1 North Pacific Research Board 1 2 Bering Sea Integrated Ecosystem Research Program 3 4 Final Report 5

1 NORTH PACIFIC RESEARCH BOARD

3 BERING SEA INTEGRATED ECOSYSTEM RESEARCH PROGRAM

5 FINAL REPORT

7 Summer Microzooplankton in the Bering Sea

8 NPRB BSIERP Project B55 Final Report

10 Diane Stoecker

12 University of Maryland Center for Environmental Science, Horn Point Laboratory

13 PO Box 775, 2020 Horns Point Road, Cambridge, MD 21613

15 December 2012

24 Abstract

25 Microzooplankton (20-200 micron size fraction of zooplankton) abundance, biomass, contribution to 26 chlorophyll a and grazing on phytoplankton were estimated as part of the BEST-BSIERP integrated 27 ecosystem project during the summers of 2008, 2009 and 2010. One celled grazers, primarily ciliates and 28 dinoflagellates, dominated the microzooplankton biomass, and at times microzooplankton biomass was 29 higher than phytoplankton biomass in surface waters on the inner and middle shelf. A confounding factor 30 was the contribution of mixotrophic (combination of feeding and photosynthesis) ciliates to chlorophyll a, 31 which is a proxy for phytoplankton biomass. On average, mixotrophic ciliates comprised 66% of the 32 ciliate biomass, and in the North Middle Domain, sometimes contributed over 50% of the chlorophyll a in 33 surface waters. Near the shelf break and in the “bloom” waters, microzooplankton biomass was about 34 one-half that on the inner and middle shelf. At the shelf break and in the outer shelf domain, 35 microzooplankton consumed 67-78% of phytoplankton daily growth but in the middle and inner shelf 36 domains, microzooplankton grazing exceeded phytoplankton daily growth. On the northern Middle 37 Shelf, there was usually a prominent deep chlorophyll maximum (DCM); microzooplankton ingestion of 38 chlorophyll was high in the DCM, indicating a role for the DCM in trophic transfer. On the eastern 39 Bering Sea shelf in summer, microzooplankton consumed most of the phytoplankton production and 40 accounted for many of the larger size cells in the plankton; indicating that they are an important trophic 41 link in food webs supporting larger zooplankton, which in turn support fish and larger organisms.

43 Keywords: planktonic ciliates, dinoflagellates, subarctic seas, subpolar continental shelf, 44 microzooplankton grazing, dilution experiments, mixotrophy, plastidic ciliates, grazing in DCM

46 Citation: Stoecker, D.K. 2012. Microzooplankton abundance, biomass, contribution to chlorophyll a 47 and grazing in the eastern Bering Sea in summers of 2008, 2009, and 2010. NPRB BSIERP Project B55 48 Final Report, XX p.

54 Table of Contents:

55 Introduction…………………………………………………………………………………………4 56

57 Overall Objectives…………………………………………………………………………………..6

58 Chapter 1. Microzooplankton: Abundance, biomass and contribution to chlorophyll in the

59 Eastern Bering Sea in summer………………………………………………………………..……9

60 Chapter 2. Microzooplankton grazing in the Eastern Bering Sea in summer…………………49

61 Conclusions…………………………………………………………………………………………94

62 BSIERP and Bering Sea Project Connections……………………………………………………95

63 Management or policy implications...... 96

64 Publications…………………………………………………………………………………………96

65 Poster and oral presentations……………………………………………………...... 96

66 Outreach…………………………………………………………………………………...... 96

67 Acknowledgements……………………………………………………………………………….....97

68 Literature cited……………………………………………………………………………………...97

72 Study Chronology: Funded from October 1, 2007 to September 30, 2010 with no cost extension to 73 December 31, 2012. Annual reports submitted for years 1, 2, and 3 (October 2009, 2010, 2011).

83 Introduction

84 The BSIERP program was designed to study ecosystem processes and the effects of inter-annual 85 and decadal variation in weather and climate on food webs that support fisheries and marine bird and 86 mammal populations in the eastern Bering Sea. Summer data on microzooplankton were a gap in the 87 program. Microzooplankton are responsible for most of the grazing on phytoplankton in the sea (Landry 88 and Calbet, 2004; Sherr and Sherr, 2009). They are an important link in the food chain between 89 phytoplankton and zooplankton, such as copepods and krill (Calbet and Saiz, 2005; Campbell et al., 90 2009), which are food for zooplanktivorous fish such as larval and juvenile walleye pollock and forage 91 fish that support top predators (Hunt et al. 2002, 2011). Microzooplankton grazing is also a key factor 92 regulating phytoplankton blooms (Landry and Calbet, 2004; Sherr and Sherr, 2009). The project 93 “Summer Microzooplankton in the Bering Sea” contributes to the goals of the program by providing data 94 for summers 2008, 2009, and 2010 on standing stocks of microzooplankton and their grazing activities. 95 Abundance, biomass, size distribution and composition of larger microzooplankton were determined. 96 Experiments were conducted at sea to measure grazing by microzooplankton on phytoplankton. 97 Microzooplankton production was estimated. Information on microzooplankton is necessary to construct 98 models linking changes in climate and weather to changes in food webs that support fish, marine birds 99 and mammals in this highly productive, but potentially vulnerable, marine ecosystem. This project 100 specifically addresses the BSIERP hypothesis that “Climate-induced changes in physical forcing will 101 modify the availability and partitioning of food for all trophic levels through bottom-up processes”. A key 102 goal was to determine how the microzooplankton link is influenced by regional differences in physical 103 forcing and climate-induced changes in Bering Sea food webs.

104 Previous to this study, there were only three summer investigations of microzooplankton in the 105 eastern Bering Sea (Liu et al., 2002; Olson and Strom, 2002; Strom and Fredrickson, 2008, Sherr et al., in 106 press), but this limited data set illustrated the inter-annual variability in microzooplankton and raised 107 interesting questions about coupling between phytoplankton and microzooplankton in this ecosystem. 108 Two of the studies were conducted in summer of 1999 (Liu et al., 2002; Olson and Strom, 2002) and the 109 third study was conducted in summer 2004 (Strom and Fredrickson, 2008). A fourth study was part of the 110 BEST program and conducted during spring sea ice conditions of 2008, 2009 and 2010 (Sherr et al., in 111 press). The first recorded bloom in the Bering Sea of the coccolithophorid alga, Emiliana huxleyi, 112 occurred in 1997, and the bloom persisted in patches into 1999. In 1999, reduced microzooplankton 113 grazing on < 10 µm phytoplankton (the size class containing Emiliana huxleyi) was observed inside 114 bloom patches (Olson and Strom, 2002). The conventional paradigm is that microzooplankton are 115 primarily the grazers of small phytoflagellates, not large diatoms. Surprisingly, Olson and Strom (2002)

116 observed a greater impact of microzooplankton grazing on net growth of the >10 µm fraction of 117 phytoplankton than on the <10 µm fraction, and on average, MZ grazing consumed 100% of the 118 phytoplankton growth in the > 10 µm size fraction of phytoplankton. Very high abundance of 119 microzooplankton (22-227 cells ml-1) and high microzooplankton biomass were observed. These data 120 suggest that in 1999 top-down control of microzooplankton by larger zooplankton was weak and 121 microzooplankton grazing was important in regulating phytoplankton composition. Microzooplankton 122 grazing appears to have promoted the persistence of the coccolithophorid bloom that channeled 123 phytoplankton production into the microbial food web. It is not known if this increased channeling of 124 production into the microbial food web was a consequence of reduced top-down control of large cell-size 125 microzooplankton by larger zooplankton or whether it was due to “bottom-up” control by the 126 environment favoring particular phytoplankton. Probably it was due to an interaction of these factors.

127 The most recent summer investigation of microzooplankton in the Bering Sea prior to the 128 BEST/BSIERP Program was in 2004, a year with low ice cover, intense summer stratification at some 129 stations, and anomalously high surface water temperatures (Strom and Fredrickson 2008). In 2004 130 phytoplankton growth rates were similar to 1999, but microzooplankton grazing mortality of 131 phytoplankton was about half that observed in 1999. Interestingly, pennate diatoms in the genus Pseudo- 132 nitzschia were the dominant diatoms at some stations (Strom and Fredrickson, 2008). Biomass of large 133 (>20 µm) MZ in 2004 was about half that observed in 1999. Strom and Fredrickson (2008) observed that 134 the intense stratification in the anomalously warm SE Bering Sea in 2004 lead to phytoplankton nutrient 135 limitation and hypothesized that this was the cause of the reduced grazing and lower microzooplankton 136 standing stock that they observed. This reduced trophic coupling at lower trophic levels may have 137 negatively affected zooplankton stocks and thus perhaps also higher trophic levels that depend on summer 138 zooplankton production.

139 The results from these prior investigations in the SE Bering Sea suggested several hypotheses 140 concerning the influence of summer sea surface temperature and stratification on trophic coupling low in 141 the food web. These were:

142 1. In “warmer” years, microzooplankton coupling to phytoplankton growth is reduced compared to more 143 average years due to increased stratification and nutrient depletion in the mixed layer (from Strom and 144 Fredrickson, 2008). Alternate hypothesis: In “warmer” years, microzooplankton coupling to 145 phytoplankton growth is increased due to a shift of production to smaller size phytoplankton that are more 146 efficiently grazed by microzooplankton.

147 2. In “warmer” years, large microzooplankton (>20 µm) biomass is low compared to more average years; 148 less microzooplankton prey is available to zooplankton during “warm” summers. Alternate hypothesis: 149 Biomass of large microzooplankton in “warm” years is > than the biomass in average years.

150 3. At stratified stations, if a deep chlorophyll layer is present at the pycnocline, microzooplankton 151 abundance and biomass per m3 is higher in this layer than in the mixed layer above. The pycnoline is a 152 potential site of enhanced trophic transfer of microzooplankton carbon to higher trophic levels under 153 stratified conditions. Alternate hypothesis: Microzooplankton abundance and biomass per m3 in the deep 154 chlorophyll layer is < abundance and biomass in the mixed layer.

155 These hypotheses were formulated and partially tested by previous investigations in the Bering 156 Sea. However, given the inter-annual variability and spatial heterogeneity of the eastern Bering Sea, there 157 was not enough data to conclusively test any of these hypotheses. The project reported herein was 158 designed to yield comparable data, as much as possible, to the existing data base to facilitate comparison 159 of results across years. It was anticipated that the 2008-2010 field years would include “warm” years with 160 reduced sea ice during spring and that comparisons could be made between “warm” and “cold” years 161 within the project data set. However all three years were cold with average stratification indices (Ladd 162 and Stabeno, 2012; Stabeno et al., 2012b). The data on microzooplankton for summers 2008, 2009 and 163 2010 demonstrate the important domain (regional) differences in microzooplankton. The 2008-2010 data 164 also document year- to- year differences within a “cold” period in microzooplankton abundance and 165 biomass and microzooplankton grazing. Thus these data provide insights into the structure and 166 functioning of lower trophic levels in the eastern Bering Sea and provide input on microzooplankton 167 abundance, biomass and rate processes useful to zooplankton ecologists and modelers interested in 168 predicting the response of the Bering Sea ecosystem to inter-annual and decadal variation in physical 169 forcing and biotic responses.

170 Overall Objectives

171 The overall objective was to contribute to the goals of BSIERP by providing data on standing stocks of 172 microzooplankton and their grazing activities. The specific objectives were to:

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174 1. Determine the abundance, biomass and composition of the larger (>20 µm) microzooplankton. 175 Describe inter-annual and spatial (domain) variability in microzooplankton.

176 We achieved this objective by sampling mixed layer microzooplankton on summer cruises in 2008, 2009, 177 and 2010 in the eastern Bering Sea and analyzing the samples microscopically (Chapter 1, sections 2.1

178 and 2.2). Stations were grouped by physical domain (Chapter 1, Table 1) and two-way analysis of 179 variance was used to test for differences among domains by year (Chapter 1, section 2.4). Results are 180 presented in Chapter 1, Table 2 and Figures 2-4).

181 2. Determine if microzooplankton biomass in the upper mixed layer is statistically associated with sea 182 surface temperature, indices of stratification, inorganic nutrient availability, chlorophyll a (< 20 and >20 183 µm size classes and total), specific phytoplankton blooms or domain (inner, middle or outer shelf).

184 We partially achieved this objective by using Pearson Product Moment Correlation analyses to determine 185 if microzooplankton biomass was significantly associated with chlorophyll a and found that it was not 186 (Chapter 1, section 3.2 and Figure 5). Because size fractionated chlorophyll was not available from the 187 CTD casts from most stations, we only included total chlorophyll. Likewise, information on 188 phytoplankton species composition was not available for most stations so we did not have the data for 189 these analyses. Instead of presenting correlations between biomass and sea surface temperature and 190 inorganic nutrient availability, we decided to examine the association between microzooplankton grazing 191 and these factors because data on these factors was available for most “process station” casts, which we 192 used for the grazing experiments, but not for most routine sampling stations that we used for the biomass 193 estimates. We found no significant correlations between microzooplankton grazing and water temperature 194 or inorganic nutrients (Chapter 2, section 3.2). We microscopically determined the abundance of 195 Phaeocystis pouchettii in samples from the experimental stations and found no statistically significant 196 correlations with microzooplankton grazing (Chapter 2, section 3.2 ). Indices of stratification were not 197 available for individual stations and thus we did not include this factor in our statistical analyses. Two- 198 way analysis of variance tested for domain differences in microzooplankton biomass (Chapter 1, Table 2 199 and Figures 2-4) and grazing (Chapter 2 Section 3.2 and Table 3).

