Saxitoxin Biosynthesis Research Articles

Biosynthesis of Saxitoxin in Marine Dinoflagellates: An Omics Perspective.

Saxitoxin is an alkaloid neurotoxin originally isolated from the clam Saxidomus giganteus in 1957. This group of neurotoxins is produced by several species of freshwater cyanobacteria and marine dinoflagellates. The saxitoxin biosynthesis pathway was described for the first time in the 1980s and, since then, it was studied in more than seven cyanobacterial genera, comprising 26 genes that form a cluster ranging from 25.7 kb to 35 kb in sequence length. Due to the complexity of the genomic landscape, saxitoxin biosynthesis in dinoflagellates remains unknown. In order to reveal and understand the dynamics of the activity in such impressive unicellular organisms with a complex genome, a strategy that can carefully engage them in a systems view is necessary. Advances in omics technology (the collective tools of biological sciences) facilitated high-throughput studies of the genome, transcriptome, proteome, and metabolome of dinoflagellates. The omics approach was utilized to address saxitoxin-producing dinoflagellates in response to environmental stresses to improve understanding of dinoflagellates gene–environment interactions. Therefore, in this review, the progress in understanding dinoflagellate saxitoxin biosynthesis using an omics approach is emphasized. Further potential applications of metabolomics and genomics to unravel novel insights into saxitoxin biosynthesis in dinoflagellates are also reviewed.

Transcriptomic Profile and Sexual Reproduction-Relevant Genes of Alexandrium minutum in Response to Nutritional Deficiency.

Alexandrium minutum is a typical marine toxic dinoflagellate responsible for producing paralytic shellfish poisoning (PSP) toxins. Until now, we know little about the genomic information of A. minutum, so a transcriptome study was conducted to clarify the physiological adaptations related to nutritional deficiency. Here, we performed RNA-Seq analysis to assess the gene expression patterns of A. minutum under N and P deficient conditions for 0 (control), 6, and 72 h. Main differences between the control and experimental groups were observed in hydrolase activity and fatty acid, lipid, protein, and P metabolism. Activities of photosystem I (PSI) and PSII were significantly down-regulated, and the endocytosis pathway (clathrin-dependent endocytosis) was significantly enriched under N and P stress compared with the control, indicating that A. minutum shifts its trophy pattern under N and P stress. We also identified several unigenes related to the process of sexual reproduction, including sex determination, sperm-egg recognition, sex differentiation, mating, and fertilization. Approximately 50% of the successfully annotated unigenes were differentially expressed between the short-term stimulated sample (6 h) and control (R). However, the expression level of most unigenes returned to normal levels after 72 h, indicating that N and P stress plays a limited role in the induction of sexual reproduction. Furthermore, the quantitative real-time PCR (qRT-PCR) results of the five representative sex-related unigenes were consistent with sequencing data, which confirmed the authenticity of transcriptomic analysis. Also, qRT-PCR analysis showed that the long and short form transcripts of the saxitoxin biosynthesis gene (sxtA) were down-regulated under the nutrient deficient condition compared with the control, indicating that N and P stress regulates sxtA expression. Overall, transcriptome analysis of A. minutum revealed that N and P deficiency induced responses associated with stress response, photosynthetic efficiency, toxin biosynthesis, and sexual reproduction. Our data indicate that algae change their trophic modes (to facultative mixotrophy) and related physiological reactions under stress conditions; this possibly represents an ecological adaption strategy in the algal life cycle.

Identification of a Novel Saxitoxin Analogue, 12β-Deoxygonyautoxin 3, in the Cyanobacterium, Anabaena circinalis (TA04).

Saxitoxin (STX) and its analogues, the potent voltage-gated sodium channel blockers, are biosynthesized by freshwater cyanobacteria and marine dinoflagellates. We previously identified several biosynthetic intermediates in the extract of the cyanobacterium, Anabaena circinalis (TA04), that are primarily produced during the early and middle stages in the biosynthetic pathway to produce STX. These findings allowed us to propose a putative biosynthetic pathway responsible for STX production based on the structures of these intermediates. In the present study, we identified 12β-deoxygonyautoxin 3 (12β-deoxyGTX3), a novel STX analogue produced by A. circinalis (TA04), by comparing the retention time and MS/MS fragmentation pattern with those of synthetic standards using LC–MS. The presence of this compound in A. circinalis (TA04) is consistent with stereoselective enzymatic oxidations at C11 and C12, and 11-O-sulfation, during the late stage of STX biosynthesis, as proposed in previous studies.

