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Triphenyltin (TPT) often coexists with tributyltin (TBT) and Cu in coastal waters worldwide. The combined toxic effect of TPT and TBT has always been assumed to be additive without any scientific proof, and the combined effect of Cu and TPT on marine organisms has not been vigorously studied. This study, therefore, investigated the acute toxicity of binary mixture of TPT/Cu and TPT/TBT to five selected marine species including Thalassiosira pseudonana , Skeletonema costatum , Tigriopus japonicus , Brachionus koreanus and Oryzias melastigma . The interaction between TPT and TBT or Cu was modeled antagonistic based on concentration addition (CA) model, while it was synergistic according to response addition (RA) model. Both model well predicted the toxicity of binary mixtures to the five organisms. As for the environmental risk assessment, CA overestimated the toxicity in most cases and thus is a more conservative model than RA model for assessing the toxicity of these chemical mixtures. [ABSTRACT FROM AUTHOR]

A 96-h exposure experiment was conducted to elucidate the toxicity responses of the marine diatom Thalassiosira pseudonana upon exposure to different temperatures and copper (Cu) concentrations. Three Cu treatments (seawater control; 200 μg/L Cu, EC 50 for the yield at 25 °C; and 1000 μg/L Cu, EC 50 for growth inhibition at 25 °C) were conducted against four temperatures (10 °C, 15 °C, 25 °C and 30 °C). Growth rate and photosynthetic responses showed a significant interacting thermal-chemical effect with strong synergistic responses observed at 30 °C treatments. Expression of heat shock protein ( hsp ) was positively modulated by increasing temperatures. Hsp 90 , hsp90–2 and sit1 (related to silica shell formation) were highly expressed at 30 °C under 1000 μg/L Cu, while the genes encoding light harvesting proteins ( 3HfcpA and 3HfcpB ) and silaffin precursor sil3 were significantly up-regulated at 15 °C under 200 μg/L Cu. Our results indicated an increase Cu toxicity to T. pseudonana under high temperature and Cu dose. [ABSTRACT FROM AUTHOR]

The photosynthetic response of the marine diatom Thalassiosira pseudonana to a matrix of dynamic light regimes is described. Ash-free dry weight and chlorophyll-a were measured as a function of dynamic irradiance having maximum intensities of 250, 500, 1000 and 2000 μmol photons m-2 s-1 with lengths of day being 6, 9, 12, 18 and 24-h periods. Incident irradiance followed a Gaussian (normal) distribution, which provided a statistical standard for the integrated quantum flux into a 20 cm water column. The matrix of conditions resulted with growth occurring in three groups (low, high and intermediate light) as a function of the amount for residual irradiance estimated at the mixing depth of 20 cm. The low light group displayed shade-limited ("linear" from self-shading) and the high light group displayed light-limited ("exponential" with no self-shading) phases of the light-controlled growth dynamic. For the same range of integrated daily incident irradiances, cultures growing under the phase of "linear" growth had higher biomass yields per quantum than cultures growing under an "exponential" phase of the growth dynamic. The difference in net quantum efficiency due to phase of the growth dynamic entails the relationship between photic zone and mixing depth and provides a new perspective for interpreting Sverdrup's Critical Depth Model. [ABSTRACT FROM AUTHOR]

We investigated the effects of iron (Fe) limitation on the elemental stoichiometry, silicic acid (Si(OH)4) uptake kinetics and cell morphology in two Southern Ocean diatoms Eucampia antarctica and Proboscia inermis and the temperate diatom Thalassiosira pseudonana. An increase in Fe-stress resulted in reductions in specific growth rate and decreases in cellular nitrogen (N) and carbon (C) content relative to cellular biogenic silica (BSi) in both Southern Ocean diatoms and a reduction in growth rate only for T. pseudonana. Both E. antarctica and P. inermis exhibited an increase in cell volume in response to Fe-limitation resulting in a decrease in the cell surface to volume ratio, while normalization of BSi content to cell surface area suggests these diatoms do not become more heavily silicified under Fe limitation. Kinetic Si(OH)4 uptake experiments performed on all three diatom species show that Si(OH)4 uptake is reduced under Fe-limited conditions. For Southern Ocean diatoms, this was manifested through a decrease in the maximum specific uptake rate of Si(OH)4 ( VSi-max), along with a decrease in the half-saturation constant for Si(OH)4 uptake ( KSi) under Fe stress for E. antarctica. Our data also show that when normalized to cell surface area, VSi-max of the three diatoms species exhibited a linear relationship with cellular growth rate, and was independent of cell morphological variations. Our results suggest that the morphological adaptations of Southern Ocean diatoms in response to Fe-stress have the potential to affect phytoplankton community dynamics and Si(OH)4 : NO3 uptake and export ratios in Southern Ocean waters. [ABSTRACT FROM AUTHOR]

Background: Flagella have been lost in the vegetative phase of the diatom life cycle, but they are still present in male gametes of centric species, thereby representing a hallmark of sexual reproduction. This process, besides maintaining and creating new genetic diversity, in diatoms is also fundamental to restore the maximum cell size following its reduction during vegetative division. Nevertheless, sexual reproduction has been demonstrated in a limited number of diatom species, while our understanding of its different phases and of their genetic control is scarce. Results: In the transcriptome of Leptocylindrus danicus, a centric diatom widespread in the world's seas, we identified 22 transcripts related to the flagella development and confirmed synchronous overexpression of 6 flagellum-related genes during the male gamete formation process. These transcripts were mostly absent in the closely related species L. aporus, which does not have sexual reproduction. Among the 22 transcripts, L. danicus showed proteins that belong to the Intra Flagellar Transport (IFT) subcomplex B as well as IFT-A proteins, the latter previously thought to be absent in diatoms. The presence of flagellum-related proteins was also traced in the transcriptomes of several other centric species. Finally, phylogenetic reconstruction of the IFT172 and IFT88 proteins showed that their sequences are conserved across protist species and have evolved similarly to other phylogenetic marker genes. Conclusion: Our analysis describes for the first time the diatom flagellar gene set, which appears to be more complete and functional than previously reported based on the genome sequence of the model centric diatom, Thalassiosira pseudonana. This first recognition of the whole set of diatom flagellar genes and of their activation pattern paves the way to a wider recognition of the relevance of sexual reproduction in individual species and in the natural environment. [ABSTRACT FROM AUTHOR]

