Veronica Beuzenberg

Complex toxin profiles in phytoplankton and Greenshell mussels (Perna canaliculus), revealed by LC–MS/MS analysis

Toxin profiles were determined in phytoplankton cell concentrates and Greenshell mussels (Perna canaliculus) exposed to a dinoflagellate bloom dominated by Dinophysis acuta and Protoceratium reticulatum. This was achieved by using a method for the simultaneous identification and quantification of a variety of micro-algal toxins by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionisation (+/-) and monitoring of daughter ions in multiple reaction modes. Plankton concentrates and shellfish contained high levels of yessotoxins (YTXs) and pectenotoxins (PTXs) and low levels of okadaic acid (OA). A high proportion (>87%) of the OA in both plankton and shellfish was released by alkaline hydrolysis. An isomer of pectenotoxin 1 (PTX1i) was nearly as abundant as pectenotoxin 2 (PTX2) in the plankton and shellfish, and the latter contained high levels of their respective seco acids. DTX1, DTX2, and PTX6 were not detected. MS-MS experiments revealed that the shellfish contained several other oxygenated metabolites of YTX in addition to 45-hydroxy yessotoxin (45OH-YTX). Gymnodimine (GYM) was present in the shellfish but not plankton and it was probably the residue from a previous GYM contamination event. Unlike the other toxins, GYM was concentrated in tissues outside the digestive gland and levels did not decrease over 5 months. The depuration rates of YTX and PTXs from mussels were modelled.

Liquid chromatography–mass spectrometry of spiroketal stereoisomers of pectenotoxins and the analysis of novel pectenotoxin isomers in the toxic dinoflagellate Dinophysis acuta from New Zealand

The acid-catalyzed inter-conversion of spiroketal isomers of pectenotoxins PTX1, PTX6 and PTX2 were studied by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS-MS). Using a C8-silica reversed-phase column and a mobile phase of aqueous acetonitrile containing 2 mM ammonium formate and 50 mM formic acid, the known spiroketal stereoisomers of PTX1 eluted in order of PTX1, PTX4 and PTX8, while those of PTX6 eluted in the order PTX6, PTX7 and PTX9. Acid treatment of PTX2 yielded two novel spiroketal stereoisomers, which have been named PTX2b and PTX2c. LC-MS-MS spectra obtained for the [M+NH4]- ions of PTX2, PTX2b and PTX2c were essentially identical. As an application of the LC-MS-MS methodology, a sample of the toxic dinoflagellate Dinophysis acuta collected from the coast of New Zealand was analyzed for pectenotoxins. PTX2 and a new pectenotoxin, which has been named PTX11, were detected as the most predominant compounds. Novel PTX2 and PTX11 isomers were also found in the D. acuta although the levels of these compounds were low.

Investigations into the cellular actions of the shellfish toxin gymnodimine and analogues

The effects of the shellfish toxin gymnodimine and its analogues (gymnodimine acetate, gymnodimine methyl carbonate and gymnodamine) on cellular viability were tested using the Neuro2a neuroblastoma cell line. Concentrations of toxins up to 10μM had variable effects on reducing cell number as determined using the MTT assay and no effects on the expression of a number of signal transduction proteins (c-Jun, ATF-2, ATF-3) which are sensitive to cellular stress. However, pre-exposure of Neuro2a cells to 10μM concentrations of toxins for 24h greatly sensitized these cells to the apoptotic effects of another algal toxin, okadaic acid. These results suggest that gymnodimine and its analogues sensitize Neuro2a cells to cytotoxins and raise the possibility that algal blooms involving the production of both okadaic acid-type molecules and gymnodimine may generate greater cytotoxicity and pose a greater public health problem. Furthermore, our studies establish the Neuro2a cell line as a potentially high-throughput cellular system sensitive to the pharmacological effects of gymnodimine and analogues, and as a potential screen for algal-derived toxins.

Brevisulcenal-F: a polycyclic ether toxin associated with massive fish-kills in New Zealand.

A novel marine toxin, brevisulcenal-F (KBT-F, from karenia brevisulcata toxin) was isolated from the dinoflagellate Karenia brevisulcata. A red tide of K. brevisulcata in Wellington Harbour, New Zealand, in 1998 was extremely toxic to fish and marine invertebrates and also caused respiratory distress in harbor bystanders. An extract of K. brevisulcata showed potent mouse lethality and cytotoxicity, and laboratory cultures of K. brevisulcata produced a range of novel lipid-soluble toxins. A lipid soluble toxin, KBT-F, was isolated from bulk cultures by using various column chromatographies. Chemical investigations showed that KBT-F has the molecular formula C(107)H(160)O(38) and a complex polycyclic ether nature. NMR and MS/MS analyses revealed the complete structure for KBT-F, which is characterized by a ladder-frame polyether scaffold, a 2-methylbut-2-enal terminus, and an unusual substituted dihydrofuran at the other terminus. The main section of the molecule has 17 contiguous 6- and 7-membered ether rings. The LD(50) (mouse i.p.) for KBT-F was 0.032 mg/kg.

