This study explores the dynamics of protist communities in the northeastern East China Sea (ECS) focusing on their responses to changing water masses and oceanographic conditions during spring, summer, and autumn in 2023. Water masses were classified using K-means clustering based on physico-chemical parameters, revealing significant seasonal variation. Protist community spatial, temporal, and vertical distributions were analyzed using 18S rRNA gene amplicon sequencing and morphological identification along the 33° N latitude, a region characterized by the confluence of multiple water masses, including Kuroshio surface (KSW) and sub-surface (KSSW) waters, Yellow Sea water (YSW), Changjiang diluted water (CDW), and ECS Shelf water. Protist diversity, community structure, and cell abundance exhibited pronounced seasonal variations influenced by temperature, salinity, and nutrient availability. In spring, YSW, characterized by phosphate deficiencies and, consequently, increased N:P ratios, exhibited a prevalence of dinoflagellates, whose phagotrophic capabilities likely conferred a competitive advantage in such nutrient-fluctuating environments. During summer, diatoms were prevalent in the ECS Shelf water, underscoring the role this water plays in shaping seasonal community structure. In contrast, CDW exhibited low species diversities, likely due to unstable salinity. In autumn, water column mixing increased overall diversity and cell abundance, particularly in YSW, where Chaetoceros socialis and other diatoms thrived. Potential phosphorus limitation, especially in the western waters, may have further influenced the spatial structuring of communities across seasons. These findings highlight complex interactions between oceanographic factors and protistan communities in the northeastern ECS, contributing to a deeper understanding of their distributions and responses to environmental changes.

Intensifying stratification in marginal seas is transforming planktonic ecosystems, yet the interaction between taxonomic composition, functional traits, and biomass remains poorly quantified. Here, we analyze eukaryotic protist communities at three depth layers across three seasonal cruises (May, July, and October 2023) in the northern East China Sea (nECS). We combined small-organelle enriched metagenomics (SoEM) with high-performance liquid chromatography (HPLC) pigment chemotaxonomy to separate two complementary signals: the richness and composition of detected operational taxonomic units (OTUs), derived from SoEM, and the size-fractionated chlorophyll-a biomass, derived from diagnostic pigments. A total of 602 eukaryotic OTUs were recovered from 61 samples. OTU richness, Faith's phylogenetic diversity (PD), and mean nearest-taxon distance-based clustering (Nearest Taxon Index [NTI]) revealed pronounced seasonal restructuring: richness and PD tripled from May and July to October, whereas all three seasons showed positive Net Relatedness Index values, indicating that environmental filtering on community assembly operated throughout the year rather than being a summer-only phenomenon. NTI was nevertheless on average lower in October than in July, consistent with autumn mixing relaxing the fine-scale co-occurrence of closest relatives. In terms of composition, nanoplankton (2-20 ㎛) accounted for the largest share of OTUs under stratified surface conditions, while microplankton (>20 ㎛), mostly diatoms, increased in the deep layer. Mixotrophic protists reached ~30% of OTUs at stratified summer surfaces, and heterotrophs expanded in October. In parallel, pigment-based size-fractionated chlorophyll-a showed that microphytoplankton biomass persisted at and below the deep-chlorophyll maximum year-round, whereas nanophytoplankton biomass dominated surface layers in May and July. Because SoEM quantifies the presence of taxa rather than their abundance, while HPLC integrates standing stock, the combined framework disentangles who is present from who dominates the biomass pool. Our findings highlight that cell size, trophic plasticity, and vertical niche partitioning, mediated by seasonal hydrography, jointly shape the assembly and biogeochemical function of protist communities in the nECS. As climate change intensifies stratification and suppresses seasonal mixing, trait- and size-based models will be essential for predicting future carbon dynamics in warming marginal seas.

Dinoflagellate harbors diverse associated bacterial communities (ABCs) that play crucial roles in host physiology and ecological interactions. Coolia malayensis is a benthic dinoflagellate distributed from temperate to tropical regions. Despite growing knowledge about the morphology, distribution, and toxicity of C. malayensis, little is known about its associated bacteria, particularly under changing environmental conditions. Given that climate change alters microbial interactions through temperature shifts, this study investigated the composition, diversity, and dynamics of ABCs across four C. malayensis strains, under varying temperatures and growth time. Despite differences in strain origin, a stable core microbiome, comprising Alteromonas, Marinobacter, Muricauda, and Ruegeria, was consistently observed, suggesting these taxa are functionally important members of the ABC. However, strain-specific differences and temperature-driven shifts were also detected, especially among low-abundance bacterial taxa. Negative interactions among abundant amplicon sequence variants, such as those between Ruegeria and Muricauda, and between Alteromonas and Marinobacter, were conserved across conditions, suggesting stable patterns of co-occurrence. Functional predictions and Kyoto Encyclopedia of Genes and Genomes pathway analysis suggested potential involvement of ABCs in chemoheterotrophy, fermentation, hydrocarbon degradation, energy metabolism, and the metabolism of cofactors and vitamins, implying potential diverse metabolic exchanges between the bacteria and C. malayensis. These findings highlight both the ecological stability and environmental sensitivity of C. malayensis culture-associated microbiomes, with important implications for host health and ecosystem dynamics under climate change.