200 3. Estimate phytoplankton growth rates (µ) and mortality (g) due to microzooplankton grazing (<200 µm 201 fraction) with the seawater dilution method. Calculate g:µ to estimate grazing as a fraction of total 202 phytoplankton production.

203 We achieved this by conducting seawater dilution grazing experiments on all three summer cruises 204 (Chapter 2, section 2.1 and 2.2, Figure 1 and Table 2). Results (µ, g, and µ/g) are given Chapter 2, section 205 3.2 and Table 3 and Figure 2.

206 4. Based on estimated phytoplankton mortality due to microzooplankton grazing (dilution experiments), 207 carbon:chlorophyll ratios and microzooplankton gross growth efficiencies from the literature, estimate 208 microzooplankton production.

209 Estimates of microzooplankton production based on chlorophyll ingestion are presented in Chapter 2, 210 section 3.2 and Figure 4.

211 5. Compare microzooplankton data from this investigation to data from the literature to assess inter- 212 annual and regional variability.

213 This was achieved for both microzooplankton biomass (Chapter 1, sections 4.3 and 4.4, Table 3) and 214 grazing (Chapter 2, section 4, Table 6).

215 6. Provide data to other BEST/BSIERP field investigators and modelers; collaborate in assessing the role 216 of MZ in the SE Bering Sea Ecosystem and in ecosystem responses to inter-annual and decadal changes 217 in weather and climate.

218 Data from all cruises has been submitted. We are collaborating with other BEST/BSIERP investigators.

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235 Chapter 1.

236 Microzooplankton: Abundance, Biomass and Contribution to Chlorophyll in the Eastern Bering

237 Sea in Summer

238 Diane K. Stoeckera*

239 Alison C. Weigela

240 Dean A. Stockwellb

241 Michael W. Lomasc,d

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243 a. University of Maryland Center for Environmental Science, Horn Point Laboratory, P.O. Box 244 775, Cambridge, MD 21613, USA. 245 b. Institute of Marine Science, University of Alaska Fairbanks, P.O. Box 757220, Fairbanks, AK 246 99775, USA. [email protected] 247 c. Bermuda Institute of Ocean Sciences, 17 Biological Station, St. George’s, GEO1, Bermuda 248 d. Current Address: Bigelow Laboratory for Ocean Sciences, 60 Bigelow Drive, East Boothbay, 249 04544. [email protected] 250

251 *Corresponding author: Tel: +1 410-221-8407; Fax +1 410 221 8490: [email protected]

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253 Keywords; Microzooplankton, Ciliates, Dinoflagellates, Mixotrophy, Bering Sea

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255 Submitted December 10, 2012 to Deep Sea Research PART II, Third Bering Sea Project special issue

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262 Abstract

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264 Summer microzooplankton abundance and biomass were determined for three years, 2008, 2009, 265 and 2010, during a four year “cold” period in the eastern Bering Sea. Average microzooplankton densities 266 ranged from 4 X 103 to 25 X 103 cells l-1 in the mixed layer. Microzooplankton biomass was 21-25 µg C l- 267 1 in the mixed layer on the middle shelf (South and North Middle Domains) which has relatively low 268 chlorophyll during summer stratification. However, microzooplankton biomass was about one-half that in 269 the less stratified waters near the shelf break, greenbelt, and in the “bloom” waters in the Pribilof Island 270 Domain. Although phytoplankton biomass was higher in deep chlorophyll maxima (DCM) than in 271 surface waters on the shelf, microzooplankton biomass was generally not elevated in the DCM. High 272 ratios (>1) of microzooplankton biomass to phytoplankton biomass were observed at chlorophyll a 273 concentrations < 1 µg l-1. At times, average microzooplankton biomass was higher than the calculated 274 phytoplankton biomass in the mixed layer in coastal (Inner Domain) and middle shelf (South and North 275 Middle Domain) waters. A confounding factor in comparing microzooplankton and phytoplankton 276 biomass was the contribution of plastid-retaining, mixotrophic, ciliates to chlorophyll a. On average, 277 mixotrophic ciliates comprised 66% of the ciliate biomass, and in the North Middle Domain, on some 278 cruises, contributed over 50% of the chlorophyll a in the mixed layer. The 2008-2009 data suggest that 279 extent of summer stratification, presence of localized blooms, and domain differences all have major 280 influences on coupling of microzooplankton to phytoplankton stocks in summer in the eastern Bering Sea.

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292 1. Introduction

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294 The eastern Bering Sea supports productive commercial and subsistence fisheries as well as 295 major marine mammal and bird populations (Hunt et al., 2008, 2011). It is also highly sensitive to inter- 296 annual and decadal-scale climate variability, with differences between “warm”, “cold” and “average” 297 years reflected in secondary productivity and fish, bird, and mammal populations, particularly on the 298 southern shelf (Aydin and Mueter, 2007; Hunt et al., 2002, 2011). The spatial extent of winter sea ice and 299 the timing of sea ice retreat have a large impact on the timing and characteristics of the spring bloom and 300 the ecology of the Bering Sea. Winter-spring conditions set up the shelf for the summer, influencing 301 summer phytoplankton and zooplankton populations (Stabeno et al., 2012b). In addition, summer 302 weather, particularly storms and wind mixing, break down stratification (Ladd and Stabeno, 2012) 303 resulting in large but ephemeral blooms (Sambrotto et al., 1986) that result in enhanced net community 304 production (Mordy et al., 2012).

305 Microzooplankton are an important link between primary production and higher trophic levels in 306 polar and sub-polar as well as temperate and tropical waters (Levinsen and Nielsen, 2002; Calbet and 307 Saiz, 2005; Campbell et al., 2009). In both “warm” and “cold” years and spring as well as summer, 308 microzooplankton are major consumers of primary production in the eastern Bering Sea (Liu et al., 2002; 309 Olson and Strom, 2002; Strom and Fredrickson, 2008; Sherr et al., accepted; Stoecker et al., this issue). 310 Microzooplankton are important prey for mesozooplankton, including large crustacean zooplankton such 311 as euphausiids and copepods (e.g., Calanus., Neocalanus, and Metridia spp.) as well as for small 312 crustacean zooplankton such as Acartia, Pseudocalanus and Oithona spp. (Gifford and Dagg, 1991; 313 Levinsen and Nielsen, 2002; Campbell et al., 2009; Stoecker 2012) and fish larvae (Fukami et al., 1999; 314 Figueiredo et al., 2007; Montagnes et al., 2010). Crustacean zooplankton are food for larvae and juveniles 315 of many important Bering Sea fish species including pollock (Howell-Kübler et al., 1996; Coyle et al., 316 2008; Hunt et al., 2011). When chlorophyll is low and dominated by small phytoplankton, 317 microzooplankton can be crucial to the survival and fecundity of mesozooplankton. Microzooplankton 318 are particularly important as a food for mesozooplankton after the spring bloom, during late spring and 319 summer stratification (Ohman and Runge, 1994; Fileman et al., 2010).

320 Studies during both cold (1999) and warm (2004) years have shown that summer 321 microzooplankton abundance and biomass in the eastern Bering Sea shelf is relatively high and 322 dominated by heterotrophic and mixotrophic dinoflagellates and ciliates. Most data are from the southern 323 Bering shelf and the productive area around the Pribilof Islands (Olson and Strom, 2002; Strom and

324 Fredrickson, 2008). However, relatively little information is available on variation in microzooplankton 325 abundance and biomass across the shelf or on the shelf north of St. Matthew Island. The Eastern Bering 326 Sea Shelf is extensive and can be divided into regions by its hydrography and bathymetry; coastal (<50m 327 depth, Inner Domain), middle shelf (50-100m; Middle Domain), and outer shelf (100-200m depth; Outer 328 Domain) (Hunt et al., 2002). Each domain varies in physical forcing, susceptibility to climate change and 329 biology, as does the northern and southern portions of the shelf (Stabeno et al., 2012a). In summer, the 330 south middle shelf is a thermally stratified system with a wind mixed surface layer typically 20-30m thick 331 and a tidally mixed bottom layer. The middle shelf is separated from the inshore, shallow well-mixed 332 coastal domain by the Inner Front, and from the offshore, outer domain by the Middle Transition Zone 333 (Kachel et al., 2002; Stabeno et al. 2012a). Thus, Inner, Middle, and Outer domains now are subdivided 334 into southern and northern regions with the transitional area corresponding to ~60oN, the position of the 335 MN line (Fig. 1) (Stabeno et al., 2012a). The area around the Pribilof Islands on the southern shelf is a 336 separate domain. The Pribilof Domain is supplied with nutrient and plankton rich water from the slope 337 and outer shelf as well as being subject to tidal mixing; it can have new production and localized 338 phytoplankton blooms in summer even when the rest of the south middle shelf is highly stratified (Hunt et 339 al., 2008, Sambrotto et al., 2008).

340 The southern region is the most susceptible part of the eastern Bering Sea to climate change due 341 to inter-annual variation in the marginal ice zone (Stabeno et al., 2012a, 2012b). During 2007-2010 (cold 342 years) the south middle shelf became well mixed after the retreat of sea ice, with thermal stratification 343 starting in May (Stabeno et al., 2012b). The two-layer structure usually persists through October, 344 however, the magnitude and extent of thermal stratification is not correlated with warm or cold years or 345 spring sea ice extent on the southern shelf (Stabeno et al., 2012b). Wind mixing determines nutrient 346 supply to the euphotic zone, and hence net community production (Mordy et al. 2012), during summer on 347 the southern middle shelf.

348 On the northern shelf, the average amount of sea ice is greater and persists later in the year than in 349 the south. The north middle domain is usually stratified in summer due to combination of salinity and 350 temperature (Stabeno et al., 2012a). Mixed layer water temperature in summer on the northern shelf is 351 several degrees cooler than in the south. In contrast to the southern middle domain, subsurface 352 phytoplankton blooms are common in summer on the northern middle shelf. The euphotic zone (~20-40 353 m deep) extends at least partially into the pycnocline (Stabeno et al., 2012a); phytoplankton growth may 354 occur in the euphotic portions of the pycnocline. However, deep chlorophyll maxima resulting from 355 sedimentation of surface blooms may also occur in the pycnocline. No data were available on the 356 association of microzooplankton with these subsurface, high chlorophyll layers.

357 As part of the BEST-BSIERP ecosystem study in the eastern Bering Sea, we sampled 358 microzooplankton in both the southern and northern shelf on approximately month long cruises in 359 summers of 2008, 2009, and 2010. This was during a four year “cold” period with extensive sea ice cover 360 in spring (Stabeno et al., 2012b). Our primary goal was to investigate summer microzooplankton 361 abundance, biomass, and composition across domains. We were also interested in documenting the 362 relative biomass of microzooplankton to phytoplankton in both the mixed layer and in the deep 363 chlorophyll maxima because this ratio is an indicator of the potential importance of microzooplankton as 364 food for mesozooplankton. The biomass of autotrophic and mixotrophic ciliates is often high in polar 365 seas during summer (Putt, 1990; Simé-Ngando et al., 1992; Sorokin et al., 1996; Suzuki and Taniguchi, 366 1998; Levinsen and Nielsen, 1999; Montagnes et al., 2008). One of our goals was to determine if plastidic 367 ciliates were important biomass components in the Eastern Bering Sea and to estimate their contribution 368 to chlorophyll a in summer.

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370 2. Materials and Methods

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372 2.1 Microzooplankton sampling and onboard observations

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374 Samples for abundance and biomass estimates of microzooplankton were collected on 375 BEST/BSIERP summer cruises in 2008 2009, and 2010 on the USCG Healy, R/V Knorr and R/V T.G. 376 Thompson, respectively. In 2008, the cruise was in June but in 2009 and 2010, the cruise was later, from 377 mid-June to mid-July (Table 1). Samples were collected from routine sampling stations along established 378 transects including the cross shelf NP, MN and SL lines and south to north mid-shelf 70-m line (Figure 379 1). In 2009 the XB and XB2 transects were added to sample the greenbelt area. Mixed layer samples 380 were routinely collected at a predetermined depth, usually 5 or 10 m. Microzooplankton samples were 381 also collected from productivity casts from the depth of 55% of surface incident PAR irradiance that 382 varied from 3-15 m depending upon the station (Lomas et al., 2012). In all cases, this depth was within 383 the surface mixed layer. At stations with a pronounced deep chlorophyll a fluorescence maximum 384 (DCM), additional samples were obtained. The fluorescence maximum samples were from depths of 14 to 385 46 m. When multiple maxima were sampled, data are presented for the depth with the highest measured 386 chlorophyll a.

