In situ community transcriptomics illuminates CO2-fixation potentials and supporting roles of phagotrophy and proton pump in plankton in a subtropical marginal sea.

Lineage-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.

Diversity and ecological function of urease-producing bacteria in the cultivation environment of Gracilariopsis lemaneiformis.

Urease-producing bacteria (UPB) provide inorganic nitrogen for primary producers by hydrolyzing urea, and play an important role in marine nitrogen cycle. However, there is still an incomplete understanding of UPB and their ecological functions in the cultivation environment of the red macroalgae Gracilariopsis lemaneiformis. This study comprehensively analyzed the diversity of culturable UPB and explored their effects on urea uptake by G. lemaneiformis. A total of 34 isolates belonging to four main bacterial phyla i.e. (Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria) were identified through 16S rRNA sequencing and were screened for UPB by urea agar chromogenic medium assay and ureC gene cloning. Our data revealed that only 8 strains contained urease. All of these UPB exhibited different urease activities, which were determined by the Berthelot reaction colorimetry assay. Additionally, the UPB strain (G13) isolated from G. lemaneiformis with higher urease activity was selected for co-culture with G. lemaneiformis to explore its role in promoting or inhibiting nitrogen uptake by macroalgae. The results showed a significant increase in urea consumption in the culture medium and the total cellular nitrogen in G. lemaneiformis in the UPB-co culture group compared to the sterile group. This suggests that the selected UPB strain positively influences nitrogen uptake by G. lemaneiformis. Similarly, isotopic assays revealed that the δ15N content of G. lemaneiformis was significantly higher in the UPB-co culture than in the control group, where δ15N-urea was the only nitrogen source in the culture medium. This indicates that the UPB helped G. lemaneiformis to absorb more nitrogen from urea. Moreover, the highest content of δ15N was found in G. lemaneiformis with epiphytic bacteria compared to sterilized (i.e. control), showing that epiphytic bacteria, along with UPB, have a compound effect in helping G. lemaneiformis absorb more nitrogen from urea. Taken together, these results provide unique insight into the ecological role of UPB and suggest that urease from macroalgae environment-associated bacteria might be an important player in marine nitrogen cycling.

Comparison and improvement of RNA extraction methods in Sargassum (Phaeophyta).

Sargassum (Sargassaceae) is widely distributed globally and plays an important role in regulating climate change, but the landscape of genomes and transcripts is less known. High-quality nucleic acids are the basis for molecular biology experiments such as high-throughput sequencing. Although extensive studies have documented methods of RNA extraction, these methods are not very applicable to Sargassum, which contains high levels of polysaccharides and polyphenols. To find a suitable method to improve the quality of RNA extracted, we compared and modified several popular RNA extraction methods and screened one practical method with three specific Sargassum spp. The results showed that three CTAB methods (denoted as Methods 1, 2, and 3) and the RNAprep Pure Plant Kit (denoted as Method 4) could, with slight modifications, effectively isolate RNA from Sargassum species, except for Method 4 used with S. fusiforme. By performing further screening, we determined Method 4 was the best choice for S. hemiphyllum and S. henslowianum, as revealed by RNA yields, RNA Integrity Number (RIN), extraction time, and unigene mapped ratio. For S. fusiforme, Methods 1, 2, and 3 showed no obvious differences among the yields, quality, or time to perform. In addition, one other method was tested, but we found the quality of the RNA extracted by TRIzol reagent methods (denoted as Method 5) performed the worst when compared with the above four methods. Therefore, our study provides four suitable methods for RNA extraction in Sargassum and is essential for future genetic exploration of Sargassum.

Unraveling the Diverse Profile of N-Acyl Homoserine Lactone Signals and Their Role in the Regulation of Biofilm Formation in Porphyra haitanensis-Associated Pseudoalteromonas galatheae.

N-acyl homoserine lactones (AHLs) are small, diffusible chemical signal molecules that serve as social interaction tools for bacteria, enabling them to synchronize their collective actions in a density-dependent manner through quorum sensing (QS). The QS activity from epiphytic bacteria of the red macroalgae Porphyra haitanensis, along with its involvement in biofilm formation and regulation, remains unexplored in prior scientific inquiries. Therefore, this study explores the AHL signal molecules produced by epiphytic bacteria. The bacterium isolated from the surface of P. haitanensis was identified as Pseudoalteromonas galatheae by 16s rRNA gene sequencing and screened for AHLs using two AHL reporter strains, Agrobacterium tumefaciens A136 and Chromobacterium violaceum CV026. The crystal violet assay was used for the biofilm-forming phenotype. The inferences revealed that P. galatheae produces four different types of AHL molecules, i.e., C4-HSL, C8-HSL, C18-HSL, and 3-oxo-C16-HSL, and it was observed that its biofilm formation phenotype is regulated by QS molecules. This is the first study providing insights into the QS activity, diverse AHL profile, and regulatory mechanisms that govern the biofilm formation phenotype of P. galatheae. These findings offer valuable insights for future investigations exploring the role of AHL producing epiphytes and biofilms in the life cycle of P. haitanensis.

