Environmental Factors Modulate Saxitoxins (STXs) Production in Toxic Dinoflagellate Alexandrium: An Updated Review of STXs and Synthesis Gene Aspects.

The marine dinoflagellate Alexandrium is known to form harmful algal blooms (HABs) and produces saxitoxin (STX) and its derivatives (STXs) that cause paralytic shellfish poisoning (PSP) in humans. Cell growth and cellular metabolism are affected by environmental conditions, including nutrients, temperature, light, and the salinity of aquatic systems. Abiotic factors not only engage in photosynthesis, but also modulate the production of toxic secondary metabolites, such as STXs, in dinoflagellates. STXs production is influenced by a variety of abiotic factors; however, the relationship between the regulation of these abiotic variables and STXs accumulation seems not to be consistent, and sometimes it is controversial. Few studies have suggested that abiotic factors may influence toxicity and STXs-biosynthesis gene (sxt) regulation in toxic Alexandrium, particularly in A. catenella, A. minutum, and A. pacificum. Hence, in this review, we focused on STXs production in toxic Alexandrium with respect to the major abiotic factors, such as temperature, salinity, nutrients, and light intensity. This review informs future research on more sxt genes involved in STXs production in relation to the abiotic factors in toxic dinoflagellates.

Development of saxitoxin biosynthesis gene sxtB-targeted qPCR assay for the quantification of toxic dinoflagellates Alexandrium catenella (group I) and A. pacificum (group IV) occurring in the Korean coast.

Toxic dinoflagellate Alexandrium can produce saxitoxins (STXs) and cause paralytic shellfish poisoning (PSP), and thus they are monitored for environmental safety management. Microscopic discrimination of dinoflagellates is difficult to distinguish between toxic and non-toxic species due to their similar morphology. Meanwhile, an alternative quantitative PCR (qPCR) assay is sensitive, rapid, and cost-effective for harmful species monitoring. Herein, we developed a novel qPCR assay to detect the STXs biosynthesis gene sxtB of Alexandrium catenella and A. pacificum, the leading cause of PSP outbreaks in Asian coasts and worldwide. The newly designed sxtB TaqMan probes target the species without any positive signal in other relative dinoflagellates. Deming regression analysis revealed that the sxtB copy number of A. catenella and A. pacificum was 3.6 and 4.1 copies per cell, respectively. During the blooming periods (April 13th-14th, 2020), only A. catenella cells were detected through the qPCR assay, ranging from 5.0 × 10 to 2.5 × 104 eq cells L-1. In addition, sxtB qPCR quantified more accurately compared to large subunit (LSU) rRNA targeting qPCR assay that overestimate cell density. Besides, the sensitivity of sxtB was higher compared to the microscope when the species were rarely present (5.0 × 102 cells L-1). These suggest that the sxtB qPCR assay can be applied to toxic Alexandrium monitoring in the Korean coast, even in the early stage of bloomings.

Polyphyletic origin of saxitoxin biosynthesis genes in the marine dinoflagellate Alexandrium revealed by comparative transcriptomics.

The marine dinoflagellate Alexandrium is known to form harmful algal blooms, and at least 14 species within the genus can produce saxitoxins (STXs). STX biosynthesis genes (sxt) are individually revealed in toxic dinoflagellates; however, the evolutionary history remains controversial. Herein, we determined the transcriptome sequences of toxic Alexandrium (A. catenella and A. pacificum) and non-toxic Alexandrium (A. fraterculus and A. fragae) and characterized their sxt by focusing on evolutionary events and STX production. Comparative transcriptome analysis revealed higher homology of the sxt in toxic Alexandrium than in non-toxic species. Notably, non-toxic Alexandrium spp. were found to have lost two sxt core genes, namely sxtA4 and sxtG. Expression levels of 28 transcripts related to eight sxt core genes showed that sxtA, sxtG, and sxtI were relatively high (>1.5) in the toxic group compared to the non-toxic group. In contrast, the non-toxic group showed high expression levels in sxtU (1.9) and sxtD (1.7). Phylogenetic tree comparisons revealed distinct evolutionary patterns between 28S rDNA and sxtA, sxtB, sxtI, sxtD, and sxtU. However, similar topology was observed between 28S rDNA, sxtS, and sxtH/T. In the sxtB and sxtI phylogeny trees, toxic Alexandrium and cyanobacteria were clustered together, separating from non-toxic species. These suggest that Alexandrium may acquire sxt genes independently via horizontal gene transfer from toxic cyanobacteria and other multiple sources, demonstrating monocistronic transcripts of sxt in dinoflagellates.

