Okadaic acid, an apoptogenic toxin for symbiotic/parasitic annelids in the demosponge Suberites domuncula

The role of okadaic acid (OA) in the defense system of the marine demosponge Suberites domuncula against symbiotic/parasitic annelids was examined. Bacteria within the mesohyl produced okadaic acid at concentrations between 32 ng/g and 58 ng/g of tissue (wet weight). By immunocytochemical methods and by use of antibodies against OA, we showed that the toxin was intracellularly stored in vesicles. Western blotting experiments demonstrated that OA also existed bound to a protein with a molecular weight of 35,000 which was tentatively identified as a galectin (by application of antigalectin antibodies). Annelids that are found in S. domuncula undergo apoptotic cell death. OA is one candidate inducer molecule of this process, since this toxin accumulated in these symbionts/parasites. Furthermore, we identified the cDNA encoding the multifunctional prosurvival molecule BAG-1 in S. domuncula; it undergoes strong expression in the presence of the annelid. Our data suggest that sponges use toxins (here, OA) produced from bacteria to eliminate metazoan symbionts/parasites by apoptosis.     

Development of an immunochromatographic strip test for the rapid detection of okadaic acid in shellfish sample

A rapid detection technology for okadaic acid (OA) in shellfish with one-step immunochromatographic assay using colloidal gold-labeled monoclonal antibody (Mab) probe was developed. OA is one of the diarrhetic shellfish toxins. Firstly, OA was conjugated to bovine serum albumin, and the conjugations as immunogen were injected into mice to raise the polyclonal antibody against OA. Hybridoma cells fused between spleen cells from immunized mouse and myeloma cells (Sp2/0) were prepared and injected into mice intraperitoneally at 1?×?10⁶?cells to produce monoclonal antibody in the ascitic fluid. With the monoclonal antibody against OA, the idc-ELISA assay was established to detect OA. The calibration curve for OA was linear over the concentration range of 0.31–50 ng mL^?1, and the detection limit for OA was 0.45 ng mL^?1. On that basis, paper test strips for detecting OA were prepared, and a fast detection method for okadaic acid using gold-labeled immunological assay was established. With the paper test strips, the detection limit was 6.25 ng mL^?1, and whole detection process for OA in shellfish samples needed only about 40 min.     

ELISA FOR DETECTING OKADAIC ACED IN MODEL SYSTEMS USING PURIFIED POLYCLONAL ANTIBODIES

Polyclonal antibodies were produced in rabbits against okadaic acid (OA) following its coupling to bovine serum albumin using standard carbodiimide condensation procedure to form the immunogen. The immunogen was dialyzed against Tris buffer pH 7.45 at 4C and used to immunize two rabbits. Each rabbit received (at four sites) 0.5 mL immunogen (i.d.) together with adjuvant followed by three repeated injections of 0.3 mL emulsified immunogen-adjuvant mixture at weekly intervals. The last injection was made 60 days after the fourth. The antiserum was collected, at intervals, the immunoglobulin fraction (IgG) isolated, purified and used in an ELISA system to capture the okadaic moiety of the immunogen. Antibody titers increased following repeated immunization and the IgG recognized low levels of OA. However, ELISA was more sensitive for detecting the immunogen than for pure OA, but using the biotinylated-IgG enhanced the titration to both and the detection limit for OA was 0.63 ng per 0.1 mL buffer-methanol and the assay linearity ranged from 0.63 to 5.0 ng OA.     

Multi-species okadaic acid contamination and human poisoning during a massive bloom of Dinophysis acuminata complex in southern Brazil

