The coupling of toxicity expression with cell-cycle phases was studied in the toxic marine prymnesiophyte Chrysochromulina polylepis Manton & Parke, Clone B1511. Cell synchronisation of cultures in exponential or early stationary growth phases under nutrient-replete conditions was achieved by manipulation of the photoperiod. Chlorophyll a (chl a) and cell number increased in a stepwise manner, but were asynchronous, with chl a increasing during the light period and cell number increasing during the dark period. In the course of the light period, nearly all cells clustered in the G1 (Gap 1) phase, which lasted for about 20 h. DNA synthesis (S phase) occurred mainly in the dark during a discrete period (about 4 h) and G2 (Gap 2) and mitosis (M) were always completed before the end of the dark period. Toxicity expression, measured by the erythrocyte lysis assay (ELA), exhibited a dramatic drop in LC50 values (increase in toxicity) during the light period, although this effect was less pronounced after the first 2 generations of cell division when the cultures had entered the stationary phase, Similarly, haemolytic activity per unit cell volume decreased by a factor of 3 to 4 during the dark period over the first 48 h, but became irregular towards the end of the experiment. In this study, the light-dependent effect on toxicity and relationship to discrete phases of the cell cycle are demonstrated for the first time in a prymnesiophyte.

Laboratory experiments were performed to determine the growth and grazing capabilities of 2 heterotrophic dinoflagellates with different feeding modes (pallium feeder: Oblea rotunda; engulfment feeder Oxyrrhis marina) when fed the raphidophyte Fibrocapsa japonica, Both dinoflagellates readily ingested prey and exhibited positive growth when feeding on monocultures of F japonica. Maximum growth rates at food saturation were 0.54 and 0.72 d(-1) for O. rotunda and O, marina, respectively. Both dinoflagellates are thus able to grow faster than their prey, for which a maximum growth rate of 0.45 d(-1) has been previously reported. In the case of O. rotunda, it was found that a rather high food concentration of 300 F japonica cells ml(-1) (corresponding to 142 mug C l(-1)) was needed to sustain half-saturated growth. This is consistent with the quantification of behavioural aspects of the feeding process, In about 55% of cases, a failure to attach the tow filament after prey encounter was recorded, and in about 83% of cases, F. japonica was able to escape from the attached tow filament, indicating that motility of F. japonica is a quite effective defence mechanism against pallium-feeding dinoflagellates. In addition, qualitative observations suggest that trichocysts of F japonica may act mechanically as a grazer deterrent.

BACKGROUND To study the fragile Prymnesiophyte species Chrysochromulina polylepis by flow cytometry (FC), we needed an effective fixation method. This method must guarantee a high yield of fixed cells to achieve acceptable measurement times by FC and to allow quick processing of many samples. Moreover, we wanted a method that allows for storage of fixed samples when FC analysis cannot be done immediately. METHODS Different aldehydes and methanol were tested at different final concentrations. Gravity sedimentation and centrifugation were applied to achieve higher cell concentrations. Storage of fixed samples was tested under different conditions. RESULTS 0.25% glutaraldehyde (GA) fixation yielded a recovery rate of about 90%. The signals obtained by FC analysis were excellent. It is possible to centrifuge GA-fixed cells and to store them for several weeks. CONCLUSIONS GA is the fixative of choice for FC analysis of C. polylepis (and possibly other small delicate species) because it yielded highly significant recovery rates and high-quality FC signals. Cells can be centrifuged to increase the cell concentration, thereby achieving short measurement times with FC. The possibility of long-term storage of fixed cells presents an additional advantage if FC analysis cannot be done immediately.

In European coastal waters toxic blooms of Prymnesiophytes occur regularly. Chrysochromulina polylepis is a highly toxic member of this taxonomic group, which formed a devastating bloom in Scandinavian waters in 1988. Blooms like these can cause severe damage to benthic and pelagic communities and can result in million dollar losses for commercial fisheries and mariculture of fin and shellfish. Effective prevention and mitigation strategies require an understanding of environmental factors that contribute to the formation of harmful algal blooms, as well as information on the regulation of algal toxicity at the molecular level. To approach the latter aspect, we investigated the variation in toxin production of synchronized C. polylepis cultures by means of a sensitive bioassay and by flow cytometry. The results so far obtained show significant variation in cellular toxin content and specific toxin production throughout the sampling period of three days. The existence of times in the cell cycle of high and low levels of toxin production will be used as the basis for the identification of differentially expressed genes and proteins using differential display and 2‐D protein gel electrophoresis. Preliminary results concerning the molecular analysis will be presented as well.