387 Duplicate samples (250 ml) for microzooplankton were collected with 30 liter Niskin bottles 388 attached to a CTD and water samples were immediately preserved with 10% (final concentration) acid 389 Lugol’s solution in amber glass bottles on HY0803 in 2008 and with 5% (final concentration) acid 390 Lugol’s solution in 2009 (KN195-10) and 2010 (TN-250). In 2008, a 10 sample subset of samples was 391 fixed with 2%, 5%, and 10% acid Lugol’s solution. The estimated abundance with the different strengths 392 were compared; the abundance estimate with 10% acid Lugol’s was not higher than with the 5% solution 393 (data not shown). We switched to 5% acid Lugol’s in 2009 and 2010 since this lower concentration 394 reduces cell shrinkage of ciliates and matched the fixative concentration used on the spring cruises (Sherr 395 et al., 2012). Lugol’s fixed samples were stored in the dark until analysis at Horn Point Laboratory.

396 Onboard, we examined “fresh” samples from some stations, either live or fixed with 1% 397 glutaraldehyde with transmitted light and/or epifluorescence microscopy in Utermöhl chambers with an 398 inverted microscope. Our objective was to examine cells for the presence or absence of plastids and 399 characterize the types of microzooplankton and microphytoplankton present. We determined which 400 morphotypes of ciliates had plastids (i.e., were mixotrophs) and which were strictly heterotrophic.

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402 2.2 Microzooplankton sample analysis

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404 At Horn Point Laboratory we enumerated > 20 µm protistan microzooplankton (> ~15 µm when 405 fixed with acid Lugol’s), using a Nikon Eclipse TE 2000-U inverted microscope, after settling 25 or 50 406 ml volumes overnight in Utermöhl chambers (Gifford and Caron, 2000). Our detection limit was ~40 407 cells liter-1. A SPOT v 4.0 RT camera and SPOT diagnostic software were used to record images and to 408 measure cells. Biovolumes were calculated from the measurements using appropriate geometric formulae. 409 In accordance with earlier microzooplankton studies in the Bering Sea (Olson and Strom, 2002; Strom 410 and Fredrickson, 2008), all dinoflagellates within the designated size range were included in abundance 411 and biomass estimates because most if not all photosynthetic dinoflagellates are also phagotrophic (Jeong 412 et al., 2010).

413 The ciliates were placed in the following functional and taxonomic categories: 1. Mesodinium 414 rubrum (sym. Myrionecta rubra); this photoautotrophic ciliate consumes cryptophytes and other small 415 prey. 2. Mixotrophic ciliates; these are non-loricate spirotrich ciliates (strombidiids) that graze on 416 phytoplankton (usually nanophytoplankton) but have chlorophyll and are also photosynthetic. Only 417 ciliates which we observed to have chlorophyll at sea and which we could recognize as the same

418 morphospecies in the Lugol’s fixed samples are included in this category. Thus, it is a minimum estimate 419 of mixotrophic ciliates. 3. Non-loricate strictly heterotrophic spirotrich ciliates (this includes 420 heterotrophic strombidiids and strobilidiids). This category may include a few mixotrophs that we were 421 not able to recognize in the Lugol’s fixed samples. The ciliates in this category are mostly grazers on 422 phytoplankton. 4. Tintinnid ciliates. These were all heterotrophs. 5. Other=All other ciliates.

423 We used the “C=0.19*V” conversion factor for ciliates from Putt and Stoecker (1989) for the 424 samples fixed in 5% acid Lugol’s solution. This conversion factor is commonly used (for example, in the 425 studies of Bering Sea microzooplankton by Olson and Strom, 2002; Strom and Fredrickson, 2008; Sherr 426 et al. 2012). To correct for shrinkage in the 2008 samples fixed with 10% acid Lugol’s solution we used a 427 correction factor of “C= 0.251*V” based on the Putt and Stoecker factor, but corrected for cell shrinkage 428 in 10% acid Lugol’s solution using data in Stoecker et al. (1994).

429 Dinoflagellates were categorized as thecate or non-thecate. Most of the dinoflagellates were non- 430 thecate and we were not able to separate many of the smaller non-thecate dinoflagellates by morphotype 431 in the Lugol’s fixed samples and thus could not classify them as plastidic or non-plastidic. Therefore, we 432 aggregated all dinoflagellates in one group. The dinoflagellates did not shrink differently in 5% and 10% 433 acid Lugol’s data (data not shown), so we used the dinoflagellate specific algorithm of Menden-Deuer 434 and Lessard (2000) to estimate the biomass of morphotypes in both the 5% and 10% acid Lugol’s 435 samples. This procedure is the same as in other recent Bering Sea investigations of microzooplankton 436 (Olson and Strom, 2002; Strom and Fredrickson, 2008; Sherr et al., 2012).

437 We also enumerated and sized other microzooplankton sized protist cells that could not be readily 438 classified taxonomically or which were rare. These included unidentified heterotrophic microflagellates, 439 testate amoebae, and the mixotrophic silicoflagellates . We did not include the colonial mixotrophic 440 flagellate, Dinobryon spp., in our estimates because it did not preserve well in the Lugol’s fixed samples. 441 Biomasses of non-ciliate and non-dinoflagellate microzooplankton were estimated from biovolume using 442 the general protist algorithm of Menden Deuer and Lessard (2000). Metazoan microzooplankton 443 (crustacea and rotifers) were rare compared to protists and are not included in our abundance and biomass 444 estimates.

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446 2.3 Chlorophyll a and determination of Phytoplankton Biomass

447 For samples collected in conjunction with productivity casts, chlorophyll data available from the 448 primary productivity study were used (Lomas et al., 2012). For other stations, we used chlorophyll data

449 collected as part of core hydrographic measurements. At stations for which our sampling depth did not 450 match the sampling depths for chlorophyll, we interpolated between depths when possible. We estimated 451 phytoplankton biomass from chlorophyll a using an empirically determined Carbon to Chlorophyll a ratio 452 of 50 (Lomas et al., 2012). Only data for stations and depths for which both microzooplankton and 453 chlorophyll data were available were used in comparing microzooplankton and phytoplankton biomasses.

454

455 2.4 Data Analyses and Statistics

456 We grouped stations by physical domain (Figure 1) (Hunt et al., 2008; Lomas et al. 2012; 457 Stabeno et al. 2012a) with the number of stations sampled in each domain each year (N) given in Table 1. 458 . Data for the MN line, a transitional area between the northern and southern shelves of the eastern Bering 459 Sea, do not belong in the other domains (Stabeno et al. 2012a) and are separate. Data are presented only 460 for domains for which microzooplankton data are available for 2 or more stations in each year. Data are 461 not available for all stations in all years and the distribution of data among domains varied from year to 462 year. For example, the representation of middle and inner shelf domains was higher in 2010 than 2008 or 463 2009 (Table 1). Over-all yearly averages of abundance and biomass are influenced by station distribution.

464 All statistical tests were run using Sigmaplot version 9.0. Two way Analysis of Variance 465 (ANOVA) was used to test for differences in the microzooplankton abundance or biomass among 466 domains by year. We applied standard transformations (log 10, square root or arcsin) when data did not 467 meet assumptions for ANOVA. In cases in which the transformed data still did not meet the assumptions, 468 one way ANOVAs were conducted on data for each domain separately or a one way ANOVA on ranks 469 was run using the combined data. Pair-wise multiple comparisons were made using the Holm-Sidak 470 method. We used the Pearson Product Moment Correlation to examine association between 471 microzooplankton abundance or biomass and chlorophyll a.

472

473 3. Results

474

475 3.1 Abundance and Composition

476 During summers 2008-2010 the abundance of protistan microzooplankton was high in the eastern 477 Bering Sea (Table 2). Protistan microzooplankton occurred at average densities of 4 X 103 to 25 X 103 478 cells l-1 in the mixed layer on the shelf. Year and the “domain X year” interaction were significant

479 sources of variation in microzooplankton abundance (Table 2). On the north middle shelf and along the 480 MN transect, the mixed layer MZ abundance was higher in 2010 than in 2009 and higher in 2009 than in 481 2008 (p<0.05). On the South Outer shelf, abundances were also higher in 2010 than in 2008 (p<0.05).

482 Ciliates and heterotrophic and mixotrophic dinoflagellates numerically dominated the 483 microzooplankton, with other taxa contributing <5% to this assemblage. Ciliates ranged in average 484 abundance in the mixed layer samples from 1.4 X 103 to 13.0 X 103 cells l-1 (Fig 2a). In the South Outer 485 domain, South Inner domain and along the MN transect, ciliates were more abundant in 2010 than 2008 486 or 2009 (p<0.05). In the North Middle domain they were more abundant in 2009 and 2010 than in 2008 487 (p<0.05). Ciliates accounted for 30-53% of the microzooplankton in surface waters.

488 Over 95% of the ciliates were non-loricate spirotrich ciliates (mostly strombidiids and 489 strobilidiids). The most common ciliates were mixotrophic strombidiids (Strombidium spp. and Laboea 490 strobilia) with algal plastids. Mixotrophic ciliates were particularly prevalent in surface waters on the 491 shelf where they numerically contributed 68-75% of the ciliate microzooplankton. The average 492 abundance of mixotrophic ciliates was 0.4 X 103 to 7.7 X 103 cells l-1 (Fig. 3). Similar to the total ciliates, 493 mixotrophic ciliates were more abundant in 2009 and 2010 than in 2008 in the North Middle Domain 494 (Fig. 3). In the south inner domain mixotrophic ciliates were more abundant in 2010 than 2008 or 2009. 495 The primarily photosynthetic ciliate, Mesodinium rubra, and tintinnid ciliates each made relatively minor 496 (1-6%) numerical contributions to the ciliate assemblage.

497 Heterotrophic and mixotrophic dinoflagellates were co-dominant with the ciliates and ranged in 498 abundance from 1.3 X 103 to 15.7 X 103 cells l-1 (Fig. 2b). In the South Outer domain, dinoflagellates 499 were more abundant in surface waters in 2009 and 2010 than in 2008 and along the MN transect and in 500 the North Middle Domain they tended to be more abundant in 2010 than in 2009 and 2008 (p<0.05). 501 Heterotrophic and mixotrophic dinoflagellates contributed an average of 45-67% of the microzooplankton 502 in the mixed layer. The dinoflagellates were dominated by non-thecate heterotrophic dinoflagellates, with 503 Gyrodinium and Gyrodinium-like species common. Thecate dinoflagellates such as Protoperidinium 504 were rare.

505

506 3.2 Biomass and correlations with Chlorophyll a

507 Average microzooplankton biomass in the mixed layer was 12-14 µg C l-1 in the south outer 508 domain, at the shelf break station and at the Pribilof Island stations. Average microzooplankton biomass 509 was 21-25 µg C l-1 on the shelf (middle domains, south inner domain) (Figure 4). Biomass along the MN

510 transect, which crossed from the inner to the middle and outer shelf between the south and north, and 511 thus contained a mixture of domains, averaged 16 µg C l-1. In the South Outer Domain, 512 microzooplankton biomass was significantly higher in 2010 than 2008 . Average microzooplankton 513 biomass was similar for the three summer cruises for the South Middle Domain, MN transect, and Shelf 514 Break. In the South Outer Domain, microzooplankton biomass was significantly higher in 2010 than 2008 515 (Figure 4).

516 Microzooplankton biomass in the mixed layer was not correlated with chlorophyll a 517 concentration (Pearson Product Moment Correlation, N=132, p>0.05). High ratios (>1) of 518 microzooplankton biomass to calculated phytoplankton biomass were observed at chlorophyll a 519 concentrations of < 1 µg Chl l-1 with ratios > 2 observed occasionally observed at chlorophyll 520 concentrations of < 0.5 µg Chl l-1 (Figure 5). Domain had a significant effect on the ratio of 521 microzooplankton to phytoplankton biomass in the mixed layer during summer (Figure 6). In the South 522 Inner and North Middle domains in summer 2008, average microzooplankton biomass in the mixed layer 523 was greater than estimated phytoplankton biomass (Figure 6). In the North and South Middle domains 524 and along the MN transect in summer 2010, microzooplankton biomass was greater than phytoplankton 525 biomass in the mixed layer (Figure 6). In summer 2009, compared to summer 2008 or 2010, the ratio of 526 microzooplankton to phytoplankton biomass was low in north middle domain. In general, ratios were 527 highest on the inner and middle shelf and lowest on the outer shelf and shelf break (Figure 6).

528 The relative contribution of ciliates and dinoflagellates to microzooplankton biomass was roughly 529 similar to their numerical contribution. Heterotrophic and mixotrophic dinoflagellates averaged 64% and 530 ciliates 35% of microzooplankton biomass. Non-thecate heterotrophic dinoflagellates contributed almost 531 all of the biomass in the dinoflagellate fraction. Among the ciliates, non-loricate choreotrich ciliates 532 contributed 88% of the ciliate biomass and were dominated by mixotrophic species, contributing ~65% of 533 the ciliate biomass. Other protistan taxa contributed < 5% of the microzooplankton biomass at all stations.

