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

Abstract(#br)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 (Fe 2+ , Zn 2+ , Cu 2+ , Mn 2+ , Ni 2+ ). 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, Fe 2+ 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

Identification and Expression Analysis of an Atypical Alkaline Phosphatase in Emiliania huxleyi

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

Acoustic wave-driven microdroplet enrichment for surface-enhanced Raman spectroscopy detection

Surface-enhanced Raman scattering (SERS) holds significant potential across environmental monitoring, materials science, and biomedical applications. However, challenges regarding ultra-sensitive detection and repeatability are bottlenecks for practical applications, especially in terms of detection uniformity. In this study, we utilized surface acoustic waves (SAW) in conjunction with Raman spectroscopy to actively enrich 5 μL of 50 nm gold nanoparticles (AuNPs), thereby achieving innovative SERS-active sensing. This dynamic enrichment process enables the dense and uniform aggregation of AuNPs in droplets, thereby facilitating reliable ultrasensitive detection. The SAW system was further optimized through hydrophobic surface treatment. Using 4-mercaptobenzoic acid as a probe analyte, our SAW-SERS method successfully detected concentrations as low as 10−8 mol/L. The surface acoustic waves had the capability to significantly amplify Raman signal intensity up to 100 compared to conventional drying methodologies. This SAW-induced AuNP clustering technology offers a rapid, label-free SERS sensing method characterized by exceptional sensitivity and uniformity

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

Physiological and transcriptomic responses to N-deficiency and ammonium: nitrate shift in <i>Fugacium kawagutii</i> (Symbiodiniaceae)

AbstractSymbiodiniaceae are the source of essential coral symbionts of reef building corals. The growth and density of endosymbiotic Symbodiniaceae within the coral host is highly 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 nitrogen 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 decreased investment in nutrient transport and assimilation. These all have important implications of N nutrient to support symbiosis in coral ecosystem, especially regarding ammonium. In addition, by integrating our current results with previous data, we identified ten highly and stably expressed genes as candidate reference genes, which will be potentially useful for gene expression studies in the future.</jats:p

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.</jats:p

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 &beta;-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 &zeta;-carotene desaturase) and CHYB (the gene of &beta;-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

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

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.</jats:p