Marine Diatom Ditylum Brightwellii Research Articles

Effect of temperature on the production and degradation of bromoform and other brominated methanes by marine microorganisms

Brominated methanes such as bromoform (CHBr3) are known to be important carriers of bromine from the ocean to the atmosphere. Bromine released from brominated methanes by photolysis has been shown to catalyze ozone depletion. Marine phytoplankton has been reported as a source of CHBr3 and marine bacteria as a sink for CHBr3. The effects of temperature on both CHBr3 production by phytoplankton and CHBr3 degradation by bacteria have yet to be investigated. We investigated the effects of temperature on CHBr3 production and CHBr3 degradation by marine microorganisms. The marine diatom Ditylum brightwellii (CCMP358) was cultured at 15 °C, 20 °C, 24 °C, and 30 °C. The maximum CHBr3 production rate at 24 °C was 1.57–2.39 pmol (μg chlorophyll a)−1 d−1, several times higher than that at 15 °C (0.25–0.41 pmol (μg chlorophyll a)−1 d−1). Higher rates of CHBr3, CHBr2Cl, and CHBrCl2 production were observed in the late exponential phase (and stationary phase) than in the early exponential phase at each temperature. These results suggest that temperature affects the rate of CHBr3 production during plankton growth. We then cultured the marine α-proteobacterium Phaeobacter gallaeciensis (JCM 21319) and the γ-proteobacterium Pseudomonas sp. HKF-4 at 10 °C, 15 °C, 20 °C, and 25 °C for up to 15 days to analyze temperature effects on spiked 13CHBr3 degradation. The degradation rate of 13CHBr3 by P. gallaeciensis increased with increasing temperature from 10 °C to 25 °C. The half-life of 13CHBr3 at 25 °C was about 1.1 d, which is about 6 times shorter than the half-life at 10 °C (about 6.9 d). On the other hand, the change in the half-life of the degradation of 13CHBr3 by HKF-1 was relatively small as the temperature increased from 10 °C (half-life: about 5.5 d) to 25 °C (half-life: about 1.8 d). Considering the rate of CHBr3 production and degradation at each temperature, we estimated how much of the CHBr3 produced by D. brightwellii for 7 days was degraded by the coexisting bacteria and how much remained after 7 days at each temperature. When coexisting with P. gallaeciensis, the residual CHBr3 concentration in the culture was relatively higher at 20–25 °C. Similarly, when coexisting with HKF-4, it was relatively higher at 20–25 °C. To estimate the impact of future warming on CHBr3 concentrations in the oceans, we assume a 5 °C increase in sea surface temperature, with two sea surface temperatures, 15 °C and 20 °C, changing to 20 °C and 25 °C, respectively. Under this assumption, the residual concentration of CHBr3 produced by D. brightwellii in seawater would be “increased” (or, less likely, “no change”) when P. gallaeciensis coexisted in these areas. Similarly, the total amount of CHBr3 residuals produced by D. brightwellii in the seawater areas where HKF-4 coexisted increased as the temperature increased by 5 °C. If, as the present results suggest, there is a temperature effect on microbial CHBr3 production and degradation in the oceans, then future increases in surface seawater temperature could result in an upward trend in CHBr3 concentrations in the open oceans, although such effects would be greatly influenced by both the phytoplankton species and coexisting bacterial species.

Effects of light intensity on the production of VSLs from the marine diatom Ditylum brightwellii

Effects of light intensity on the production of VSLs from the marine diatom Ditylum brightwellii

Different transcriptional responses of heat shock protein 70/90 in the marine diatom Ditylum brightwellii exposed to metal compounds and endocrine-disrupting chemicals

Environmental hazard assessments using diatoms have been well documented; however, their molecular toxicology has not been sufficiently studied. In this study, we characterized heat shock protein (HSP) 70/90 of the diatom Ditylum brightwellii (Db) and evaluated their transcriptional profiles in response to various environmental stresses (e.g., thermal shocks and metal and non-metal pollutants). Putative DbHSP70 (658aa, 71.7kDa) and DbHSP90 (707aa, 80.2kDa) proteins had conserved HSP family motifs but different C-terminus motifs, that is, “EEVD” in DbHSP70 and “MEEVD” in DbHSP90. Phylogenetic analyses of both proteins showed that D. brightwellii was well clustered with other diatoms. Real-time PCR analysis showed that thermal stress considerably upregulated DbHSP70 and DbHSP90. As for chemical pollutants, DbHSP70 greatly responded to CuSO4 and NiSO4 exposure, but not CuCl2 or NiCl2. However, DbHSP90 was significantly upregulated by all the metal compounds tested (CuSO4, NiSO4, CuCl2, and NiCl2). Strikingly, the expression of both genes was not induced by the organic pollutants tested, such as endocrine-disrupting chemicals. These data suggest that DbHSP70 and DbHSP90 are differentially involved in the defense response against various environmental stressors. Moreover, metal toxicity may be specifically affected by the conjugated anion in the metal compounds (e.g., SO42- and Cl−).

