How relevant are sterols in the mode of action of prymnesins?

Prymnesins, produced by the haptophyte Prymnesium parvum, are considered responsible for fish kills when this species blooms. Although their toxic mechanism is not fully understood, membrane disruptive properties have been ascribed to A-type prymnesins. Currently it is suggested that pore-formation is the underlying cause of cell disruption. Here the hypothesis that A-, B-, and C-type prymnesins interact with sterols in order to create pores was tested. Prymnesin mixtures containing various analogs of the same type were applied in hemolysis and cytotoxicity assays using Atlantic salmon Salmo salar erythrocytes or rainbow trout RTgill-W1 cells. The hemolytic potency of the prymnesin types reflected their cytotoxic potential, with approximate concentrations reaching 50 % hemolysis (HC50) of 4 nM (A-type), 54 nM (C-type), and 600 nM (B-type). Variabilities in prymnesin profiles were shown to influence potency. Prymnesin-A (3 Cl) + 2 pentose + hexose was likely responsible for the strong toxicity of A-type samples. Co-incubation with cholesterol and epi-cholesterol pre-hemolysis reduced the potential by about 50 % irrespective of sterol concentration, suggesting interactions with sterols. However, this effect was not observed in RTgill-W1 toxicity. Treatment of RTgill-W1 cells with 10 µM lovastatin or 10 µM methyl-ß-cyclodextrin-cholesterol modified cholesterol levels by 20-30 %. Regardless, prymnesin cytotoxicity remained unaltered in the modified cells. SPR data showed that B-type prymnesins likely bound with a single exponential decay while A-types seemed to have a more complex binding. Overall, interaction with cholesterol appeared to play only a partial role in the cytotoxic mechanism of pore-formation. It is suggested that prymnesins initially interact with cholesterol and stabilize pores through a subsequent, still unknown mechanism possibly including other membrane lipids or proteins.

Sterolysin from a 1950s culture of Karlodinium veneficum (aka Gymnodinium veneficum Ballantine) forms lethal sterol dependent membrane pores.

In 1957 Abbott and Ballantine described a highly toxic activity from a dinoflagellate isolated from the English Channel in 1949 by Mary Park. From a culture maintained at Plymouth Laboratory since 1950, we have been able to isolate two toxic molecules (abbotoxin and 59-E-Chloro-abbotoxin), determine the planar structures by analysis of HRMS and 1D and 2D NMR spectra, and found them to be karlotoxin (KmTx) congeners. Both toxins kill larval zebrafish with symptoms identical to those described by Abbot and Ballantine for gobies (Gobius virescens). Using surface plasma resonance the sterol binding specificity of karlotoxins is shown to require desmethyl sterols. Our results with black lipid membranes indicate that karlotoxin forms large-conductance channels in the lipid membrane, which are characterized by large ionic conductance, poor ionic selectivity, and a complex gating behavior that exhibits strong voltage dependence and multiple gating patterns. In addition, we show that KmTx 2 pore formation is a highly targeted mechanism involving sterol-specificity. This is the first report of the functional properties of the membrane pores formed by karlotoxins and is consistent with the initial observations of Abbott and Ballantine from 1957.

Long-Read Sequencing Unlocks New Insights into the Amphidinium carterae Microbiome.

Dinoflagellates are one of the largest groups of marine microalgae and exhibit diverse trophic strategies. Some dinoflagellates can produce secondary metabolites that are known to be toxic, which can lead to ecologically harmful blooms. Amphidinium carterae is one species of dinoflagellate that produces toxic compounds and is used as a model for dinoflagellate studies. The impact of the microbiome on A. carterae growth and metabolite synthesis is not yet fully understood, nor is the impact of bacterial data on sequencing and assembly. An antibiotic cocktail was previously shown to eliminate 16S amplification from the dinoflagellate culture. Even with drastically reduced bacterial numbers during antibiotic treatment, bacterial sequences were still present. In this experiment, we used novel Nanopore long-read sequencing techniques on A. carterae cultures to assemble 15 full bacterial genomes ranging from 2.9 to 6.0 Mb and found that the use of antibiotics decreased the percentage of reads mapping back to bacteria. We also identified shifts in the microbiome composition and identified a potentially deleterious bacterial species arising in the absence of the antibiotic treatment. Multiple antibiotic resistance genes were identified, as well as evidence that the bacterial population does not contribute to toxic secondary metabolite synthesis.