On June 2016, a major bloom of Dinophysis acuminata complex was noticed over the coast of Paraná State (PR), southern Brazil, an area unprotected by any official monitoring program. Here we report the results of an extensive sampling effort that ultimately led PR authorities to issue the first State shellfish-harvesting ban due to multi-species okadaic acid (OA) contamination. During its peak, the bloom covered an area of 201 km² (∼2.0–3.5 × 54.0 km), attaining unprecedentedly high cell densities along the shallow (<15 m) continental shelf (mean 2.2 × 10⁵, maximum 2.1 × 10⁶ cells L⁻¹) and adjacent sandy beaches (mean 2.8 × 10⁵, maximum 5.2 × 10⁶ cells L⁻¹). Only OA was detected in suspension (max. 188 ng L⁻¹). Toxin levels measured in bivalves were several times greater than the regulatory limit of 160 ng g⁻¹, reaching up to 3600 ng g⁻¹ in Crassostrea gasar, by far the highest OA concentrations ever reported in oysters worldwide, 7700 ng g⁻¹ in brown mussels, Perna perna, and lower levels in clams, Anomalocardia brasiliana, and mangrove mussels, Mytella spp. Nine cases of human intoxication were officially reported and five people were hospitalized with typical symptoms of Diarrhetic Shellfish Poisoning linked to the consumption of contaminated bivalves. All bivalves quickly converted most of the OA into its esterified form, DTX-3, and eliminated the toxins only a few weeks following the bloom, with C. gasar being the slowest-detoxifying species. Lower OA levels were accumulated in zooplankton, gastropods and several novel toxin vectors, including benthic organisms such as sand dollars Mellita quinquiesperforata and the ghost-shrimp Callichirus major, which may act as a good indicator of the presence of toxins in sandy beaches, and pelagic fish species that can serve as potential alternative sources of OA to humans (Chaetodipterus faber and Mugil liza). Monitoring toxin contamination in seafood other than bivalves is thus recommended to ensure comprehensive human health protection during massive Dinophysis blooms. Additionally, since OA was also present at low concentrations in the liver of Guiana dolphins Sotalia guianensis and penguins Spheniscus magellanicus, exposure to biotoxins should be considered in conservation actions involving threatened and near-threatened marine organisms in this region.     

Production and characteristics of monoclonal antibodies to the diphtheria toxin

Monoclonal antibodies to the diphtheria toxin were produced without cross reactivity with the thermolabile toxin (LT) from Escherichia coli; ricin; choleraic toxin; the SeA, SeB, SeE, SeI, and SeG toxins of staphylococcus; the lethal factor of the anthrax toxin; and the protective antigen of the anthrax toxin. A pair of antibodies for the quantitative determination of the diphtheria toxin in the sandwich variation of enzyme-linked immunosorbent assay (ELISA) was chosen. The determination limit of the toxin was 0.7 ng/ml in plate and 1.6 ng/ml in microchip ELISA. The presence of a secretion from the nasopharynx lavage did not decrease the sensitivity of the toxin determination by sandwich ELISA. The immunization of mice with the diphtheria toxin and with a conjugate of the diphtheria toxin with polystyrene microspheres demonstrated that the conjugate immunization resulted in the formation of hybridoma clones which produced antibodies only to the epitopes of the A fragment of the diphtheria toxin. The immunization with the native toxin caused the production of hybridoma clones which predominantly produced antibodies to the epitopes of the B fragment.     

A screening lateral flow immunochromatographic assay for on-site detection of okadaic acid in shellfish products

A lateral flow immunochromatographic (LFIC) test strip based on a colloidal gold-monoclonal antibody (McAb) conjugate was developed for on-site rapid detection of okadaic acid (OA) in shellfish. It applies a competitive format using an immobilized toxin conjugate and free toxin present in samples. The McAb against OA was conjugated with 20-nm colloidal gold as detector reagent. The toxin in the sample competed with the immobilized toxin to bind to the gold conjugated with McAb. The colloidal gold/McAb/toxin mobile complex was not captured by OA-bovine serum albumin (BSA) on the test line, but it was captured by goat anti-mouse immunoglobulin G (IgG) on the control line. The color density of the test line correlated with the concentration of toxin in the range of 10-50 ng ml(-1). The qualitative detection limit of 150 μg kg(-1) sample was close to the European Union (EU) regulatory limit (160 μg kg(-1)). Therefore, these strips were able to directly and qualitatively estimate the consuming safety of shellfish. They require no equipment because of available visual results, and they screened numerous samples within 10 min. The results were further confirmed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). As a food safety screening tool, the test strips are convenient and useful to rapidly on-site test the presence of OA in shellfish products.     