Diatoms are marine primary producers that sink in part due to the density of their silica frustules. Sinking of these phytoplankters is crucial for both the biological pump that sequesters carbon to the deep ocean and for the life strategy of the organism. Sinking rates have been previously measured through settling columns, or with fluorimeters or video microscopy arranged perpendicularly to the direction of sinking. These side-view techniques require large volumes of culture, specialized equipment and are difficult to scale up to multiple simultaneous measures for screening. We established a method for parallel, large scale analysis of multiple phytoplankton sinking rates through top-view monitoring of chlorophyll a fluorescence in microtitre well plates. We verified the method through experimental analysis of known factors that influence sinking rates, including exponential versus stationary growth phase in species of different cell sizes; Thalassiosira pseudonana CCMP1335, chain-forming Skeletonema marinoi RO5A and Coscinodiscus radiatus CCMP312. We fit decay curves to an algebraic transform of the decrease in fluorescence signal as cells sank away from the fluorometer detector, and then used minimal mechanistic assumptions to extract a sinking rate (m d-1) using an RStudio script, SinkWORX. We thereby detected significant differences in sinking rates as larger diatom cells sank faster than smaller cells, and cultures in stationary phase sank faster than those in exponential phase. Our sinking rate estimates accord well with literature values from previously established methods. This well plate-based method can operate as a high throughput integrative phenotypic screen for factors that influence sinking rates including macromolecular allocations, nutrient availability or uptake rates, chain-length or cell size, degree of silification and progression through growth stages. Alternately the approach can be used to phenomically screen libraries of mutants. [ABSTRACT FROM AUTHOR]

Diatoms are important components of marine ecosystems and contribute greatly to the world’s primary production. Despite their important roles in ecosystems, the molecular basis of how diatoms cope with oxidative stress caused by nutrient fluctuations remains largely unknown. Here, an isobaric tags for relative and absolute quantitation (iTRAQ) proteomic method was coupled with a series of physiological and biochemical techniques to explore oxidative stress- and cell fate decision-related cellular and metabolic responses of the diatom Thalassiosira pseudonana to nitrate (N) and inorganic phosphate (P) stresses. A total of 1151 proteins were detected; 122 and 56 were significantly differentially expressed from control under N- and P-limited conditions, respectively. In N-limited cells, responsive proteins were related to reactive oxygen species (ROS) accumulation, oxidative stress responses and cell death, corresponding to a significant decrease in photosynthetic efficiency, marked intracellular ROS accumulation, and caspase-mediated programmed cell death activation. None of these responses were identified in P-limited cells; however, a significant up-regulation of alkaline phosphatase proteins was observed, which could be the major contributor for P-limited cells to cope with ambient P deficiency. These findings demonstrate that fundamentally different metabolic responses and cellular regulations are employed by the diatom in response to different nutrient stresses and to keep the cells viable. [ABSTRACT FROM AUTHOR]

Background: An inducible promoter for recombinant protein expression provides substantial benefits because under induction conditions cellular energy and metabolic capability can be directed into protein synthesis. The most widely used inducible promoter for diatoms is for nitrate reductase, however, nitrogen metabolism is tied into diverse aspects of cellular function, and the induction response is not necessarily robust. Silicon limitation offers a means to eliminate energy and metabolic flux into cell division processes, with little other detrimental effect on cellular function, and a protein expression system that works under those conditions could be advantageous. Results: In this study, we evaluate a number of promoters for recombinant protein expression induced by silicon limitation and repressed by the presence of silicon in the diatoms Thalassiosira pseudonana and Cyclotella cryptica. In addition to silicon limitation, we describe additional strategies to elevate recombinant protein expression level, including inclusion of the 5' fragment of the coding region of the native gene and reducing carbon flow into ancillary processes of pigment synthesis and formation of photosynthetic storage products. We achieved yields of eGFP to 1.8% of total soluble protein in C. cryptica, which is about 3.6-fold higher than that obtained with chloroplast expression and ninefold higher than nuclear expression in another well-established algal system. Conclusions: Our studies demonstrate that the combination of inducible promoter and other strategies can result in robust expression of recombinant protein in a nuclear-based expression system in diatoms under silicon limited conditions, separating the protein expression regime from growth processes and improving overall recombinant protein yields. [ABSTRACT FROM AUTHOR]

Temperature and nutrients are fundamental, highly nonlinear drivers of biological processes, but we know little about how they interact to influence growth. This has hampered attempts to model population growth and competition in dynamic environments, which is critical in forecasting species distributions, as well as the diversity and productivity of communities. To address this, we propose a model of population growth that includes a new formulation of the temperature-nutrient interaction and test a novel prediction: that a species' optimum temperature for growth, Topt, is a saturating function of nutrient concentration. We find strong support for this prediction in experiments with a marine diatom, Thalassiosira pseudonana: Topt decreases by 3-6 °C at low nitrogen and phosphorus concentrations. This interaction implies that species are more vulnerable to hot, low-nutrient conditions than previous models accounted for. Consequently the interaction dramatically alters species' range limits in the ocean, projected based on current temperature and nitrate levels as well as those forecast for the future. Ranges are smaller not only than projections based on the individual variables, but also than those using a simpler model of temperature-nutrient interactions. Nutrient deprivation is therefore likely to exacerbate environmental warming's effects on communities. [ABSTRACT FROM AUTHOR]