A Toxic Sterolysin From a 1950s Culture of Gymnodinium Veneficum Ballantine.

In 1957 Abbott and Ballentine described a highly toxic activity from a dinoflagellate isolated from the English Channel. in 1949 by Mary Park. From a culture maintained at Plymouth Laboratory since 1950, we have been able to isolate two toxic molecules (Abbotoxin and 59-E-Chloro-Abbotoxin), determine the planar structures by analysis of HRMS and 1D and 2D NMR spectra and found them to be karlotoxin (KmTx) congeners. Both toxins kill larval zebrafish with symptoms identical to that described by Abbot and Ballantine for gobies (Gobius virescens). Using surface plasma resonance the sterol binding specificity of karlotoxins is shown to require desmethyl sterols. Our results with black lipid membranes indicate that karlotoxin forms large-conductance channels in the lipid membrane, which are characterized by large ionic conductance, poor ionic selectivity, and a complex gating behavior that exhibits strong voltage dependence and multiple gating patterns. In addition, we show that KmTx 2 pore formation is a highly targeted mechanism involving sterol-specificity. This is the first report of the functional properties of the membrane pores formed by karlotoxins and are consistent with the intial observations of Abbott and Ballentine from 1957.

Division time (td) for in situ growth measurements demonstrates thermal ecotypes of Karlodinium veneficum.

The toxic dinoflagellate Karlodinium veneficum forms fish killing blooms in temperate estuaries worldwide. These blooms have variable toxicity which may be related to bloom stage and in situ growth rates of the constituent K. veneficum cells. Measurement of in situ growth rates is challenging and methods such as the mitotic index technique require knowledge of the dynamics of cell division. In order to better understand these dynamics, we determined the duration of cell division (td) in four geographically distinct laboratory strains of K. veneficum at three different environmentally relevant temperatures. The results demonstrated that the td value for each strain, growing at strain-specific optimal temperatures, was 1.6 ± 0.1 h. This value corresponded to a range of growth rates from 0.17 ± 0.08 d-1 to 0.62 ± 0.07 d-1. Equivalent values of td spread across four geographically distinct laboratory strains and a nearly fourfold range of growth rates implies that 1.6 h represents the td value of K. veneficum. Additionally, temperature conditions yielding this value for td and the highest growth rates varied among strains, indicating cold-adapted (Norway), warm-adapted (Florida, USA), and eurythermally-adapted (Maryland, USA) strains. These differences have been apparently retained in culture over many years, indicating a conserved genetic basis that suggests distinct thermal ecotypes of the morphospecies K. veneficum. This knowledge together with the first estimate of td for K. veneficum will be useful in future field studies aimed at correlating bloom toxicity with in situ growth rate using the mitotic index technique.

Investigating A Multi-Domain Polyketide Synthase in Amphidinium carterae.

Dinoflagellates are unicellular organisms that are implicated in harmful algal blooms (HABs) caused by potent toxins that are produced through polyketide synthase (PKS) pathways. However, the exact mechanisms of toxin synthesis are unknown due to a lack of genomic segregation of fat, toxins, and other PKS-based pathways. To better understand the underlying mechanisms, the actions and expression of the PKS proteins were investigated using the toxic dinoflagellate Amphidinium carterae as a model. Cerulenin, a known ketosynthase inhibitor, was shown to reduce acetate incorporation into all fat classes with the toxins amphidinol and sulpho-amphidinol. The mass spectrometry analysis of cerulenin-reacted synthetic peptides derived from ketosynthase domains of A. carterae multimodular PKS transcripts demonstrated a strong covalent bond that could be localized using collision-induced dissociation. One multi-modular PKS sequence present in all dinoflagellates surveyed to date was found to lack an AT domain in toxin-producing species, indicating trans-acting domains, and was shown by Western blotting to be post-transcriptionally processed. These results demonstrate how toxin synthesis in dinoflagellates can be differentiated from fat synthesis despite common underlying pathway.

Co-occurrence of marine and freshwater phycotoxins in oysters, and analysis of possible predictors for management.