534

535 3.3 Microzooplankton in the Deep Chlorophyll Maxima (DCM)

536 On the middle shelf, there was often a pronounced deep chlorophyll maximum layer (DCM). In 537 the South Middle Domain a DCM was observed at 41% of the stations and occurred at average depth of 538 24-26 m; in the North Middle Domain, a pronounced DCM was observed at 70% of the stations sampled 539 and occurred at average depths of 30-34 m. The DCMs were usually located at or below the base of the 540 thermocline and were rich in chain-forming diatoms. Although phytoplankton were elevated in

541 abundance in the DCM, microzooplankton were not. Estimated biomass of microzooplankton in the south 542 or north middle domain DCMs was not different from average biomass in the mixed layer for the domain 543 and year (Figure 7). As a result, the ratio of microzooplankton to phytoplankton biomass in the DCM was 544 lower than the ratio in the corresponding mixed layer. In the South Middle Domain, the microzooplankton 545 biomass ranged from an average of 33 to 103% in the mixed layer and 25 to 49% of phytoplankton 546 biomass in the DCM depending on year (Figure 7a). In the North Middle Domain, the microzooplankton 547 biomass ranged from an average of 31 to 281% in the mixed layer and 5 to 22% in the DCM of 548 phytoplankton biomass depending on year (Figure 7b).

549

550 3.4 Estimated contribution of ciliates to chlorophyll a.

551 Using the mean chlorophyll a to carbon conversion factor of 0.0164 pg chl (pg C)-1 derived for 552 ciliates, we estimated mixotrophic ciliates (strombidiid species) chlorophyll in mixed layer samples. The 553 mean estimated ciliate chlorophyll a l-1 was 0.09 (SD, 0.094), the median 0.07, and the range 0.00 to 0.49 554 (Fig. 8). Estimated ciliate chlorophyll was not correlated with total chlorophyll (Pearson Product 555 Moment Correlation, N=135; p>0.05). Estimated ciliate contribution to total chlorophyll was highly 556 variable across years and domains (Figure 8). In the North Middle Domain, the contribution was 557 sometimes over 50%, with the estimated contribution greater in 2008 and 2010 than in 2009 when it was 558 ~11%. In 2010, the contribution of ciliates to chlorophyll in the North Middle Domain was significantly 559 higher than in the South Middle Domain or South Outer Domain, all other comparisons were not 560 statistically significant (p>0.05).

561

562 4. Discussion

563

564 4.1. Confounding factors

565 Several factors confound the comparison of microzooplankton among seasons and years (Table 566 3). Timing of cruises is undoubtedly important. The Olson and Strom (2002) and Strom and Fredrickson 567 (2008) data were from cruises in late July through August, about a month later than our cruises. Domains 568 in the eastern Bering Sea differ in their physics and biology as well as the timing of events (Hunt et al., 569 2008; Stabeno et al., 2012a). The mix of stations sampled varied among studies and years, with some 570 domains being better represented in some studies and years than others. For example, in 1999 and 2004,

571 sampling was focused around the Pribilof Domain and the southern shelf (Olson and Strom, 2002; Strom 572 and Fredrickson, 2008) whereas our investigation included the northern domains (Figure 1). Even 573 within our investigation, the cruise track and distribution of stations among domains varied from year to 574 year (Table 1). The spring cruises focused on the marginal ice zone, with the distribution of stations 575 depending on year-to-year variation in ice conditions (Sherr et al. accepted). In addition, although the 576 methods were quite similar in all the studies (Table 3), there were differences in sampling schemes, 577 fixation and counting procedures that may have influenced abundance and biomass estimates.

578 All three of our summer cruises were within a four year “cold” period with extensive sea ice in 579 winter and spring (Stabeno et al., 2012b). However, there were year to year differences in the summer 580 distribution and biomass of microzooplankton among our cruises. In 2009, the average ratio of 581 microzooplankton to phytoplankton biomass in the mixed layer was low, particularly in the North Middle 582 Domain. The contribution of mixotrophic ciliates to biomass was also relatively low in 2009. The 583 average temperature for our mixed layer samples in the North Middle Domain was colder (3.7+ 0.76oC) 584 in summer 2009 than in summer 2008 or (6.3+ 0.51 oC) or 2010 (5.9+ 0.22 oC). Although 2009 is 585 classified as an average year in terms of the August stratification index, stratification set-up was unusually 586 late (4 June) and breakdown early at M2 (station on south middle shelf) compared to most other years 587 (Ladd and Stabeno, 2012). It appears that the typically summer microzooplankton assemblage, dominated 588 by mixotrophic ciliates and heterotrophic dinoflagellates adapted to low chlorophyll, stratified waters, 589 was not well developed by the time of our cruise in 2009.

590 Particularly in 2009 we noted small blooms of gelatinous colonial phytoplankton, Phaeocystis 591 pouchettii and Chaetoceros socialis, on parts of the middle and outer shelf. These colonial phytoplankton 592 are often poor food for microzooplankton (Nejstgaard et al., 2007). The presence of these bloom species 593 may partially explain the relatively low ratio of microzooplankton to phytoplankton biomass in summer 594 2009. Strom and Fredrickson (2008) also noted that few large ciliates and dinoflagellates were present at 595 high chlorophyll “green belt” stations. Localized mixing events and storms can lead to episodic blooms 596 and relative low ratios of microzooplankton grazing to phytoplankton growth on the shelf (Stoecker et al., 597 this issue).

598

599 4.2. Comparison between spring and summer

600 During spring sea ice conditions, microzooplankton biomass was more variable than during 601 summer, particularly at bloom stations, however, average microzooplankton biomass during spring and

602 summer were similar (Table 3). The ratio of microzooplankton to phytoplankton is higher in summer 603 than in spring (Figure 6; Sherr et al., accepted). In spring, microzooplankton biomass was positively 604 related to chlorophyll a (Sherr et al., accepted), but in summer we found no such relationship. In early 605 spring, microzooplankton do not increase in abundance until phytoplankton populations are high enough 606 to support their growth and thus there is temporary “uncoupling” of microzooplankton growth from 607 phytoplankton growth at the onset of blooms (Sherr and Sherr, 2009; Sherr et al., accepted). Although 608 integrated rates of Net Primary Production (NPP) are lower in summer than in spring, estimated growth 609 rates of phytoplankton are about twice as high in summer (Lomas et al., 2012); phytoplankton turn-over 610 rapidly in summer and thus can support relatively high populations of microzooplankton for the observed 611 chlorophyll a standing stock. Another factor that leads to persistence of high biomass of 612 microzooplankton relative to phytoplankton after blooms is acquired phototrophy in mixotrophic ciliates 613 (Dolan and Perez, 2000; Stoecker et al., 2009).

614

615 4.3 Comparisons among Domains

616 Summer microzooplankton abundance and biomass were surprisingly high in the low chlorophyll 617 water found in the mixed layer over the middle shelf, with average estimates of microzooplankton 618 biomass greater than in higher chlorophyll waters in the Pribilof Domain and at frontal and shelf break 619 stations. In the North Middle Domain there was usually a well-developed DCM, but the biomass of 620 microzooplankton was not usually elevated in the DCM relative to the mixed layer.

621 Both top down and bottom up factors may be responsible for the high biomass of 622 microzooplankton in these relatively low chlorophyll waters. Top down control of microzooplankton may 623 be lower on the middle shelf compared to the more productive Pribilof Domain and “Green Belt” areas 624 that have higher mesozooplankton populations (Coyle et al., 2008; Hunt et al., 2008). In the Pribilof 625 Domain and in the “Green Belt”, large oceanic crustacean zooplankton are advected onto the shelf along 626 with nutrient rich slope waters (Hunt et al., 2008). In polar waters, copepod grazing can have a strong top- 627 down impact on microzooplankton populations, particularly large cell-size ciliates (Levinsen and Nielsen, 628 2002; Campbell et al., 2009).

629 Secondly, although phytoplankton biomass was higher at “bloom” stations in the Pribilof Domain 630 and in the “Green Belt” and frontal areas, the phytoplankton may not have been as suitable a food for 631 microzooplankton as the mixed populations of phytoflagellates and small diatoms on the middle shelf. 632 Particularly in 2009, blooms of Phaeocystis pouchettii were present at the high chlorophyll stations,

633 including some Shelf Break, Pribiliof Island, South Outer, and North Middle domain stations. The food 634 value of P. pouchettii, particularly the colonies, is a subject of debate (reviewed in Nejstgaard et al., 635 2007). Although P. pouchettii is consumed by some microzooplankton, it may, in general, be a poor food 636 for microzooplankton (Calbet et al., 2011 and references cited therein). Most microzooplankton may not 637 be able to consume large colonies (Nejstgaard et al., 2007; Calbet et. al., 2011), which dominated at some 638 “bloom” stations. At other “bloom” stations, we observed Chaetoceros socialis, a colonial gelatinous 639 diatom that also may not be readily ingested by most microzooplankton.

640 Likewise, although phytoplankton were more abundant in DCMs than in the mixed layer above, 641 microzooplankton usually were not more abundant in the DCM. Mesozooplankton grazers may 642 congregate in the DCM and/or the Phaeocystis pouchettii and Chaetoceros socialis colonies that form 643 many DCMs may be poor foods for microzooplankton. Some DCMs contained “old” phytoplankton, 644 including sea ice diatoms left-over from the spring, which may be poor quality foods for micro-grazers.

645

646 4.4 Comparison between warm and cold years

647 The average biomass and composition of microzooplankton we found were within the ranges 648 reported previously for microzooplankton in surface waters of the eastern Bering Sea (Table 3). The 649 average biomass of microzooplankton we observed appears to be somewhat lower than observed by 650 Strom and Fredrickson (2008) during a warm year with a high stratification index (2004), however, 651 differences in sampling locations confound this broad comparison by cruise averages. A higher 652 proportion of their stations were in slope waters, the “green belt” or Pribilof Domain than in our study in 653 which more sampling was on the middle shelf. If comparisons are by domain, the interpretation is 654 different. In summer 2004, at the middle south domain station (M2), microzooplankton biomass was 655 consistently <15 µg C l-1 (Strom and Fredrickson 2008); this value is lower than the 21-25 µg C l-1 that 656 we observed in the middle and inner domains during 2008-2010 (Table 3). In the summer of 1999, a cold 657 year with low stratification, microzooplankton biomass estimates were perhaps slightly higher than for 658 summer 2004, however the biomass ranges overlap (Table 3). There is no good evidence that 659 microzooplankton biomass is higher in summers of “warm” than “cold” years. Although for the one 660 warm year for which there are data on microzooplankton, summer stratification was also high (Table 3), 661 over-all the strength of summer stratification is not correlated with “warm” or “cold” years as defined by 662 depth averaged water temperature (Ladd and Stabeno, 2012). Domain, stratification, intensity, and timing 663 of cruises in relationship to mixing events may have a greater effect on observed microzooplankton 664 biomass and coupling to phytoplankton than temperature.

665

666 4.5 Contributions of mixotrophic ciliates to chlorophyll a

667 One of the surprising results of our cruises is the high abundance and biomass of mixotrophic 668 strombidiid ciliates (Laboea strobilia, Strombidium spp.) and their estimated contribution to total 669 chlorophyll. The primarily autotrophic ciliate, Mesodinium rubrum (Myrionecta rubra) was also present, 670 but not abundant. On average, mixotrophic ciliates were 62% of ciliate abundance and 66% of estimated 671 ciliate biomass in the mixed layer. Sorokin et al. (1996) estimated that in late spring-early summer, 30- 672 40% of the ciliates on the shelf in the western Bering Sea were mixotrophs; Booth et al. (1993) estimated 673 that in the western sub-Arctic, 30-50% of the ciliates were mixotrophs. In the subarctic Atlantic (Iceland, 674 Greenland and Barents Seas), Putt (1990) observed that 58-65% of the ciliates in surface waters were 675 mixotrophs. In summer, in stratified sub-arctic seas, mixotroph ciliates are generally very abundant and 676 important components of the microzooplankton.

677 Calculation of estimated contribution of ciliates to chlorophyll is very sensitive to the factors used 678 to estimate chlorophyll per unit biovolume or biomass. In the North Middle Domain, where chlorophyll 679 levels were very low in the mixed layer in 2008 and 2010, average estimated contribution of mixotrophic 680 ciliates to total chlorophyll was over 50% using the mean value of 3.12 fg Chl a µm3 calculated for 681 mixotrophic ciliates (refer to methods). Putt (1990), based on determination of the chlorophyll content of 682 ciliates collected at sea, estimated that ciliates accounted for 4-15% of total chlorophyll a in summer in 683 surface waters of the Nordic Seas. This is similar to our estimate of 5-14% for the south shelf domains, 684 but lower than our estimate of 22-35% for the more northern shelf (MN transect and North Middle 685 Domain).

686 About 64% of the chlorophyll in the Bering Sea in summer is due to cells < 5 µm in size (Lomas 687 et al., 2012). Assuming 36% of the chlorophyll in the mixed layer in all domains is > 5 µm and using the 688 mean value for chlorophyll content of mixotrophic ciliates, the average contribution of ciliates to 689 chlorophyll in the > 5 µm size class should range from 15% to 96%. It is clear that the contribution of 690 ciliates to chlorophyll (and probably photosynthesis), particularly in the size fraction that is readily 691 accessible to crustacean zooplankton, is considerable, particularly in the northern domains. This also 692 implies that bulk chlorophyll measurements overestimate the biomass of phytoplankton and that the ratios 693 of microzooplankton biomass to chlorophyll a or phytoplankton biomass are underestimates.

