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Caused by both eukaryotic dinoflagellates and prokaryotic cyanobacteria, harmful algal blooms are events of severe ecological, economic, and public health consequence, and their incidence has become more common of late. Despite coordinated research efforts to identify and characterize the genomes of harmful algal bloom-causing organisms, the genomic basis and evolutionary origins of paralytic shellfish toxins produced by harmful algal blooms remain at best incomplete. The paralytic shellfish toxin saxitoxin has an especially complex genomic architecture and enigmatic phylogenetic distribution, spanning dinoflagellates and multiple cyanobacterial genera. Using filtration and extraction techniques to target the desired cyanobacteria from nonaxenic culture, coupled with a combination of short- and long-read sequencing, we generated a reference-quality hybrid genome assembly for Heteroscytonema crispum UTEX LB 1556, a freshwater, paralytic shellfish toxin-producing cyanobacterium thought to have the largest known genome in its phylum. We report a complete, novel biosynthetic gene cluster for the paralytic shellfish toxin saxitoxin. Leveraging this biosynthetic gene cluster, we find support for the hypothesis that paralytic shellfish toxin production has appeared in divergent Cyanobacteria lineages through widespread and repeated horizontal gene transfer. This work demonstrates the utility of long-read sequencing and metagenomic assembly toward advancing our understanding of paralytic shellfish toxin biosynthetic gene cluster diversity and suggests a mechanism for the origin of paralytic shellfish toxin biosynthetic genes., (Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution 2024.)

In 2015, the largest recorded harmful algal bloom (HAB) occurred in the Northeast Pacific, causing nearly 100 million dollars in damages to fisheries and killing many protected marine mammals. Dominated by the toxic diatom Pseudo-nitzschia australis , this bloom produced high levels of the neurotoxin domoic acid (DA). Through molecular and transcriptional characterization of 52 near-weekly phytoplankton net-tow samples collected at a bloom hotspot in Monterey Bay, California, we identified active transcription of known DA biosynthesis ( dab ) genes from the three identified toxigenic species, including P. australis as the primary origin of toxicity. Elevated expression of silicon transporters ( sit1 ) during the bloom supports the previously hypothesized role of dissolved silica (Si) exhaustion in contributing to bloom physiology and toxicity. We find that coexpression of the dabA and sit1 genes serves as a robust predictor of DA one week in advance, potentially enabling the forecasting of DA-producing HABs. We additionally present evidence that low levels of iron could have colimited the diatom population along with low Si. Iron limitation represents an overlooked driver of both toxin production and ecological success of the low-iron-adapted Pseudo-nitzschia genus during the 2015 bloom, and increasing pervasiveness of iron limitation may fuel the escalating magnitude and frequency of toxic Pseudo-nitzschia blooms globally. Our results advance understanding of bloom physiology underlying toxin production, bloom prediction, and the impact of global change on toxic blooms., Competing Interests: Competing interests statement:The authors declare no competing interest.

The catastrophic loss of aquatic life in the Central European Oder River in 2022, caused by a toxic bloom of the haptophyte microalga Prymnesium parvum (in a wide sense, s.l.), underscores the need to improve our understanding of the genomic basis of the toxin. Previous morphological, phylogenetic, and genomic studies have revealed cryptic diversity within P. parvum s.l. and uncovered three clade-specific (types A, B, and C) prymnesin toxins. Here, we used state-of-the-art long-read sequencing and assembled the first haplotype-resolved diploid genome of a P. parvum type B from the strain responsible for the Oder disaster. Comparative analyses with type A genomes uncovered a genome-size expansion driven by repetitive elements in type B. We also found conserved synteny but divergent evolution in several polyketide synthase (PKS) genes, which are known to underlie toxin production in combination with environmental cues. We identified an approximately 20-kbp deletion in the largest PKS gene of type B that we link to differences in the chemical structure of types A and B prymnesins. Flow cytometry and electron microscopy analyses confirmed diploidy in the Oder River strain and revealed differences to closely related strains in both ploidy and morphology. Our results provide unprecedented resolution of strain diversity in P. parvum s.l. and a better understanding of the genomic basis of toxin variability in haptophytes. The reference-quality genome will enable us to better understand changes in microbial diversity in the face of increasing environmental pressures and provides a basis for strain-level monitoring of invasive Prymnesium in the future., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Dinoflagellate species that form some of the most frequent toxic blooms are also bioluminescent, yet the two traits are rarely linked when studying bloom development and persistence. P. bahamense is a toxic, bioluminescent dinoflagellate that previously bloomed in Florida with no known record of saxitoxin (STX) production. Over the past 20 years, STX was identified in P. bahamense populations. The goal of this study was to examine toxin dynamics and associated molecular mechanisms in spatially and temporally distinct P. bahamense populations from the Indian River Lagoon, FL. SxtA4 is a key gene required for toxin biosynthesis. SxtA4 genotype analysis was performed on individual cells from multiple sites. Cell abundance, toxin quota cell -1 , and sxtA4 and RubisCo ( rbcL ) transcript abundance were also measured. There was a significant negative correlation between cell abundance and toxin quota cell -1 . While the sxtA4+ genotype was dominant at all sites, its frequency varied, but it occurred at 90-100% in many samples. The underlying mechanism for toxin decrease with increased cell abundance remains unknown. However, a strong, statistically significant negative correlation was found between stxA4 transcripts and the sxtA4/rbcL ratio, suggesting cells make fewer sxtA4 transcripts as a bloom progresses. However, the influence of sxtA4- cells must also be considered. Future plans include bioluminescence measurements, normalized to a per cell basis, at sites when toxicity is measured along with concomitant quantification of sxtA4 gene and transcript copy numbers as a means to elucidate whether changes in bloom toxicity are driven more at the genetic (emergence of sxtA4- cells) or transcriptional (repression of sxtA4 in sxtA4+ cells) level. Based on the results of this study, a model is proposed that links the combined traits of toxicity and bioluminescence in P. bahamense bloom development.

Blooms of Prymnesium parvum, a unicellular alga globally distributed in marine and brackish environments, frequently result in massive fish kills due to the production of toxins called prymnesins by this haptophyte. In August 2022, a harmful algal bloom (HAB) of this species occurred in the lower Oder River (Poland and Germany), which caused mass mortalities of fish and other organisms. This HAB was linked to low discharge of the Oder and mining activities that caused a significant increase in salinity. In this context, we report on the molecular detection and screening of this haptophyte and its toxins in environmental samples and clonal cultures derived thereof. Both conventional PCR and droplet digital PCR assays reliably detected P. parvum in environmental samples. eDNA metabarcoding using the V4 region of the 18S rRNA gene revealed a single Prymnesium sequence variant, but failed to identify it to species level. Four clonal cultures established from environmental samples were unambiguously identified as P. parvum by molecular phylogenetics (near full-length 18S rRNA gene) and light microscopy. Phylogenetic analysis (ITS1-5.8S-ITS2 marker region) placed the cultured phylotype within a clade containing other P. parvum strains known to produce B-type prymnesins. Toxin-screening of the cultures using liquid chromatography-electrospray ionization - time of flight mass spectrometry identified B-type prymnesins, which were also detected in extracts of filter residues from water samples of the Oder collected during the HAB. Overall, our investigation provides a detailed characterization of P. parvum, including their prymnesins, during this HAB in the Oder River, contributing valuable insights into this ecological disaster. In addition, the droplet digital PCR assay established here will be useful for future monitoring of low levels of P. parvum on the Oder River or any other salt-impacted and brackish water bodies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Dinoflagellates of the genus Alexandrium are responsible for harmful algal blooms and produce paralytic shellfish toxins (PSTs). Their very large and complex genomes make it challenging to identify the genes responsible for toxin synthesis. A family-based genomic association study was developed to determine the inheritance of toxin production in Alexandrium minutum and identify genomic regions linked to this production. We show that the ability to produce toxins is inheritable in a Mendelian way, while the heritability of the toxin profile is more complex. We developed the first dinoflagellate genetic linkage map. Using this map, several major results were obtained: 1. A genomic region related to the ability to produce toxins was identified. 2. This region does not contain any polymorphic sxt genes, known to be involved in toxin production in cyanobacteria. 3. The sxt genes, known to be present in a single cluster in cyanobacteria, are scattered on different linkage groups in A. minutum . 4. The expression of two sxt genes not assigned to any linkage group, sxtI and sxtG , may be regulated by the genomic region related to the ability to produce toxins. Our results provide new insights into the organization of toxicity-related genes in A. minutum , suggesting a dissociated genetic mechanism for the production of the different analogues and the ability to produce toxins. However, most of the newly identified genes remain unannotated. This study therefore proposes new candidate genes to be further explored to understand how dinoflagellates synthesize their toxins.

