Microzooplankton grazing impact in the Western Arctic Ocean

ARTICLE IN PRESS Deep-Sea Research II 56 (2009) 1264–1273

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Microzooplankton grazing impact in the Western Arctic Ocean Evelyn B. Sherr , Barry F. Sherr, Aaron J. Hartz College of Oceanic and Atmospheric Sciences, Oregon State University, Corvallis, OR 97331-5503, USA

a r t i c l e in f o

a b s t r a c t

Article history: Accepted 29 October 2008 Available online 25 November 2008

Microzooplankton grazing impact on phytoplankton was assessed using the Landry–Hassett dilution technique in the Western Arctic Ocean during spring and summer 2002 and 2004. Forty experiments were completed in a region encompassing productive shelf regions of the Chukchi Sea, mesotrophic slope regions of the Beaufort Sea off the North Slope of Alaska, and oligotrophic deep-water sites in the Canada Basin. A variety of conditions were encountered, from heavy sea-ice cover during both spring cruises, moderate sea-ice cover during summer of 2002, and light to no sea ice during summer of 2004, with a concomitant range of trophic conditions, from low chlorophyll-a (Chl-a; o0.5 mg L1) during heavy ice cover in spring and in the open basin, to late spring and summer shelf and slope open-water diatom blooms with Chl-a 45 mg L1. The microzooplankton community was dominated by large naked ciliates and heterotrophic gymnodinoid dinoflagellates. Significant, but low, rates of microzooplankton herbivory were found in half of the experiments. The maximum grazing rate was 0.16 d1 and average grazing rate, including experiments with no significant grazing, was 0.0470.06 d1. Phytoplankton intrinsic growth rates varied from the highest values of about 0.4 d1 to the lowest values of zero to slightly negative growth, on average 0.1670.15 d1. Light limitation in spring and post-bloom senescence during summer were likely explanations of observed low phytoplankton growth rates. Microzooplankton grazing consumed 0–120% (average 22726%) of phytoplankton daily growth. Grazing and growth rates found in this study were low compared to rates reported in another Arctic system, the Barents Sea, and in major geographic regions of the world ocean. & 2008 Elsevier Ltd. All rights reserved.

Keywords: Microzooplankton Herbivory Dilution technique Phytoplankton growth Western Arctic Ocean

1. Introduction Microzooplankton, mainly phagotrophic protists o200 mm in size, have a central role in pelagic food webs as herbivores (Sherr and Sherr, 2002; Calbet and Landry, 2004) and as food for larger zooplankton such as copepods (Stoecker and Capuzzo, 1990; Gifford and Dagg, 1991; Fessenden and Cowles, 1994; Levinsen et al., 2000; Olson et al., 2006). However, there have been few studies to date of the significance of microzooplankton in Arctic food webs. Phagotrophic ciliates and dinoflagellates are known to be abundant in Arctic marine systems based on prior research in the central Arctic Ocean (Sherr et al., 1997, 2003), in coastal waters of the Canadian Archipelago (Bursa, 1961) and of Western Greenland (Nielsen and Hansen, 1995; Levinsen et al., 1999, 2000; Levinsen and Nielsen, 2002), and in the Barents Sea (Verity et al., 2002). Bursa (1961) observed heterotrophic dinoflagellates consuming diatoms in the Canadian Arctic. Sherr et al. (2003) reported that the biomass of microzooplankton increased along with the biomass of phytoplankton during spring and summer in the central Arctic and had the potential to consume a large

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E-mail address: [email protected] (E.B. Sherr). 0967-0645/$ - see front matter & 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.dsr2.2008.10.036

fraction of phytoplankton production. Abundant large-sized ciliates and heterotrophic dinoflagellates in Disko Bay, West Greenland, and in Young Sound, NE Greenland, were found to be important as grazers of phytoplankton (Nielsen and Hansen, 1995; Hansen et al., 1999; Levinsen et al., 1999; Rysgaard et al., 1999; Levinsen and Nielsen, 2002) and as food for copepods (Levinsen et al., 2000; Levinsen and Nielsen, 2002). In the Barents Sea during early summer, phytoplankton growth and microzooplankton grazing were closely coupled, and grazing losses accounted for 64–97% of growth (Verity et al., 2002). These studies suggest that microzooplankton may be as important in Arctic ecosystems as they are in other parts of the world ocean. Here we report the first study of microzooplankton grazing impact on phytoplankton in the Western Arctic Ocean using the seawater dilution technique. During spring and summer Shelf– Basin Interactions (SBI) process cruises in 2002 and 2004, we assessed microzooplankton herbivory in the euphotic zone of shelf, slope, and deep-water regions of an area encompassing parts of the Chukchi Sea, Beaufort Sea, and Canada Basin. Our goal was to estimate the flux of carbon through the microzooplankton component of the pelagic food webs in this region of the Arctic Ocean, and thus to be able to compare the grazing impact of microzooplankton on phytoplankton with that of mesozooplankton, determined separately by Campbell et al. (2009).

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2. Methods 2.1. Sampling Microzooplankton grazing impact was assessed as part of the SBI project during four process cruises in the Western Arctic Ocean during May/June and July/August 2002 and 2004. Water for grazing experiments was collected at a subset of stations occupied during these cruises along established transects from shelf to open basin: along Barrow Canyon, off the northern coast of Alaska east of Point Barrow, east of Hanna Shoals, and west of Hanna Shoals in the Chukchi Sea (Fig. 1). A total of 40 dilution experiments were completed (Table 1). Except for the first experiment, only one depth was selected for each experiment. Sampling for dilution experiments was coordinated with sampling for primary productivity assays (Kirchman et al., 2009) and for mesozooplankton grazing experiments (Campbell et al., 2009).

