Growth and photosynthetic activity of diatom Thalassiosira weissflogii at decreasing salinity

The relative yield of variable chlorophyll fluorescence (F _v/F _m), rate of photosynthetic carbon fixation (P), growth rate, and production of extracellular photosynthates (P _ec) was studied in diatom T. weissflogii in seawater with salinity decreasing from 35 to 15 and 5‰. After incubation at 5‰ for 1 day, the diatom abundance (N) decreased as a result of death of a fraction of cells, while viable cells demonstrated decreased F _v/F _m and P, P _ec was detectable, and cell division was likely inhibited. After incubation for 2 days, population started to grow, while exponential growth rate and the abundance by day 8 were lower compared to 35‰. After incubation at 15‰ for 1 day, P was higher and F _v/F _m was lower compared to 35‰. No cell death was observed and exponential growth rate insignificantly differed from that at 5‰. The value of N by day 8 was lower compared to 35‰ but higher compared to 5‰. The dependence of photosynthetic parameters and population dynamics of T. weissflogii on the relative salinity is discussed.     

Effect of mercuric chloride and methylmercury on photosynthetic activity of the diatom Thalassiosira weissflogii by a fluorescent method

It was shown by the pulse-amplitude modulation fluorescent method that, at a weak illumination (6 microE m-2.s-1), methylmercury at a concentration of 10(-6)-10(-7) M decreases the photochemical activity of the reaction centers of photosystem II in cells of microalgae Thalassiosira weissflogii after a prolonged lag phase. Cells resistant to methylmercury at these low concentrations were detected by the microfluorimetric method. Chloride mercury decreased the activity of photosystem II of the algae only when at higher concentrations. Both toxicants at a concentration of 10(-6) M decreased the rate of recovery of photoinduced damage of centers of photosystem II and led to an increase in the energization component of nonphotochemical fluorescence quenching. These results indicate that the complex of fluorescent methods can be used to monitor early changes in the photosynthetic apparatus of algae in response to the toxic action of heavy metals.     

Comparative phosphorus nutrition of the marine cyanobacterium Synechococcus WH7803 and the marine diatom Thalassiosira weissflogii

Phosphate uptake kinetics of Synechococcus sp. WH7803 and Thalassiosiraweissflogii were studied in axenic batch culture. Phosphate-repleteSynechococcus sp. WH7803 cells have a lower affinity for inorganicphosphate (Pi) (K_s = 67 µmol l^–1) than Pi-starvedcells (K_s = 3.1 µmol l^–1). The K_s of Pi-starvedcells increased     

Structural and inhibition insights into carbonic anhydrase CDCA1 from the marine diatom Thalassiosira weissflogii

Carbonic anhydrases (CAs) catalyze with high efficiency the reversible hydration of carbon dioxide, an essential reaction for many biological processes, such as photosynthesis, respiration, renal tubular acidification, and bone resorption. Diatoms, which are one of the most common types of phytoplankton and are widespread in oceans, possess CAs fundamental for acquisition of inorganic carbon. Recently, in the marine diatom Thalassiosira weissflogii a novel enzyme, CDCA1, naturally using Cd in its active site, has been isolated and categorized in a new CA class, namely zeta-CA. This enzyme, which consists of three repeats (R1, R2 and R3), is a cambialistic carbonic anhydrase that can spontaneously exchange Zn or Cd at its active centre, presumably an adaptative advantage for diatoms that grow fast in the metal-poor environment of the surface ocean. In this paper we completed the characterization of this enzyme, reporting the X-ray structure of the last repeat, CDCA1-R3 in its cadmium-bound form, and presenting a model of the full length protein obtained by docking approaches. Results show that CDCA1 has a quite compact not symmetric structure, characterized by two covalently linked R1-R2 and R2-R3 interfaces and a small non-covalent R1-R3 interface. The three dimensional arrangement shows that most of the non-conserved aminoacids of the three repeats are located at the interface regions and that the active sites are far from each other and completely accessible to the substrate. Finally, a detailed inhibition study of CDCA1-R3 repeat in both cadmium- and zinc- bound form has been performed with sulfonamides and sulfamates derivatives. The results have been compared with those previously reported for other CA classes, namely alpha- and beta-classes, and correlated with the structural features of these enzymes.     

