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The dinoflagellate Alexandrium pacificum can produce paralytic shellfish toxins and is mainly distributed in the Pacific. Blooms of A. pacificum have been frequently reported in offshore areas of the East China Sea, but not along the coast. To investigate the bloom dynamics of A. pacificum and their potential origins in the Taiwan Strait, we performed intensive sampling of both water and sediments from 2017 to 2020. Ellipsoidal cysts were identified as A. pacificum and enumerated based on microscopic observation. Their abundances were quite low but there was a maximum of 9.6 cysts cm−3 in the sediment near the Minjiang River estuary in May 2020, consistent with the high cell abundance in the water column in this area. Cells of A. pacificum were examined using a quantitative polymerase chain reaction, and they appeared to be persistent in the water column across the seasons. High densities of A. pacificum (103 cells L−1) were observed near the Jiulongjiang and Minjiang River estuary in early May 2020, where high nutrients (dissolved inorganic nitrogen and phosphate), and relatively low temperatures (20–21 °C) were also recorded. Strains isolated from the East and South China Sea exhibited the highest division rate (0.63 and 0.93 divisions d−1) at 20 and 23 °C, respectively, but the strain from the Yellow Sea showed the highest division (0.40 divisions d−1) at 17–23 °C. Strains from the East and South China Sea shared similar toxin profiles dominated by the N-sulfocarbamoyl toxins C1/2, but the strain from the Yellow Sea predominantly produced the carbamoyl toxins GTX1/4 and no C1/2. Our results suggest that both cyst germination and persistent cells in the water column might contribute to the bloom formation in the Taiwan Strait. Our results also indicate that the East and South China Sea populations are connected genetically through similar toxin formation but separated from the Yellow Sea population geographically.

Planktonic dinoflagellate records for New Zealand are substantial due to intense monitoring programmes that have taken place on behalf of New Zealand’s biotoxin regulators and the shellfish...

In recent years, blooms of toxic Alexandrium ostenfeldii strains have been reported from around the world. In 2013, the species formed a red tide in a shallow lagoon in western Japan, which was the first report of the species in the area. To investigate the genetic relatedness of Japanese A. ostenfeldii and global isolates, the full-length SSU, ITS and LSU sequences were determined, and phylogenetic analyses were conducted for isolates from western and northern Japan and from the Baltic Sea. Genotyping and microsatellite sequence comparison were performed to estimate the divergence and connectivity between the populations from western Japan and the Baltic Sea. In all phylogenetic analyses, the isolates from western Japan grouped together with global isolates from shallow and low saline areas, such as the Baltic Sea, estuaries on the east coast of U.S.A. and from the Bohai Sea, China. In contrast, the isolates from northern Japan formed a well-supported separate group in the ITS and LSU phylogenies, indicating differentiation between the Japanese populations. This was further supported by the notable differentiation between the sequences of western and northern Japanese isolates, whereas the lowest differentiation was found between the western Japanese and Chinese isolates. Microsatellite genotyping revealed low genetic diversity in the western Japanese population, possibly explained by a recent introduction to the lagoon from where it was detected. The red tide recorded in the shallow lagoon followed notable changes in the salinity of the waterbody and phytoplankton composition, potentially facilitating the bloom of A. ostenfeldii.

8 pages, 2 tables, A recently published study analyzed the phylogenetic relationship between the genera Centrodinium and Alexandrium, confirming an earlier publication showing the genus Alexandrium as paraphyletic. This most recent manuscript retained the genus Alexandrium, introduced a new genus Episemicolon, resurrected two genera, Gessnerium and Protogonyaulax, and stated that: “The polyphyly [sic] of Alexandrium is solved with the split into four genera”. However, these reintroduced taxa were not based on monophyletic groups. Therefore this work, if accepted, would result in replacing a single paraphyletic taxon with several non-monophyletic ones. The morphological data presented for genus characterization also do not convincingly support taxa delimitations. The combination of weak molecular phylogenetics and the lack of diagnostic traits (i.e., autapomorphies) render the applicability of the concept of limited use. The proposal to split the genus Alexandrium on the basis of our current knowledge is rejected herein. The aim here is not to present an alternative analysis and revision, but to maintain Alexandrium. A better constructed and more phylogenetically accurate revision can and should wait until more complete evidence becomes available and there is a strong reason to revise the genus Alexandrium. The reasons are explained in detail by a review of the available molecular and morphological data for species of the genera Alexandrium and Centrodinium. In addition, cyst morphology and chemotaxonomy are discussed, and the need for integrative taxonomy is highlighted, Support to DMA from the NOAA ECOHAB program (Grant #NA15NOS4780181) is gratefully acknowledged. Support to EG, AR, NS from the COPAs project (CTM2017-86121-R) is acknowledged. IGL and CJBS are COFFA-IPN and EDI-IPN fellows, With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