The bloom-forming toxic dinoflagellate Alexandrium catenella was first detected in southern Chile (39.5-55° S) 50 years ago and is responsible for most of the area's cases of paralytic shellfish poisoning (PSP). Given the complex life history of A. catenella , which includes benthic sexual cysts, in this study, we examined the potential link between latitude, toxicity, and sexual compatibility. Nine clones isolated from Chilean Patagonia were used in self- and out-crosses in all possible combinations ( n = 45). The effect of latitude on toxicity, reproductive success indexes, and cyst production was also determined. Using the toxin profiles for all strains, consisting of C1, C2, GTX4, GTX1, GTX3, and NeoSTX, a latitudinal gradient was determined for their proportions (%) and content per cell (pg cell -1 ), with the more toxic strains occurring in the north (-40.6° S). Reproductive success also showed a latitudinal tendency and was lower in the north. None of the self-crosses yielded resting cysts. Rather, the production of resting cysts was highest in pairings of clones separated by distances of 1000-1650 km. Our results contribute to a better understanding of PSP outbreaks in the region and demonstrate the importance of resting cysts in fueling new toxic events. They also provide additional evidence that the introduction of strains from neighboring regions is a cause for concern.

As the secondary metabolites of cyanobacterial harmful algal blooms (Cyano-HABs), microcystins (MCs) were generated under various environmental and cellular conditions. The understanding of the causes of MCs generation is of great interest in the field of water treatment and environmental science. In this work, we studied how Microcystis aeruginosa (FACHB-905) cell densities affect the MCs synthetase genes (mcy) expression, microcystin-LR (MC-LR) and quorum sensing molecules (Acyl-homoserine lactones (AHLs)) production. An electrochemical sensor was developed here for sensitive and quantitative detection of MC-LR that cultured at different cell densities. The results showed that mcy expression and MC-LR concentration started to increase when the cell density reached ca. 22 × 10 6 cells/mL, and was significantly increased with increasing cell densities. Moreover, the up-regulation of AHLs with increasing cell densities revealed that MC-LR is quorum sensing-mediated. Our results undoubtedly confirmed that MC-LR was produced in a cell density-dependent way that mimics quorum sensing, and the minimum cell density (ca. 22 × 10 6 cells/mL) that was required to produce MC-LR was provided and offered a reference standard for the prevention and control of MCs pollution in the actual water environment., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Due to increased frequency of cyanobacterial blooms and emerging evidence of cyanotoxicity in biofilm, reliable methods for early cyanotoxin threat detection are of major importance for protection of human, animal and environmental health. To complement the current methods of risk assessment, this study aimed to evaluate selected qPCR assays for detection of potentially toxic cyanobacteria in environmental samples. In the course of one year, 25 plankton and 23 biofilm samples were collected from 15 water bodies in Slovenia. Three different analyses were performed and compared to each other; qPCR targeting mcyE , cyrJ and sxtA genes involved in cyanotoxin production, LC-MS/MS quantifying microcystin, cylindrospermopsin and saxitoxin concentration, and microscopic analyses identifying potentially toxic cyanobacterial taxa. qPCR analyses detected potentially toxic Microcystis in 10 lake plankton samples, and potentially toxic Planktothrix cells in 12 lake plankton and one lake biofilm sample. A positive correlation was observed between numbers of mcyE gene copies and microcystin concentrations. Potential cylindrospermopsin- and saxitoxin-producers were detected in three and seven lake biofilm samples, respectively. The study demonstrated a potential for cyanotoxin production that was left undetected by traditional methods in both plankton and biofilm samples. Thus, the qPCR method could be useful in regular monitoring of water bodies to improve risk assessment and enable timely measures.

Diarrheic shellfish poisoning (DSP) is caused by the consumption of shellfish contaminated with a group of phycotoxins that includes okadaic acid (OA), dinophysistoxin-1 (DTX-1), and dinophysistoxin-2 (DTX-2). These toxins are inhibitors of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A), but show distinct levels of toxicity. Aside from a difference in protein phosphatases (PP) inhibition potency that would explain these differences in toxicity, others mechanisms of action are thought to be involved. Therefore, we investigated and compared which mechanisms are involved in the toxicity of these three analogues. As the intestine is one of the target organs, we studied the transcriptomic profiles of human intestinal epithelial Caco-2 cells exposed to OA, DTX-1, and DTX-2. The pathways specifically affected by each toxin treatment were further confirmed through the expression of key genes and markers of toxicity. Our results did not identify any distinct biological mechanism for OA and DTX-2. However, only DTX-1 induced up-regulation of the MAPK transduction signalling pathway, and down-regulation of gene products involved in the regulation of DNA repair. As a consequence, based on transcriptomic results, we demonstrated that the higher toxicity of DTX-1 compared to OA and DTX-2 was consistent with certain specific pathways involved in intestinal cell response.

Background: We successfully captured a kind of gelatinous organism DA-6 from Antarctic water, extracted its total RNAs and proteins, and performed species identification through a combination of transcriptomics and proteomics in this study., Methods: The gelatinous organism DA-6 was captured 200 m underwater in Antarctica. Total RNA was extracted to construct the transcriptome and the proteins were identified by LC-MS/MS., Results: DA-6 was identified as an Antarctic Salpa sp. through morphological examination and MT-CO1 phylogenetic analysis. A total of 47,183 unigenes were harvested through transcriptome. We also successfully annotated 11,954 (25.34%), 10,006 (22.21%), 4469 (9.47%) and 4901 (9.71%) unigenes with NR, SwissProt, GO and KEGG databases, respectively. In the proteomic analysis, a total of 4680 peptides and 1280 proteins were harvested using the transcriptome as the reference database. A number of both 549 (31.98%) proteins reannotated against the GO and KEGG databases. Moreover, a number of 5 toxic proteins matched from the 89 toxin-related unigenes were successfully screened, including 2 metalloproteinases, 1 serine protease, 1 serine protease inhibitor and 1 aflatoxin., Conclusion: Our study is the first to identify an Antarctic Salpa sp. according to the combination of de novo transcriptomics and proteomics, which can further be served as a public database for the identification of potential polar Salpa-derived lead compounds. In addition to morphology and CO1, the combined analysis of transcriptome and proteome can also be used as a value method for new species identify e.g. Salpa sp., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Nemerteans (ribbon worms) employ toxins to subdue their prey, but research thus far has focused on the small-molecule components of mucus secretions and few protein toxins have been characterized. We carried out a preliminary proteotranscriptomic analysis of putative toxins produced by the hoplonemertean Amphiporus lactifloreus (Hoplonemertea, Amphiporidae). No variants were found of known nemertean-specific toxin proteins (neurotoxins, cytotoxins, parbolysins or nemertides) but several toxin-like transcripts were discovered, expressed strongly in the proboscis, including putative metalloproteinases and sequences resembling sea anemone actitoxins, crown-of-thorn sea star plancitoxins, and multiple classes of inhibitor cystine knot/knottin family proteins. Some of these products were also directly identified in the mucus proteome, supporting their preliminary identification as secreted toxin components. Two new nemertean-typical toxin candidates could be described and were named U-nemertotoxin-1 and U-nemertotoxin-2. Our findings provide insight into the largely overlooked venom system of nemerteans and support a hypothesis in which the nemertean proboscis evolved in several steps from a flesh-melting organ in scavenging nemerteans to a flesh-melting and toxin-secreting venom apparatus in hunting hoplonemerteans.