Identification of a new gene family expressed during the onset of sexual reproduction in the centric diatom Thalassiosira weissflogii.

An intriguing feature of the diatom life cycle is that sexual reproduction and the generation of genetic diversity are coupled to the control of cell size. A PCR-based cDNA subtraction technique was used to identify genes that are expressed as small cells of the centric diatom Thalassiosira weissflogii initiate gametogenesis. Ten genes that are up-regulated during the early stages of sexual reproduction have been identified thus far. Three of the sexually induced genes, Sig1, Sig2, and Sig3, were sequenced to completion and are members of a novel gene family. The three polypeptides encoded by these genes possess different molecular masses and charges but display many features in common: they share five highly conserved domains; they each contain three or more cysteine-rich epithelial growth factor (EGF)-like repeats; and they each display homology to the EGF-like region of the vertebrate extracellular matrix glycoprotein tenascin X. Interestingly, the five conserved domains appear in the same order in each polypeptide but are separated by variable numbers of nonconserved amino acids. SIG1 and SIG2 display putative regulatory domains within the nonconserved regions. A calcium-binding, EF-hand motif is found in SIG1, and an ATP/GTP binding motif is present in SIG2. The striking similarity between the SIG polypeptides and extracellular matrix components commonly involved in cell-cell interactions suggests that the SIG polypeptides may play a role in sperm-egg recognition. The SIG polypeptides are thus important molecular targets for determining when and where sexual reproduction occurs in the field.     

Effects of fundamental nutrient stresses on the lipid accumulation profiles in two diatom species Thalassiosira weissflogii and Chaetoceros muelleri

Bioprocess and Biosystems Engineering - Microalgae are considered as attractive feedstocks for biofuel production nowadays because of their high lipid contents and easy cultivation. In the present...     

Cultivation and biomass production of the diatom Thalassiosira weissflogii as a live feed for white-leg shrimp in hatcheries and commercial farms in Vietnam

This study investigated the biomass production process from the laboratory to the pilot scale in order to use the nutrient-rich biomass of the diatom Thalassiosira weissflogii as live feed for white-leg shrimp (Litopenaeus vannamei) at larval stages (zoeal, mysis, and postlarval) and in commercial production in hatcheries in Vietnam. Our results showed that T. weissflogii was successfully cultured in 1–2 L Erlenmeyer flasks, 0.2–3.5 m³ composite tanks, and 6.5 m³ tubular photobioreactors, with the highest cell density of 1.6 × 10⁶ cells mL^?1 reached after 6 days of culture. Under optimal culture conditions, the protein, lipid, and carbohydrate contents in this algal biomass were 13.2%, 20.0%, and 10.0% of dry cell weight, respectively. The fatty acid composition contains high amount of palmitic acid (C16:0, 43.11% of total fatty acid), and polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, C20:5ω-3), approximated 16.5% of total fatty acid. In a 50 L larval rearing tank, at the optimal stocking density of 125 nauplii L^?1, the survival percentage (75.55%), the total body length (from 5.376 ± 0.007 to 10.860 ± 0.030 mm), and weight (at from PL₁ to PL₁₂ stages) (from 0.145 ± 0.002 to 1.158 ± 0.005 g) of the white-leg shrimp larvae reached the highest values but the metamorphosis time (234 h) was shortest compared with the other stocking densities. Further, adding living T. weissflogii biomass to the diet of white-leg shrimp larvae at the nauplii 6 stage led to an increase in the body length, weight, and survival percentage of white-leg shrimp larvae of 21.17%, 35.7%, and 33% higher compared with those of larvae fed the control diet (without the addition of T. weissflogii), respectively. At the same time, the metamorphosis time of larvae (from Z₁ to PL₁) decreased by 4 h compared to the control group. In intensive ponds (area of 6400 m² pond^?1), using seed stocks at the postlarvae 12 stage that had been fed T. weissflogii, the final weight, yield, and survival percentage of the shrimp were increased by 7.3%, 14.2%, and 16.3%, respectively, compared with those of the control group. There were no statistically significant differences in the protein and carbohydrate contents in the shrimp flesh among the experimental and control group (p > 0.05). The lipid, omega-3, omega-6, and omega-9 fatty acid contents of shrimp flesh in experiment formula (per 100 g shrimp) were 1.21 g, 72.9 mg, 114 mg, and 86.1 mg, 11%, 29%, 21.6%, and 17.7% higher than that those in control, respectively. The obtained results show the great potential of using T. weissflogii as live feed on white-leg shrimp farms in Vietnam.