Biocatalytic synthesis of α-amino ketones.

Stereospecific generation of α-amino ketones from common α-amino acids is difficult to achieve, often employing superstoichiometric alkylating reagents and requiring multiple protecting group manipulations. In contrast, the α-oxoamine synthase protein family performs this transformation stereospecifically in a single step without the need for protecting groups. Herein, we detail the characterization of the 8-amino-7-oxononanoate synthase (AONS) domain of the four-domain polyketide-like synthase SxtA, which natively mediates the formation of the ethyl ketone derivative of arginine. The function of each of the four domains is elucidated, leading to a revised proposal for the initiation of saxitoxin biosynthesis, a potent neurotoxin. We also demonstrate the synthetic potential of SxtA AONS, which is applied to the synthesis of a panel of novel α-amino ketones.

Identification of the Gene sxtA (Domains sxtA1 and sxtA4) in Mexican Strains of Gymnodinium catenatum (Dinophyceae) and Their Evolution

Saxitoxin (STX) and its analogs are a broad group of natural neurotoxic alkaloids, commonly known as paralytic shellfish toxins. SxtA is the initial gene in the biosynthesis of saxitoxin. It has been proposed that the genes for STX biosynthesis had a bacterial origin and were acquired in the dinoflagellates by a horizontal gene transfer (HGT). In Gymnodinium catenatum, the origin of the STX genes is not well established. In this paper, we sequenced sxtA gene (domains sxtA1 and sxtA4) and determined the gene copy number in the genome in four Mexican strains of G. catenatum. We compare them with sequences of G. catenatum, Pyrodinium bahamense, and Alexandrium spp. from other geographic regions, and non-toxic producing dinoflagellates. Amplifications were performed for domains sxtA1 and sxtA4 from strains of G. catenatum and the phylogenetic analyses was done by maximum likelihood and Bayesian inference. The copy number determination was carried out using qPCR. The phylogenetic tree of domain sxtA4 showed the formation of two clades where G. catenatum sequences separated from the Alexandrium/Pyrodinium clade. The domain sxtA1 formed a higher number of clades than sxtA4. Sequences of G. catenatum were grouped together with sequences of Alexandrium. Dinoflagellates sequences that do not produce saxitoxin formed a separate clade. The gene copy number was 64 ± 30 and 110 ± 50 copies of sxtA1 and sxtA4 respectively. The identification of the gene sxtA of G. catenatum shows that the sequences are similar to those of Alexandrium species with low variations between species. These results may indicate that the acquisition of the gene sxtA was an early HGT event in the evolution of dinoflagellates. The possible loss of the ability to produce STX in some species suggests that the HGT from Alexandrium species towards G. catenatum is not possible.

Paralytic shellfish toxin producing Aphanizomenon gracile strains isolated from Lake Iznik, Turkey

Aphanizomenon gracile is one of the most widespread Paralytic Shellfish Toxin (PST) producing cyanobacteria in freshwater bodies in the Northern Hemisphere. It has been shown to produce various PST congeners, including saxitoxin (STX), neosaxitoxin (NEO), decarbamoylsaxitoxin (dcSTX) and gonyautoxin 5 (GTX5) in Europe, North America and Asia. Three cyanobacteria strains were isolated in Lake Iznik in northwestern Turkey. Morphological characterization of these strains suggested all three strains conformed to classical taxonomic identification of A. gracile with some differences such as clumping of filaments, partially hyaline cells in some filaments and longer than usual vegetative cells. Sequences of 16S rRNA gene of these strains were placed within an A. gracile cluster including the majority of PST producing strains, confirming the identification of these strains as A. gracile. These new strains possessed saxitoxin biosynthesis genes sxtA, sxtG and their sequences clustered with those of other A. gracile. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis demonstrated the presence of NEO, STX, dcSTX and decarbamoylneosaxitoxin (dcNEO) in all strains. This is the first report of a PST producer in any water body in Turkey and first observation of dcNEO in an A. gracile culture.

