L. Peperzak

Sexual reproduction, mating system, chloroplast dynamics and abrupt cell size reduction in Pseudo-nitzschia pungens from the North Sea (Bacillariophyta)

Clonal cultures of Pseudo-nitzschia pungens were isolated at various times from seven sites in the North Sea. During the mitotic cell cycle, the two plate-shaped chloroplasts were girdle-appressed during interphase and mitosis. After cytokinesis, the chloroplasts moved onto the parental valve and remained there during the formation of the new hypovalve and until separation and re-arrangement of the sibling cells within the cell chain had been completed. Clones were almost always heterothallic and cultures of opposite mating type isolated from different localities were compatible. Meiosis I was cytokinetic and accompanied by chloroplast division. Meiosis II involved karyokinesis but not cytokinesis and preceded the rearrangement and contraction of the two gametes. Sexual reproduction involved physiological anisogamy. With one exception, gamete behaviour was clone-specific, gametes being active in clones of one mating type but passive in clones of the other mating type. Auxospore development was accompanied by deposition of a transverse and then a longitudinal perizonium. Infrequently, triploid auxospores and presumably haploid auxospores were produced. The four chloroplasts of diploid auxospores did not divide, and behaved synchronously during the two acytokinetic mitotic cycles accompanying the deposition of the initial thecae. Just before the first division of the initial cell, the chloroplasts shifted onto the valves (two per valve). The division of the initial cell was not accompanied by chloroplast division and so the two daughter cells received two chloroplasts each. Two modes of abrupt cell size reduction were detected. One occurred during initial cell formation when part of the expanded auxospore aborted. The other pattern was more gradual and was observed in growing cultures; during successive cell divisions a frustule constriction appeared and intensified, one chloroplast split into two, and part of the protoplast aborted. A simple naming system is proposed for mating types in pennate diatoms.

Nutrient limitation in the North Sea: A bioassay approch

A differential nutrient enrichment bioassay with Skeletonema costatum showed that phytoplankton in the continental Dutch coastal waters was potentially phosphorus and silicon limited in 1988. In the central North Sea, potential N limitation predominated. The bioassay results depended on the ratio of the dissolved nutrients in the test water and on the optimum atomic ratio for the test diatom species. The nutrient concentrations were often so low that actual limitations of nutrient uptake rates were possible. In the stratified region of the North Sea dinoflagellates and μ-flagellates were dominant, while diatoms were absent in June and July in that area. In the mixed parts of the study area both diatoms and flagellates were present. The absence of diatoms during the summer stratification in the central North Sea cannot be explained by Si limitation. It seems more probable that it is caused by a combination of N depletion and losses from sedimentation.

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.

Identification of a domoic acid-producing pseudo-Nitzschia species (Bacillariophyceae) in the Dutch Wadden sea with electron microscopy and molecular probes

Biological monitoring in the Dutch Wadden Sea between November 1993 and July 1994 revealed Pseudo-nitzschia-like pennate chain-forming diatom species with cell numbers ranging from 102 to 105 per litre. Cultured isolates and field samples were examined by electron microscopy, which revealed the majority to be Pseudo-nitzschia pungens. This species dominated over other diatoms in the phytoplankton population during November 1993 and at the end of June 1994. At the beginning of June 1994, P. fraudulenta was also present; occasionally, P. delicatissima was observed. One isolate showed the characteristic morphology of P. multiseries. Species-specific polyclonal antibodies and large-subunit (LSU) rRNA-targeted oligonucleotides for North American strains of P. multiseries and P. pungens applied to the European isolates, confirmed species designations based on electron microscopy. The isolate of P. multiseries from the Dutch Wadden Sea produced domoic acid; after 55 days of growth about 19 pg per cell was measur...

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.

Flow cytometry: fast and quantitative characterization of particles in suspension

Abstract The application of flow cytometers for automated and rapid measurements of particles in suspension is presently widely accepted for biomedical applications. Flow cytometers, however, also offer many advantages for determination of (properties of) particles in other application areas. In this paper the potential of flow cytometry for environmental analysis is demonstrated, using examples taken from the analysis of phytoplankton in sea water. Flow cytometry is applied for automated measurement of the species abundance of phytoplankton in sea water, on the basis of the characteristic optical properties of the various algal species. More specific determination of, for instance, toxic phytoplankton could be achieved by making use of selective stains. Another application area is that of functional analysis of phytoplankton species. Here also specific fluorescent stains are used. Examples are presented of viability analysis and of cell cycle analysis, both of phytoplankton species. Other applications of flow cytometry in analytical sciences are discussed.

Flow cytometry and other optical methods for characterization and quantification of phytoplankton in seawater

New, optical, approaches to automatize and to accelerate the analysis of algae are discussed. Firstly, the application of flow cytometric analysis will be presented. This technique is based on classification of particles in flow by measuring several optical properties per individual particle. Characteristics of the Optical Plankton Analyser, a flow cytometer constructed specifically for the analysis of phytoplankton, will be presented. Two experiments that demonstrate the suitability of this instrument for the analysis of coastal phytoplankton will be presented. Subsequently, the applicability of flow cytometry and other optical approaches to the measurement of phytoplankton, in particular for monitoring purposes, will be discussed. Microscopy, as well as in-situ measurement and optical remote sensing are approaches that each offer specific advantages for the determination of phytoplankton. An optimal and integrated picture of the development of the distribution of phytoplankton species in space and time can be obtained by the combined application of these techniques.

Exotic harmful algae in marine ecosystems

Harmful algal blooms (HABs) are the cause of important damages to marine living resources and human beings. HABs are generated by micro-algae. These marine species are primarily introduced through ballast water of ships and, to a lesser extent, through import of living fish, in particular shellfish. Effective and efficient regulation of HABs requires an integration of insights from biological, economic and legal sciences. Such an integration consists of (a) a clear identification of the bio-ecological pathways and overall consequences related to the damages of HABs; (b) an assessment of monetary costs of HABs; and (c) an understanding of the set of complementary legal-institutional and economic instruments dealing with HABs through prevention, restoration and amelioration. This paper discusses each element in detail, in which biological, economic and legal aspects come together, drawing conclusions for decision making in marine management. In order to move away from the general level of discussion, an example of HABs is presented in which, biological, economic and legal aspects are combined.