2.2. Dilution experiments Since Landry and Hassett (1982) first proposed the dilution technique to quantify the rate of consumption of phytoplankton by microzooplankton, the method has since been widely used in various regions of the world ocean (Calbet and Landry, 2004). The approach is to dilute whole seawater with 0.2-mm-filtered (particle-free) water by varying amounts. The phytoplankton will, in theory, have the same intrinsic growth rate, m, across the dilution series, while grazing mortality, g, will be decreased in proportion to the dilution. For each dilution treatment, realized phytoplankton growth rate, r, is estimated from change in chlorophyll-a (Chl-a) concentration over the time of incubation. The y-intercept of a regression of r as a function of dilution approximates m, the growth rate of phytoplankton in the absence of grazing, and the slope of the regression is equivalent to g, the microzooplankton grazing rate, both in units of time1. Considerations regarding the dilution technique have been discussed by Gifford (1988), Gallegos (1989), Landry (1993), Neuer and Cowles (1994), Dolan et al. (2000), and Olson and Strom (2002). Two common manipulations in the method are to gently pre-screen whole seawater to exclude grazers 4200 mm, and to

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add nutrients to the dilution series to minimize potential nutrient limitation of phytoplankton growth (Landry, 1993). An additional concern for conducting dilution experiments in the Arctic Ocean is the high light sensitivity of polar phytoplankton (Smith and Sakshaug, 1990). Caron et al. (2000) reported that significant Chl-a bleaching of phytoplankton cells during dilution experiments carried out in the Ross Sea, Antarctica, precluded estimates of phytoplankton growth rates. In order to address this problem, we carried out most experiments at low simulated light levels, and also quantified cell-specific fluorescence (FL) of phytoplankton at the beginning and end time of each incubation via flow cytometry. Dilution experiments were carried out following the protocol of Landry (1993). All carboys, bottles, and tubing used in setting up dilution assays were pre-soaked in 10% HCl and thoroughly rinsed with deionized water. Nitex gloves were worn during experimental set-up. Water for the dilution assays was collected in 30-L Niskin bottles at a pre-determined depth, either the Chl-a maximum or a depth in the upper mixed layer, usually at the 5% or 15% light level, corresponding to a depth sampled for phytoplankton production (Kirchman et al., 2009). Seawater was gently transferred into 50-L carboys through silicon tubing with 200-mm Nitex mesh pouches zip tied to the ends to screen out copepods and other larger grazers following the method of Olson and Strom (2002); care was taken to keep the mesh pouches below the water surface and to avoid bubbles in the tubing as the carboys were filled. The 200-mm-screened water was used as whole seawater in setting up the dilution series. After collection of seawater, all other preparation steps were carried out in a temperature-controlled environmental chamber set at 1 1C under dim light (approximately 0.1% of incident light). For dilutions, particle-free seawater was prepared by gravity filtration through a Pall 0.2-mm filter that was pre-soaked in 10% HCl and thoroughly rinsed with deionized water. Five to seven liters of seawater were passed through the 0.2-mm filter before beginning collection of particle-free water for the dilutions. Experimental bottles were filled within 2–3 h of sample collection. Particle-free water was added to 2-L polycarbonate bottles using a set of bottles of known volume to yield replicate treatments of 100%, 80%, 60%, 40%, 20%, and 8% or 12% whole seawater. To ensure non-nutrient-limited growth of phytoplankton, ammonium nitrate and sodium phosphate were added to

Fig. 1. Location of stations in the Canada Basin and Chukchi Sea at which water was collected to set up dilution assay experiments during the four SBI process cruises. Left map shows collection stations during spring and right map during summer. Triangles indicate 2002 locations and circles indicate 2004 locations.

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Table 1 Summary of dates, station locations, bottom depths, sampling depth, water temperature, and nitrate concentration at the sampling depth, and light level during incubation as percent of incident light, for dilution assay experiments conducted during SBI process cruises.. Bottom depth (m)

Sampling depth (m)

Water temp. (1C)

Nitrate (mM)

Incubation light (% Io)

168.76 168.76 160.16 157.58 158.51 159.81 153.90 154.28

50 50 1170 2850 400 45 50 1980

2.2 6.2 12 31 31 18 11 7.6

1.7 1.7 1.6 1.5 1.6 1.7 1.7 1.6

17.1 17.1 0.50 0.71 1.6 4.9 5.6 2.7

50 15 Darka Darka 5 15 15 5

71.62 72.48 72.33 71.98 71.55 71.28 72.62 73.05 73.39 73.68 72.77 73.27

155.99 153.45 151.94 152.22 152.32 152.53 158.68 158.16 157.40 159.21 161.15 164.56

180 3160 3330 2120 190 50 120 1680 3180 2830 50 70

25 42 19 22 21 14 22.5 23 36 36 19 32

1.4 1.6 1.5 1.4 0.8 1.2 1.3 1.1 1.6 0.6 1.5 1.6

0.09 2.6 0.55 0.46 0.11 0.06 0.01 0.06 2.2 0.15 0.12 8.3

15 5 15 5 5 5 15 15 5 15 15 5

Spring 2004 5/25/04 5/26/04 5/30/04 6/03/04 6/04/04 6/08/04 6/12/04 6/15/04 6/16/04 6/18/04