Untargeted metabolomics datasets provide ample opportunity for discovery of novel metabolites. The major challenge is focusing data analysis on a short list of metabolites. Here, we apply a combination of computational tools that serve to reduce complex mass spectrometry data in order allow us to focus on new environmentally-relevant metabolites. In the first portion of the project, we explored mass spectrometry data from intracellular metabolites extracted from a model marine diatom, Thalassiosira pseudonana . The fragmentation data from these samples were analyzed using molecular networking, an on-line tool that clusters metabolites based on shared structural similarities. The features within each metabolite cluster were then putatively annotated using MetFrag, an in silico fragmentation tool. Using this combination of computational tools, we observed multiple lyso-sulfolipids, organic compounds not previously known to exist within cultured marine diatoms. In the second stage of the project, we searched our environmental data for these lyso-sulfolipids. The lyso-sulfolipid with a C14:0 fatty acid was found in dissolved and particulate samples from the western Atlantic Ocean, and a culture of cyanobacteria grown in our laboratory. Thus, the putative lyso-sulfolipids are present in both laboratory experiments and environmental samples. This project highlights the value of combining computational tools to detect and putatively identify organic compounds not previously recognized as important within T. pseudonana or the marine environment. Future applications of these tools to emerging metabolomics data will further open the black box of natural organic matter, identifying molecules that can be used to understand and monitor the global carbon cycle. [ABSTRACT FROM AUTHOR]

Background: Biological mineral formation (biomineralization) proceeds in specialized compartments often bounded by a lipid bilayer membrane. Currently, the role of membranes in biomineralization is hardly understood. Results: Investigating biomineralization of SiO2 (silica) in diatoms we identified Silicanin-1 (Sin1) as a conserved diatom membrane protein present in silica deposition vesicles (SDVs) of Thalassiosira pseudonana. Fluorescence microscopy of GFP-tagged Sin1 enabled, for the first time, to follow the intracellular locations of a biomineralization protein during silica biogenesis in vivo. The analysis revealed incorporation of the N-terminal domain of Sin1 into the biosilica via association with the organic matrix inside the SDVs. In vitro experiments showed that the recombinant N-terminal domain of Sin1 undergoes pH-triggered assembly into large clusters, and promotes silica formation by synergistic interaction with long-chain polyamines. Conclusions: Sin1 is the first identified SDV transmembrane protein, and is highly conserved throughout the diatom realm, which suggests a fundamental role in the biomineralization of diatom silica. Through interaction with long-chain polyamines, Sin1 could serve as a molecular link by which the SDV membrane exerts control on the assembly of biosilica-forming organic matrices in the SDV lumen. [ABSTRACT FROM AUTHOR]

Continuous cultures of the marine diatom Thalassiosira pseudonana were grown at irradiances between 6 and 47 µmol m −2 s −1 in order to evaluate the effect of light on hydrogen isotope fractionation in lipids. δ 2 H values increased with irradiance in phytol by 1.1‰ (µmol m −2 s −1 ) −1 and by 0.3‰ (µmol m −2 s −1 ) −1 in the C14:0 fatty acid, but decreased by 0.8‰ (µmol m −2 s −1 ) −1 in the sterol 24-methyl-cholesta-5,24(28)-dien-3β-ol (C 28 Δ 5,24(28) ). The anticorrelation between δ 2 H values in C 28 Δ 5,24(28) and irradiance is attributed to enhanced sterol precursor synthesis via the plastidic methylerythritol phosphate (MEP) pathway at high irradiance, relative to the cytosolic mevalonic acid (MVA) pathway, and the supposition that MEP precursors are 2 H-depleted compared to MVA precursors because they incorporate a greater proportion of hydrogen from photosynthetically produced NADPH. Increasing δ 2 H values of phytol and C14:0 with irradiance is attributed to a greater proportion of pyruvate, the last common precursor to both lipids, being sourced from glycolysis in the mitochondria and cytosol, where enhanced incorporation of metabolic NADPH and further hydrogen exchange with cell water can enrich pyruvate with 2 H relative to pyruvate from the chloroplast. Irrespective of the biosynthetic mechanisms responsible for the 2 H/ 1 H fractionation response to light, the high sensitivity of lipid δ 2 H values in T. pseudonana continuous cultures would result in −30‰ to +40‰ variations in δ 2 H over a 40 µmol m −2 s −1 range in sub-saturating irradiance if expressed in the environment, depending on the lipid. [ABSTRACT FROM AUTHOR]

After the exponential growth phase, variability in the scattering efficiency of phytoplankton cells over their complete life cycle is not well characterised. Bulk measurements are impacted by senescent cells and detritrus. Thus the analysis of the evolution of the optical properties thanks to their morphological and/or intra-cellular variations remains poorly studied. Using the Cytosense flow cytometer (CytoBuoy b.v., NL), the temporal course of the forward and sideward efficiencies of two phytoplankton species (Thalassiosira pseudonana and Chlamydomonas concordia) were analyzed during a complete life-cycle. These two species differ considerably from a morphological point of view. Over the whole experiment, the forward and sideward efficiencies of Thalassiosira pseudonana were, on average, respectively 2.2 and 1.6 times higher than the efficiencies of Chlamydomonas concordia. Large intra-species variability of the efficiencies were observed over the life cycle of the considered species. It highlights the importance of considering the optical properties of phytoplankton cells as a function of the population growth stage of the considered species. Furthermore, flow cytometry measurements were combined with radiative transfer simulations and biogeochemical and optical measurements. Results showed that the real refractive index of the chloroplast is a key parameter driving the sideward signal and that a simplistic two-layered model (cytoplasm-chloroplast) seems particularly appropriate to represent the phytoplankton cells. [ABSTRACT FROM AUTHOR]