In paleoecological studies, molecular markers are being used increasingly often to reconstruct community structures, environmental conditions and ecosystem changes. In this work, nodularin, anabaenopeptins and selected DNA sequences were applied as Nodularia spumigena markers to reconstruct the history of the cyanobacterium in the Norwegian fjords. For the purpose of this study, three sediment cores collected in Oslofjorden, Trondheimsfjorden and Balsfjorden were analyzed. The lack of nodularin in most recent sediments is consistent with the fact that only one report on the sporadic occurrence and low amounts of the cyanobacterium in Norwegian Fjords in 1976 has been published. However, analyses of species-specific chemical markers in deep sediments showed that thousands of years ago, N. spumigena constituted an important component of the phytoplankton community. The content of the markers in the cores indicated that the biomass of the cyanobacterium increased during the warmer Holocene periods. The analyses of genetic markers were less conclusive; they showed the occurrence of microcystin/nodularin producing cyanobacteria of Nostocales order, but they did not allow for the identification of the organisms at a species level.

Cyanobacteria produce an array of toxins that pose serious health risks to humans and animals. The closely related diazotrophic genera, Anabaena, Dolichospermum , and Aphanizomenon , frequently form poisonous blooms in lakes and brackish waters around the world. These genera form a complex now termed the Anabaena, Dolichospermum , and Aphanizomenon (ADA) clade and produce a greater array of toxins than any other cyanobacteria group. However, taxonomic confusion masks the distribution of toxin biosynthetic pathways in cyanobacteria. Here we obtained 11 new draft genomes to improve the understanding of toxin production in these genera. Comparison of secondary metabolite pathways in all available 31 genomes for these three genera suggests that the ability to produce microcystin, anatoxin-a, and saxitoxin is associated with specific subgroups. Each toxin gene cluster was concentrated or even limited to a certain subgroup within the ADA clade. Our results indicate that members of the ADA clade encode a variety of secondary metabolites following the phylogenetic clustering of constituent species. The newly sequenced members of the ADA clade show that phylogenetic separation of planktonic Dolichospermum and benthic Anabaena is not complete. This underscores the importance of taxonomic revision of Anabaena, Dolichospermum , and Aphanizomenon genera to reflect current phylogenomic understanding., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Ciguatera fish poisoning (CFP) is a human illness caused via consumption of seafood contaminated with neurotoxins produced by some species from the epiphytic dinoflagellate genus Gambierdiscus. In this study, we describe two new species of Gambierdiscus isolated from Heron Island in the Southern Great Barrier Reef, Queensland, Australia. These new species were analysed using light microscopy, scanning electron microscopy, and phylogenetic analyses of nuclear encoded ribosomal ITS, SSU as well as D1-D3 and D8-D10 of the LSU gene regions. Gambierdiscus lewisii sp. nov. (Po, 3', 0a, 7″, 6c,? s, 5‴, 0p, 2'‴) is distinguished by its strong reticulate-foveate ornamentation and is genetically distinct from its sister species, G. pacificus. Gambierdiscus holmesii sp. nov. (Po, 3', 0a, 7″, 6c, 6s?, 5‴, 0p, 2'‴) is morphologically distinct from the genetically similar species G. silvae because of a strongly ventrally displaced apical pore complex and a characteristic fold at the anterior edge of the sulcus. Both G. lewisii and G. holmesii produce putative Maitotoxin-(44-Methylgambierone) and compounds which show ciguatoxin and maitotoxin-like activities. Identification of two new Gambierdiscus species will enable us to more accurately assess the risk of CFP in Australia and internationally., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Hydra actinoporin-like toxin 1 (HALT-1) was previously shown to cause cytolysis and haemolysis in a number of human cells and has similar functional properties to the actinoporins equinatoxin and sticholysin. In addition to HALT-1, five other HALTs (HALTs 2, 3, 4, 6 and 7) were also isolated from Hydra magnipapillata and expressed as recombinant proteins in this study. We demonstrated that recombinant HALTs have cytolytic activity on HeLa cells but each exhibited a different range of toxicity. All six recombinant HALTs bound to sulfatide, while rHALT-1 and rHALT-3 bound to two additional sphingolipids, lysophosphatidic acid and sphingosine-1-phosphate as indicated by the protein-lipid overlay assay. When either tryptophan 133 or tyrosine 129 of HALT-1 was mutated, the mutant protein lost binding to sulfatide, lysophosphatidic acid and sphingosine-1-phosphate. As further verification of HALTs' binding to sulfatide, we performed ELISA for each HALT. To determine the cell-type specific gene expression of seven HALTs in Hydra, we searched for individual HALT expression in the single-cell RNA-seq data set of Single Cell Portal. The results showed that HALT-1, 4 and 7 were expressed in differentiating stenoteles. HALT-1 and HALT-6 were expressed in the female germline during oogenesis. HALT-2 was strongly expressed in the gland and mucous cells in the endoderm. Information on HALT-3 and HALT-5 could not be found in the single-cell data set. Our findings show that subfunctionalisation of gene expression following duplication enabled HALTs to become specialized in various cell types of the interstitial cell lineage., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The emergence of marine biotoxins in geographical areas where they have never been reported before is a concern of considerable impact on seafood contamination, and consequently, on public health [...].

Background: A major cause of phytoplankton mortality is predation by zooplankton. Strategies to avoid grazers have probably played a major role in the evolution of phytoplankton and impacted bloom dynamics and trophic energy transport. Certain species of the genus Pseudo-nitzschia produce the neurotoxin, domoic acid (DA), as a response to the presence of copepod grazers, suggesting that DA is a defense compound. The biosynthesis of DA comprises fusion of two precursors, a C10 isoprenoid geranyl pyrophosphate and L-glutamate. Geranyl pyrophosphate (GPP) may derive from the mevalonate isoprenoid (MEV) pathway in the cytosol or from the methyl-erythritol phosphate (MEP) pathway in the plastid. L-glutamate is suggested to derive from the citric acid cycle. Fragilariopsis, a phylogenetically related but nontoxic genus of diatoms, does not appear to possess a similar defense mechanism. We acquired information on genes involved in biosynthesis, precursor pathways and regulatory functions for DA production in the toxigenic Pseudo-nitzschia seriata, as well as genes involved in responses to grazers to resolve common responses for defense strategies in diatoms., Results: Several genes are expressed in cells of Pseudo-nitzschia when these are exposed to predator cues. No genes are expressed in Fragilariopsis when treated similarly, indicating that the two taxa have evolved different strategies to avoid predation. Genes involved in signal transduction indicate that Pseudo-nitzschia cells receive signals from copepods that transduce cascading molecular precursors leading to the formation of DA. Five out of seven genes in the MEP pathway for synthesis of GPP are upregulated, but none in the conventional MEV pathway. Five genes with known or suggested functions in later steps of DA formation are upregulated. We conclude that no gene regulation supports that L-glutamate derives from the citric acid cycle, and we suggest the proline metabolism to be a downstream precursor., Conclusions: Pseudo-nitzschia cells, but not Fragilariopsis, receive and respond to copepod cues. The cellular route for the C10 isoprenoid product for biosynthesis of DA arises from the MEP metabolic pathway and we suggest proline metabolism to be a downstream precursor for L-glutamate. We suggest 13 genes with unknown function to be involved in diatom responses to grazers.

Shellfish is a highly valuated natural food product that is usually consumed minimally processed. Some foodborne pathogens have been associated to marine products and isolated from aquatic environments. Helicobacter pylori emerges as one of the most concerning human pathogens associated to water and, thereby, it could be present in raw and slightly treated marine food products. The present research work aimed to detect the presence of H. pylori in Spanish commercial samples of shellfish (mussels, clams, and cockles) by means of a quantitative real-time polymerase chain reaction (qPCR) approach based on the vacuolating cytotoxin A (vacA) gene specificity. Putative H. pylori amplicons were confirmed by sequencing. qPCR was positive for 12 out of the 100 samples, being 67% (8/12) from mussels, 25% (3/12) from clams, and only 8% (1/12) from cockles. After sequencing, three of the amplicons showed 97-99% homology with the H. pylori vacA gene. Quantitative results indicate that the levels of contamination remained below 10 2 log 10 colony forming units per mL (CFU/mL). The present research shows for the first time the effectiveness of the optimized qPCR in the identification of potentially H. pylori contaminated shellfish products. Our results confirm the presence of H. pylori in shellfish from the Spanish western seacoast and verify the possible relationship between the presence of H. pylori in seawater and the role of contaminated seafoods as vehicles of H. pylori entrance into the food chain.

