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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 algaeTisochrysis luteain 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. [ABSTRACT FROM PUBLISHER]

A capture mechanism observed in a culture of the dinoflagellate Dinophysis acuta when preying on the ciliate Mesodinium rubrum (also sometimes referred to as Myrionecta rubra ) is described. The dinoflagellate released cohesive clumps of mucilage into the culture media. When M . rubrum cells came into contact with this mucilage, they were immediately immobilized, but remained alive for a short period of time. Observations of D. acuta cells ‘visiting and probing’ trapped M . rubrum cells were made and at a critical point D. acuta cells removed individual M . rubrum cells from the mucus to swim away with them. The removal of M. rubrum from the mucus coincided with the cells losing all their cilia and becoming swollen, presumably signifying the death of the cell. These changes may enable the D. acuta peduncle to penetrate the ciliate cell cortex. It is hypothesized that toxins produced by D. acuta play a role in the immobilization process within the mucilage trap. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Because of its novel bioactive properties the production of gymnodimine for use as a pharmaceutical precursor has aroused interest. The dinoflagellate, Karenia selliformis produces gymnodimine when grown in bulk culture using GP+selenium medium but the growth rates (μ) and levels of gymnodimine are low (μ, 0.05days−1; gymnodimine 250μgL−1 max). We describe the effects of organic acid additions (acetate, glycolate, alanine and glutamate additions and combinations of these) in enhancing growth and gymnodimine production in axenic cultures. The most effective organic acid combinations in decreasing order were: glycolate/alanine>acetate>glycolate. Glycolate/alanine optimised gymnodimine production by prolonging growth (maximum cell yield, 1.76×105 cellsmL−1; gymnodimine, 1260μgL−1; growth rate (μ), 0.2days−1) compared to the control (growth maximum cell yield, 7.8×104 cellsmL−1; gymnodimine, 780μgL−1; μ, 0.17days−1). Acetate enhanced gymnodimine by stimulating growth rate (μ, 0.23days−1) and the large concentration of gymnodimine per cell (16pgcell−1 cf. 9.8pgcell−1 for the control) suggests a role for this compound in gymnodimine biosynthesis. Amending culture media with Mn2+ additions resulted in slightly decreased growth in control cultures and increased the gymnodimine while in glycolate/alanine cultures growth was stimulated but gymnodimine production decreased. The results suggest that the organic acid can enhance gymnodimine production by either enhancing growth maximum or the biosynthetic pathway. [Copyright &y& Elsevier]

The major pectenotoxin and okadaic acid group toxins in Dinophysis acuta and Dinophysis acuminata cell concentrates, collected from various locations around the coast of the South Island of New Zealand (NZ), were determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS). PTX2 and PTX11 were the major polyether toxins in all Dinophysis spp. cell concentrates. D. acuta contained PTX11 and PTX2 at concentrations of 4.7–64.6 and 32.5–107.5 pg per cell, respectively. The amounts of PTX11 and PTX2 in D. acuminata were much lower at 0.4–2.1 and 2.4–25.8 pg per cell, respectively. PTX seco acids comprised only 4% of the total PTX content of both D. acuta and D. acuminata. D. acuta contained low levels of OA (0.8–2.7 pg per cell) but specimens from the South Island west coast also contained up to 10 times higher levels of OA esters (7.0–10.2 pg per cell). Esterified forms of OA were not observed in D. acuta specimens from the Marlborough Sounds. D. acuta did not contain any DTX1 though all D. acuminata specimens contained DTX1 at levels of 0.1–2.4 pg per cell. DTX2 was not present in any New Zealand Dinophysis spp. specimens. Although the total toxin content varied spatially and temporally, the relative proportions of the various toxins in different specimens from the same location appeared to be relatively stable. The total PTX/total OA ratios in different isolates of D. acuta were very similar (mean±S.E.: 14.9±1.9), although the Marlborough Sounds D. acuminata isolates had a higher total PTX/total OA ratio (mean±S.E.: 22.7±2.4) than the Akaroa Harbour isolates (8.0). No evidence of azaspiracids were detected in these specimens. These results show that the LC–MS/MS monitoring of plankton for PTX group toxins (e.g. PTX2) and their derivatives (e.g. PTX2 seco acid) may provide a sensitive, semi-quantitative, indicator of the presence of more cryptic OA group toxins (e.g. OA esters). [Copyright &y& Elsevier]

