Computational protocol: Pico and nanoplankton abundance and carbon stocks along the Brazilian Bight

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Protocol publication

[…] Flow cytometry analysis was performed as previously described in and using a BD FACSCanto II™(Becton Dickinson, San Jose, CA) flow cytometer equipped with a blue laser (488 nm, air-cooled, 20 mW, solid state). Emitted light was collected through the following set of filters: 488/10 band pass for side scatter, 533/30 band pass for green SYBR fluorescence (FL1), 585/42 band pass for orange phycoerythrin fluorescence (FL2), and 670 long pass for red chlorophyll fluorescence (FL3). Samples were thawed at room temperature, and 0.95 µm beads (0.95 G Fluoresbrite® Polysciences, Warrington, PA, USA) were used for FCM calibration. A first analysis of 3 min at a rate of 70 µL min−1 was performed to enumerate phytoplankton cells. Acquisition was triggered on chlorophyll fluorescence (FL3-H ), which therefore excluded any heterotrophic cell, using a threshold of 200. A second analysis was performed in order to enumerate heterotrophic prokaryotes: SYBR Green® (Molecular Probes, Leiden, Netherlands) was added at a final concentration of 1/10,000 and samples were incubated for at least 15 min at room temperature in the dark. Flow cytometry acquisition was triggered on FL1 with a threshold value of 500 and performed for 2 min with a flow rate of 60 µL min−1. Data were analyzed with the Flowing Software® 2.5 (http://www.flowingsoftware.com). For phytoplankton, chlorophyll and phycoerythrin fluorescence, as well as forward and side scatter were used to distinguish between four major groups: Prochlorococcus, Synechococcus, pico-phytoeukaryotes and nano-phytoeukaryotes (). For SYBR Green® stained samples, only prokaryotes (called throughout the paper heterotrophic bacteria, including possibly Archaea) were included in the analysis to the exclusion of any heterotrophic eukaryotes.Picoplankton biomass (heterotrophic bacteria, Prochlorococcus, Synechococcus and picoeukaryotes) was calculated from flow cytometry abundance data, using cell-to-carbon conversion factors from the literature: 20 fgC cell−1 for heterotrophic bacteria (), 36 fgC cell−1 for Prochlorococcus, 255 fgC cell−1 for Synechococcus, and 2,590 fgC cell−1 for picoeukaryotes (). Nano-phytoplankton biomass was not calculated due the lack of robust conversion factors to carbon content. [...] Statistical analyses were made with the STATISTICA 12.5® software (Version 13; StatSoft, Inc., Tulsa, OK, USA) in order to explore relationships between abiotic and biotic data. A Spearman correlation analysis was performed considering both environmental (temperature, fluorescence, salinity, phosphates and nitrates) and biotic data (heterotrophic bacteria, Prochlorococcus, Synechococcus, picoeukaryote and nanoeukaryote abundances). A Principal Component Analysis (PCA, N = 72) was performed with abiotic and biotic data computed as active and supplementary variables, respectively. Graphic interpolations were produced with the DIVA Gridding algorithm from the software Ocean Data View® version 4.7.6 (). Flow cytometry and environmental data (Table S1) can be found at https://dx.doi.org/10.6084/m9.figshare.3492098.v2. […]

Pipeline specifications

Software tools Flowing Software, Statistica
Applications Miscellaneous, Flow cytometry
Diseases Overbite
Chemicals Carbon