© 2020 Elsevier B.V. Similarities and differences between Australia and New Zealand in Harmful Algal species occurrences and Harmful Algal Events impacting on human society (HAEDAT) are reported and factors that explain their differences explored. Weekly monitoring of harmful phytoplankton and biotoxins commenced in Australia in 1986 and in New Zealand in 1993. Anecdotal historic HAB records in both countries are also catalogued. In Australia, unprecedented highly toxic Paralytic Shellfish Toxin (PST)-producing blooms of Alexandrium catenella have impacted the seafood industry along the 200 km east coast of Tasmania from 2012 to present. Toxic blooms in 1986-1993 by Gymnodinium catenatum in Tasmania were effectively mitigated by closing the affected area for shellfish farming, while a bloom by this same species in 2000 in New Zealand caused significant economic damage from restrictions on the movement of greenshell mussel spat. The biggest biotoxin event in New Zealand was an unexpected outbreak of Neurotoxic Shellfish Poisoning (NSP) in 1993 in Hauraki Gulf (putatively due to Karenia cf. mikimotoi) with 180 reported cases of human poisonings as well as reports of respiratory irritation north of Auckland. Strikingly, NSP never recurred in New Zealand since and no NSP events have ever been reported in Australia. In New Zealand, Paralytic Shellfish Poisoning (PSP) was the predominant seafood toxin syndrome, while in Australia Ciguatera Fish Poisoning (CFP) was the major reported seafood toxin syndrome, while no CFP has been recorded from consumption of New Zealand fish. In Australia, Diarrhetic Shellfish Poisoning (DSP) illnesses were recorded from two related outbreaks in 1997/98 following consumption of beach harvested clams (pipis) from a previously non-monitored area, whereas in New Zealand limited DSP illnesses are known. No human illnesses from Amnesic Shellfish Poisoning (ASP) have been reported in either Australia or New Zealand. Selected examples of HABs appearing and disappearing (NSP in New Zealand, Alexandrium catenella in Tasmania), species expanding their ranges (Noctiluca, Gambierdiscus), and reputed ballast water introductions (Gymnodinium catenatum) are discussed. Eutrophication has rarely been invoked as a cause except for confined estuaries and fish ponds and estuarine cyanobacterial blooms. No trend in the number of HAEDAT events from 1985 to 2018 was discernible.

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.

Berdalet, Elisa ... et al.-- 23 pages, 3 figures, The idea for establishment of the GlobalHAB Programme arose at the final Synthesis Open Science Meeting (OSM) of the Global Ecology and Oceanography of Harmful Algal Blooms (GEOHAB) Programme in Paris, in April 2013 (GEOHAB 2014). Participants at the meeting evaluated and synthesized the outcomes of GEOHAB and agreed that international coordination of HAB science was still needed to increase understanding of these events and how to mitigate their impacts on humans and aquatic ecosystems. In 2015, the GlobalHAB Programme was formally adopted by the two cosponsors, the Intergovernmental Oceanographic Commission (IOC) of UNESCO [through the Intergovernmental Panel on Harmful Algal Blooms (IPHAB)] and the Scientific Committee on Oceanic Research (SCOR), and was also endorsed by the International Atomic Energy Agency (IAEA). IOC and SCOR appointed a Scientific Steering Committee (SSC) for the new GlobalHAB Programme in 2015, which met for the first time in March 2016 to initiate the development of the GlobalHAB Science and Implementation Plan that is summarized in this chapter. [...]

Berdalet, Elisa ... et al.-- Special issue on International Cooperation in Harmful Algal Bloom Science.-- 12 pages, 6 figures, From 1998 to 2013, the international community of scientists researching harmful algal blooms (HABs) in marine systems worked through the Intergovernmental Oceanographic Commission (IOC) of UNESCO and the Scientific Committee on Oceanic Research (SCOR) to better understand the ecological and oceanographic controls on these natural events that cause harm to humans and ecosystems. During this period, IOC and SCOR cosponsored the Global Ecology and Oceanography of Harmful Algal Blooms (GEOHAB) program to facilitate progress in HAB research, observations, and modeling. In 2016, building on the foundation provided by GEOHAB, IOC and SCOR launched a new HAB project design to extend research into freshwater systems and address several topics related to the effects of HABs on human society now and in a rapidly changing world, The GlobalHAB Scientific Steering Committee acknowledges the financial support provided by IOC and by SCOR through Grant OCE-1546580 from the US National Science Foundation, and from ICES, IPHAB, ISSHA, PICES, and the Scottish Association of Marine Science