A comparative study reveals the relative importance of prokaryotic and eukaryotic proton pump rhodopsins in a subtropical marginal sea.

Proton-pump rhodopsin (PPR) in marine microbes can convert solar energy to bioavailable chemical energy. Whereas bacterial PPR has been extensively studied, counterparts in microeukaryotes are less explored, and the relative importance of the two groups is poorly understood. Here, we sequenced whole-assemblage metatranscriptomes and investigated the diversity and expression dynamics of PPR in microbial eukaryotes and prokaryotes at a continental shelf and a slope site in the northern South China Sea. Data showed the whole PPRs transcript pool was dominated by Proteorhodopsins and Xanthorhodopsins, followed by Bacteriorhodopsin-like proteins, dominantly contributed by prokaryotes both in the number and expression levels of PPR unigenes, although at the continental slope station, microeukaryotes and prokaryotes contributed similarly in transcript abundance. Furthermore, eukaryotic PPRs are mainly contributed by dinoflagellates and showed significant correlation with nutrient concentrations. Green light-absorbing PPRs were mainly distributed in >3 µm organisms (including microeukaryotes and their associated bacteria), especially at surface layer at the shelf station, whereas blue light-absorbing PPRs dominated the <3 µm (mainly bacterial) communities at both study sites, especially at deeper layers at the slope station. Our study portrays a comparative PPR genotype and expression landscape for prokaryotes and eukaryotes in a subtropical marginal sea, suggesting PPR's role in niche differentiation and adaptation among marine microbes.

Enhanced Carotenoid Production in Chlamydomonas reinhardtii by Overexpression of Endogenousand Exogenous Beta-Carotene Ketolase (BKT) Genes.

Chlamydomonas reinhardtii is a unicellular green alga that can grow heterotrophically by using acetate as a carbon source. Carotenoids are natural pigments with biological activity and color, which have functions such as antioxidant, anti-inflammatory, vision protection, etc., and have high commercial value and prospects. We transformed Chlamydomonas reinhardtii with the BKT genes from Phaffia rhodozyma (PrBKT) and Chlamydomonas reinhardtii (CrBKT) via plasmid vector, and screened out the stable transformed algal strains C18 and P1. Under the condition that the cell density of growth was not affected, the total carotenoid content of C18 and P1 was 2.13-fold and 2.20-fold higher than that of the WT, respectively. CrBKT increased the levels of ß-carotene and astaxanthin by 1.84-fold and 1.21-fold, respectively, while PrBKT increased them by 1.11-fold and 1.27-fold, respectively. Transcriptome and metabolome analysis of C18 and P1 showed that the overexpression of CrBKT only up-regulated the transcription level of BKT and LCYE (the gene of lycopene e-cyclase). However, in P1, overexpression of PrBKT also led to the up-regulation of ZDS (the gene of ζ-carotene desaturase) and CHYB (the gene of ß-carotene hydroxylase). Metabolome results showed that the relative content of canthaxanthin, an intermediate metabolite of astaxanthin synthesis in C18 and P1, decreased. The overall results indicate that there is a structural difference between CrBKT and PrBKT, and overexpression of PrBKT in Chlamydomonas reinhardtii seems to cause more genes in carotenoid pathway metabolism to be up-regulated.

In Situ Molecular Ecological Analyses Illuminate Distinct Factors Regulating Formation and Demise of a Harmful Dinoflagellate Bloom.

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.

Eukaryotic plankton community assembly and influencing factors between continental shelf and slope sites in the northern South China Sea.