Chemistry of a Unique Polyketide-like Synthase.

Like many complex natural products, the intricate architecture of saxitoxin (STX) has hindered full exploration of this scaffold's utility as a tool for studying voltage-gated sodium ion channels and as a pharmaceutical agent. Established chemical strategies can provide access to the natural product; however, a chemoenzymatic route to saxitoxin that could provide expedited access to related compounds has not been devised. The first step toward realizing a chemoenzymatic approach toward this class of molecules is the elucidation of the saxitoxin biosynthetic pathway. To date, a biochemical link between STX and its putative biosynthetic enzymes has not been demonstrated. Herein, we report the first biochemical characterization of any enzyme involved in STX biosynthesis. Specifically, the chemical functions of a polyketide-like synthase, SxtA, from the cyanobacteria Cylindrospermopsis raciborskii T3 are elucidated. This unique megasynthase is comprised of four domains: methyltransferase (MT), GCN5-related N-acetyltransferase (GNAT), acyl carrier protein (ACP), and the first example of an 8-amino-7-oxononanoate synthase (AONS) associated with a multidomain synthase. We have established that this single polypeptide carries out the formation of two carbon-carbon bonds, two decarboxylation events and a stereospecific protonation to afford the linear biosynthetic precursor to STX (4). The synthetic utility of the SxtA AONS is demonstrated by the synthesis of a suite of α-amino ketones from the corresponding α-amino acid in a single step.

Temperature Influences the Production and Transport of Saxitoxin and the Expression of sxt Genes in the Cyanobacterium Aphanizomenon gracile.

The cyanobacterium Aphanizomenon gracile is the most widely distributed producer of the potent neurotoxin saxitoxin in freshwaters. In this work, total and extracellular saxitoxin and the transcriptional response of three genes linked to saxitoxin biosynthesis (sxtA) and transport (sxtM, sxtPer) were assessed in Aphanizomenon gracile UAM529 cultures under temperatures covering its annual cycle (12 °C, 23 °C, and 30 °C). Temperature influenced saxitoxin production being maximum at high temperatures (30 °C) above the growth optimum (23 °C), concurring with a 4.3-fold increased sxtA expression at 30 °C. Extracellular saxitoxin transport was temperature-dependent, with maxima at extremes of temperature (12 °C with 16.9% extracellular saxitoxin; and especially 30 °C with 53.8%) outside the growth optimum (23 °C), coinciding with a clear upregulation of sxtM at both 12 °C and 30 °C (3.8–4.1 fold respectively), and yet with just a slight upregulation of sxtPer at 30 °C (2.1-fold). Nitrate depletion also induced a high extracellular saxitoxin release (51.2%), although without variations of sxtM and sxtPer transcription, and showing evidence of membrane damage. This is the first study analysing the transcriptional response of sxtPer under environmental gradients, as well as the effect of temperature on putative saxitoxin transporters (sxtM and sxtPer) in cyanobacteria in general.

Use of three monitoring approaches to manage a major Chrysosporum ovalisporum bloom in the Murray River, Australia, 2016.

An unusual bloom of Chrysosporum ovalisporum (basionym Aphanizomenon ovalisporum) occurred for the first time in the Murray River and distributary rivers in New South Wales, Australia, from mid-February to early June 2016. At its greatest extent, it contaminated a combined river length of ca. 2360km. Chrysosporum ovalisporum usually comprised >99% of the total bloom biovolume at most locations sampled, which at times exceeded 40mm3l-1. The origins of the bloom were most likely reservoirs on the upper Murray River, with cyanobacterial-infested water released from them contaminating the river systems downstream. An integrated approach using three analytical methods: (1) identification and enumeration by microscopy, (2) multiplex quantitative polymerase chain reaction (qPCR), and (3) toxin analysis, was used to obtain data for the assessment of risk to water users and management of the bloom. qPCR indicated some cyrA and stxA genes responsible for cylindrospermopsin and saxitoxin biosynthesis respectively were present, but mostly below the level of quantification. No mcyE genes for microcystin biosynthesis were detected. Toxin analysis also revealed that cylindrospermopsin, saxitoxin and microcystin were all below detection. Lack of measurable toxicity in a species usually considered a cylindrospermopsin producer elsewhere meant the possibility of relaxing management guidelines; however, high (Red) alerts needed to be maintained due to risk to water users from other biohazards potentially produced by the cyanobacteria such as contact irritants. A three-tiered monitoring strategy is suggested for monitoring cyanobacterial blooms to provide enhanced data for bloom management.

