Responses of Prorocentrum cordatum (Ostenfeld) Dodge, 1975 (Dinoflagellata) to copper nanoparticles and copper ions effect.

In the present study, changes were determined in morphological, structural-functional, and fluorescent parameters of Prorocentrum cordatum with the addition of CuO nanoparticles (NPs) and copper ions (CuSO4). A stimulating effect of low Cu2+ concentrations (30 µg L-1) on algal growth characteristics was observed. Higher Cu2+ concentration of 60-600 µg L-1 and CuO NPs concentration of 100-520 µg L-1 inhibited algal growth. Ionic copper is more toxic to P. cordatum than NPs. After 72 h of algae cultivation in the medium supplemented with CuSO4 and CuO NPs, EC50 values (calculated based on cell abundance) were of 60 and 300 µg L-1 (in terms of copper ions), respectively. Reduction in algal growth rate is due to disruption in cell cycle, changes in nuclear morphology, chromatin dispersion, and DNA damage. The studied pollutants slightly affected the efficiency of P. cordatum photosynthetic apparatus. Addition of the pollutants resulted in an increased production of reactive oxygen species (ROS). At a concentration of Cu2+ of 120 µg L-1 and a concentration of CuO NPs 0-300 µg L-1 of CuO NPs increase in ROS production is short-term with a decrease at later stages of the experiment. This is probably due to the activation of antioxidant mechanisms in cells and an increase in the concentration of carotenoids (peridinin) in cells. The high values of ROS production persisted throughout the experiment at sublethal copper concentrations (400-600 µg L-1 of CuSO4 and 520 µg L-1 of CuO NPs). Sublethal concentrations of pollutants caused restructuring of cell membranes in P. cordatum. Shedding of cell membranes (ecdysis) and formation of immobile stages (temporary or resting cysts) were recorded. The pronounced mechanical impact of NPs on the cell surface was observed such as-deformation and damage of a cell wall, its "wrinkling" and shrinkage, and adsorption of NP aggregates.

Multifaceted Dinoflagellates and the Marine Model Prorocentrum cordatum.

BACKGROUND: Dinoflagellates are a monophyletic group within the taxon Alveolata, which comprises unicellular eukaryotes. Dinoflagellates have long been studied for their organismic and morphologic diversity as well as striking cellular features. They have a main size range of 10-100 µm, a complex "cell covering", exceptionally large genomes (∼1-250 Gbp with a mean of 50,000 protein-encoding genes) spread over a variable number of highly condensed chromosomes, and perform a closed mitosis with extranuclear spindles (dinomitosis). Photosynthetic, marine, and free-living Prorocentrum cordatum is a ubiquitously occurring, bloom-forming dinoflagellate, and an emerging model system, particularly with respect to systems biology. SUMMARY: Focused ion beam/scanning electron microscopy (FIB/SEM) analysis of P. cordatum recently revealed (i) a flattened nucleus with unusual structural features and a total of 62 tightly packed chromosomes, (ii) a single, barrel-shaped chloroplast devoid of grana and harboring multiple starch granules, (iii) a single, highly reticular mitochondrion, and (iv) multiple phosphate and lipid storage bodies. Comprehensive proteomics of subcellular fractions suggested (i) major basic nuclear proteins to participate in chromosome condensation, (ii) composition of nuclear pores to differ from standard knowledge, (iii) photosystems I and II, chloroplast complex I, and chlorophyll a-b binding light-harvesting complex to form a large megacomplex (>1.5 MDa), and (iv) an extraordinary richness in pigment-binding proteins. Systems biology-level investigation of heat stress response demonstrated a concerted down-regulation of CO2-concentrating mechanisms, CO2-fixation, central metabolism, and monomer biosynthesis, which agrees with reduced growth yields. KEY MESSAGES: FIB/SEM analysis revealed new insights into the remarkable subcellular architecture of P. cordatum, complemented by proteogenomic unraveling of novel nuclear structures and a photosynthetic megacomplex. These recent findings are put in the wider context of current understanding of dinoflagellates.

The enigmatic nucleus of the marine dinoflagellate Prorocentrum cordatum.

The marine, bloom-forming dinoflagellate Prorocentrum cordatum CCMP 1329 (formerly P. minimum) has a genome atypical of eukaryotes, with a large size of ~4.15 Gbp, organized in plentiful, highly condensed chromosomes and packed in a dinoflagellate-specific nucleus (dinokaryon). Here, we apply microscopic and proteogenomic approaches to obtain new insights into this enigmatic nucleus of axenic P. cordatum. High-resolution focused ion beam/scanning electron microscopy analysis of the flattened nucleus revealed highest density of nuclear pores in the vicinity of the nucleolus, a total of 62 tightly packed chromosomes (~0.4-6.7 µm3), and interaction of several chromosomes with the nucleolus and other nuclear structures. A specific procedure for enriching intact nuclei was developed to enable proteomic analyses of soluble and membrane protein-enriched fractions. These were analyzed with geLC and shotgun approaches employing ion-trap and timsTOF (trapped-ion-mobility-spectrometry time-of-flight) mass spectrometers, respectively. This allowed identification of 4,052 proteins (39% of unknown function), out of which 418 were predicted to serve specific nuclear functions; additional 531 proteins of unknown function could be allocated to the nucleus. Compaction of DNA despite very low histone abundance could be accomplished by highly abundant major basic nuclear proteins (HCc2-like). Several nuclear processes including DNA replication/repair and RNA processing/splicing can be fairly well explained on the proteogenomic level. By contrast, transcription and composition of the nuclear pore complex remain largely elusive. One may speculate that the large group of potential nuclear proteins with currently unknown functions may serve yet to be explored functions in nuclear processes differing from those of typical eukaryotic cells. IMPORTANCE Dinoflagellates form a highly diverse group of unicellular microalgae. They provide keystone species for the marine ecosystem and stand out among others by their very large, unusually organized genomes embedded in the nuclei markedly different from other eukaryotic cells. Functional insights into nuclear and other cell biological structures and processes of dinoflagellates have long been hampered by the paucity of available genomic sequences. The here studied cosmopolitan P. cordatum belongs to the harmful algal bloom-forming, marine dinoflagellates and has a recently de novo assembled genome. We present a detailed 3D reconstruction of the P. cordatum nucleus together with comprehensive proteogenomic insights into the protein equipment mastering the broad spectrum of nuclear processes. This study significantly advances our understanding of mechanisms and evolution of the conspicuous dinoflagellate cell biology.