The origin of novel traits can promote expansion into new niches and drive speciation. Ctenophores (comb jellies) are unified by their possession of a novel cell type: the colloblast, an adhesive cell found only in the tentacles. Although colloblast-laden tentacles are fundamental for prey capture among ctenophores, some species have tentacles lacking colloblasts and others have lost their tentacles completely. We used transcriptomes from 36 ctenophore species to identify gene losses that occurred specifically in lineages lacking colloblasts and tentacles. We cross-referenced these colloblast- and tentacle-specific candidate genes with temporal RNA-Seq during embryogenesis in Mnemiopsis leidyi and found that both sets of candidates are preferentially expressed during tentacle morphogenesis. We also demonstrate significant upregulation of candidates from both data sets in the tentacle bulb of adults. Both sets of candidates were enriched for an N-terminal signal peptide and protein domains associated with secretion; among tentacle candidates we also identified orthologs of cnidarian toxin proteins, presenting tantalizing evidence that ctenophore tentacles may secrete toxins along with their adhesive. Finally, using cell lineage tracing, we demonstrate that colloblasts and neurons share a common progenitor, suggesting the evolution of colloblasts involved co-option of a neurosecretory gene regulatory network. Together these data offer an initial glimpse into the genetic architecture underlying ctenophore cell-type diversity.

Blue biotechnologies implement marine bio-resources for addressing practical concerns. The isolation of biologically active molecules from marine animals is one of the main ways this field develops. Strikingly, cnidaria are considered as sustainable resources for this purpose, as they possess unique cells for attack and protection, producing an articulated cocktail of bioactive substances. The Mediterranean sea anemone Anemonia viridis has been studied extensively for years. In this short review, we summarize advances in bioprospecting of the A. viridis toxin arsenal. A. viridis RNA datasets and toxin data mining approaches are briefly described. Analysis reveals the major pool of neurotoxins of A. viridis , which are particularly active on sodium and potassium channels. This review therefore integrates progress in both RNA-Seq based and biochemical-based bioprospecting of A. viridis toxins for biotechnological exploitation., Competing Interests: The authors declare no conflict of interest.

Swinholides are 42-carbon ring polyketides with a 2-fold axis of symmetry. They are potent cytotoxins that disrupt the actin cytoskeleton. Swinholides were discovered from the marine sponge Theonella sp. and were long suspected to be produced by symbiotic bacteria. Misakinolide, a structural variant of swinholide, was recently demonstrated to be the product of a symbiotic heterotrophic proteobacterium. Here, we report the production of swinholide A by an axenic strain of the terrestrial cyanobacterium Nostoc sp. strain UHCC 0450. We located the 85-kb trans -AT polyketide synthase (PKS) swinholide biosynthesis gene cluster from a draft genome of Nostoc sp. UHCC 0450. The swinholide and misakinolide biosynthesis gene clusters share an almost identical order of catalytic domains, with 85% nucleotide sequence identity, and they group together in phylogenetic analysis. Our results resolve speculation around the true producer of swinholides and demonstrate that bacteria belonging to two distantly related phyla both produce structural variants of the same natural product. In addition, we described a biosynthesis cluster from Anabaena sp. strain UHCC 0451 for the synthesis of the cytotoxic and antifungal scytophycin. All of these biosynthesis gene clusters were closely related to each other and created a group of cytotoxic macrolide compounds produced by trans -AT PKSs of cyanobacteria and proteobacteria. IMPORTANCE Many of the drugs in use today originate from natural products. New candidate compounds for drug development are needed due to increased drug resistance. An increased knowledge of the biosynthesis of bioactive compounds can be used to aid chemical synthesis to produce novel drugs. Here, we show that a terrestrial axenic culture of Nostoc cyanobacterium produces swinholides, which have been previously found only from marine sponge or samples related to them. Swinholides are polyketides with a 2-fold axis of symmetry, and they are potent cytotoxins that disrupt the actin cytoskeleton. We describe the biosynthesis gene clusters of swinholide from Nostoc cyanobacteria, as well as the related cytotoxic and antifungal scytophycin from Anabaena cyanobacteria, and we study the evolution of their trans -AT polyketide synthases. Interestingly, swinholide is closely related to misakinolide produced by a symbiotic heterotrophic proteobacterium, demonstrating that bacteria belonging to two distantly related phyla and different habitats can produce similar natural products., (Copyright © 2018 Humisto et al.)

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

More than a century of research on sea anemone venoms has shown that they contain a diversity of biologically active proteins and peptides. However, recent omics studies have revealed that much of the venom proteome remains unexplored. We used, for the first time, a combination of proteomic and transcriptomic techniques to obtain a holistic overview of the venom arsenal of the well-studied sea anemone Stichodactyla haddoni. A purely search-based approach to identify putative toxins in a transcriptome from tentacles regenerating after venom extraction identified 508 unique toxin-like transcripts grouped into 63 families. However, proteomic analysis of venom revealed that 52 of these toxin families are likely false positives. In contrast, the combination of transcriptomic and proteomic data enabled positive identification of 23 families of putative toxins, 12 of which have no homology known proteins or peptides. Our data highlight the importance of using proteomics of milked venom to correctly identify venom proteins/peptides, both known and novel, while minimizing false positive identifications from non-toxin homologues identified in transcriptomes of venom-producing tissues. This work lays the foundation for uncovering the role of individual toxins in sea anemone venom and how they contribute to the envenomation of prey, predators, and competitors., Biological Significance: Proteomic analysis of milked venom combined with analysis of a tentacle transcriptome revealed the full extent of the venom arsenal of the sea anemone Stichodactyla haddoni. This combined approach led to the discovery of 12 entirely new families of disulfide-rich peptides and proteins in a genus of anemones that have been studied for over a century., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Microcystins are globally the most commonly occurring freshwater cyanotoxins, causing acute poisoning and chronically inducing hepatocellular carcinoma. However, the detection and toxicological study of microcystins is hampered by the limited availability and high cost of pure toxin standards. Biosynthesis of microcystin variants in a fast-growing heterologous host offers a promising method of achieving reliable and economically viable alternative to isolating toxin from slow-growing cyanobacterial cultures. Here, we report the heterologous expression of recombinant microcystin synthetases in Escherichia coli to produce [d-Asp 3 ]microcystin-LR and microcystin-LR. We assembled a 55 kb hybrid polyketide synthase/nonribosomal peptide synthetase gene cluster from Microcystis aeruginosa PCC 7806 using Red/ET recombineering and replaced the native promoters with an inducible Ptet O promoter to yield microcystin titers superior to M. aeruginosa. The expression platform described herein can be tailored to heterologously produce a wide variety of microcystin variants, and potentially other cyanobacterial natural products of commercial relevance.

The globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus contains several biologically active proteins. We have cloned the cDNA of one of the toxin components, SUL-I, which is a rhamnose-binding lectin (RBL) that acts as a mitogen through binding to carbohydrate chains on target cells. Recombinant SUL-I (rSUL-I) was produced in Escherichia coli cells, and its carbohydrate-binding specificity was examined with the glycoconjugate microarray analysis, which suggested that potential target carbohydrate structures are galactose-terminated N-glycans. rSUL-I exhibited mitogenic activity for murine splenocyte cells and toxicity against Vero cells. The three-dimensional structure of the rSUL-I/l-rhamnose complex was determined by X-ray crystallographic analysis at a 1.8 Å resolution. The overall structure of rSUL-I is composed of three distinctive domains with a folding structure similar to those of CSL3, a RBL from chum salmon (Oncorhynchus keta) eggs. The bound l-rhamnose molecules are mainly recognized by rSUL-I through hydrogen bonds between its 2-, 3-, and 4-hydroxy groups and Asp, Asn, and Glu residues in the binding sites, while Tyr and Ser residues participate in the recognition mechanism. It was also inferred that SUL-I may form a dimer in solution based on the molecular size estimated via dynamic light scattering as well as possible contact regions in its crystal structure., (© 2017 The Protein Society.)