Evaluation of Pakistanian Bacillus thuringiensis isolates against Scirpophaga incertulas and Cnaphalocrocis medinalis

A large number of Bacillus thuringiensis (Bt) isolates separated from different ecological regions of Pakistan were characterized for crystal protein gene composition and pesticidal activity against two lepidopteran rice insect pests, the yellow stem borer (Scirpophaga incertulas) and the rice leaf folders (Cnaphalocrocis medinalis). A representative seventeen isolates were selected on the basis of initial screening and further characterization of pesticidal activity was performed according to following criteria; colony and parasporal inclusion morphology, SDS-PAGE, western blot analysis and comparative biotoxicity assays to determine LC₅₀ values. All isolates produced parasporal inclusion bodies and spores in their cells. Immunoblotting results showed that Pakistanian isolates synthesized entomocidal proteins belonging to Cry1A and Cry2A toxin groups. The biological activity of local isolates demonstarted a wide range of LC₅₀ values against both target insects pests. The most potent isolates, INS 1.13, INS 2.25 and NW 4.1 against S. incertulas showed LC₅₀ values of 29.83, 30.37 and 24.77 ng/ml of toxin, respectively. The LC₅₀ values of 57.37 and 73.09 ng/ml of toxin were exhibited by local isolates, INS 2.25 and RL 4.8 against C. medinalis, respectively.     

The Mitochondrial Toxin Produced by Streptomyces griseus Strains Isolated from an Indoor Environment Is Valinomycin

Actinomycete isolates from indoor air and dust in water-damaged schools and children’s day care centers were tested for toxicity by using boar spermatozoa as an indicator. Toxicity was detected in extracts of four strains which caused a loss of sperm motility, and the 50% effective concentrations (EC₅₀) were 10 to 63 ng (dry weight) ml of extended boar semen⁻¹. The four strains were identified as Streptomyces griseus strains by 16S ribosomal DNA and chemotaxonomic methods. The four S. griseus strains had similar effects on sperm cells, including loss of motility and swelling of mitochondria, but we observed no loss of plasma membrane integrity or depletion of cellular ATP. None of the effects was observed with sperm cells exposed to extracts of other indoor actinomycete isolates at concentrations of ≥5,000 to 72,000 ng ml⁻¹. The toxin was purified from all four strains and was identified as a dodecadepsipeptide, and the fragmentation pattern obtained by tandem mass spectrometry was identical to that of valinomycin. Commercial valinomycin had effects in sperm cells that were identical to the effects of the four indoor isolates of S. griseus. The EC₅₀ of purified toxin from the S. griseus strains were 1 to 3 ng ml of extended boar semen⁻¹, and the EC₅₀ of commercial valinomycin was 2 ng ml of extended boar semen⁻¹. To our knowledge, this is the first report of the presence of ionophoric toxin producers in an indoor environment and the first report of valinomycin-producing strains identified as S. griseus.     

Cell cycle regulation of the mixotrophic dinoflagellate Dinophysis acuminata: Growth,photosynthetic efficiency and toxin production

The mixotrophic dinoflagellate Dinophysis acuminata is a widely distributed diarrhetic shellfish poisoning (DSP) producer. Toxin variability of Dinophysis spp. has been well studied, but little is known of the manner in which toxin production is regulated throughout the cell cycle in these species, in part due to their mixotrophic characteristics. Therefore, an experiment was conducted to investigate cell cycle regulation of growth, photosynthetic efficiency, and toxin production in D. acuminata. First, a three-step synchronization approach, termed “starvation-feeding-dark”, was used to achieve a high degree of synchrony of Dinophysis cells by starving the cells for 2 weeks, feeding them once, and then placing them in darkness for 58 h. The synchronized cells started DNA synthesis (S phase) 10 h after being released into the light, initiated G2 growth stage eight hours later, and completed mitosis (M phase) 2 h before lights were turned on. The toxin content of three dominant toxins, okadaic acid (OA), dinophysistoxin-1 (DTX1) and pectenotoxin-2 (PTX2), followed a common pattern of increasing in G1 phase, decreasing on entry into the S phase, then increasing again in S phase and decreasing in M phase during the diel cell cycle. Specific toxin production rates were positive throughout the G1 and S phases, but negative during the transition from G1 to S phase and late in M phase, the latter reflecting cell division. All toxins were initially induced by the light and positively correlated with the percentage of cells in S phase, indicating that biosynthesis of Dinophysis toxins might be under circadian regulation and be most active during DNA synthesis.     

