Dataset: Protist Carbon
Deployment: NF1704

This dataset is from CTD hydrocasts in the Gulf of Mexico from R/V Nancy Foster cruises in May 2017 and May 2018, which were part of a NOAA RESTORE project (aka: BLOOFINZ-GoM) to investigate the epipelagic marine nitrogen cycle, plankton dynamics, and impacts on growth and survival of larval Atlantic Bluefin Tuna (ABT). These data are meant to be used in inter-species, interregional comparisons to data from the BLOOFIN-IO study of larval Southern Bluefin Tuna in the Indian Ocean spawning region.

Seawater samples (500 mL) for analysis by epifluorescence microscopy (EPI) were preserved with 260 µL of alkaline Lugol's solution, 10 mL 0.08M borax-buffered 10% formalin and 500 µL 0.19M sodium thiosulfate (Sherr and Sherr, 1993), and stained with 1 mL of proflavin (0.33% w/v) and 1 mL of DAPI (0.01 mg mL-1) prior to filtering. Subsamples of 50 mL were filtered onto 25-mm, black, 0.8-µm pore polycarbonate filters to enumerate small cells at 630X magnification. The remaining 450 mL was filtered onto 25-mm, black, 8.0-µm pore polycarbonate filters to count larger cells at 200X. Each filter was mounted onto a glass slide using Type DF immersion oil and a No. 2 cover slip.

The slides were imaged and digitized using an automated Zeiss Axiovert 200M inverted epifluorescence microscope, with an AxioCam MRc black and white 8-bit CCD camera (Taylor et al., 2016). Fifty random positions were imaged for each slide, with each position consisting of four fluorescent channels: Chla, DAPI (DNA stain), FITC (proflavin protein stain, cell outline) and phycoerythrin (PE). In addition, 6–7 Z-plane images were acquired at each position for each fluorescence channel. The resulting z-stack images were combined using an extended depth of field algorithm to produce one in-focus image for each position and channel (Chla, DAPI, FITC, PE). These were then false colored (red, blue, green and orange, respectively) and combined into a single composite 24-bit RGB image for each position. Cell biovolumes (BV; µm3) were determined from length (L) and width (W) measurements according to Taylor et al. (2011) from images that passed quality inspection. Image processing and analysis was carried out in Image Pro software. Carbon (C; pg cell-1) biomass was computed from BV from the equations: C = 0.216 * BV0.939 for non-diatoms, and C = 0.288 * BV0.811 for diatoms (Menden-Deuer and Lessard, 2000).

Seawater samples (150 mL) were also preserved with 5% acid Lugol's solution for separate analyses of ciliates, concentrated onto 25-mm 8.0-μm polycarbonate membranes and prepared as slides according to the protocol of Freibott et al. (2014). The slides were imaged on a Zeiss AxioVert 200 M inverted microscope at 200X magnification using brightfield illumination and processed using Image Pro software as described for EPI microscopy. Length and width measurements were used to calculate cell biovolumes (BV, µm3) based on the most appropriate cell shape, and carbon biomass was calculated as pg C = 0.19 x BV (Putt and Stoecker, 1989).