     

Regulation of carbonic anhydrase expression by zinc, cobalt, and carbon dioxide in the marine diatom Thalassiosira weissflogii

TWCA1 is the major Zn-requiring isoform of carbonic anhydrase (CA) in the marine diatom Thalassiosira weissflogii. We have examined the roles that trace metals and CO(2) play in the regulation of TWCA1 expression over ranges of concentrations that bracket those encountered in the marine environment. Both steady-state levels of TWCA1 and the kinetics of induction were measured by western analysis. TWCA1 levels correlated well with cellular CA activity levels. TWCA1 was induced at a low CO(2) concentration but the level of induction, as determined by western analysis, was dependent on the availability of Zn. Co effectively substituted for Zn in regulating TWCA1 expression and promoting TWCA1 activity. Upon shift from low to high CO(2), the concentration of TWCA1 decreased. The expression of TWCA1 is diel cycle regulated, and cellular TWCA1 decreased during the dark phase. These results provide the basis for studying the expression of CA in field populations and, taken together with previous radiolabeling studies, provide strong evidence of in vivo metal substitution of Co for Zn in a CA. Our data also support the conclusion that TWCA1 plays a central role in carbon acquisition in T. weissflogii.     

Two-photon excitation chlorophyll fluorescence lifetime imaging: a rapid and noninvasive method for in vivo assessment of cadmium toxicity in a marine diatom Thalassiosira weissflogii

We demonstrate that a two-photon excitation fluorescence lifetime imaging technology can rapidly and noninvasively assess the cadmium (Cd)-induced toxic effects in a marine diatom Thalassiosira weissflogii. The chlorophyll, an intrinsic fluorophore, was used as a contrast agent for imaging of cellular structures and for assessment of cell toxicity. The assessment is based on an imaging-guided statistical analysis of chlorophyll fluorescence decay. This novel label-free imaging method is physically based and free of tedious preparation and preprocessing of algal samples. We first studied the chlorophyll fluorescence quenching induced by the infrared two-photon excitation laser and found that the quenching effects on the assessment of Cd toxicity could be well controlled and calibrated. In the toxicity study, chlorophyll fluorescence lifetime images were collected from the diatom samples after exposure to different concentrations of Cd. The alteration of chloroplast structure at higher Cd concentration was clearly identified. The decay of chlorophyll fluorescence extracted from recorded pixels of high signal-to-noise ratio in the fluorescence lifetime image was analyzed. The increase of average chlorophyll fluorescence lifetime following Cd treatment was observed, indicating the Cd inhibition effect on the electron transport chain in photosynthesis system. The findings of this study show that the temporal characteristics of chlorophyll fluorescence can potentially be utilized as a biomarker for indicating Cd toxicity noninvasively in algal cells.     

Direct impact of silicate on the photosynthetic performance of the diatom Thalassiosira weissflogii assessed by on- and off-line PAM fluorescence measurements

Pulse-amplitude-modulation (PAM) fluorometry was used to investigate the effects of varying the silicate concentration on different fluorescence characteristics of batch and chemostat cultures of the diatom Thalassiosira weissflogii. The fluorescence signals, measured both on- and off-line, were used to calculate the actual (P) and potential (P0) photochemical efficiencies of the reaction centres of photosystem II (PSII). Also the relative electron transport rate of the reaction centres of PSII (Je) was calculated. Fluorescence decreased in silicate-limited cells and increased rapidly after silicate addition. The decrease was caused by strong non-photochemical quenching (qN) in silicate-limited cells. Continuous recording (on-line) of the minimum and maximum fluorescence provided data with high temporal resolution, revealing that the first recovery of silicate-limited cells occurred 20 min after the addition of silicate. Based on these observations, we assume a strong influence of silicate metabolism on the photosynthetic efficiency of the reaction centres of PSII assessed by PAM fluorescence. This implies that silicate, as well as other nutrients, has to be considered as a possible cause for variable photosynthetic efficiency of diatoms.      