Characterization of the major proteins in the crystalline styles of Perna canaliculus and Mytilus galloprovincialis was carried out to attempt to identify the molecular basis of differences in the physical structure of the styles and their biochemical significance. The bivalve P. canaliculus has a crystalline style that is typical of those with permanent hard-type styles, whereas the style of M. galloprovincialis is typical of bivalves with transient soft-type styles. The consistency of the styles is clearly related to their respective moisture content, and both style types contain a suite of high-molecular weight (>800 kDa and ∼95–170 kDa) glycolproteins and medium-molecular weight (40–50 kDa) protein duplexes. Total carbohydrates (as determined by the phenol-sulfuric acid method) make up a small proportion of the mass of the styles. It is suggested that these 40–50 kDa proteins play a role in the formation of the protein gel that establishes the structural integrity of the style. Their solubili...

Assays of chitinase, cellulase, and amylase activities in the crystalline styles of selected fast- and slow-growing families of the mussel Perna canaliculus were carried out on 2 occasions over 6 mo, during which time the shellfish grew in a normal mussel cultivation situation. The data did not support the hypothesis that differences in growth rate by these families could be related to differences in the expression of digestive enzymes in the styles, leading to improved nutritional efficiency by faster growing families. On the contrary, the styles of faster growing families in general exhibited lower rates of enzyme activity. Differences in enzyme activities between families appeared to be related to the size of the individuals rather than their genetic heritage. The crystalline styles of larger, more mature shellfish contained less moisture and less soluble protein, and the lower rates of enzyme activity in larger individuals may be indicative of a reallocation of style protein synthesis toward higher levels of insoluble structural proteins.

Specimens of Munida gregaria were collected within and in the vicinity of a bloom of the toxic dinoflagellate Alexandrium catenella in Queen Charlotte Sound, New Zealand. The crustacean contained paralytic shellfish toxin (PST) with an analogue profile dominated by N-sulfocarbamoyl analogues (C1,2 and GTX5) and carbamate gonyautoxins (GTX1,4), similar to that of the dinoflagellate. A feeding experiment showed that M. gregaria is capable of actively grazing on A. catenella and it may play a role in controlling population growth of the dinoflagellate. This is the first account of the accumulation of PST by M. gregaria. When it is periodically abundant, M. gregaria is an important food item for fish, birds and other marine fauna and they are a vector by which PST may be transferred to higher trophic levels.

New Zealand's reputation as a supplier of high quality food products is vital to the national economy; international consumers are acutely aware of food safety issues and markets are increasingly demanding higher standards. Filter feeding bivalves are particularly sensitive to the nature of the environment in which they are grown, and quality assurance is a major preoccupation of the shellfish aquaculture industry. With the exception of a couple of incidents, most notably the Gymnodinium catenatum blooms in 2000–2003, paralytic shellfish toxin (PST) contamination has, to date, not had an important effect on the economics and sustainability of the industry. However, the dinoflagellate species responsible for producing these toxins are not uncommon in New Zealand coastal phytoplankton communities, and it is important that awareness of the potential risk is maintained. This review summarises what we know about the causes and incidence of PST contamination from research and monitoring over the last 20 years, ...

Blooms of the dinoflagellate Dinophysis acuminata occur every year in an important mussel cultivation area in Port Underwood, Marlborough Sounds, New Zealand. Annual maximum cell numbers range from 1500&ndash, 75,000 cells L&minus, 1 and over 25 years of weekly monitoring the D. acuminata bloom has never failed to exhibit peaks in abundance at some time between spring and autumn. During winter (June&ndash, August) the dinoflagellate is often undetectable, or at low levels (&le, 100 cells L&minus, 1), and the risk of diarrhetic shellfish poisoning (DSP)-toxin contamination over this period is negligible. Bloom occurrence may be coupled to the abundance of D. acuminata prey (Mesodinium sp.) but the mechanism by which it maintains its long-term residence in this hydrologically dynamic environment is unknown. The toxin profile of D. acuminata is dominated by pectenotoxin-2 (PTX-2) and dinophysistoxin-1 (DTX-1), but the cellular toxin content is low. It is rare that free DTX-1 is detected in mussels as this is invariably exclusively present as fatty acid-esters. In only five out of &gt, 2500 mussel samples over 16 years have the levels of total DTX-1 marginally exceeded the regulated level of 0.16 mg kg&minus, 1. It is also rare that free PTX-2 is detected in mussels, as it is generally only present in its hydrolysed non-toxic PTX-2 seco acid form. The D. acuminata alert level of 1000 cells L&minus, 1 is often exceeded without DTX-1 residues increasing appreciably, and this level is considered too conservative.

