Transcriptomic investigation into polyketide toxin synthesis in Ostreopsis (Dinophyceae) species.

In marine ecosystems, dinoflagellates can become highly abundant and even dominant at times, despite their comparatively slow growth. Their ecological success may be related to their production of complex toxic polyketide compounds. Ostreopsis species produce potent palytoxin-like compounds (PLTX), which are associated with human skin and eye irritations, and illnesses through the consumption of contaminated seafood. To investigate the genetic basis of PLTX-like compounds, we sequenced and annotated transcriptomes from two PLTX-producing Ostreopsis species; O. cf. ovata, O. cf. siamensis, one non-PLTX producing species, O. rhodesae and compared them to a close phylogenetic relative and non-PLTX producer, Coolia malayensis. We found no clear differences in the presence or diversity of ketosynthase and ketoreductase transcripts between PLTX producing and non-producing Ostreopsis and Coolia species, as both groups contained >90 and > 10 phylogenetically diverse ketosynthase and ketoreductase transcripts, respectively. We report for the first-time type I single-, multi-domain polyketide synthases (PKSs) and hybrid non-ribosomal peptide synthase/PKS transcripts from all species. The long multi-modular PKSs were insufficient by themselves to synthesize the large complex polyether backbone of PLTX-like compounds. This implies that numerous PKS domains, including both single and multi-, work together on the biosynthesis of PLTX-like and other related polyketide compounds.

Role of Modular Polyketide Synthases in the Production of Polyether Ladder Compounds in Ciguatoxin-Producing Gambierdiscus polynesiensis and G. excentricus (Dinophyceae).

Gambierdiscus, a benthic dinoflagellate, produces ciguatoxins that cause the human illness Ciguatera. Ciguatoxins are polyether ladder compounds that have a polyketide origin, indicating that polyketide synthases (PKS) are involved in their production. We sequenced transcriptomes of Gambierdiscus excentricus and Gambierdiscus polynesiensis and found 264 contigs encoding single domain ketoacyl synthases (KS; G. excentricus: 106, G. polynesiensis: 143) and ketoreductases (KR; G. excentricus: 7, G. polynesiensis: 8) with sequence similarity to type I PKSs, as reported in other dinoflagellates. In addition, 24 contigs (G. excentricus: 3, G. polynesiensis: 21) encoding multiple PKS domains (forming typical type I PKSs modules) were found. The proposed structure produced by one of these megasynthases resembles a partial carbon backbone of a polyether ladder compound. Seventeen contigs encoding single domain KS, KR, s-malonyltransacylase, dehydratase and enoyl reductase with sequence similarity to type II fatty acid synthases (FAS) in plants were found. Type I PKS and type II FAS genes were distinguished based on the arrangement of domains on the contigs and their sequence similarity and phylogenetic clustering with known PKS/FAS genes in other organisms. This differentiation of PKS and FAS pathways in Gambierdiscus is important, as it will facilitate approaches to investigating toxin biosynthesis pathways in dinoflagellates.

Both modular and single-domain Type I polyketide synthases are expressed in the brevetoxin-producing dinoflagellate, Karenia brevis (Dinophyceae).

Dinoflagellates are prolific producers of polyketide compounds, many of which are potent toxins with adverse impacts on human and marine animal health. To identify polyketide synthase (PKS) genes in the brevetoxin-producing dinoflagellate, Karenia brevis, we assembled a transcriptome from 595 million Illumina reads, sampled under different growth conditions. The assembly included 125,687 transcripts greater than 300 nt in length, with over half having >100× coverage. We found 121 transcripts encoding Type I ketosynthase (KS) domains, of which 99 encoded single KS domains, while 22 contained multiple KS domains arranged in 1-3 protein modules. Phylogenetic analysis placed all single domain and a majority of multidomain KSs within a monophyletic clade of protist PKSs. In contrast with the highly amplified single-domain KSs, only eight single-domain ketoreductase transcripts were found in the assembly, suggesting that they are more evolutionarily conserved. The multidomain PKSs were dominated by trans-acyltransferase architectures, which were recently shown to be prevalent in other algal protists. Karenia brevis also expressed several hybrid nonribosomal peptide synthetase (NRPS)/PKS sequences, including a burA-like sequence previously reported in a wide variety of dinoflagellates. This contrasts with a similarly deep transcriptome of Gambierdiscus polynesiensis, which lacked NRPS/PKS other than the burA-like transcript, and may reflect the presence of amide-containing polyketides in K. brevis and their absence from G. polynesiensis. In concert with other recent transcriptome analyses, this study provides evidence for both single domain and multidomain PKSs in the synthesis of polyketide compounds in dinoflagellates.