Evolution of conflict and cooperation of nematodes associated with solitary and social sweat bees

Parasites and mutualists can wield great influence on the fitness of social organisms, yet the effect that the host’s social structure has on the evolution of parasites, commensals, and mutualists (collectively referred to here as symbionts) is poorly known. Evolutionary theory suggests that host social structure may select for more cooperative symbiont strains in comparison to symbionts of solitary hosts. We compared the productivity of one social and one solitary bee species (Halictus ligatus and Augochlora pura) in the family Halictidae with and without the presence of their nematode symbionts (Acrostichus halicti and Acrostichus puri, respectively). We measured the number of offspring produced, the number of cells provisioned, and nesting activity (for Au. pura) to test the hypothesis that symbionts specific to a social host exhibit greater cooperation than symbionts specific to a solitary host. Infected and uninfected nests of both species did not differ in any fitness estimates indicating that: (1) Acrostichus species are commensals, or at least lack large fitness effects on their hosts, and (2) the transition from association with a solitary host to association with a social host that lives in small colonies does not have detectable effects on the evolution of conflict and cooperation in this system. This is the first comparative study to test the idea that host social structure may influence the evolution of symbionts; future work should compare closely related mutualists and parasites of more advanced eusocial insects to mutualists and parasites of solitary insects.     

Characterization and comparison of toxin‐producing isolates of Dinophysis acuminata from New England and Canada

Following the identification of the first toxic isolate of Dinophysis acuminata from the northwestern Atlantic, we conducted detailed investigations into the morphology, phylogeny, physiology, and toxigenicity of three isolates from three sites within the northeastern U.S./Canada region: Eel Pond and Martha's Vineyard, Massachusetts, and the Bay of Fundy. Another isolate, collected from the Gulf of Mexico, was grown under the same light, temperature, and prey conditions for comparison. Despite observed phenotypic heterogeneity, morphometrics and molecular evidence classified the three northwestern Atlantic isolates as D. acuminata Claparède & Lachmann, whereas the isolate from the Gulf of Mexico was morphologically identified as D. cf. ovum. Physiological and toxin analyses supported these classifications, with the three northwestern Atlantic isolates being more similar to each other with respect to growth rate, toxin profile, and diarrhetic shellfish poisoning (DSP) toxin content (okadaic acid + dinophysistoxin 1/cell) than they were to the isolate from the Gulf of Mexico, which had toxin profiles similar to those published for D. cf. ovum F. Schütt. The DSP toxin content, 0.01–1.8 pg okadaic acid (OA) + dinophysistoxin (DTX1) per cell, of the three northwestern Atlantic isolates was low relative to other D. acuminata strains from elsewhere in the world, consistent with the relative scarcity of shellfish harvesting closures due to DSP toxins in the northeastern U.S. and Canada. If this pattern is repeated with the analyses of more geographically and temporally dispersed isolates from the region, it would appear that the risk of significant DSP toxin outbreaks in the northwestern Atlantic is low to moderate. Finally, the morphological, physiological, and toxicological variability within D. acuminata may reflect spatial (and/or temporal) population structure, and suggests that sub‐specific resolution may be helpful in characterizing bloom dynamics and predicting toxicity.     

Antibacterial Activity of Sirodesmin PL Phytotoxin: Application to the Selection of Phytotoxin-Deficient Mutants

Sirodesmin PL, a phytotoxin and mycotoxin produced by Leptosphaeria maculans, the causal agent of stem-canker disease of crucifers, exhibited antibacterial activity against gram-positive bacteria and particularly Bacillus subtilis. The importance of the disulfide bridge of the molecule in antibacterial activity was demonstrated. A simple and reliable bioassay based on the antibacterial activity of the toxin was performed for screening sirodesmin PL-deficient mutants when grown on solid culture medium. A mutant was selected and found to produce 3,700-fold less toxin than did the wild-type strain. A sensitive procedure for quantification of the toxin by high-pressure liquid chromatography was developed. Levels of product as low as 100 ng could be detected by this procedure.     