Eukaryotic plankton are pivotal members of marine ecosystems playing crucial roles in marine food webs and biogeochemical cycles. However, understanding the patterns and drivers of their community assembly remains a grand challenge. A study was conducted in the northern South China Sea (SCS) to address this issue. Here, 49 samples were collected and size-fractionated from discrete depths at continental shelf and continental slope in the northern SCS over a diel cycle. From high throughput sequencing of the 18S rDNA gene V4 region, 2463 operational taxonomic units (OTUs) were retrieved. Alveolata and Opisthokonta overwhelmingly dominated the assemblages in the abundance (44.76%, 31.08%) and species richness (59%, 12%). Biodiversity was higher in the slope than the shelf and increased with depth. Temperature and salinity appeared to be the most important deterministic drivers of taxon composition. Community structure was influenced by multiple factors in the importance order of: environmental factors (temperature + salinity) > spatial factor > water depth > sampling time. Furthermore, the neutral model explained more variations in the smaller-sized (0.22-3 µm) community (24%) than larger-sized (3-200 µm) community (16%) but generally explained less variations than did deterministic processes. Additionally, our data indicated that the larger plankton might be more environmentally filtered and less plastic whereas the smaller plankton had stronger dispersal ability. This study sheds light on the differential contributions of the deterministic process and stochastic process and complexities of assembly mechanisms in shaping the community assembly of micro-nano and pico-eukaryotic biospheres in a subtropical ocean.

Molecular phylogeny of the spiny-surfaced species of the dinoflagellate Prorocentrum with the description of P. thermophilum sp. nov. and P. criophilum sp. nov. (Prorocentrales, Dinophyceae).

Spiny-surfaced species of Prorocentrum form harmful algal blooms, and its taxonomic identity is obscure due to the size and shape variability. Molecular phylogenies reveal two major clades: one for P. cordatum with sequences mainly retrieved as P. minimum, and the other for P. shikokuense with sequences also retrieved as P. dentatum and P. donghaiense. Several closely related clades still need to be characterized. Here, we provide nuclear SSU and LSU rRNA genes, and nuclear ITS region (ITS1-5.8S gene-ITS2) sequences of the strain CCMP3122 isolated from Florida (initially named P. donghaiense) and strains Prorocentrum sp. RCC6871-2 from the Ross Sea, Antarctica. We describe Prorocentrum thermophilum sp. nov. based on the strain CCMP3122, a species also distributed in the open waters of the Gulf of Mexico, New Zealand, and the Arabian Gulf; and Prorocentrum criophilum sp. nov. based on the strain RCC6872, which is distributed in the Antarctic Ocean and Arctic Sea. Prorocentrum thermophilum is roundish (~14 µm long, ~12 µm wide), with an inconspicuous anterior spine-like prolongation under light microscopy, valves with tiny, short knobs (5-7 per µm2 ), and several (<7) large trichocyst pores (~0.3 µm) in the right valve, as well as smaller pores (~0.15 µm). Prorocentrum criophilum is round in valve view (~11 µm long, 10 µm wide) and asymmetrically roundish in lateral view, the periflagellar area was not discernible under light microscopy, valves with very tiny, short knobs (6-10 per µm2 ), and at least 12 large pores in the right valve. Other potentially undescribed species of spiny-surfaced Prorocentrum are discussed.

Industry chain and challenges of microalgal food industry-a review.

Currently, the whole world is facing hunger due to the increase in the global population and the rising level of food consumption. Unfortunately, the impact of environmental, climate, and political issues on agriculture has resulted in limited global food resources. Thus, it is important to develop new food sources that are environmentally friendly and not subject to climate or space limitations. Microalgae represent a potential source of nutrients and bioactive components for a wide range of high-value products. Advances in cultivation and genetic engineering techniques provide prospective approaches to widen their application for food. However, there are currently problems in the microalgae food industry in terms of assessing nutritional value, selecting processes for microalgae culture, obtaining suitable commercial strains of microalgae, etc. Additionally, the limitations of real data of market opportunities for microalgae make it difficult to assess their actual potential and to develop a better industrial chain. This review addresses the current status of the microalgae food industry, the process of commercializing microalgae food and breeding methods. Current research progress in addressing the limitations of microalgae industrialization and future prospects for developing microalgae food products are discussed.

Endoplasmic reticulum-quality control pathway and endoplasmic reticulum-associated degradation mechanism regulate the N-glycoproteins and N-glycan structures in the diatom Phaeodactylum tricornutum.

Tunicamycin inhibits the first step of protein N-glycosylation modification. However, the physiological, transcriptomic, and N-glycomic effects of tunicamycin on important marine diatom Phaeodactylum tricornutum are still unknown. In this study, comprehensive approaches were used to study the effects of tunicamycin stress. The results showed that cell growth and photosynthesis were significantly inhibited in P. tricornutum under the tunicamycin stress. The soluble protein content was significantly decreased, while the soluble sugar and neutral lipid were dramatically increased to orchestrate the balance of carbon and nitrogen metabolisms. The stress of 0.3 µg ml-1 tunicamycin resulted in the differential expression of ERQC and ERAD related genes. The upregulation of genes involved in ERQC pathway, the activation of anti-oxidases and the differential expression of genes related with ERAD mechanism might be important for maintaining homeostasis in cell. The identification of N-glycans, especially complex-type N-glycan structures enriched the N-glycan database of diatom P. tricornutum and provided important information for studying the function of N-glycosylation modification on proteins. As a whole, our study proposed working models of ERQC and ERAD will provide a solid foundation for further in-depth study of the related mechanism and the diatom expression system.