Normalization genes for mRNA expression in the marine diatom Ditylum brightwellii following exposure to thermal and toxic chemical stresses

Toxicity assessments using the diatom Ditylum brightwellii are well documented; however, analysis of their toxicogenomics has been little attempted. Currently, quantitative real-time PCR is the most accurate and widely applied method to detect differential gene expression, including that of specific genes induced by environmental contaminants. This method requires internal reference genes to normalize expression levels, and their selection is a critical factor for the correct analysis of the results. Here, we assessed the gene expression stability of nine housekeeping genes (HKGs), including 18S rRNA, ACT, TUA, EF2, MDH, UBQ, UCE, PCNA, and GAPDH, in 28 RNA samples of D. brightwellii. All the tested HKGs displayed different expression patterns under different experimental conditions such as heat shock and exposure to metals and non-metals. Analysis of CT values showed that at least two genes were required for proper normalization according to the tested conditions. Overall, TUA, followed by ACT, was the most stable gene under all conditions. Furthermore, we examined the expression of the HSP70 gene in D. brightwellii when exposed to heat shock and chemicals by using the most stable references and found that the gene was significantly up-regulated during the stress period. This study has evaluated, for the first time, the normalization genes in D. brightwellii, providing potential references for gene expression studies of diatoms.

우리나라 황해의 해산 돌말류 Ditylum brightwellii (West) Grunow의 형태학적 특징에 의한 두 유형

This study deals with the taxonomy of the marine diatom Ditylum brightwellii (West) Grunow: shape, size and ratio, marginal ridge, labiate process, areolation, and seasonal distribution are all considered. Ditylum brightwellii were divided into two type; prism- and cylinder-shaped. Comparisons between specimens from the Yellow Sea were made on, (1) shape- prism types are prismatic-shaped and/or regular square or right-angled tetragon, cylinder types are cylinder-shaped and/or long right-angled tetragon in the girdle view, respectively; (2) size and ratiodiameters of prism types are 82.1 ± 2 µm, while those of cylinder types are 21.2 ± 5 µm and ratio of pervalvar axis/diameter- prism types cells are 1.0-1.5, but cylinder types are 3.9-5.5; (3) marginal ridges- prism types have dotted, while those of the cylinder-type cells have piece, slotted or fimbriated on the marginal ridge; (4) labiate process- both prism- and cylinder-types are central part in valve face, but length of labiate processes of prism types are longer than those of cylinder types; (5) cell margin- the girdle margin show many shallow furrows in prism type and smooth in cylinder type; (6) areolation- both prism- and cylinder-types are a radial pattern in the valve center, but areolae of prism types are larger than those of cylinder types; (7) seasonal distribution- cylinder types are dominated in summer and prism types in the other seasons.

Spring bloom development, genetic variation, and population succession in the planktonic diatom Ditylum brightwellii

We determined spatial and temporal variation in abundance, distribution, and genetic composition of the planktonic marine diatom Ditylum brightwellii in 21 water samples collected between February and June from five locations in Puget Sound and the Strait of Juan de Fuca, Washington. Four blooms with cell abundances of >1,000 cells L-1 were detected during the sampling period. The blooms were comprised of two genetically distinct populations identified by analyzing up to three microsatellite loci from 707 individual cells. One population was detected at all locations from February to April and comprised three blooms. In May and June, it was gone, replaced by a second, genetically distinct population that comprised a different bloom. Both populations had high levels of diversity: on average, 94% of cells in each sample were genetically distinct. The two populations contained cells with identical 18S and 5.8S rDNA sequences and 1.1% sequence divergence at the internal transcribed spacer regions, which suggests that both populations were members of the same species. The populations detected in early and late spring were composed of cells with average valve diameters of 22 and 69 mm, respectively. The population sampled in late spring, comprised of large-diameter cells, had a distinct genetic composition, indicating that it did not arise from a sexual reproduction event in the population sampled in early spring. Both populations were associated with significantly different exposures to solar irradiance and silicic acid concentrations, suggesting that environmental selection may regulate bloom dynamics of distinct populations.