694

695 4.6. Importance of microzooplankton to crustacean zooplankton

696 Crustacean zooplankton, particularly large crustacean zooplankton, are important in food webs 697 that support higher trophic levels in the southeastern Bering Sea (Coyle et al., 2008; Hunt et al., 2008; 698 Stabeno et al., 2012b). Crustacean zooplankton consume both phytoplankton and microzooplankton, but 699 often have higher clearance rates for microzooplankton than phytoplankton, particularly when 700 phytoplankton are not abundant and/or are small in size (Gifford and Dagg, 1991; Calbet and Saiz, 2005; 701 Campbell et al., 2009). After the spring bloom, when phytoplankton are scarce, predation on 702 microzooplankton can maintain fecundity of copepods (Ohman and Runge, 1994). Large, mixotrophic 703 ciliates are particularly important as prey for copepods (Dutz and Peters, 2008) and young fish larvae 704 (Figuieredo et al., 2007). Under these conditions, photosynthesis in ciliates may “boost” microbial food 705 web efficiency and hence carbon availability to higher trophic levels (Stoecker et al., 2009). This may be 706 important in stratified Bering Sea shelf waters where chlorophyll levels were relatively low (mean total 707 Chl a ~0.9 µg l-1) and composed of small cells (~64% of chl a < 5 µm) in summer (Lomas et al., 2012). 708 Most crustacean zooplankton do not effectively graze on cells < 5 µm in size. Almost all cells larger than 709 10 µm in size in the mixed layer on the inner and middle shelf (with the exception of the Pribilof Domain) 710 were microzooplankton (personal obs). Estimated microzooplankton biomass in the mixed layer was 98% 711 (SD 166%) of estimated phytoplankton biomass with a median value of 55% (Maximum 155%, 712 Minimum 2%, N=132). In low chlorophyll surface waters, such as on the middle shelf in summer, about 713 50% of the diet of copepods is ciliates (Calbet and Saiz, 2005). Although microzooplankton probably are 714 very important in the diet of crustacean zooplankton on Bering Sea Shelf in summer, data on predation 715 rates on microzooplankton for this season are lacking.

716

717 4.7. Climate variability and the microzooplankton trophic link

718 Climate, weather and ocean acidification all have the potential to influence the future ecology of 719 the eastern Bering Sea (Mathis et al 2010; Overland et al., 2012; Stabeno et al., 2012b; Wang et al 2012). 720 It is not clear if the pattern of the last few years of decreased annual variability in temperature will 721 continue or if the Eastern Bering Sea will return to strong inter-annual variability (Overland et al. 2012). 722 During the “warm” years of 1998 and 2001-2005, surface water temperatures on the southern middle 723 shelf rose above 10oC during the summer, although during “average” and “cold” years surface waters 724 temperatures remain below 10oC (Stabeno et al., 2012b). The timing and strength of stratification controls 725 the availability of nutrients for phytoplankton growth and hence has a strong influence on new 726 production, phytoplankton species composition, and perhaps the suitability of phytoplankton as food for 727 microzooplankton (Sambrotto et al., 1986, 2008; Strom and Fredrickson, 2008; Mathis et al., 2010).

728 During July and August 2004, intense stratification in the southeastern Bering Sea led to phytoplankton 729 nutrient limitation and reduced microzooplankton grazing (Strom and Fredrickson, 2008). Contrary to 730 expectations, stratification index does not correlate with “warm” and “cold” years in the eastern Bering 731 Sea (Ladd and Stabeno, 2012). In the eastern Bering Sea, weather and tides are important in controlling 732 stratification. Advection of water onto the shelf and wind and tidal mixing make nutrients available to 733 phytoplankton and can lead to localized blooms even during summer (Sambrotto et al. 1986, 2008).

734 However, in the Bering Sea Shelf, where surface water temperatures during “cold” and “normal” 735 years are below 10oC even in summer, there might be a direct, positive effect of increased temperature on 736 coupling between microzooplankton and phytoplankton growth. Based on a compendium of laboratory 737 studies of cultured protists, Rose and Caron (2007) found that the maximum growth rate of herbivorous 738 microzooplankton was lower than the maximum growth rates of phytoplankton below 10oC. If this 739 relationship holds in natural communities, then the coupling of microzooplankton to phytoplankton would 740 be expected to increase with increases in water temperature. However, modeling of the general responses 741 of microzooplankton herbivory to warming suggest that it may increase the ratio of microzoozooplankton 742 grazing to phytoplankton growth under eutrophic but not oligotrophic conditions (Chen et al., 2012). This 743 indicates that stratification and mixing, by largely controlling nutrient availability, may control the 744 coupling of microzooplankton and phytoplankton. Our knowledge of how climate variability will affect 745 key phytoplankton groups, and their interactions with microzooplankton, is very limited, but it is clear 746 that interactive effects of temperature, stratification/mixing and nutrients are important (Olson and Strom, 747 2002; Rose et al 2009; Boyd et al., 2010).

748 Ocean acidification is another climate change phenomenon that has the potential to affect 749 phytoplankton and hence the microzooplankton food web link in the Bering Sea. Ocean acidification 750 induced changes in calcium carbonate mineral saturation states are most evident in high latitude seas, 751 such as the Bering Sea (Mathis et al., 2010). Most of the microzooplankton in the Bering Sea are not 752 calcifying organisms, and thus unlikely to be directly influenced by ocean acidification. However, 753 indirect effects due to changes in net primary production, phytoplankton species composition or 754 biochemical composition of phytoplankton are possible (Suffrian et al., 2008; Hopkinson et al., 2010; 755 Yoshimura et al., 2010). Hare et al. (2007) addressed synergistic effects of “greenhouse ocean”

756 conditions (i.e., increased temperature and pCO2 in combination) on plankton communities in 757 manipulative, shipboard experiments conducted on the Bering Sea shelf. They found that simulated 758 “greenhouse ocean” conditions decreased phytoplankton biomass but increased biomass-specific 759 photosynthetic rates and cause a shift from diatoms towards nanophytoplankton (Hare et al. 2007), 760 changes which may favor microzooplankton grazers. Somewhat similar experiments conducted in

761 temperate waters during the North Atlantic spring bloom suggest a transient positive effect of increased

762 temperature and pCO2 on microzooplankton abundance and grazing, but after the initial stimulation, 763 microzooplankton abundance decreased (Rose et al., 2009). The changes in microzooplankton 764 assemblages were attributed to changes in phytoplankton composition rather than the direct effects of

765 temperature or pCO2 on microzooplankton, although no direct measurements on microzooplankton were 766 made (Rose et al., 2009).

767

768 5. Conclusions

769

770

771 a. Microzooplankton biomass equaled or exceeded summer phytoplankton biomass in the mixed 772 layer in some domains. Microzooplankton were relatively more important in stratified shelf 773 waters than in the “green belt”, Pribilof Island Domain or at the Shelf Break. 774 775 b. Mixotrophic strombidiid ciliates were the dominant component of the ciliate microzooplankton. 776 They made important contributions to both microzooplankton biomass and chlorophyll, 777 especially in the larger size classes. 778 779 c. Based on our data and previous investigations, the range of summer microzooplankton biomass 780 was similar in “warm” and “cold” years, with differences among years obscured by differences 781 among domains, derived in part by differences in mixing, nutrient supply and phytoplankton 782 composition. 783

784 Acknowledgements

785 We thank the officers and crew of the USCG Icebreaker Healy, R/V Knorr and R/V T.G. 786 Thompson for their support and John Casey of BIOS, Dave Kachel of PMEL, Nancy Kachel of 787 UW/JISAO, Jessica Cross of UAF for CTD sampling, advice and good cheer. We thank Kristin Blattner 788 for excellent technical assistance during the first 2 years of the project. We thank Evelyn Sherr for helpful 789 comments on an early draft of the manuscript. This research was supported primarily by NPRB project 790 award B55 (DKS and ACW). This is BSIERP contribution number…..and UMCES contribution 791 number……

792

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831 Fileman, E.S., Petropavlovsky, A., Harris, R.P., 2010. Grazing by the copepods Calanus helgolandicus 832 and Acartia clause on the protozooplankton community at station L4 in the Western English Channel. J. 833 Plankton Res. 32,709–724.

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835 Fukami, K., Watanabe, A., Fujita, S., Yamaoka, K., Nishijima,T., 1999. Predation on naked protozoan 836 microzooplankton by fish larvae. Mar. Ecol. Prog. Ser. 185, 285–291.

837

838 Gifford, D.J., Caron, D.A., 2000. Chapter 5. Sampling, preservation, enumeration and biomass of marine 839 protozooplankton, in: Harris, R., Wiebe, P., Lenz, J., Skjoldal, H.R., Huntley, M. (Eds.) ICES 840 Zooplankton Methodology Manual. Academic Press, San Diego, pp 193-221.

841

842 Gifford, D.J., Dagg, M.J., 1991. The microzooplankton-mesozooplankton link: consumption of 843 planktonic protozoa by the calanoid copepods Acartia tonsa Dana and Neocalanus plumchrus Murukawa. 844 Mar. Microbial Food Webs 5, 161-177.

845

846 Hare, C.E., LeBlanc, K., DiTullio, G.R., Kudela, R.M., Zhang, Y., Lee , P.A., Riseman, S., Hutchins,

847 D.A., 2007. Consequences of increased temperature and CO2 for phytoplankton community structure in 848 the Bering Sea. Mar. Ecol. Prog. Ser. 352, 9-16.

849

850 Hopkinson, B.M., Xu, Y., Shi, D.L., McGinn, P.J., Morel, F.M.M., 2010. The effect of CO2 on the 851 photosynthetic physiology of phytoplankton in the Gulf of Alaska. Limnol. Oceanogr. 55, 2011-2024.

852

853 Howell-Kübler, A.N., Lessard, E.J., Napp, J.M., 1996. Springtime microprotozoan abundance and 854 biomass in the southeastern Bering Sea and Shelikof Strait, Alaska. J. Plankton Res. 18, 731-745.

855

856 Hunt Jr., G.L., Coyle K.O., Eisner, L.B., Farley, E.V., Heintz, R.A., Mueter, F., Napp, J.M., Overland, 857 J.E., Ressler, P.H., Salo, S., Stabeno, P.J., 2011. Climate impacts on eastern Bering Sea food webs: a 858 synthesis of new data and an assessment of the Oscillating Control Hypothesis. ICES J. Mar. Sci. 68, 859 1230-1243.

860

861 Hunt Jr., G.L., Stabeno, P., Strom, S., Napp, J.M., 2008. Patterns of spatial and temporal variation in the 862 marine ecosystem of the southeastern Bering sea, with special reference to the Pribilof Domain. DeepSea 863 Res. II 55, 1919-1944.

864

865 Hunt Jr., G.L., Stabeno, P., Walters, G., Sinclair, E., Brodeur, R.D., Napp, J.M., Bond, N.A., 2002. 866 Climate change and control of the southeastern Bering Sea pelagic ecosystem. Deep Sea Res. II 49, 5821- 867 5853.

868

869 Jeong, H. J., Yoo, Y. D., Kim, J. S., Seong, K.A., Kand, N.S., Kim, T.H. , 2010. Growth, feeding and 870 ecological roles of the mixotrophic and heterotrophic dinoflagellates in marine planktonic food webs. 871 Ocean. Sci. J. 45, 65-91.

872

873 Kachel, N.B., Hunt, G.L., Salo, S.A., Schumacher, J.D., Stabeno, P.J., Whitledge, T.E., 2002. 874 Characteristics and variability of the inner front of the southeastern Bering Sea. Deep Sea Res. II 49, 875 5889-5909.

876

877 Ladd, C., Stabeno, P.J., 2012. Stratification on the Eastern Bering Sea shelf revisited. Deep Sea Res. II 878 65-70, 72-83.

879

880 Levinsen, H., Nielsen, T.G., 1999. Plankton community structure and carbon cycling on the western coast 881 of Greenland during the stratified summer situation. II. Heterotrophic dinoflagellates and ciliates. Aquat. 882 Microb. Ecol. 16, 217-232.

883

884 Levinsen, H., Nielsen, T.G., 2002. The trophic role of marine pelagic ciliates and heterotrophic 885 dinoflagellates in arctic and temperate coastal ecosystems: a cross-latitude comparison. Limnol. 886 Oceanogr. 47, 427-439.

887

888 Liu, H., Suzuki, K., Saino, T., 2002. Phytoplankton growth and microzooplankton grazing in the subarctic 889 Pacific Ocean and the Bering Sea during summer 1999. Deep Sea Res. I 49, 363-375.

890

891 Lomas, M.W., Moran, S.B., Casey, J.R., Bell, D.W., Tiahlo, M., Whitefield, J., Kelly, R.P., Mathis, J.T., 892 Cokelet, E.D., 2012. Spatial and seasonal variability of primary production on the Eastern Bering Sea 893 shelf. Deep-Sea Res. II 65-70: 126-140.