Active meiosis during dinoflagellate blooms: A 'sex for proliferation' hypothesis.

In dinoflagellates, sexual reproduction is best known to be induced by adverse environmental conditions and culminate in encystment for survival ('sex for encystment'). Although increasing laboratory observations indicate that sex can lead to production of vegetative cells bypassing encystment, the occurrence of this alternative pathway in natural populations and its ecological roles remain poorly understood. Here we report evidence that sex in dinoflagellates can potentially be an instrument for bloom proliferation or extension. By bloom metatranscriptome profiling, we documented elevated expression of meiosis genes in two evolutionarily distinct species (Prorocentrum shikokuense and Karenia mikimotoi) during bloom, a timing unexpected of the 'sex for encystment' scenario. To link these genes to meiosis, we induced encystment and cyst germination in the cyst-forming species Scrippsiella acuminata, and found that five of these genes were upregulated during cyst germination, when meiosis occurs. Integrating data from all three species revealed that SPO11, MND1, and DMC1 were likely common between cyst-forming and non-encysting sex in dinoflagellates. Furthermore, flow cytometric analyses revealed consecutive rounds of DNA halving during blooms of P. shikokuense and K. mikimotoi, evidencing meiosis. These data provided novel evidence that sexual reproduction in dinoflagellates might serve to promote cell proliferation, and along with the consequent enhancement of genetic diversity facilitating resistance against pathogens and environmental stress, to boost or extend a bloom ('sex for proliferation'). The putative meiosis-specific genes and insights reported here will prove to be helpful for rigorously testing the hypothesis and addressing whether the two modes of sex are genetically predisposed (i.e. species-specific) or environmentally induced (switchable within species), and if the latter what triggers the switch.

Diversity Distribution, Driving Factors and Assembly Mechanisms of Free-Living and Particle-Associated Bacterial Communities at a Subtropical Marginal Sea.

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.

Laminarin, a Major Polysaccharide in Stramenopiles.

During the processes of primary and secondary endosymbiosis, different microalgae evolved to synthesis different storage polysaccharides. In stramenopiles, the main storage polysaccharides are ß-1,3-glucan, or laminarin, in vacuoles. Currently, laminarin is gaining considerable attention due to its application in the food, cosmetic and pharmaceuticals industries, and also its importance in global biogeochemical cycles (especially in the ocean carbon cycle). In this review, the structures, composition, contents, and bioactivity of laminarin were summarized in different algae. It was shown that the general features of laminarin are species-dependence. Furthermore, the proposed biosynthesis and catabolism pathways of laminarin, functions of key genes, and diel regulation of laminarin were also depicted and comprehensively discussed for the first time. However, the complete pathways, functions of genes, and diel regulatory mechanisms of laminarin require more biomolecular studies. This review provides more useful information and identifies the knowledge gap regarding the future studies of laminarin and its applications.

Physiological and transcriptomic responses to N-deficiency and ammonium: Nitrate shift in Fugacium kawagutii (Symbiodiniaceae).

Symbiodiniaceae are the source of essential coral symbionts of reef building corals. The growth and density of endosymbiotic Symbiodiniaceae within the coral host is dependent on nutrient availability, yet little is known about how Symbiodiniaceae respond to the dynamics of the nutrients, including switch between different chemical forms and changes in abundance. In this study, we investigated physiological, cytometric, and transcriptomic responses in Fugacium kawagutii to nitrogen (N)-nutrient deficiency and different chemical N forms (nitrate and ammonium) in batch culture conditions. We mainly found that ammonium was consumed faster than nitrate when provided separately, and was preferentially utilized over nitrate when both N compounds were supplied at 1:2, 1:1 and 2:1 molarity ratios. Besides, N-deficiency caused decreases in growth, energy production, antioxidative capacity and investment in photosynthate transport but increased energy consumption. Growing on ammonium produced a similar cell yield as nitrate, but with a reduced investment in nutrient transport and assimilation; yet at high concentrations ammonium exhibited inhibitory effects. These findings together have important implications in N-nutrient regulation of coral symbiosis. In addition, we identified ten highly and stably expressed genes as candidate reference genes, which will be potentially useful for gene expression studies in the future.

