Effects of copper and butyltin compounds on the growth, photosynthetic activity and toxin production of two HAB dinoflagellates: The planktonic Alexandrium catenella and the benthic Ostreopsis cf. ovata.

Controlled laboratory experiments were conducted to test the effects of copper (Cu2+) and butyltins (BuT) on the growth, photosynthetic activity and toxin content of two HABs (Harmful Algal Blooms) dinoflagellates, the planktonic Alexandrium catenella and the benthic Ostreopsis cf. ovata. Microalgae were exposed to increasing concentrations of Cu2+ (10-4 to 31 nM) or BuT (0.084 to 84 nM) for seven days. When considering the growth, EC50 values were 0.16 (±0.09) nM and 0.03 (±0.02) nM of Cu2+ for A. catenella and O. cf. ovata, respectively. Regarding BuT, EC50 was 14.2 (±6) nM for O. cf. ovata, while A. catenella growth inhibition appeared at BuT concentrations ≥27 nM. Photosynthetic activity of the studied dinoflagellates decreased with increasing Cu and BuT concentrations. For O. cf. ovata, the response of this physiological parameter to contamination was less sensitive than the biomass. Cu exposure induced the formation of temporary cysts in both organisms that could resist adverse conditions. The ovatoxin-a and -b concentrations in O. cf. ovata cells increased significantly in the presence of Cu. Altogether, the results suggest a better tolerance of the planktonic A. catenella to Cu and BuT. This could result in a differentiated selection pressure exerted by these metals on phytoplankton species in highly polluted waters. The over-production of toxins in response to Cu stress could pose supplementary health and socio-economic threats in the contaminated marine ecosystems where HABs develop.

Toxin production, growth kinetics and molecular characterization of Ostreopsis cf. ovata isolated from Todos os Santos Bay, tropical southwestern Atlantic.

The toxin profile and hemolytic activity of a strain of Ostreopsis cf. ovata (UFBA013) isolated from Todos os Santos Bay (northeastern Brazil) were evaluated under different levels of N and P. Phylogenetic analyses based on ITS rDNA region (ITS1-5.8S-ITS2) placed UFBA013 within the Atlantic/Mediterranean/Pacific clade of O. cf. ovata. Growth experiments were conducted in f/2 medium modified by adding N and P (P: 0-36 µM; N: 0-882 µM). The growth kinetics was adequately described by logistic equations. The best growth (highest Gm) was recorded under levels of N/P = 0/18, 129/5 and 441/36, while one of the lowest Gm was obtained under P-depletion. The maximum and specific maximum growth rates (as vm; cells mL-1 d-1 and µm; d-1) were achieved with N limitation (N/P = 441/36) and P-limitation/depletion (753/5.3 and 441/0) and are the highest values reported in the literature, most similar to isolates from Pacific and Mediterranean areas. The control experiment (N/P = 441/18) also yielded similar values to those from some Mediterranean isolates, but higher than formerly reported for Brazilian isolates. In all conditions assayed, no palytoxin (PLTX) was detected. The ovatoxins (OVTXs) a, b, c, d and e did not show significant differences in cell quota between exponential and stationary phases. A significant relationship was detected between OVTXs concentration and hemolytic activity.

cDNA cloning and expression of Contractin A, a phospholipase A2-like protein from the globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus.

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.

Eutrophic urban ponds suffer from cyanobacterial blooms: Dutch examples.