894

895 Mathis, J.T., Cross, J.N., Bates, N.R., Morman, S.B., Lomas, M.W., Mordy, C.W., Stabeno, P.J., 2010. 896 Seasonal distribution of dissolved inorganic carbon and net community production on the Bering Sea 897 shelf. Biogeosciences 7, 1769-1787.

898

899 Menden-Deurer, S., Lessard, E.J., 2000. Carbon to volume relationships for dinoflagellates, diatoms, and 900 other protist plankton. Limnol. Oceanogr. 45, 569-579.

901

902 Montagnes, D.J.S., Allen, J., Brown, L., Bulit, C., Davidson, R., Diaz-Avalos, C., Fielding, S., Heath, M., 903 Holliday, N.P., Rasmussen, J., Sanders, R., Waniek, J.J., Wilson, D., 2008. Factors controlling the 904 abundance and size distribution of the phototrophic ciliate Myrionecta rubra in open waters of the North 905 Atlantic. J. Eukaryot. Microbiol. 55,457-465.

906

907 Montagnes, D.J.S., Dower, J.F., Figueiredo, G.M., 2010. The protozooplankton–ichthyoplankton trophic 908 link: an overlooked aspect of aquatic food webs. J. Eukaryot. Microbiol. 57, 223–228.

909

910 Moran, S.B., Lomas, M.W., Kelly, R.P., Gradinger, R., Iken, K., Mathis, J.T., 2012. Seasonal succession 911 of net primary productivity, particulate organic carbon export and autotrophic community composition in 912 the eastern Bering Sea. Deep Sea Res. II 65-70, 84-97.

913

914 Mordy, C.W., Cokelet, E.D., Ladd, C., Menzia, F.A., Proctor, P., Stabeno, P.J., Wisegarver, E., 2012. Net 915 community production on the middle shelf of the eastern Bering Sea. Deep Sea Res. II 65-70, 110-125.

916

917 Nejstgaard, J., Tang, K., Steinke, M., Dutz J., Koski M., Antajan, E., Long, J.D., 2007. Zooplankton 918 grazing on Phaeocystis: a quantitative review and future challenges. Biogeochemistry 83, 147-172.

919

920 Ohman, M.D., Runge, J.A., 1994. Sustained fecundity when phytoplankton resources are in short supply: 921 Omnivory by Calanus finmarchicus in the Gulf of St. Lawrence. Limnol. Oceanogr. 39, 21-36.

922

923 Olson, M.B., Strom, S.L., 2002. Phytoplankton growth, microzooplankton herbivory and community 924 structure in the southeast Bering Sea: insight into the formation and temporal persistence of an Emiliania 925 huxleyi bloom. Deep-Sea Res II 49, 5969-5990.

926

927 Overland, J.E., Wang, M., Wood, K.R., Percival, D.B., Bond, N.A., 2012. Recent Bering Sea warm and 928 cold events in a 95-year context. Deep Sea Res. II 65-70, 6-13.

929

930 Putt, M., 1990. Abundance, chlorophyll content and photosynthetic rates of ciliates in the Nordic Seas 931 during summer. Deep-Sea Res. A 37, 1713-1731.

932

933 Putt, M., Stoecker, D.K., 1989. An experimentally determined carbon:volume ratio for marine 934 “oligotrichous” ciliates from estuarine and coastal waters. Limnol. Oceanogr. 34, 1097-1103.

935

936 Rose, J.M., Caron D.A., 2007. Does low temperature constrain the growth rates of heterotrophic protists? 937 Evidence and implications for algal blooms in cold waters. Limnol. Oceanogr. 52, 886-895.

938

939 Rose, J.M., Feng, Y., Gobler, C.J., Gutierrez, R., Hare, C.E., LeBlanc, K., Hutchins, D.A., 2009. Effects 940 of increased pCO2 and temperature on the North Atlantic spring bloom. II. Microzooplankton abundance 941 and grazing. Mar. Ecol. Prog. Ser. 388, 27-40.

942

943 Sambrotto, R.N., Mordy, C., Zeeman, S.I., Stabeno, P.J., Macklin, S.A., 2008. Physical forcing and 944 nutrient conditions associated with patterns of Chl a and phytoplankton productivity in the southeastern 945 Bering Sea during summer. Deep-Sea Res. II 55, 1745-1760.

946

947 Sambrotto, R.N., Niebauer, H.J., Goering, J.J., Iverson, R.L., 1986. Relationships among vertical mixing, 948 nitrate uptake, and phytoplankton growth during the spring bloom in the southeast Bering Sea middle 949 shelf. Continental Shelf Res. 5, 161-198.

950

951 Sherr E.B., Sherr B.F, 2009. Capacity of herbivorous protists to control initiation and development of 952 mass phytoplankton blooms. Aquat. Microb. Ecol. 57, 253-262.

953

954 Sherr E.B., Sherr B.F., Ross, C., accepted. Microzooplankton grazing impact in the Bering Sea during 955 spring sea ice conditions. Deep Sea Research II……………

956

957 Simé-Ngando, T., Juniper, K., Vézina, A., 1992. Ciliated protozoan communities over Cobb Seamount: 958 increase in biomass and spatial patchiness. Mar. Ecol. Prog. Ser. 89, 37-51.

959

960 Sorokin, Y.I., Sorokin, P.Y., Mamaeva, T.I., 1996. Density and distribution of bacterioplankton and 961 planktonic ciliates in the Bering Sea and North Pacific. J. Plankton Res. 18, 1-16.

962

963 Stabeno, P.J., Farley Jr., E.V., Kachel, N.B., Moore, S., Mordy, C.W., Napp, J.M., Overland, J.E., 964 Pinchuk, A.I., Sigler, M.F., 2012a. A comparison of the physics of the northern and southern shelves of 965 the eastern Bering Sea and some implications for the ecosystem. Deep Sea Res. II 65-70, 14-30.

966

967 Stabeno, P.J., Kachel, N.B., Moore, S.E., Napp, J.M., Sigler, M., Yamaguchi, A., Zerbini, A.N., 2012b. 968 Comparison of warm and cold years on the southeastern Bering Sea shelf and some implications for the 969 ecosystem. Deep Sea Res. II 65-70, 31-45.

970

971 Stoecker, D.K., 2012. Chapter 5, Predators of tintinnids, in: Dolan, J.R., Montagnes, D.J.S., Agatha, S., 972 Coats, D.W., Stoecker, D.K. (Eds), The Biology and Ecology of Tintinnid Ciliates: Models for Marine 973 Plankton. John Wiley & Sons, Ltd., Oxford, pp. 123-144.

974

975 Stoecker, D.K., Gifford, D.J., Putt, M., 1994. Preservation of marine planktonic ciliates: losses and cell 976 shrinkage during fixation. Mar. Ecol. Prog. Ser. 110, 293-299.

977

978 Stoecker, D.K., Johnson, M.D., de Vargas, C., Not, F., 2009. Acquired phototrophy in aquatic protists. 979 Aquat. Microb. Ecol. 57, 279-310.

980

981 Stoecker, D.K., Weigel, A., Goes, J.I., This issue. Microzooplankton grazing in the eastern Bering Sea in 982 summer. Deep-Sea Res II……..

983

984 Strom, S.L., Fredrickson, K.A., 2008. Intense stratification leads to phytoplankton nutrient limitation and 985 reduced microzooplankton grazing in the southeastern Bering Sea. Deep Sea Res. II 55, 1761-1774.

986

987 Suffrian, K., Simonelli, P., Nejstgaard, J.C., Putzeys, S., Carotenuto, Y., Antia, A.N., 2008.

988 Microzooplankton grazing and phytoplankton growth in marine mesocosms with increased CO2 levels. 989 Biogeosciences 5, 1145-1156.

990

991 Suzuki, T., Taniguchi, A., 1998. Standing crops and vertical distribution of four groups of marine 992 planktonic ciliates in relation to phytoplankton chlorophyll a. Mar. Biol. 132, 375-382.

993

994 Turner, J.T., 1984. The feeding ecology of some zooplankters that are important prey items of larval fish. 995 NOAA Technical Report NMFS 7, U.S. Department of Commerce, National Oceanic and Atmospheric 996 Administration, National Marine Fisheries Service.

997

998 Yoshimura, T., Nishioka, J., Suzuki, K., Hattori, H., Kiyosawa, H., Watanabe, Y.W., 2010. Impacts of

999 elevated CO2 on organic carbon dynamics in nutrient depleted Okhotsk Sea surface waters. J. Exp. Mar. 1000 Biol. Ecol. 395, 191-198.

1001

1002 Wang, M., Overland, J.E., Stabeno, P., 2012. Future climate of the Bering and Chukchi Seas projected by 1003 global climate models. Deep-Sea Res. II 65-70, 46-57.

1004

1005

1006

1007

1008 Table 1. Number of microzooplankton samples by domain, cruise and layer (ML=mixed layer; 1009 DCM=chlorophyll max) on summer BEST/BSIERP cruises in SE Bering Sea in 2008 (HLY-08-03, July 3 1010 to July 31), 2009 (KNORR 195-10, June 14 to July 13) and 2010 (TN-250, June 16 to July 14). See 1011 Figure 1 for station locations; not all stations sampled on all cruises.

1012

Domain Layer 2008 2009 2010

South Outer ML 6 8 6

South Middle ML 10 13 15

DCM 7 8 11

South Inner ML 3 2 3

MN Transect ML 6 13 9

DCM

North Middle ML 8 15 9

DCM 8 14 8

Pribilof Islands ML 4 2 3

Shelf Break ML 4 3 3

1013

1014

1015 Table 2. Summer mixed layer microzooplankton abundance in the eastern Bering Sea (cells X 103 l-1).

1016

2008 2009 2010

Domain Mean SD Mean SD Mean SD

South Outer 5.23 2.956 13.21 7.986 24.85 8.525

South 12.57 10.459 12.33 3.706 16.506 6.280 Middle

South Inner 6.82 3.794 7.68 2.574 24.58 16.564

North 3.96 2.750 15.58 5.317 24.40 8.852 Middle

MN 4.10 2.676 11.35 5.820 19.05 8.066 Transect

Pribilofs 17.15 5.915 9.10 5.398 17.70 6.963

Shelf Break 6.17 3.772 8.44 3.26 12.08 6.17

1017 2-WAY ANOVA:

1018 Source of Variation df p

1019 Domain 6 0.230

1020 Year 2 <0.001

1021 Interaction 12 0.001

1022 Total 144

1023

1024 Table 3. Microzooplankton biomass and composition in “warm” and “cold” years1 and in years with 1025 “high” and “low”2 summer stratification index in the eastern Bering Sea.

1026

Year Season Biomass (µg C l-1) Composition of Ref microzooplankton

1992 Spring 1-10 (Slope Dinoflagellates 48- Howell-Kubler et waters) 54% of integrated al. (1996) biomass; remainder mostly ciliates

1999 (cold; Summer 57 (18-164) Dinoflagellates ~ Olson and Strom summer SI low2 ) (Emiliania huxleyi 50%, ciliates (2002) bloom year) ~50%

2004 (warm; high Summer 38 (11-118) ~70% biomass HD Strom and summer SI) (included Fredrickson (2008) <15 (M2, South dinoflagellates Middle domain); <20µm); remainder mostly Higher values in “green belt”, slope ciliates and Pribilof Domain

2008, 2009, 2010 Spring (sea ice Profiles: 11 +/- 17 Heterotrophic Sherr et al. 2012 (cold) conditions) (non-bloom dinoflagellates 65- conditions) 75% MZ biomass; remainder mostly 23+/-22 (bloom ciliates conditions)

2008, 2009, 2010 Summer 12-14 (outer Heterotrophic and This study (cold; average domain, shelf mixotrophic summer SI) break and dinoflagellates Pribilofs) 64% biomass ; remainder mostly 21-25 (middle and ciliates inner domains)

1027 1. “Warm” and “cold” years as defined by depth averaged water temperature (Stabeno et al. 2012a). 1028 2. SI=Stratification index at station M2 (middle south domain) in August. SI is compared to average 1029 values + 1 SD in August (Ladd & Stabeno 2012). 1030

1031

1032

1033

1034

1035

1036

1037

1038

1039

1040

1041

1042

1043

1044

1045

1046

1047 Figure Legends

1048

1049 Fig. 1. Location of transects and stations. The MN transect stations lie between the S and N domains. 1050 All stations were not sampled in all years (refer to Table 1)

1051

1052 Fig. 2. Abundance of ciliates (A) and heterotrophic/mixotrophic dinoflagellates (B) in the mixed layer 1053 samples by domain/transect (SO=South Outer, SM=South Middle, SI=South Inner, MN=MN transect, 1054 NM=North Middle, SB=Shelf Break, Pri=Pribilof Islands) and year. Ciliate abundances were greater in 1055 2010 than 2008 or 2009 (p<0.05) with the year X domain interaction significant. Abundances of 1056 dinoflagellates were greater in 2010 than 2008 and in 2009 than 2008 (p<0.05) with the year X domain 1057 interaction significant. Across years, domain was not a significant source of variation for abundance of 1058 ciliates (p=0.416) or dinoflagellates (p=0.081). Within a domain, different letters indicate significant 1059 differences among years (p<0.05). 2-Way ANOVA on square root transformed data; df=144).