The aim of this study was to develop and validate a high resolution melting (HRM) method for the rapid, accurate identification of the various harmful diatom Pseudo-nitzschia species in marine environments. Pseudo-nitzschia has a worldwide distribution and some species are toxic, producing the potent domoic acid toxin, which poses a threat to both human and animal health. Hence, it is important to identify toxic Pseudo-nitzschia species. A pair of primers targeting the LSU rDNA of the genus Pseudo-nitzschia was designed for the development of the assay and its specificity was validated using 22 control DNAs of the P. calliantha, P. delicatissima/P. arenysensis complex and P. pungens. The post-PCR HRM assay was applied to numerous unidentified Pseudo-nitzschia strains isolated from the northwestern Adriatic Sea (Mediterranean Sea), and it was able to detect and discriminate three distinct Pseudo-nitzschia taxa from unidentified samples. Moreover, the species-specific identification of Pseudo-nitzschia isolates by the HRM assay was consistent with phylogenetic analyses. The HRM assay was specific, robust and rapid when applied to high numbers of cultured samples in order to taxonomically identify Pseudo-nitzschia isolates recovered from environmental samples.

Fish venom remains a virtually untapped resource. There are so few fish toxin sequences for reference, which increases the difficulty to study toxins from venomous fish and to develop efficient and fast methods to dig out toxin genes or proteins. Here, we utilized Chinese yellow catfish (Pelteobagrus fulvidraco) as our research object, since it is a representative species in Siluriformes with its venom glands embedded in the pectoral and dorsal fins. In this study, we set up an in-house toxin database and a novel toxin-discovering protocol to dig out precise toxin genes by combination of transcriptomic and proteomic sequencing. Finally, we obtained 15 putative toxin proteins distributed in five groups, namely Veficolin, Ink toxin, Adamalysin, Za2G and CRISP toxin. It seems that we have developed a novel bioinformatics method, through which we could identify toxin proteins with high confidence. Meanwhile, these toxins can also be useful for comparative studies in other fish and development of potential drugs.

Paralytic shellfish toxins (PSTs) are a group of water soluble neurotoxic alkaloids produced by two different kingdoms of life, prokaryotic cyanobacteria and eukaryotic dinoflagellates. Owing to the wide distribution of these organisms, these toxic secondary metabolites account for paralytic shellfish poisonings around the world. On the other hand, their specific binding to voltage-gated sodium channels makes these toxins potentially useful in pharmacological and toxicological applications. Much effort has been devoted to the biosynthetic mechanism of PSTs, and gene clusters encoding 26 proteins involved in PST biosynthesis have been unveiled in several cyanobacterial species. Functional analysis of toxin genes indicates that PST biosynthesis in cyanobacteria is a complex process including biosynthesis, regulation, modification and export. However, less is known about the toxin biosynthesis in dinoflagellates owing to our poor understanding of the massive genome and unique chromosomal characteristics [1]. So far, few genes involved in PST biosynthesis have been identified from dinoflagellates. Moreover, the proteins involved in PST production are far from being totally explored. Thus, the origin and evolution of PST biosynthesis in these two kingdoms are still controversial. In this review, we summarize the recent progress on the characterization of genes and proteins involved in PST biosynthesis in cyanobacteria and dinoflagellates, and discuss the standing evolutionary hypotheses concerning the origin of toxin biosynthesis as well as future perspectives in PST biosynthesis., Scientific Question: Paralytic shellfish toxins (PSTs) are a group of potent neurotoxins which specifically block voltage-gated sodium channels in excitable cells and result in paralytic shellfish poisonings (PSPs) around the world. Two different kingdoms of life, cyanobacteria and dinoflagellates are able to produce PSTs. However, in contrast with cyanobacteria, our understanding of PST biosynthesis in dinoflagellates is extremely limited owing to their unique features. The origin and evolution of PST biosynthesis in these two kingdoms are still controversial., Technical Significance: High-throughput omics technologies, such as genomics, transcriptomics and proteomics provide powerful tools for the study of PST biosynthesis in cyanobacteria and dinoflagellates, and have shown their powerful potential with regard to revealing genes and proteins involved in PST biosynthesis in two kingdoms., Scientific Significance: This review summarizes the recent progress in PST biosynthesis in cyanobacteria and dinoflagellates with focusing on the novel insights from omics technologies, and discusses the evolutionary relationship of toxin biosynthesis genes between these two kingdoms., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Venomous sea urchins contain various biologically active proteins that are toxic to predators. Contractin A is one such protein contained within the globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus. This protein exhibits several biological activities, such as smooth muscle contraction and mitogenic activity. N-terminal amino acid residues of Contractin A have been determined up to 37 residues from the purified protein. In this study, we cloned cDNA for Contractin A by reverse transcription-PCR using degenerate primers designed on the basis of its N-terminal amino acid sequence. Analysis of the cDNA sequence indicated that Contractin A is composed of 166 amino acid residues including 31 residues of a putative signal sequence, and has homology to the sequence of phospholipase A2 from various organisms. In this study, recombinant Contractin A was expressed in Escherichia coli cells, and the protein was subjected to an assay to determine lipid-degrading activity using carboxyfluorescein-containing liposomes. As a result, Contractin A was found to exhibit Ca(2+)-dependent release of carboxyfluorescein from the liposomes, suggesting that Contractin A has phospholipase A2 activity, which may be closely associated with its biological activities., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The heteronemertine Parborlasia corrugatus contains a cytolytic protein, parborlysin, which after extensive purification was found by Edman sequencing to be a mixture of several homologues. To investigate this microheterogeneity and enable the analysis of single toxins, we have obtained seven parborlysin isoform genes from P. corrugatus collected in Antarctica. Total RNA was isolated from the homogenized head region and parborlysin genes were identified from a cDNA library using degenerate primers. The translated sequences reveal that the isoforms are ∼ 10 kDa basic (pI ∼ 10) proteins of which all but one harbour six cysteine residues. We generated a model of the three dimensional structure of parborlysins, which suggests that they are composed of five alpha-helical segments that include large, exposed hydrophobic surfaces. Finally, we constructed plasmids and inserted them into Escherichia coli to obtain overexpressed amino- or carboxy-terminal polyhistidine-tagged parborlysin isoforms fused to the third domain of the E. coli periplasmic-protein TolA to facilitate toxin isolation. One of the isoforms adversely affected growth in the E. coli expressing it. Although we succeeded in isolating one of the recombinant parborlysin constructs, it lacked haemolytic activity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Using a partial transcriptome of the snakelocks anemone (Anemonia sulcata) we identify toxin gene candidates that were incorrectly assembled into several Trinity components. Our approach recovers hidden diversity found within some toxin gene families that would otherwise go undetected when using Trinity, a widely used program for venom-focused transcriptome reconstructions. Unidentified hidden transcripts may significantly impact conclusions made regarding venom composition (or other multi-copy conserved genes) when using Trinity or other de novo assembly programs., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Purpose: The objective of this study is to investigate the antiproliferative activity and mechanism of integrin-binding rLj-RGD4 in a Hep-2 human laryngeal carcinoma-bearing nude mouse model., Methods: Human laryngeal squamous carcinoma cells (Hep-2) were inoculated subcutaneously into the axilla of nude mice to generate a Hep-2 human laryngeal carcinoma-bearing nude mouse model. When the Hep-2 xenograft model was successfully established, the animals were randomly separated into five groups. Three groups were treated with different dosages of rLj-RGD4. Cisplatin was administered to the positive control group, and normal saline (NaCl) was administered to the negative control group for 3 weeks. The body weights and the survival of the nude mice were evaluated, and the volumes and weights of the solid tumours were measured. The mechanism underlying rLj-RGD4 inhibition of tumour growth in transplanted Hep-2 human laryngeal carcinoma-bearing nude mice was evaluated by haematoxylin-eosin (HE) staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL), measurement of intratumoural microvessel density (MVD), Western blotting, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR)., Results: The tumour volumes and weights of the treatment groups were reduced compared with the model group, and survival times were improved by rLj-RGD4 treatment in Hep-2 human laryngeal carcinoma-bearing nude mice. The number of apoptotic Hep-2 human cells and intratumoural MVD significantly decreased after the administration of rLj-RGD4. In the xenograft tissue of animals treated with rLj-RGD4, FAK, PI3K, and Akt expression was unaltered, whereas P-FAK, P-PI3K, Bcl-2, P-Akt, and VEGF levels were down-regulated. In addition, activated caspase-3, activated caspase-9, and Bax levels were up-regulated., Conclusion: rLj-RGD4 exhibits potent in vivo activity and inhibits the growth of transplanted Hep-2 human laryngeal carcinoma cells in a nude mouse model. Thus, these results indicate that the recombinant RGD toxin protein rLj-RGD4 may serve as a potent clinical therapy for human laryngeal squamous carcinoma., (Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Paralytic shellfish toxins (PSTs) are a group of potent neurotoxic alkaloids produced by cyanobacteria and dinoflagellates. The PST biosynthesis gene cluster and several toxin-related proteins have been unveiled in cyanobacteria, yet little is known about dinoflagellates. Here, we compared the protein profiles of a toxin-producing dinoflagellate Alexandrium catenella (ACHK-T) and its non-toxic mutant (ACHK-NT), and characterized differentially displayed proteins using a combination of the iTRAQ-based proteomic approach and the transcriptomic database. Totally 3488 proteins were identified from A. catenella, and proteins involved in carbohydrate, amino acid and energy metabolism were the most abundant. Among them, 185 proteins were differentially displayed: proteins involved in amino acid biosynthesis, protein and carbohydrate metabolism and bioluminescence were more abundant in ACHK-T, while proteins participating in photosynthesis, fatty acid biosynthesis, and the processes occurring in peroxisome displayed higher abundances in ACHK-NT. Seven toxin-related proteins were identified but they varied insignificantly between the two strains. Different carbon and energy utilization strategies were potentially related to the toxin producing ability, and the regulation mechanism of PST biosynthesis was more complex in dinoflagellates. Our study provides the first comprehensive dataset on the dinoflagellate proteome and lays the groundwork for future proteomic study., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: The use of venom in intraspecific aggression is uncommon and venom-transmitting structures specifically used for intraspecific competition are found in few lineages of venomous taxa. Next-generation transcriptome sequencing allows robust characterization of venom diversity and exploration of functionally unique tissues. Using a tissue-specific RNA-seq approach, we investigate the venom composition and gene ontology diversity of acrorhagi, specialized structures used in intraspecific competition, in aggressive and non-aggressive polyps of the aggregating sea anemone Anthopleura elegantissima (Cnidaria: Anthozoa: Hexacorallia: Actiniaria: Actiniidae)., Results: Collectively, we generated approximately 450,000 transcripts from acrorhagi of aggressive and non-aggressive polyps. For both transcriptomes we identified 65 candidate sea anemone toxin genes, representing phospholipase A2s, cytolysins, neurotoxins, and acrorhagins. When compared to previously characterized sea anemone toxin assemblages, each transcriptome revealed greater within-species sequence divergence across all toxin types. The transcriptome of the aggressive polyp had a higher abundance of type II voltage gated potassium channel toxins/Kunitz-type protease inhibitors and type II acrorhagins. Using toxin-like proteins from other venomous taxa, we also identified 612 candidate toxin-like transcripts with signaling regions, potentially unidentified secretory toxin-like proteins. Among these, metallopeptidases and cysteine rich (CRISP) candidate transcripts were in high abundance. Furthermore, our gene ontology analyses identified a high prevalence of genes associated with "blood coagulation" and "positive regulation of apoptosis", as well as "nucleoside: sodium symporter activity" and "ion channel binding". The resulting assemblage of expressed genes may represent synergistic proteins associated with toxins or proteins related to the morphology and behavior exhibited by the aggressive polyp., Conclusion: We implement a multifaceted approach to investigate the assemblage of expressed genes specifically within acrorhagi, specialized structures used only for intraspecific competition. By combining differential expression, phylogenetic, and gene ontology analyses, we identify several candidate toxins and other potentially important proteins in acrorhagi of A. elegantissima. Although not all of the toxins identified are used in intraspecific competition, our analysis highlights some candidates that may play a vital role in intraspecific competition. Our findings provide a framework for further investigation into components of venom used exclusively for intraspecific competition in acrorhagi-bearing sea anemones and potentially other venomous animals.