Abstract: The uptake of paralytic shellfish poisoning (PSP) toxins and spirolides by the paddle crab (Ovalipes catharus) was investigated in two laboratory feeding trials using Greenshell™ mussels (Perna canaliculus), which had been fed toxic strains of either Alexandrium catenella or A. ostenfeldii, as a vector. Toxin uptake by crabs occurred in both feeding trials and was limited to the visceral tissue; no toxins were detected in the body meat or the gills. The first trial utilized a strain of A. catenella that had high total PSP toxin content, 442.3±91.6fmol/cell, that was dominated by low toxicity N-sulfocarbamoyl toxins resulting in a low cellular toxicity, 5.5±1.6pgSTXequiv./cell. In this trial, toxin accumulation in the crabs was highly variable and ranged from 3.8 to 221.5μgSTXequiv./100g, with 3/4 of the crabs exceeding the regulatory limit of 80μgSTXequiv./100g. Eight days after feeding on toxic mussels the crabs still retained high levels of toxin suggesting that depuration rates in this species may be slow. In the second feeding trial, the A. ostenfeldii strain fed to mussels produced low levels of both PSP toxins (52.0±19.5fmol/cell; 1.4±0.3pgSTXequiv./cell) and spirolides (1.8pg/cell) and, as a result, the concentration transferred to crabs via the mussels was very low-PSP toxins ranged from 2.5 to 6.8μgSTXequiv./100g and spirolides from 6 to 7μg/kg. The results of our study demonstrate that paddle crabs are capable of acquiring both PSP toxins and spirolides and suggest that this may occur in the wild during a toxic shellfish event. It also highlights the need to remove the viscera before consumption. [Copyright &y& Elsevier]

At about 20-year intervals, since at least the 1860s, there have been accounts of the accumulation of large quantities of mucilage in the waters off the northern coast of the South Island of New Zealand. On a few occasions these events have been associated with harmful effects, such as the mass mortality of marine fauna and the impediment of fishing activities, though previous attempts to identify the causative organism(s) or the environmental conditions which promote its development have been unsuccessful. In January 2000, there was a moderate re-occurrence of mucilage accumulation in Tasman Bay that led to the identification of the primary origin of this material as the planktonic, thecate, dinoflagellate Gonyaulax hyalina (Ostenfeld et Schmidt). Macro-aggregate accumulation was occurring in the water column when dinoflagellate cell numbers were relatively low. Experimental work on cultures of this organism showed that the dinoflagellate produced transparent exo-polymers (TEPs) composed primarily of galactose and glucose monomers (44.8 and 29.6 mol%, respectively). Analysis of natural and cultured mucilage showed this had a high inorganic content (>60% (w/w)). A substantial proportion (40% (w/w)) of this inorganic fraction was composed of calcium that apparently played an important role in stabilizing the gel. Phosphorus was also enriched in the mucilage. It is hypothesized that the high inorganic N/P ratio observed in the water column at the time of mucilage formation was an effect rather than a cause of this event, which resulted in the scavenging of particulate material from the water column and enrichment of sediments and bottom waters with organic material and phosphorus. [Copyright &y& Elsevier]