The reproductive strategy of diatoms includes asexual and sexual phases, but in many species, including the model centric diatom Thalassiosira pseudonana, sexual reproduction has never been observed. Furthermore, the environmental factors that trigger sexual reproduction in diatoms are not understood. Although genome sequences of a few diatoms are available, little is known about the molecular basis for sexual reproduction. Here we show that ammonium reliably induces the key sexual morphologies, including oogonia, auxospores, and spermatogonia, in two strains of T. pseudonana, T. weissflogii, and Cyclotella cryptica. RNA sequencing revealed 1,274 genes whose expression patterns changed when T. pseudonana was induced into sexual reproduction by ammonium. Some of the induced genes are linked to meiosis or encode flagellar structures of heterokont and cryptophyte algae. The identification of ammonium as an environmental trigger suggests an unexpected link between diatom bloom dynamics and strategies for enhancing population genetic diversity. [ABSTRACT FROM AUTHOR]

The study investigated the effect of the thylakoid membrane lipids monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulphoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) on the structure of two algal light-harvesting complexes (LHCs). In contrast to higher plants whose thylakoid membranes are characterized by an enrichment of the neutral galactolipids MGDG and DGDG, both the green alga Mantoniella squamata and the centric diatom Thalassiosira pseudonana contain membranes with a high content of the negatively charged lipids SQDG and PG. The algal thylakoids do not show the typical grana-stroma differentiation of higher plants but a regular arrangement. To analyze the effect of the membrane lipids, the fucoxanthin chlorophyll protein (FCP) complex of T. pseudonana and the LHC of M. squamata (MLHC) were prepared by successive cation precipitation using Triton X-100 as detergent. With this method, it is possible to isolate LHCs with a reduced amount of associated lipids in an aggregated state. The results from 77 K fluorescence and photon correlation spectroscopy show that neither the neutral galactolipids nor the negatively charged lipids are able to significantly alter the aggregation state of the FCP or the MLHC. This is in contrast to higher plants where SQDG and PG lead to a strong disaggregation of the LHCII whereas MGDG and DGDG induce the formation of large macroaggregates. The results indicate that LHCs which are integrated into thylakoid membranes with a high amount of negatively charged lipids and a regular arrangement are less sensitive to lipid-induced structural alterations than their counterparts in membranes enriched in neutral lipids with a grana-stroma differentiation. [ABSTRACT FROM AUTHOR]

The article discusses research on the expression of Histophilus somni IbpA DR2 protective antigen in the diatom Thalassiosira pseudonana. Topics include the development of expression systems in order to produce a vaccine against bovine respiratory disease, the efficiency of using unicellular microalgae as an expression platform, and modest immune response successfully exhibited in mice exposed to diatom-expressed DR2 in whole or sonicated cells.

Fast Repetition and Relaxation chlorophyll fluorescence induction is used to estimate the effective absorption cross section of PSII (σ PSII ), to analyze phytoplankton acclimation and electron transport. The fitting coefficient ρ measures excitation transfer from closed PSII to remaining open PSII upon illumination, which could theoretically generate a progressive increase in σ PSII for the remaining open PSII. To investigate how ρ responds to illumination we grew marine phytoplankters with diverse antenna structures ( Prochlorococcus , Synechococcus , Ostreococcus and Thalassiosira pseudonana ) under limiting or saturating growth light. Initial ρ varied with growth light in Synechococcus and Thalassiosira . With increasing actinic illumination PSII closed progressively and ρ decreased for all four taxa, in a pattern explicable as an exponential decay of ρ with increasing distance between remaining open PSII reaction centers. This light-dependent down-regulation of ρ allows the four phytoplankters to limit the effect of increasing light upon σ PSII . The four structurally distinct taxa showed, however, distinct rates of response of ρ to PSII closure, likely reflecting differences in the spacing or orientation among their PSII centers. Following saturating illumination recovery of ρ in darkness coincided directly with PSII re-opening in Prochlorococcus . Even after PSII had re-opened in Synechococcus a transition to State II slowed dark recovery of ρ. In Ostreococcus sustained NPQ slowed dark recovery of ρ. In Thalassiosira dark recovery of ρ was slowed, possibly by a light-induced change in PSII spacing. These patterns of ρ versus PSII closure are thus a convenient probe of comparative PSII spacings. [ABSTRACT FROM AUTHOR]

The presence of CO2-concentrating mechanisms (CCMs) is believed to be one of the characteristics that allows diatoms to thrive in many environments and to be major contributors to global productivity. Here, the type of CCM and the responses to variable CO2 concentration were studied in marine and freshwater diatoms. At 400 ppm, there was a large diversity in physiological and biochemical mechanisms among the species. While Phaeodactylum tricornutum mainly used HCO3-, Thalassiosira pseudonana mainly used CO2. Carbonic anhydrase was an important component of the CCM in all species and C4 metabolism was absent, even with T. weissflogii. For all species, at 20 000 ppm, the affinity for dissolved inorganic carbon was lower than at 400 ppm CO2 and the reliance on CO2 was higher. Despite the difference in availability of inorganic carbon in marine and fresh waters, there were only small differences in CCMs between species from the two environments, and Navicula pelliculosa behaved similarly when grown in the two environments. The results suggest that species-specific differences are great, and more important than environmental differences in determining the nature and effectiveness of the CCM in diatoms. [ABSTRACT FROM AUTHOR]