Polyketide synthesis genes associated with toxin production in two species of Gambierdiscus (Dinophyceae).

BACKGROUND: Marine microbial protists, in particular, dinoflagellates, produce polyketide toxins with ecosystem-wide and human health impacts. Species of Gambierdiscus produce the polyether ladder compounds ciguatoxins and maitotoxins, which can lead to ciguatera fish poisoning, a serious human illness associated with reef fish consumption. Genes associated with the biosynthesis of polyether ladder compounds are yet to be elucidated, however, stable isotope feeding studies of such compounds consistently support their polyketide origin indicating that polyketide synthases are involved in their biosynthesis. RESULTS: Here, we report the toxicity, genome size, gene content and transcriptome of Gambierdiscus australes and G. belizeanus. G. australes produced maitotoxin-1 and maitotoxin-3, while G. belizeanus produced maitotoxin-3, for which cell extracts were toxic to mice by IP injection (LD50 = 3.8 mg kg(-1)). The gene catalogues comprised 83,353 and 84,870 unique contigs, with genome sizes of 32.5 ± 3.7 Gbp and 35 ± 0.88 Gbp, respectively, and are amongst the most comprehensive yet reported from a dinoflagellate. We found three hundred and six genes involved in polyketide biosynthesis, including one hundred and ninety-two ketoacyl synthase transcripts, which formed five unique phylogenetic clusters. CONCLUSIONS: Two clusters were unique to these maitotoxin-producing dinoflagellate species, suggesting that they may be associated with maitotoxin biosynthesis. This work represents a significant step forward in our understanding of the genetic basis of polyketide production in dinoflagellates, in particular, species responsible for ciguatera fish poisoning.

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.

Cob gene pyrosequencing enables characterization of benthic dinoflagellate diversity and biogeography.

Dinoflagellates in marine benthic habitats living epiphytically on macroalgae are an important but highly understudied group of protists. Many produce toxins that can have severe economic impacts on marine-based economies, and improved monitoring tools are required to enhance the management of toxin-related hazards. We analysed the distribution and diversity of epibenthic dinoflagellates inhabiting eight sites in Cocos (Keeling) Islands, Papua New Guinea, and Broome and Exmouth, Western Australia. We used pyrosequencing approaches based on two DNA barcoding marker genes - 18S ribosomal RNA (rRNA) and mitochondrial cytochrome b (cob) - and compared these to an approach based on clone libraries (197 sequences) using the cob gene. Dinoflagellate sequences accounted for 133 [64 unique operational taxonomic units (OTU)] out of 10 529 18S rRNA gene sequences obtained from all samples. However, using the dinoflagellate specific assay targeting the cob gene marker, we obtained 9748 (1217 unique OTU) dinoflagellate sequences from the same environmental samples, providing the largest, to date, set of dinoflagellate cob gene sequences and reliable estimates of total dinoflagellate richness within the samples and biogeographic comparisons between samples. This study also reports the presence of potentially toxic species of the genera Gambierdiscus, Ostreopsis, Coolia, Prorocentrum and Amphidinium from the above-mentioned geographical regions.

Response of planktonic microbial assemblages to disturbance in an urban sub-tropical estuary.