Effects of periodic, low UVA radiation on cell characteristics and oxidative stress in the marine planktonic diatom Ditylum brightwellii

A continuous culture experiment was conducted with the marine diatom Ditylum brightwellii to assess the photo-oxidative effects of transition from dim light to high light at three successive levels. The focus was on temporary stress by ambient UVA at the water surface, via a simulation of the periodic ascent of diatoms caused by vertical mixing. Within a 14 h dim-light period, cells were exposed daily to 4 h of : (A) moderate PAR (100 μmol photon m−2 s−1), (B) high PAR (400 μmol photon m−2 s−1) and (C) high PAR (400–700 nm) plus 6·7 μmol photon m−2 s−1 (2·2 W m−2) UVA. Cell shape, life cycle and sinking rates were not affected by high PAR per se (mode B). The daily 4 h high-irradiance mode C, with additional UVA (UVA:PAR = 0·016), caused an increase in cell diameter, a decrease in length axes, and a production of vegetative resting stages. Superoxide dismutase (SOD) activities, oxidized glutathione (GSSG) pools and malondialdehyde (MDA) contents increased as well, which indicates that this biologically effective UVA dose (0·78 W m−2) promoted active oxygen production, oxidative stress and lipid peroxidation. Sinking rates in UVA-exposed D. brightwellii were higher than in those grown without UVA. A close correlation between sinking rates and MDA contents suggests that UVA-induced lipid peroxidation has altered the membrane functions which regulate buoyancy in D. brightwellii. Although UVA-induced formation of resting cells will contribute to accelerated sinking, it is not evident that this can be considered as a stress avoidance mechanism under adverse light (UVA) conditions. D. brightwellii proved to be sensitive to transitions from shade to natural surface irradiance conditions. In coastal waters, such large diatom species may experience oxidative stress after a few hours' residence near the water surface at ambient UVA levels.

Speciation of copper and responses of the marine diatom Ditylum brightwellii upon increasing copper concentrations

In continuous culture, cells of the marine diatom Ditylum brightwellii were subjected to increasing Cu concentrations. Cu speciation due to production of dissolved organic ligands by the diatom and changes in adsorption characteristics of the cell walls were studied. With increasing total dissolved Cu concentrations, four phenomena were observed: (1) the rate of cell division decreased; and (2) the concentration of organic ligands with a conditional stability constant of K′ = 10 10 increased; (3) Cu complexed with relatively hydrophobic ligands ( K′ = 10 9.6) increased; a positive relationship between the biovolume of the broken cells and the concentration of hydrophobic Cu-complexes was found; and (4) the adsorption capacity of the cell walls increased at high total dissolved Cu concentrations. Calculations showed that the Cu 2+ concentration was buffered at 0.2 nM by the production of organic ligands up to a total dissolved Cu concentration of 110 nM. Further addition of Cu, however, could not be compensated, and resulted in the collapse of the culture and large increases in both ligand concentrations and adsorption capacity of the cell walls. The resulting free Cu concentration decreased to 0.08 nM.

Impact of UV-B Radiation on the Lipid and Fatty Acid Composition of Synchronized Ditylum brightwellii (West) Grunow

Abstract The marine diatom Ditylum brightwellii (West) Grunow isolated from the Baltic Sea could be synchronized by a light/dark rhythm of 6.5:17.5 h (white light intensity 8 W m-2) at 18 °C and 0.035 vol.% CO2. Content of protein, DNA and RNA increased linearly up to the end of the cell cycle. Pigments (chlorophyll a, chlorophyll c1 + c2, carotenoids) and galactolipids were synthesized in the light period only. A lag phase of 2 h was observed in the biosynthesis of sulphoquinovosyl diacylglycerol and phosphatidylglycerol. Formation of phosphatidylglycerol and phosphatidylcholin continued in the dark period (30% and 28%, respectively). The pattern of major fatty acids (C14:0, C16:1, C16:0, C18:1 and C20:5) varied during the cell cycle of Ditylum. Biosynthesis of acyl lipids was reduced in dependence on the UV-B dose. The most sensitive lipid was digalactosyl diacylglycerol (total inhibition at 585 J m-2), whereas phosphatidylcholin was less affected (20% reduction). UV-B radiation during the dark period had no effect on the lipid and pigment content. Strongest inhibitory effect of UV-B on cell division, synthesis of protein, pigments, sulphoquinovosyl diacylglycerol and phosphatidylglycerol was found after UV-B radiation at the beginning of the cell cycle (0.-2. h). An exposure time at the end of the light period (4.-6. h) led to a marked damage on the synthesis of monogalactosyl diacylglycerol and phosphatidylglycerol. These findings indicate a stage-dependent response of Ditylum to UV-B irradiance. The impact of UV-B resulted in an increase of unsaturated long chained fatty acids (C18, C20) and in a diminution of short chained fatty acids (C14, C16). Content of ATP was not affected by UV-B radiation under the used conditions. The inhibitory effect of UV-B on synthesis of DNA, RNA, protein and acyl lipids was mainly reversible. Results were discussed with reference to UV-B damage on the enzymes involved in the biosynthesis of acyl lipids and by a reduction of available metabolites.