SAGER: a database of Symbiodiniaceae and Algal Genomic Resource.

Symbiodiniaceae dinoflagellates are essential endosymbionts of reef building corals and some other invertebrates. Information of their genome structure and function is critical for understanding coral symbiosis and bleaching. With the rapid development of sequencing technology, genome draft assemblies of several Symbiodiniaceae species and diverse marine algal genomes have become publicly available but spread in multiple separate locations. Here, we present a Symbiodiniaceae and Algal Genomic Resource Database (SAGER), a user-friendly online repository for integrating existing genomic data of Symbiodiniaceae species and diverse marine algal gene sets from MMETSP and PhyloDB databases. Relevant algal data are included to facilitate comparative analyses. The database is freely accessible at http://sampgr.org.cn. It provides comprehensive tools for studying gene function, expression and comparative genomics, including search tools to identify gene information from Symbiodiniaceae species, and BLAST tool to find orthologs from marine algae and protists. Moreover, SAGER integrates transcriptome datasets derived from diverse culture conditions of corresponding Symbiodiniaceae species. SAGER was developed with the capacity to incorporate future Symbiodiniaceae and algal genome and transcriptome data, and will serve as an open-access and sustained platform providing genomic and molecular tools that can be conveniently used to study Symbiodiniaceae and other marine algae. Database URL: http://sampgr.org.cn.

High throughput sequencing of 18S rRNA and its gene to characterize a Prorocentrum shikokuense (Dinophyceae) bloom.

Comparing phytoplankton non-bloom and bloom communities using rRNA and its coding gene can help understand the shift of dominant species and its driving processes (e.g., intrinsic growth or grazing). Here we conducted high-throughput sequencing of 18S rRNA and its coding gene for studying non-bloom and bloom plankton communities in East China Sea. The non-bloom community was dominated by diatoms whereas during the bloom it was dominated by the dinoflagellate Prorocentrum shikokuense (formerly P. donghaiense). P. shikokuense rRNA abundance and rRNA:rRNA gene ratio both increased markedly in the bloom, indicating that the bloom arose from active growth. In contrast, some non-bloom species showed low DNA abundances during the bloom albeit high rRNA:rRNA gene ratios, suggesting that cell loss processes such as grazing might have prevented these species from blooming or that these species might be at an early stage of bloom development. Furthermore, Pearson's correlation analysis showed that dinoflagellate abundance was positively correlated with temperature and negatively related to dissolved inorganic phosphate (DIP) concentrations, suggesting warm and DIP-poor environment as a niche space for P. shikokuense. Our results demonstrate the usefulness of combined analysis of rRNA and its gene in characterizing phytoplankton bloom development to shed light on the complex phytoplankton dynamics and regulating mechanisms in the course of bloom development.

RNA-seq profiling of Fugacium kawagutii reveals strong responses in metabolic processes and symbiosis potential to deficiencies of iron and other trace metals.

A healthy symbiotic relationship between corals and Symbiodiniaceae relies on suitable temperature and adequate nutrients including trace metals. Besides global warming, trace metal deficiency has been shown to cause coral bleaching, a phenomenon responsible for extensive coral reef degradation around the world. How trace metal deficiency impacts Symbiodiniaceae and coral symbiosis is poorly understood, however. In this study, we applied RNA-seq to investigate how Fugacium kawagutii responds to the deficiency of five trace metals (Fe2+, Zn2+, Cu2+, Mn2+, Ni2+). We identified 685 to 2805 differentially expressed genes (DEGs) from these trace metal deficiency conditions, among which 372 were commonly regulated by all the five trace metals and were significantly enriched in energy metabolism (e.g. fatty acid synthesis). Furthermore, genes associated with extracellular matrix (ECM), cell surface structure and cell adhesion were impacted, suggesting that the ability of recognition and adhesion of F. kawagutii may be altered by trace metal deficiencies. In addition, among the five metals, Fe2+ deficiency exhibited the strongest influence, with Fe-rich redox elements and many antioxidant synthesis genes being markedly down-regulated, indicative of adaptive reduction of Fe demand but a compromised ability to combat oxidative stress. Overall, deficiency of trace metals (especially Fe) seems to repress growth and ability of ROS scavenging, elevate energy metabolism and innate immunity, and alter cell adhesion capability, with implications in symbiosis disruption and coral bleaching.

Genome Improvement and Core Gene Set Refinement of Fugacium kawagutii.

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.

Non-Conventional Metal Ion Cofactor Requirement of Dinoflagellate Alkaline Phosphatase and Translational Regulation by Phosphorus Limitation.

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.