Biosynthesis of Saxitoxin

Biosynthesis of Saxitoxin

Column switching combined with hydrophilic interaction chromatography-tandem mass spectrometry for the analysis of saxitoxin analogues, and their biosynthetic intermediates in dinoflagellates

Hydrophilic-interaction chromatography (HILIC) is reportedly useful for the analysis of saxitoxin (STX) analogues, collectively known as paralytic shellfish toxins. Column switching and two-step gradient elution using HILIC combined with mass spectrometry enabled the simultaneous analysis of the 15 primary STX analogues and their biosynthetic intermediates, arginine, Int-A’, and Int-C’2, and the shunt product, Cyclic-C’. Crude extracts of toxin-producing dinoflagellates can be injected without any treatment except filtration. Enrichment of the compounds using this method was highly reproducible with respect to retention times (% RSD was under 1%) and highly sensitive (limits of detection (LODs) were in the range 0.9 (Int-C’2) - 116 (C3) μM) in terms of avoiding matrix effects associated with co-eluting substances. Validation studies demonstrated acceptable performance of this method for specificity, repeatability, linearity and recovery. A comparison of the quantitative results for STX analogues in Alexandrium tamarense using HPLC with post-column fluorescent derivatization and the column-switching HILIC–MS method revealed good agreement. The presence of Int-A’, Int-C’2, and Cyclic-C’ in toxic dinoflagellate species with different toxin profiles was confirmed using this method. Our data support the hypothesis that the early stages of the STX biosynthesis and shunt pathways are the same in dinoflagellates and cyanobacteria.

Molecular genetic identification and seasonal succession of toxigenic cyanobacteria in phytoplankton of the Rybinsk Reservoir (Russia)

Data on the hepato- and neurotoxigenic cyanobacteria in phytoplankton of the Rybinsk Reservoir have been obtained for the first time. Different methods for revealing toxigenic cyanobacteria (light microscopy, PCR analysis, and enzyme-linked immunosorbent assay) demonstrate the same results. Hepatotoxins microcystins and for the first time neurotoxins saxitoxins were detected in the reservoir, whereas cylindrospermopsin and anatoxin-a were not revealed. The presence of mcyE and stxA genes responsible for microcystin and saxitoxin biosynthesis in total phytoplanktonic DNA is demonstrated. The following three genera of cyanobacteria containing mcyE gene are identified: Microcystis (M. aeruginosa, M. viridis), Planktothrix (P. agardhii), and Dolichospermum (Anabaena). It is hypothesized that saxitoxin-producing cyanobacteria Dolichospermum (Anabaena) inhabit the Rybinsk Reservoir.

First detection of benthic cyanobacteria in Lake Baikal producing paralytic shellfish toxins

Cyanobacteria were screened from the surface of diseased sponges, stone and bedrock in Lake Baikal for the presence of saxitoxin using enzyme-linked immunosorbent assay. In sequel, eight paralytic shellfish toxin (PST) variants were identified using a MALDI mass spectrometry. Microscopic examination found that Tolypothrix distorta dominated in the biofouling samples. PCR and sequencing detected sxtA gene involved in saxitoxin biosynthesis, thereby providing evidence of the PST producing potential of Baikal cyanobacterial communities inhabiting different substrates.