Salinity Affects Saxitoxins (STXs) Toxicity in the Dinoflagellate Alexandrium pacificum, with Low Transcription of SXT-Biosynthesis Genes sxtA4 and sxtG.

Salinity is an important factor for regulating metabolic processes in aquatic organisms; however, its effects on toxicity and STX biosynthesis gene responses in dinoflagellates require further elucidation. Herein, we evaluated the physiological responses, toxin production, and expression levels of two STX synthesis core genes, sxtA4 and sxtG, in the dinoflagellate Alexandrium pacificum Alex05 under different salinities (20, 25, 30, 35, and 40 psu). Optimal growth was observed at 30 psu (0.12 cell division/d), but cell growth significantly decreased at 20 psu and was irregular at 25 and 40 psu. The cell size increased at lower salinities, with the highest size of 31.5 µm at 20 psu. STXs eq was highest (35.8 fmol/cell) in the exponential phase at 30 psu. GTX4 and C2 were predominant at that time but were replaced by GTX1 and NeoSTX in the stationary phase. However, sxtA4 and sxtG mRNAs were induced, and their patterns were similar in all tested conditions. PCA showed that gene transcriptional levels were not correlated with toxin contents and salinity. These results suggest that A. pacificum may produce the highest amount of toxins at optimal salinity, but sxtA4 and sxtG may be only minimally affected by salinity, even under high salinity stress.

Microcystis Sp. Co-Producing Microcystin and Saxitoxin from Songkhla Lake Basin, Thailand.

The Songkhla Lake Basin (SLB) located in Southern Thailand, has been increasingly polluted by urban and industrial wastewater, while the lake water has been intensively used. Here, we aimed to investigate cyanobacteria and cyanotoxins in the SLB. Ten cyanobacteria isolates were identified as Microcystis genus based on16S rDNA analysis. All isolates harbored microcystin genes, while five of them carried saxitoxin genes. On day 15 of culturing, the specific growth rate and Chl-a content were 0.2-0.3 per day and 4 µg/mL. The total extracellular polymeric substances (EPS) content was 0.37-0.49 µg/mL. The concentration of soluble EPS (sEPS) was 2 times higher than that of bound EPS (bEPS). The protein proportion in both sEPS and bEPS was higher than the carbohydrate proportion. The average of intracellular microcystins (IMCs) was 0.47 pg/cell on day 15 of culturing, while extracellular microcystins (EMCs) were undetectable. The IMCs were dramatically produced at the exponential phase, followed by EMCs release at the late exponential phase. On day 30, the total microcystins (MCs) production reached 2.67 pg/cell. Based on liquid chromatograph-quadrupole time-of-flight mass spectrometry, three new MCs variants were proposed. This study is the first report of both decarbamoylsaxitoxin (dcSTX) and new MCs congeners synthesized by Microcystis.

Molecular detection of some toxogenic cyanobacteria in Tigris River in Baghdad-Iraq.

Cyanobacteria and their pollution are being increasingly commonly reported worldwide that cause a serious hazard to environmental and human health. Cyanotoxin was the most algal toxin reported to be produced by several orders of cyanobacteria. This study aimed to provide a technique to detect cylindrosprmopsin and saxitoxin biosynthesis genes in the river. In November, December 2019, and January 2020. Cyanobacteria were isolated from freshwater of Tigris River and identified by compound microscope also conventional PCR. Five isolates of cyanobacteria that successfully amplified a gene fragment from the phycocyanin were found in all cyanobacteria (Microcystis flosaquae, Microcystis sp, anabaena circinalis, nostoc commune and westiellopsis prolifica) and all isolates successfully amplified aoaC gene to detecting the cylidrospemopsin and the saxitoxin. Our results concluded that PCR assay can be used for early detection of cylidrospemopsin and the saxitoxin producing cyanobacteria in river water that useful to stations responsible for the preparation of drinking water to public.

