Wwc Gieskes

Silicon deposition in diatoms: control by the pH inside the silicon deposition vesicle

To test the hypothesis that silicification occurs under acid conditions in the silicon deposition vesicle (SDV), the acidity of the SDV of the pennate diatoms Navicula pelliculosa (Brébisson et Kützing) Hilse, N. salinarum (Grunow) Hustedt, and Nitzschia sigma (Kützing) Smith was determined during development of new frustule valves. Cells were incubated with the weak base 3‐(2,4‐dinitroanilino)‐3′‐amino‐N‐methylpropylamine (DAMP) followed by immunocytochemical localization in whole cells and on ultrathin sections. After resupplying silicate to cells synchronized by silicon depletion, the uptake of this nutrient from the medium was the same with or without DAMP; new valves developed without morphological aberrations that could conceivably have been caused by the probe. DAMP was found in cellular compartments known to be acidic, such as vacuoles active as lysosomes, the lumen of thylakoids, and microbodies. In the nucleus and mitochondria, which are circumneutral and basic compartments, the probe did not appear. Besides its presence in acidic compartments, DAMP was specifically accumulated within the SDV during formation of new valves; during the process of valve maturation, the SDV seemed to become increasingly acidic. In control experiments using the ionophores chloroquine, valinomycin, and nigericin, the compartmental location of DAMP was clearly disturbed, resulting in a random intracellular distribution. Accumulation of the fluorescent probe rhodamine 123, which can be translocated over membranes by a reducing potential, confirmed that the SDV can translocate weak bases. The results with DAMP suggest that the pH of the SDV is important in the silicification of diatoms: It facilitates a fast nucleation and aggregation of silica particles, thus increasing the rate of formation of the mature frustules. In addition, the acidic environment might protect the newly formed valves against dissolution before completion and coverage by the organic casing prior to their secretion.

Monitoring ultraviolet-B-induced DNA damage in individual diatom cells by immunofluorescent thymine dimer detection

We developed a method to investigate the effect of ultraviolet‐B radiation (UVBR) on the formation of thy‐mine dimers in microalgal DNA that can be used for both laboratory and in situ research. Antibody labeling of dimers was followed by a secondary antibody (fluorescein isothiocyanate) staining to allow visualization of DNA damage with flow cytometry or fluorescence microscopy. Thymine dimer‐specific fluorescence in nuclear DNA of the marine diatom Cyclotella sp. was linearly related to the UVBR dose. Simultaneous measurements of cellular DNA content showed that the vulnerability of G2 cells to DNA damage did not differ significantly from the vulnerability of G1 cells. The formation and removal of thymine dimers in Cyclotella sp. cells was monitored for 3 consecutive days at two realistic UVBR irradiance levels. Thy‐mine dimers were removed within 24 h when exposed to a saturating photosynthetically active radiation intensity following the UVBR treatment. This new method allows the study of UVBR‐induced DNA damage on a cell‐to‐cell basis. It is also feasible for field studies because cells remain intact and can be recognized readily after antibody treatment.

Diatom silicon biomineralization as an inspirational source of new approaches to silica production

The demand for new materials and products is still growing and the interest in naturally formed biopolymers and biominerals, such as chitin, calcium precipitates and silica is increasing. Photosynthesizing microalgae of the family Bacillariophyceae (diatoms) produce silica exoskeletons with a potential to be used in specific industrial or technological processes, they also are an excellent model in studies of silicon biomineralization. In contrast to geologically aged diatomaceous earth, the freshly prepared silica of cultured or harvested natural diatoms has been characterized insufficiently with respect to the properties (e.g. purity, specific surface area, porosity) required for technological and industrial application. In this contribution we summarize aspects of cellular processes that are involved in silicon biomineralization of diatoms and the current knowledge of the characterization of diatomaceous silica, following methods used for synthetically derived silica-based materials.

Chattonella and Fibrocapsa (Raphidophyceae): First observation of, potentially harmful, red tide organisms in Dutch coastal waters

Species of the potentially toxic and red-tide-forming marine-phytoplankton genera Chattonella and Fibrocapsa (Raphidophyceae) were observed for the first time in 1991 in samples taken in Dutch coastal waters; they were again recorded and enumerated in the following years. Chattonella spp. cell numbers varied with the season, with a maximum in May or June in the Dutch Wadden Sea. Cell numbers of Chattonella and F. japonica Toriumi et Takano were up to 6.0 . 10(3) cells . dm(-3) in the Dutch Wadden Sea, except at one station in June 1993 when over 10(4) cells . dm(-3) Chattonella were counted. In May 1993, a minor bloom (over 2.0 . 10(5) cells . dm(-3)) was observed at a station in the southern central North Sea, 100 km northwest of the island of Terschelling. The potentially neurotoxic species Chattonella marina (Subrahmanyan) Hara et Chihara was identified and discriminated from morphologically related species within the class of Raphidophyceae by immunofluorescence. F. japonica could only be clearly identified in live samples; in fixed samples cell morphology was severely affected. The identification of this species was supported by the presence of mucocysts, structures that can be observed readily by optical and electron microscopy.