Influence of nitrogen availability on the expression of genes involved in the biosynthesis of saxitoxin and analogs in Cylindrospermopsis raciborskii

The development of cyanobacterial blooms in inland aquatic ecosystems is greatly promoted by nutrient availability, especially nitrogen and phosphorous. When blooms are dominated by toxigenic species the harmful effects of nutrient loading becomes particularly relevant. Among toxic species, Cylindrospermopsis raciborskii found in South American ecosystems is characterized by the production of saxitoxin and analogs (Paralytic Shellfish Poisoning, PSP), for which the factors that trigger their production have not been elucidated. In this study, the effect of nitrate availability on the relative transcript abundance of two genes (sxtU and sxtI), both involved in different steps of PSP biosynthetic pathway, was addressed in C. raciborskii MVCC19 by qPCR. The relative transcript abundance of both genes significantly increased from the beginning to the end of growth, independent of nitrate availability in the culture medium. Differences between the genes in terms of the levels of relative expression were also found, implying that during growth in nitrate-rich or nitrate-deprived conditions C. raciborskii MVCC19 has the ability to produce different kind of PSP molecules. The presence of nifH transcripts in the nitrogen-depleted treatment confirmed that in the absence of nitrate C. raciborskii fixed atmospheric N2. Moreover, after transferring filaments to nitrate-rich conditions the synthesis of nifH mRNA continued for few hours, suggesting that cell adjustments enabling the utilization of soluble nitrogen sources are not immediate. Our results show that biosynthesis of saxitoxin and analogs in C. raciborskii is not related to nitrate availability, but rather is linked to cyanobacteria growth rate.

Abiotic variables affect STX concentration in a meso-oligotrophic subtropical coastal lake dominated by Cylindrospermopsis raciborskii (Cyanophyceae)

The cyanobacterium Cylindrospermopsis raciborskii is capable of producing toxins including saxitoxin (STX). Few studies have verified the influence of environmental variables on the production of STX and most have only been studied in the laboratory. The goal of this work was to identify the abiotic variables related to STX concentration in situ. The relationship among STX concentration and the physical variables, nutrients and chlorophyll-a (chl-a) concentration was examined in a meso-oligotrophic subtropical coastal lake dominated by C. raciborskii. A generalized linear model was developed, incorporating all variables measured monthly over a 45-month monitoring period. Conductivity and dissolved inorganic nitrogen (DIN) concentration provided the greatest explanatory power for STX concentration in situ. Previous studies suggested that C. raciborskii cells exposed to stress associated with higher ionic concentrations appear to activate the biosynthesis of STX suggesting that STX can elicit changes cell permeability and may contribute to the homeostasis of this organism. An increase of DIN concentration results in a higher concentration of STX which may be related to a reduced metabolic demand, since the uptake of inorganic nitrogen requires less energy than N2-fixation. Thus, increased DIN can favor the growth of C. raciborskii population or improve cellular homeostasis, both potentially increasing STX concentration in the aquatic system, which was observed through a delayed response pattern. The developed model, while providing only a moderate predictive power, can assist in the understanding of the environmental variables associated with increases in STX concentration, and in monitoring and minimizing the risks of toxic blooms.

Molecular Detection of theSxtaGene from Saxitoxin-ProducingAlexandrium minutumin Commercial Oysters

ABSTRACT The production of toxic secondary metabolites by marine phytoplankton and their accumulation in molluscs and fish has ecosystem-wide and human health impacts. The potent neurotoxin saxitoxin and its analogs, which can cause paralytic shellfish poisoning, are produced by species of the dinoflagellate genus Alexandrium. These toxins can accumulate in filter-feeding molluscs, including commercially grown species of shellfish. A feeding experiment was designed to assess the use of quantitative polymerase chain reaction to detect a gene involved in saxitoxin biosynthesis (sxtA) in two commercial oyster stocks. Both diploid and triploid Crassostrea gigas were fed with toxic cultures of Alexandrium minutum over a period of 12 days to allow toxin accumulation. A barcoding gene specific to Alexandrium (ITS 5.8s rRNA) and the sxtA gene, domain 4, which is specific to the saxitoxin synthesis pathway, were assayed. Both targets were detected in oysters collected after 6 and 12 days feeding with A. minutum, a...

Proteogenomics of a saxitoxin-producing and non-toxic strain of Anabaena circinalis (cyanobacteria) in response to extracellular NaCl and phosphate depletion.

