Bioactive substances in marine phytoplankton: salinity effects on growth and toxin production of the dinophyte Alexandrium ostenfeldii

Alexandrium ostenfeldii is one of the most intensely studied toxic dinophyta in the world’s oceans and coastal waters. Its toxins are highly potent neurotoxins and causatives of food intoxications by contaminated sea food. In the last few years harmful algal blooms (HAB) of A. ostenfeldii have become a recurrent phenomenon in coastal waters. In 2012 such a dense bloom occurred in the Ouwerkerkse Kreek. This creek system in The Netherlands is discharging water in the Oosterschelde estuary, a large stock of mussels, oysters and fishery. Only little information is available about this A. ostenfeldii population, both in terms of its toxin profile and concerning its physiological characteristics in a highly variable ecosystem. We used multiple isolates of a bloom population for a thorough characterization of the phycotoxin profile and its variability in the population. A total of 68 A. ostenfeldii isolates were analyzed and revealed the presence of both, paralytic shellfish toxins (PSP) and cyclic imine toxins. Whereas the relative composition of PSP-toxins, consisting of saxitoxin, gonyautoxin and C-toxins, was almost identical among isolates, cyclic imine toxins were quite diverse. We detected a total of 23 different compounds with both spirolides (mainly 13-desmethyl spirolide C) and gymnodimines (both gymnodimine A and 12-methyl gymnodimine A) present and with a high variability in cyclic imine toxin profile among all isolates. Toxin cell quota was found to range 8-fold for total PSP-toxins and 11-fold for total cyclic imine toxins. Furthermore, for one selected isolate, the impact of variable salinities from 3 to 34 on growth and toxin content was determined. With similar growth rates from 0.13 to 0.2 d-1 over a salinity range from 6 to 34, a broad salinity tolerance of the Dutch A. ostenfeldii population was demonstrated. Furthermore, toxin composition and the expression of single PSP- and cyclic imine toxins were depended on salinity. Highest toxin cell quota was observed for exceptionally low and high salinities. In addition, isolates were found to produce lytic compounds with EC50 values (i.e. the concentration of A. ostenfeldii causing 50 % cell lysis of the target species Rhodomonas salina) of about 659 cells ml-1. A comparison of lytic activity in various fractions (whole cultures, culture supernatant, cell extract) revealed that whole cultures and also cell-free supernatants were about 5-fold more lytic than cell extracts. The lytic potency was susceptible for variable salinities: at very low salinities lytic effects of extracellular compounds were larger. At very high salinities, a larger amount of lytic substances was measured inside the cells. During the toxin screening a potentially new spirolide (m/z 696) was detected. LC-MS data allowed a hypothetical characterization of the compound as 23-hydroxy-13,19-didesmethyl spirolide D. For further NMR-analyzes, A. ostenfeldii was mass cultured and a total of 140 μg of the new compound was collected