Paralytic shellfish toxins or spirolides? The role of environmental and genetic factors in toxin production of the Alexandrium ostenfeldii complex

Dinoflagellates of the Alexandrium ostenfeldii complex (A. ostenfeldii, A. peruvianum) are capable of producing different types of neurotoxins: paralytic shellfish toxins (PSTs), spirolides and gymnodimines, depending on the strain and its geographic origin. While Atlantic and Mediterranean strains have been reported to produce spirolides, strains originating from the brackish Baltic Sea produce PSTs. Some North Sea, USA and New Zealand strains contain both toxins. Causes for such intraspecific variability in toxin production are unknown. We investigated whether salinity affects toxin production and growth rate of 5 A. ostenfeldii/peruvianum strains with brackish water (Baltic Sea) or oceanic (NE Atlantic) origin. The strains were grown until stationary phase at 7 salinities (6–35), and their growth and toxin production was monitored. Presence of saxitoxin (STX) genes (sxtA1 and sxtA4 motifs) in each strain was also analyzed. Salinity significantly affected both growth rate and toxicity of the individual strains but did not change their major toxin profile. The two Baltic Sea strains exhibited growth at salinities 6–25 and consistently produced gonyautoxin (GTX) 2, GTX3 and STX. The two North Sea strains grew at salinities 20–35 and produced mainly 20-methyl spirolide G (20mG), whereas the strain originating from the northern coast of Ireland was able to grow at salinities 15–35, only producing 13-desmethyl spirolide C (13dmC). The effects of salinity on total cellular toxin concentration and distribution of toxin analogs were strain-specific. Both saxitoxin gene motifs were present in the Baltic Sea strains, whereas the 2 North Sea strains lacked sxtA4, and the Irish strain lacked both motifs. Thus sxtA4 only seems to be specific for PST producing strains. The results show that toxin profiles of A. ostenfeldii/peruvianum strains are predetermined and the production of either spirolides or PSTs cannot be induced by salinity changes. However, changes in salinity may lead to changed growth rates, total cellular toxin concentrations as well as relative distribution of the different PST and spirolide analogs, thus affecting the actual toxicity of A. ostenfeldii/peruvianum populations.     

Characterization of saxitoxin production and release and phylogeny of sxt genes in paralytic shellfish poisoning toxin-producing Aphanizomenon gracile

The freshwater cyanobacterium Aphanizomenon gracile is one of the most widely distributed producers of the potent neurotoxins saxitoxin (STX) and its derivatives (paralytic shellfish poisoning toxins, PSP toxins). However, the phylogeny of STX biosynthesis genes and the regulation of STX production and release remain poorly studied in the genus Aphanizomenon. In this study, two A. gracile strains from Spanish freshwaters were grown in semi-continuous cultures under three temperatures (15, 20 and 28 °C) and their STX production and release were determined by Enzyme-Linked ImmunoSorbent Assay (ELISA). STX production was stable along the temperature range, with 1.4–2.3-fold shifts in biomass-standardized STX contents, and maxima of 0.22 μg equivalent STX mg⁻¹ dry weight 15.3 fg equiv STX cell⁻¹ and 15.1 μg equiv STX mg⁻¹ Chl a. The extracellular fraction was remarkably high (13.6–35.3%), not clearly affected by temperature but with nitrate-depleted medium (BG11₀) inducing a 2-fold increase in extracellular content. STX production and release were not directly related to growth rates. The 16S rRNA phylogenetic analyses in sixteen A. gracile strains from Spanish and German freshwaters showed that PSP-producing A. gracile grouped within a monospecific and highly supported cluster, together with PSP-producing Aphanizomenon sp. NH-5 and clearly separated from a monospecific Aphanizomenon flos-aquae cluster. The sixteen A. gracile strains formed also monospecific and highly supported clusters for PSP-biosynthesis genes (sxtG, sxtI, sxtH and sxtX) together with Aphanizomenon sp. NH-5. This study evidences an elevated extracellular proportion of STX in A. gracile with importance for risk assessment, and supports the identification of Aphanizomenon sp. NH-5 as A. gracile.     