Microbes are sensitive indicators of estuarine processes because they respond rapidly to dynamic disturbance events. As most of the world's population lives in urban areas and climate change-related disturbance events are becoming more frequent, estuaries bounded by cities are experiencing increasing stressors, at the same time that their ecosystem services are required more than ever. Here, using a multidisciplinary approach, we determined the response of planktonic microbial assemblages in response to seasonality and a rainfall disturbance in an urban estuary bounded by Australia's largest city, Sydney. We used molecular barcoding (16S, 18S V4 rRNA) and microscopy-based identification to compare microbial assemblages at locations with differing characteristics and urbanisation histories. Across 142 samples, we identified 8,496 unique free-living bacterial zOTUs, 8,175 unique particle associated bacterial zOTUs, and 1,920 unique microbial eukaryotic zOTUs. Using microscopy, we identified only the top <10% abundant, larger eukaryotic taxa (>10 µm), however quantification was possible. The site with the greater history of anthropogenic impact showed a more even community of associated bacteria and eukaryotes, and a significant increase in dissolved inorganic nitrogen following rainfall, when compared to the more buffered site. This coincided with a reduced proportional abundance of Actinomarina and Synechococcus spp., a change in SAR 11 clades, and an increase in the eukaryotic microbial groups Dinophyceae, Mediophyceae and Bathyoccocaceae, including a temporary dominance of the harmful algal bloom dinoflagellate Prorocentrum cordatum (syn. P. minimum). Finally, a validated hydrodynamic model of the estuary supported these results, showing that the more highly urbanised and upstream location consistently experienced a higher magnitude of salinity reduction in response to rainfall events during the study period. The best abiotic variables to explain community dissimilarities between locations were TDP, PN, modelled temperature and salinity (r = 0.73) for the free living bacteria, TP for the associated bacteria (r = 0.43), and modelled temperature (r = 0.28) for the microbial eukaryotic communities. Overall, these results show that a minor disturbance such as a brief rainfall event can significantly shift the microbial assemblage of an anthropogenically impacted area within an urban estuary to a greater degree than a seasonal change, but may result in a lesser response to the same disturbance at a buffered, more oceanic influenced location. Fine scale research into the factors driving the response of microbial communities in urban estuaries to climate related disturbances will be necessary to understand and implement changes to maintain future estuarine ecosystem services.

Assessment of salinity-induced photorespiratory glycolate metabolism in Anabaena sp. PCC 7120.

This paper reports an investigation of salinity-induced glycolate metabolism in the cyanobacterium Anabaena sp. PCC 7120 (hereafter Anabaena PCC 7120). Quantitative analysis of transcripts for the photosynthesis-associated genes encoding ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco), phosphoribulokinase and transketolase, as well as those involved in glycolate metabolism (phosphoglycolate phosphatase, glycolate oxidase, alanine-glyoxylate aminotransferase and serine hydroxymethyltransferase) was performed. The expression of all investigated photosynthesis-associated genes except Rubisco was downregulated after 24 h NaCl treatment. However, under the same conditions, the transcripts encoding enzymes involved in glycolate metabolism were overexpressed. This was further confirmed by the quantitative analysis of the intermediates involved in glycolate metabolism. The intracellular levels of organic acids (glyceric, glycolic and glyoxylic acids) and amino acids (glycine and serine) were elevated in salt-treated cells as compared to those in the control cells. Transcriptional inhibition of photosynthesis-associated genes, and upregulation of genes and enhanced synthesis of intermediates associated with glycolate metabolism, indicate the occurrence of this photorespiratory metabolic pathway metabolism in Anabaena PCC 7120 under salt stress.

Evaluation of sxtA and rDNA qPCR assays through monitoring of an inshore bloom of Alexandrium catenella Group 1.