In Australia, saxitoxin production is strain dependent within the bloom-forming freshwater cyanobacterium Anabaena circinalis. Freshwater cyanobacteria are exposed to rapid fluctuations in environmental nutrient concentrations, and their adaption is vital for competition, succession and dominance. Two elements of environmental significance, phosphorus and sodium chloride, are proposed to play a role in bloom development and saxitoxin biosynthesis respectively. The aim of our study was to comparatively analyse the model saxitoxin-producing A. circinalis AWQC131C and non-toxic A. circinalis AWQC310F at the genomic level and proteomic level, in response to phosphate depletion and increased extracellular NaCl. When challenged, photosynthesis, carbon/nitrogen metabolisms, transcription/translation, oxidative stress and nutrient transport functional categories demonstrated the largest changes in protein abundance. In response to increased NaCl, SxtC, a protein conserved in all known saxitoxin biosynthetic pathways, was downregulated. Additionally, toxin quantification revealed a decrease in total saxitoxin and decarbomoyl-gonyautoxin2/3 content in response to the NaCl treatment. In response to phosphate depletion, the toxic and non-toxic strain displayed similar proteomic profiles, although the toxic strain did not alter the abundance of as many proteins as the non-toxic strain. These findings have important implications for the future, since response and adaption mechanisms are directly related to in situ dominance of cyanobacteria.

Gene duplication, loss and selection in the evolution of saxitoxin biosynthesis in alveolates

A group of marine dinoflagellates (Alveolata, Eukaryota), consisting of ∼10 species of the genus Alexandrium, Gymnodinium catenatum and Pyrodinium bahamense, produce the toxin saxitoxin and its analogues (STX), which can accumulate in shellfish, leading to ecosystem and human health impacts. The genes, sxt, putatively involved in STX biosynthesis, have recently been identified, however, the evolution of these genes within dinoflagellates is not clear. There are two reasons for this: uncertainty over the phylogeny of dinoflagellates; and that the sxt genes of many species of Alexandrium and other dinoflagellate genera are not known. Here, we determined the phylogeny of STX-producing and other dinoflagellates based on a concatenated eight-gene alignment. We determined the presence, diversity and phylogeny of sxtA, domains A1 and A4 and sxtG in 52 strains of Alexandrium, and a further 43 species of dinoflagellates and thirteen other alveolates. We confirmed the presence and high sequence conservation of sxtA, domain A4, in 40 strains (35 Alexandrium, 1 Pyrodinium, 4 Gymnodinium) of 8 species of STX-producing dinoflagellates, and absence from non-producing species. We found three paralogs of sxtA, domain A1, and a widespread distribution of sxtA1 in non-STX producing dinoflagellates, indicating duplication events in the evolution of this gene. One paralog, clade 2, of sxtA1 may be particularly related to STX biosynthesis. Similarly, sxtG appears to be generally restricted to STX-producing species, while three amidinotransferase gene paralogs were found in dinoflagellates. We investigated the role of positive (diversifying) selection following duplication in sxtA1 and sxtG, and found negative selection in clades of sxtG and sxtA1, clade 2, suggesting they were functionally constrained. Significant episodic diversifying selection was found in some strains in clade 3 of sxtA1, a clade that may not be involved in STX biosynthesis, indicating pressure for diversification of function.

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.

Gene expression and molecular evolution of sxtA4 in a saxitoxin producing dinoflagellate Alexandrium catenella

Dinoflagellates of the genus Alexandrium produce the neurotoxin saxitoxin (STX), responsible for paralytic shellfish poisoning (PSP) and accumulates in marine invertebrates. The recent identification of STX biosynthesis genes allowed us to investigate the expression of sxtA4 at different growth stages in Alexandrium catenella Group IV. We found no significant differences in expression of sxtA4, despite significant differences in STX levels at different growth stages (P < 0.023). Three reference genes were tested for normalisation: actin, cytochrome b (cob), and the large subunit ribosomal RNA (LSU rDNA). cob was most stably expressed but the combination of two reference genes, actin and cob, resulted in the best stability factor. Most genomic sequences of sxtA4 from A. catenella were in a clade that included sequences from Alexandrium fundyense Group I, however, one paralogue was not related to the others, suggesting recombination or lateral transfer. A comparison of the sxtA4 cDNA sequences with genomic DNA sequences indicated the possibility of transcript editing and the preferential transcription of certain genomic DNA loci. The results show that, in dinoflagellates, post-transcriptional mechanisms play a major role in the regulation of saxitoxin biosynthesis.