Actinoporins are small 18.5 kDa pore-forming toxins. A family of six actinoporin genes has been identified in the genome of Hydra magnipapillata, and HALT-1 (Hydra actinoporin-like toxin-1) has been shown to have haemolytic activity. In this study, we have used site-directed mutagenesis to investigate the role of amino acids in the pore-forming N-terminal region and the conserved aromatic cluster required for cell membrane binding. A total of 10 mutants of HALT-1 were constructed and tested for their haemolytic and cytolytic activity on human erythrocytes and HeLa cells, respectively. Insertion of 1-4 negatively charged residues in the N-terminal region of HALT-1 strongly reduced haemolytic and cytolytic activity, suggesting that the length or charge of the N-terminal region is critical for pore-forming activity. Moreover, substitution of amino acids in the conserved aromatic cluster reduced haemolytic and cytolytic activity by more than 80%, suggesting that these aromatic amino acids are important for attachment to the lipid membrane as shown for other actinoporins. The results suggest that HALT-1 and other actinoporins share similar mechanisms of pore formation and that it is critical for HALT-1 to maintain an amphipathic helix at the N-terminus and an aromatic amino acid-rich segment at the site of membrane binding.

Cnidarians rely on their nematocysts and the venom injected through these unique weaponry systems to catch prey and protect themselves from predators. The development and physiology of the nematocysts of Hydra magnipapillata, a classic model organism, have been intensively studied, yet the composition and biochemical activity of their venom components are mostly unknown. Here, we show that hydra actinoporin-like toxins (HALTs), which have previously been associated with Hydra nematocysts, belong to a multigene family comprising six genes, which have diverged from a single common ancestor. All six genes are expressed in a population of Hydra magnipapillata. When expressed recombinantly, HALT-1 (Δ-HYTX-Hma1a), an actinoporin-like protein found in the stenoteles (the main penetrating nematocysts used in prey capture), reveals hemolytic activity, albeit about two-thirds lower than that of the anemone actinoporin equinatoxin II (EqTII, Δ-AITX-Aeq1a). HALT-1 also differs from EqTII in the size of its pores, and likely does not utilize sphingomyelin as a membrane receptor. We describe features of the HALT-1 sequence which may contribute to this difference in activity, and speculate on the role of this unusual family of pore-forming toxins in the ecology of Hydra., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Nemerteans are one of few animal groups that have evolved the ability to utilize toxins for both defense and subduing prey, but little is known about specific nemertean toxins. In particular, no study has identified specific toxin genes even though peptide toxins are known from some nemertean species. Information about toxin genes is needed to better understand evolution of toxins across animals and possibly provide novel targets for pharmaceutical and industrial applications. We sequenced and annotated transcriptomes of two free-living and one commensal nemertean and annotated an additional six publicly available nemertean transcriptomes to identify putative toxin genes. Approximately 63-74% of predicted open reading frames in each transcriptome were annotated with gene names, and all species had similar percentages of transcripts annotated with each higher-level GO term. Every nemertean analyzed possessed genes with high sequence similarities to known animal toxins including those from stonefish, cephalopods, and sea anemones. One toxin-like gene found in all nemerteans analyzed had high sequence similarity to Plancitoxin-1, a DNase II hepatotoxin that may function well at low pH, which suggests that the acidic body walls of some nemerteans could work to enhance the efficacy of protein toxins. The highest number of toxin-like genes found in any one species was seven and the lowest was three. The diversity of toxin-like nemertean genes found here is greater than previously documented, and these animals are likely an ideal system for exploring toxin evolution and industrial applications of toxins., (© The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Ostreopsis sp. is a toxic marine benthic dinoflagellate that causes high biomass blooms, posing a threat to human health, marine biota and aquaculture activities, and negatively impacting coastal seawater quality. Species-specific identification and enumeration is fundamental because it can allow the implementation of all the necessary preventive measures to properly manage Ostreopsis spp. bloom events in recreational waters and aquaculture farms. The aim of this study was to apply a rapid and sensitive qPCR method to quantify Ostreopsis cf. ovata abundance in environmental samples collected from Mediterranean coastal sites and to develop site-specific environmental standard curves. Similar PCR efficiencies of plasmid and environmental standard curves allowed us to estimate the LSU rDNA copy number per cell. Moreover, we assessed the effectiveness of mitochondrial COI and cob genes as alternative molecular markers to ribosomal genes in qPCR assays for Ostreopsis spp. quantification., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Chronic vascular inflammation is driven by interactions between activated leukocytes and the endothelium. Leukocyte β2-integrins bind to endothelial intercellular adhesion molecule 1 (ICAM-1), which allows leukocyte spreading, crawling and transendothelial migration. Leukocytes scan the vascular endothelium for permissive sites to transmigrate, which suggests that there is apical membrane heterogeneity within the endothelium. However, the molecular basis for this heterogeneity is unknown. Leukocyte adhesion induces ICAM-1 clustering, which promotes its association to the actin-binding proteins filamin B, α-actinin-4 and cortactin. We show that these endothelial proteins differentially control adhesion, spreading and transmigration of neutrophils. Loss of filamin B, α-actinin-4 and cortactin revealed adaptor-specific effects on a nuclear-to-peripheral gradient of endothelial cell stiffness. By contrast, increasing endothelial cell stiffness stimulates ICAM-1 function. We identify endothelial α-actinin-4 as a key regulator of endothelial cell stiffness and of ICAM-1-mediated neutrophil transmigration. Finally, we found that the endothelial lining of human and murine atherosclerotic plaques shows elevated levels of α-actinin-4. These results identify endothelial cell stiffness as an important regulator of endothelial surface heterogeneity and of ICAM-1 function, which in turn controls the adhesion and transmigration of neutrophils., (© 2014. Published by The Company of Biologists Ltd.)