Diarrheic toxins in field-sampled and cultivated Dinophysis spp. cells from southern Brazil

In southern Brazil, mixotrophic dinoflagellates belonging to the Dinophysis acuminata complex have recently been involved in diarrheic shellfish poisoning episodes through the production of lipophilic toxins such as okadaic acid (OA) and dinophysistoxin-1 (DTX-1). The present investigation used a combination of laboratory cultures and field surveys at three large estuarine systems in that region to examine toxin retention in Dinophysis spp. cells under optimum or growth-limiting conditions. This study represents the first successful culture of a Dinophysis isolate from the Atlantic South America region. Starved D. acuminata complex cells reached 5.6-fold higher cellular OA quotas (up to 18 pg cell^?1) than Mesodinium rubrum-fed cultures 20 days after inoculation in the laboratory. Moreover, in field samples, light-limited cells at the bottom of a stratified water column were less abundant, yet 6.6- to 11-fold more toxic (up to 26.4 pg OA and 1.7 pg DTX-1 cell^?1) than those located at the illuminated surface. This phenomenon of toxin retention by slow-dividing cells may partially explain the enormous variation in cell toxin quota found within Dinophysis spp. populations from a single location, and it may have serious implications for cell count-based monitoring program in bivalve aquaculture areas. In fact, only low to moderate OA levels were detected in the digestive glands of oysters Crassostrea spp. (up to 17.8 ng g^?1) and the guts and livers of filter-feeding fish (44.7 ng g^?1) during the present study, despite the relatively high Dinophysis cell densities (up to 19,500 cells L^?1) found in the field.     

Identification of small molecule inhibitors of Pseudomonas aeruginosa exoenzyme S using a yeast phenotypic screen

Pseudomonas aeruginosa is an opportunistic human pathogen that is a key factor in the mortality of cystic fibrosis patients, and infection represents an increased threat for human health worldwide. Because resistance of Pseudomonas aeruginosa to antibiotics is increasing, new inhibitors of pharmacologically validated targets of this bacterium are needed. Here we demonstrate that a cell-based yeast phenotypic assay, combined with a large-scale inhibitor screen, identified small molecule inhibitors that can suppress the toxicity caused by heterologous expression of selected Pseudomonas aeruginosa ORFs. We identified the first small molecule inhibitor of Exoenzyme S (ExoS), a toxin involved in Type III secretion. We show that this inhibitor, exosin, modulates ExoS ADP-ribosyltransferase activity in vitro, suggesting the inhibition is direct. Moreover, exosin and two of its analogues display a significant protective effect against Pseudomonas infection in vivo. Furthermore, because the assay was performed in yeast, we were able to demonstrate that several yeast homologues of the known human ExoS targets are likely ADP-ribosylated by the toxin. For example, using an in vitro enzymatic assay, we demonstrate that yeast Ras2p is directly modified by ExoS. Lastly, by surveying a collection of yeast deletion mutants, we identified Bmh1p, a yeast homologue of the human FAS, as an ExoS cofactor, revealing that portions of the bacterial toxin mode of action are conserved from yeast to human. Taken together, our integrated cell-based, chemical-genetic approach demonstrates that such screens can augment traditional drug screening approaches and facilitate the discovery of new compounds against a broad range of human pathogens.     

Evidence of freshwater algal toxins in marine shellfish: Implications for human and aquatic health

The occurrence of freshwater harmful algal bloom toxins impacting the coastal ocean is an emerging threat, and the potential for invertebrate prey items to concentrate toxin and cause harm to human and wildlife consumers is not yet fully recognized. We examined toxin uptake and release in marine mussels for both particulate and dissolved phases of the hepatotoxin microcystin, produced by the freshwater cyanobacterial genus Microcystis. We also extended our experimental investigation of particulate toxin to include oysters (Crassostrea sp.) grown commercially for aquaculture. California mussels (Mytilus californianus) and oysters were exposed to Microcystis and microcystin toxin for 24 h at varying concentrations, and then were placed in constantly flowing seawater and sampled through time simulating riverine flushing events to the coastal ocean. Mussels exposed to particulate microcystin purged the toxin slowly, with toxin detectable for at least 8 weeks post-exposure and maximum toxin of 39.11 ng/g after exposure to 26.65 μg/L microcystins. Dissolved toxin was also taken up by California mussels, with maximum concentrations of 20.74 ng/g after exposure to 7.74 μg/L microcystin, but was purged more rapidly. Oysters also took up particulate toxin but purged it more quickly than mussels. Additionally, naturally occurring marine mussels collected from San Francisco Bay tested positive for high levels of microcystin toxin. These results suggest that ephemeral discharge of Microcystis or microcystin to estuaries and the coastal ocean accumulate in higher trophic levels for weeks to months following exposure.     