Oysters (Crassostrea virginica) were screened for 12 phycotoxins over two years in nearshore waters to collect baseline phycotoxin data and to determine prevalence of phycotoxin co-occurrence in the commercially and ecologically-relevant species. Trace to low concentrations of azaspiracid-1 and -2 (AZA1, AZA2), domoic acid (DA), okadaic acid (OA), and dinophysistoxin-1 (DTX1) were detected, orders of magnitude below seafood safety action levels. Microcystins (MCs), MC-RR and MC-YR, were also found in oysters (maximum: 7.12 µg MC-RR/kg shellfish meat wet weight), warranting consideration of developing action levels for freshwater phycotoxins in marine shellfish. Oysters contained phycotoxins that impair shellfish health: karlotoxin1-1 and 1-3 (KmTx1-1, KmTx1-3), goniodomin A (GDA), and pectenotoxin-2 (PTX2). Co-occurrence of phycotoxins in oysters was common (54%, n = 81). AZAs and DA co-occurred most frequently of the phycotoxins investigated that are a concern for human health (n = 13) and PTX2 and KmTxs co-occurred most frequently amongst the phycotoxins of concern for shellfish health (n = 9). Various harmful algal bloom (HAB) monitoring methods and tools were assessed for their effectiveness at indicating levels of phycotoxins in oysters. These included co-deployed solid phase adsorption toxin tracking (SPATT) devices, toxin levels in particulate organic matter (POM, >1.5 µm) and whole water samples and cell concentrations from water samples as determined by microscopy and quantitative real-time PCR (qPCR). The dominant phycotoxin varied between SPATTs and all other phycotoxin sample types, and out of the 11 phycotoxins detected in oysters, only four and seven were detected in POM and whole water respectively, indicating phycotoxin profile mismatch between ecosystem compartments. Nevertheless, there were correlations between DA in oysters and whole water (simple linear regression [LR]: R2 = 0.6, p < 0.0001, n = 40), and PTX2 in oysters and SPATTs (LR: R2 = 0.3, p = 0.001, n = 36), providing additional monitoring tools for these phycotoxins, but oyster samples remain the best overall indicators of seafood safety.

A Comparison of Dinoflagellate Thiolation Domain Binding Proteins Using In Vitro and Molecular Methods.

Dinoflagellates play important roles in ecosystems as primary producers and consumers making natural products that can benefit or harm environmental and human health but are also potential therapeutics with unique chemistries. Annotations of dinoflagellate genes have been hampered by large genomes with many gene copies that reduce the reliability of transcriptomics, quantitative PCR, and targeted knockouts. This study aimed to functionally characterize dinoflagellate proteins by testing their interactions through in vitro assays. Specifically, nine Amphidinium carterae thiolation domains that scaffold natural product synthesis were substituted into an indigoidine synthesizing gene from the bacterium Streptomyces lavendulae and exposed to three A. carterae phosphopantetheinyl transferases that activate synthesis. Unsurprisingly, several of the dinoflagellate versions inhibited the ability to synthesize indigoidine despite being successfully phosphopantetheinated. However, all the transferases were able to phosphopantetheinate all the thiolation domains nearly equally, defying the canon that transferases participate in segregated processes via binding specificity. Moreover, two of the transferases were expressed during growth in alternating patterns while the final transferase was only observed as a breakdown product common to all three. The broad substrate recognition and compensatory expression shown here help explain why phosphopantetheinyl transferases are lost throughout dinoflagellate evolution without a loss in a biochemical process.

A Strategy for Gene Knockdown in Dinoflagellates.

Dinoflagellates are unicellular protists that display unusual nuclear features such as large genomes, condensed chromosomes and multiple gene copies organized as tandem gene arrays. Genetic regulation is believed to be controlled at the translational rather than transcriptional level. An important player in this process is initiation factor eIF4E which binds the 7-methylguanosine cap structure (m7G) at the 5'-end of mRNA. Transcriptome analysis of eleven dinoflagellate species has established that each species encodes between eight to fifteen eIF4E family members. Determining the role of eIF4E family members in gene expression requires a method of knocking down their expression. In other eukaryotes this can be accomplished using translational blocking morpholinos that bind to complementary strands of RNA, therefore inhibiting the mRNA processing. Previously, unmodified morpholinos lacked the ability to pass through cell membranes, however peptide-based reagents have been used to deliver substances into the cytosol of cells by an endocytosis-mediated process without damaging the cell membrane. We have successfully delivered fluorescently-tagged morpholinos to the cytosol of Amphidinium carterae by using a specific cell penetrating peptide with the goal to target an eIF4e-1a sequence to inhibit translation. Specific eIF4e knockdown success (up to 42%) has been characterized via microscopy and western blot analysis.