Ecophysiological Aspects and sxt Genes Expression Underlying Induced Chemical Defense in STX-Producing Raphidiopsis raciborskii (Cyanobacteria) against the Zooplankter Daphnia gessneri.

Cyanobacteria stand out among phytoplankton when they form massive blooms and produce toxins. Because cyanotoxin genes date to the origin of metazoans, the hypothesis that cyanotoxins function as a defense against herbivory is still debated. Although their primary cellular function might vary, these metabolites could have evolved as an anti-predator response. Here we evaluated the physiological and molecular responses of a saxitoxin-producing Raphidiopsis raciborskii to infochemicals released by the grazer Daphnia gessneri. Induced chemical defenses were evidenced in R. raciborskii as a significant increase in the transcription level of sxt genes, followed by an increase in saxitoxin content when exposed to predator cues. Moreover, cyanobacterial growth decreased, and no significant effects on photosynthesis or morphology were observed. Overall, the induced defense response was accompanied by a trade-off between toxin production and growth. These results shed light on the mechanisms underlying zooplankton-cyanobacteria interactions in aquatic food webs. The widespread occurrence of the cyanobacterium R. raciborskii in freshwater bodies has been attributed to its phenotypic plasticity. Assessing the potential of this species to thrive over interaction filters such as zooplankton grazing pressure can enhance our understanding of its adaptive success.

Low Temperature and Cold Stress Significantly Increase Saxitoxins (STXs) and Expression of STX Biosynthesis Genes sxtA4 and sxtG in the Dinoflagellate Alexandrium catenella.

Toxic dinoflagellate Alexandrium spp. produce saxitoxins (STXs), whose biosynthesis pathway is affected by temperature. However, the link between the regulation of the relevant genes and STXs' accumulation and temperature is insufficiently understood. In the present study, we evaluated the effects of temperature on cellular STXs and the expression of two core STX biosynthesis genes (sxtA4 and sxtG) in the toxic dinoflagellate Alexandrium catenella Alex03 isolated from Korean waters. We analyzed the growth rate, toxin profiles, and gene responses in cells exposed to different temperatures, including long-term adaptation (12, 16, and 20 °C) and cold and heat stresses. Temperature significantly affected the growth of A. catenella, with optimal growth (0.49 division/day) at 16 °C and the largest cell size (30.5 µm) at 12 °C. High concentration of STXs eq were detected in cells cultured at 16 °C (86.3 fmol/cell) and exposed to cold stress at 20→12 °C (96.6 fmol/cell) compared to those at 20 °C and exposed to heat stress. Quantitative real-time PCR (qRT-PCR) revealed significant gene expression changes of sxtA4 in cells cultured at 16 °C (1.8-fold) and cold shock at 20→16 °C (9.9-fold). In addition, sxtG was significantly induced in cells exposed to cold shocks (20→16 °C; 19.5-fold) and heat stress (12→20 °C; 25.6-fold). Principal component analysis (PCA) revealed that low temperature (12 and 16 °C) and cold stress were positively related with STXs' production and gene expression levels. These results suggest that temperature may affect the toxicity and regulation of STX biosynthesis genes in dinoflagellates.

Exposure of Nile Tilapia (Oreochromis niloticus) Fingerlings to a Saxitoxin-Producing Strain of Raphidiopsis (Cylindrospermopsis) raciborskii (Cyanobacterium) Reduces Growth Performance and Increases Mortality Rate.

