and HTML versions of the Article.

The theoretical limit of link prediction is a fundamental problem in this field. Taking the network structure as object to research this problem is the mainstream method. This paper proposes a new viewpoint that link prediction methods can be divided into single or combination methods, based on the way they derive the similarity matrix, and investigates whether there a theoretical limit exists for combination methods. We propose and prove necessary and sufficient conditions for the combination method to reach the theoretical limit. The limit theorem reveals the essence of combination method that is to estimate probability density functions of existing links and nonexistent links. Based on limit theorem, a new combination method, theoretical limit fusion (TLF) method, is proposed. Simulations and experiments on real networks demonstrated that TLF method can achieve higher prediction accuracy.

The marine epiphytic dinoflagellate Gambierdiscus is a toxicologically important genus responsible for ciguatera fish poisoning, the principal cause of non-bacterial illness associated with fish consumption. The genus currently contains species exhibiting either globular or anterior-posteriorly compressed morphologies with marked differences in cell shape and plate arrangement. Here we report a third globular, epiphytic and tychoplanktonic species from the coasts of Ubatuba, Brazil. The new species can be distinguished from G. yasumotoi and G. ruetzleri by its broader first apical plate that occupies a larger portion of the epitheca. Accordingly, phylogenetic trees from small subunit (SSU) and large subunit (LSU) ribosomal DNA sequences also showed strongly supported separation of the new species from the G. yasumotoi/G. ruetzleri group albeit with short distance. The molecular phylogenies, which included new sequences of the planktonic species Goniodoma polyedricum, further indicated that the globular species of Gambierdiscus formed a tight clade, clearly separated (with strong bootstrap support) from the clade of lenticular species including the type for Gambierdiscus. The morphological and molecular data in concert support the split of Gambierdiscus sensu lato into two genera. Gambierdiscus sensu stricto should be reserved for the species with lenticular shapes, highly compressed anterioposteriorly, with short-shank fishhook apical pore plate, large 2' plate, low and ascending cingular displacement, and pouch-like sulcal morphology. The new genus name Fukuyoa gen. nov. should be applied to the globular species, slightly laterally compressed, with long-shank fishhook apical pore plate, large 1' plate, greater and descending cingular displacement, and not pouch-like vertically-oriented sulcal morphology. Fukuyoa contains the new species Fukuyoa paulensis gen. et sp. nov., and F. yasumotoi comb. nov. and F. ruetzleri comb. nov.

Marine phytoplankton are highly diverse with different species possessing different cell coverings, posing challenges for thoroughly breaking the cells in DNA extraction yet preserving DNA integrity. While quantitative molecular techniques have been increasingly used in phytoplankton research, an effective and simple method broadly applicable to different lineages and natural assemblages is still lacking. In this study, we developed a bead-beating protocol based on our previous experience and tested it against 9 species of phytoplankton representing different lineages and different cell covering rigidities. We found the bead-beating method enhanced the final yield of DNA (highest as 2 folds) in comparison with the non-bead-beating method, while also preserving the DNA integrity. When our method was applied to a field sample collected at a subtropical bay located in Xiamen, China, the resultant ITS clone library revealed a highly diverse assemblage of phytoplankton and other micro-eukaryotes, including Archaea, Amoebozoa, Chlorophyta, Ciliphora, Bacillariophyta, Dinophyta, Fungi, Metazoa, etc. The appearance of thecate dinoflagellates, thin-walled phytoplankton and "naked" unicellular organisms indicates that our method could obtain the intact DNA of organisms with different cell coverings. All the results demonstrate that our method is useful for DNA extraction of phytoplankton and environmental surveys of their diversity and abundance.