72.01 72.08 72.67 72.87 73.14 71.44 71.78 72.26 71.92 71.66

159.60 159.63 158.77 158.25 157.79 154.32 154.82 154.52 154.86 156.20

40 45 160 1160 2430 35 190 1830 550 105

8.8 6.8 10 46 31 11 12 25.3 32 28

1.6 1.6 1.6 1.6 1.3 1.4 1.5 1.4 1.5 1.5

3.4 2.7 0.38 2.1 0.56 4.3 0.39 0.18 0.19 12.8

5 5 5 Darka Darka 5 5 5 5 5

Summer 2004 7/22/04 7/26/04 7/30/04 8/01/04 8/03/04 8/06/04 8/08/04 8/11/04 8/14/04 8/17/04

71.06 71.94 71.54 72.52 72.02 71.64 72.45 72.65 72.86 73.763

159.44 154.82 152.42 152.15 152.27 152.32 153.55 158.50 158.30 156.78

75 690 146 3690 2260 420 3075 170 930 3600

19 21 26 53 29 12 15 34 44 31

0.8 1.1 4.2 0.4 0.6 4.3 5.2 1.4 0.9 0.8

3.4 0.18 0.15 0.12 1.8 0.05 0.16 4.0 0.08 0.09

5 5 Darka 5 Darka 15 15 5 5 5

Date

Latitude (1N)

Spring 2002 5/10/02 5/10/02 5/19/02 5/23/02 5/27/02 5/30/02 6/04/02 6/07/02

67.51 67.51 73.29 73.45 72.85 72.24 71.49 72.14

Summer 2002 7/22/02 7/27/02 7/29/02 7/31/02 8/03/02 8/04/02 8/06/02 8/10/02 8/12/02 8/13/02 8/17/02 8/19/02

a

Longitude (1W)

Experiment run in the dark at either 1 or 2 1C due to excessive temperature variation in the on-deck incubator.

experimental bottles to yield concentrations of 5 mM N and 0.25 mm P. Duplicate whole seawater treatments without added nutrients were also included in most of the dilution experiments. A carboy filled with whole seawater was gently mixed for several minutes using a plexiglass rod with a small plexiglass disc attached to the end. Then, while the carboy continued to be gently mixed, whole seawater was siphoned out of the carboy to fill the experimental bottles and an additional 2-L bottle for initial samples. Parafilm was placed on top of each bottle prior to securing the cap, in order to minimize air bubbles in the bottles, since protist cells can lyse in contact with air (Gifford, 1988). The experimental bottles were placed into plexiglass cylinders covered with combinations of neutral-density grey, Scrim, and blue plastic film to mimic the approximate in situ light intensity and quality of the water depths sampled. An incubation light level corresponding to 5%, 15%, or 50% of incident light was chosen for each experiment, depending on the water depth at which the

sample was collected and information on actual light level profiles under the ice provided to us by Rohf Gradinger (pers. comm.) during the cruises. The cylinders were secured in on-deck incubators cooled with flowing surface seawater, which ranged in temperature from 1 to 6 1C depending on the season and location in the SBI sampling region. In summer 2004 there was less ice cover compared to summer 2002, so that the respective light levels occurred at deeper sampling depths in 2004. In 2002 the duration of dilution experiments was 60–75 h. The fact that there was no strong diel light variation during the arctic summer minimized concern about incubation periods not conforming to strict multiples of 24-h days. In 2004 a shorter incubation time of approximately 48 h for experiments was used, since results from the 2002 field season suggested that 2-day incubations would yield adequate growth and grazing rates, and a shorter time minimized potential for temperature variation during the incubation. Temperature in the on-deck incubators was continually

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monitored using a Hobo temperature recorder immersed in one of the incubators. In six experiments in which there were problems with the seawater flow to the incubators or in which the ship was expected to travel through water masses of varying surface temperature during an experiment, dilution experiment bottles were incubated at constant temperature in the dark in either the 1 1C environmental chamber or in a walk-in refrigerator set at 2 1C. Initial samples were taken from whole seawater samples for the determination of Chl-a concentration, for flow cytometric analysis of phytoplankton cell abundance, light scattering properties, and FL, and for microscopic enumeration of microzooplankton abundance, biomass, and general taxonomic composition. Depending on the phytoplankton concentration, from 25- to 150-mL quadruplicate volumes were settled via vacuum filtration onto GFF filters in dim light. The filters were extracted in 6 mL of 90% acetone in 13  100 mm glass culture tubes at 20 1C for 18–24 h. At the end of the extraction period, the filter was carefully removed from each tube, and the Chl-a concentration determined using a calibrated Turner Designs fluorometer. A solid chlorophyll standard was used to check for fluorometer drift at the beginning of each reading of Chl-a samples. For flow cytometry samples, one 3-mL aliquot per dilution bottle was pipetted into a 4-mL cryovial and preserved with 0.2% (w/v) final concentration of freshly made paraformaldehyde. The samples were gently mixed and placed in a dark at room temperature for 10 min before freezing and stored at 80 1C until flow cytometric analysis was performed. Two hundred-milliliter subsamples for determination of microzooplankton biomass and abundance were preserved with 10% (2002) or 5% (2004) final concentration acid Lugol solution for inverted microscopy. Separate subsamples were preserved for inspection via epifluorescence microscopy with a three-step alkaline Lugol-sodium thiosulfate-2% final concentration formalin fixation protocol (Sherr and Sherr, 1993). Formalinpreserved samples were held at 2 1C for 12–24 h, and then settled onto 0.8-mm or 3.0-mm black membrane filters, stained with DAPI (5 mg mL1 final concentration), and mounted onto glass slides, which were stored at temperatures of 20 1C or lower until analysis. At the end of the dilution incubations, final samples were taken from each bottle for Chl-a concentration and for flow cytometric analysis of phytoplankton. Depending on the initial phytoplankton concentration and dilution, from 25 to 500-mL replicate or triplicate subsamples from each bottle were filtered for Chl-a determination. Phytoplankton growth and grazing rates were calculated from dilution by change in Chl-a concentration corrected for change in cell-specific FL determined via flow cytometry (see Section 2.3). Initial Chl-a concentrations in the dilutions were estimated from whole seawater Chl-a concentrations and the known dilution. Phytoplankton growth rates (r) were calculated for each experimental bottle using a logistic growth model based on initial and final Chl-a concentrations. Based on change in cell-specific red FL during the incubation period, measured via flow cytometry, final time Chl-a concentrations were adjusted as necessary, using a simple proportional formula (corrected Tf Chl-a ¼ Tf Chl-a(To FL/Tf FL)), in each dilution experiment. Change in cell-specific FL affected estimates of phytoplankton growth rates, but not of microzooplankton grazing rates. Regression statistics of the plots of fraction of nutrient-amended whole seawater versus growth rate were used to estimate m (y-intercept), the intrinsic growth rate of the phytoplankton with no grazing mortality, and g (slope), the microzooplankton grazing rate, both in units of day1 (Landry, 1993). In a few cases, there was non-linearity in the regression slopes in which increase in r was observed only in dilution treatments of o60%. For these plots, regressions to determine m