Blooms of toxin-producing cyanobacteria have been more frequent and lasting because of the eutrophication of freshwater ecosystems, including those used for aquaculture. The aim of the present study was to investigate the effects of chronic exposure to a saxitoxin-producing strain of Raphidiopsis (Cylindrospermopsis) raciborskii on the performance of Nile tilapia (Oreochromis niloticus) fingerlings over a 60-d period. The fingerlings were cultivated under the following conditions: 1) water without cyanobacterium (WATER), 2) R. raciborskii in ASM-1 culture medium (CYANO), and 3) ASM-1 culture medium without cyanobacterium (ASM). Exposure to the CYANO treatment led to a significant increase in the mortality rate (p < 0.05) and a significant reduction in growth (p < 0.05) compared to fingerlings submitted to the ASM and WATER treatments, in which similar survival and growth were found (p > 0.05). Saxitoxin toxicity was dependent on the weight of the fingerling (p < 0.05), with maximum mortality caused by the ingestion of 13.66 µg saxitoxin equivalent L-1 g-1 . The present results clearly show the harm caused by saxitoxins to the production of Nile tilapia fingerlings in the early growth phase. These findings underscore the importance of maintaining adequate water quality in aquaculture activities to minimize the risk of saxitoxin-producing cyanobacterial blooms and avoid economic losses among producers. Environ Toxicol Chem 2020;39:1409-1420. © 2020 SETAC.

Transcriptome survey and toxin measurements reveal evolutionary modification and loss of saxitoxin biosynthesis genes in the dinoflagellates Amphidinium carterae and Prorocentrum micans.

In the present study, we characterized the potential toxin genes for polyketide synthase (PKS) and saxitoxin (STX) biosynthesis using the transcriptomes of two non-STX producing dinoflagellates Amphidinium carterae and Prorocentrum micans. RNA sequencing revealed 94 and 166 PKS contigs in A. carterae and P. micans, respectively. We first detected type III PKS, which was closely related to bacteria. In addition, dozens of homologs of 20 STX biosynthesis genes were identified. Interestingly, the core STX-synthesizing genes sxtA and sxtB were only found in P. micans, whereas sxtD was detected in A. carterae alone. Bioinformatic analysis showed that the first two core genes (sxtA and sxtG) had a low sequence similarity (37.0-67.6%) and different domain organization compared to those of other toxigenic dinoflagellates, such as Alexandrium pacificum. These might result in the breakdown of the initial reactions in STX production and ultimately the loss of the ability to synthesize the toxins in both dinoflagellates. Our findings suggest that toxin-related PKS and sxt genes are commonly found in non-STX producing dinoflagellates. In addition to their involvement in the synthesis of toxins, our result indicates that genes may also have other molecular metabolic functions.

Identification of promoter elements in the Dolichospermum circinale AWQC131C saxitoxin gene cluster and the experimental analysis of their use for heterologous expression.

BACKGROUND: Dolichospermum circinale is a filamentous bloom-forming cyanobacterium responsible for biosynthesis of the paralytic shellfish toxins (PST), including saxitoxin. PSTs are neurotoxins and in their purified form are important analytical standards for monitoring the quality of water and seafood and biomedical research tools for studying neuronal sodium channels. More recently, PSTs have been recognised for their utility as local anaesthetics. Characterisation of the transcriptional elements within the saxitoxin (sxt) biosynthetic gene cluster (BGC) is a first step towards accessing these molecules for biotechnology. RESULTS: In D. circinale AWQC131C the sxt BGC is transcribed from two bidirectional promoter regions encoding five individual promoters. These promoters were identified experimentally using 5' RACE and their activity assessed via coupling to a lux reporter system in E. coli and Synechocystis sp. PCC 6803. Transcription of the predicted drug/metabolite transporter (DMT) encoded by sxtPER was found to initiate from two promoters, PsxtPER1 and PsxtPER2. In E. coli, strong expression of lux from PsxtP, PsxtD and PsxtPER1 was observed while expression from Porf24 and PsxtPER2 was remarkably weaker. In contrast, heterologous expression in Synechocystis sp. PCC 6803 showed that expression of lux from PsxtP, PsxtPER1, and Porf24 promoters was statistically higher compared to the non-promoter control, while PsxtD showed poor activity under the described conditions. CONCLUSIONS: Both of the heterologous hosts investigated in this study exhibited high expression levels from three of the five sxt promoters. These results indicate that the majority of the native sxt promoters appear active in different heterologous hosts, simplifying initial cloning efforts. Therefore, heterologous expression of the sxt BGC in either E. coli or Synechocystis could be a viable first option for producing PSTs for industrial or biomedical purposes.