Response of the Ubiquitous Pelagic Diatom Thalassiosira weissflogii to Darkness and Anoxia

Thalassiosira weissflogii, an abundant, nitrate-storing, bloom-forming diatom in the world’s oceans, can use its intracellular nitrate pool for dissimilatory nitrate reduction to ammonium (DNRA) after sudden shifts to darkness and anoxia, most likely as a survival mechanism. T. weissflogii cells that stored 4 mM ¹⁵N-nitrate consumed 1.15 (±0.25) fmol NO₃ ^- cell^-1 h^-1 and simultaneously produced 1.57 (±0.21) fmol ¹⁵NH₄ ⁺ cell^-1 h^-1 during the first 2 hours of dark/anoxic conditions. Ammonium produced from intracellular nitrate was excreted by the cells, indicating a dissimilatory rather than assimilatory pathway. Nitrite and the greenhouse gas nitrous oxide were produced at rates 2-3 orders of magnitude lower than the ammonium production rate. While DNRA activity was restricted to the first few hours of darkness and anoxia, the subsequent degradation of photopigments took weeks to months, supporting the earlier finding that diatoms resume photosynthesis even after extended exposure to darkness and anoxia. Considering the high global abundance of T. weissflogii, its production of ammonium and nitrous oxide might be of ecological importance for oceanic oxygen minimum zones and the atmosphere, respectively.     

Oxidative metabolism by Thalassiosira weissflogii (Bacillariophyceae) of a diol-ester of okadaic acid, the diarrhetic shellfish poisoning

Previous investigations into the comparative toxicity of the diarrhetic shellfish poisoning (DSP) toxins to Thalassiosira weissflogii (Grun.) Fryxell et Hasle found that this diatom oxidatively metabolized okadaic acid diol‐ester (OA diol‐ester) to a more water‐soluble product. This oxidative transformation of OA diol‐ester by the diatom is significant for two reasons. First, it is known that dinophysistoxin‐4 (DTX‐4), the primary DSP toxin produced by the dinoflagellate Exuviaella lima (Ehr.) Butschli, will be hydrolyzed to the diol‐ester following cell rupture (e.g. ingestion by a predator). Second, it implies that the ester, an uncharged, lipophilic intermediate, can easily enter cells and therefore may play an important role in the uptake and transfer of DSP toxins through the food web. It has been suggested that the water soluble DTX‐4 may also be the form in which DSP toxins are excreted from the producing cell. Therefore, the stability of DTX‐4 was examined when incubated either in fresh seawater medium into which washed cells of E. lima were introduced or in seawater medium conditioned by E. lima cells. Rapid hydrolysis of DTX‐4 to the diol‐ester took place in both cases. Thus, regardless of the route by which DTX‐4 is liberated from the cell, either by cell disruption or excretion, the diol‐ester will be the dominant form of the toxin to challenge associated organisms. To examine the metabolism of OA diol‐ester by T. weissflogii in more detail, serial cultures of the diatom were challenged with OA diol‐ester at a concentration of 2.0 μg·mL^?1. The metabolism and fate of the diol‐ester in both cellular and medium fractions were monitored over 3 days using liquid chromatography with either ultraviolet (LC‐UV) or mass spectrometric (LC‐MS) detection. During the course of the experiment, all of the diol‐ester was metabolized. LC‐MS analysis revealed the presence of multiple oxidative products of OA diol‐ester in the medium fraction, including a carboxylic acid derivative. The major metabolites were isolated in sufficient quantity to permit structural elucidation by NMR and MS. All the metabolites identified resulted from oxidation of the diol‐ester side chain with the primary sites of attack at the terminal, subterminal, and unsaturated carbons. OA was found in both cellular and medium fractions, and its production was directly correlated with the metabolism of the diol‐ester. The relative partitioning of both OA diol‐ester and its oxidation products between cells and medium supports the contention that OA diol‐ester can readily enter cells, be metabolized, and then excreted in more water‐soluble forms.     