In 2010 the Cawthron Institute adopted AOAC official method 2005.06 (Lawrence method) for regulatory testing of paralytic shellfish toxins. This included adapting the method to a UPLC format and developing a rapid periodate screen to eliminate the vast majority of samples with no PSTs present. The method gained New Zealand regulatory approval and has since been used to test >2000 samples. Soon after implementation a major HAB of the toxic dinoflagellate Alexandrium catenella occurred in a prime shellfish growing area of New Zealand. This event was the most serious to date in this country with extremely high cell concentrations observed in some locations (>4 × 106 cells L−1). Toxin levels observed in Greenshell™ mussels (Perna canaliculus) and Flat oysters (Ostrea chilensis) exceeded the regulatory level of 0.8 mg/kg shellfish meat as saxitoxin equivalents. Closures of commercial shellfish harvesting areas were enforced for a period of up to three months as toxin levels remained above the regulatory level for an extended period, even after the bloom had crashed. Analysis of several hundred positive shellfish samples during this event allowed us to better understand the technical performance of the method during a bloom event. The periodate screen substantially overestimated the true PST level in the samples because several PSTs gave co-eluting oxidation products, and it was assumed that the entire peak was due to the presence of the more toxic congener. The ratio between the screen and confirmation test results remained relatively constant throughout the bloom events. This information supports an amendment to the overly conservative regulatory control scheme employed in New Zealand for PST testing. Despite overestimation, the periodate screen has proved highly useful as it allows a quick determination of PST-free samples and provides a high level of security against harvesting contaminated products.

An enzyme capable of hydrolysing pectenotoxins (PTXs) and okadaic acid (OA) esters within the hepatopancreas of the Greenshell™ mussel Perna canaliculus was isolated and characterized. The enzyme was purified by sequential polyethylene glycol fractionation, anion exchange, hydrophobic interaction, gel filtration and hydroxyapatite chromatography. The enzyme was an acidic (pI ∼ 4.8), monomeric, 67 kDa, serine esterase with optimum activity at pH 8.0 and 25 °C. PTX2 and PTX1 were hydrolysed but the enzyme was inactive against PTX11, PTX6 and acid isomerised PTX2 and PTX11. PTX11 and PTX2b competitively inhibited PTX2 hydrolysis. The enzyme also hydrolysed short and medium chain length (C2-C10) 4-nitrophenyl-esters, okadaic acid C8-C10 diol esters and DTX1 7-O-palmitoyl ester (DTX3). MALDI-Tof MS/MS analysis showed that the enzyme had some homology with a juvenile hormone esterase from the Red Flour Beetle Tribolium castaneum, although BLAST searches of several data bases using de novo amino acid sequences failed to identify any homology with known proteins.

Over a 6 day period in mid winter (June) 2010 about 200 tonnes of Chinook salmon ( Oncorhynchus tshawytscha ) on a sea-cage farm at Ruakaka Bay, Queen Charlotte Sound died as a result of exposure to a bloom of the dictyochophyte Pseudochattonella verruculosa . The alga was isolated and its identity confirmed by morphology and large subunit ribosomal DNA sequencing. Cells densities of P. verruculosa over several tens of thousands of cells per litre were sufficient to cause fish mortalities. The bloom originated in the inland reaches of the sound in the Grove Arm and the farm was located near its seaward extension. Fish were affected and recovered as the bloom front moved on and off the farm site under the influence of tidal currents. Before the bloom reached its peak (at about 1.0 × 10 5 cells L −1 ) the farm was relocated to a site outside the affected area where surviving fish quickly recovered. The bloom took place when day length and seawater temperatures were near their annual minimum (9 h and 12 °C respectively), and after its peak it was rapidly displaced by the in situ development of a diatom bloom dominated by Skeletonema costatum . The P. verruculosa bloom arose after several weeks of persistent rainfall. This may have assisted in the development of a circulation pattern that led to the movement of increasingly buoyant, relatively warm, nutrient-enriched bottom water into the Grove Arm, shoaling to shallow depths at the head of the inlet. The high productivity of waters in this area at the time is believed to have been largely driven by this mechanism. Larger size (∼3.5 kg) fish were mainly killed by the bloom and examination of affected salmon tissues showed pathologies in the liver and kidneys. There are ambitious plans for the expansion of sea cage fish farming in New Zealand and this event is a timely reminder of the hazard these events pose to sea cage fish farming operations.