While phytoplankton are a major component of coastal ecosystems and experience a wide range of oxygen conditions, the survival and physiological responses of common phytoplankton species under hypoxia remain insufficiently understood. To explore these effects, cell densities of the two dinoflagellate species Prorocentrum triestinum and Amphidinium carterae, and the two diatom species Cylindrotheca closterium and Chaetoceros curvisetus-isolated from waters with a dissolved oxygen (DO) concentration of 1.5 mg O₂ L^-1-were measured under target DO concentrations of 0.5, 1.0, 1.5, 4.0, and >7.0 mg O₂ L^-1. Using these data, the growth rate, net photosynthesis rate, and dark respiration rate of each species were determined. While A. carterae, Cy. closterium, and Ch. curvisetus survived at all tested DO concentrations, P. triestinum survived only at ≥1.0 mg O₂ L^-1. The two dinoflagellate species examined here, which possess active swimming ability, exhibited the highest growth rates at >7.0 mg O₂ L^-1, whereas the two diatom species examined here showed peak growth rate at 1.5 mg O₂ L^-1. Net photosynthesis rates of all four species peaked at 1.5 mg O₂ L^-1, while dark respiration rates were highest at >7.0 mg O₂ L^-1. These findings suggest that although these phytoplankton species can grow under hypoxic conditions, dinoflagellate species achieve maximal growth at higher oxygen concentrations, possibly due to the energetic demands of motility. This study provides a useful basis for understanding species-specific responses of phytoplankton to varying oxygen conditions in coastal environments.

While γ-aminobutyric acid (GABA) promotes nutrient accumulation in green microalgae, its effects on marine diatoms-critical aquaculture feed-remain understudied. Understanding the physiological response of diatoms to exogenous GABA is vital for optimizing high-quality feed production. In this study, we investigated the impact of GABA on diatom growth, photosynthetic performance, proximate composition (lipids, proteins, carbohydrates, and fatty acids), and oxidative stress markers of two commonly used feed diatoms Phaeodactylum tricornutum and Chaetoceros muelleri. GABA effects were species-specific: low doses (0.2-1.0 pmol cell^-1) inhibited P. tricornutum at the exponential onset, while C. muelleri remained resilient to higher doses (1.5-16.2 pmol cell^-1). Notably, this resilience in C. muelleri shifted to growth promotion when GABA was applied at the late stage, demonstrating clear stage-dependency. In P. tricornutum, supplementation with 0.2-1.0 pmol cell^-1 GABA triggered a significant accumulation of cellular carbohydrates, proteins, and lipids accompanied by a broad-spectrum increase in various cellular fatty acid concentrations including polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid and docosahexaenoic acid, important PUFAs. Notably, 1.0 pmol cell^-1 GABA induced a three-fold increase in carbohydrate and lipid levels over the control. GABA triggered significant oxidative stress and photosynthetic inhibition in P. tricornutum in a dose-dependent manner, while C. muelleri exhibited high physiological tolerance with no marked changes in reactive oxygen species and superoxide dismutase or photosynthetic efficiency. A two-stage cultivation strategy involving initial biomass maximization and subsequent GABA-induced nutrient enrichment can be effectively utilized to biofortify P. tricornutum for use as aquaculture feed.

Zeaxanthin epoxidase (ZEP) plays a central role in carotenoid metabolism, but its evolution and function in red algae remain unclear. Here, we comprehensively identified ZEP homologs across red algal lineages through phylogenetic reconstruction, conserved motif analysis, structural comparison, and branch-specific selection pressure analyses to uncover adaptive evolutionary patterns. The ZEP homolog from Pyropia haitanensis (PhZEP) was selected for functional characterization, including promoter cis-element analysis, heterologous expression, in vitro enzymatic assays, and expression profiling under high light and desiccation-rehydration stress. Thirteen ZEP homologs were identified and clustered into three clades, indicating early divergence and a cyanobacterial origin. Lineage-specific structural divergence and purifying selection with relaxed constraints in Bangiophyceae were observed. Recombinant PhZEP specifically converted zeaxanthin to antheraxanthin, but not further to violaxanthin. PhZEP expression was moderately induced by high light and strongly upregulated during desiccation, peaking at 9.52-fold at 4.8% relative water content. These results demonstrate that PhZEP exhibits specific zeaxanthin epoxidase activity and may be associated with abiotic stress responses, providing insights into carotenoid pathway evolution and photoprotection strategies in red algae.

Prasiola japonica is a freshwater green alga distributed in Northeast Asia. This study investigated the effects of 70% ethanol extract of P. japonica (Pj-EE) under testosterone-induced hair loss conditions using C57BL/6 mice and human dermal papilla (HDP) cells. Topical application of Pj-EE promoted hair regrowth, increased hair length, and enhanced follicular density and activity in vivo. Pj-EE treatment also partially restored hair type distribution by reducing the proportion of zigzag hairs and increasing guard and awl hairs. Although these effects were less pronounced than those observed with minoxidil, consistent improvements were detected across multiple parameters. Mechanistic studies showed that Pj-EE inhibited 5α-reductase activity and reduced dihydrotestosterone (DHT) production in HDP cells, accompanied by downregulation of androgen receptor expression. Additionally, Pj-EE decreased pro-apoptotic Bax levels while increasing proliferation-associated markers (Ki67 and proliferating cell nuclear antigen) and the anti-apoptotic protein Bcl-2 in a dose-dependent manner. Functionally, Pj-EE enhanced HDP cell proliferation and migration and attenuated testosterone-induced apoptotic responses, indicating improved follicular cell viability under androgen-stimulated conditions. High-performance liquid chromatography analysis based on comparison with an authentic phytol standard identified phytol as a representative constituent of Pj-EE, and its high abundance was associated with 5α-reductase inhibitory activity. Collectively, these findings suggest that Pj-EE may attenuate androgen-responsive hair loss conditions by modulating 5α-reductase/DHT signaling and supporting follicular cell proliferation and survival in a testosterone-induced model.