Diatoms operate a CO2-concentrating mechanism (CCM) that drives upwards of 20% of annual global primary production. Recent progress in CCM research in the marine pennate diatom Phaeodactylum tricornutum revealed that this diatom directly takes up HCO3- from seawater through low-CO2-inducible plasma membrane HCO3- transporters, which belong to the solute carrier (SLC) 4 family. Apart from this, studies of carbonic anhydrases (CAs) in diatoms have revealed considerable diversity in classes and localization among species. This strongly suggests that the CA systems, which control permeability and flux of dissolved inorganic carbon (DIC) by catalysing reversible CO2 hydration, have evolved from diverse origins. Of particular interest is the occurrence of low-CO2-inducible external CAs in the centric marine diatom Thalassiosira pseudonana, offering a strategy of CA-catalysed initial CO2 entry via passive diffusion, contrasting with active DIC transport in P. tricornutum. Molecular mechanisms to transport DIC across chloroplast envelopes are likely also through specific HCO3- transporters, although details have yet to be elucidated. Furthermore, recent discovery of a luminal θ-CA in the diatom thylakoid implied a common strategy in the mechanism to supply CO2 to RubisCO in the pyrenoid, which is conserved among green algae and some heterokontophytes. These results strongly suggest an occurrence of convergent coevolution between the pyrenoid and thylakoid membrane in aquatic photosynthesis. [ABSTRACT FROM AUTHOR]

The present research investigated the effect of diuron on sinking rate and the physiochemical changes in two marine diatoms, Thalassiosira pseudonana (single-celled species) and Skeletonema marinoi-dohrnii complex (chain-forming species). The results revealed that the sinking rate of both diatoms exposed to diuron at a level of 50% effective concentration for growth (EC50) decreased significantly compared with the control. Photosynthetic performance (Fv/Fm and PI ABS ) of both diatoms also decreased significantly with diuron exposure. The number of cells per chain in S. marinoi-dohrnii decreased significantly with diuron treatment, but T. pseudonana cell diameter remained stable. Neutral lipid concentration per cell was significantly higher compared with control at 72 h in both diatom species exposed to EC50 level diuron. And water-soluble protein concentration per cell at 72 h was lower than control in the T. pseudonana EC50 group only. These biochemical changes may decrease specific gravity of cells and seems to cause a decreased sinking rate in diatoms. The positive significant correlation between the numbers of cells per chain and sinking rate in S. marinoi-dohrnii indicated that chain length is also an important factor in sinking rate regulation for chain-forming diatoms. Thus, our present study suggested that suppression of photosynthetic performance and the resultant physiochemical changes induced the decreased sinking rate that may inhibit the normal survival strategy (avoidance from the surface layer where strong light either causes photo-inhibition or interrupts resting cell formation). Therefore, the use of antifouling agents should be considered for the sustainable marine environment. [ABSTRACT FROM AUTHOR]

Ocean acidification due to atmospheric CO rise is expected to influence marine phytoplankton. Diatoms are responsible for about 40% of the total primary production in the ocean. In order to investigate the physiological response of marine diatom Thalassiosira pseudonana to ocean acidification, we grew the cells under ambient CO level (380 µatm) versus the elevated CO level (800 µatm) at a light level of 180 µmol m s for 30 generations. Our results showed that the elevated CO concentration caused a decrease of the effective photochemical efficiency of PSII $$\left( {{{F_{{\text{v}}}^{\prime } } \mathord{\left/ {\vphantom {{F_{{\text{v}}}^{\prime } } {F_{{\text{m}}}^{\prime } }}} \right. \kern-\nulldelimiterspace} {F_{{\text{m}}}^{\prime } }}} \right)$$ and increase of the dark respiration in T. pseudonana. The intracellular carbonic anhydrase activity was suppressed and the photosynthetic affinity for CO was lowered in the high CO-grown cells, reflecting a downregulation of the CO concentrating mechanism (CCM). PSI activity was enhanced to support an increase in ATP synthesis by cyclic electron transfer as required for transport of inorganic carbon and regulation of intracellular pH. The energetic benefit from the downregulation of CCM to growth as reported in other diatom species was not observed here in T. pseudonana. [ABSTRACT FROM AUTHOR]

Occurrence of algal blooms in coastal waters is predicted to be more prevalent in future. To minimize their occurrence, manipulating the grazing pressure by suspension feeders is a potential management strategy, but its effectiveness may depend on their feeding preference. Therefore, we assessed the clearance rate of a widespread serpulid polychaete Hydroides elegans in larval and adult stages on various coastal phytoplankton. Additionally, the growth and development of H. elegans after consuming these phytoplankton were determined to reflect its sustainability to counter algal blooms. Results showed that H. elegans can consume and utilize different phytoplankton, except diatom Thalassiosira pseudonana , for growth and development in both life stages. Given the fast-colonizing ability which allows easy manipulation of abundance, H. elegans is considered practically and biologically ideal for tackling algal blooms. Other suspension feeders with different feeding niches could be used in combination to maximize the versatility of the top-down control. [ABSTRACT FROM AUTHOR]

Light drives phytoplankton productivity, so phytoplankton must exploit variable intensities and durations of light exposure, depending upon season, latitude, and depth. We analyzed the growth, photophysiology and composition of small, Thalassiosira pseudonana, and large, Thalassiosira punctigera, centric diatoms from temperate, coastal marine habitats, responding to a matrix of photoperiods and growth light intensities. T. pseudonana showed fastest growth rates under long photoperiods and low to moderate light intensities, while the larger T. punctigera showed fastest growth rates under short photoperiods and higher light intensities. Photosystem II function and content responded primarily to instantaneous growth light intensities during the photoperiod, while diel carbon fixation and RUBISCO content responded more to photoperiod duration than to instantaneous light intensity. Changing photoperiods caused species-specific changes in the responses of photochemical yield (e−/photon) to growth light intensity. These photophysiological variables showed complex responses to photoperiod and to growth light intensity. Growth rate also showed complex responses to photoperiod and growth light intensity. But these complex responses resolved into a close relation between growth rate and the cumulative daily generation of reductant, across the matrix of photoperiods and light intensities. [ABSTRACT FROM AUTHOR]

