Claire Marcaillou-Le Baut

ISSR as new markers for genetic characterization and evaluation of relationships among phytoplankton

In order to increase the molecular tools and markers needed for the identification of phytoplankton species, the inter simple sequence repeat (ISSR) fingerprinting was adapted to micro-algae and its use in genetic analysis was demonstrated. Twelve strains, 6 Alexandrium, 4 Pseudo-nitzschia, 1 Skeletonema and 1 Tetraselmis were analysed for the first time with ISSR amplifications. The patterns were highly polymorphic and very reproducible. The 6 primers gave 223 polymorphic markers that clearly and easily distinguished all 12 strains (mainly toxic ones) and gave 187 polymorphic markers among the Alexandrium and the Pseudo-nitzschia species. ISSR amplifications also indicated a large occurrence of simple sequence repeat (SSR) in phytoplankton genomes, especially in Pseudo-nitzschia, and show their usefulness to cluster intra and inter species. ISSR markers were found to be good markers for genetic characterization and diversity study and led to consider them as new tools for the survey of phytoplankton.

DEVELOPMENT OF SEQUENCE CHARACTERIZED AMPLIFIED REGION MARKERS FROM INTERSIMPLE SEQUENCE REPEAT FINGERPRINTS FOR THE MOLECULAR DETECTION OF TOXIC PHYTOPLANKTON ALEXANDRIUM CATENELLA (DINOPHYCEAE) AND PSEUDO-NITZSCHIA PSEUDODELICATISSIMA (BACILLARIOPHYCEAE) FROM FRENCH COASTAL WATERS1

Harmful algal blooms are a serious threat to shellfish farming and human health all over the world. The monitoring of harmful algae in coastal waters originally involved morphological identification through microscopic examinations, which was often difficult unless performed by specialists and even then often did not permit identification of toxic species. More recently, specific molecular markers have been used to identify specific phytoplankton species or strains. Here we report on the use of the intersimple sequence repeat (ISSR) technique to develop specific sequence characterized amplified region markers (SCAR) and to identify with these tools two toxic species in French coastal waters, the diatom Pseudo‐nitzschia pseudodelicatissima (Hasle) Hasle and the dinoflagellate Alexandrium catenella (Whedon and Kofoid 1936), Balech 1985. Six polymorphic ISSR regions were selected among amplified fingerprints of a representative sample of phytoplankton species. After cloning and sequencing the selected polymorphic ISSR regions, pairs of internal primers were designed to amplify a unique and specific sequence designed as a SCAR marker. Of the six selected SCAR markers, three were specific to P. pseudodelicatissima and one for A. catenella. The SCAR marker specificity was confirmed by using basic local alignment search tool comparison, by experimental assays on different strains from 11 countries, and by checking that the sequence amplified was the expected one. When tested on water samples collected along the French shores, the four specific SCAR markers proved to be efficient tools for fast and low‐cost detection of toxic phytoplankton species.

Specificity of the test based on modification of cell morphology for detection of lipophilic inhibitors of protein phosphatases

The test based on morphological changes in KB cells was assayed with different toxins. Only lipophilic inhibitors of protein phosphatases, such as okadaic acid or calyculin A, induced visible changes in cell morphology. The activity of contaminated mussel extracts on KB cells was evaluated comparatively by direct interpretation of morphological changes and by a colorimetric method estimating the number of viable cells after staining. The latter technique revealed interferences (not detected by the former) with mussel cytotoxins. These results show that the technique, based on determination of the minimal active concentration of toxic extracts inducing morphological changes, is more specific, faster and preferable to the determination of IC50 for the detection of protein phosphatase inhibitors in shellfish.

Effect of an unknown toxin isolated from dinophysis sp.-contaminated French mussels, on the electrical and mechanical activity of frog heart

The effects of an unknown toxin, isolated along with okadaic acid from the hepatopancreas of French mussels contaminated by Dinophysis sp., producing ataxia, neurologic symptoms, bradycardia, arrhythmias, electrocardiographic changes, and cardiac arrest, have been studied in terms of the electrical and mechanical activity of frog atrial heart muscle. The toxin, in a dose-dependent manner, decreased the amplitude of the stimulated peak tension of isolated fibers. The toxin (1-36 micrograms/ml) did not modify the membrane resting potential but decreased the amplitude of the plateau and shortened the duration of the action potential. The toxin inhibited the Cd-sensitive L-type Ca current and increased a 4-aminopyridine-sensitive outward current in voltage-clamped cardiac myocytes. The data show that the cardiac effect of the toxin is markedly different from that of okadaic acid.