Abstract: Karenia brevisulcata (Chang), a new toxic dinoflagellate of the genus Karenia was isolated from a harmful algal bloom that occurred in Wellington Harbour, New Zealand in 1998. The bloom severely affected most marine biota resulting in long-term ecological damage and causing respiratory distress in harbour bystanders. Cultures of K. brevisulcata produced a range of novel toxins including ten lipid-soluble K. brevisulcata toxins (KBTs) and six water-soluble brevisulcatic acids (BSXs). Brevetoxins were not detected. KBT-F, KBT-G, BSX-1 and BSX-2 were isolated from 1450L of bulk cultures and purified in mg quantities. Preliminary chemical and toxicological investigations show that KBT-F (M 2054 C107H160O38) and KBT-G (M 2084 C108H162O39) are complex polycyclic ethers with UVmax at 227nm. NMR data gave characteristics of ladder frame polyether structures and a 2-methylbut-2-enal side chain, similar to gymnocins. The mouse i.p. LD50s for KBT-F and -G were 0.032 and 0.040mgkg−1, respectively. These KBTs were also highly cytotoxic and haemolytic. BSX-1 (M 916 C49H72O16) and BSX-2 (M 872 C47H68O15) are polycyclic ether dicarboxylates with UVmax 196nm. BSX-4 and BSX-5, the lactone ring-closed analogues and the presumed primary toxins in the algal cells, were isolated in smaller quantities. Preliminary structural information from NMR and MS showed a carboxylated side chain and some similarities to brevetoxin-A. However, the structures have not yet been fully elucidated due to conformers confounding the NMR. The mouse i.p. LD50 for BSX-1 was 3.9mgkg−1 while no deaths were seen in mice injected with BSX-2 at 6.6mgkg−1. The LD50s for the lactones BSX-4 and -5 were 1.4 and 1.6mgkg−1 respectively. BSX-4 and -5 were agonists of voltage-gated sodium channels but only weakly haemolytic. Activities in the Neuro-2a cytotoxicity assay were ca 10% of dihydrobrevetoxin-2 and were fully antagonised by saxitoxin. [Copyright &y& Elsevier]

Abstract: A cis-isomer of a C8-diol ester of okadaic acid (1) was isolated during large-scale purification of pectenotoxins (PTXs) from extracts of Dinophysis acuta collected from the west coast of South Island, New Zealand. The compound was identified by NMR spectroscopic and liquid chromatography-mass spectrometry (LC-MS) studies, and is the first reported cis-isomer of an okadaic acid C8-diol-ester identified in Dinophysis. The more abundant trans-C8-diol ester of okadaic acid (2) isolated from the same Dinophysis extract was rapidly hydrolyzed to okadaic acid in vitro by the supernatant from green-lipped mussel hepatopancreas. [Copyright &y& Elsevier]

Abstract: Yessotoxin (YTX) contamination of Greenshell™ mussels (Perna canaliculus) in the Marlborough Sounds, New Zealand, occurred in May 2004 with no known YTX-producing algae present in associated seawater samples. Gonyaulax spinifera, previously considered a non-YTX producer, was isolated from the seawater samples and production of YTX by the cells grown in culture was determined by identification of YTXs by ELISA. The algal identification was based on the type description and cyst form. DNA sequence data for G. spinifera differed from the morphologically similar G. membranacea, G. digitale, and G. cf. spinifera. Protoceratium reticulatum was isolated from the same site as G. spinifera 3 weeks later, but was not a YTX-producer by LC-MS and YTX ELISA analyses. Implications of the discovery that YTX is produced by G. spinifera are that this micro-alga will need to be included in future risk assessments for YTX based on the phytoplankton composition. [Copyright &y& Elsevier]

Morphological descriptions, toxicity data and an analysis of LSU rRNA gene sequences are presented for seven species within the marine dinoflagellate genus Alexandrium (Halim), identified in New Zealand coastal waters. All species were established in culture and comparison of their morphology with descriptions from the literature showed these isolates to correspond to the previously described taxa: A. catenella, A. tamarense, A. fraterculus, A. concavum, A. ostenfeldii, A. margalefi and A. pseudogoniaulax. With the exception of A. ostenfeldii, none of these species has previously been recorded in New Zealand. Most of these species are widespread and common, though they are rarely abundant, A. fraterculus has been the most frequent bloom former. Three species, A. catenella, A. tamarense, A. ostenfeldii, produced paralytic shellfish poisoning (PSP) toxins but to date only A. catenella has been associated with a significant shellfish-toxin contamination event. A. catenella and A. tamarense isolates produced toxin profiles predominating in low specific toxicity N-sulfo-carbamoyl analogues, and had identical LSU rRNA gene sequences which place them within the Pacific/Asian clade. The formation of putative hypnozygotes in mating experiments between A. tamarense and some A. catenella isolates suggested these were sexually compatible. However, although >70% of these cysts germinated, the survival of the progeny was poor. A. pseudogoniaulax and A. concavum are the most distantly related to other species within the genus. [Copyright &y& Elsevier]