Interaction of toxic trace metals and mechanisms of detoxification in the planktonic diatoms Ditylum brightwellii and Thalassiosira pseudonana.

Effects of cadmium (10 nM), copper (80 nM) and zinc (150 nM) additions were studied in the marine diatom Ditylum brightwellii and the riverine diatom Thalassiosira pseudonana. Defense against oxidative stress via cellular thiol (SH) pools and superoxide dismutase (SOD) activation, detoxification via phytochelatins and cell damage were monitored in metal-exposed exponential-phase cells and controls, grown in estuarine medium. Total SH and reduced+oxidized glutathione (GSH+GSSG) in T. pseudonana were much higher than in D. brightwellii. In T. pseudonana, total SH and GSH decreased at 322 nM Zn, and GSH increased at 80 nM Cu but decreased at 119 nM Cu. GSH:GSSG ratios were low, while phytochelatins were not detectable in metal-exposed D. brightwellii. Cd-exposed T. pseudonana made more phytochelatins than Cu-exposed cells, and in different proportions. At 322 nM Zn, SOD activity decreased in T. pseudonana. Zn caused a major, and Cu a minor increase of SOD activity in D. brightwellii; inhibition of photosynthesis was observed in Cu-exposed D. brightwellii, probably due to oxidative damage. The C:N ratios were higher and protein contents lower in Cu-exposed cells of both species, which might indicate excretion due to a loss of cell membrane integrity. From these results, it is hypothesized that T. pseudonana has evolved an effective detoxification mechanism as a result of a more severe exposure to toxic metals in rivers and estuaries. In contrast, D. brightwellii, a marine-estuarine species, cannot adjust well to metal exposure. Its poor defense against metal toxicity was marked by low SH-contents.

Interaction of toxic trace metals and mechanisms of detoxification in the planktonic diatoms Ditylum brightwellii and Thalassiosira pseudonana

Effects of cadmium (10 nM), copper (80 nM) and zinc (150 nM) additions were studied in the marine diatom Ditylum brightwellii and the riverine diatom Thalassiosira pseudonana. Defense against oxidative stress via cellular thiol (SH) pools and superoxide dismutase (SOD) activation, detoxification via phytochelatins and cell damage were monitored in metal-exposed exponential-phase cells and controls, grown in estuarine medium. Total SH and reduced + oxidized glutathione (GSH + GSSG) in T. pseudonana were much higher than in D. brightwellii. In T. pseudonana, total SH and GSH decreased at 322 nM Zn, and GSH increased at 80 nM Cu but decreased at 119 nM Cu. GSH:GSSG ratios were low, while phytochelatins were not detectable in metal-exposed D. brightwellii. Cd-exposed T. pseudonana made more phytochelatins than Cu-exposed cells, and in different proportions. At 322 nM Zn, SOD activity decreased in T. pseudonana. Zn caused a major, and Cu a minor increase of SOD activity in D. brightwellii; inhibition of photosynthesis was observed in Cu-exposed D. brightwellii, probably due to oxidative damage. The C:N ratios were higher and protein contents lower in Cu-exposed cells of both species, which might indicate excretion due to a loss of cell membrane integrity. From these results, it is hypothesized that T. pseudonana has evolved an effective detoxification mechanism as a result of a more severe exposure to toxic metals in rivers and estuaries. In contrast, D. brightwellii, a marine–estuarine species, cannot adjust well to metal exposure. Its poor defense against metal toxicity was marked by low SH-contents.

Does energy control the sinking rates of marine diatoms?