1060

1061 Fig. 3. Abundance of mixotrophic ciliates in the mixed layer by domain/transect (SO=South Outer, 1062 SM=South Middle, SI=South Inner, MN=MN transect, NM=North Middle, SB=Shelf Break, Pri=Pribilof 1063 Islands) and year. Mixotrophic ciliates were more abundant in 2010 than 2008 and more abundant in 2009 1064 than 2008 with domain X year interaction significant (p<0.05). Across years, domain was not a 1065 significant source of variation for abundance of mixotrophic ciliates (p=0.326). Within a domain, 1066 different letters indicate significant differences among years (p<0.05). 2-Way ANOVA on log 10 1067 transformed data; df=144).

1068

1069 Fig. 4. Biomass of microzooplankton in the mixed layer samples by domain/transect (SO=South Outer, 1070 SM=South Middle, SI=South Inner, MN=MN transect, NM=North Middle, SB=Shelf Break, Pri=Pribilof 1071 Islands). 1-way ANOVAs tested the effect of year for each domain except SI and NM, for which the data 1072 did not meet assumptions for ANOVA. Within a domain, different letters indicate significant differences 1073 among years (p<0.05).

1074

1075 Fig. 5. Ratio of estimated microzooplankton biomass to phytoplankton biomass versus Chlorophyll a 1076 (Chl a) in mixed layer, eastern Bering Sea summers 2008, 2009 and 2010. Data are from all samples for 1077 which there were both microzooplankton data and estimates of chlorophyll a (N=132).

1078

1079 Fig. 6. Ratio of estimated microzooplankton biomass to phytoplankton biomass in the mixed layer 1080 samples by domain/transect (SO=South Outer, SM=South Middle, SI=South Inner, MN=MN transect, 1081 NM=North Middle, SB=Shelf Break, Pri=Pribilof Islands). The ratio of microzooplankton biomass to 1082 phytoplankton biomass was greater in 2010 than in 2008 or 2009. The ratio in the Middle domains and 1083 along the MN transect was greater than in the South Outer domain and the ratio in North Middle Domain 1084 was greater than at the Shelf Break (2-Way ANOVA). Within a domain, different letters indicate 1085 significant differences among years (p<0.05).

1086

1087 Fig.7. Comparison of estimated biomass of microzooplankton and phytoplankton in the mixed layer and 1088 in the deep chlorophyll maximum (DCM) in the: A. South Middle Domain, and B. North Middle 1089 Domain. Note difference in y-axes between A and B. There were no significant differences in 1090 microzooplankton biomass between the mixed layer and the DCM in the south or north middle domains 1091 (2-Way ANOVAs of year and layer; p>0.05). Mean +/- Standard deviation.

1092

1093 Fig. 8. Estimated contribution of mixotrophic ciliates to chlorophyll a in the mixed layer samples by 1094 domain/transect (SO=South Outer, SM=South Middle, SI=South Inner, MN=MN transect, NM=North 1095 Middle, SB=Shelf Break, Pri=Pribilof Islands). Ciliate chlorophyll was estimated using an average of 1096 3.12 fg chl a/um3 for mixotrophic strombidiids. Mean +/- Standard deviation.

1097 In 2010, estimated contribution of mixotrophic ciliates to chlorophyll a was higher in North Middle 1098 domain than in South Middle Domain or South Outer Domain (p<0.05). For the North Middle Domain, 1099 the percent contribution of ciliates to chlorophyll a was higher in 2008 and 2010 than in 2009 (p<0.05).

1100

1101

1102

1103 Fig. 1.

1104

1105

1106

1107

1108

1109

1110

1111

1112

1113

1114

1115

1116

1117 Fig. 2A

30x103

2008 25x103 2009 2010 b ) b -1 20x103

b a 15x103 a a b b a

10x103 a a a Abundance (Ciliates l Abundance a a a a a 5x103 a a a a 0 SO SM SI MN NM SB Pri

1118 1119

1120

1121

1122 Fig. 2B

30x103 2008 2009 25x103 2010 )

-1 a c

20x103 a a c a

3 15x10 a bc a bc b a 10x103 a a

a a Abundance (Dinoflagellates l Abundance a 3 a 5x10 a a a

0 SO SM SI MN NM SB Pri

1123 1124

1125

1126

1127

1128

1129

1130 Fig. 3

30x103

2008 2009 ) 25x103

-1 2010

20x103

ac 15x103 b a b a b 10x103 a

b a a a 3 a Abundance (Mixotrophic Ciliates l Abundance 5x10 a a a a a a a a ab 0 SO SM SI MN NM SB Pri

1131

1132

1133

1134 Fig. 4

2008 60 2009 2010 )

-1 50 a

g C l g a µ 40 a a a

30 b a a b ab a b 20 a a MZ Biomass ( Biomass MZ

a 10

0 SO SM SI MN NM SB Pri

1135

1136

1137

1138

1139

1140

1141

1142

1143

1144

1145 Fig. 5

Microzooplankton Biomass/Phytoplankton Biomass Biomass/Phytoplankton Microzooplankton 0 0 1 2 3 4 5

-1 1146 Chlorophyll a (µg l )

1147

1148

1149

1150

1151

1152

1153

1154

1155

1156

1157

1158 Fig. 6

5 2008 2009 2010 4

3 a a a b 2 a a a a 1 aba a b a a a a a a a a Microzooplankton Biomass/Phytoplankton Biomass 0 SO SM SI NM MN Pri SB

1159

1160

1161

1162

1163

1164

1165

160 A 140 Phytoplankton Microzooplankton

120 ) -1 100 g C l µ 80

60 Biomass ( 40

0 2008 2009 2010 2008 2009 2010

Mixed Layer Deep Chlorophyll Maximum

1166 Fig.7a

1167

1168

1169

1170

1171

1172

1173

1174

800 B

) 600 -1 Phytoplankton Microzooplankton g C l µ 400 Biomass ( 200

0 2008 2009 2010 2008 2009 2010

Mixed Layer Deep Chlorophyll Maximum

1175 Fig. 7b

1176

1177

1178

1179

1180

1181

1182

1183

1184 Fig. 8

2008 200 2009

α 2010

150

100

Estimated % Total Chl. Chl. Total % Estimated 50

SO SM SI MN NM Pri SB

1185

1186

1187

1188

1189

1190

1191

1192

1193

1194

1195

1196 Chapter 2. Microzooplankton Grazing in the Eastern Bering Sea in Summer

1197

1198 Diane K. Stoeckera,*, Alison Weigelb, Joaquim I. Goesc

1199

1200 aUniversity of Maryland Center for Environmental Science, Horn Point Laboratory

1201 P.O. Box 775

1202 Cambridge, Maryland 21613, USA

1203 [email protected]

1204 bUniversity of Maryland Center for Environmental Science, Horn Point Laboratory

1205 P.O. Box 775

1206 Cambridge, Maryland 21613, USA

1207 c10 Marine Biology, 1208 Department of Marine Biology and Paleoenvironment 1209 Lamont Doherty Earth Observatory at Columbia University, 1210 61 Route 92 1211 Palisades, New York, 10964, USA 1212 [email protected]

1213

1214 *Corresponding author. Tel: +1 410 2218407; fax +1 410221 8490.

1215

1216

1217 Submitted November 28, 2012 to Deep Sea Research Part II, Third Bering Sea Project special volume

1218

1219

1220 ABSTRACT

1221

1222 Dilution experiments to estimate microzooplankton grazing on phytoplankton were conducted during the

1223 summers of 2008, 2009, and 2010 in the eastern Bering Sea as part of the BEST-BSIERP integrated

1224 ecosystem project. All three summers followed cold springs in the Bering Sea. Average

1225 microzooplankton grazing coefficients were relatively similar among regions, ranging from 0.16 to 0.34

-1 1226 d in simulated in situ incubations with mixed layer water collected from the depth of the 55% Io isolume.

1227 In off shelf and outer shelf domains, microzooplankton consumed 67-78% of phytoplankton daily growth

1228 but in the middle and inner shelf domains, microzooplankton grazing exceeded phytoplankton daily

1229 growth. Regional estimates of microzooplankton ingestion of phytoplankton carbon ranged from 4.4 to

1230 11.0 µg C d-1, with highest ingestion in the Off Shelf, Outer Shelf, and Alaska Peninsula regions and,

1231 lower ingestion in the Middle Shelf and Inner Shelf regions. On the northern Middle Shelf, a deep

1232 chlorophyll maximum (DCM) occurred at most stations. Grazing coefficients in the DCM were similar in

1233 magnitude to coefficients in the corresponding mixed layer. However, because of the higher

1234 phytoplankton biomass in the DCM, estimated microzooplankton ingestion and secondary production l-1

1235 were higher in the DCM than in the mixed layer. Measurements of variable fluorescence in whole

1236 seawater and diluted treatments indicated that with some plankton assemblages, dilution has a negative

1237 effect on phytoplankton physiology and could compromise their growth rates. This could also result in an

1238 underestimation of microzooplankton grazing. Nevertheless, it is clear that microzooplankton grazing

1239 consumes most of the phytoplankton production in summer, and that microzooplankton must be an

1240 important link in food webs supporting larger zooplankton and in carbon flow in the eastern Bering Sea.

1241

1242 Keywords: Bering Sea, microzooplankton grazing, dilution method, deep chlorophyll maximum

1243

1244 1. Introduction

1245

1246 Microzooplankton (defined as the <200 µm fraction which includes nanozooplankton) are an

1247 important link between primary producers and higher trophic levels in sub-polar and polar waters as well

1248 as in temperate and tropical waters (Levinsen and Nielsen, 2002; Calbet and Saiz, 2005; Campbell et al.,

1249 2009; Sherr et al., submitted). They are important grazers on pico, nano and microplankton, including

1250 large diatoms (Sherr et al., 2009, submitted). Previous studies have shown microzooplankton (defined as

1251 the <200 µm fraction, which includes nanozooplankton, for dilution experiments) are the major

1252 consumers of primary production in summer in the eastern Bering Sea (Liu et al., 2002; Olson and Strom,

1253 2002; Strom and Fredrickson, 2008). In Arctic and sub-Arctic as well as temperate and tropical seas,

1254 microzooplankton are important prey for mesozooplankton, including both small and large crustacean

1255 zooplankton (Levinsen and Nielsen, 2002; Campbell et al., 2009) and hence are a significant component

1256 of the food web and carbon cycle.

1257 Although microzooplankton can graze on large as well as small phytoplankton, including chain

1258 forming dinoflagellates (Strom et al., 2007; Sherr et al., submitted) their grazing rates, particularly

1259 microzooplankton biomass specific rates, can be influenced by phytoplankton species composition,

1260 physiological state and cell size (Olson and Strom, 2002; Strom and Fredrickson, 2008). In the eastern

1261 Bering Sea, phytoplankton < 5 µm comprise ~70% of the chlorophyll a in summer and autotrophic

1262 biomass is dominated by phytoflagellates (Lomas et al., 2012; Moran et al., 2012). Sporadic blooms of

1263 chain forming diatoms (mostly Chaetoceros and Thalassiosira spp.) and blooms of the solitary or

1264 colonial phytoflagellate Phaeocystis pouchetti occur in response to tidal and storm mixing or intrusion of

1265 deeper water onto the shelf (Sukhanova et al., 1999; Sambrotto et al., 2008). In late spring and summer,

1266 prolonged blooms of diatoms and P. pouchetti are associated with the shelf break and shelf partition

1267 fronts (Flint et al., 2002). During the anomalously warm and stratified spring of 1997, a bloom of the

1268 coccolithophorid, Emiliana huxleyi, developed on the southeastern shelf and also during summers 1998-

1269 2000 the bloom was present (Stockwell et al., 2001; Merico et al., 2004). In summer, particularly on the

1270 northern shelf, a deep chlorophyll maximum (DCM) composed primarily of diatoms has often been

1271 encountered. It is not clear if the DCM is a resident low light population or physiologically inactive

1272 settled material (Moran et al., 2012; Stabeno et al., 2012a), and to what extent it is grazed by

1273 microzooplankton.

1274 Microzooplankton were sampled as part of the BEST-BSIERP ecosystem study in the eastern

1275 Bering Sea in summers of 2008, 2009, and 2010 (Stoecker et al., this issue), all part of a four year “cold”

1276 period characterized by extensive sea ice in spring (Stabeno et al., 2012b). Average summer

1277 microzooplankton (defined as 20-200 µm size range) densities ranged from 4 X 103 to 25 X 103 cells l-1 in

1278 the mixed layer in stratified shelf waters but were about half that concentration in less stratified waters

1279 near the shelf break. High ratios (>1) of microzooplankton biomass to phytoplankton biomass were

1280 observed when chlorophyll concentrations were below 1 µg l-1 in the mixed layer (Stoecker et al., this

1281 issue). In coastal (inner domain) and middle shelf (middle domain) waters, the average biomass of

1282 microzooplankton in the mixed layer was often equal to or higher than that of phytoplankton.