Ponds play an important role in urban areas. However, cyanobacterial blooms counteract the societal need for a good water quality and pose serious health risks for citizens and pets. To provide insight into the extent and possible causes of cyanobacterial problems in urban ponds, we conducted a survey on cyanobacterial blooms and studied three ponds in detail. Among 3,500 urban ponds in the urbanized Dutch province of North Brabant, 125 showed cyanobacterial blooms in the period 2009-2012. This covered 79% of all locations registered for cyanobacterial blooms, despite the fact that urban ponds comprise only 11% of the area of surface water in North Brabant. Dominant bloom-forming genera in urban ponds were Microcystis, Anabaena and Planktothrix. In the three ponds selected for further study, the microcystin concentration of the water peaked at 77 µg l(-1) and in scums at 64,000 µg l(-1), which is considered highly toxic. Microcystin-RR and microcystin-LR were the most prevalent variants in these waters and in scums. Cyanobacterial chlorophyll-a peaked in August with concentrations up to 962 µg l(-1) outside of scums. The ponds were highly eutrophic with mean total phosphorus concentrations between 0.16 and 0.44 mg l(-1), and the sediments were rich in potential releasable phosphorus. High fish stocks dominated by carp lead to bioturbation, which also favours blooms. As urban ponds in North Brabant, and likely in other regions, regularly suffer from cyanobacterial blooms and citizens may easily have contact with the water and may ingest cyanobacterial material during recreational activities, particularly swimming, control of health risk is of importance. Monitoring of cyanobacteria and cyanobacterial toxins in urban ponds is a first step to control health risks. Mitigation strategies should focus on external sources of eutrophication and consider the effect of sediment P release and bioturbation by fish.

[Seasonal dynamics of genus Alexandrium (potentially toxic dinoflagellate) in the lagoon of Bizerte (North of Tunisia) and controls by the abiotic factors]. / Dynamique saisonnière du genre Alexandrium (dinoflagellé potentiellement toxique) dans la lagune de Bizerte (Nord de la Tunisie) et contrôle par les facteurs abiotiques environnants.

Some species of the genus Alexandrium are known as potential producers of saxitoxin, a neurotoxin that causes the paralytic shellfish poisoning (PSP) syndrome. Blooming of these species, especially in shellfish farms can affect the aquaculture production and harm human health. Seasonal dynamics of Alexandrium spp. abundance in relationship to environmental factors was investigated from November 2007 to February 2009 at six stations in the Bizerte lagoon, an important shellfish farming area situated in SW Mediterranean. The sampling stations represented different hydrological and trophic conditions: one station TJ (Tinja) is affected by the river plume; two stations (Chaara [Ch] and Canal [Ca]) are influenced by marine inflow (particularly in summer), industrial and urban effluents; and the three other stations (Menzel Abdelrahmen [MA], Menzel Jemil [MJ] and Douaouda [Do]) are located close to shellfish farms. Cell abundance of Alexandrium spp. varied among stations and months. Species of this genus showed a sporadic appearance, but they reached high concentration (0.67-7 × 10(5)cells L(-1)). Maximal cell density was detected in autumn (November 2007; station MA), at salinity of 37.5, temperature of 16 °C and NH(4)(+) level of 55.45 µM. During this month, Alexandrium spp. abundance accounted for a large fraction (61%) of the harmful phytoplankton. The statistical analysis revealed that Alexandrium concentrations were positively correlated with N:P ratio and NH4+ levels. Thus, the eutrophic waters of the lagoon favour the growth of Alexandrium, which seemed to have preference for N-nutrient loading from antrophogenic activities, as ammonium. Blooms of these potential harmful algae may constitute a potential threat in this coastal lagoon of the southern Mediterranean. Consequently, it is necessary to be well vigilant and to do regular monitoring of Alexandrium species.

Screening and cDNA cloning of Kv1 potassium channel toxins in sea anemones.

When 21 species of sea anemones were screened for Kv1 potassium channel toxins by competitive inhibition of the binding of (125)I-α-dendrotoxin to rat synaptosomal membranes, 11 species (two species of Actiniidae, one species of Hormathiidae, five species of Stichodactylidae and three species of Thalassianthidae) were found to be positive. Furthermore, full-length cDNAs encoding type 1 potassium channel toxins from three species of Stichodactylidae and three species of Thalassianthidae were cloned by a combination of RT-PCR, 3'RACE and 5'RACE. The precursors of these six toxins are commonly composed of signal peptide, propart and mature peptide portions. As for the mature peptide (35 amino acid residues), the six toxins share more than 90% sequence identities with one another and with κ(1.3)-SHTX-She1a (Shk) from Stichodactyla helianthus but only 34-63% identities with the other type 1 potassium channel toxins.