Glucose Uptake in Enterocytes: A Test for Molecular Targets of Okadaic Acid

Abstract

The main diarrheic shellfish poisoning (DSP) toxin is okadaic acid (OA). Although OA is a protein phosphatase 1 and 2A inhibitor less is known about the involvement of the toxin in diarrhea. The initial statement was that OA, by altering the phosphorylation state of proteins, might modify glucose uptake and consequently ionic and water reabsorption across the small intestine. This report presents studies of glucose transport in isolated rabbit enterocytes by using a fluorescent derivative of D‐glucose. The dye allowed examining the relation between the toxic effect of OA and cellular mechanisms involved in glucose transport. The central findings are: (i) OA potentiates decrease on glucose uptake due to protein kinase A (PKA) inhibitors such as H89; and (ii) the increase of sugar uptake induced by the protein kinase C (PKC) inhibitor chelerythrine is enhanced by OA. Importance of this work is justified by the need to determine molecular targets of diarrheic toxins in intestinal cells.     

Development and Characterization of Recombinant Antibody Fragments That Recognize and Neutralize In Vitro Stx2 Toxin from Shiga Toxin-Producing Escherichia coli

Background

Stx toxin is a member of the AB₅ family of bacterial toxins: the active A subunit has N-glycosidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB) bind to globotria(tetra)osylceramide receptors (Gb3/Gb4) on the cell membrane. Shiga toxin-producing Escherichia coli strains (STEC) may produce Stx1 and/or Stx2 and variants. Strains carrying Stx2 are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans. The development of tools for the detection and/or neutralization of these toxins is a turning point for early diagnosis and therapeutics. Antibodies are an excellent paradigm for the design of high-affinity, protein-based binding reagents used for these purposes.

Methods and Findings

In this work, we developed two recombinant antibodies; scFv fragments from mouse hybridomas and Fab fragments by phage display technology using a human synthetic antibody library. Both fragments showed high binding affinity to Stx2, and they were able to bind specifically to the GKIEFSKYNEDDTF region of the Stx2 B subunit and to neutralize in vitro the cytotoxicity of the toxin up to 80%. Furthermore, the scFv fragments showed 79% sensitivity and 100% specificity in detecting STEC strains by ELISA.

Conclusion

In this work, we developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for the first time, we showed a human monovalent molecule, produced in bacteria, able to neutralize the cytotoxicity of Stx2 in vitro.     

Production and Characterization of Antibodies against Each of the Three Subunits of the Bacillus cereus Nonhemolytic Enterotoxin Complex

The nonhemolytic enterotoxin (Nhe) is one of the two three-component enterotoxins which are responsible for diarrheal food poisoning syndrome caused by Bacillus cereus. To facilitate the detection of this toxin, consisting of the subunits NheA, NheB, and NheC, a complete set of high-affinity antibodies against each of the three components was established and characterized. A rabbit antiserum specific for the C-terminal part (15 amino acids) of NheC was produced using a respective synthetic peptide coupled to a protein carrier for immunization. Using purified B. cereus exoprotein preparations as immunogens, one monoclonal antibody against NheA and several antibodies against NheB were obtained. No cross-reactivity with other proteins produced by different strains of B. cereus was observed. Antibodies against the NheB component were able to neutralize the cytotoxic activity (up to 98%) of Nhe. Based on indirect enzyme immunoassays, the antibodies developed in this study were successfully used in the characterization of the enterotoxic activity of several B. cereus strains. For the first time, it could be shown that strains carrying the nhe genes usually express the complete set of the three components, including NheC. However, the amount of toxin produced varies considerably between the different strains.     