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and g were based on 8% or 12%, 20%, and 40% dilution bottle data. In order to estimate daily phytoplankton growth and grazing loss in terms of carbon biomass, we first calculated the daily phytoplankton intrinsic growth and portion of growth consumed by microzooplankton in terms of Chl-a using the logistics equation, and then converted these values to daily increase or consumption of phytoplankton carbon biomass using the C:Chl-a ratio of 30 empirically determined for this region of the Arctic Ocean (Booth and Horner, 1997; Sherr et al., 2003). 2.3. Post-cruise sample analysis Samples for FCM were thawed and kept on ice in a dark container until run on a Becton–Dickinson FACSCaliber flow cytometer with a 488-nm laser. Five hundred-microliter subsamples were processed as described in Sherr et al. (2005). Each subsample was spiked immediately before processing with a known amount of 3.0 mm Polysciences Fluoresbrite yellowgreen beads from a stock solution that had been precalibrated with Becton–Dickinson True-Count beads. The number of beads enumerated in each sample run was used to accurately determine the sample volume processed, and thus the abundance of photosynthetic cells. Primarily red-fluorescing phytoplankton were found in SBI samples, and only a negligible number of orange-fluorescing cells. These cells were probably cryptophyte algae as they had higher values for light scatter properties (related to cell size) than do coccoid cyanobacteria, which are routinely observed in flow cytograms in other regions of the sea (Li, 2002; Sherr et al., 2005). Microzooplankton abundance and biomass were determined in initial whole seawater samples for 20 experiments, including experiments set up in shelf, slope, and basin regions of the study area. From 50 to 100 mL of Lugol-preserved samples were settled for a minimum of 24 h and then the whole slide inspected by inverted light microscopy. All ciliate and dinoflagellate cells in each sample were counted, sized, and categorized into the general taxonomic groups of choreotrichous ciliates, oligotrichous ciliates, didinid ciliates, tintinnids, athecate dinoflagellates, and thecate dinoflagellates. From 200 to 600 protist cells were counted and sized in each sample inspected. Samples preserved for epifluorescence microscopy were inspected to determine whether dinoflagellates counted in Lugol-preserved samples were heterotrophic or autotrophic; only heterotrophic dinoflagellate morphotypes were included in the microzooplankton data. The biovolume of each enumerated cell was determined using algorithms for spherical, conical, and ellipsoidal shapes. Cell biomass was estimated using the algorithm of Menden-Deuer and Lessard (2000). Significance of relationships between growth rates and grazing rates estimated in the dilution experiments and environmental parameters was assessed by Spearman Rank Correlation Matrix using the NCSS Statistical Software for Windows developed by Dr. J.L. Hintze, Kaysville, Utah. Using this statistical approach prevented potential problems of outliers and non-linearity in the data set.

3. Results 3.1. General conditions The region of the Western Arctic Ocean covered by the SBI process cruises included highly productive shelf regions of the Chukchi Sea, mesotrophic slope regions of the Beaufort Sea off the North Slope of Alaska, and highly oligotrophic deep-water sites in

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the Canada Basin (Fig. 1). A variety of conditions were encountered, from heavy sea-ice cover during both spring cruises, moderate sea-ice cover during summer of 2002, and light to no sea ice during summer of 2004, and a concomitant range of trophic conditions, from low Chl-a (o0.5 mg L1) during heavy ice cover in spring and in the open basin, to late spring–summer shelf and slope open-water diatom blooms with Chl-a 45 mg L1. Nutrient concentrations were relatively high (Codispoti et al., 2009); for example, nitrate concentrations in water samples taken for dilution assay experiments averaged 2.574.3 mM, with a range of 0–17 mM (Table 1). Assessment of phytoplankton communities by epifluorescence microscopic inspection of water samples taken during the cruises indicated that sea-ice algae, either sloughed off from local sea ice or advected north in the Alaska Coastal Current, were an important component of algal biomass in the water column during May to June. In July and August, pelagic diatom species, e.g., Chaetocerous spp and Thalassiosira spp. dominated the phytoplank-

ton, where Chl-a was 45 mg L1. In low-Chl-a slope and basin regions, there was a mixed species assembly of phytoflagellates, including Micromonas sp., single-cell Phaeocystis sp., and occasionally Dinobryon sp., along with smaller sized diatoms.