[Effects of different phosphorus sources on the growth and toxin production of Prorocentrum lima].

To explore the nutrient properties of Prorocentrum lima and biosynthesis mechanism of diarrhetic shellfish poison (DSP), the growth and activities of alkaline phosphatase of Prorocentrum lima were observed under different phosphorus sources. DSP productions were also analyzed. The maximum growth rate (micro(max)) was slightly lower under beta-sodium glycerophosphate than those under NaH2PO4 and ATP as phosphorus sources, respectively. The maximum biomass (X) under ATP was higher than those under NaH2PO4 and beta-sodium glycerophosphate as the phosphorus sources, respectively. When the concentration of NaH2PO4 was below 2 micromol/L, the activity of alkaline phosphatase increased significantly. However, the activities were much low in the all treatments when beta-sodium glycerophosphate used as phosphorus source, whereas the activities increased with the concentration of ATP when ATP used as phosphorus source. The level of okadaic acid (OA) in Prorocentrum lima at the stationary phase under beta-sodium glycerophosphate was higher than those under NaH2PO4 and ATP. These suggested that beta-sodium glycerophosphate could be utilized directly by Prorocentrum lima with lower efficiency; ATP could induce alkaline phosphatase to produce inorganic phosphate for algae. DSP production in Prorocentrum lima were different under various phosphate sources, beta-sodium glycerophosphate enhanced production of DSP. The difference in DSP production might be related with the physiological state of Prorocentrum lima.

Nitrogen:Phosphorus supply ratio may control the protein and total toxin of dinoflagellate Alexandrium tamarense.

The protein and total toxin of dinoflagellate Alexandrium tamarense at the exponential growth phase were studied at four N:P supply ratios=8, 16, 24 and 48 by maintaining the N concentration at 880 microM with variable P concentrations without nutrient limitation. Because A. tamarense grew well at all N:P supply ratios, they might synthesize RNA which contains high phosphorus and consequently low N:P atomic ratio of cells might be retained during exponential growth phase. Cellular protein:C ratio and toxin:C ratio depended on N:P supply ratio, suggesting that intracellular biochemical composition of A. tamarense might vary due to N:P supply conditions. These biochemical changes could not be detected by only investigations of cellular N:C atomic ratio which was independent on N:P supply ratio. Total cellular toxin contents of A. tamarense increased with increasing N:P supply ratio, indicated that total cellular toxin contents of A. tamarense might be stimulated by relative P-deficiency. In situ P concentration of the Seto Inland Sea of Japan has been reduced since 1980s the environmental regulation issued by Japanese Government, and therefore N:P supply ratio of input water from adjacent rivers has became higher than the Redfield ratio. The present study may suggest that the reduction of P supply into ambient sea water might cause A. tamarense to accumulate more toxin within the cell, in result, noxious A. tamarense would be more influential to marine organisms in coastal ecosystem.

Studies of the biosynthesis of DTX-5a and DTX-5b by the dinoflagellate Prorocentrum maculosum: regiospecificity of the putative Baeyer-Villigerase and insertion of a single amino acid in a polyketide chain.

The biosynthetic origins of the diarrhetic shellfish poisoning toxins DTX-5a and DTX-5b have been elucidated by supplementing cultures of the producing organism Prorocentrum maculosum with stable isotope labeled precursors and determining the incorporation patterns by 13C NMR spectroscopy. The amino acid residue in the sulfated side chain is found to originate from glycine, and oxygen insertion in the chain is shown to occur after polyketide formation.

Ichthyotoxic ARF after fish gallbladder ingestion: a large case series from Vietnam.