A bioreactor system for biotoxin production was appraised against traditional methods of growing dinoflagellate cultures. In an optimised bioreactor culture (5.4 L) operated in batch mode, growth of Karenia selliformis was more efficient than in 15-L bulk carboy culture in terms of growth rate (μ = 0.07 day−1 versus 0.05 day−1) and growth maximum (G max, 169.106 versus 41.106 cells L−1). Maximal gymnodimine concentration (1200 μg L−1) in bioreactor culture was 8-fold higher than in bulk carboy culture, and the yield per cell (pg cell−1) was 2-fold higher. Similarly the bioreactor batch culture of Alexandrium ostenfeldii performed more efficiently than carboy cultures in terms of growth rate (1.6-fold higher), growth maximum (15-fold higher) and desmethyl C spirolide (SPX-desMe-C) yield (5-fold higher [μg L−1], though the yield [pg cell−1basis] was lower). When bioreactor cultures of K. selliformis were operated in continuous mode, the yield of gymnodimine was substantially higher than a carboy or the bioreactor run in batch mode to growth max (793 μg day−1 over 58 days in continuous culture was achieved versus an average of 60 μg day−1 [carboy over 40 days] or 249 μg day−1 [batch mode] over 26 days). Likewise in continuous bioreactor cultures of A. ostenfeldii run over 25 days, the yield of SPX-desMe-C (29 μg day−1) was substantially higher than in same cultures run in batch mode or carboys (10.2 day−1 and 7.7 μg day−1 respectively). Similarly 5.4 L bioreactor batch cultures of K. brevisulcata reached 3.8-fold higher cell densities than carboy cultures, and when operated in continuous mode, the brevisulcatic acids were more efficiently produced than in batch culture (12 μg day−1 versus 7 μg day−1). When the bioreactor system was upscaled to 52 L, the maximum cell densities and toxin yields of K. brevisulcata cultures were somewhat less than those achieved in the smaller reactor, which was attributed to reduced light penetration.

The analysis of a variety of environmental samples and the installation of passive solid phase adsorption devices (SPATT) in two warm-temperate estuaries (Rangaunu and Parengarenga Harbours), during consecutive summers (2009 and 2010), revealed the pervasive influence of bioactive polyether compounds secreted by benthic dinoflagellates within the mangrove and sea-grass habitats. Pinnatoxin (PnTx) analogues PnTx-E and PnTx-F and okadaic acid (OA) and its esters were the most abundant, though traces of other polyether compounds (dinophysistoxins, pectenotoxin, spirolides), were also detected. In sediments, algal mats and micro-algal films, the parent compound PnTx-F was the predominate analogue. In bivalves and gastropods PnTx-E and PnTx-F were either present in equivalent amounts or there was a predominance of the former, indicative of in vivo metabolism. Esterified OA was the predominant form of this toxin in the deposit feeding sea hare Bursatella leachii, however OA-toxins were only about 14% as abundant as the combined PnTx analogues in this animal. Conversely levels of PnTxs accumulated within the SPATT bags were only 50% of total-OA, about 10% of which was in the form of OA-esters. Neither OA nor OA-esters were observed in the oyster Crassotrea gigas. The levels of total-PnTxs in C. gigas were about 8% of that observed in B. leachii (200 and 2580 μg/kg respectively). Although there was abundant OA in this environment, cultured oysters did not incorporate this toxin presumably because they were not exposed to Prorocentrum lima cells in the water column or were unable to digest these cells. The low levels of pinnatoxins sequestered by oysters in these estuaries, despite persistent and very abundant populations of the causative dinoflagellate, is probably also due to the general inaccessibility of these cells to the shellfish and therefore there appears to be a low risk to human consumers

Sampling and monitoring for cyanotoxins can be problematic as concentrations change with environmental and hydrological conditions. Current sampling practices (e.g. grab samples) provide data on cyanotoxins present only at one point in time and may miss areas or times of highest risk. Recent research has identified the widespread distribution of anatoxin-producing benthic cyanobacteria in rivers highlighting the need for development of effective sampling techniques. In this study we evaluated the potential of an in situ method known as solid phase adsorption toxin tracking (SPATT) for collecting and concentrating anatoxin-a (ATX) and homoanatoxin-a (HTX) in river water. Fifteen different adsorption substrates were screened for efficiency of ATX uptake, nine of which retained high proportions (>70%) of ATX. Four substrates were then selected for a 24-h trial in a SPATT bag format in the laboratory. The greatest decrease in ATX in the water was observed with powdered activated carbon (PAC) and Strata-X (a polymeric resin) SPATT bags. A 3-d field study in a river containing toxic benthic cyanobacterial mats was undertaken using PAC and Strata-X SPATT bags. ATX and HTX were detected in all SPATT bags. Surface grab samples were taken throughout the field study and ATX and HTX were only detected in one of the water samples, highlighting the limitations of this currently used method. Both Strata-X and PAC were found to be effective absorbent substrates. PAC has the advantage that it is cheap and readily available and appears to continue to sorb toxins over longer periods than Strata-X. SPATT has the potential to be integrated into current cyanobacterial monitoring programmes and would be a very useful and economical tool for early warning of ATX and HTX contamination in water.