Isolation and characterisation of halo-tolerant Dunaliella strains from Lake Grassmere/Kapara Te Hau, New Zealand

Strains of the chlorophyte genus Dunaliella were isolated from Dominion Salt NZ Ltd evaporation ponds at Lake Grassmere/Kapara Te Hau, New Zealand. Five halo-tolerant groups were characterised using morphology, molecular phylogenetic analysis and physiological response to varying culture conditions. Species identification based on morphological characteristics of cell size, shape and flagella to cell length ratio was supported by molecular internal transcribed spacer one and two regions (ITS-1, ITS-2) and 5.8S ribosomal RNA gene sequences. Investigations into salinity and photostress response were performed using pulse amplitude modulation (PAM) fluorometry, and high-performance liquid chromatography (HPLC) quantification of pigment profiles with an emphasis on carotenoid production are presented. Cryo-preservation storage was successfully applied to all strains.

Optimising conditions for growth and xanthophyll production in continuous culture of Tisochrysis lutea using photobioreactor arrays and central composite design experiments

ABSTRACT The production of valuable metabolites from microalgae represents a potentially sustainable source of a range of products that can be difficult to synthesise directly. Microalgae respond to the dynamic and often subtly shifting growth environment in a complex way. The optimal conditions for growth can be quite different to those needed for optimal product generation, depending on the nature of the biosynthesis of that product. This is especially so for secondary metabolites. A combination of a multi-vessel photobioreactor array, where certain growth conditions can be monitored and controlled precisely together with an experimental design matrix has been used to determine the optimal combination of temperature, irradiance and pH for a group of xanthophylls including fucoxanthin in the algae Tisochrysis lutea in continuous culture. Continuous culture as a mode is more suited for industrial production than batch mode in which the media constituents and algal population change dramatically over time. The central composite design experiment matrix has a range of set values for each parameter being investigated that bracket the optimal conditions. The three parameters investigated in this work (temperature, irradiance and pH) are major factors influencing algal growth. The method can be applied to other parameters that might affect growth or might affect production of a metabolite of interest, such as a nutrient level. The combined approach has been used previously to indicate optimal growth conditions for biomass generation and this work is one of the first to apply it to the generation of an algal product of interest.

Measuring the influence of nutrients and river water on the photosynthetic efficiency of Didymosphenia geminata using pulse amplitude modulated fluorometry

Over the past three decades the freshwater diatom Didymosphenia geminata (Lyngbye) M. Schmidt has expanded its range globally. In some rivers D. geminata has become invasive, forming expansive and thick polysaccharide-dominated mats. Techniques to maintain and study D. geminata in the laboratory are limited. In this study, a 96-well plate format assay using pulse amplitude modulated (PAM) fluorometry was developed to study D. geminata under controlled conditions. The PAM assay and morphological assessments were used to investigate the addition of sodium nitrate (NaNO3) to a previously developed D. geminata-specific growth medium (Didymo Medium; DM). Addition of low concentrations (ca. 0.003–0.018 µM) enhanced cell survival and health. Central-composite design (CCD) experiments coupled with response surface methodology were then used to investigate optimal concentrations of six key chemicals in DM (magnesium sulphate, calcium chloride, mono-potassium phosphate, sodium metasilicate pentahydrate, ferric sodium ethylenediaminetetraacetic acid (EDTA) and NaNO3). An optimised DM recipe is provided. The PAM assay was also used to analyse the influence of maintaining D. geminata cells in river waters sourced from locations with and without the diatom. A NaNO3 spiking experiment was undertaken using water from one location. The maximum quantum yields of cells maintained in all river waters remained relatively constant and higher than those maintained in DM or Milli-Q water for the seven day test period. The results of the NaNO3 river water spiking experiment provided contrasting results to the culture medium trials, with minimal impact on photosynthetic efficiency. These data, coupled with the results of the CCD experiment, suggest complex interactions among nutrients that have varying effects on D. geminata cell health. Together with microscopical observations, the 96-well plate PAM assay provides a useful tool for improving knowledge of D. geminata biology and growth requirements.