3.2. Dilution experiments A total of 40 dilution experiments were completed: 20 experiments during the 2002 process cruises and 20 experiments during the 2004 process cruises (Table 1). Initial Chl-a concentrations in the experiments varied from o0.2 mg L1 during spring of 2002 to 415 mg L1 during summer of 2004 (Table 2). Significant diatom blooms composed of a variety of species were encountered during 2004. Flow cytometric analysis of phytoplankton demonstrated either a slightly positive increase or up to a 30% decrease in

Table 2 Summary of dilution experiment results.. Date

Spring 2002 5/10/02 5/10/02 5/19/02a 5/23/02a 5/27/02 5/30/02 6/04/02 6/07/02

Chl-a (mg L1)

0.32 0.34 0.15 0.22 0.27 0.23 0.50 0.20

Phytoplankton growth rate (d1)

Microzooplankton grazing rate (d1)

Regression slope r

Ratio of grazing to growth rate

0.425 0.371 0.030 0.018 0.045 0.301 0.421 0.267

0.000 0.000 0.000 0.000 0.000 0.024 0.084 0.151

NS NS NS NS NS NS 0.82 0.91

0.00 0.00 Dark Dark 0.00 0.08 0.20 0.56

Summer 2002 7/22/02 7/27/02 7/29/02 7/31/02 8/03/02 8/04/02 8/06/02 8/10/02 8/12/02 8/13/02 8/17/02 8/19/02

7.2 1.0 0.21 1.5 0.55 1.2 0.41 0.35 1.1 0.24 0.87 2.5

0.148 0.191 0.261 0.203 0.230 0.127 0.169 0.278 0.150 0.076 0.217 0.105

0.013 0.038 0.150 0.059 0.047 0.009 0.169 0.093 0.064 0.033 0.026 0.030

NS 0.59 0.90 0.87 0.77 NS 0.76 0.85 0.81 0.66 NS NS

0.08 0.20 0.57 0.29 0.21 0.00 1.01 0.33 0.42 0.00 0.00 0.00

Spring 2004 5/25/04 5/26/04 5/30/04 6/03/04a 6/04/04a 6/08/04 6/12/04 6/15/04 6/16/04 6/18/04

1.5 2.2 1.5 3.28 0.57 4.7 7.2 1.3 5.38 7.3

0.299 0.370 0.087 0.058 0.032 0.192 0.312 0.281 0.026 0.259

0.087 0.161 0.000 0.117 0.034 0.027 0.041 0.010 0.014 0.148

NS 0.65 NS 0.70 NS NS NS NS NS 0.75

0.29 0.44 0.00 Dark Dark 0.14 0.13 0.03 0.00 0.57

Summer 2004 7/22/04 7/26/04 7/30/04a 8/01/04 8/03/04a 8/06/04 8/08/04 8/11/04 8/14/04 8/17/04

14 18.0 0.79 0.92 16 0.66 1.4 2.9 2.3 0.16

0.383 0.028 0.031 0.144 0.097 0.106 0.066 0.011 0.032 0.120

0.120 0.002 0.044 0.038 0.013 0.122 0.060 0.024 0.038 0.056

0.69 NS 0.90 0.74 NS 0.79 0.90 NS 0.87 0.75

0.31 0.00 Dark 0.26 Dark 1.16 0.00 0.00 0.00 0.46

‘‘NS’’ ¼ not significant at the ro0.2 level. a Experiment run in the dark at either 1 1C or 2 1C due to excessive temperature variation in the on-deck incubator.

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cell-specific FL during incubations. We concluded that independent determination of growth and grazing rates of small-sized phytoplankton using flow cytometric assessment of change in cell number in the dilution series would not be a useful addition to the data set. Sufficient numbers (43000 mL1) of small (o5 mm) phytoplankton in the initial water sample, needed for accurate assessment of growth rate at the higher dilutions, were found in only a few of the experiments, mainly in open-basin samples. In these experiments, parameters determined for small-sized cells by flow cytometry were similar to growth and grazing rates determined via analysis of change in Chl-a. Large-sized phytoplankton cells (4 about 5 mm) were never sufficiently abundant (i.e., 43000 mL1) in the experiments to reliably determine relative growth at higher dilutions using flow cytometric counts.

