Data files of figures 1, 2 and 3

We investigated optimal conditions for characterization of bioactivity of lytic compound(s) excreted by Alexandrium tamarense based on a cell-bioassay system. Allelochemical response of the cryptophyte Rhodomonas salina indicated the presence oflytic compound(s) in a reliable and reproducible way and allows for quantification of this lytic effect. The parameters tested were the incubation time of putatively lytic extracts or fractions with the target organism R. salina, different techniques for cell harvest from A. tamarense cultures and the optimal harvest time. A three hour incubation time was found to be optimal to yield a rapid response while accurately estimating effective concentration (ECso) values. Harvest of A. tamarense cultures by filtration resulted in loss of lytic activity in most cases and centrifugation was most efficient in terms of recovery of lytic activity. Maximum yield of extracellular lytic activity of A. tamarense cultures was achieved in the stationary phase. Such optimized bioassay guided fractionation techniques are a valuable asset in the isolation and eventual stmctural elucidation of the unknown lytic substances.

Files of figures 2, 3 and table

Certain allelochemicals of the marine dinoflagellate Alexandrium tamarense cause lysis of a broad spectrum of target protist cells but the lytic mechanism is poorly defined. We first hypothesized that membrane sterols serve as molecular targets of these lytic compounds, and that differences in sterol composition among donor and target cells may cause insensitivity of Alexandrium and sensitivity of targets to lytic compounds. We investigated Ca2+ influx after application of lytic fractions to a model cell line PC12 derived from a pheochromocytoma of the rat adrenal medulla to establish how the lytic compounds affect ion flux associated with lysis of target membranes. The lytic compounds increased permeability of the cell membrane for Ca2+ ions even during blockade of Ca2+ channels with cadmium. Results of a liposome assay suggested that the lytic compounds did not lyse such target membranes non-specifically by means of detergent-like activity. Analysis of sterol composition of isolates of A. tamarense and of five target protistan species showed that both lytic and non-lytic A. tamarense strains contain cholesterol and dinosterol as major sterols, whereas none of the other tested species contain dinosterol. Adding sterols and phosphatidylcholine to a lysis bioassay with the cryptophyte Rhodomonas salina for evaluation of competitive binding indicated that the lytic compounds possessed apparent high affinity for free sterols and phosphatidylcholine. Lysis of protistan target cells was dose-dependently reduced by adding various sterols or phosphatidylcholine. For three tested sterols, the lytic compounds showed highest affinity towards cholesterol followed by ergosterol and brassicasterol. Cholesterol comprised a higher percentage of total sterols in plasma membrane fractions of A. tamarense than in corresponding whole cell fractions. We conclude therefore that although the molecular targets of the lytic compounds are likely to involve sterol components of membranes, A. tamarense must have a complex self-protective mechanism that still needs to be addressed.

Files related to figures 1 to 13

Members of the marine dinoflagellate genus Alexandrium are known to exude allelochemicals, unrelated to well-known neurotoxins (PSP-toxins, spirolides), with negative effects on other phytoplankton and marine grazers. Physico/chemical characterization of extracellular lytic compounds of A. tamarense, quantified by Rhodomonas salina bioassay, showed that the lytic activity, and hence presumably the compounds were stable over wide ranges of temperatures and pH and were refractory to bacterial degradation. Two distinct lytic fractions were collected by reversed-phase solid-phase extraction. The more hydrophilic fraction accounted for about 2% of the whole lytic activity of the A. tamarense culture supernatant, while the less hydrophilic one accounted for about 98% of activity. Although temporal stability of the compounds is high, substantial losses were evident during purification. Lytic activity was best removed from aqueous phase with chloroform-methanol (3:1). A "pseudo-loss" of lytic activity in undisturbed and low-concentrated samples and high activity of an emulsion between aqueous and n-hexane phase after liquid-liquid partition are strong evidence for the presence of amphipathic compounds. Lytic activity in the early fraction of gel permeation chromatography and lack of activity after 5 kD ultrafiltration indicate that the lytic agents form large aggregates or macromolecular complexes.

Benthic foraminiferal assemblages and paleoproductivity reconstructions for the last 550 kyr of the Ontong Java Plateau, western equatorial Pacific

We analyzed foraminiferal and nannofossil assemblages and stable isotopes in samples from ODP Hole 807A on the Ontong Java Plateau in order to evaluate productivity and carbonate dissolution cycles over the last 550 kyr (kilo year) in the western equatorial Pacific. Our results indicate that productivity was generally higher in glacials than during interglacials, and gradually increased since MIS 13. Carbonate dissolution was weak in deglacial intervals, but often reached a maximum during interglacial to glacial transitions. Carbonate cycles in the western equatorial Pacific were mainly influenced by changes of deep-water properties rather than by local primary productivity. Fluctuations of the estimated thermocline depth were not related to glacial to interglacial alternations, but changed distinctly at ~280 kyr. Before that time the thermocline was relatively shallow and its depth fluctuated at a comparatively high amplitude and low frequency. After 280 kyr, the thermocline was deeper, and its fluctuations were at lower amplitude and higher frequency. These different patterns in productivity and thermocline variability suggest that thermocline dynamics probably were not a controlling factor of biological productivity in the western equatorial Pacific Ocean. In this region, upwelling, the influx of cool, nutrient-rich waters from the eastern equatorial Pacific or of fresh waters from rivers have probably never been important, and their influence on productivity has been negligible over the studied period. Variations in the inferred productivity in general are well correlated with fluctuations in the eolian flux as recorded in the northwestern Pacific, a proxy for the late Quaternary history of the central East Asian dust flux into the Pacific. Therefore, we suggest that the dust flux from the central East Asian continent may have been an important driver of productivity in the western Pacific.