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.

Metabolomic study of saxitoxin analogues and biosynthetic intermediates in dinoflagellates using 15N-labelled sodium nitrate as a nitrogen source.

A stable-isotope-labelling method using 15N-labelled sodium nitrate as a nitrogen source was developed for the toxic dinoflagellate Alexandrium catenella. The labelled saxitoxin analogues (STXs), their precursor, and the biosynthetic intermediates were analyzed by column-switching high-resolution hydrophilic interaction liquid chromatography with mass spectrometry. The low contents on Day 0, high 15N incorporation % of Int-C'2 and Int-E' suggested that their turn-over rates are high and that the sizes of the pool of these compounds are smaller than those of the other intermediates. The experimentally determined isotopomer distributions showed that arginine, Int-C'2, 11-hydroxy-Int-C'2, Int-E', GTX5, GTX4, C1, and C2, each existed as a combination of three populations that consisted of the non-labelled molecules and the labelled isotopomers representing molecules newly synthesized by incorporation of 15N assimilated from the medium with two different incorporation rates. The order of 15N incorporation % values of the labelled populations predicted by this model largely agreed with the proposed biosynthetic route. The stable-isotope-labelling method will be useful for understanding the complex mechanism of nitrogen flux in STX-producing dinoflagellates.

Pyridoxal phosphate-dependent reactions in the biosynthesis of natural products.

Covering: up to mid-2018 Pyridoxal 5'-phosphate (PLP) is a versatile organic cofactor used to catalyze diverse reactions on amino acid, oxoacid, and amine substrates. Here we review the reactions catalyzed by PLP-dependent enzymes, highlighting enzymes reported in the natural product biosynthetic literature. We describe enzymes that catalyze transaminations, Claisen-like condensations, and ß- and γ-eliminations and substitutions, along with epimerizations, decarboxylations, and transaldolations. Finally, we describe a newly reported group of O2-, PLP-dependent enzymes. Altogether, natural product biosynthesis showcases the incredible versatility of PLP-dependent transformations for building chemical complexity.

Nitrogen limitation, toxin synthesis potential, and toxicity of cyanobacterial populations in Lake Okeechobee and the St. Lucie River Estuary, Florida, during the 2016 state of emergency event.

Lake Okeechobee, FL, USA, has been subjected to intensifying cyanobacterial blooms that can spread to the adjacent St. Lucie River and Estuary via natural and anthropogenically-induced flooding events. In July 2016, a large, toxic cyanobacterial bloom occurred in Lake Okeechobee and throughout the St. Lucie River and Estuary, leading Florida to declare a state of emergency. This study reports on measurements and nutrient amendment experiments performed in this freshwater-estuarine ecosystem (salinity 0-25 PSU) during and after the bloom. In July, all sites along the bloom exhibited dissolved inorganic nitrogen-to-phosphorus ratios < 6, while Microcystis dominated (> 95%) phytoplankton inventories from the lake to the central part of the estuary. Chlorophyll a and microcystin concentrations peaked (100 and 34 µg L-1, respectively) within Lake Okeechobee and decreased eastwards. Metagenomic analyses indicated that genes associated with the production of microcystin (mcyE) and the algal neurotoxin saxitoxin (sxtA) originated from Microcystis and multiple diazotrophic genera, respectively. There were highly significant correlations between levels of total nitrogen, microcystin, and microcystin synthesis gene abundance across all surveyed sites (p < 0.001), suggesting high levels of nitrogen supported the production of microcystin during this event. Consistent with this, experiments performed with low salinity water from the St. Lucie River during the event indicated that algal biomass was nitrogen-limited. In the fall, densities of Microcystis and concentrations of microcystin were significantly lower, green algae co-dominated with cyanobacteria, and multiple algal groups displayed nitrogen-limitation. These results indicate that monitoring and regulatory strategies in Lake Okeechobee and the St. Lucie River and Estuary should consider managing loads of nitrogen to control future algal and microcystin-producing cyanobacterial blooms.