Phytoplankton growth r, d-1

0.3

0.2

0.1 µ = 0.203, g = -0.059, r = 0.77

0.0

Phytoplankton growth r, d-1

0

0.2

0.4

0.6

0.8

1

0.8

1

0.3

0.2

0.1 µ = 0.281, g = -0.010, ns

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Thus, only the change in Chl-a in the nutrient-amended dilution series was used to estimate growth rates. There was a significant negative slope and thus a positive microzooplankton grazing rate in half (20 out of 40) of the experiments (Fig. 2A, Table 2). In most of the other experiments, a similar phytoplankton growth rate was obtained for all dilution treatments (Fig. 2B), which indicated that there was negligible microzooplankton grazing. In some experiments not incubated in the dark, phytoplankton growth rates were slightly negative (Table 2); these were likely due to post-bloom senescence. In a few cases, there was non-linearity in the regression slopes such that increase in r was observed only in dilution treatments of o60% of whole seawater, as originally observed in dilution assays by Gallegos (1989). For these plots, such as the one in Fig. 2C, regressions to determine m and g were based on the three higher dilutions of whole seawater. Phytoplankton intrinsic growth rates varied from the highest values of about 0.4 d1 to the lowest values of zero to slightly negative growth (Table 2). Examination of the entire data set showed a negative relation of intrinsic growth rate to sample depth (Fig. 3). Spearman Rank Correlation analysis of relation of phytoplankton intrinsic growth, m, to various parameters showed no significant relation of m to initial Chl-a concentration. There were, however, significant relations of m to initial temperature (0.42, ro0.006), initial nitrate concentration (0.32, ro0.05), and incubation light level (0.45, ro0.004). The strongest rank correlation in the data set was the negative relation (0.56, ro0.0002) between growth rate and sample depth. Inspection of epifluorescent slides suggested that during spring cruises, the Chla maximum at depths 420 m was due to senescent ice algae, and during summer, to senescent pelagic diatoms. Suspended diatoms were judged to be in a senescent state based on low pigment FL in cells and a large amount of clumped diatoms associated with detrital particles in epifluorescence slide preparations. Grazing mortality as a fraction of phytoplankton growth ranged from zero to a grazing mortality slightly higher than phytoplankton growth rate (Table 2). Grazing rate, g, was significantly correlated only to phytoplankton growth rate, m (ro0.005), and not to Chl-a concentration or other measured environmental parameters. Comparison of mean values for seawater temperature, initial Chl-a concentrations, carbon-based phytoplankton growth rates, and carbon-based microzooplankton

0.0 0

0.2

0.4

0.6

0.4 Phytoplankton growth µ, d-1

Phytoplankton growth r, d-1

0.2

0.1

µ = 0.168, g = -0.169, r = 0.76

0.2

0.0

0.0 0

0.2

0.4 0.6 0.8 Fraction whole seawater

1

Fig. 2. Examples of dilution experiment plots of realized phytoplankton growth, r, as a function of the fraction of seawater in the dilution series. (A) Plot with significant slope—7/31/2002, y-intercept (m) ¼ 0.203, slope (g) ¼ 0.059, regression r ¼ 0.77; (B) plot with non-significant slope—6/15/2004, y-intercept (m) ¼ 0.281, slope (g) ¼ 0.010; (C) plot with non-linearity in lower dilutions, regression based on data from higher dilutions—8/06/2002, y-intercept (m) ¼ 0.168, slope (g) ¼ 0.169, regression r ¼ 0.76. Highest dilution in plots A and C were 12% WSW, and highest dilution in plot B was 8% WSW.

-0.2 0

10

20 30 Experimental sample depth, m

40

50

Fig. 3. Phytoplankton intrinsic growth rate, m, estimated from dilution experiments as a function of initial sample water depth for the entire data set. Triangles indicate 2002 data and circles indicate 2004 data. Values that fall below the dotted line represent negative intrinsic phytoplankton growth, i.e., decline in biomass with time not resulting from grazing mortality.

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Table 3 Mean seawater temperature, Chl-a concentration, phytoplankton production, and microzooplankton grazing rate as mg C m3 d1, and fraction of phytoplankton production grazed by microzooplankton during spring and summer of 2002 and 2004; mean values7one standard deviation.. Season and year

Seawater temperature (1C)

Chl-a (mg L1)

Phyto. prod. rate (mg C m3 d1)

Microzoop. grazing rate (mg C m3 d1)

Fraction of prod. grazed

Spring ‘02 Summer ‘02 Spring ‘04 Summer ‘04

1.670.1 1.370.3 1.570.1 0.872.6

0.2870.11 1.471.9 3.572.5 5.777.2

2.772.8 6.679.2 25726 20761

0.3570.61 1.271.1 8.2713.4 13.0724.6

0.1470.22 0.2870.30 0.2070.21 0.4470.440

For phytoplankton production rate and fraction of production grazed, data from experiments run in dark were excluded.

grazing rates suggested a difference in the 2002 and 2004 field years (Table 3). In 2004, summer water temperatures in the experiments had a larger range and were on average higher, Chl-a stocks were higher during both spring and summer, and carbonbased growth rates were more variable and on average higher, compared to 2002. The mean fraction of daily phytoplankton production grazed varied from 0.1470.22 in spring 2002 to 0.4470.44 in summer 2004 (Table 3).

100 µm

3.3. Microzooplankton composition and biomass Microzooplankton biomass and general taxonomic composition were analyzed for a subset of initial water samples from the dilution experiments. There were abundant ciliates and heterotrophic dinoflagellates in all samples inspected. Ciliates were dominated by naked spirotrichs, both choreotrichous and oligotrichous species, including species in the genera Strombidium, Strobilidium, Leegardiella, and Laboea. A few tintinnid species were observed, the most common of which was a Tintinnopsis sp. A Ptychocylis sp. was abundant in one sample. However, tintinnids composed only a small fraction of the ciliate biomass. Some ciliate morphotypes were very large, 4100 to 200 mm in length (example in Fig. 4A). Heterotrophic dinoflagellates, including thecate and athecate forms, occurred in all samples. Large athecate gymnodinoid dinoflagellates, e.g., morphotypes similar to Gyrodinium spirale, 4100 mm in length (example in Fig. 4B), were especially abundant in association with diatom blooms. Total microzooplankton protist biomass varied from 1.6 up to 60 mg C L1, and averaged 13716 mg C L1. Ciliate biomass was on average 4.475.4 mg C L1 (range 0.5–25 mg C L1), and dinoflagellate biomass averaged 9712 mg C L1 (range 0.8–53 mg C L1). While thecate dinoflagellates were present, they were usually a small fraction (on average 12%) of total dinoflagellate biomass. Epifluorescence microscopic inspection of samples confirmed that the dinoflagellates enumerated did not have chloroplasts, and thus were not autotrophic. There was no strong relation between protist biomass and Chl-a concentration (Fig. 5A). We did note that athecate heterotrophic dinoflagellates often composed 450% of total heterotrophic protist biomass, particularly at higher Chl-a concentrations (Fig. 5B). Instances of high proportion of athecate heterotrophic dinoflagellate biomass at lower Chl-a concentrations (Fig. 5B) were mainly associated with post-bloom senescent diatoms.