Alexandrium catenella (formerly A. tamarense Group 1, or A. fundyense) is the leading cause of Paralytic Shellfish Poisoning in North and South America, Europe, Africa, Australia and Asia. The quantification of A.catenella via sxtA, a gene involved in Paralytic Shellfish Toxin synthesis, may be a promising approach, but has not been evaluated in situ on blooms of A. catenella, in which cell abundances may vary from not detectable to in the order of 106 cells L-1. In this study, we compared sxtA assay performance to a qPCR assay targeted to a species-specific region of ribosomal DNA (rDNA) and an established fluorescent in situ hybridization (FISH) microscopy method. Passing-Bablok regression analyses revealed the sxtA assay to overestimate abundances when <5 cell equivalents A. catenella DNA were analysed, but otherwise was closer to microscopy estimates than the rDNA assay, which overestimated abundance across the full range of concentrations analysed, indicative of a copy number difference between the bloom population and a culture used for assay calibration a priori. In contrast, the sxtA assay performed more consistently, indicating less copy number variation. The sxtA assay was generally reliable, fast and effective in quantifying A. catenella and was predictive of PST contamination of shellfish.

An aerobic eukaryotic parasite with functional mitochondria that likely lacks a mitochondrial genome.

Dinoflagellates are microbial eukaryotes that have exceptionally large nuclear genomes; however, their organelle genomes are small and fragmented and contain fewer genes than those of other eukaryotes. The genus Amoebophrya (Syndiniales) comprises endoparasites with high genetic diversity that can infect other dinoflagellates, such as those forming harmful algal blooms (e.g., Alexandrium). We sequenced the genome (~100 Mb) of Amoebophrya ceratii to investigate the early evolution of genomic characters in dinoflagellates. The A. ceratii genome encodes almost all essential biosynthetic pathways for self-sustaining cellular metabolism, suggesting a limited dependency on its host. Although dinoflagellates are thought to have descended from a photosynthetic ancestor, A. ceratii appears to have completely lost its plastid and nearly all genes of plastid origin. Functional mitochondria persist in all life stages of A. ceratii, but we found no evidence for the presence of a mitochondrial genome. Instead, all mitochondrial proteins appear to be lost or encoded in the A. ceratii nucleus.

Characterization of a novel multidrug resistance plasmid pSGB23 isolated from Salmonella enterica subspecies enterica serovar Saintpaul.

BACKGROUND: Salmonella enterica subspecies enterica serovar Saintpaul (S. Saintpaul) is an important gut pathogen which causes salmonellosis worldwide. Although intestinal salmonellosis is usually self-limiting, it can be life-threatening in children, the elderlies and immunocompromised patients. Appropriate antibiotic treatment is therefore required for these patients. However, the efficacy of many antibiotics on S. enterica infections has been greatly compromised due to spreading of multidrug resistance (MDR) plasmids, which poses serious threats on public health and needs to be closely monitored. In this study, we sequenced and fully characterized an S. enterica MDR plasmid pSGB23 isolated from chicken. RESULTS: Complete genome sequence analysis revealed that S. Saintpaul strain SGB23 harbored a 254 kb megaplasmid pSGB23, which carries 11 antibiotic resistance genes responsible for resistance to 9 classes of antibiotics and quaternary ammonium compounds that are commonly used to disinfect food processing facilities. Furthermore, we found that pSGB23 carries multiple conjugative systems, which allow it to spread into other Enterobacteriaceae spp. by self-conjugation. It also harbors multiple types of replicons and plasmid maintenance and addictive systems, which explains its broad host range and stable inheritance. CONCLUSIONS: We report here a novel MDR plasmid pSGB23 harboured by S. enterica. To our knowledge, it carried the greatest number of antibiotic resistance genes with the broadest range of resistance spectrum among S. enterica MDR plasmids identified so far. The isolation of pSGB23 from food sources is worrisome, while surveillance on its further spreading will be carried out based on the findings reported in this study.

Predation by Bdellovibrio bacteriovorus significantly reduces viability and alters the microbial community composition of activated sludge flocs and granules.

