Dataset: MV1015 Prochlorococcus FCM QPCR

These data include Prochlorococcus cell concentrations from samples collected during the C-MORE Biogeochemical Gradients Role in Arranging Planktonic Assemblages (BiG-RAPA) expedition (Cruise MV1015) in the Peru Current and Eastern South Pacific Subtropical Gyre between dates 2010-11-19 and 2010-12-10 along a zonal transect from the northern coast of Chile to the island of Rapa Nui.

Seawater Collection:  For both flow cytometry and qPCR samples, 500 milliliters (mL) of seawater from each depth was collected in a clean HDPE bottle (10% bleach for 20 minutes, followed by thorough rinsing with ship's tap water). Each clean HDPE bottle was rinsed 3 times with seawater from the Niskin bottle before filling.

Flow Cytometry:  For each of the 7 stations along the transect, 9 samples were collected across the euphotic zone; from these, water samples were also collected for ecotype qPCR. Depths at each station were chosen based on the location of the deep chlorophyll maximum, the mixed layer depth, and light attenuation profiles. At station 7, samples were also collected from high-resolution deep-chlorophyll maximum casts (63 and 69), and counts for these are included. Fixed samples for flow cytometry were prepared by adding 5 microliters (ul) of 25% glutaraldehyde to 1 mL whole seawater, mixing, and allowing the sample to fix in the dark for 10 minutes, followed by flash freezing in liquid nitrogen. The samples were stored in liquid nitrogen or at -80C for 2-4 months before running flow cytometry. Two or three flow cytometry samples were preserved from each Niskin sampled, but only one of these sampling replicates was run. For each site and depth collected, a sample was run on a BD/Cytopeia Influx flow cytometer with 488 and 457 lasers both illuminating the sample and 2 um fluorescent bead standards mixed in. No stains were used. Detector sensitivity ranges were optimized for Prochlorococcus and held constant throughout all runs. Each sample was thawed in the dark and run twice (flow cytometry technical duplicates). Replicates were run back to back, flushing the lines in between, so that samples weren't left thawed for longer than 1 hour or put through extra freeze-thaw cycles. Values in the table are averages of the pair of technical duplicates.  As measurements, these values are good to ~2-3 significant digits (depending on the length of time a sample was run and the number of Prochlorococcus cells actually detected).

Ecotype quantitative PCR:  Samples for qPCR were collected and preserved as described  (Zinser et al., 2006). 100 milliliters (mL) of seawater was filtered onto 25 millimeter (mm) diameter, 0.2 micrometer (um) pore size, polycarbonate filters (in triplicate) and chased with 3 mL of preservation solution (10 millimolar (mM) Tris pH=8, 100 mM EDTA, 500 mM NaCl).  Filters were placed dry inside 2 mL screw cap polypropylene bead beater tubes and stored at -80 degress C until use. DNA from field samples was extracted from filters as previously described (Zinser et al., 2006). Cells were resuspended by adding 650 microliters (uL) of 10 mM Tris pH=8 to the bead beater tube containing the filter and bead beated at maximum speed (~4800 rpm) for 2 minutes. 500 uL of the respuspended cells were transfered to a 1.5 mL centrifuge tube and the cells were heat lysed at 95 degrees C for 15 minutes. DNA samples were then stored at -80 degrees C until analysis.

The qPCR assay was performed as previously described (Ahlgren et al., 2006; Zinser et al., 2006; Malmstrom et al., 2010; Malmstrom et al., 2012) using the same standards and reaction conditions. Technical duplicates of three replicate filters were analyzed for BiG RAPA (i.e. 6 data points per sample). Data quality was assessed using the percent coefficient of variation for the 6 data points. When cell concentrations fell below the detection limit of the assay (indicated by the associated quality flag), cell concentrations were set to the theoretical detection limit of 0.65 cells/mL.

General Issue:  In most cases these water samples are not from the same bottles or depths as cruise core measurement casts. 

Flow Cytometry:  At station 1, the mixed layer samples do not have technical replicates. In the first few attempted flow cytometry runs Prochlorococcus cells were not visible - not because their fluorescence was low (these waters are relatively turbid), but because the cells were scarce. Later, the remainders of these samples were used for single, long slow runs, and we were able to detect a small Prochlorococcus population - these counts are not robust, representing small numbers with background noise, but they round out the depth profile for a rough idea.

Field samples were collected by Jessie Berta-Thompson and Paul Berube, flow cytometry data were collected and analyzed by Jessie Berta-Thompson and Allison Coe, and quantitative PCR data were collected and analyzed by Allison Coe and Paul Berube.

ProChl values were calculated by normalizing chlorophyll fluorescence of the target population to chlorophyll fluorescence of the standard in the following manner: the mean of red fluorescence (680 nm emmission wavelength) for the gated Prochlorococcus was divided by the mean of red fluorescence (680 nm emmission wavelength) for the gated fluorescent bead standard.

ProFSC values were calculated by normalizing forward scatter of the target population to forward scatter of the standard in the following manner: the mean of forward scatter for the gated Prochlorococcus was divided by the mean of forward scatter for the gated fluorescent bead standard.

QUALITY FLAGS column header = variable[QUALITY]
0 = Less than detection limit
1 = Good data
2 = Single measurement - no assessment of measurement variation
3 = >5% difference of techinical duplicates (flow cytometry)
4 = >50 %CV (ecotype qPCR)