Fish gallbladders are consumed in rural areas of Asia as a traditional medicine to improve symptoms of arthritis, decreased visual acuity, and impotence. Consumption of large amounts of this traditional medicine can result in systemic toxicities; in particular, acute renal failure. We reviewed records of all admissions to Cho Ray Hospital (Ho Chi Minh City, Vietnam) between January 1995 and December 2000 after this ingestion. Clinical courses and outcomes were similar in 16 of 17 patients. Within hours, patients experienced profuse vomiting (n = 16) and diarrhea (n = 15). All developed acute renal failure, with a mean serum creatinine concentration of 14.7 +/- 3.9 mg/dL (1,299.5 +/- 344.8 micromol/L). Four patients administered intravenous fluid (IVF) developed extracellular fluid volume overload, as did 1 patient not administered IVF. Time to peak creatinine concentration was 8.6 +/- 3.0 days, which was accompanied by decreased urine volume (174.7 +/- 161.6 mL/24 h). Blood pressure remained normal, with a mean arterial pressure of 91 +/- 12 mm Hg. Twelve patients required renal replacement therapy. A mean of 1.9 +/- 1.1 hemodialysis sessions was performed per patient. Sixteen patients recovered renal function; 1 patient died of fulminant hepatic failure. Kidney biopsies showed features of acute tubular injury. Acute renal failure after fish gallbladder ingestion is characterized by a failure to respond to IVF, an 8.6-day interval to peak creatinine level, frequent need for dialysis therapy, and findings on renal biopsy consistent with acute tubular necrosis. Acute renal failure after fish gallbladder ingestion has an excellent prognosis. However, death from fulminant hepatic failure can occur.

Dynamics of C2 toxin and chlorophyll-a formation in the dinoflagellate Alexandrium tamarense during large scale cultivation.

The production of paralytic shellfish toxins (PSTs) by the dinoflagellate Alexandrium tamarense ATCI01, a toxigenic strain isolated from South China coastal waters, was studied in batch cultures in relatively large volumes (20l). Under nutrient-replete conditions, this strain produced C2 toxin (C2T) as a predominant PST. In a 15-day production culture, phosphate was depleted by day 4, the stationary phase began at day 6, and the toxin productivity peaked at day 10, in which the cell content of C2T reached 76 fmol per cell. Much of the toxin was produced after the depletion of phosphate in the medium suggesting that C2T is a secondary metabolite. Aeration with small bubbles was useful in increasing cell mass and toxin yield. Chlorophyll-a (Chl-a) was formed in algal cells until the culture entered the stationary phase, after which Chl-a began to disappear rapidly from the culture while the C2T content continued to rise. These results suggest a metabolic relationship between Chl-a and C2T.

Toxin compartmentation and delivery in the Cnidaria: the nematocyst's tubule as a multiheaded poisonous arrow.

With the aid of dialysis and ion exchange chromatography, a new polypeptide toxin was purified from the tentacles of the Mediterranean jellyfish Rhopilema nomadica. The amino acid sequence of the N-terminal segment of the new toxin revealed that it is a phospholipase A2 (PhA2) toxin closely resembling those previously isolated from reptile and hymenopterous venoms. The occurrence of a PhA2 toxin in the jellyfish tentacles may explain both their local (dermanecrotic) and systemic (cardiac-respiratory) effects upon human envenomation. We used an antibody raised against the above toxin as a probe to explore, for the first time, the site of toxin allocation in cnidarian nematocysts and its morphological route of delivery. Our immunocytochemical approach revealed that the toxin is stored on the outer ("cytoplasmic") surface of the inverted tubule folded in the capsule of the resting nematocyst. During discharge the toxin is translocated to the internal surface surrounding the lumen of the everting tubule, and its delivery via extended spirally arrayed barbs is apparently propelled by the high hydrostatic pressure of the capsule. This is a unique example where subcellular translocation and transfer of a polypetide is driven by mechanical forces.