Figure 1. Sampling stations for the GLOBEC LTOP cruises	Figure 2. Sample flow cytometer cytogram plotting cells based on red fluorescence (chl-a), versus orange fluorescence (phycobiliproteins), showing "clouds" of coccoid cyanobacteria (cells in orange oval) and of nano-phytoplankton (cells in green rectangle).

Ciliates: samples were preserved with 10% final concentration of acid Lugol solution for settling and enumeration/ sizing via inverted light microscopy. Heterotrophic dinoflagellates and other flagellates: samples were preserved with formalin, stained with DAPI, and settled onto 3.0 um black-stained filters for enumeration via epifluorescence microscopy. We enumerated cells larger than about 10 um in size. Carbon biomass of protists was determined from biovolume estimation of each cell counted, using algorithms for carbon:biovolume ratios (Menden-Deuer and Lessard, 2000).

Phytoplankton: 3 ml samples were preserved with paraformal-dehyde, quick-frozen and stored in liquid nitrogen until thawed and analysed using a Becton-Dickinson FACSCalibur flow cytometer. Coccoid cyanobacteria (Synechococcus) and eukaryotic phytoplankton in two size ranges were enumerated based on orange and red fluorescence, respectively (Figure 2).). Distributions of cells were compared to sigma-t (as a proxy for upwelling) and to in situ fluorescence (as a proxy for phytoplankton biomass) from LTOP CTD data collected on each cruise. We also compared carbon biomass of Synechococcus (100 fg C/cell) and of nano-eukaryotic phytoplankton (1.5 pg C/cell) to total phytoplankton biomass (chlorophyll-a x 40 mg C/ug chl-a) (Zubkov et al. 2000).

Results:

- We found a distinctive pattern of distribution of smaller-sized phytoplankton during the 2001 GLOBEC LTOP cruises. Based on chlorophyll-a concentrations,Total phytoplankton, mainly diatoms, tended to be most abundant in inshore regions of upwelling. In contrast, highest abundances of both coccoid cyanobacteria (Synechococcus) (1 to 5 x 10⁵ cells/ml) and of nano-sized eukaryotic phytoplankton (1 to 8 x 10⁴ cells/ml) were often found in slope waters, usually in the region of the offshore upwelling front, based on sigma-t surfaces. Smaller-sized phytoplankton also showed peaks in abundance at the outermost stations of the transects.

The fraction of total phytoplankton carbon biomass due to coccoid cyanobacterial biomass +nano-eukaryotic phytoplankton biomass was highly variable, but in general was > 0.1 when chl-a concentrations were < 5 µg/liter (Figure 3) Figure 3. Fraction of phytoplankton biomass due to the sum of coccoid cyanobacteria + nano-eukaryotic cell biomass in relation to total phytoplankton stock (chlorophyll-a concentration), September 2001. Colored dots denote individual transect lines.

- Both ciliates and heterotrophic dinoflagellates were common components of the microzooplankton community in the upper water column of the CCS (Figure 4-A & -B). In epifluorescence preparations, ciliates and dinoflagellates were often observed with coccoid cyanobacteria and small eukaryotic phytoplankton in food vacuoles. In the euphotic zone, ciliate abundances ranged from 1 to 14 per ml, and the assemblage was dominated by choreotrichs and oligotrichs with an average cell size of about 20 um ESD.

Figure 4 A. Examples of microzooplankton protists observed in the California Current system: four pelagic ciliates visualized via inverted microscopy; 15 to 40 µm oligotrich and choreotrich species such as these were the most abundant components of the ciliate assemblage. All bars are 20 µm in length.	Figure 4 B. Two heterotrophic dinoflagellates visualized via epifluorescence microscopy; these dinoflagellates have food vacuoles full of recently ingested coccoid cyanobacteria (bright red-orange cells in the vacuoles) (blue organelle is the DAPI-stained nucleus).

- Distribution of ciliates across individual transects showed variable patterns. For the Newport Hydroline, September 2001, ciliate biomass was high both inshore and offshore, but low at slope stations where pico- and nano-phyto-plankton biomass was highest (Figure 5). In contrast, for the Five Mile Hydroline, high ciliate abundance was confined to the upper 10 m at the slope station, where there was a locally intense bloom of small phytoplankton (Figure 6).

Figure 5. Newport Hydroline, September, 2001: Depth distribution of of A) chlorophyll-a (color) compared to sigma-t surfaces (contour lines); B) coccoid cyanobacteria plus nano-eukaryotic biomass (color) compared to in situ fluorescence (contour lines); and C) ciliates (blue color) compared to in situ fluorescence (contour lines).

Figure 6. Five Mile Line, September, 2001: Depth distribution of cell abundances of A) coccoid cyanobacteria plus nano-eukaryotic biomass (color) compared to sigma-t surfaces (contour lines); B) ciliates (blue color) compared to in situ fluorescence (contour lines).