4. Discussion 4.1. Microzooplankton grazing impact in the Western Arctic Ocean Algal biomass and primary production in the Western Arctic Ocean is dominated by diatoms, including sea-ice diatoms and pelagic diatoms (Booth and Horner, 1997; Gradinger, 1999; Gosselin et al., 1997). The bulk of both sea-ice and pelagic diatom

50 µm

Fig. 4. Examples of large microzooplankton protists observed in the Western Arctic Ocean: (A) 220-mm-long choreotrichous ciliate; (B) 130-mm-long heterotrophic gymnodinoid dinoflagellate, morphotype similar to Gyrodinium spirale. Protists were from initial experimental samples preserved with 5% final concentration acid Lugol solution and viewed via transmitted light. Micrographs were taken at a magnification of 200  using an image analysis system consisting of a Cooke Sensicam QE CCD camera with Image Pro Plus software, mated to an Olympus BX61 microscope.

primary production in the Western Arctic Ocean either sediments out of the euphotic zone for consumption and remineralization in the benthos, or advects off the shelf to slope and basin systems (Gradinger, 2009; Kirchman et al., 2009). A central question addressed by this study, coupled with the estimates of grazing on phytoplankton by mesozooplankton (Campbell et al., 2009), is the extent to which planktonic grazers consume phytoplankton production in situ, before export. In virtually all regions of the world ocean examined to date, microzooplankton have been found to exert a significant grazing impact on phytoplankton biomass and primary production (Putland, 2000; Olson and Strom, 2002; Calbet and Landry,

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2004; Leising et al., 2005a). However, our growth and grazing data based on dilution experiments were low both as compared to values from another Arctic region, the Barents Sea, at the same general latitude range (4701N) as the SBI study area, or as compared to average values for general oceanographic regions (Table 4). The only other report of similarly low microzooplankton grazing impact in the world ocean was that of Caron et al. (2000) in the Ross Sea, Antarctica. In that study, only 13 out of 51 dilution experiments resulted in microzooplankton grazing rates significantly different from zero, and the highest grazing rate was only 0.26 d1. While we found significant grazing rates in half of our

Heterotrophic protist biomass µg C l-1

60

40

20

0 0

5

10

15

20

25

Fraction athecate heterotrophic dinoflagellates of total protist biomass

1.0 0.8 0.6 0.4 0.2

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experiments, our highest grazing rate, 0.16 d1, was even lower than that found for Ross Sea microzooplankton. Average grazing mortality for phytoplankton in the SBI study region of the Western Arctic Ocean was 21.5725.5% of phytoplankton growth. Unfortunately, Caron et al. (2000) were unable to estimate phytoplankton growth rates and thus the fraction of phytoplankton growth grazed due to bleaching of Chl-a during their on-deck incubations. 4.2. Phytoplankton growth rates The average phytoplankton growth rate found in this study, 0.1670.15 d1, was also lower than average growth rates estimated using the dilution technique reported in the Barents Sea and in various geographic regions of the ocean (Table 4). These low growth rates are in part due to the extensive seasonal and regional coverage during the SBI process cruises. During both spring cruises, and in summer 2004, we often sampled in regions with heavy sea-ice cover. The strong light limitation of phytoplankton in the Arctic Ocean is well documented (Smith and Sakshaug, 1990; Sherr et al., 2003). During spring cruises, some of our dilution experiments were set up using samples from depths at which most phytoplankton were post-bloom ice algae; during summer cruises, we at times encountered what appeared to be senescing pelagic diatom blooms. The physiological state of phytoplankton has not been generally considered in studies of in situ phytoplankton growth. The low and sometimes negative values for intrinsic growth rate found at deeper sampling depths (Fig. 3) were likely due in part to both low light level and to ice algae or phytoplankton in post-bloom condition. There was marked inter-annual variability in environmental conditions during this study. In 2002, sea ice covered much of the sampling region both in spring and summer, but 2004 was a year characterized by extensive retreat of sea ice during summer (Codispoti et al., 2009). In 2004, there were both warmer seawater temperatures during the summer cruise, and higher phytoplankton biomass as measured by Chl-a concentration during both spring and summer cruises, compared to 2002 (Table 3). Average rates of carbon-based phytoplankton growth and grazing mortality, mg C m3 d1, were also higher in 2004 compared to 2002 due to the greater phytoplankton biomass (Table 3). 4.3. Microzooplankton biomass and composition

0.0 0

5

10

15

20

25

Chl-a, µg l-1 Fig. 5. Relation of microzooplankton biomass to phytoplankton stocks: (A) total microzooplankton biomass, mg C L1, as a function of Chl-a and (B) fraction of athecate heterotrophic dinoflagellate biomass of total protist biomass as a function of Chl-a. Triangles indicate 2002 data and circles indicate 2004 data.

