A Selwood

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.

Toxic dinoflagellates (Dinophyceae) from Rarotonga, Cook Islands

Dinoflagellate species isolated from the green calcareous seaweed, Halimeda sp. J.V. Lamouroux, growing in Rarotongan lagoons, included Gambierdiscus australes Faust & Chinain, Coolia monotis Meunier, Amphidinium carterae Hulburth, Prorocentrum lima (Ehrenberg) Dodge, P. cf. maculosum Faust and species in the genus Ostreopsis Schmidt. Isolates were identified to species level by scanning electron microscopy and/or DNA sequence analysis. Culture extracts of G. australes isolate CAWD149 gave a response of 0.04 pg P-CTX-1 equiv. per cell by an N2A cytotoxicity assay (equivalent to ca 0.4 pg CTX-3C cell(-1)). However, ciguatoxins were not detected by LC-MS/MS. Partitioned fractions of the cell extracts potentially containing maitotoxin were found to be very toxic to mice after intraperitoneal (i.p.) injection. A. carterae was also of interest as extracts of mass cultures caused respiratory paralysis in mice at high doses, both by i.p. injection and by oral administration. The Rarotongan isolate fell into a different clade to New Zealand A. carterae isolates, based on DNA sequence analysis, and also had a different toxin profile. As A. carterae co-occurred with G. australes, it may contribute to human poisonings attributed to CTX and warrants further investigation. A crude extract of C. monotis was of low toxicity to mice by i.p. injection, and an extract of Ostreopsis sp. was negative in the palytoxin haemolysis neutralisation assay.

Production of pinnatoxins E, F and G by scrippsielloid dinoflagellates isolated from Franklin Harbour, South Australia

Abstract A pinnatoxin producing dinoflagellate was isolated in cyst form from sediments from Franklin Harbour, South Australia, December 2009. One isolate (CAWD180) produced pinnatoxin G, E, F and A (87, 10, 41 and 1.3 pg/cell respectively; liquid chromatograph–mass spectrometer, LC-MS, analysis) and another (CAWD183) produced pinnatoxin G only (13 pg/cell). Australian strains were identical to pinnatoxin E and F producers isolated from Northland, New Zealand (2008), based on large subunit (LSU) rDNA and ITS spacer region sequence data. Cysts were capable of division and produced more toxin per cell than the excysted motile form. Crude extracts of mass cultures were tested for toxicity in mice by intraperitoneal (i.p.) injection, gavage and voluntary consumption, and toxicity ratios were 1.0:1.8:4.5 (CAWD180) and 1.0:2.9:7.8 (CAWD183). This is similar to the ratios for New Zealand isolates, but differs from other cyclic imines for which oral toxicity can be 10–1000-fold less than i.p. administration.

The first report of Vulcanodinium rugosum (Dinophyceae) from the South China Sea with a focus on the life cycle

An individual non-motile (NM) cell was isolated from a surface sediment sample collected in Guangxi, China and subsequently established as a dinoflagellate strain in culture. The motile cells are 22.5–32.5 µm long and 20.0–30.0 µm wide, with a plate formula of Po, X, 4′, 3a, 7″, 6c, 5(?)s, 5″′, 2″″, fitting the description of Vulcanodinium rugosum. The Chinese strain shares 99.8%, 97.4% and 96.7% similarity (LSU sequence) with those from Australasia, France and Japan. Asexual division of V. rugosum takes place either in the thecate motile stage or within a NM division cell. Motile cells divided by binary fission inside the parent cell and transformed to NM division cells within 24 h. The NM cell underwent one to three consecutive divisions within the parent wall. The divisions were not always synchronous and neither was the release of motile cells from the NM cells. It generally took 4 to 6 days for the NM cells to complete one division. NM cells survived for 1 month at 4 °C in the dark, suggesting that they might play an important role in species dispersal. A novel pinnatoxin was detected at 20 pg cell−1 and no other known pinnatoxins (A–G) were detected.

Trace metal effects on the production of biotoxins by microalgae

Mass production of bioactive compounds was enhanced by the addition of particular trace metals to specific microalgae. An increase in domoic acid isomer-C production by Pseudo-nitzschia australis was achieved by adding copper (Cu) or Zinc (0.1–0.2μmol l−1) to standard growth media. The combined addition of selenium (Se) and Magnesium (0.1μmol l−1 and 3.6μmol l−1 respectively) elicited a 58% increase in gymnodimine production by Karenia selliformis. Addition of Cu (0.16μmol l−1) resulted in a 100% increase in the production of palytoxin-related compounds by Ostreopsis siamensis and a 50% increase in okadaic acid diol esters produced by Prorocentrum lima. Yessotoxin production by Protoceratium reticulatum was increased by adding Se (0.1μmol l−1). Enhanced toxin production in culture is important for the isolation of novel bioactive compounds, for the preparation of certified toxin standards and for toxicological studies. The effects of trace metals on biotoxin production are of concern given the substantial inputs into coastal waters from road and land run-off. Subsequent increases in algal bloom toxicity could impact on shellfish aquaculture.