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.

Molecular and morphological survey of saxitoxin-producing cyanobacterium Dolichospermum circinale (Anabaena circinalis) isolated from geographically distinct regions of Australia.

The cyanobacterium Dolichospermum circinale (formerly Anabaena circinalis) is responsible for neurotoxic saxitoxin-producing blooms in Australia. Previous studies have reported distinct isolates of toxic D. circinale producing different saxitoxin analogues at varying amounts, but the mechanisms responsible remain poorly understood. To assess the characteristics that may be responsible for this variance, a morphological, molecular and chemical survey of 28 Anabaena isolates was conducted. Morphological characteristics, presence or absence of saxitoxin biosynthetic genes and toxin amount and profile were assessed. The 28 isolates were collected from 16 locations. A correlation between the size of the isolates and its reported toxicity or geographical location could not be found. Molecular screening for the presence of several sxt genes revealed eight out of the 28 strains harboured the sxt gene cluster and all tailoring genes except sxtX. Furthermore, the presence of PSTs was correlated with the presence of the sxt cluster using quantitative pre-column oxidation high performance liquid chromatography with fluorescence detection (HPLC-FLD) and LC-MS/MS. Interestingly, isolates differed in the amount and type of toxins produced, with the eight toxic strains containing the core and tailoring biosynthetic genes while non-toxic strains were devoid of these genes. Moreover, the presence of sxt tailoring genes in toxic strains correlated with the biosynthesis of analogues. A greater understanding of toxin profile/quantity from distinct sites around Australia will aid the management of these at-risk areas and provide information on the molecular control or physiological characteristics responsible for toxin production.

Ocean warming since 1982 has expanded the niche of toxic algal blooms in the North Atlantic and North Pacific oceans.

Global ocean temperatures are rising, yet the impacts of such changes on harmful algal blooms (HABs) are not fully understood. Here we used high-resolution sea-surface temperature records (1982 to 2016) and temperature-dependent growth rates of two algae that produce potent biotoxins, Alexandrium fundyense and Dinophysis acuminata, to evaluate recent changes in these HABs. For both species, potential mean annual growth rates and duration of bloom seasons significantly increased within many coastal Atlantic regions between 40°N and 60°N, where incidents of these HABs have emerged and expanded in recent decades. Widespread trends were less evident across the North Pacific, although regions were identified across the Salish Sea and along the Alaskan coastline where blooms have recently emerged, and there have been significant increases in the potential growth rates and duration of these HAB events. We conclude that increasing ocean temperature is an important factor facilitating the intensification of these, and likely other, HABs and thus contributes to an expanding human health threat.

Synthesis and Identification of Key Biosynthetic Intermediates for the Formation of the Tricyclic Skeleton of Saxitoxin.

Saxitoxin (STX) and its analogues are potent voltage-gated sodium channel blockers biosynthesized by freshwater cyanobacteria and marine dinoflagellates. We previously identified genetically predicted biosynthetic intermediates of STX at early stages, Int-A' and Int-C'2, in these microorganisms. However, the mechanism to form the tricyclic skeleton of STX was unknown. To solve this problem, we screened for unidentified intermediates by analyzing the results from previous incorporation experiments with 15 N-labeled Int-C'2. The presence of monohydroxy-Int-C'2 and possibly Int-E' was suggested, and 11-hydroxy-Int-C'2 and Int-E' were identified from synthesized standards and LC-MS. Furthermore, we observed that the hydroxy group at C11 of 11-hydroxy-Int-C'2 was slowly replaced by CD3 O in CD3 OD. Based on this characteristic reactivity, we propose a possible mechanism to form the tricyclic skeleton of STX via a bicyclic intermediate from 11-hydroxy-Int-C'2.