The genus Pseudo-nitzschia includes a number of species responsible for blooms in coastal and open waters worldwide. P. delicatissima, a species reported as a potential source of amnesic shellfish poisoning (ASP), reaches high concentrations in the Gulf of Naples (Mediterranean Sea), where it regularly blooms in spring and, at times, in autumn. We assessed both intra-and interindividual genetic diversity of this species before and during a bloom (February to April 2001) by sequencing the internal transcribed spacer regions (ITS1 and ITS2) and the 5.8S gene of the nuclear ribosomal DNA. PCR products obtained from 70 strains were cloned and several ITS copies were sequenced for each strain to assess intra-individual polymorphism. Phylogenies showed the presence of 5 distinct, well-supported lineages within what was considered to be a single morphospecies. Genetic diversity was higher in pre-bloom conditions, while all strains collected at the height of the bloom clustered within a single major clade. Ultrastructural investigations carried out on selected strains revealed morphological features slightly different from the ones typical for P. delicatissima only in 1 strain, outside the major clade. Our results, supported by the analysis of the hypervariable domains of LSU (large subunit) rDNA carried out on selected strains, suggest the presence of cryptic diversity within P. delicatissima. Such diversity could in fact explain the existence of toxic and nontoxic strains within the same species and the occasional mismatches between 'species-specific' molecular probes and target species. KEY WORDS: Diatoms · Pseudo-nitzschia delicatissima · ITS nDNA · Cryptic diversity · BloomResale or republication not permitted without written consent of the publisher

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The frequency and intensity of harmful algal blooms (HABs) appear to be on the rise globally. There is also evidence of the geographic spreading of toxic strains of these algae. Consequently, methods had to be established and new ones are still needed for the evaluation of possible hazards caused by increased algal toxin production in the marine food chain. Different clinical effects of algae-related poisoning have attracted scientific attention; paralytic shellfish poisoning, diarrhetic shellfish poisoning, and amnesic shellfish poisoning are among the most common. Additionally, cyanobacteria (blue-green algae) in brackish waters often produce neurotoxic and hepatotoxic substances. Bioassays with mice or rats are common methods to determine algal and cyanobacterial toxins. However, biological tests are not really satisfactory because of their low sensitivity. In addition, there is growing public opposition to animal testing. Therefore, there has been increasing effort to determine algal toxins by chemical methods. Plankton samples from different European marine and brackish waters were taken during research cruises and analyzed on board directly. The ship routes covered marine areas in the northwest Atlantic, Orkney Islands, east coast of Scotland, and the North and Baltic seas. The first results on the occurrence and frequency of harmful algal species were obtained in 1997 and 1998. During the 2000 cruise an HPLC/MS coupling was established on board, and algal toxins were measured directly after extraction of the plankton samples. In contrast to earlier cruises, the sampling areas were changed in 2000 to focusing on coastal zones. The occurrence of toxic algae in these areas was compared to toxin formation during HABs in the open sea. It was found that the toxicity of the algal blooms depended on the prevailing local conditions. This observation was also confirmed by monitoring cyanobacterial blooms in the Baltic Sea. Optimal weather conditions, for example, during the summers of 1997 and 2003, favored blooms of cyanobacteria in all regions of the Baltic. The dominant species regarding the HABs in the Baltic was Nodularia spumigena. However, in addition to high concentrations of Nodularia spumigena in coastal zones, other blue-green algae are involved in bloom formation, with changes in plankton communities influencing both toxin profiles and toxicity.

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The toxins of cyanobacteria include potent neurotoxins and hepatotoxins. Deaths after exposure by ingestion of cyanobacterial cells and toxins from freshwater sources have been reported. The hepatotoxins are cyclic peptides, which comprise microcystins (MC) and nodularin (NOD), and they cause poisonings with an established syndrome of human and animal illnesses. The identification and determination of these hepatotoxic peptides are challenges for analytical chemists. Methods based on solid phase extraction and size exclusion chromatography are available for the clean up of raw extracts. Both principles are also used for the enrichment of MC from low contaminated sample material. The separation of MC is achieved by high‐pressure liquid chromatography methods with ultraviolet (UV) detection or mass spectrometric detection. The unambiguous identification of MC not available as standards is possible by mass spectrometry (MS) or, under optimal conditions, by UV spectrometry using diode array detection. To date, 60 different MC and four NOD have been described, but new compounds are frequently identified. Therefore, powerful tools for the elucidation of the structure of unknown MC are an important need. In addition to classical methods for structure elucidation (e.g. nuclear magnetic resonance spectrometry), microwave‐assisted hydrolysis of cyclic peptides followed by enantio‐selective determination of the amino acid profiles can be used. More recently, MS with collision‐activated dissociation has become an important tool for obtaining structure information. The present paper discusses the advantages and disadvantages of the various analytical techniques used for monitoring of cyanobacteria in lakes, optimized or developed at the Institute of Nutrition, Jena, Germany. A pathway for handling MC‐containing samples with regard to different analytical tasks (i.e. the identification and elucidation of structure followed by qualitative and quantitative determination) is proposed.