We recently isolated and characterised a predatory Bdellovibrio bacteriovorus strain from activated sludge (Ulu Pandan Water Reclamation Plant, Singapore), and this strain, B. bacteriovorus UP, was able to prey upon a broad spectrum of bacterial isolates from the activated sludge when grown as planktonic cells or as biofilms. Here, we have tested the effect of Bdellovibrio predation on floccular and granular sludge to determine if the spatial organisation, loosely or tightly aggregated communities, was protective from predation. The effect of predation was assessed using a combination of biomass quantification, cellular activity measurement and microscopic image analysis to determine community viability. Additionally, changes in the microbial communities due to predation by B. bacteriovorus UP were analysed through total RNA sequencing. Predation led to a significant reduction in microbial activity and total biomass for both floccular and granular sludge communities. Predation was also associated with significant changes in the microbial community composition in both communities, with >90% of the community members reduced in relative abundance after 24 h. Of those community members, the dominant organisms, such as Proteobacteria and Bacteroidetes, were the most affected phylotypes. This suggests that predatory bacteria, which display indiscriminant feeding, could significantly shift the species composition and thus, may disturb the operational performance of wastewater treatment systems.

Qualitative and quantitative assessment of the presence of ciguatoxin, P-CTX-1B, in Spanish Mackerel (Scomberomorus commerson) from waters in New South Wales (Australia).

Ciguatera Fish Poisoning (CFP) is a tropical disease caused by the consumption of fish contaminated with ciguatoxins (CTXs). Currently, the only feasible prevention methods for CFP are to avoid the consumption of fish of certain species from some regions, avoid larger fish of certain species, or avoid all fish caught from specific regions. Here, we quantified levels of P-CTX-1B in Spanish Mackerel (Scomberomorus commerson), which is the main fish species that causes CFP in New South Wales and Queensland, Australia, using LC-MS detection against a toxin standard. We found detectable P-CTX-1B in both flesh and liver tissues in fish from New South Wales (n = 71, 1.4% prevalence rate, with a confidence interval of 1%-4%, and 7% prevalence, 1%-12%, in flesh and liver, respectively). In the small sample of fish from Queensland, there was a 46% prevalence (19-73%, n = 13). Toxin levels found were 0.13 µg kg-1 to <0.1 µg kg-1 in flesh, and 1.39 µg kg-1 to <0.4 µg kg-1 in liver, indicating that liver tissue had a significantly higher concentration (∼5 fold) of P-CTX-1B. No apparent relationship was observed between the length or weight of S. commerson and the detection of P-CTX-1B in this study. Footnote.

A new species of Gambierdiscus (Dinophyceae) from the south-west Pacific: Gambierdiscus honu sp. nov.

Two isolates of a new tropical, epiphytic dinoflagellate species, Gambierdiscus honu sp. nov., were obtained from macroalgae sampled in Rarotonga, Cook Islands, and from North Meyer Island, Kermadec Islands. Gambierdiscus honu sp. nov. had the common Gambierdiscus Kofoidian plate formula: Po, 3', 6″, 6C?, 6 or 7S, 5‴, 1p and 2⁗. The characteristic morphological features of this species were its relatively small short dorsoventral length and width and the shape of individual plates, in particular the combination of the hatchet-shaped 2' and pentagonal 3' plates and the length to width ratio of the antapical 1p plate. The combination of these characteristics plus the smooth thecal surface and equal sized 1⁗ and 2⁗ plates differentiated this species from other Gambierdiscus species. The phylogenetic analyses supported the unique description. Both isolates of G. honu produced the putative maitotoxin (MTX)-3 analogue, but neither produced ciguatoxin (CTX) or MTX. Extracts of G. honu were shown to be highly toxic to mice by intraperitoneal injection (0.2mg/kg), although less toxic by gavage. It is possible that toxins other than putative MTX-3 are produced.

Reduced Intracellular c-di-GMP Content Increases Expression of Quorum Sensing-Regulated Genes in Pseudomonas aeruginosa.