Location and expression of frustulins in the pennate diatoms Cylindrotheca fusiformis, Navicula pelliculosa, and Navicula salinarum (Bacillariophyceae)

A detailed immunocytochemical and biochemical study of the location and expression of frustulins, a family of proteins associated with the frustules of diatoms, has been performed for Cylindrotheca fusiformis Reimann et Lewin, Navicula pelliculosa (Brébisson et Kützing) Hilse, and Navicula salinarum (Grunow) Husted. Immunocytochemistry revealed that frustulins, which share homologous epitopes but are different in size, were predominantly located in the organic casing. Based on timed immunolocalization experiments and Western blotting analysis of cell extracts obtained sequentially after repleting silicate to Si‐synchronized cells, the continuous presence of the frustulins in the mature and parental organic casing of the examined species was observed. The frustulins of N. pelliculosa appeared as proteins similar to those of C. fusiformis, sharing identical epitopes. The extractions, however, yielded a markedly lower abundance of frustulins in N. pelliculosa. Peak concentrations of extracted frustulins appeared to be expressed just ahead of the silicification process in C. fusiformis, whereas the level of expression in N. pelliculosa increased along with maturation of the new valves. For N. salinarum, the presence of the frustulins could not be confirmed properly by Western blotting, most probably because of the small sample volumes, inefficient extraction, and a lower amount of homologous frustulins in the casing of this species. It is concluded that the frustulins of these species are not associated with the silicalemma of the newly formed silica deposition vesicles and therefore do not seem to be involved in the silicification process itself. Overall, the results imply a structural role of the frustulins in the casing of diatoms rather than a regulation of the silicification process.

Effects of iron limitation on photosynthesis and carbohydrate metabolism in the Antarctic diatom Chaetoceros brevis (Bacillariophyceae)

Iron, one of the structural elements of organic components that play an essential role in photosynthesis and nitrogen assimilation of plants, is available at extremely low concentrations in large parts of the Southern Oceans surface waters. We tested the hypothesis that photosynthesis is the primary target of iron stress in phytoplankton living in this specific environment, resulting in a reduced carbohydrate production. Cultures of a small Antarctic diatom, Chaetoceros brevis, were exposed to two different photon irradiances under iron-rich and iron-poor conditions. Under both light regimes growth rate was reduced only slightly by iron starvation, as expected because the iron requirement of a small-celled species such as C. brevis is low. Even so, iron-starved cells differed markedly from iron-replete cells: for low and high irradiance, respectively, they had a 20 and 27% lower content of light-harvesting pigments (chlorophyll a and c 2 and fucoxanthin), a 8 and 15% decrease in light absorption and a 15 and 17% decrease in quantum yield of photosystem II. The diurnal production of water-extractable carbohydrates was reduced by 28 and 31%, which resulted in a low supply of energy and carbon skeletons from these storage products. This may well have influenced protein synthesis. The nocturnal consumption of carbohydrates was also reduced, which, together with the almost proportional decrease in cellular C and N content, suggests that the C and N metabolism were tightly tuned in iron-stressed cultures. The decrease in C and N content correlated with a decrease in cell volume. Our results indicate that iron limitation is likely to affect the ability of phytoplankton to maintain high rates of protein synthesis within the deep wind-mixed layer of the Southern Ocean. In addition, growth at the surface could be inhibited by too much light: iron-poor cultures of C. brevis grown at low irradiance showed enhanced sensitivity to photoinhibition.

Monsoonal differences in primary production in the eastern Banda Sea (Indonesia)

Abstract 14C incorporation into phytoplankton of the eastern Banda Sea was registered during incubations of samples, both in situ and in an incubator equipped with blue filters. Light conditions throughout the euphotic zone were apparently simulated quite well in the deck incubator: the agreement between the two series of measurements was very good. Primary production was 1.85 g C·m−2..dt-1 in August 1984, after upwelling forced by the southeast monsoon; values ranged from 0.5 g C·m−2..dt-1 in the open Banda Sea up to 7 g C·m−2..dt-1 south of Irian Jaya. In February 1985, when the upper part of the water column was stratified due to persistent monsoonal winds from the northwest, primary production was less: 0.91 g C·m−2..dt-1. In August 1984 production was also measured with the oxygen method. The photosynthetic quotient was 1.05 near the surface and up to 1.86 near the bottom of the euphotic zone. Extrapolation of all measurements to an estimate of annual primary production resulted in a value as high as 5·102 g C·m−2..dt-1. The assimilation ratio in surface samples was locally up to 8 mg C·(mg chl. a)−1.h−1 (in the photoperiod) during both cruises, with a mean of 5.85 in August 1984 and 5.95 in February 1985. The surprisingly high values during the oligotrophic conditions of February underline the importance of rapid recycling of nutrients in the euphotic zone.