The isolation and structural determination of new marine ladder-frame polyethers, brevisulcatic acids-1 (1) and -4 (2) are reported. Brevisulcatic acids were isolated from the dinoflagellate Karenia brevisulcata, which was identified as the causative species of a major red tide event in New Zealand in 1998. The ether ring composition and a β-hydroxy, γ-methylene valeric acid side chain of 1 and 2 are common, but 2 has a γ-lactone as the 5-membered A-ring while 1 is the seco acid analogue. Compound 2 has structural and bioactivity similarities to brevetoxin A.

Yessotoxins from a large-scale culture (226 L) of Protoceratium reticulatum strain CAWD129 were harvested by filtration followed by solid-phase extraction. The extract was purified by column chromatography over basic alumina and reverse-phase flash chromatography to afford pure yessotoxin (193 mg). Isolation of yessotoxin was greatly facilitated by selection of a strain which did not produce analogues that interfered with yessotoxin isolation. In addition to yessotoxin, numerous minor yessotoxins were detected by LC-MS in other fractions. From one of these, an early eluting minor analogue with the same molecular weight as yessotoxin and a similar mass spectrometric fragmentation pattern was isolated. This analogue was identified by NMR and mass spectrometry as a novel yessotoxin analogue containing a furan ring in the side chain. This finding reveals biosynthetic flexibility of the yessotoxin pathway in P. reticulatum and confirms earlier findings of production of many minor yessotoxin analogues by this alga. Production of these analogues appeared to be a constitutive trait of P. reticulatum CAWD129.

Pectenotoxins (PTXs) accumulate in shellfish feeding on dinoflagellates of the genus Dinophysis, so that humans can be exposed to these toxins through shellfish consumption. Some PTXs are toxic to experimental animals, whereas others are of much lower toxicity. Pectenotoxin-2, the most abundant PTX from most Dinophysis spp., is rapidly metabolized by most shellfish to a mixture of pectenotoxin-2 seco acid (2) and 7-epi-pectenotoxin-2 seco acid (1). A mixture of 1 and 2 was produced during purification of an extract from in vitro enzymatic hydrolysis of pectenotoxin-2. These were separated by preparative HPLC, and the structure of 1 was confirmed by one- and two-dimensional 1H and 13C NMR spectroscopy and LC-MS3 analyses. No toxic changes were recorded in mice injected intraperitoneally with 1 or 2 at a dose of 5000 microg/kg. PTX seco acids are therefore unlikely to be of consequence to human consumers at the concentrations found in contaminated shellfish.

A new pectenotoxin, which has been named pectenotoxin-11 (PTX11), was isolated from the dinoflagellate Dinophysis acuta collected from the west coast of New Zealand. The structure of PTX11 was determined as 34S-hydroxypectenotoxin-2 by tandem mass spectrometry and UV and NMR spectroscopy. PTX11 appears to be only the third pectenotoxin identified as a natural biosynthetic product from algae after pectenotoxin-2 and pectenotoxin-12. The LD50 of PTX11 determined by mouse intraperitoneal injection was 244 microg/kg. The LD(min) of PTX11 in these experiments was 250 microg/kg. No signs of toxicity were recorded in mice following an oral dose of PTX11 at 5000 microg/kg. No diarrhea was observed in any of the animals administered with the test substance by either route of administration. Unlike pectenotoxin-2 (PTX2), PTX11 was not readily hydrolyzed to its corresponding seco acid by enzymes from homogenized green-lipped mussel (Perna canaliculus) hepatopancreas.