Dinoflagellate Phosphopantetheinyl Transferase (PPTase) and Thiolation Domain Interactions Characterized Using a Modified Indigoidine Synthesizing Reporter.

Photosynthetic dinoflagellates synthesize many toxic but also potential therapeutic compounds therapeutics via polyketide/non-ribosomal peptide synthesis, a common means of producing natural products in bacteria and fungi. Although canonical genes are identifiable in dinoflagellate transcriptomes, the biosynthetic pathways are obfuscated by high copy numbers and fractured synteny. This study focuses on the carrier domains that scaffold natural product synthesis (thiolation domains) and the phosphopantetheinyl transferases (PPTases) that thiolate these carriers. We replaced the thiolation domain of the indigoidine producing BpsA gene from Streptomyces lavendulae with those of three multidomain dinoflagellate transcripts and coexpressed these constructs with each of three dinoflagellate PPTases looking for specific pairings that would identify distinct pathways. Surprisingly, all three PPTases were able to activate all the thiolation domains from one transcript, although with differing levels of indigoidine produced, demonstrating an unusual lack of specificity. Unfortunately, constructs with the remaining thiolation domains produced almost no indigoidine and the thiolation domain for lipid synthesis could not be expressed in E. coli. These results combined with inconsistent protein expression for different PPTase/thiolation domain pairings present technical hurdles for future work. Despite these challenges, expression of catalytically active dinoflagellate proteins in E. coli is a novel and useful tool going forward.

Mitigation of CyanoHABs Using Phoslock® to Reduce Water Column Phosphorus and Nutrient Release from Sediment.

Cyanobacterial blooms can be stimulated by excessive phosphorus (P) input, especially when diazotrophs are the dominant species. A series of mesocosm experiments were conducted in a lake dominated by a cyanobacteria bloom to study the effects of Phoslock®, a phosphorus adsorbent. The results showed that the addition of Phoslock® lowered the soluble reactive phosphate (SRP) concentrations in water due to efficient adsorption and mitigated the blooms. Once settled on the sediments, Phoslock® serves as a barrier to reduce P diffusion from sediments into the overlying waters. In short-term (1 day) incubation experiments, Phoslock® diminished or reversed SRP effluxes from bottom sediments. At the same time, the upward movement of the oxic-anoxic interface through the sediment column slightly enhanced NH4+ release and depressed N2 release, suggesting the inhibition of nitrification and denitrification. In a long-term (28 days) experiment, Phoslock® hindered the P release, reduced the cyanobacterial abundance, and alleviated the bloom-driven enhancements in the pH and oxygen. These results suggest that, through suppression of internal nutrient effluxes, Phoslock® can be used as an effective control technology to reduce cyanobacteria blooms common to many freshwater systems.

A Global Approach to Estimating the Abundance and Duplication of Polyketide Synthase Domains in Dinoflagellates.

Many dinoflagellate species make toxins in a myriad of different molecular configurations but the underlying chemistry in all cases is presumably via modular synthases, primarily polyketide synthases. In many organisms modular synthases occur as discrete synthetic genes or domains within a gene that act in coordination thus forming a module that produces a particular fragment of a natural product. The modules usually occur in tandem as gene clusters with a syntenic arrangement that is often predictive of the resultant structure. Dinoflagellate genomes however are notoriously complex with individual genes present in many tandem repeats and very few synthetic modules occurring as gene clusters, unlike what has been seen in bacteria and fungi. However, modular synthesis in all organisms requires a free thiol group that acts as a carrier for sequential synthesis called a thiolation domain. We scanned 47 dinoflagellate transcriptomes for 23 modular synthase domain models and compared their abundance among 10 orders of dinoflagellates as well as their co-occurrence with thiolation domains. The total count of domain types was quite large with over thirty-thousand identified, 29 000 of which were in the core dinoflagellates. Although there were no specific trends in domain abundance associated with types of toxins, there were readily observable lineage specific differences. The Gymnodiniales, makers of long polyketide toxins such as brevetoxin and karlotoxin had a high relative abundance of thiolation domains as well as multiple thiolation domains within a single transcript. Orders such as the Gonyaulacales, makers of small polyketides such as spirolides, had fewer thiolation domains but a relative increase in the number of acyl transferases. Unique to the core dinoflagellates, however, were thiolation domains occurring alongside tetratricopeptide repeats that facilitate protein-protein interactions, especially hexa and hepta-repeats, that may explain the scaffolding required for synthetic complexes capable of making large toxins. Clustering analysis for each type of domain was also used to discern possible origins of duplication for the multitude of single domain transcripts. Single domain transcripts frequently clustered with synonymous domains from multi-domain transcripts such as the BurA and ZmaK like genes as well as the multi-ketosynthase genes, sometimes with a large degree of apparent gene duplication, while fatty acid synthesis genes formed distinct clusters. Surprisingly the acyl-transferases and ketoreductases involved in fatty acid synthesis (FabD and FabG, respectively) were found in very large clusters indicating an unprecedented degree of gene duplication for these genes. These results demonstrate a complex evolutionary history of core dinoflagellate modular synthases with domain specific duplications throughout the lineage as well as clues to how large protein complexes can be assembled to synthesize the largest natural products known.

