Lincoln MacKenzie

Identification of Protoceratium reticulatum as the biogenetic origin of yessotoxin

Yessotoxin (YTX), a disulfated polyether toxin, was isolated from cultured cells of the marine dinoflagellate Protoceratium reticulatum and unambiguously identified by high-performance liquid chromatography, 1H NMR, and MS data. The result is the first to confirm toxigenicity of this species and demonstrate it as one of the biogenetic origins of YTX found in shellfish.

Gymnodimine, a new marine toxin of unprecedented structure isolated from New Zealand oysters and the dinoflagellate, Gymnodinium sp.

Abstract A new marine toxin, gymnodimine, was isolated from New Zealand oysters, Tiostrea chilensis, and the dinoflagellate Gymnodinium cf. mikimotoi. Its unique structure was elucidated by spectroscopic methods.

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.

Comparative morphology and molecular phylogenetic analysis of three new species of the genus Karenia (Dinophyceae) from New Zealand

Three new dinoflagellate species, Karenia papilionacea sp. nov., Karenia selliformis sp. nov., and Karenia bidigitata sp. nov., were compared with the toxic species Karenia mikimotoi (Miyake & Kominami ex Oda) G. Hansen & Moestrup, Karenia brevis (Davis) G. Hansen & Moestrup, and Karenia brevisulcata (Chang) G. Hansen & Moestrup using the same fixative. Distinguishing morphological characters for the genus Karenia included a smooth theca and a linear apical groove. The new species can be distinguished on the basis of morphological characters of vegetative cells that include the location and shape of the nucleus; the relative excavation of the hypotheca; the characteristics of apical and sulcal groove extensions on the epitheca; the cellular shape, size, and symmetry; the degree of dorsoventral compression; and the presence of an apical protrusion or carina. Species with pronounced dorsoventral compression swim in a distinctive fluttering motion. An intercingular tubular structure traversing the proximal and distal ends of the cingulum is common to the species of Karenia, Karlodinium micrum (Leadbeater & Dodge) J. Larsen, Gymnodinium pulchellum J. Larsen, and Gyrodinium corsicum Paulmier. Molecular phylogenetic analyses of rDNA sequence alignments show that the new species are phylogenetically distinct but closely related to K. mikimotoi and K. brevis.

Pectenotoxin-2 seco acid: a toxin converted from pectenotoxin-2 by the New Zealand Greenshell mussel, Perna canaliculus

Comparison of pectenotoxin (PTX) profiles of toxic dinoflagellate Dinophysis acuta, Greenshell mussels (Perna canaliculus) and Blue mussels (Mytilus galloprovincialis) collected from Wedge Point, Queen Charlotte Sound, New Zealand was carried out by liquid chromatography-mass spectrometry with turbo-ionspray ionization. Although the major PTX homologue in D. acuta was pectenotoxin-2 (PTX2), both Greenshell and Blue mussels contained pectenotoxin-2 seco acid (PTX2SA) as the predominant toxin. More than 90% of PTX2 isolated from D. acuta was rapidly converted to PTX2SA and its epimer 7-epi-pectenotoxin-2 seco acid (7-epi-PTX2SA) in the Greenshell mussel extracts. The conversion from PTX2 to PTX2SA and 7-epi-PTX2SA was not observed in phosphate buffers at various pH ranging from 4.1 to 9.1. These findings indicate that PTX2SA and 7-epi-PTX2SA are not artifact toxins resulting from hydrolysis of PTX2, but arise from the conversion of PTX2 by mussel tissues.

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.

In situ passive solid-phase adsorption of micro-algal biotoxins as a monitoring tool.

Laboratory and field studies of the passive solid-phase adsorption toxin tracking (SPATT) method have been carried out around the world. A wide range of marine micro-algal toxins have been detected and the potential of the method to provide reliable, sensitive, time-integrated sampling to monitor the occurrence of toxic algal bloom events has been demonstrated. The method has several important advantages over current phytoplankton and shellfish monitoring methods. Trials of various adsorption substrates have been carried out and the best candidates have been selected for the lipophilic marine biotoxin groups; however, research continues to locate suitable substrates for the more polar water-soluble compounds such as domoic acid and the saxitoxins. The technique has also been successfully applied to the detection of a range of freshwater cyanobacterial toxins.

Toxic and noxious phytoplankton in Big Glory Bay, Stewart Island, New Zealand

Diurnal vertical profile sampling of the water column, during a fish killing bloom of the raphidophycean alga Heterosigma akashiwo, revealed a phytoplankton population otherwise composed almost entirely of a variety of dinoflagellates. Of these Glenodinium danicum, Dinophysis acuta, Polykrikos schwartzii, Ceratium furca and Gyrodinium spirale were predominant. The distribution of the major species within the phytoplankton were documented and evidence of synchronous vertical migration of H. akashiwo, G. danicum and P. schwartzii was observed. Extracts of shellfish obtained during the bloom and tested by mouse bioassay showed no PSP toxicity but a marginal degree of DSP toxicity. During a subsequent one year phytoplankton monitoring programme another potentially noxious species (Chaetoceros convolutus) appeared and the seasonal reoccurrence of species present during the bloom (e.g. H. akashiwo) was observed. Important year to year differences in the summer phytoplankton (diatom versus flagellate dominated populations) were apparent and analysis of climate data showed that these differences related to different weather conditions prevailing during the two summer periods sampled. The data suggest the fish killing bloom was giving a chance to develop by a prolonged period of warm, calm weather (during which several heavy rainfall events occurred) leading to stable hydrographic conditions (i.e. stratification) and an increase in the retention time of water within the bay.

Detection of tetrodotoxin from the grey side-gilled sea slug - Pleurobranchaea maculata, and associated dog neurotoxicosis on beaches adjacent to the Hauraki Gulf, Auckland, New Zealand

Investigations into a series of dog poisonings on beaches in Auckland, North Island, New Zealand, resulted in the identification of tetrodotoxin (TTX) in the grey side-gilled sea slug, Pleurobranchaea maculata. The levels of TTX in P. maculata, assayed by liquid chromatography-mass spectrometry (LC-MS) ranged from 91 to 850 mg kg(-1) with a median level of 365 mg kg(-1) (n = 12). In two of the dog poisoning cases, vomit and gastrointestinal contents were found to contain TTX. Adult P. maculata were maintained in aquaria for several weeks. Levels of TTX decreased only slightly with time. While in the aquaria, P. maculata spawned, with each individual producing 2-4 egg masses. The egg masses and 2-week old larvae also contained TTX. Tests for other marine toxins were negative and no other organisms from the area contained TTX. This is the first time TTX has been identified in New Zealand and the first detection of TTX in an opisthobranch.

DOES DINOPHYSIS (DINOPHYCEAE) HAVE A SEXUAL LIFE CYCLE?1

A variety of morphotypes (of two size classes) within two wild populations of Dinophysis acuta Ehrenberg and D. cf. acuminata are described. The observation that these two cell types engaged in the formation of couplets (possibly leading to the engulfment of the smaller by the larger cell) suggests that these species may undergo a process of sexual reproduction involving the fusion of anisogamous gametes. This behavior and asexual cell division involved a small portion of the population at any one time and took place rapidly at specific subsurface depths and time of day, the former during the evening, the latter during the morning. Even though definite proof of sexuality by Dinophysis was not obtained, the possiblity that sexual dimorphism exists in several species of the genus is discussed.