The raphidophyte Heterosigma akashiwo is a globally distributed harmful alga that has been associated with fish kills in coastal waters. To understand the mechanisms of H. akashiwo bloom formation, gene expression analysis is often required. To accurately characterize the expression levels of a gene of interest, proper reference genes are essential. In this study, we assessed ten of the previously reported algal candidate genes (rpL17-2, rpL23, cox2, cal, tua, tub, ef1, 18S, gapdh, and mdh) for their suitability as reference genes in this species. We used qRT-PCR to quantify the expression levels of these genes in H. akashiwo grown under different temperatures, light intensities, nutrient concentrations, and time points over a diel cycle. The expression stability of these genes was evaluated using geNorm and NormFinder algorithms. Although none of these genes exhibited invariable expression levels, cal, tub, rpL17-2 and rpL23 expression levels were the most stable across the different conditions tested. For further validation, these selected genes were used to normalize the expression levels of ribulose-1, 5-bisphosphate carboxylase/oxygenase large unite (HrbcL) over a diel cycle. Results showed that the expression of HrbcL normalized against each of these reference genes was the highest at midday and lowest at midnight, similar to the diel patterns typically documented for this gene in algae. While the validated reference genes will be useful for future gene expression studies on H. akashiwo, we expect that the procedure used in this study may be helpful to future efforts to screen reference genes for other algae.

Knowledge of in situ copepod diet diversity is crucial for accurately describing pelagic food web structure but is challenging to achieve due to lack of an easily applicable methodology. To enable analysis with whole copepod-derived DNAs, we developed a copepod-excluding 18S rDNA-based PCR protocol. Although it is effective in depressing amplification of copepod 18S rDNA, its applicability to detect diverse eukaryotes in both mono- and mixed-species has not been demonstrated. Besides, the protocol suffers from the problem that sequences from symbiotic ciliates are overrepresented in the retrieved 18S rDNA libraries. In this study, we designed a blocking primer to make a combined primer set (copepod/symbiotic ciliate-excluding eukaryote-common: CEEC) to depress PCR amplification of symbiotic ciliate sequences while maximizing the range of eukaryotes amplified. We firstly examined the specificity and efficacy of CEEC by PCR-amplifying DNAs from 16 copepod species, 37 representative organisms that are potential prey of copepods and a natural microplankton sample, and then evaluated the efficiency in reconstructing diet composition by detecting the food of both lab-reared and field-collected copepods. Our results showed that the CEEC primer set can successfully amplify 18S rDNA from a wide range of isolated species and mixed-species samples while depressing amplification of that from copepod and targeted symbiotic ciliate, indicating the universality of CEEC in specifically detecting prey of copepods. All the predetermined food offered to copepods in the laboratory were successfully retrieved, suggesting that the CEEC-based protocol can accurately reconstruct the diets of copepods without interference of copepods and their associated ciliates present in the DNA samples. Our initial application to analyzing the food composition of field-collected copepods uncovered diverse prey species, including those currently known, and those that are unsuspected, as copepod prey. While testing is required, this protocol provides a useful strategy for depicting in situ dietary composition of copepods.

The discovery of microbial rhodopsins in marine proteobacteria changed the dogma that photosynthesis is the only pathway to use the solar energy for biological utilization in the marine environment. Although homologs of these rhodopsins have been identified in dinoflagellates, the diversity of the encoding genes and their physiological roles remain unexplored. As an initial step toward addressing the gap, we conducted high-throughput transcriptome sequencing on Oxyrrhis marina to retrieve rhodopsin transcripts, rapid amplification of cDNA ends to isolate full-length cDNAs of dominant representatives, and quantitative reverse-transcription PCR to investigate their expression under varying conditions. Our phylogenetic analyses showed that O. marina contained both the proton-pumping type (PR) and sensory type (SR) rhodopsins, and the transcriptome data showed that the PR type dominated over the SR type. We compared rhodopsin gene expression for cultures kept under light: dark cycle and continuous darkness in a time course of 24 days without feeding. Although both types of rhodopsin were expressed under the two conditions, the expression levels of PR were much higher than SR, consistent with the transcriptomic data. Furthermore, relative to cultures kept in the dark, rhodopsin expression levels and cell survival rate were both higher in cultures grown in the light. This is the first report of light-dependent promotion of starvation survival and concomitant promotion of PR expression in a eukaryote. While direct evidence needs to come from functional test on rhodopsins in vitro or gene knockout/knockdown experiments, our results suggest that the proton-pumping rhodopsin might be responsible for the light-enhanced survival of O. marina, as previously demonstrated in bacteria.