Description: Amphidinols are polyketides produced by dinoflagellates suspected of causing fish kills. Here, we demonstrate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the identification and quantification of amphidinols (AM). Novel AM were detected by neutral loss (NL) scan and then quantified together with known AM by selection reaction monitoring (SRM). With the new method, AM were detected in four of eight analyzed strains with a maximum of 3680 fg toxin content per cell. In total, sixteen novel AM were detected by NL scan and characterized via their fragmentation patterns. Of these, two substances are glycosylated forms. This is the first detection of glycosylated AM.

Description: The dinoflagellate genus Prorocentrum is globally represented by a wide variety of species found upon benthic and/or epiphytic substrates. Many epibenthic Prorocentrum species produce lipophilic polyether toxins, some of which act as potent protein phosphatase inhibitors and tumor-promoters associated with Diarrheic Shellfish Poisoning (DSP). Most members of the Prorocentrum lima species complex (PLSC) commonly found in the tropics and sub-tropics are toxigenic. Epiphytic and planktonic bacteria co-occur with toxigenic Prorocentrum but reciprocal allelochemical interactions are under-investigated. The aim of the present study was to identify the culturable bacteria collected together with isolates of the PLSC from seagrass (Thalassia testudinum) and macroalgae along tropical Atlantic coasts of Mexico, and to explore potential species interactions with selected isolates. Twenty-one bacterial genera belonging to Proteobacteria, Actinobacteria, and Bacteroidetes were identified by amplification of the 16S rRNA gene marker from nine clonal Prorocentrum cultures, with g-proteobacteria comprising the dominant class. A positive correlation was found between the bacterial genera associated with two Prorocentrum clones and the esterified toxin analog DTX1a-D8, but there was no apparent correlation between the other PLSC clones and their associated bacteria with the other five DSP toxins detected. No bacteriostatic or allelochemical response was found for cell- and culture medium extracts of five Prorocentrum isolates assayed for bioactivity against Staphylococcus sp. DMBS2 and Vibrio sp. HEL66. Bulk cell-washing of Prorocentrum PA1, followed by growth with antibiotics, was only effective in reducing bacterial load in the initial growth stages, but did not yield axenic cultures or lower bacterial cell densities throughout the culture cycle. Antibiotic treatment did not impair growth or survival of the dinoflagellate, or apparently affect DSP toxin production. There was no significant correlation between Prorocentrum cell volume, growth rate, bacterial cell counts, or cellular toxin concentration over the entire time-series culture cycle. Benthic Prorocentrum and associated bacterial communities comprise highly diverse and characteristic microbiomes upon substrates, and among compartments in culture, but this study provides little evidence that allelochemical interactions among Prorocentrum cells and associated bacteria originating from epibenthic substrates play a definable role in growth and toxigenicity.

Description: Saxitoxin (STX) and its analogs are paralytic alkaloid neurotoxins that block the voltage-gated sodium channel pore (Nav), impeding passage of Na+ ions into the intracellular space, and thereby preventing the action potential in the peripheral nervous system and skeletal muscle. The marine dinoflagellate Gymnodinium catenatum produces an array of such toxins, including the recently discovered benzoyl analogs, for which the mammalian toxicities are essentially unknown. We subjected STX and its analogs to a theoretical docking simulation based upon two alternative tri-dimensional models of the Nav1.4 to find a relationship between the binding properties and the known mammalian toxicity of selected STX analogs. We inferred hypothetical toxicities for the benzoyl analogs from the modeled values. We demonstrate that these toxins exhibit different binding modes with similar free binding energies and that these alternative binding modes are equally probable. We propose that the principal binding that governs ligand recognition is mediated by electrostatic interactions. Our simulation constitutes the first in silico modeling study on benzoyl-type paralytic toxins and provides an approach towards a better understanding of the mode of action of STX and its analogs.