Development and assessment of radioreceptor binding assays for the detection of saxitoxin binding proteins in biological extracts

Several radioreceptor assays using tritiated saxitoxin ([(3)H]STX) were developed to identify a suitable primary screening method for the detection and characterization of soluble saxitoxin binding proteins from biological extracts. Assays using anion and cation exchange, protein binding, and traditional charcoal radioreceptor methods were compared with two previously reported formats. A protein binding assay incorporating filters of mixed cellulose esters (MCE) outperformed all other assay strategies with maximal signal, low background, exceptional reproducibility, minimal matrix effects, and high throughput. Binding site titrations verified that an increase in total protein in the assay led to a concomitant linear increase in the amount of specifically bound [(3)H]STX within the range of 1-90microg total protein. Saturation binding experiments demonstrated that the binding sites were saturable and that nonspecific binding was linear. The MCE assay was unaffected by 600mM NaCl and 500mM KCl. Likewise, minimal variation of specific binding was observed between pH 5 and pH 9, but inhibition was observed below pH 5.     

Use of two-dimensional gel electrophoresis to differentiate morphospecies of Alexandrium minutum, a paralytic shellfish poisoning toxin-producing dinoflagellate of harmful algal blooms

Contamination of shellfish with paralytic shellfish poisoning toxins (PST) produced by toxic harmful algal blooms (HABs) have been negatively affecting the shellfish and aquaculture industries worldwide. Therefore, accurate and early identification of toxic phytoplankton species is crucial in HABs surveillance programs that allow fish-farmers to take appropriate preventive measures in shellfish harvesting and other aquaculture activities to overcome the negative impacts of HABs on human health. The identification of toxic dinoflagellates present in the water is currently a time-consuming operation since it requires skillful taxonomists and toxicologists equipped with optical and scanning electron microscopes as well as sophisticated equipment, for example, high-performance liquid chromotography-fluorescence detection. In this paper, a two-dimensional gel electrophoresis (2-DE)-based proteomic approach was applied to discriminate between toxic and nontoxic strains of Alexandrium minutum. Variation in morphological features between toxic and nontoxic strains was minimal and not significant. Also, variation in 2-DE protein patterns within either toxic or nontoxic strains was low, but pronounced differences were detected between toxic and nontoxic strains. The most notable differences between these strains were several abundant proteins with pIs ranging from 4.8 to 5.3 and apparent molecular masses between 17.5 and 21.5 kDa. Groups of proteins, namely NT1, NT2, NT3, and NT4, were consistently found in all nontoxic strains, while T1 and T2 were prominent in the toxic strains. These specific protein spots characteristic for toxic and nontoxic strains remained clearly distinguishable irrespective of the various growth conditions tested. Therefore, they have the potential to serve as "taxonomic markers" to distinguish toxic and nontoxic strains within A. minutum. Initial studies revealed that the expression pattern of T1 was tightly correlated to toxin biosynthesis in the examined alga and may be used to serve as a potential toxin indicator.     

The presence of 12β-deoxydecarbamoylsaxitoxin in the Japanese toxic dinoflagellate Alexandrium determined by simultaneous analysis for paralytic shellfish toxins using HILIC-LC–MS/MS

A differential screening study using high-resolution (HR)-hydrophilic interaction chromatography (HILIC)-electrospray ionization (ESI)–quadrupole time-of-flight mass spectrometry (Q-TOF MS) was conducted to identify saxitoxin (STX) analogues in the marine dinoflagellate toxic sub-clone Alexandrium tamarense Axat-2 and the non-toxic sub-clone UAT-014-009 derived from the same Japanese isolate. One unknown compound was identified only in the toxic sub-clone and was found to have the molecular formula C₉H₁₆N₆O₂. This structure differed from that of decarbamoyl STX (dcSTX; C₉H₁₆N₆O₃) by the loss of a single oxygen. A 12-deoxy-dcSTX standard (a mixture of 12α- and β-deoxy-dcSTX) was chemically prepared from dcSTX by reduction with sodium borohydride. The unknown compound in the toxic strain of A. tamarense was identified as 12β-deoxy-dcSTX by comparison of its HR-HILIC-LC–MS retention time and HR–MS/MS spectrum with those of the chemically prepared standard, and the identification was confirmed by high-sensitivity HPLC analysis with post-column fluorescent derivatization. Moreover, two Japanese isolates of A. catenella showing toxin profiles different from that of A. tamarense were also found to contain 12β-deoxy-dcSTX. Previously, 12β-deoxy-dcSTX was isolated from the freshwater cyanobacterium Lyngbya wollei, which produces a unique set of STX analogues. This study is the first evidence of the presence of 12β-deoxy-dcSTX in marine dinoflagellates.     