Spatiotemporal distribution of phycotoxins and their co-occurrence within nearshore waters.

Harmful algal blooms (HABs), varying in intensity and causative species, have historically occurred throughout the Chesapeake Bay, U.S.; however, phycotoxin data are sparse. The spatiotemporal distribution of phycotoxins was investigated using solid-phase adsorption toxin tracking (SPATT) across 12 shallow, nearshore sites within the lower Chesapeake Bay and Virginia's coastal bays over one year (2017-2018). Eight toxins, azaspiracid-1 (AZA1), azaspiracid-2 (AZA2), microcystin-LR (MC-LR), domoic acid (DA), okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2), and goniodomin A (GDA) were detected in SPATT extracts. Temporally, phycotoxins were always present in the region, with at least one phycotoxin group (i.e., consisting of OA and DTX1) detected at every time point. Co-occurrence of phycotoxins was also common; two or more toxin groups were observed in 76% of the samples analyzed. Toxin maximums: 0.03 ng AZA2/g resin/day, 0.25 ng DA/g resin/day, 15 ng DTX1/g resin/day, 61 ng OA/g resin/day, 72 ng PTX2/g resin/day, and 102,050 ng GDA/g resin/day were seasonal, with peaks occurring in summer and fall. Spatially, the southern tributary and coastal bay regions harbored the highest amount of total phycotoxins on SPATT over the year, and the former contained the greatest diversity of phycotoxins. The novel detection of AZAs in the region, before a causative species has been identified, supports the use of SPATT as an explorative tool in respect to emerging threats. The lack of karlotoxin in SPATT extracts, but detection of Karlodinium veneficum by microscopy, however, emphasizes that this tool should be considered complementary to, but not a replacement for, more traditional HAB management and monitoring methods.

A Screening Tool for the Direct Analysis of Marine and Freshwater Phycotoxins in Organic SPATT Extracts from the Chesapeake Bay.

Many detection methods for phycotoxins, bioactive compounds produced by harmful algae, focus on one compound or a class of related compounds. Multiple harmful algal species often co-occur in the environment, however, emphasizing the need to analyze for the presence of multiple groups of marine and freshwater phycotoxins in environmental samples, e.g., extracts from solid phase adsorption toxin tracking (SPATT). Two methods were developed to screen for 13 phycotoxins (microcystin-RR, -LR, -YR, azaspiracid-1, -2, karlotoxin 3, goniodomin A, brevetoxin-2, yessotoxin, pectenotoxin-2, dinophysistoxin-1, -2, and okadaic acid) in organic SPATT extracts using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) equipped with a trapping dimension (trap) and at-column dilution (ACD). The performance of each compound under 36 combinations of chromatographic conditions was characterized, and two final methods, acidic and basic, were selected based on peak shapes, signal intensities, resolution, and the separation in time of positive and negative MS ionization modes. Injection volumes of up to 1 mL were possible through trap/ACD technology, resulting in limits of detection between 0.001 and 0.05 µg/L across the analytes. Benefits highlighted in this study, beyond the improved detection limits and co-detection of multiple toxin groups, include the ability to inject samples of 100% organic solvent, ensuring analyte stability and streamlining workflow through the elimination of laborious sample preparation steps.

Effects of dietary taurine level on visual function in European sea bass (Dicentrarchus labrax).

