[1] A combination of 15 N 2 labeling, Tyramide Signal Amplification -Fluorescent in Situ Hybridization (TSA-FISH) assay, and chemical analyses were performed along a trophic gradient (8000 km) in the equatorial Pacific. Nitrogen fixation rates were low (0.06 ± 0.02 to 2.8 ± 2.1 nmol L À1 d À1 ) in HNLC waters, higher in the warm pool (0.11 ± 0.0 to 18.2 ± 2.8 nmol L À1 d À1 ), and extremely high close to Papua New Guinea (38 ± 9 to 610 ± 46 nmol L À1 d À1). Rates attributed to the <10-mm fraction accounted for 74% of total activity. Both unicellular and filamentous diazotrophs were detected and reached 17 cells mL À1 and 1.85 trichome mL À1 . Unicellular diazotrophs were found to be free-living in <10-mm fraction or in association with mucilage, particles, or eukaryotes in the >10-mm fraction, leading to a possible overestimation of this fraction to total N 2 fixation. In oceanic waters, 98% of the unicellular diazotrophs were picoplanktonic. Finally, we found a clear longitudinal pattern of niche partitioning between diazotroph groups: while unicellular diazotrophs were present all along the transect, Trichodesmium spp. were detected only in coastal waters, where nitrogen fixation associated to both size fractions was greatly stimulated.

Picoeukaryotes (cells of <3 m in diameter) contribute significantly to marine plankton biomass and productivity, and recently molecular studies have brought to light their wide diversity. Among the methods that have been used so far to quantify aquatic microorganisms, fluorescence in situ hybridization of oligonucleotide probes combined with flow cytometry offers the advantages of both high resolution for taxonomic identification and automated cell counting. However, cell losses, cell clumps, and low signal-to-background ratio have often been mentioned as major problems for routine application of this combination of techniques. We developed a new protocol associating tyramide signal amplification-fluorescence in situ hybridization and flow cytometry, which allows the detection of picoeukaryotes in cultures during both the exponential and stationary phases. The use of surfactant and sonication proved to be essential for the detection and quantification of picoeukaryotes from the natural environment, with as little as a few tenths of a milliliter of 3-m-pore-size prefiltered sea water. The routine application of the technique was tested along a coastal transect off Brittany (France), where the different groups of picoeukaryotes were investigated using already published specific probes and a newly designed probe that targets the order Mamiellales (Prasinophyceae, Chlorophyta). Among the picoeukaryotes, Mamiellales outnumbered by 1 order of magnitude both the cyanobacteria and the non-Chlorophyta, which were represented mainly by the Pelagophyceae class. Picoeukaryote abundance increased from open toward more estuarine water, probably following changes in water temperature and stability.Picoeukaryotes (cells smaller than 3 m in diameter) (22) are widely distributed in aquatic environments. Their important biomass and their high productivity suggest that they play a major role in oceanic, coastal, and freshwater ecosystems (27,28,48). Because of their small size and their simple morphology, a detailed study of these organisms is difficult. It is only recently that their diversity has been revealed, in particular by phylogenetic studies based on 18S rDNA sequence analysis (31, 34). It is necessary to quantify the dominating phylogenetic groups of picoeukaryotes in the natural environment in order to understand their contribution to the microbial food web and biogeochemical cycles.So far, the abundance of marine picoeukaryotes has been estimated based on their natural fluorescence by flow cytometry (28, 54) or by analysis of their pigment composition by high-performance liquid chromatography (56). The taxonomic resolution of these techniques is limited (e.g., class level only for high-performance liquid chromatography) and they do not allow the quantification of heterotrophic organisms. Molecular techniques based on full or partial sequence analysis of 18S rDNA allow us to determine species composition very precisely but are labor-intensive, and only a few samples can be analyzed at once (31,34

Photosynthetic picoeukaryotes (phytoplankton cells with a diameter smaller than 2 to 3 µm) contribute significantly to both biomass and primary production in the oligotrophic open ocean and coastal waters, at certain times of the year. The identification of these organisms is difficult because of their small size and simple morphology, therefore hindering detailed ecological studies of their distribution and role. In this paper, we demonstrate the use of oligonucleotide probes specific to algal classes or to lower order taxa in combination with fluorescent in situ hybridization and tyramide signal amplification (FISH-TSA) to determine eukaryotic picophytoplankton diversity. Target cells were detected and enumerated using epifluorescence microscopy. The sensitivity of the technique and the specificity of the probes were tested on pure and mixed picoplanktonic strains, as well as on natural samples from the English Channel. In these samples, the community was dominated by cells belonging to the division Chlorophyta. Haptophyta, Bolidophyceae and Pelagophyceae were also detected at low abundance. The FISH-TSA method is readily applicable to the study of picoplankton diversity in natural communities. KEY WORDS: Fluorescent in situ hybridization · Tyramide signal amplification · Picoplankton · Eukaryotes · Coastal waters · DiversityResale or republication not permitted without written consent of the publisher Aquat Microb Ecol 28: 157-166, 2002 samples permit culture-independent assessments of diversity and indicate that there is a considerable number of yet uncultured species in eukaryotic picoplankton communities (Lopez-Garcia et al. 2001, Moon-van der Staay et al. 2001. The enumeration of specific taxonomic groups and the estimation of their contribution to eukaryotic picoplankton, however, are more difficult. Photosynthetic pigments have been widely used to estimate the contribution of algal classes to total chlorophyll a (chl a) biomass (Latasa & Bidigare 1998). However, this method is limited for several reasons. First, it cannot resolve diversity below the class level. Second, it is based on the assumption that the relative cellular content of diagnostic pigments is constant for a given algal group. This assumption is invalid because pigment content varies between species, as well as within species. For a given strain, it is also affected by environmental conditions (e.g. Stolte et al. 2000).Whole cell fluorescent in situ hybridization (FISH) with rRNA-targeted nucleic acid probes has been used more and more extensively to detect bacteria (Amann 1995). This method, which combines identification with quantitative determination of cell number, has been successfully applied to complex bacteria communities such as biofilms (Brümmer et al. 2000), marine sediments (Llobet-Brossa et al. 1998) and soils (Ravenschlag et al. 2000). Fluorescent rRNA probes have not been used as widely for eukaryotic phytoplankton (Simon et al. 1995, Scholin et al. 1996. Attempts to use mono-labeled oligonucleotide probes for...

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