Single-cell analysis of paralytic shellfish toxins in Alexandrium tamarense by HPLC with post-column fluorescent derivatization

We developed a methodology for analyzing the C-toxin (C2) content in single Alexandrium tamarense cells; this method was based on high performance liquid chromatography (HPLC). C2 is the main paralytic shellfish toxin (PST) detected in a clonal culture of A. tamarense, which is a common causative organism in cases of paralytic shellfish poisoning in Japan. This HPLC method employs post-column fluorescent derivatization (FL). Mobile phase, column size, flow rate, reagent concentrations, and lamp type for the fluorescent detector were all optimized for the detection of C2. With this improved methodology, we could measure 1 fmol of C2 with a signal to noise ratio (S/N) = 2. Clonal heterogeneity within the toxic strain, which was maintained for 13 years after re-isolation from the original clonal culture, ranged from <1 fmol to 700 fmol cell⁻¹. This report is the first to demonstrate definitively that PST content varies on a cell-by-cell basis in a clonal culture of a dinoflagellate that causes paralytic shellfish poisoning.     

Growth of the toxic dinoflagellate Alexandrium minutum (Dinophyceae) using high biomass culture systems

Toxic dinoflagellates are important in natural ecosystems and are ofglobal economic significance because of the impact of toxic blooms onaquaculture and human health. Both the organisms and the toxins they producehave potential for biotechnology applications. We investigated autotrophicgrowth of a toxic dinoflagellate, Alexandrium minutum, inthree different high biomass culture systems, assessing growth, productivityandtoxin production. The systems used were: aerated and non-aerated2-L Erlenmeyer flasks; 0.5-L glass aerated tubes; anda 4-L laboratory scale alveolar panel photobioreactor. A range ofindicators was used to assess growth in these systems. Alexandriumminutum grew well in all culture conditions investigated, with amarked increase in both biomass and productivity in response to aeration. Thehighest cell concentration (4.9 × 10⁵ cellsmL^–1) and productivity (2.6 ×10⁴cells mL^–1d^–1) was achieved inthe aerated glass culture tubes. Stable growth of A.minutum in the laboratory scale alveolar panel photobioreactor wasmaintained over a period of five months, with a maximum cell concentration of3.3 × 10⁵ cells mL^–1, a meanproductivity of 1.4 × 10⁴ cells mL^–1d^–1, and toxin production of approximately 20g L^–1 d^–1 with weeklyharvesting.     

Bioactive compounds of marine dinoflagellate isolates from western Greenland and their phylogenetic association within the genus Alexandrium

The diversity and biogeography of populations of the toxigenic marine dinoflagellate genus Alexandrium, a major global cause of paralytic shellfish poisoning (PSP), are represented by only a few studies based upon a low number of cultured isolates and remain poorly described in Arctic and sub-Arctic waters. Multiple clonal isolates (n = 22) of the Alexandrium tamarense species complex, and a single isolate of A. tamutum, were collected from the water column while on board an oceanographic expedition to the west coast of Greenland. After culturing of these isolates under controlled conditions, their phylogenetic affinities within the genus Alexandrium were characterized by sequence analysis of nuclear large sub-unit (LSU) rDNA. Based upon morphological and molecular genetic criteria, all isolates of the A. tamarense species complex were consistent with membership in the Group I ribotype (previously known as the North American ribotype). Phenotypic signatures were also analyzed based upon their respective profiles of paralytic shellfish toxins (PST) and allelochemical interactions against a target cryptophyte Rhodomonas, as determined by lytic potency. All isolates conforming to the A. tamarense Group I produced PST, but no toxins were detected in A. tamutum P2E2. Unusually, only carbamoyl toxins were produced among the A. tamarense Group I isolates from Greenland; sulfocarbamoyl derivatives, generally present in A. tamarense population from other locations, including the Arctic, North Pacific and North Atlantic, were absent from all isolates. Allelochemical activity, causing cell lysis of Rhodomonas, but generally being unrelated to cellular PST, was expressed by all A. tamarense isolates and also by A. tamutum, but varied widely in potency. Comparison of the genotypic (rDNA) and phenotypic (PST profile, allelochemical activity) characteristics of Greenland isolates with those of other Arctic populations reveals a complex pattern of intra-specific diversity. Estimation of diversity relationships is problematic because of the distinct patterns of divergence and lack of evidence of linkage among the alternative biomarkers and morphology. Nevertheless, such studies are necessary as the basis for constructing hindcasting scenarios and predicting changes in Alexandrium species distribution in the Arctic from the regional to the global scale.     