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.

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 , 0.05 days −1 ; gymnodimine 250 μg L −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 × 10 5 cells mL −1 ; gymnodimine, 1260 μg L −1 ; growth rate ( μ ), 0.2 days −1 ) compared to the control (growth maximum cell yield, 7.8 × 10 4 cells mL −1 ; gymnodimine, 780 μg L −1 ; μ , 0.17 days −1 ). Acetate enhanced gymnodimine by stimulating growth rate ( μ , 0.23 days −1 ) and the large concentration of gymnodimine per cell (16 pg cell −1 cf. 9.8 pg cell −1 for the control) suggests a role for this compound in gymnodimine biosynthesis. Amending culture media with Mn 2+ 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.

Yessotoxin 32-O-[beta-L-arabinofuranosyl-(5'--1'')-beta-L-arabinofuranoside] (3) was isolated from extracts of Protoceratium reticulatum during a large scale isolation of yessotoxin (1). The structure was characterized by mass spectrometry and NMR spectroscopy. Di-glycoside-3, along with the corresponding mono-glycoside (2) were detected in cultures of P. reticulatum originating from Europe and New Zealand, suggesting that production of arabinosides of 1 is a normal feature of this alga. Formation of multiply charged anions and fragmentation of 3 occurred much more readily than for 1 and 2 under the LC-MS conditions used in this study.

44,55-Dihydroxyyessotoxin (1) was isolated from extracts of Protoceratium reticulatum and identified by analysis of its one- and two-dimensional NMR and mass spectra. In addition, LC–MS methods revealed the presence of compounds tentatively identified as (44-R,S)-44,55-dihydroxy-41a-homoyessotoxin (2) and (44-R,S)-44,55-dihydroxy-9-methyl-41a-homoyessotoxin (3). LC–MS analyses indicate that 1 is a constituent of P. reticulatum in New Zealand and Norway, and it was present in three species of mussels from New Zealand, Norway, and Canada.

New Zealand has been subject to massive blooms of gymnodinoid dinoflagellates over the last decade and in some instances marine biota mortalities. Respiratory problems for people impacted by aerosols from these blooms have occurred. However, because of the difficulty in definitively identifying gymnodinoid dinoflagellates, the identities of the causative organisms have not always been established with certainty. This paper documents the occurrence of several newly described species in the genera Karenia and Takayama in New Zealand's coastal waters as determined by analysis of DNA sequence data. The species include Karenia umbella de Salas, Bolch & Hallegraeff, Takayama helix de Salas, Bolch, Botes & Hallegraeff, and T. tasmanica de Salas, Bolch & Hallegraeff, which are known to have caused fish kills in Tasmanian (Australia) waters in the past. Liquid chromatography‐mass spectrometry analyses of mass cultures of these dinoflagellates have been negative for neurotoxic brevetoxins. The species desi...

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).

A simple and sensitive in situ method for monitoring the occurrence of toxic algal blooms and shellfish contamination events has been developed. The technique involves the passive adsorption of biotoxins onto porous synthetic resin filled sachets (SPATT bags) and their subsequent extraction and analysis. The success of the method is founded on the observation that during algal blooms significant amounts of toxin, including the low polarity lipophilic compounds such as the pectenotoxins and the okadaic acid complex toxins, are dissolved in the seawater. The results of field trials during Dinophysis acuminata and Protoceratium reticulatum blooms are presented. These data prove the concept and demonstrate that the technique provides a means of forecasting shellfish contamination events and predicting the net accumulation of polyether toxins by mussels. As an early warning method it has many advantages over current monitoring techniques such as shellfish-flesh testing and phytoplankton monitoring. In contrast to the circumstantial evidence provided by genetic probe technologies and conventional phytoplankton monitoring methods, it directly targets the toxic compounds of interest. The extracts that are obtained for analysis lack many of the extraneous lipophilic materials in crude shellfish extracts so that many of the matrix problems associated with chemical and biological analysis of these extracts are eliminated. Analyses can confidently target parent compounds only, because analytical and toxicological uncertainties associated with the multiplicity of toxin analogues produced by in vivo biotransformation in shellfish tissues are reduced. Time integrated sampling provides a good simulation of biotoxin accumulation in filter feeders and the high sensitivity provides lengthy early warning and conservative estimates of contamination potential. The technique may reduce monitoring costs and provide improved spatial and temporal sampling opportunities. When coupled with appropriate analytical techniques (e.g. LC-MS/MS multi-toxin screens, ELISA assays, receptor binding assays), the technique has the potential to offer a universal early warning method for marine and freshwater micro-algae toxins.