Description: Neurotoxins belonging to the group of saxitoxin (STX) and tetrodotoxin (TTX) analogs are guanidinium alkaloids that share a common high affinity and ion flux blockage capacity for voltage-gated sodium ion channels (Nav. Members of the STX group, also known as paralytic shellfish toxins (PST), are produced among three genera of marine dinoflagellate and several genera of phylogenetically distant and primarily freshwater filamentous cyanobacteria. The origin of the biosynthetic genes in dinoflagellates remains controversial and may represent single or multiple horizontal gene transfer (HGT) events from progenitor eubacteria and/or cyanobacteria. The TTXs occur primarily among marine puffer fish and a host of terrestrial amphibians. The biosynthetic pathway has not been completely elucidated and the origin of tetrodotoxicity,including the syndrome puffer fish poisoning (PFP) in human seafood consumers,remains somewhat enigmatic. Although symbiotic bacteria are most often invoked as the source of TTX in macrofauna, endogenous biosynthesis independent of bacteria cannot be excluded. Integration of knowledge on the biogenic origins, linked to heterogeneity of the biogeographical and phylogenetic distribution of these respective toxin groups, provides the basis for rational inferences and reasonable speculation about the functional role in aquatic and terrestrial ecosystems. Recent identification of the biosynthetic genes for STX analogs in both cyanobacteria and dinoflagellates has yielded insights into biosynthetic mechanisms of toxin heterogeneity among strains and the evolutionary origins of their respective elements of the toxin gene clusters. Although it is not fully understood how or why these molecules are produced in nature, development of improved detection methods will make possible the discovery of new sources and analogs. Once genetic mechanisms for toxin biosynthesis are fully incorporated with modeling of receptor binding interactions and the structural–functional affinities of the ion channels, this will facilitate further biotechnological exploitation of these exquisite bioactive compounds and point the way toward future development of pharmaceuticals and therapeutic applications.

Description: The paralytic shellfish toxin (PST) profiles of Gymnodinium catenatum Graham have been reported for several strains from the Pacific coast of Mexico cultured under different laboratory conditions, as well as from natural populations. Up to 15 saxitoxin analogues occurred and the quantity of each toxin depended on the growth phase and culture conditions. Previous analysis of toxin profiles of G. catenatum isolated from Mexico have been based on post-column oxidation liquid chromatography with fluorescence detection (LC-FLD), a method prone to artefacts and non-specificity, leading to misinterpretation of toxin composition. We describe, for the first time, the complete toxin profile for several G. catenatum strains from diverse locations of the Pacific coast of Mexico. The new results confirmed previous reports on the dominance of the less potent sulfocarbamoyl toxins (C1/2); significant differences, however, in the composition (e.g., absence of saxitoxin, gonyautoxin 2/3 and neosaxitoxin) were revealed in our confirmatory analysis. The LC-MS/MS analyses also indicated at least seven putative benzoyl toxin analogues and provided support for their existence. This new toxin profile shows a high similarity (〉 80%) to the profiles reported from several regions around the world, suggesting low genetic variability among global populations.

Description: The marine dinoflagellate genus Prorocentrum Ehrenberg comprises many species occupying primarily benthic or epiphytic habitats, particularly in tropical and sub-tropical waters. Despite concerted efforts to establish phylogenetic associations, there remain unresolved issues in defining morphospecies and membership in species complexes. The study described herein addressed the inter- and infraspecific relationships of members of the Prorocentrum lima and Prorocentrum hoffmannianum species complexes (PLSC and PHSC, respectively) by applying multivariate approaches in morphotaxonomy, molecular phylogenetics and chemodiversity to establish affinities among multiple clonal isolates. Morphotaxonomic analysis showed consistency with classical morphospecies descriptors, and high variability in cell size and dimensions, but did not challenge current species complex concepts. Phylogenetic analysis of ITS/5.8S rDNA sequences from isolates from the Gulf of California, Caribbean Sea, and Gulf of Mexico coasts compared with archived global GenBank sequences served to define five consistent clades with separation of the PLSC and PHSC. Secondary structure modeling of ITS2 rRNA variation based on compensatory base changes (CBC) was effective in resolving details of the respective species complexes and even indicated putative incipient or cryptic speciation due to potential hybridization barriers. This study represents the largest (n = 67 isolates) chemodiversity analysis of polyketide-derived toxins associated with diarrheic shellfish poisoning (DSP) from a benthic dinoflagellate genus. Relative composition of some analogs (OA, OA-D8, DTX1, DTX1a, and DTX1a-D8), including two new undescribed isomers, distinguished P. lima from P. hoffmannianum sensu lato, but without clear associations with substrate type or geographical origin. Although all P. lima and most (one exception) P. hoffmannianum were toxigenic, the total cell toxin content could not be linked at the species level. This research demonstrates that clonal chemodiversity in toxin composition cannot yet be effectively applied to define ecological niches or species interactions within local assemblages. Phylogenetic analysis of the ITS/5.8 rDNA, particularly when combined with secondary structure modeling, rather than only a comparison of LSU rDNA sequences, is a more powerful approach to identify cryptic speciation and to resolve species complexes within benthic dinoflagellate groups.