Cyclic-di-GMP (c-di-GMP) is an intracellular secondary messenger which controls the biofilm life cycle in many bacterial species. High intracellular c-di-GMP content enhances biofilm formation via the reduction of motility and production of biofilm matrix, while low c-di-GMP content in biofilm cells leads to increased motility and biofilm dispersal. While the effect of high c-di-GMP levels on bacterial lifestyles is well studied, the physiology of cells at low c-di-GMP levels remains unclear. Here, we showed that Pseudomonas aeruginosa cells with high and low intracellular c-di-GMP contents possessed distinct transcriptome profiles. There were 535 genes being upregulated and 432 genes downregulated in cells with low c-di-GMP, as compared to cells with high c-di-GMP. Interestingly, both rhl and pqs quorum-sensing (QS) operons were expressed at higher levels in cells with low intracellular c-di-GMP content compared with cells with higher c-di-GMP content. The induced expression of pqs and rhl QS required a functional PqsR, the transcriptional regulator of pqs QS. Next, we observed increased production of pqs and rhl-regulated virulence factors, such as pyocyanin and rhamnolipids, in P. aeruginosa cells with low c-di-GMP levels, conferring them with increased intracellular survival rates and cytotoxicity against murine macrophages. Hence, our data suggested that low intracellular c-di-GMP levels in bacteria could induce QS-regulated virulence, in particular rhamnolipids that cripple the cellular components of the innate immune system.

The rapid in vivo evolution of Pseudomonas aeruginosa in ventilator-associated pneumonia patients leads to attenuated virulence.

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe airway infections in humans. These infections are usually difficult to treat and associated with high mortality rates. While colonizing the human airways, P. aeruginosa could accumulate genetic mutations that often lead to its better adaptability to the host environment. Understanding these evolutionary traits may provide important clues for the development of effective therapies to treat P. aeruginosa infections. In this study, 25 P. aeruginosa isolates were longitudinally sampled from the airways of four ventilator-associated pneumonia (VAP) patients. Pacbio and Illumina sequencing were used to analyse the in vivo evolutionary trajectories of these isolates. Our analysis showed that positive selection dominantly shaped P. aeruginosa genomes during VAP infections and led to three convergent evolution events, including loss-of-function mutations of lasR and mpl, and a pyoverdine-deficient phenotype. Specifically, lasR encodes one of the major transcriptional regulators in quorum sensing, whereas mpl encodes an enzyme responsible for recycling cell wall peptidoglycan. We also found that P. aeruginosa isolated at late stages of VAP infections produce less elastase and are less virulent in vivo than their earlier isolated counterparts, suggesting the short-term in vivo evolution of P. aeruginosa leads to attenuated virulence.

Evolutionary distinctiveness of fatty acid and polyketide synthesis in eukaryotes.

Fatty acids, which are essential cell membrane constituents and fuel storage molecules, are thought to share a common evolutionary origin with polyketide toxins in eukaryotes. While fatty acids are primary metabolic products, polyketide toxins are secondary metabolites that are involved in ecologically relevant processes, such as chemical defence, and produce the adverse effects of harmful algal blooms. Selection pressures on such compounds may be different, resulting in differing evolutionary histories. Surprisingly, some studies of dinoflagellates have suggested that the same enzymes may catalyse these processes. Here we show the presence and evolutionary distinctiveness of genes encoding six key enzymes essential for fatty acid production in 13 eukaryotic lineages for which no previous sequence data were available (alveolates: dinoflagellates, Vitrella, Chromera; stramenopiles: bolidophytes, chrysophytes, pelagophytes, raphidophytes, dictyochophytes, pinguiophytes, xanthophytes; Rhizaria: chlorarachniophytes, haplosporida; euglenids) and 8 other lineages (apicomplexans, bacillariophytes, synurophytes, cryptophytes, haptophytes, chlorophyceans, prasinophytes, trebouxiophytes). The phylogeny of fatty acid synthase genes reflects the evolutionary history of the organism, indicating selection to maintain conserved functionality. In contrast, polyketide synthase gene families are highly expanded in dinoflagellates and haptophytes, suggesting relaxed constraints in their evolutionary history, while completely absent from some protist lineages. This demonstrates a vast potential for the production of bioactive polyketide compounds in some lineages of microbial eukaryotes, indicating that the evolution of these compounds may have played an important role in their ecological success.

A new Gambierdiscus species (Dinophyceae) from Rarotonga, Cook Islands: Gambierdiscus cheloniae sp. nov.

