From Jungbluth et al. 2017 – MEPS

Prey size spectra and abundance (Coulter Counter)

Initial and final time-point Coulter Counter (CC) samples were taken for prey particle spectra by gently pouring 20 ml from each incubation bottle through a 35 µm cap filter into a clean beaker, then gently back-washing the filter into the experimental bottle to return any nauplii using a small volume of 0.2 µm filtered seawater. From this subsample, triplicate 2 ml volumes were measured with a Beckman Coulter Multisizer III CC with a 100 µm orifice tube, yielding a spectrum of particle sizes from 2–35 µm ESD, as well as quantitative abundance data. These raw data were further processed in R (R Core Team 2016) to streamline binning of prey size groups, for calculations of clearance and ingestion rates, and for statistical analyses.

Prey ESD was converted to biovolume (BV; µm 3), then to carbon (C; pg C cell–1), using the relationship C = 0.216 × BV0.939, which applies well to taxonomically diverse protists (Menden-Deuer & Lessard 2000). Averages of cell abundance and biomass were binned into 5 prey size groupings (2–5, 5–10, 10–15, 15–20, and 20–35 µm). The binned, averaged data for initial and final time points for each control and treatment bottle were used to calculate carbon ingestion (I; ng C nauplius–1 h–1) and clearance rates (F, ml nauplius–1 h–1) on each prey size group using the equations of Frost (1972).

Photosynthetic eukaryotes (flow cytometry)

Flow cytometry (FCM) samples (1.5 ml) for photosynthetic eukaryote (PEUK) abundance were taken from the 20 ml subsamples (as described above), preserved in 0.4% paraformaldehyde (final concentration), flash-frozen in liquid nitrogen and transferred to a –80°C freezer until processing. Preserved, frozen FCM samples were thawed in batches, stained with the DNA dye Hoechst 34442 (1 µg ml–1, final concentration) (Campbell & Vaulot 1993, Monger & Landry 1993), and analyzed using a Beckman-Coulter Altra flow cytometer for phytoplankton population abundances using fluorescence signals from DNA, phycoerythrin and chl a. Data were grouped into relevant populations using FlowJo (Treestar). PEUK cell abundances were converted to biomass using data from parallel microscopy samples, which showed that the eukaryotic phytoplankton in these samples were dominated by 2 to 3 µm ESD spherical cells, with an average biomass of 1.55 pg C cell–1 (biomass conversions as in Menden-Deuer & Lessard 2000).

Nano- and microplankton abundance and biomass (microscopy)

Initial and final samples for nano- and microplankton abundance by epifluorescence microscopy (EPI) were preserved (0.4% paraformaldehyde, final concentration), and kept in the dark and cold (4°C) until filtered within 24 to 48 h. EPI samples (25 or 50 ml) were stained with 0.5 nM proflavin (1 to 2 h prior to filtration), then filtered onto 0.8 µm black polycarbonate filters (Midland Scientific), stained with 4’,6-diamidino-2-phenylindole (DAPI) for 2 min and mounted on a slide. These slides were frozen at –80°C until digitally imaged within 2 mo of collection.

Digital images of the slides were taken using a color camera (Olympus U-LH100HGAPO) attached to an epifluorescence microscope (Olympus Model BX51 TRF, 400× total magnification), and the software program MicrofireTM (Optronics). For each slide, 3 sequential digital images were taken of 30 random fields, using 3 different excitation/emission filters; one each to illuminate chl a/proflavin (EX450-480; DM500, EM≥515), phycoerythrin (primarily due to Synechococcus), and DNA (EX330-385, DM400, EM>420) fluorescence. Living cells were distinguished from dead cells and debris by the presence of nuclei, and autotrophic and heterotrophic cells were distinguished by the presence of chl a.

Images were analyzed by sizing, counting, and identifying autotrophic and heterotrophic cells 2–10 µm in size until >100 cells were characterized. To evaluate the large (>10 µm) cell abundance and biomass, all >10 µm cells on ¼ of one randomly selected control and treatment slide from each experiment were counted, identified as an autotroph or heterotroph, and measured using a calibrated ocular micrometer. Cell dimensions were used to estimate biovolume (oblate spheroid), and converted to biomass (Menden-Deuer & Lessard 2000). Diatoms >10 µm were also quantified, but they were never abundant during our experiments (see Table 2).

Ciliate biomass and abundance was estimated by inverted microscopy on samples pre-screened through a <35 µm mesh, preserved with a 1/20 dilution of acid Lugol’s solution (Throndsen 1978), and kept in the dark at room temperature until analysis (~1 yr later) by the Utermöhl technique (Sherr & Sherr 1993). Aliquots of 28 ml from randomly selected control and experimental treatments were settled and their entire contents examined (18 to 87 total cells in sample volume, median 55) with a Zeiss inverted microscope (400× magnification), with digital images taken (Moticam camera and software) for subsequent dimensional analyses. The measured length and width of each cell was converted to biovolume based on the appropriate geometric shapes, and converted to carbon biomass (Menden-Deuer & Lessard 2000).

Chl a determinations

For chl a, triplicate 305 ml samples were filtered onto GF/Fs (Whatman), flash-frozen (LN2), and kept at –80°C freezer until measurements were made 4 mo later. Chl a (and phaeopigment) was measured using a Turner Designs (model 10AU) fluorometer, using the standard extraction and acidification technique (Yentsch & Menzel 1963, Strickland & Parsons 1972).

For complete methodology, see the Supplemental Files section.

init_prey_MEPS.csv