Distribution and composition of microplankton along the SE Pacific Ocean Fernando Gómez 1 * & Hervé Claustre 2 2 Laboratoire d’Océanographie de Villefranche-sur-Mer 1 Department of Aquatic Biosciences, The University of Tokyo * address until February 2004 Title

To study the distribution (vertical and longitudinal) and composition of microplankton from the eutrophic (coastal upwelling of Chile) to ultra-oligotrophic regimes (South Pacific gyre). To study the temporal variations on the distribution (diel cycle) in selected stations To investigate the occurrence and distribution of N 2 -fixers (Trichodesmium spp.) and N 2 -fixer symbiotic associations (Richelia and others) To contribute to the study of marine phytoplankton taxonomy and biodiversity in one of the less studied major oceanic entities of the world ocean Objectives

Sample collection 3 ml Lugol Polyestyrene plastic bottle # station, depth Storage: dark, cool and quiet place seawater 500 ml Niskin bottle 0, 5, 10, 20, 30, 50, 70, 90, 100, 120, 150, 175, 200 m depth

Pre-concentration Settling in counting chambers 500 ml Lugol fixed sea-water samples Microscopical analysis (inverted microscope) Methodo Methodology

The specimens of interest will be isolated with a capillary from the chambers: transferred to a glass slide High magnification microphotographs (×1000) Nomarski Differential Interference Contrast (D.I.C.) Morphology and location of organelles such as chloroplasts, pyrenoids, nuclei, cellulose thecal plates, flagella, etc Taxonomical studies: 1. Light microscopy staining compounds (flourochromes: DAPI, Fluorescent Brightener, etc ) Fluorescence microscopy (UV light or blue light according to the staining compound)

Taxonomical studies: 2. SEM Scanning Electron Microscopy The specimens of interest will be isolated with a capillary from the chambers: Adhered on the poly-L-lysine coated glass plate rinse in distilled water; dehydrate through an ethanol series Ion spatter coating with Au or Au-Pd Scanning Electron Microscopy Takayama method

Taxonomical studies: 3. Gene sequencing After to complete the morphological characterization of the species of interest, the forthcoming specimens will be picked and keep for single-cell molecular analysis (phylogeny) The protocol of extraction is quite similar, the gene amplification protocol varies according to the instruments and reagents suppliers Discrepancies on the most appropriate fixative (methanol) for Polymerase Chain Reaction (PCR) appear in the literature Nucleotide sequences of coding (small subunit (SSU), partial large subunit (LSU) (specific diversity) and internal transcribed spacer region (ITS1-5.8SSU-ITS2) parts of the rRNA operon (intraspecific diversity)