A method using high-performance liquid chromatography coupled with tandem mass spectrometry was developed for the simultaneous determination of organic acids in microalgae. o-Benzylhydroxylamine was used to derivatize the analytes, and stable isotope-labeled compounds were used as internal standards for precise quantification. The proposed method was evaluated in terms of linearity, recovery, matrix effect, sensitivity, and precision. Linear calibration curves with correlation coefficients >0.99 were obtained over the concentration range of 0.4-40 ng/mL for glycolic acid, 0.1-10 ng/mL for malic acid and oxaloacetic acid, 0.02-2 ng/mL for succinic acid and glyoxylic acid, 4-400 ng/mL for fumaric acid, 20-2000 ng/mL for isocitric acid, 2-200 ng mL−1 for citric acid, 100-10000 ng mL−1 for cis-aconitic acid, and 1-100 ng mL−1 for α-ketoglutaric acid. Analyte recoveries were between 80.2 and 115.1%, and the matrix effect was minimal. Low limits of detection (0.003-1 ng/mL) and limits of quantification (0.01-5 ng/mL) were obtained except cis-aconitic acid. Variations in reproducibility for standard solution at three different concentrations levels were <9%. This is the first report of the simultaneous analysis of ten organic acids in microalgae, which promotes better understanding of their growth state and provides reference value for high-yield microalgae cultures. [ABSTRACT FROM AUTHOR]

Among marine phytoplankton groups, diatoms span the widest range of cell size, with resulting effects upon their nitrogen uptake, photosynthesis and growth responses to light. We grew two strains of marine centric diatoms differing by ~4 orders of magnitude in cell biovolume in high (enriched artificial seawater with ~500 µmol L µmol L NO ) and lower-nitrogen (enriched artificial seawater with <10 µmol L NO ) media, across a range of growth light levels. Nitrogen and total protein per cell decreased with increasing growth light in both species when grown under the lower-nitrogen media. Cells growing under lower-nitrogen media increased their cellular allocation to RUBISCO and their rate of electron transport away from PSII, for the smaller diatom under low growth light and for the larger diatom across the range of growth lights. The smaller coastal diatom Thalassiosira pseudonana is able to exploit high nitrogen in growth media by up-regulating growth rate, but the same high-nitrogen growth media inhibits growth of the larger diatom species. [ABSTRACT FROM AUTHOR]

Mangroves are productive ecosystems that are highly threatened by anthropogenic activities. We investigated the environmental quality of the Serinhaém river estuary located in a legally protected area. Through chemical analysis of sediments and tissues of Cardisoma guanhumi , in addition to bioassays with elutriate involving Nitokra sp. and Thalassiosira pseudonana , we determined the contamination status and risk factors related to trace metals in the estuary. For the sediment, the concentrations of Cr and Ni were above the limit established by CONAMA n° 454/2012 in the "City" site, and Cr above the TEL in all sampling sites. Ecotoxicological tests showed high toxicity in samples from "City" and "Tributary". The elements Cr, Mn, Ni and Zn were also higher in crabs from these sites. Cr levels exceeded the Brazilian limit for food consumption. The bioaccumulation factor was not significant. However, the overall analysis proved that this estuary is increasingly impacted by anthropogenic pressure. • The estuary is located in a protected area that was hit by the biggest oil spill accident in the history of Brazil. • The Cr contents were above the TEL for all sample points, indicating a moderate contamination in the sediment. • The bioassay with elutriate showed high toxicity with growth inhibition of Thalassiosira pseudonana. • There was less Nitokra sp spawning after exposure to elutriate from the point close to the urban area. • The Cr content in tissues of Cardisoma guanhumi is above that established for human consumption by Brazilian legislation. [ABSTRACT FROM AUTHOR]

Diatoms are an important group of eukaryotic microalgae, which play key roles in marine biochemical cycling and possess significant biotechnological potential. Despite the importance of diatoms, their regulatory mechanisms of protein synthesis at the translational level remain largely unexplored. Here, we describe the detailed development of a ribosome profiling protocol to study translation in the model diatom Thalassiosira pseudonana, which can easily be adopted for other diatom species. To isolate and sequence ribosome-protected mRNA, total RNA was digested, and the ribosome-protected fragments were obtained by a combination of sucrose-cushion ultracentrifugation and polyacrylamide gel electrophoresis for size selection. To minimize rRNA contamination, a subtractive hybridization step using biotinylated oligos was employed. Subsequently, fragments were converted into sequencing libraries, enabling the global quantification and analysis of changes in protein synthesis in diatoms. The development of this novel ribosome profiling protocol represents a major expansion of the molecular toolbox available for diatoms and therefore has the potential to advance our understanding of the translational regulation in this important group of phytoplankton. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Ribosome profiling in Thalassiosira pseudonana Alternate Protocol: Ribosome profiling protocol for diatoms using sucrose gradient fractionation., (© 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.)