There is an energy requirement for the maintenance of low sinking rates (<0.1 m d−1) in the marine diatom Ditylum brightwellii. The lowest sinking rates occurred when cells were grown under irradiances saturating for growth. The highest sinking rates occurred when cells grown at growth‐limiting irradiances were placed in the dark, or in cells treated with a respiratory inhibitor. Small cells sank more rapidly than large cells under saturating light, but under severe energy limitation sinking rates were directly proportional to cell volume. When cells were grown under saturating irradiance and then placed in the dark, their sinking rate increased as their respiration rate decreased over time. We suggest that when light is unavailable, a diatom’s ability to use energy gained from respiration may be the principal determinant of its sinking rate. We provide the first estimate of the dependence of cell sinking rate on cell respiration and suggest that the sinking‐respiration relationship may be useful in predicting the sinking dynamics of diatoms during a bloom. We speculate that energetic sinking control may have evolved to allow diatoms to colonize the photic zone and that larger diatoms with lower sinking rates may have an ecological advantage over smaller diatoms with higher sinking rates early in a bloom.

Copper toxicity and adaptation in the marine diatom Ditylum brightwellii

1. 1. The planktonic diatom Ditylum brightwellii, grown in light-limited resp. nitrogen-limited continuous culture, has been exposed to Cu levels, comparable with those in the Scheldt estuary. 2. 2. At increasing levels D. brightwellii initially detoxified Cu, producing metal-binding ligands (amino acids), and increasing its cell volume. 3. 3. In light-limited D. brightwellii, photosynthesis could be adjusted to increasing Cu stress, division rates remained constant, and cells proved to be adaptable to 200 nM dissolved Cu. 4. 4. Nitrogen-limited D. brightwellii detoxified Cu inadequately: it stored large amounts of Cu (30–60 μM) that inhibited cell division.

Implications of salinity fluctuation for growth and nitrogen metabolism of the marine diatom Ditylum brightwellii in comparison with Skeletonema costatum

In 1987 effects of salinity fluctuations on growth of Ditylum brightwellii (West) Grunow, isolated from the Eastern Scheldt estuary (SW Netherlands) in 1981, were studied. D. brightwellii was grown in a 12 h light: dark cycle at constant salinity in brackish media. Ammonium-limited cultures were subjected to a salinity fluctuation. By decreasing the salinity to 4.8‰ photosynthesis and cell division were inhibited; cells were deformed. Protein and carbohydrate contents increased slightly, dark respiration was stimulated and cellular levels of glucose decreased at low salinity; this indicated a possible role of sugars in osmoregulation. Ammonium was accumulated in cultures, amino acids may have been stored; the role of the vacuole as a storage compartment was discussed. Both the ammonium uptake capacity and the affinity for ammonium decreased. Nitrogen limitation was relieved in the transient state. [With the activity of the nitrogen assimilation enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT) being uninhibited by lower salinity.] Recovery from hypo-osmotic stress during a salinity increase was initiated by stimulated photosynthesis; chlorophyll a increased, but persistant contractions of cytoplasm (with chloroplasts) may have delayed cell growth. The glutamate dehydrogenase (GDH) activity decreased further whereas the cellular level of alanine increased in the presence of large ammonium pools; this may indicate a temporary activity of ADH (alanine dehydrogenase). Skeletonema costatum (Greville) Cleve, recovered faster from hypoosmotic stress than did D. brightwellii. Due to an osmotic shock from 13.6 to 7.1‰ S both species excreted amino acids and glucose; S. costatum accumulated more glucose, D. brightwellii accumulated more amino acids. S. costatum may with the competition for nitrogen in waters with an unstable salinity; it will replace D. brightwellii.

Effect of EDTA on growth of the marine diatom Ditylum brightwellii (West) Grunow : Canterford, G. S., 1979. Aust. J. mar. Freshwat. Res., 30(6): 765–772. Department of Chemical Engineering, Monash University, Clayton, Vic. 3168, Australia

Effect of EDTA on growth of the marine diatom Ditylum brightwellii (West) Grunow : Canterford, G. S., 1979. Aust. J. mar. Freshwat. Res., 30(6): 765–772. Department of Chemical Engineering, Monash University, Clayton, Vic. 3168, Australia

Effect of EDTA on Growth of the Marine Diatom Ditylum brightwellii (West) Grunow

The presence of a chelating agent such as EDTA was shown to be necessary for growth of D. brightwellii in SiO2, Stosch-modified Schreiber medium irrespective of initial cell concentration, temperature and illuminance. Growth increased with increasing EDTA concentration up to about 2 × 10-6M above which growth was independent of EDTA concentration. The reason for lack of growth of D. brightwellii in the absence of EDTA was investigated using various methods of sterilization, filtration and modification of the composition of culture media. Filtration of seawater may either remove essential constituents which may be replaced by EDTA or introduce contaminants such as heavy metals. From a model describing the speciation of heavy metals in the culture medium, it was concluded that the beneficial effect of added EDTA was not due to a reduction in metal toxicity.