Nutrients have the capacity to change cyanobacterial toxin loads via growth-related toxin production, or shifts in the dominance of toxic and nontoxic strains. This study examined the effect of nitrogen (N) and phosphorus on cell division and strain-related changes in production of the toxins, cylindrospermopsins (CYNs) by the cyanobacterium, Cylindrospermopsis raciborskii. Two short-term experiments were conducted with mixed phytoplankton populations dominated by C. raciborskii in a subtropical reservoir where treatments had nitrate (NO3 ), urea (U) and inorganic phosphorus (P) added alone or in combination. Cell division rates of C. raciborskii were only statistically higher than the control on day 5 when U and P were co-supplied. In contrast, cell quotas of CYNs (QCYNS ) increased significantly in treatments where P was supplied, irrespective of whether N was supplied, and this increase was not necessarily related to cell division rates. Increased QCYNS did correlate with an increase in the proportion of the cyrA toxin gene to 16S genes in the C. raciborskii-dominated cyanobacterial population. Therefore, changes in strain dominance are the most likely factor driving differences in toxin production between treatments. Our study has demonstrated differential effects of nutrients on cell division and strain dominance reflecting a C. raciborskii population with a range of strategies in response to environmental conditions., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Conotoxins from cone snails are valuable in physiology research and therapeutic applications. Evolutionary mechanisms of conotoxins have been investigated in several superfamilies, but there is no phylogenetic analysis on M-superfamily conotoxins. In this study, we characterized identical sequences, gene structure, novel cysteine frameworks, functions and evolutionary mechanisms of M-superfamily conotoxins. Identical M-superfamily conotoxins can be found in different Conus species from the analysis of novel 467 M-superfamily conotoxin sequences and other published M-superfamily conotoxins sequences. M-superfamily conotoxin genes consist of two introns and three exons from the results of genome walking. Eighteen cysteine frameworks were identified from the M-superfamily conotoxins, and 10 of the 18 may be generated from framework III. An analysis between diet types and phylogeny of the M-superfamily conotoxins indicate that M-superfamily conotoxins might not evolve in a concerted manner but were subject to birth-and-death evolution. Codon usage analysis shows that position-specific codon conservation is not restricted to cysteines, but also to other conserved residues. By analysing primary structures and physiological functions of M-superfamily conotoxins, we proposed a hypothesis that insertions and deletions, especially insertions in the third cysteine loop, are involved in the creation of new functions and structures of the M-superfamily conotoxins., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Anemonia viridis is a widespread and extensively studied Mediterranean species of sea anemone from which a large number of polypeptide toxins, such as blood depressing substances (BDS) peptides, have been isolated. The first members of this class, BDS-1 and BDS-2, are polypeptides belonging to the β-defensin fold family and were initially described for their antihypertensive and antiviral activities. BDS-1 and BDS-2 are 43 amino acid peptides characterised by three disulfide bonds that act as neurotoxins affecting Kv3.1, Kv3.2 and Kv3.4 channel gating kinetics. In addition, BDS-1 inactivates the Nav1.7 and Nav1.3 channels. The development of a large dataset of A. viridis expressed sequence tags (ESTs) and the identification of 13 putative BDS-like cDNA sequences has attracted interest, especially as scientific and diagnostic tools. A comparison of BDS cDNA sequences showed that the untranslated regions are more conserved than the protein-coding regions. Moreover, the KA/KS ratios calculated for all pairwise comparisons showed values greater than 1, suggesting mechanisms of accelerated evolution. The structures of the BDS homologs were predicted by molecular modelling. All toxins possess similar 3D structures that consist of a triple-stranded antiparallel β-sheet and an additional small antiparallel β-sheet located downstream of the cleavage/maturation site; however, the orientation of the triple-stranded β-sheet appears to differ among the toxins. To characterise the spatial expression profile of the putative BDS cDNA sequences, tissue-specific cDNA libraries, enriched for BDS transcripts, were constructed. In addition, the proper amplification of ectodermal or endodermal markers ensured the tissue specificity of each library. Sequencing randomly selected clones from each library revealed ectodermal-specific expression of ten BDS transcripts, while transcripts of BDS-8, BDS-13, BDS-14 and BDS-15 failed to be retrieved, likely due to under-representation in our cDNA libraries. The calculation of the relative abundance of BDS transcripts in the cDNA libraries revealed that BDS-1, BDS-3, BDS-4, BDS-5 and BDS-6 are the most represented transcripts.

Phytoflagellates of the genus Pseudochattonella (Dictyochophyceae, Ochrophyta) form blooms in marine coastal waters in northern Europe, Japan, and New Zealand that at times cause fish kills with severe losses for the aquaculture industry. The aim of this study was to develop molecular probes for the detection and identification of Pseudochattonella at the genus and species level. A variety of probes were developed and applied to either dot blot hybridization, (q)PCR, or microarray format. In the dot blot hybridization assay, five different oligonucleotide probes targeting the small subunit (SSU) rDNA were tested against DNA from 18 microalgal strains and shown to be specific to the genus Pseudochattonella. A genus-specific PCR assay was developed by identifying an appropriate primer pair in the SSU-internal transcribed spacer 1 (ITS1) rDNA region. Its specificity was tested by screening against both target and non-target strains, and the assay was used to confirm the presence or absence of Pseudochattonella species in environmental samples. In order to distinguish between the two species of the genus, two PCR primer pairs each biased towards one of the species were designed in the large subunit (LSU) rDNA D1 domain and used for quantitative real-time PCR. Five selected probes (three SSU and two LSU rDNA) were adapted for the use on microarrays and included on a prototype multi-species microarray for the detection of harmful algae ( http://www.midtal.com ). Finally, microarrays and qPCR were used for the monthly monitoring of a sampling site in outer Oslofjorden during a 1-year period. Members of Pseudochattonella are difficult to identify by light microscopy in Lugol's preserved samples, and the two species Pseudochattonella verruculosa and Pseudochattonella farcimen can be morphologically distinguished only by transmission electron microscopy. The molecular probes designed in this study will be a valuable asset to microscopical detection methods in the monitoring of harmful algae and for biogeographical and ecological studies of this genus.