Thalassiosira pseudonana es utilizada como alimento en acuicultura, pero su valor nutricional está influenciado por las condiciones de crecimiento. Sistemas semicontinuos, en fase de estabilización, con tres irradiancias (60, 120 y 180μE/m².s) y dos medios de cultivo (Algal y Humus) fueron las condiciones en las que se determinó el crecimiento y componentes bioquímicos de T. pseudonana. En Humus a 180μE/m².s se obtuvieron las máximas densidades celulares (entre 3.86 y 4.62x10(6)cel/mL) y mayores concentraciones de lípidos y carbohidratos, con porcentajes de 20.3±2.28 y 16.6±2.43, respectivamente. A 180μE/m².s en Algal se observaron los mayores valores de proteínas (45.0±5.05) y carotenoides totales (0.5±0.01). El contenido de clorofila a fue favorecido por baja intensidad de luz, principalmente en Algal, con máximos de 0.9±0.04%. El grado de insaturación de los ácidos grasos disminuyó por incremento de la irradiancia en ambos medios, estando mayoritariamente representados por el ácido eicosapentaenoico, 20:5 n-3 (AEP), con porcentajes máximos (6.20%) en Algal a 60μE/m².s. Los resultados muestran múltiples respuestas fisiológicas de T. pseudonana frente a cambios en las condiciones de crecimiento, las cuales pueden ser aprovechadas para mejorar su valor nutricional como alimento de organismos cultivados, utilizando medios de cultivo alternativos, que disminuyan los costos en la producción microalgal.

Diatoms are heterokont algae derived from a secondary symbiotic event in which a eukaryotic host cell acquired an eukaryotic red alga as plastid. The multiple endosymbiosis and horizontal gene transfer processes provide diatoms unusual opportunities for gene mixing to establish distinctive biosynthetic pathways and metabolic control structures. Diatoms are also known to have significant impact on global ecosystems as one of the most dominant phytoplankton species in the contemporary ocean. As such their metabolism and growth regulating factors have been of particular interest for many years. The publication of the genomic sequences of two independent species of diatoms and the advent of an enhanced experimental toolbox for molecular biological investigations have afforded far greater opportunities than were previously apparent for these species and re-invigorated studies regarding the central carbon metabolism of diatoms. In this review we discuss distinctive features of the central carbon metabolism of diatoms and its response to forthcoming environmental changes and recent advances facilitating the possibility of industrial use of diatoms for oil production. Although the operation and importance of several key pathways of diatom metabolism have already been demonstrated and determined, we will also highlight other potentially important pathways wherein this has yet to be achieved.

Background: Diatoms are able to acclimate to frequent and large light fluctuations in the surface ocean waters. However, the molecular mechanisms underlying these acclimation responses of diaotms remain elusive. Results: In this study, we investigated the mechanism of high light protection in marine diatom Thalassiosira pseudonana using comparative proteomics in combination with biochemical analyses. Cells treated under high light (800 μmol photons m-2s-1) for 10 h were subjected to proteomic analysis. We observed that 143 proteins were differentially expressed under high light treatment. Light-harvesting complex proteins, ROS scavenging systems, photorespiration, lipid metabolism and some specific proteins might be involved in light protection and acclimation of diatoms. Non-photochemical quenching (NPQ) and relative electron transport rate could respond rapidly to varying light intensities. High-light treatment also resulted in increased diadinoxanthin + diatoxanthin content, decreased Fv/Fm, increased triacylglycerol and altered fatty acid composition. Under HL stress, levels of C14:0 and C16:0 increased while C20:5ω3 decreased. Conclusions: We demonstrate that T. pseudonana has efficient photoprotective mechanisms to deal with HL stress. De novo synthesis of Ddx/Dtx and lipid accumulation contribute to utilization of the excess energy. Our data will provide new clues for in-depth study of photoprotective mechanisms in diatoms. [ABSTRACT FROM AUTHOR]

There is relatively little choice in cultivation methods for growing algae outdoors, either in open pond systems or closed photobioreactors-as batch, continuous, or semi-continuous culture. Algal batch culture grown in a nutrient replete environment with adequate sunlight will become self-shaded with sufficient cell density and enter a stage in the growth dynamic known as the 'phase of linear growth.' It is during this phase of linear growth that primary production is at maximum and that the highest biomass is harvested. The inherent problem with batch culture is that the exponential (and possibly lag) phases necessary to achieve densities required prior to the phase of linear growth consume time and waste surface area, and thereby make this an inefficient method to grow algae. Semi-continuous culture can be forced into shade-limiting conditions by reducing growth rate from maximum through dilution, whereby phases of lag and exponential growth are skipped, and culture growth is put into a state similar to a perpetual phase of linear growth with an appropriate culture harvest/dilution cycle. Importantly, semi-continuous culture can increase net growth efficiency over batch culture when compared by shade-limited growth rate. However, scientific study and theory covering shade-limited algal growth under semi-continuous culture conditions are nearly non-existent, which currently makes its application to phycological technologies impractical through 'hit and miss' strategies. This laboratory study compares shade-limited growth dynamics for batch and semi-continuous cultures of Thalassiosira pseudonana (small-sized, marine diatom). Theory for optimizing production of mass algal culture with semi-continuous culture technique through cycle period and harvest volume is developed, and guidelines to practical industrial applications are provided. [ABSTRACT FROM AUTHOR]

Background: CRISPR-Cas is a recent and powerful addition to the molecular toolbox which allows programmable genome editing. It has been used to modify genes in a wide variety of organisms, but only two alga to date. Here we present a methodology to edit the genome of Thalassiosira pseudonana, a model centric diatom with both ecological significance and high biotechnological potential, using CRISPR-Cas. Results: A single construct was assembled using Golden Gate cloning. Two sgRNAs were used to introduce a precise 37 nt deletion early in the coding region of the urease gene. A high percentage of bi-allelic mutations (.61.5%) were observed in clones with the CRISPR-Cas construct. Growth of bi-allelic mutants in urea led to a significant reduction in growth rate and cell size compared to growth in nitrate. Conclusions: CRISPR-Cas can precisely and efficiently edit the genome of T. pseudonana. The use of Golden Gate cloning to assemble CRISPR-Cas constructs gives additional flexibility to the CRISPR-Cas method and facilitates modifications to target alternative genes or species. [ABSTRACT FROM AUTHOR]