The effect of positive selection on a sexual reproduction gene in Thalassiosira weissflogii (Bacillariophyta): results obtained from maximum-likelihood and parsimony-based methods

Maximum-Likelihood-based and parsimony-based methods were used to test for potential effects of positive selection on the sexually induced gene 1 (Sig1) in Thalassiosira weissflogii. The Sig proteins are thought to play a role in mediating sperm-egg recognition during the sexual reproduction phase. The results obtained from parsimony-based analyses showed that none of the amino acid sites were influenced by positive selection. Maximum-likelihood analyses indicated that positive selection was affecting a maximum of seven and a minimum of four amino acid sites in the polypeptide derived from Sig1. It was concluded that the results obtained from the maximum-likelihood-based method are more reliable than those obtained from the parsimony-based approach. This is apparently the first study that has shown that reproductive proteins in unicellular eukaryotes are influenced by positive selection.     

Thalassiosira antarctica (Bacillariophyceae): Vegetative and resting stage ultrastructure of an ice-related marine diatom

Summary The ultrastructure of T. antarctica var. antarctica vegetative and resting stages are compared using light and transmission electron microscopy. Resting spores contain noticeably more lipid reserves than do vegetative cells. Numerous mitochondria and generally fewer numbers of other organelles are eliminated from spores into an abortive daughter cell when the spore formation division sequence is terminated. The remaining spore contents are a compact arrangement of organelles with lipid bodies predominating. These two stages are thus ultrastructurally distinct, and differences in their chemical composition can be manifested as cytological modifications.     

False-positive selection identified by ML-based methods: examples from the Sig1 gene of the diatom Thalassiosira weissflogii and the tax gene of a human T-cell lymphotropic virus

Sexually induced gene 1 (Sig1) in the centric diatom Thalassiosira weissflogii is considered to encode a gamete recognition protein. Sorhannus (2003) analyzed nucleotide sequences of Sig1 using parsimony analysis and the maximum-likelihood (ML)-based Bayesian method for inferring positive selection at single amino acid sites and reported that positively selected sites were detected by the latter method but not by the former. He then concluded that for this type of study, the ML-based method is more reliable than parsimony analysis. Here we show that his results apparently represent false-positive cases of the ML-based method and that there is no solid evidence that this gene contains positively selected sites. We further demonstrate that in the tax gene of human T-cell lymphotropic virus type I (HTLV-I), all codon sites, including invariable sites, can be inferred as positively selected sites by the ML-based method. These observations indicate that the ML-based method may produce many false-positive sites. One of the main reasons for the occurrence of false positives is that in the ML-based method, codon sites are grouped into several categories, with different nonsynonymous/synonymous rate ratios (omegas), on a purely statistical basis, and positive selection is inferred indirectly by examining whether the average omega for each category is greater than 1. In parsimony analysis, however, the evolutionary change of nucleotides at each codon site is examined. For this reason, parsimony-based methods rarely produce false positives and are safer than ML-based methods for detecting positive selection at individual codon sites, although a large number of sequences are necessary.     

Selenocysteyl-tRNA occurs in the diatom Thalassiosira and in the ciliate Tetrahymena

Selenocysteyl-tRNAs that decode UGA were identified previously in animal and bacterial cells and the genes for these tRNAs have been shown to be widespread in animals and eubacteria. In the present study, we identify a selenocysteyl-tRNA that codes for UGA in Thalassiosira pseudonana, which is a diatom, and in Tetrahymena borealis, which is a ciliate. The fact that these very diverse unicellular organisms also contain a selenocysteyl-tRNA suggests that selenocysteine-containing proteins and the use of UGA as a codon for selenocysteine are widespread, if not ubiquitous, in nature.     