The microzooplankton biomass in initial water samples was similar to that found in previous Arctic studies and in other regions of the sea characterized by diatom blooms. Although, in our study, total microzooplankton biomass was mainly o15 mg C L1, in a few samples, biomass was 30–60 mg C L1. In these samples either Chl-a concentrations were 415 mg L1 or phytoplankton appeared be in a state of senescence. In the Barents Sea

Table 4 Comparison of phytoplankton growth and microzooplankton grazing rates, and percent of phytoplankton production grazed, found in this study with values for these parameters determined by the dilution technique in another Arctic system, and in general geographic regions of the world ocean.. Region

Chl-a (mg L1)

Phyto. growth m (d1)

MZP grazing g (d1)

Phyto growth grazed (%)

Reference

Arctic systems Western Arctic Ocean spring and summer Barents Sea early summer

2.874.3 0.6670.20

0.1670.15 0.3270.13

0.0470.06 0.2470.11

21.5725.5 76.878.2

This study Verity et al. (2002)

Other ocean regions Oceanic Tropical/subtropical Temperate/subpolar Polar (Southern Ocean)

0.5870.03 1.0170.21 5.1870.66 0.6270.06

0.5970.02 0.7270.02 0.6970.03 0.4470.05

0.3970.01 0.5070.02 0.4170.02 0.4170.16

69.671.5 74.572.0 60.871.8 59.273.3

Calbet Calbet Calbet Calbet

and and and and

Landry Landry Landry Landry

(2004) (2004) (2004) (2004)

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during early summer (post-bloom), with Chl-a concentrations on average 0.6670.2 mg C L1, Verity et al. (2002) reported a total microzooplankton biomass averaging 12.473.4 mg C L1. In subarctic and temperate marine systems characterized by diatom blooms, microzooplankton biomass can be as great or greater than found in this study. Olson and Strom (2002) found microzooplankton biomasses of 9–132 mg C L1 in the southeast Bering Sea during summer, with the highest microzooplankton biomass associated with blooms consisting of diatoms and the coccolithophorid Emiliania huxleyi. Leising et al. (2005a) reported total microzooplankton biomass in the range of 20–60 mg C L1 during diatom blooms (5–20 mg Chl-a L1) in Dabob Bay, Washington, USA. In Washington coastal waters during early autumn, Olson et al. (2006) found microzooplankton biomass in the upper water column of 30715 mg C L1, with a range of 20–52 mg C L1. Irigoien et al. (2005) analyzed data on microzooplankton biomass in 12 regions of the sea and concluded that microzooplankton biomass in general increases with phytoplankton biomass, but plateaus at about 50 mg C L1, a finding that our data supports. The high fraction of athecate gymnodinoid dinoflagellates of total microzooplankton biomass found in this study (Fig. 5B) also supports previous reports of significant biomass of these phagotrophic dinoflagellates in marine systems where mass diatom blooms occur (Hansen, 1991; Archer et al., 1996; Putland, 2000; Olson and Strom, 2002; Verity et al., 2002; Horner et al., 2005; Leising et al., 2005a). The dominance of heterotrophic dinoflagellates in the microzooplankton in the Western Arctic Ocean is also important in regard to the review of Rose and Caron (2007), in which they suggest that temperature constraint on intrinsic maximum growth rate of herbivorous protists is a factor in the initiation and development of mass blooms of phytoplankton in highlatitude, cold-water regions of the ocean. While we did find relatively low rates of protist herbivory in the cold-water environment of the Western Arctic Ocean, which appears to support the hypothesis of Rose and Caron (2007), there is a caveat. These authors considered only growth rates of ciliates and nanoflagellates, and not of heterotrophic dinoflagellates, in their comparison of temperature–growth relationships of herbivorous protists versus phytoplankton. It may be that maximum growth rates of heterotrophic dinoflagellates do not decline with temperature in the same way as do growth rates of the protistan species used to represent herbivores in their analysis. Empirical determination of the relative growth rates of heterotrophic dinoflagellates and of their diatom prey in polar systems will be required to resolve this issue.

4.4. Potential top–down control of herbivorous protists by mesozooplankton Microzooplankton can be a significant food resource for mesozooplankton in marine systems (Stoecker and Capuzzo, 1990; Kleppel, 1993; Suzuki et al., 1999; Levinsen et al., 2000; Vincent and Hartmann, 2001; Liu et al., 2005; Leising et al., 2005b, c). There is also evidence that feeding on microzooplankton along with phytoplankton can enhance copepod fecundity (Kleppel, 1993; Klein Breteler et al., 1999; Bonnet and Carlotti, 2001; Castellani et al., 2005). A concurrent set of experiments on mesozooplankton grazing in the Western Arctic Ocean have shown that microzooplankton are generally preferred over phytoplankton as prey (Campbell et al., 2009). It is possible that mesozooplankton grazing curtailed accumulation of biomass of microzooplankton stocks sufficiently to limit grazing impact of herbivorous protists on phytoplankton in this region.

5. Conclusions We found that in the Western Arctic Ocean, microzooplankton grazing impact was highly variable and accounted, on average, for only about one fifth of daily phytoplankton production, rather than the 60–70% of production found in other marine systems (Table 4). A potential explanation for this observation is the strong top–down control of microzooplankton stocks due to preferential predation on ciliates and heterotrophic dinoflagellates by Arctic copepods (Levinsen et al., 2000; Campbell et al., 2009). In addition, at times we found high protist biomass but low rates of herbivory in the presence of senescing sea-ice diatoms or pelagic diatoms. In these samples, microscopic inspection showed abundant nanoflagellates, including choanoflagellates. Since heterotrophic flagellates are a food resource for both ciliates (Verity, 1991) and heterotrophic dinoflagellates (Jeong et al., 2007) it may be that microzooplankton were consuming non-algal prey in those experiments. Further studies in polar ecosystems are needed to confirm our results of generally lower rates of grazing by microzooplankton than has been reported for marine systems at lower latitudes. If our observations regarding low rates of microzooplankton herbivory in the Western Arctic Ocean prove to be generally the case, then a larger proportion of phytoplankton production may be available for export in this region compared to other oceanic systems.

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