In Mexican waters, there is no a formal and well-established monitoring program of harmful algal blooms (HAB) events. Until now, most of the work has been focused on the characterization of organisms present in certain communities. Therefore, the development of new techniques for the rapid detection of HAB species is necessary. Capillary electrophoresis finger print technique (CE-SSCP) is a fingerprinting technique based on the identification of different conformers dependent of its base composition. This technique, coupled with capillary electrophoresis, has been used to compare and identify different conformers. The aim of this study was to determine if CE-SSCP analysis of ribosomal RNA (rRNA) gene fragments could be used for a rapid identification of toxic and harmful HAB species to improve monitoring activities along the coasts of Baja California Sur, Mexico.Three different highly variable regions of the 18S and 28S rRNA genes were chosen and their suitability for the discrimination of different dinoflagellate species was assessed by CE-SSCP.The CE-SSCP results obtained for the LSU D7 fragment has demonstrated that this technique with this gene region could be useful for the identification of the ten dinoflagellates species of different genera.We have shown that this method can be used to discriminate species and the next step will be to apply it to natural samples to achieve our goal of molecular monitoring for toxic algae in Mexican waters. This strategy will offer an option to improve an early warning system of HAB events for coastal BCS, allowing the possible implementation of mitigation strategies. A monitoring program of HAB species using molecular methods will permit the analysis of several samples in a short period of time, without the pressure of counting with a taxonomic expert in phytoplankton taxonomy.

A multiplex surface plasmon resonance (SPR) biosensor method for the detection of paralytic shellfish poisoning (PSP) toxins, okadaic acid (and analogues) and domoic acid was developed. This method was compared to enzyme-linked immunosorbent assay (ELISA) methods. Seawater samples (n=256) from around Europe were collected by the consortia of an EU project MIcroarrays for the Detection of Toxic Algae (MIDTAL) and evaluated using each method. A simple sample preparation procedure was developed which involved lysing and releasing the toxins from the algal cells with glass beads followed by centrifugation and filtering the extract before testing for marine biotoxins by both multi-SPR and ELISA. Method detection limits based on IC20 values for PSP, okadaic acid and domoic acid toxins were 0.82, 0.36 and 1.66 ng/ml, respectively, for the prototype multiplex SPR biosensor. Evaluation by SPR for seawater samples has shown that 47, 59 and 61 % of total seawater samples tested positive (result greater than the IC20) for PSP, okadaic acid (and analogues) and domoic acid toxins, respectively. Toxic samples were received mainly from Spain and Ireland. This work has demonstrated the potential of multiplex analysis for marine biotoxins in algal and seawater samples with results available for 24 samples within a 7 h period for three groups of key marine biotoxins. Multiplex immunological methods could therefore be used as early warning monitoring tools for a variety of marine biotoxins in seawater samples.

Many chemically complex cyanobacterial polyketides and nonribosomal peptides are of great pharmaceutical interest, but the levels required for exploitation are difficult to achieve from native sources. Here we develop a framework for the expression of these multifunctional cyanobacterial assembly lines in Escherichia coli using the lyngbyatoxin biosynthetic pathway, derived from a marine microbial assemblage dominated by the cyanobacterium Moorea producens. Heterologous expression of this pathway afforded high titers of both lyngbyatoxin A (25.6 mg L(-1)) and its precursor indolactam-V (150 mg L(-1)). Production, isolation, and identification of all expected chemical intermediates of lyngbyatoxin biosynthesis in E. coli also confirmed the previously proposed biosynthetic route, setting a solid chemical foundation for future pathway engineering. The successful production of the nonribosomal peptide lyngbyatoxin A in E. coli also opens the possibility for future heterologous expression, characterization, and exploitation of other cyanobacterial natural product pathways.

Sea anemones represent one of the emerging groups of interest concerning venomous animals in toxinology and the goal of the present work was the prospection, and the structural and functional characterization of the compounds present in the secretion of the sea anemone Stichodactyla duerdeni from Brazilian coast. We used a combination of offline RPC-MALDI-TOF and online nano-RPC-ESI-LTQ-Orbitrap proteomic techniques as well as functional bioassays. The mucus was milked by electric stimulation and fractionated by gel filtration on Sephadex G-50 yielding 5 main fractions. The low molecular weight fractions were further submitted to RP-HPLC resulting in 35 new subfractions that were subsequently analyzed by offline MALDI-TOF mass spectrometry. MALDI peptide mass fingerprinting yielded up to 134 different molecular masses, ranging from m/z 901 to 10,833. Among these subfractions, a new peptide of 3431Da, named U-SHTX-Sdd1, was purified and completely sequenced by automated Edman's degradation and tandem mass spectrometry. An analysis of U-SHTX-Sdd1 revealed a modified O-HexNAc-Threonine at position 1, which, at the best of our knowledge, constitutes the first sea anemone toxin reported with such post-translational modification. Because of its sequence similarity with other sea anemone toxins, the pharmacological activity of U-SHTX-Sdd1 was assessed by electrophysiological measurements using the two electrode voltage-clamp technique on cloned voltage-gated potassium channel subtypes, expressed in Xenopus laevis oocytes. However, U-SHTX-Sdd1 did not show activity on these channels. A large-scale proteomic approach was also employed to shed lights on the sea anemone compounds, and a total 67 proteins and peptides were identified., Biological Significance: In this manuscript, we report an extensive characterization of S. duerdeni secretion by means of peptide mass fingerprinting and high-throughput proteome analyses. Also, we report the structure of a new glycopeptide by a combination of biochemical techniques. Despite the previous studies that described proteinaceous compounds present in sea anemone secretions, the number of reported primary sequences is still low. Thus, to access the scenery of protein components from S. duerdeni mucus, including their biological functions, a robust proteomic approach was used together with bioinformatic tools. The demonstrated strategy of analysis is perfectly suitable to other sea anemone secretions and animal venoms. Moreover, new peptide structures can arise contributing to the knowledge of the diversity of these animal peptides., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The Alexandrium tamarense/catenella/fundyense complex is the major causative agent responsible for harmful algal blooms and paralytic shellfish poisoning around the world. However, taxonomy of the A. tamarense complex is contentious and the evolutionary relationships within the complex are unclear. This study compared protein profiles of the A. tamarense complex collected from different geographic regions using the two dimensional fluorescence difference gel electrophoresis (2-D DIGE) approach, and identified species-specific peptides using MALDI-TOF/TOF mass spectrometry. The results showed that three Alexandrium morphotypes presented significantly different protein expression patterns with about 30-40% shared proteins. However, ecotypes from different geographic regions within a species exhibited the same expression patterns, although a few proteins were altered in abundance. Several proteins, i.e. ribulose-1,5-bisphosphate carboxylase oxygenase form II, plastid protein NAP50, methionine S-adenosyltransferase, and peridinin-chlorophyll a-binding protein, were identified and presented different shift patterns in isoelectric point and/or molecular weight in the 2-D DIGE gels, indicating that amino acid mutation and/or posttranslational modification of these proteins had occurred. The species-specific peptide mass fingerprint and amino acid sequence of ribulose-1,5-bisphosphate carboxylase oxygenase were characterized in the A. tamarense complex, and amino acid substitution occurred among them. This study indicated that evolutionary divergence had occurred at the proteomic level in the A. tamarense complex, and that the species-specific peptides could be used as potential biomarkers to distinguish the three morphotypes., Biological Significance: Scientific question: The Alexandrium tamarense/catenella/fundyense complex is the major causative agent responsible for harmful algal blooms and paralytic shellfish poisoning around the world. However, taxonomy of the A. tamarense complex is contentious and the evolutionary relationships within the complex are unclear, which has seriously impeded our understanding of Alexandrium-causing HABs and, consequently, the monitoring, mitigation and prevention. Technical significance: This study, for the first time, compared the global protein expression patterns of eight ecotypes from the A. tamarense complex and identified species-specific peptides using a quantitative proteomic approach combining 2-D DIGE and MALDI-TOF/TOF MS., Scientific Significance: This study demonstrated that the evolutionary divergence had occurred in the A. tamarense complex at the proteomic level, and the complex should be classified into three species, i.e. A. tamarense, A. catenella, and A. fundyense. Moreover, the species-specific peptide mass fingerprints could be used as potential biomarkers to distinguish the three morphotypes., (Copyright © 2013 Elsevier B.V. All rights reserved.)