Ciguatera fish poisoning (CFP) has been reported for many years in Rarotonga, Cook Islands, and has had the world's highest reported incidence of this illness for the last 20 years. Following intensive sampling to understand the distribution of the causative organisms of CFP, an undescribed Gambierdiscus species was isolated from the Rarotongan lagoon. Gambierdiscus cheloniae sp. nov. has the common Gambierdiscus Kofoidian plate formula (except for a variability in the number of precingular plates in aberrant cells): Po, 3', 6″ (7″), 6C?, 6 or 7S, 5'″, 1p and 2″″. The 2' plate is hatchet shaped and the dorsal end of 1p is pointed and the relatively narrow 1p plate. Morphologically G. cheloniae is similar to the genetically closely related species G. pacificus, G. toxicus and G. belizeanus, although smaller (depth and length) than G. toxicus. The apical pore plate varies from those of G. belizeanus and G. pacificus, which are shorter and narrower, and from G. toxicus, which is larger. G. cheloniae also differs from G. pacificus in the shape of the 2' plate. The description of this new species is supported by phylogenetic analyses using three different gene regions. G. cheloniae produced the putative maitotoxin-3 analogue, MTX-3, but neither maitotoxin or monitored ciguatoxin. Extracts of G. cheloniae were shown to be highly toxic to mice by intraperitoneal (i.p.) injection, although they were less toxic by gavage. It is possible that this species produces toxins other than putative MTX-3.

A fish kill associated with a bloom of Amphidinium carterae in a coastal lagoon in Sydney, Australia.

We report on a dense bloom (~1.80 × 105 cells mL-1) of the marine dinoflagellate species Amphidinium carterae (Genotype 2) in a shallow, small intermittently open coastal lagoon in south eastern Australia. This bloom co-occurred with the deaths of >300 individuals of three different species of fish. The opening of the lagoon to the ocean, as well as localized high nutrient levels, preceded the observations of very high cell numbers. A. carterae is usually benthic and sediment-dwelling, but temporarily became abundant throughout the water column in this shallow (<2 m) sandy habitat. Histopathological results showed that the Anguilla reinhardtii individuals examined had damage to epithelial and gill epithelial cells. An analysis of the bloom water indicated the presence of a compound with a retention time and UV spectra similar to Luteophanol A, a compound known from a strain of Amphidinium. Assays with a fish gill cell line were conducted using a purified compound from cells concentrated from the bloom, and was found to cause a loss of 87% in cell viability in 6 h. The fish deaths were likely due to the low dissolved oxygen levels in the water and/or the presence of Luteophanol A-like compounds released during the bloom.

Biosynthesis of toxic naturally-occurring seafood contaminants.

Outbreaks of human illness caused by the consumption of contaminated seafood, continues to be a major problem particularly for the shellfish industry. Toxins from marine, brackish and freshwater environments, which are often produced as a result of harmful algal blooms, have been implicated as the causative agents of these poisonings. Commonly, poisoning events have been grouped into one of six classes, Paralytic Shellfish Poisoning (PSP), Diarrhetic Shellfish Poisoning (DSP), Neurotoxic Shellfish Poisoning (NSP), Ciguatera Fish Poisoning (CFP), Azaspiracid Shellfish Poisoning (AZP), and Amnesiac Shellfish Poisoning (ASP). The causative agents of these specific poisonings along with their biosyntheses are discussed in this review. The highly unusual and complex structures of most common seafood toxins have made them interesting targets for biosynthetic studies. Many of the toxins presented are biosynthesized via complex pathways that have been elucidated either through isotope labelled precursor feeding studies and/or characterization of the genes encoding the producing organism's biosynthetic machinery. Feeding studies key to our understanding of a particular toxin's biosynthesis, such as the incorporation of unusual precursors, as well as unique biosynthetic pathways and rare chemical mechanisms involved in the assembly process are highlighted. More recently, however, modern genomics-based techniques have been used for the elucidation of biosynthetic pathways and these are presented in the context of polyketide, non-ribosomal peptide, and hybrid pathway derived, toxin assembly.