Quantitative Assessment of Picoeukaryotes in the Natural Environment by Using Taxon-Specific Oligonucleotide Probes in Association with Tyramide Signal Amplification-Fluorescence In Situ Hybridization and Flow Cytometry

Abstract

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

Significant CO2 fixation by small prymnesiophytes in the subtropical and tropical northeast Atlantic Ocean

“…For Synechococcus, the probes used targeted the following specific lineages: clade I (SYN1006), clade II (SYN1006RS), clade III (SYN262), clade IV (SYN635) or more generally clades I-VII and X (SYN1258). The nuclear 18S rRNA oligonucleotide probes used were EUK1209r, NCHLO01 and CHLO02 to target all eukaryotes and the class-specific probes PRAS01, PRAS02, PRAS05 (targeting prasinophytes), and CRYPTO13, PRYM02 and PELA01 targeting cryptophytes, prymnesiophytes and pelagophytes, respectively (Giovannoni et al, 1988;Simon et al, 1995Simon et al, , 2000Biegala et al, 2003;Not et al, 2002Not et al, , 2004. The chrysophytespecific probe (CHRYSO1037) targeted the chloroplast 16S rRNA gene, as used previously for dot blot hybridization analysis (Fuller et al, 2006b) and validated here for tyramide signal amplification (TSA)-FISH analysis (see below).…”

Phenotypic and genetic diversification of Pseudanabaena spp. (cyanobacteria)

“…Once obtained, monoclonal axenic strains were maintained in their specific growth medium in the Culture Collection Yerseke (CCY; Table 1). Morphology and microscopy Cells were collected from exponential or stationary liquid cultures and were fixed in a mixture of 1% (w/v) formaldehyde and 0.05% (w/v) glutaraldehyde and subsequently stored at À80 1C until they were analyzed (Biegala et al, 2003). Microscope slides were prepared by covering them with a thin layer of 1% (w/v) molten agarose (50 1C; SigmaAldrich, Zwijndrecht, The Netherlands) that was allowed to solidify shortly before cells were applied.…”

Morphological and molecular characteristics of selected species ofMinidiscus(Bacillariophyta, Thalassiosiraceae)

“…Until recently, eukaryotic picoplankton received much less scientific attention (Lo´pez-Garcı´a et al, 2001a, b;Biegala et al, 2003). A multitude of flagellates, including prasinophytes, dinoflagellates and non-cultured groups dominate eukaryotic picoplankton communities when characterized molecularly (Moon van der Staay et al, 2001;Vaulot, 2008).…”