Development of a LC-MS/MS method for the quantification of goniodomins A and B and its application to Alexandrium pseudogonyaulax strains and plankton field samples of Danish coastal waters.

An external standard of goniodomin A (GDA) was prepared from a strain of Alexandrium pseudogonyaulax originating from New Zealand and its chemical structure was confirmed by nuclear magnetic resonance (NMR) spectroscopy. Using the GDA standard, an ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method in selected reaction monitoring (SRM) mode was developed for separation and quantification of GDA. This method was successfully applied to planktonic field samples collected during an oceanographic expedition conducted with R/V Uthörn along the Danish west coast, Limfjord and Kattegat in June 2016. In addition, this method was used to characterize goniodomin (GD) profiles of 17 A. pseudogonyaulax strains from the coastal North Sea and from Limfjord. Highest GDA levels were found in Limfjord (up to 590 ng NT-1 m-1), but GDA was also detected in the North Sea appearing at the latitude of Sylt Island northwards and in Kattegat from the eastern mouth of Limfjord down to the Kiel Bight, but at lower abundances than within Limfjord. This is the first reported detection of GDA in planktonic field samples. Chemical analysis of 17 strains of A. pseudogonyaulax revealed that all strains were producers of GDA (5-35 pg cell-1) as well as in most cases minor amounts (0.01-0.07 pg cell-1, expressed as GDA equivalents) of goniodomin B (GDB).

Mode of action of membrane-disruptive lytic compounds from the marine dinoflagellate Alexandrium tamarense.

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 Ca(2+) 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 Ca(2+) ions even during blockade of Ca(2+) 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.