Food selection by 2 dominant calanoid copepods, Acartia sp. and Temora longicornis, was studied during mass occurrence of N-2-mixing cyanobacteria in June/July 1993 and 1994 in the Gotland Sea (Baltic Proper). The aim of this study was to assess the importance of N-2-fixing cyanobacteria in the diet of calanoid copepods. Two different methods were used: firstly the analysis of marker carotenoids by HPLC (high-performance Liquid chromatography), and secondly the analysis of delta(15)N signals of copepods by mass spectrometry. The first method provides a 'snapshot' of autotrophic material ingested; the second method summarises a longer period, and gives evidence that a certain food source is not only ingested but also assimilated. In 1994, mass occurrence of cyanobacteria showed a higher concentration in the euphotic layer than 1993 (97 mu g C l(-1) in 1994, 57 mu g C l(-1) in 1993), which was reflected in higher food uptake of N-2-fixing cyanobacteria in 1994. The average relative amount of myxoxanthophyll, the specific carotenoid of N-2-fixing cyanobacteria, in the copepod guts showed high values in 1994 (Acartia sp. 37 %, T. longicomis 41%) and low values in 1993 (1% for both copepods). The low delta(15)N values of both Acartia sp. and T. longicornis in 1994 (9 parts per thousand) compared to those in 1993 (10.5 parts per thousand) support the results of HPLC analyses, because Nz-fixing cyanobacteria have a lower delta(15)N (average 0.7 parts per thousand) than eukaryotic phytoplankton (average 12 parts per thousand). The low delta(15)N values in 1994 indicate that N-2-fixing cyanobacteria were not only ingested but also assimilated by the copepods to a higher extent in 1994 than 1993.

A cocktail of the s-triazines, simazine and atrazine, was introduced into three of four experimental tanks (volume=900 l) that contained a natural estuarine pelagic community from the Pomeranian Bight. Herbicide concentrations in the spiked tanks corresponded to about 10, 100 and 1000 times the background of the control with absolute values of each compound of 0.005 μgl−1 (control) and around 0.04, 0.6 and 6 μgl−1 (spiked tanks). Herbicide concentrations, as well as the response of phytoplankton and bacterial stock and turnover, were monitored over a period of 10 days. Both triazines remained at the initial levels in each system. In spite of high activity of both bacteria and microalgae, no herbicide-specific effect could be observed. This resistance is attributed to the inherent stability of a well established and balanced multi-species regenerating system. The low level of algae–herbicide interaction shows, however, that the coastal system under study has no degradative capacity with regard to those compounds. Riverine inputs will be transferred to the open Baltic with no major decontamination effects.

The grazing pressure of micro- and nanozooplankton on phytoplankton was estimated in serial dilution experiments in the northwestern Arabian Sea and its adjacent areas (the Somali Current, the Somali Basin, the Gulf of Aden and the southern Red Sea) during the NE monsoon 1992-1993. Microzooplankton grazing rates (g) on total phytoplankton (analyzed as chi a) were generally exceeded by phytoplankton growth rates (g = 0.2 to 1.19 d(-1), mean 0.48 d(-1); mu = 0.52 to 1.12 d(-1), mean 0.72 d(-1)), resulting in an average daily consumption of 38 % of the phytoplankton standing stock and 67 % of the primary production. Microzooplankton grazing on 4 picophytoplankton groups (Prochlorococcus spp., Synechococcus spp., and 2 picoeukaryotes) analyzed by flow cytometry showed growth (mu = 0.27 to 0.92 d(-1), mean 0.68 d(-1)) and grazing mortality rates (g = 0.26 to 0.73 d(-1), mean 0.67 d(-1)) well in balance, with an average of 49 % of the standing stock and 102% of the primary production grazed per day. Picophytoplankton growth and grazing mortality rates increased dramatically when grazers >10 mu m were removed. These results suggest a control of the small grazers by larger ones (trophic cascade) and a close coupling between picoautotrophic prey and small grazers. The trophic cascade within the microbial food web of the nanoplankton encompasses 3 trophic levels: picoplankton - small HNF - larger flagellates and ciliates.

Laboratory experiments were carried out to investigate the effect of protozoan, copepod and combined grazing on Phaeocystis biomass. Phaeocystis cf. globosa single cells were offered to 3 different protozoan species, to the calanoid copepod Temora longicornis, as well as to mixtures of both grazer types. The heterotrophic dinoflagellate Oxyrrhis marina and the oligotrich ciliate Strombidinopsis acuminatum ingested Phaeocystis at much higher rates than did the copepod. Nevertheless, protozoan growth and ingestion rates were submaximal, indicating Phaeocystis to be suboptimal food. The oligotrich ciliate Strombidium elegans did not feed on Phaeocystis. In grazing experiments with mixtures of both predator types, the decline of Phaeocystis single cells could be explained by protozoan grazing alone, implying no grazing by the copepods on Phaeocystis. Instead, copepods ingested the protozoans at high rates. Predation on 0. marina and S. acuminatum by T longicornis resulted in a reduction of the total grazing pressure on Phaeocystis of 21 and 67 % respectively. We conclude that mesozooplankton predation on herbivorous ciliates and heterotrophic dinoflagellates, which consumed Phaeocystis cells, can considerably reduce the overall grazing pressure and may enhance Phaeocystis blooming.