The microbiome of the dinoflagellate Prorocentrum cordatum in laboratory culture and its changes at higher temperatures.

In the ocean, phytoplankton are dependent on communities of bacteria living in the phycosphere, a hot spot of metabolic and genetic exchange. Many types of interactions between phytoplankton and phycosphere bacteria have been shown, but it is unclear if the microbial communities associated with microalgae strains in culture collections are beneficial or harmful to the host strain. Here, we studied the microbial communities associated with four strains of the dinoflagellate Prorocentrum cordatum that had been isolated from distant geographical locations and maintained in culture collection for hundreds of generations. Community composition was determined by 16S rRNA gene amplicon sequencing. The dinoflagellate host strain was the strongest parameter separating communities, while growth phase, lifestyle (particle-attached versus free-living) and temperature had only a modulating effect. Although the strains had been isolated from distant locations in the Atlantic and Pacific Ocean, 14 ASVs were shared among all strains, the most abundant ones being Gilvibacter, Marivita, uncultivated Rhodobacteraceae, Marinobacter, Hyphomonadaceae, Cupriavidus, Variovorax, and Paucibacter. Adaptation to higher temperatures resulted in specific changes in each phycosphere microbiome, including increased abundance of rare community members. We then compared the growth of the four xenic cultures to that of the axenic P. cordatum CCMP1329. At 20°C, growth of the xenic cultures was similar or slower than that of CCMP1329. At 26°C, all four xenic cultures experienced a death phase, while the axenic culture stably remained in the stationary phase. At 30°C, only two of the xenic cultures were able to grow. A shift of dinoflagellate metabolism from autotrophy to mixotrophy and competition between dinoflagellate and bacteria for limiting nutrients, including essential vitamins, may contribute to these differences in growth patterns.

Oyster hatchery breakthrough of two HABs and potential effects on larval eastern oysters (Crassostrea virginica).

Harmful algal bloom (HAB) dinoflagellate species Karlodinium veneficum and Prorocentrum cordatum (prev. P. minimum) are commonly found in Chesapeake Bay during the late spring and early summer months, coinciding with the spawning season of the eastern oyster (Crassostrea virginica). Unexplained larval oyster mortalities at regional commercial hatcheries prompted screening of oyster hatchery water samples for these HAB species. Both HAB species were found in treated hatchery water during the oyster spawning season, sometimes exceeding bloom cell concentrations (≥ 1,000 cells/mL). To investigate the potential for these HAB species, independently or in co-exposure, to affect larval oyster mortality and activity, 96-h laboratory single and dual HAB bioassays with seven-day-old oyster larvae were performed. Treatments for the single HAB bioassay included fed and unfed controls, K. veneficum at 1,000; 5,000; 10,000; and 50,000 cells/mL, P. cordatum at 100; 5,000; 10,000; and 50,000 cells/mL. Subsequently, the 1,000 cells/mL K. veneficum and 50,000 cells/mL P. cordatum treatments were combined in a co-exposure treatment for the dual HAB bioassay. At all cell concentrations tested, K. veneficum swarmed oyster larvae and caused significant larval oyster mortality by 96 h (Karlo1,000: 21 ± 5%; Karlo5,000: 93 ± 2%; Karlo10,000: 85 ± 3%; Karlo50,000: 83 ± 5%, SE). In contrast, there was no significant difference in larval oyster mortality between the control treatments and any of the P. cordatum treatments by 96 h. By 24 h, larval oysters were significantly less active (immotile) in the presence of either HAB species as compared to control treatments (e.g., Karlo1,000: 37.8 ± 4.1%; Proro100: 47.3 ± 7.4%; Fed: 10.8 ± 3.2%; Unfed: 10.1 ± 4.9%, SE). In the dual HAB bioassay, larval oyster mortality associated with 1,000 cells/mL K. veneficum (44 ± 9%, SE) was not changed by the addition of 50,000 cells/mL P. cordatum (55 ± 7%, SE), demonstrating that K. veneficum was primarily responsible for the observed mortality. This study demonstrated that even low cell concentrations of K. veneficum and P. cordatum are harmful to larval oysters, and could contribute to reductions in oyster hatchery production through impacts on this critical life stage.