Elisabeth Antoine

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.

Isolation and characterization of extremely thermophilic archaebacteria related to the genus Thermococcus from deep-sea hydrothermal Guaymas Basin

During the “Guaynaut” oceanographic cruise performed by IFREMER in November 1991, sediment cores were collected from high-temperature and petroleum-rich deposits in an active hydrothermal zone, at the Guaymas basin (Central gulf of California). Those samples were collected by the French deep-sea manned submersible “Nautile” at a depth of 2000 meters. Four sediment cores of 20–40-cm length were drilled at the bottom of a block assemblage of active smokers inside sediments whose temperatures were 3.5°C on the top to 105°C at 20 cm depth. They were subsampled in 22 slices of 5-cm thickness and used for isolation of heterotrophic hyperthermophilic microorganisms, after inoculation in sulfur-free SME liquid medium. From those enrichments 18 isolates were obtained, 2 growing at 95°C and 16 at 80°C, and their taxonomic characterization was undertaken. Lipid analysis indicated the presence of diethers and tetraethers in the cell walls and membranes, characteristic of Archaebacteria. Examinations by scanning electron microscopy showed that isolates were cocci of heterogeneous sizes (diameter from 0.1 to 0.5 micrometers) or thick, piled-up discs 0.5 μm thick and 1 μm in diameter. Both forms were embedded in a dense fiber network. Physiologically they were found to be anaerobic, heterotrophic, and hyperthermophilic (80°–95°C). Determination of the DNA base composition resulted in G + C mol % values ranging from 36 to 57. Qualitative hybridizations of the 18 isolate DNAs with hyperthermophilic Archaebacteria reference strain DNAs showed that hybridizations occurred neither with the two species of Pyrococcus nor with the two species of Desulfurococcus, nor with Staphylothermus marinus. On the other hand, all isolates hybridized with at least one of the three species of Thermococcus tested (T. celer, T. stetteri, T. litoralis). Restriction polymorphism on a PCR-amplified fragment of the rrn operon showed that 12 isolates had the same profile as T. celer and T. stetteri, 4 isolates had the same profile as T. litoralis, and 2 had new profiles, suggestive that they are new species.

Toward detection of harmful algae blooms by in situ surface plasmon resonance spectroscopy

Among marine algae species, Alexandrium minutum produces a phycotoxin called paralytic shellfish poisoning (PSP) that is introduced in the food chain through the ingestion of phytoplankton by shellfishs, and later by human consumers. Thus, in situ monitoring of A. minutum proliferation in coastal seawater is of great economical importance for marine resources exploitation. Here, we propose a rapid test for the detection of A. minutum by surface plasmon resonance spectroscopy. First, whole genomic DNA is extracted from the algae. Second, a 677 bp long portion of the 28S ribosomal DNA is amplified by PCR. Third, the PCR product is detected by surface plasmon resonance spectroscopy onto a DNA functionalized gold substrate.

Toward in situ detection of algae species

For the last few years there is a more and more pressing need to develop system for detecting HAB at their early stage. Researchers have then been working on new in situ sensor. In this paper a submarine transducer based on surface plasmon resonance is reported. It demonstrated a refractive index resolution of 3.10−6, which is promising for later phytoplankton detection.