Interpretation of fluorometric chlorophyll registrations with algal pigment analysis along a ferry transect in the southern North Sea

Abstract In 1991 chlorophyll a was measured continously by fluorometry on the ferry ‘Norstar’, between Zeebrugge and Hull. In order to assess the usefulness of this platform for monitoring of long-term changes in phytoplankton distribution in the southern North Sea, calibration cruises were carried out in January, April, June and August with RV ‘Holland’ to relate algal pigment fingerprints to the fluorescence record. Chlorophyll a concentrations, determined after HPLC separation, allowed a quantification of the continuous fluorescence signal. The relation between fluorescence and chlorophyll a content was found to be rather constant in June and August and allowed a prediction of the chlorophyll a concentration from fluorescence measurements within 21%. In Janaury and April, however, this relation was different for separate hydrographic regions. In April, a major chlorophyll derivative, an allomer indicative of degradation of algal matter, was abundant near the Belgian coast. During the decay of the Phaeocystis bloom, this high concentration of allomerized chlorophyll a increased the ‘background’ fluorescence signal. In January, enhancement of fluorescence caused by light scattering of non-algal suspended matter could be observed most clearly near the English coast, where the algal concentration was low (0.5 μg·dm −3 ) while suspended (inorganic) matter concentration was high (30 mg·dm −3 ). By applying linear relations, calculated for every hydrographic region, prediction of the chlorophyll a concentration could be improved to an accuracy of 38%. An analysis of taxon-specific chlorophylls and carotenoids reveals that the distribution of individual pigments and of pigment ratios was indicative of taxonomic composition of the phytoplankton. Patterns in pigment concentrations along the section suggest the same phytoplankton abundance gradients and patchiness as revealed by the continuous fluorescence measurement. A succession in the phytoplankton population was apparent: from diatoms and traces of green algae in January, to a spring bloom of Phaeocystis in April close to the Belgian coast, to diatom dominance in June, to a mixed population of green algae, diatoms and Prymnesiophyceae (probably Coccolithophorids) in August, at the end of the vegetative season. The presence, even occasional abundance, of green algae (carrying chlorophyll b ) in the southern North Sea has not been reported earlier. The allomerized chlorophyll a concentration was most abundant near the coastal ends of the section during all cruises. Apparently, the phytoplankton is an important source of detritus in the North Sea.

THE EFFECT OF LABELING INTENSITY, ESTIMATED BY REAL-TIME CONFOCAL LASER SCANNING MICROSCOPY, ON FLOW CYTOMETRIC APPEARANCE AND IDENTIFICATION OF IMMUNOCHEMICALLY LABELED MARINE DINOFLAGELLATES

Two different fluorescein isothiocyanate (FITC) conjugates were used to analyze the effect of labeling intensity on the flow cytometric appearance of marine dinoflagellates labeled with antibodies that specifically recognized the outer cell wall. Location of the labeling was revealed by epifluorescence and real‐time confocal laser scanning microscopy using an anti‐rabbit IgG/FITC‐conjugated secondary antiserum. Flow cytometric measurements showed that cells of Prorocentrum species labeled this way could not always be distinguished from unlabeled cells. The labeling intensity increased several times when a biotinylated anti‐rabbit IgG secondary antiserum was used in combination with a streptavidin/FITC conjugate. Flow cytometry indicated that the labeling intensity had increased 50%, which resulted in an improved separation of clusters of labeled and unlabeled cells.

Silicon biomineralisation: towards mimicking biogenic silica formation in diatoms.

Silica is by far the most abundant compound in the earthi¯s crust. It serves various functions in nature: organisms ranging from lower eukaryotes to plants use it as one of the biominerals that provide support, protection, strength, or tools in various functions. Biogenic silica is the least studied of the biominerals. The best-studied ones (Hunter 1996) are composed of calcium (apatite, enamel, calcite); these are present in the bone tissue and teeth of mammals and in the shells of shellfish. In contrast to calcium minerals, relatively little is known about the formation of biogenic silica. The most likely function of biogenic silica in unicellular organisms seems to be giving strength to the living cell; good examples from the aquatic environment are siliceous (exo)skeletons or cell casings of the microalgal groups of diatoms (Fig.1) and silicoflagellates, and of Radiolaria (Simpson and Volcani 1981; Pickett-Heaps et al. 1990; Round et al. 1990).