- For the September 2001 GLOBEC cruise, we were able to compare distribution patterns of the 0-50 m integrated biomass of coccoid cyanobacteria- and nano-eukaryotic phytoplankton (Figure 7-A), and of the integrated abundance of ciliates (Figure 7-B) with respect to surface CTD fluorescence.

Figure 7 A. September 2001 GLOBEC LTOP survey: comparison of 0-50 m integrated biomass (gC/m2 of coccoid cyanobacteria- and nano-eukaryotic phytoplankton (ultraphytoplankton).	Figure 7 B.Comparison of 0-50 m integrated abundance of ciliates, number per m2 (colors), with respect to surface (0-5 m) CTD fluorescence (contours)

- We used the full data set for microzooplankton (abundance and biomass) for the July 2001 Newport Hydroline to compare biomass, relative size, and potential grazing impact of three components of the microzooplankton: ciliates (Lugols samples), heterotrophic dinoflagellates, and other flagellates > ~ 10 ESD in size (Table 1). To estimate grazing impact, we used literature values for clearance rates of ciliates, dinoflagellates, and other flagellates (Neuer & Cowles 1995, Hansen et al. 1997). We calculated that, based on our cell abundances and assumed clearance rates, the microzooplankton community could clear on average about 2/3 of the water column per day, and at times could clear > 100% of the water column per day. These estimates compare favorably to the grazing rates that Neuer & Cowles (1994) empirically determined for microzooplankton in the Oregon upwelling system: 16 to 121 % of phytoplankton production grazed per day. We also found, as did Neuer & Cowles (1994, 1995), that both ciliates and heterotrophic dinoflagellates were important in terms of phytoplankton grazing (Table 1).

Table 1. Estimate of microzooplankton grazing impact in the upper 70 m of the Newport Line, June 2001: Abundance (cells/ml), average cell size (equivalent spherical diameter, ESD, um), and biomass (ugC/liter) and grazing impact (% of water volume cleared per day) for ciliates, heterotrophic dinoflagellates, other flagellates, and grazing impact for total microzooplankton. Assumed mean cell-specific clearance rates, based on literature values, were 4.6 ul/cell/hr for ciliates, 0.6 ul/cell/hr for heterotrophic dinoflagellates, and 0.1 ul/cell/hr for other heterotrophic flagellates. Mean value ± one standard deviation, range of values in parentheses.

Parameter Ciliates Heterotrophic dinoflagellates Other heterotrophic flagellates Total grazing Cells / ml 3.5 ± 2.0 (0.5 – 9.2) 17.5 ± 6.5 (8 – 32) 23.3 ± 16.1 (6 – 48) Biomass, ug C / liter 2.0 ± 1.7 (0.1 – 4.2) 2.0 ± 1.2 (0.3 – 4.4) 2.3 ± 2.0 (0.4 – 10) Equivalent spherical diameter, um 19.3 ± 5.5 (14 – 29) 11.4 ± 1.6 (9.5 – 16) 11.1 ± 2.1 (9 – 21) Clearance, % water volume/day 36.7 ± 22.5% (10 – 42%) 25.2 ± 9.3% (10 – 42%) 5.6 ± 3.9% (1 – 14%) 67.5 ± 27.4% (15 – 136%)

References cited:

Menden-Deuer, S., Lessard. E.J., 2000. Carbon to volume relationships for dinoflagellates, diatoms, and other protist plankton. Limnolology and Oceanography 45, 569-579.

Neuer, S., and T.J. Cowles. 1994. Protist herbivory in the Oregon upwelling system. Mar. Ecol. Prog. Ser. 113:147-162.

Neuer, S., and T.J. Cowles. 1995. Comparative size-specific grazing rates in field populations of ciliates and dinoflagellates. Mar. Ecol. Prog. Ser. 125:259-267.

Hansen, P.J., P. K. Bjornsen, and B.W. Hansen. 1997. Zooplankton grazing and growth: Scaling within the 2-2,000-um body size range. Limnol. Oceanogr. 42:687-704.

Zubkov, M.V., M.A. Sleigh, and P.H. Burkill. 2000. Assaying picoplankton distribution by flow cytometry of underway samples collected along a meridional transect across the Atlantic Ocean. Aquat. Microb. Ecol. 21:13-20).

Acknowlegments: We thank Carlos Lopez for technical assistance in collection of the samples on cruises and for protist enumeration via epifluorescence microscopy, Pat Wheeler and Mike Wetz for the chlorophyll-a data, and the crew of the R/V Wecoma and LTOP project personnel for invaluable support. This material is based upon work supported by the National Science Foundation under Grant No. OCE-0101294 to B. & E. Sherr.

See also a recently manuscript on distribution of coccoid cyanobacteria and small eukaryotic phytoplankton in the Oregon upwelling systems