Dietary insufficiencies have been well documented to decrease growth rates and survival (and therefore overall production) in fish aquaculture. By contrast, the effects of dietary insufficiencies on the sensory biology of cultured fish remains largely unstudied. Diets based solely on plant protein sources could have advantages over fish-based diets because of the cost and ecological effects of the latter, but plant proteins lack the amino acid taurine. Adequate levels of taurine are, however, necessary for the development of a fully functional visual system in mammals. As part of ongoing studies to determine the suitability of plant-based diets, we investigated the effects of normal and reduced taurine dietary levels on retinal anatomy and function in European sea bass (Dicentrarchus labrax). We could not demonstrate any effects of dietary taurine level on retinal anatomy, nor the functional properties of luminous sensitivity and temporal resolution (measured as flicker fusion frequency). We did, however, find an effect on spectral sensitivity. The peak of spectral sensitivity of individuals fed a 5% taurine diet was rightward shifted (i.e., towards longer wavelengths) relative to that of fish fed a 0% or 1.5% taurine diet. This difference in in spectral sensitivity was due to a relatively lower level of middle wavelength pigment (maximum absorbance .500 nm) in fish fed a 5% taurine diet. Changes in spectral sensitivity resulting from diets containing different taurine levels are unlikely to be detrimental to fish destined for market, but could be in fishes that are being reared for stock enhancement programs.

The chemodiversity of algal dissolved organic matter from lysed Microcystis aeruginosa cells and its ability to form disinfection by-products during chlorination.

Algal-derived dissolved organic matter (ADOM) originating from lysed Microcystis aeruginosa cells was investigated as precursor material to form disinfection by-products upon disinfection with free chlorine. Non-targeted ultrahigh resolution 12 T negative mode electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) revealed high molecular diversity in solid-phase extracted and ionizable components of Microcystis aeruginosa ADOM. The toxin microcystin LR was effectively degraded by free chlorine, which was expected. However, we found a high diversity of disinfection by-products associated with the addition of free chlorine to the water-soluble and solid-phase extractable fraction of ADOM and of double-bond moieties in abundant and known unsaturated fatty acids. Aromatic DOM precursors were absent from known metabolites of Microcystis aeruginosa and no evidence for aromatic disinfection by-products (DBPs) was found, despite N-containing compounds. A large diversification of N-containing molecular formulas was observed after chlorination, which seems indicative for the breakdown and oxidation of larger peptides. Additionally, a diverse group of N-compounds with presumed chloramine functional groups was observed. This study highlights the importance to evaluate ADOM and its ability to form different DBPs when compared to allochthonous or terrestrially-derived DOM.

LC-MS/MS Detection of Karlotoxins Reveals New Variants in Strains of the Marine Dinoflagellate Karlodinium veneficum from the Ebro Delta (NW Mediterranean).

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the detection and quantitation of karlotoxins in the selected reaction monitoring (SRM) mode. This novel method was based upon the analysis of purified karlotoxins (KcTx-1, KmTx-2, 44-oxo-KmTx-2, KmTx-5), one amphidinol (AM-18), and unpurified extracts of bulk cultures of the marine dinoflagellate Karlodinium veneficum strain CCMP2936 from Delaware (Eastern USA), which produces KmTx-1 and KmTx-3. The limit of detection of the SRM method for KmTx-2 was determined as 2.5 ng on-column. Collision induced dissociation (CID) spectra of all putative karlotoxins were recorded to present fragmentation patterns of each compound for their unambiguous identification. Bulk cultures of K. veneficum strain K10 isolated from an embayment of the Ebro Delta, NW Mediterranean, yielded five previously unreported putative karlotoxins with molecular masses 1280, 1298, 1332, 1356, and 1400 Da, and similar fragments to KmTx-5. Analysis of several isolates of K. veneficum from the Ebro Delta revealed small-scale diversity in the karlotoxin spectrum in that one isolate from Fangar Bay produced KmTx-5, whereas the five putative novel karlotoxins were found among several isolates from nearby, but hydrographically distinct Alfacs Bay. Application of this LC-MS/MS method represents an incremental advance in the determination of putative karlotoxins, particularly in the absence of a complete spectrum of purified analytical standards of known specific potency.

Use of Antibiotics for Maintenance of Axenic Cultures of Amphidinium carterae for the Analysis of Translation.