Gene regulation in the marine diatom Thalassiosira pseudonana upon exposure to polycyclic aromatic hydrocarbons (PAHs)

Diatoms are eukaryotic algae, which can be found worldwide in oceans and freshwaters. These organisms are ecologically relevant due to their key role in the global carbon cycle, contributing to about 25% to the global primary production [Falciatore, A., Bowler, C., 2002. Revealing the molecular secrets of marine diatoms. Annu. Rev. Plant Biol. 53, 109-130]. We investigated the effects of three polycyclic aromatic hydrocarbons (PAHs, pyrene, fluoranthene, and benzo[a]pyrene), either as single compound or as mixture, at molecular level. Dose-response curves for growth inhibition were determined and four concentrations eliciting from "no effect" up to a severe growth inhibition were chosen for further investigation to detect alterations at gene expression level by Real-Time PCR. Among the eight selected genes, two were strongly influenced by the PAH treatment. lacsA, which is involved in the fatty acid metabolism, was found to be strongly up-regulated by all single PAHs, as well as by the mixture. sil3, involved in the formation of the silica shell, was repressed by a factor up to three even at low PAH concentrations not eliciting any growth inhibition. For other genes, involved e.g. in photosynthesis, a slight down-regulation was detected. Based on the effects at gene expression level it can be assumed that PAHs impair the fatty acid metabolism and silica shell formation.     

Chemotaxis of Marinobacter adhaerens and Its Impact on Attachment to the Diatom Thalassiosira weissflogii

Alga-bacterium interactions are crucial for aggregate formation and carbon cycling in aquatic systems. To understand the initiation of these interactions, we investigated bacterial chemotaxis within a bilateral model system. Marinobacter adhaerens HP15 has been demonstrated to attach to the diatom Thalassiosira weissflogii and induce transparent exopolymeric particle and aggregate formation. M. adhaerens possesses one polar flagellum and is highly motile. Bacterial cells were attracted to diatom cells, as demonstrated by addition of diatom cell homogenate or diatom culture supernatant to soft agar, suggesting that chemotaxis might be important for the interaction of M. adhaerens with diatoms. Three distinct chemotaxis-associated gene clusters were identified in the genome sequence of M. adhaerens, with the clusters showing significant sequence similarities to those of Pseudomonas aeruginosa PAO1. Mutations in the genes cheA, cheB, chpA, and chpB, which encode histidine kinases and methylesterases and which are putatively involved in either flagellum-associated chemotaxis or pilus-mediated twitching motility, were generated and mutants with the mutations were phenotypically analyzed. ΔcheA and ΔcheB mutants were found to be swimming deficient, and all four mutants were impaired in biofilm formation on abiotic surfaces. Comparison of the HP15 wild type and its chemotaxis mutants in cocultures with the diatom revealed that the fraction of bacteria attaching to the diatom decreased significantly for mutants in comparison to that for the wild type. Our results highlight the importance of M. adhaerens chemotaxis in initiation of its interaction with the diatom. In-depth knowledge of these basic processes in interspecies interactions is pivotal to obtain a systematic understanding of organic matter flux and nutrient cycling in marine ecosystems.     

Thalassiosira punctigera (Castr.) comb, nov., a widely distributed marine planktonic diatom

Examination of type material showed that 8 taxa must be reduced to synonomy with Thalassiosira punctigera (Castr.) comb, nov., an extremely variable species as to size and valve structure. Preliminary experiments indicated a certain relationship between the areola size and the temperature at which clonal cultures had been grown. While previously known from the North Pacific and the South Atlantic Oceans in addition to a single record from the Caribbean Sea it has since 1979 been recorded regularly in the Skagerrak (between Denmark and Norway).     

Effect of Si-status on diel variation of intracellular free amino acids in Thalassiosira weissflogii under low-light intensity

Twenty-one intracellular free amino acids were analysed during a 12-12 h light-dark cycle, on duplicate axenic cultures of Thalassiosira weissflogii (clone Actin, Provasoli-Guillard CCMP) under either Si-sufficient or Si-starved conditions. Total concentrations ranged between 40 and 165 fmol/cell. Total level as well as individual levels of amino acids decreased during the dark period, and GLN/GLU ratio was lower during the dark period. All these results were correlated with the light-dark carbon metabolism of the algae and related to the protein synthesis at night. The Si-starved cultures showed a lower total level of FAA compare to the Si-sufficient cultures, especially in the light period. Silica status of the cells affected more the metabolites of the dark respiration than the photorespiratory metabolites SER and GLY. Si deprivation induced higher range of ALA and VAL, and a decrease of the TCA metabolites GLU & ASP. Additionally, the relative percentage of ASP increased under Si starvation, at the expense of GLU, and this shift was emphasized in the dark period.