Detection and quantification of Prymnesium parvum (Haptophyceae) by real-time PCR.

AIMS: The ichthyotoxic species Prymnesium parvum (Haptophyceae) is difficult to quantify in a microscopy-based monitoring programme, because the cells are very small, fragile and their morphology can be distorted by the use of fixatives. In the attempt to overcome these problems, a real-time PCR-based method for the rapid and sensitive identification and quantification of P. parvum was developed. METHODS AND RESULTS: A quantitative real-time PCR assay was optimized with primers designed on the internal transcribed spacer 2 rDNA region of P. parvum. This PCR assay was specific, showing no amplification of DNA extracted from closely related species, and sensitive. Moreover, this method was able to detect and reliably quantify P. parvum cells in preserved environmental samples artificially spiked with known amounts of cultured cells. CONCLUSIONS: Considering the specificity, sensitivity and applicability to preserved environmental samples, this method may be a useful tool for the monitoring of this toxic species. SIGNIFICANCE AND IMPACT OF THE STUDY: The real-time PCR method described in this study may represent a progress towards the rapid detection and quantification of P. parvum cells in water-monitoring programmes, allowing the early application of strategies to control bloom events, such as the use of clay minerals.

Naphthoquinones as broad spectrum biocides for treatment of ship's ballast water: toxicity to phytoplankton and bacteria.

Current UN International Maritime Organization legislation mandates the phased introduction of ballast water treatment technologies capable of complying with rigorous standards related to removal of waterborne organisms. Doubts concerning mechanical treatments at very high ballasting rates have renewed interest in chemical treatment for very large vessels. High removal rates for biota require broad spectrum biocides that are safe to transport and handle and pose no corrosion problems for ships' structure. The current study focuses on the naphthoquinone group of compounds and extends a previously reported set of screening bioassays with an investigation of the toxicity of four naphthoquinones to select protists and prokaryotes, representative of typical ballast water organisms. Vegetative dinoflagellate cysts exposed to 2.0 mg/L of the naphthoquinones juglone, plumbagin, menadione and naphthazarin showed varying degrees of chloroplast destruction, with menadione demonstrating the most potency. Laboratory and mesocosm exposures of various phytoplankton genera to menadione showed toxicity at 1.0 mg/L. Juglone demonstrated the most bactericidal activity as judged by a Deltatox assay (Vibrio fischeri) and by acridine orange counts of natural bacterial populations.

Estimating the accumulation and transfer of Nodularia spumigena toxins by the blue mussel Mytilus edulis: an appraisal from culture and mesocosm experiments.

Accumulation of Nodularia spumigena toxins by Mytilus edulis was studied during laboratory and mesocosm experiments in order to investigate the possible pathways of nodularin in mussels and calculate toxin budgets. Mussels were exposed to 0.2-15.6 microg nodularin l(-1), fed for up to 5 days with Nodularia cells from culture, or blooming in different nutrient-treated seawater. Toxin concentration was monitored with LC-ESI-MS. During different exposures, the amount of nodularin detected in mussels increased linearly with increasing toxin concentration in food and attained 0.28-13.8 microg of nodularin g dw(-1) of the mussel whole body tissue after 12 h. The digestive gland was found to be the tissue with the highest toxin concentration. Nodularin concentration in faeces was not proportional to faeces production or to toxin concentration in food; however, it seemed to be mostly related to food quality as well as to food availability. The percentage of nodularin taken up by the mussels, relative to the amount contained in the offered food, varied from 10% to 20%, depending on food quality. During a 5-day toxin accumulation experiment, the acute reduction of the toxin in mussel tissues the second day and the following stabilization, showed that probably mussels maintain low toxin levels via efficient elimination and/or toxin metabolism. After a 72 h depuration period, mussels showed 75% reduction in their toxin content.

Different methods for toxin analysis in the cyanobacterium Nodularia spumigena (Cyanophyceae).

The brackish water cyanobacterium Nodularia spumigena produce the hepatotoxic cyclic pentapeptide nodularin. Intoxications for both human as well as animal may arise when water reservoirs are contaminated with potentially toxic Nodularia species. Here, results of three independent methods for the determination of nodularin in different strains of N. spumigena are presented. The results obtained with a protein phosphatase assay and a HPLC/UV/MS method are compared with the results obtained with a bioluminescence assay, which is successfully introduced here for nodularin determination. Statistical evaluation of the three applied methods revealed a good comparability towards the detected toxin content. The methods were evaluated taking into consideration the parameters: handling, efficiency, sensitivity and selectivity. The detection limit in the protein phosphatase assay is highest (0.05ng nodularin) and lowest (250ng nodularin) in the bioluminescence assay- it was determined with 5ng (MS) and 25ng (UV) for the HPLC/UV/MS methods. The different selectivities and sensitivities are critically discussed and an analytical pathway for the determination of the biotoxin nodularin from Nodularia samples is proposed.

Evidence for multiple species within the endoparasitic dinoflagellate Amoebophrya ceratii as based on 18S rRNA gene-sequence analysis.

Parasitism within the group of dinoflagellates is a widespread phenomenon. Whether the parasitic dinoflagellates exhibit specificity in their infection is not well known, but this possibility has become an important issue in the development of biological control of harmful algal blooms. The 18S rDNA sequences from the parasite Amoebophrya sp. and its dinoflagellate host Dinophysis norvegica were determined and compared with the published sequence of Amoebophrya sp. infecting Gymnodinium sanguineum and other dinoflagellates. The results showed that the sequence from the parasite within D. norvegica was clustered with that of the one from G. sanguineum with 100% bootstrap support in a maximum-likelihood analysis. The observed identity between these two sequences was 93%, which indicates that they are not identical species. The two sequences from Amoebophrya sp. were deeply branched within the group of dinoflagellate sequences and represent the earliest diverging dinoflagellates. The sequence from the parasite Parvilucifera infectans, also infecting D. norvegica, was not closely related to the Amoebophrya sp. sequences. The sequence from D. norvegica appeared as a sister group to a cluster containing Prorocentrum lima and Alexandrium spp. without significant bootstrap support. The data presented herein support the hypothesis that A. ceratii comprises more than one species, and this opens the possibility that infections of harmful algal species might involve more than one Amoebophrya species.