Production and excretion of okadaic acid,pectenotoxin-2 and a novel dinophysistoxin from the DSP-causing marine dinoflagellate Dinophysis acuta – Effects of light,food availability and growth phase

Diarrhetic shellfish poisoning (DSP) toxins constitute a severe economic threat to shellfish industries and a major food safety issue for shellfish consumers. The prime producers of the DSP toxins that end up in filter feeding shellfish are species of the marine mixotrophic dinoflagellate genus Dinophysis. Intraspecific toxin contents of Dinophysis spp. vary a lot, but the regulating factors of toxin content are still poorly understood. Dinophysis spp. have been shown to sequester and use chloroplasts from their ciliate prey, and with this rare mode of nutrition, irradiance and food availability could play a key role in the regulation of toxins contents and production. We investigated toxin contents, production and excretion of a Dinophysis acuta culture under different irradiances, food availabilities and growth phases. The newly isolated strain of D. acuta contained okadaic acid (OA), pectenotoxins-2 (PTX-2) and a novel dinophysistoxin (DTX) that we tentatively describe as DTX-1b isomer. We found that all three toxins were excreted to the surrounding seawater, and for OA and DTX-1b as much as 90% could be found in extracellular toxin pools. For PTX-2 somewhat less was excreted, but often >50% was found extracellularly. This was the case both in steady-state exponential growth and in food limited, stationary growth, and we emphasize the need to include extracellular toxins in future studies of DSP toxins. Cellular toxin contents were largely unaffected by irradiance, but toxins accumulated both intra- and extracellularly when starvation reduced growth rates of D. acuta. Toxin production rates were highest during exponential growth, but continued at decreased rates when cell division ceased, indicating that toxin production is not directly associated with ingestion of prey. Finally, we explore the potential of these new discoveries to shed light on the ecological role of DSP toxins.     

Ubiquitins (polyubiquitin and ubiquitin extension protein) in marine sponges: cDNA sequence and phylogenetic analysis

The complete nucleotide sequences of two Suberites domuncula cDNAs and one Sycon raphanus cDNA, all encoding ubiquitin, have been determined. One cDNA from S. domuncula codes for polyubiquitin with four tandemly repeated monomeric units and the second cDNA encodes ubiquitin fused to a ribosomal protein of 78 amino acids (aa). S. domuncula possesses at least one additional polyubiquitin gene, from which the last two monomers were also sequenced. All analysed genes from S. domuncula encode identical ubiquitin proteins, with only one aa difference (Ala 19) to the human/higher animals ubiquitin (Pro 19). Ubiquitin in S. domuncula is identical with the ubiquitin found in another Demospongia, Geodia cydonium. The cDNA from S. raphanus encodes polyubiquitin with seven tandemly repeated units. All these gene monomers code for the same ubiquitin, which differs from the human/higher animals ubiquitin only at position 24 (Asp in Sycon, Glu in others). However, ubiquitin from S. raphanus (Calcarea) shows two aa differences (positions 19 and 24), when compared with the ubiquitin sequences from the two Demospongiae. In a phylogenetic tree constructed by multiple sequence alignment of all sponge ubiquitin gene monomers so far identified, all monomers from the same species cluster together, with the clear exception of the monomer from S. domuncula ribosomal protein fusion gene. This monomer branches off first from the tree and forms a separate line; this gives evidence for a very ancient split of ubiquitin-ribosomal-protein fusion genes from polyubiquitin encoding genes and their long separate coexistence in eukaryotes. The ubiquitin extension protein from S. domuncula is 78 aa long, displays all characteristics of 76–81 aa long ribosomal fusion proteins and shows 78% identity in the first 73 aa with the human S27a protein. However, its C-terminal sequence: 69-GLTYVYKKSD-78 is more similar to the plant consensus (69-GLTYVYQ/NK-76), than to the higher animal consensus (69-CLTYCFNK-76). This protein isolated from a sponge, belonging to the phylogenetically oldest multicellular animals, the Porifera, branches off first from the phylogenetic tree of metazoan ubiquitin extension proteins of the small ribosomal subunits.