To improve understanding of the temporal and spatial variability of biological productivity relevant to fisheries enhancement within Tasman Bay, New Zealand, it is necessary to evaluate the importance of variations in river inflows on inorganic nutrient dynamics. To achieve this, a series of synoptic surveys of surface and water column salinity fields and inorganic nutrient concentrations were carried out over a 1‐year period, at seasonally representative times, during, and soon after, a range of flow regimes in the major freshwater tributaries of Tasman Bay. An assessment was made of the relative importance of external (i.e., river borne) and internal (i.e., recycling) nutrient sources that control the fertility of the Tasman Bay water column. Flood events in the bay's catchment cause significant changes in the levels of dissolved nitrogen, phosphorus, and silica in surface waters, and on an annual basis 60–70% of the input of “new” dissolved inorganic nitrogen from freshwater tributaries is con...

Temporal changes in the abundance and horizontal and vertical distribution of phytoplankton biomass (chlorophyll a) in Tasman Bay, New Zealand were associated with changes in water column stratification, because of seasonal climatic effects and variations in the magnitude of freshwater inflows. The water column throughout the bay was usually strongly stratified. Vertical gradients in salinity, the result of outwelling river plumes overlying deeper more saline waters, usually played the major role in stabilising the water column. However, during summer low river‐flow periods, temperature stratification was also important. A persistent east to west gradient in Simpson's potential energy anomaly (an index of the strength of stratification) existed across the bay at most times, because of the dominant influence of the Motueka River plume. From late spring to late summer most of the nutrient depleted euphotic zone contained low chlorophyll a (Chl. a) concentrations while below the base of the pycnocli...

The 1,3-enone isomer ( 1 ) of heptanor-41-oxoyessotoxin ( 2 ) was isolated from extracts of Protoceratium reticulatum during large-scale production of yessotoxin ( 4 ). We found that 2 readily isomerizes to 1 in the presence of dilute ammonia and present evidence for the existence of 40- epi - 2 ( 3 ) that also isomerizes to 1 . 1 – 3 were detected by LC–MS methods both in extracts of P. reticulatum cultures and in mussels contaminated with yessotoxins. The isomerization of 2 and 3 into 1 occurs so readily that purification on basic alumina needs to be conducted carefully. No toxic effects were recorded in mice injected intraperitoneally with 1 at a dose of 5000 μg/kg.

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.

A new species, Alexandrium camurascutulum sp. nov. MacKenzie et Todd, is described from specimens collected from Tasman Bay and the Marlborough Sounds New Zealand. These small (26–28 μm long × 21–24 μm wide) cells can be discriminated from other species in the Alexandrium minutum group by three distinctive morphological features. The sixth pre-cingular plate (6′′) is up to 1.6 times wider than high and the left side of the plate is concave resulting in a markedly ‘hooked’ appearance. In all specimens observed, the first apical plate (1′) does not directly connect with the apical pore plate (Po) and the posterior sulcal plate (S.p.) is markedly different from the usual A. minutum form and may contain a posterior attachment pore (pap) connected to the right side plate margin. The cells may or may not have an anterior attachment pore (aap) in the apical pore plate (Po). The cells display a prominent list along the left sulcal margin and the thecal surface is perforated with numerous areolated pores. A. camurascutulum sp. nov. has been observed occasionally over a number of years in coastal waters of the northern South Island of New Zealand. There is circumstantial evidence that suggests it is not toxic.

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.

SUMMARY: Comparison of pectenotoxin (PTX) profiles between the toxic dinoflagellate Dinophysis acuta and scallops Pecten novaezelandiae collected at Wedge Point, Queen Charlotte Sound, New Zealand was carried out by liquid chromatography–mass spectrometry (LC-MS) with turbo-ionspray ionization. Although the major PTX homolog in D. acuta was pectenotoxin-2 (PTX2), the scallops contained pectenotoxin-2 seco acid (PTX2SA) as the predominant toxin. Pectenotoxin-2 isolated from D. acuta was rapidly converted to PTX2SA and its epimer 7-epi-pectenotoxin-2 seco acid (7-epi-PTX2SA) in the scallop extracts. These results indicate that PTX2SA and 7-epi-PTX2SA arose from the conversion of PTX2 by scallop tissue. The results indicate that New Zealand scallops have an ability to reduce the cytotoxicity of PTX2 by conversion to PTX2SA.