Most dinoflagellates in culture are bacterized, complicating the quantification of protein synthesis, as well as the analysis of its regulation. In bacterized cultures of Amphidinium carterae Hulbert, up to 80% of protein synthetic activity appears to be predominantly bacterial based on responses to inhibitors of protein synthesis. To circumvent this, axenic cultures of A. carterae were obtained and shown to respond to inhibitors of protein synthesis in a manner characteristic of eukaryotes. However, these responses changed with time in culture correlating with the reappearance of bacteria. Here we show that culture with kanamycin (50 µg/mL), carbenicillin (100 µg/mL), and streptomycin sulfate (50 µg/mL) (KCS), but not 100 units/mL of penicillin and streptomycin (PS), prevents the reappearance of bacteria and allows A. carterae protein synthesis to be quantified without the contribution of an associated bacterial community. We demonstrate that A. carterae can grow in the absence of a bacterial community. Furthermore, maintenance in KCS does not inhibit the growth of A. carterae cultures but slightly extends the growth phase and allows accumulation to somewhat higher saturation densities. We also show that cultures of A. carterae maintained in KCS respond to the eukaryotic protein synthesis inhibitors cycloheximide, emetine, and harringtonine. Establishment of these culture conditions will facilitate our ability to use polysome fractionation and ribosome profiling to study mRNA recruitment. Furthermore, this study shows that a simple and fast appraisal of the presence of a bacterial community in A. carterae cultures can be made by comparing responses to cycloheximide and chloramphenicol rather than depending on lengthier culture-based assessments.

Assimilation efficiency of sediment-bound PCBs ingested by fish impacted by strong sorption.

Uptake of polychlorinated biphenyls (PCBs) by fish is controlled by the bioavailability of ingested PCBs in the gut and the freely dissolved concentration in the water moving across the gills. The prediction of bioaccumulation in fish relies on models that account for these exposure routes; however, these models typically do not account for incidental ingestion of sediment by fish, which is not well studied. The literature values for the PCB assimilation efficiency in the gut have been reported for compounds in food matrices and not associated with sediment particles. It is also unclear how mitigation strategies that alter PCB bioavailability in sediments affect predictions made by the bioaccumulation models when sediment ingestion is involved. To test the bioavailability of PCBs from treated and untreated sediments, dietary assimilation efficiencies were measured for 16 PCB congeners in mummichogs (Fundulus heteroclitus) that were fed 4 experimental diets. Diets consisted of PCB-spiked earthworms, spiked untreated sediment mixed with earthworms, spiked activated carbon-treated sediment mixed with earthworms, and spiked activated carbon mixed with earthworms. Assimilation efficiencies were determined by calculating the ratio of PCB mass in the fish tissue to the PCB mass in the food after a pulse feeding experiment. Assimilation efficiencies of PCBs associated with earthworm diet were similar to the values reported in the literature. Fish that were fed the PCB-spiked untreated sediment and activated carbon particles exhibited the highest and lowest assimilation efficiencies, respectively, over a wide KOW range. Assimilation efficiencies of sediment-bound PCBs were significantly reduced (31-93% reduction for different congeners) after amendment with activated carbon. The present study indicates that assimilation of PCBs can be reduced by sorption to black carbon. Environ Toxicol Chem 2017;36:3480-3488. © 2017 SETAC.

Toxin-Antitoxin Systems in Estuarine Synechococcus Strain CB0101 and Their Transcriptomic Responses to Environmental Stressors.

Bacterial toxin-antitoxin (TA) systems are genetic elements composed of a toxin gene and its cognate antitoxin, with the ability to regulate growth. TA systems have not previously been reported in marine Synechococcus or Prochlorococcus. Here we report the finding of seven TA system pairs (Type II) in the estuarine Synechococcus CB0101, and their responses of these TA genes to under different stress conditions, which include; nitrogen and phosphate starvation, phage infection, zinc toxicity, and photo-oxidation. Database searches discovered that eight other marine Synechococcus strains also contain at least one TA pair but none were found in Prochlorococcus. We demonstrate that the relB/relE TA pair was active and resulted in RNA degradation when CB0101 was under oxidative stress caused by either zinc toxicity or high light intensities, but the growth inhibition was released when the stress was removed. Having TA systems allows Synechococcus CB0101 to adapt to the low light and highly variable environments in the Chesapeake Bay. We propose that TA systems could be more important for picocyanobacteria living in the freshwater and estuarine environments compared to those living in the open ocean.