Alexandrium (Dinophyceae) species in Malaysian waters

A study was carried out to determine the presence of paralytic shellfish poisoning (PSP) toxin-producing dinoflagellates in the coastal waters of Peninsula Malaysia. This followed first ever occurrences of PSP in the Straits of Malacca and the northeast coast of the peninsula. The toxic tropical dinoflagellate Pyrodinium bahamense var. compressum was never encountered in any of the plankton samples. On the other hand, five species of Alexandrium were found. They were Alexandrium affine, Alexandrium leei, Alexandrium minutum, Alexandrium tamarense and Alexandrium tamiyavanichii. Not all species were present at all sites. A. tamiyavanichii was present only in the central to southern parts of the Straits of Malacca. A. tamarense was found in the northern part of the straits, while A. minutum was only found in samples from the northeast coast of the peninsula. A. leei and A. affine were found in both the north and south of the straits. Cultured isolates of A. minutum and A. tamiyavanichii were proven toxic by the receptor binding assay for PSP toxins but A. tamarense clones were not toxic. Mean toxin content for the A. tamiyavanichii and A. minutum clones were 26 and 15 fmol per cell STX equivalent, respectively. This study has provided evidence on the presence of PSP toxin-producing Alexandrium species in Malaysian waters which suggests that PSP could increase in importance in the future.     

Detection of cyanobacterial sxt genes and paralytic shellfish toxins in freshwater lakes and brackish waters on Åland Islands,Finland

Harmful cyanobacteria are a globally growing concern. They produce a large variety of toxic compounds, including saxitoxin and its many structural variants, a group of potent neurotoxins collectively called paralytic shellfish toxins or PST. Nucleic acid based detection methods, such as qPCR, have been proposed as potential screening and monitoring tools for toxic cyanobacteria, but it is not clear how well the presence and quantity of saxitoxin biosynthesis (sxt) genes can be used to predict the production of PST in the environment. In this study, the prevalence of three sxt genes and their co-occurrence with paralytic shellfish toxins in the environment was investigated. The sxtA, sxtG and sxtB genes were present on average in 31% of the samples collected from lakes and brackish coastal waters on Åland Islands, Finland, during the three-year monitoring period. PST detection frequency varied from 13% to 59% from year to year, and concentrations were generally low. On average higher sxtB copy numbers were associated with PST detection, and although a positive correlation between gene copy numbers and toxin concentrations was observed (Spearman rank correlation, ρ = 0.53, P = 0.012), sxt gene presence or quantity didn’t reliably predict PST production. Sequencing of sxtA fragments and identification of main cyanobacteria indicated that the likely candidate responsible for PST production in the samples belonged to the genus Anabaena.     

水华束丝藻NH-5株产生麻痹性贝毒毒素几个相关问题的研究

近年来,有害赤潮在我国沿海养殖区发生越来越频繁,对水产养殖业造成的经济损失越来越严重,并且对人类的健康构成了威胁。引起了我国重视,并开始了研究[1、2]。有害赤潮所产生的毒素中,麻痹性贝毒毒素在世界范围内分布最广,危害最大,引起人们加倍重视[3]。由于麻痹性贝毒毒素在国际间的传送受到了严格限制,而我国处于研究起始阶段,缺乏标准毒素,缺乏易于进行培养的赤潮产毒藻株,给研究工作带来了一定困难。一种能产生麻痹性贝毒毒素的淡水蓝藻水华束丝藻NH-5株受到了注意。