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.

In this study the cross‐reactivity of an antibody raised against cell surface antigens of Alexandrium minutum Halim, a dinoflagellate isolated from the Bay of Plenty, New Zealand, during the 1993 toxic shellfish outbreaks, was tested on other strains of the same species isolated from a variety of locations, as well as a wide range of unrelated phytoplankton using an indirect immunofluorescence staining technique. The antibody showed positive reactions to all four isolates of A. minutum from different localities in New Zealand and one from South Australia. The antibody also showed a positive, but weaker, reaction to A. ostenfeldii (two isolates) from New Zealand but no reaction with A. lusitanicum, a species which is morphologically similar to A. minutum. No cross‐reactivity was observed with 51 species or strains from 10 different algal classes tested. The antibody bound to cells of A. minutum preserved with either 2.5% glutaraldehyde or 3.5% formaldehyde. Recognition of cells in stationary growt...

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. Nat. Toxins 5:164–167, 1997. © 1997 Wiley-Liss, Inc.

With the growth of aquaculture, toxic phytoplankton have become a significant problem. At present, the identification of specific algal species within blooms requires considerable expertise, and the methods are both costly and time consuming. It is clear that alternative diagnostic tests must be developed that allow rapid and simple assessments to be made on site. We report on the identification of unique sequences within the SSU and LSU rDNA genes of Alexandrium species that provide data for the design of species- and genus-specific molecular probes. In addition we examine some aspects of the phylogeny of Alexandrium isolates from New Zealand waters.

Harmful algal bloom (HAB) research and monitoring has traditionally been based on ecological and microbiological measurements which are laborious, time‐consuming, and reliant on experienced operators. Recent developments in oligonucleotide probe technology and immunofluorescence research have revealed several potential applications and techniques that may be transposable to laboratory and field‐based monitoring and research. Field trials are currently underway for fluorescent in situ hybridisation and sandwich hybridisation assays. The former is particularly suited for laboratory‐based research on harmful algal bloom (HAB) population dynamics and structure, whereas the sandwich hybridisation assays based on a portable robotics workstation, offers the potential of quick and reliable laboratory and possibly field‐based screening for HAB species. Initial development is underway for molecular beacons and the Qs replicase detection system, both offer the potential of simple and cost effective strategi...

Thecal plate morphology and paralytic shellfish poisoning (PSP) toxin spectra produced by three strains of a small Alexandrium species isolated from the Marlborough Sounds, New Zealand were examined. Although these cells displayed minor morphological features described in the literature as characteristic of both A. minutum and A. lusitanicum, the former designation was adopted. High performance liquid chromatography (HPLC) analysis of culture extracts showed the toxin profiles to consist predominantly of various proportions of the carbamate toxins GTX1, GTX2, GTX4, neoSTX, and STX. These toxin profiles were similar to those observed in other New Zealand isolates of A. minutum. They were however rather different from those observed in this species elsewhere in the world though the specific toxicity of these cells (1.8–2.2 pg STX equivalents per cell) was similarly low.

We report on the identification of unique sequences within the highly variable rDNA ITS regions and more conserved 5.8S regions which provide data for the design of species- and genus-specific probes, and we examine some aspects of the phylogeny of Alexandrium isolates from New Zealand waters.

The systematic relationships of the Class Raphidophyceae within the chromophyte algae are uncertain. The ‘V4’ domain small-subunit rDNA gene (‘V4’ domain SSU rDNA gene) was sequenced for the taxa Heterosigma carterae and Chattonella antiqua to provide characters for phylogenetic analyses. These analyses showed that the Classes Raphidophyceae and Eustigamatophyceae form a sister clade. A close systematic relationship for these two classes has not been shown before. Numerous phylogentic analyses in this study have failed to provide resolution of the deep branches of the five major evolutionary assemblages—Classes Raphidophyceae and Eustigamatophyceae; Classes Synurophyceae and Chrysophyceae; Classes Phaeophyceae and Xanthophyceae; Class Bacillariophyceae; Class Prymnesiophyceae. Transition frequency analysis shows that there is a major loss of phylogenetic signal within the chromophyte algae‘V4’ domain SSU rDNA gene sequences.

Examination of dinoflagellate cysts in sediments around the New Zealand coast revealed that a previously undescribed resting cyst (hypnozygote) of Alexandrium ostenfeldii (Paulsen) Balech et Tangen...