Description: The genus Amphidinium has been the subject of recent attention due to the production of polyketide metabolites. Some of these compounds have shown significant bioactivities and could be related to species interactions in the natural benthic microenvironment. Among these compounds, amphidinols (AMs) are suspected to be related to fish kills and probably implicated in ciguatera symptoms associated with the occurrence of benthic harmful algal blooms (bHABs). Here, we present the first report of a variety of AMs produced by cultured strains from several species from the Mexican Pacific, the Gulf of California, and the Gulf of Mexico. Through ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS), ten previously known AMs (AM02, -04, -05, -06, -07, -09, -11, -14, -15, and -17), four recently reported AMs (N7, N8/N9, N12, and N13), and three new variants (U1, U2, and U3) were identified. Of the twelve analyzed Amphidinium cultures, five were not AM producers, and the cell quotas of the remaining seven strains ranged from close to nondetectable to a maximum of 1694 fg cell-1, with many intermediate levels in between. The cultures from the Mexican North Pacific coast produced AMs in a higher quantity and variety than those from worldwide locations. This is the first study of AMs from Mexican Amphidinium strains, and our results confirm the relevance of continuing the investigation of the genus bioactive metabolites.

Description: Naturally occurring neurotoxins belonging to two structurally distinct groups of guanidinium alkaloids known collectively as saxitoxins (STXs) and tetrodotoxins (TTXs) share a high affinity and ion flux blockage capacity for voltage-gated sodium ion channels (NaV). Both toxin groups are produced by marine microorganisms and widely distributed among vector species in the oceans, but are also found in terrestrial species. The STXs are often referred to as paralytic shellfish toxins (PSTs) based on their accumulation in shellfish and the symptoms in humans after consumption of toxic seafood. Biosynthesis of STXs is confirmed in four genera of marine dinoflagellates and among about a dozen species of primarily freshwater and brackish water strains of filamentous cyanobacteria. The origin of the STX biosynthetic genes in dinoflagellates remains controversial and may represent multiple horizontal gene transfer (HGT) events from progenitor bacteria and/or cyanobacteria. The recent identification of the biosynthetic genes for STX analogs in both cyanobacteria and dinoflagellates has yielded insights into mechanisms of toxin heterogeneity among species and the evolutionary origins of the respective elements of the toxin gene cluster. The biogenic origins of TTXs and tetrodotoxicity remain even more enigmatic. The TTXs occur primarily in marine pufferfish species, and hence tetrodotoxicity is frequently described as pufferfish poisoning (PFP) after the toxin syndrome in human consumers of such toxic fish. In marine environments, TTXs also appear in invertebrate species, particularly of benthic feeders on suspended particulates and carnivorous vector species. Symbiotic colonizing bacteria or free-living bacteria sequestered via feeding from the water column or sediments are most often invoked as proximal sources of TTXs in marine macrofauna, but endogenous biosynthesis independent of bacteria cannot be excluded. The TTX biosynthetic pathway has not been completely elucidated, and the biosynthetic genes are unknown.