The hydrogen isotopic ( 2 H/ 1 H) composition of lipids in microalgae is significantly depleted relative to extracellular water. While a variety of growth conditions influence the magnitude of 2 H-depletion, the effect of salinity is of particular interest due to the paleohydrological applications of lipid 2 H/ 1 H. In previous studies, lipid–water 2 H/ 1 H fractionation was shown to decrease as salinity increased, a response largely independent of lipid type, species, or setting. The mechanism responsible for this response remains uncertain, primarily because salinity is rarely isolated as the sole variable in laboratory cultivation experiments investigating hydrogen isotope systematics in microalgae. Here we report the lipid–water 2 H/ 1 H fractionation response to salinity in nutrient-replete continuous cultures of the centric diatom Thalassiosira pseudonana . In six cultures with the same growth rate at salinities between 14–40 ppt, lipid–water 2 H/ 1 H fractionation decreased linearly as salinity increased by 1.3‰/ppt in fatty acids (C 14:0 , C 16:0 , C 16:1 ) and by 1.0‰/ppt in the sterol 24-methyl-cholesta-5,24(28)-dien-3β-ol. A constant growth rate between cultures reveals that the fractionation response to salinity is independent of growth rate. Sensitivity tests using a simple hydrogen flux model indicated that at high salinity a greater proportion of metabolic NAD(P)H in lipids at the expense of photosynthetic NADPH can cause 2 H-enrichment. Additionally, increased exudate release or decreased hydrogen transport can enrich both lipids and cell–water in 2 H. The 1.0–1.3‰/ppt increase in lipid–water 2 H/ 1 H fractionation observed in T. pseudonana is within the 0.8–2‰/ppt range observed in field studies and culture studies, supporting the application of algal lipid 2 H/ 1 H as a paleosalinity proxy. [ABSTRACT FROM AUTHOR]

Marine dissolved organic matter (DOM) is a key source of carbon and nutrients to microbial life in the oceans, but rapid biological utilization of labile DOM confounds its compositional characterization. In order to characterize potentially bioavailable DOM produced by phytoplankton, DOM from axenic cultures of Thalassiosira pseudonana cultivated in phosphorus (P) replete and low P conditions was extracted using high-recovery electrodialysis (ED) techniques, which resulted in an average dissolved organic carbon (DOC) recovery of 76% ± 7% from all cultures. Low P concentrations resulted in greater cell-normalized production of DOC relative to P replete culture controls at the same growth phase. Despite the different nutrient conditions, DOC composition and DOM molar ratios of carbon to nitrogen (C : N) were similar in all cultures. In contrast, low P concentrations influenced DOM molar carbon to phosphorus (C : P) ratios and dissolved organic phosphorus (DOP) composition. Under P replete and low P conditions, DOM C : P ratios were 130 (± 22) and 2446 (± 519), respectively. 31P Nuclear Magnetic Resonance (NMR) spectroscopy identified P esters (> 90% of DOP) as the dominant P species in DOM produced under P replete conditions, with small or negligible contributions from phosphonates or glycerol P and polyphosphates. However, based on direct fluorometric analysis, DOP from low P cultures was greater than 8 times enriched in dissolved polyphosphate compared to DOP from replete cultures, which is consistent with the growing evidence that polyphosphate is a dynamic component of total P in low P ocean regions. [ABSTRACT FROM AUTHOR]

Increased manufacture of TiO nanoproducts has caused concern about the potential toxicity of these products to the environment and in public health. Identification and confirmation of the presence of TiO nanoparticles derived from consumer products as opposed to industrial TiO NPs warrant examination in exploring the significance of their release and resultant impacts on the environment. To this end, we examined the significance of the release of these particles and their toxic effect on the marine diatom algae Thalassiosira pseudonana. Our results indicate that nano-TiO sunscreen and toothpaste exhibit more toxicity in comparison to industrial TiO and inhibited the growth of the marine diatom T. pseudonana. This inhibition was proportional to the exposure time and concentrations of nano-TiO. Our findings indicate a significant effect, and therefore, further research is warranted in evaluation and assessment of the toxicity of modified nano-TiO derived from consumer products and their physicochemical properties. [ABSTRACT FROM AUTHOR]

Many consumer products containing ZnO have raised concern for safety in regard to environmental impact and the public health. Widely used sunscreens for protecting against UV and avoiding sunburns represent a great exposure to nano-ZnO, one of the ingredients commonly applied in sunscreens. Applying nanoproducts on beaches may release nanoparticles unintentionally into the ocean. Despite the accumulation of such nanoproducts in the ocean harming or being detrimental to critical marine organisms, few studies have investigated the release and potential toxicity of nanoparticles extracted from products and compared them with those from industrial-type nanoparticles. Results show that the cytotoxicity of both industrial- and sunscreen-derived nano-ZnO to the marine diatom algae, Thalassiosira pseudonana, increased as exposure increases over time, as measured by growth inhibition (%) of the algae at a constant concentration of nano-ZnO (10 mg/L). The extent of toxicity appeared to be higher from industrial-type nano-ZnO compared with sunscreen-extracted nano-ZnO, though the extent becomes similar when concentrations increase to 50 mg/L. On the other hand, at a fixed exposure time of 48 h, the cytotoxicity increases as concentrations increase with the higher toxicity shown from the industrial-type compared with sunscreen-induced nano-ZnO. Results indicate that while industrial-type nano-ZnO shows higher toxicity than sunscreen-derived nano-ZnO, the release and extent of toxicity from nano-ZnO extracted from sunscreen are not trivial and should be monitored for the development of safe manufacturing of nanomaterials-induced products. [ABSTRACT FROM AUTHOR]