1283 Microzooplankton were also found in the deep chlorophyll maxima (DCM) on the shelf; densities of 20-

1284 200 µm microzooplankton in these high chlorophyll layers were usually similar to densities in the lower

1285 chlorophyll, mixed layer. Microzooplankton abundance and biomass data from summers 2008-2010,

1286 along with results from previous studies during both “warm” and “cold” years in the Eastern Bering Sea

1287 (Liu et al., 2002; Olson and Strom, 2002; Strom and Fredrickson, 2008), indicate that summer

1288 microzooplankton population differences among domains are far greater than differences due to year-to-

1289 year variations in sea ice extent and water temperature.

1290 We report on dilution grazing experiments conducted in conjunction with microzooplankton

1291 sampling on the BEST-BSIERP summer cruises in 2008, 2009, 2010. Prior to our study, data on

1292 microzooplankton grazing for the eastern Bering Sea were limited to the southern shelf and the productive

1293 waters around the Pribilof Islands (Olson and Strom, 2002; Hunt et al., 2008; Strom and Fredrickson,

1294 2008). Our goal was to conduct dilution grazing experiments across a spectrum of environments and to

1295 compare microzooplankton grazing among domains. We also wanted to determine if microzooplankton

1296 grazing and its impact on phytoplankton were correlated with the biomass of microzooplankton (Stoecker

1297 et al., this issue) and/or to dominance of certain phytoplankton taxa. Low microzooplankton grazing can

1298 occur during blooms of coccolithophorids (Olson and Strom, 2002) and Phaeocystis pouchetti (reviewed

1299 in Nejstgaard et al., 2007). We were also interested in determining if microzooplankton grazing was

1300 important in the DCM since this is a characteristic feature of the northern shelf in summer (Stabeno et al.,

1301 2012a).

1302

1303 2. Materials and Methods

1304

1305 2.1. Sampling

1306

1307 Microzooplankton grazing experiments were conducted on BEST/BSIERP summer cruises in

1308 2008, 2009 and 2010 on the USCG Healy (HLY-08-03, July 3 to July 31), R/V Knorr (KNORR 195-10,

1309 June 14 to July 13) and R/V T.G. Thompson 2010 (TN-250, June 16 to July 14). The stations at which

1310 dilution grazing experiments were conducted are shown in Figure 1; the stations and depths of

1311 experiments for each year are given in Table 1. In most cases the dilution grazing experiments were

1312 undertaken in conjunction with the phytoplankton biomass and primary productivity casts with water

1313 collected from the depth of the 55% of surface PAR irradiance level (Lomas et al., 2012). At all stations

1314 the 55% irradiance level was in the surface mixed layer, with the sampling depth for mixed layer

1315 incubations ranging from 3-10 m (Table 1). In 2010 we also undertook microzooplankton grazing

1316 experiments using assemblages from DCM which was located based on chlorophyll fluorescence profiles

1317 from the core CTD casts. Supporting information including water temperature, salinity, chlorophyll a,

1318 inorganic nutrients and irradiance were obtained from core program measurements or from the

1319 productivity casts (Lomas et al., 2012).

1320

1321 2.2. Dilution Experiments

1322

1323 Dilution grazing experiments are the only method available for estimating microzooplankton

1324 community grazing on the whole phytoplankton community. This method is used universally to estimate

1325 phytoplankton growth rates (µ) and mortality of phytoplankton due to microzooplankton grazing (g)

1326 (Landry, 1993). Dilution grazing experiments include all grazers < 200 µm in size, including small

1327 heterotrophic and mixotrophic flagellates, as well as the larger ciliates and dinoflagellates. Whole

1328 seawater (WSW), containing natural assemblages of phytoplankton and microzooplankton, is diluted with

1329 filtered, particle free seawater (FSW) from the same sample. Dilution reduces microzooplankton

1330 encounter rates with phytoplankton prey; in highly dilute treatments net growth rate (NGR) of

1331 phytoplankton approaches the intrinsic growth rate (µ). Phytoplankton mortality due to microzooplankton

1332 grazing is calculated as µ-NGR. A modified dilution method, the two-point method (Landry et al., 2008)

1333 was used because it is more efficient than the original method. The original and the two-point method

1334 have been compared in grazing experiments conducted in coastal Gulf of Alaska (Strom et al., 2006) and

1335 in the SE Bering Sea (Strom and Fredrickson, 2008) and found to provide similar results.

1336 In nutrient limited waters, which` can occur in summer on the Bering Sea Shelf (Strom and

1337 Fredrickson, 2008), nutrient regeneration due to micrograzers can be important in supplying inorganic

1338 nutrients for phytoplankton growth. This would violate the first assumption of the dilution technique, that

1339 phytoplankton growth rate is not influenced by dilution (Landry, 1993). To solve this problem, inorganic

1340 nutrients are added to all the bottles. However, then estimated phytoplankton growth rates are no longer

1341 similar to in situ rates. In this situation, it is usual to run incubations with and without nutrient addition.

1342 In situ phytoplankton growth rate, µ, is estimated from the incubation without added nutrients and, if

1343 nutrient limitation is important, g can be determined from NGR and µ in the incubations with added

1344 nutrients. To evaluate this, we undertook some, but not all, incubations with and without the addition of

1345 nutrients. In 2008, nutrient additions were 5 µM N as NaNO3. In 2009, nutrient additions were as 5 µM N

1346 as as NH4Cl, and in 2010 nutrient additions were 5 µM N as NaNO3 combined with 0.3 µM P as

1347 Na2HPO4. These additions are similar to those used by Strom and Fredrickson (2008) in earlier

1348 experiments in the Bering Sea, which found no difference between N addition as NH4Cl and NaNO3.

1349 All tubing, carboys, filter cartridges and incubation bottles were cleaned with 10% HCl, and

1350 rinsed 3 or more times with de-ionized water, and then rinsed with filtered seawater prior to use and

1351 between experiments (Landry, 1993). As mentioned earlier, in “mixed layer” experiments, water was

1352 collected with 30 L Niskin bottles on a CTD rosette from the depth corresponding to 55% surface

1353 irradiance level and in “DCM” experiments WSW was collected from the depth of chlorophyll

1354 fluorescence maximum. Seawater was gently siphoned (using silicone tubing) from Niskin bottles into

1355 black plastic covered polycarbonate carboys in the CTD bay. During siphoning, water was prescreened

1356 through a 200 µm Nitex mesh to remove larger zooplankton. This prescreened “whole seawater”

1357 contained phytoplankton and microzooplankton, however when phytoplankton >200 µm (long diatom

1358 chains and large colonial phytoplankton) were present, the pre-screening removed them. Thus, the

1359 “WSW” contains a variable fraction of the total in situ chlorophyll. Filtered seawater (FSW) was

1360 prepared using gravity filtration and 0.2 µm pore size sterile Pall Capsule Filters. Treatments consisted of

1361 100% WSW and diluted whole seawater. In early experiments we used 5% WSW for the diluted

1362 treatments (95% FSW), but found that in low chlorophyll waters it was difficult to obtain consistent

1363 chlorophyll measurements due to the low chlorophyll in the diluted treatment. We switched to using 20%

1364 WSW (80% FSW and 20% WSW) when in situ chlorophyll levels were low. Silicon tubing was used to

1365 gently transfer, without bubbling, water (WSW or diluted seawater) from 20 liter polycarbonate carboys

1366 to triplicate 1 liter polycarbonate incubation bottles. If a nutrient addition series was included in an

1367 experiment, nutrient stock solution was added with a micropipette directly to triplicate WSW and diluted

1368 WSW incubation bottles.

1369 At the beginning of each experiment, triplicate samples for total chlorophyll (in effect, <200 µm

1370 chlorophyll because of the prescreening) and size fractionated (<20 µm) chlorophyll were taken from the

1371 WSW and diluted seawater carboys. Samples for microzooplankton and Phaeocystis enumeration were

1372 siphoned directly from the Niskin bottles into 125 ml sampling bottles and fixed with acid Lugol’s

1373 solution as described (Stoecker et al., this volume). In mixed layer experiments (Table 1), the triplicate

1374 bottles for each treatment were incubated on deck in flowing sea-water with neutral density screening to

1375 approximate 55% surface irradiance. In DCM experiments, the bottles were incubated in the low light

1376 either on ice or in a 4oC cold room (Table 1). At the end of the incubations, chlorophyll samples (total

1377 chlorophyll a and < 20 µm chlorophyll a) were taken from each bottle. Chlorophyll samples were filtered

1378 onto 25 mm GF/F filters using gentle vacuum filtration, extracted in 90% acetone at -20o C for 24 h, and

1379 analyzed at sea with a pre-calibrated fluorometer (Turner Designs TD -700).

1380 Chlorophyll a was used as a proxy for phytoplankton biomass. Based on Landry et al. (2008),

1381 microzooplankton grazing (g) and phytoplankton growth rates (µ) were calculated from net growth rates

1382 of phytoplankton in the WSW (K) and diluted treatment (Kd) and the fraction of WSW in the diluted

1383 treatment (x). K or Kd values were calculated for each triplicate using average initial values of chlorophyll

1384 for each treatment. A one-way ANOVA was used to determine if the K and Kd values for an experimental

1385 series were statistical different (p<0.05), indicating phytoplankton mortality. The instantaneous rate of

-1 1386 phytoplankton mortality (d ), or grazing coefficient, was calculated as g = (Kd - K)/(1 - x). The

1387 instantaneous rate of phytoplankton growth (µ, d-1) was calculated as µ = K + g. Calculations were done

1388 separately for experiments with and without added nutrients (Landry 1993, Olson & Strom 2002). The

1389 fraction of phytoplankton growth grazed per day was estimated as g/µ. Daily ingestion (I) of

1390 phytoplankton biomass (µg C l-1 d-1) was estimated from chlorophyll consumption using a C:Chl ratio of

1391 50 for the Bering Sea (Lomas et al., 2012) as I= (Chl a)( 50)(g). Assuming an average gross growth

1392 efficiency of 35% for strictly heterotrophic microzooplankton, secondary production based on herbivory

1393 was estimated (Landry and Calbet, 2004).

1394

1395 2.3. Variable fluorescence measurements

1396

1397 In several studies, insignificant grazing coefficients and /or statistically significant “negative”

1398 microzooplankton grazing rates have been reported from a proportion of the stations in the Bering Sea

1399 and also from other sub-Arctic and Arctic seas (Olson and Strom, 2002; Strom and Fredrickson, 2008;

1400 Calbet et al., 2011; Sherr et al.,submitted). “Negative grazing” is impossible, but calculation of negative

1401 “g” results when the µ of phytoplankton in the diluted treatment is lower than in the WSW. Lower growth

1402 of phytoplankton in the diluted treatments than in WSW is usually attributed to lack of regenerated

1403 nutrients due to low numbers of micrograzers in the diluted treatments. However, nutrient addition

1404 usually did not eliminate the effect in our experiments. To explore the possibility that dilution itself, or

1405 chemicals released from plankton into the dilution water during preparation of FSW, have a negative

1406 effect on phytoplankton we measured variable fluorescence (Fv/Fm) of phytoplankton in the undiluted

1407 WSW and diluted treatments. Fv/Fm was measured after incubation of subsamples in the dark for ~ 1 h

1408 in a Automated Laser Fluorometer (ALF)(Chekalyuk and Hafez, 2008). Fv/Fm was measured in samples

1409 from the triplicate incubation bottles in the 100%WSW, 20%WSW, 100%WSW+nuts, 20%WSW+nuts

1410 treatments at the end of the 24 h incubations in mixed layer experiments at 14 stations (C-55, W-4b, SL-8,

1411 NP-7, NP-14, NP-11, LS-16, P14-4.5, P14-2, MN-6, MN-12, MN-19 and SL-14) in 2008 (Table 1).

1412 Samples were collected in 500 ml amber glass bottles, and stored in the dark for about 30 min, to

1413 minimize the impacts of non-photochemical quenching before analysis in the ALF.

1414

1415 2.4. Enumeration of Phaeocystis pouchettii

1416

1417 The bloom forming phytoplankter P. pouchettii has been associated with inhibition of grazing in

1418 dilution experiments (Calbet et al., 2011). To determine if low grazing coefficients were associated with

1419 abundance of Phaeocystis cells, we enumerated this phytoplankter in water samples fixed with acid

1420 Lugol’s (refer to Stoecker et al., this volume) using a 1-ml capacity Sedgwick-Rafter counting chamber at

1421 400X magnification.

1422

1423 2.5. Statistical Analyses

1424

1425 Analysis of variance (ANOVA) was used to test statistical significance (p<0.05) of differences

1426 between coefficients from incubations with and without addition of nutrients, mixed layer versus DCM

1427 incubations, and among domains. T–tests were used to compare variable fluorescence measurements

1428 between diluted and undiluted treatments within an experiment. Pearson product-moment correlation

1429 tested for statistically significant associations between factors. If the data did not fulfill the assumptions

1430 for ANOVA, we applied appropriate transformations. We used ANOVA on ranks if data still did not

1431 meet the assumptions. We used Sigmaplot version 9.0 for all statistical analyses.

1432

1433 3. Results

1434

1435 3.1. Effect of nutrient additions

1436

1437 We compared the results of paired dilution experiments conducted with and without the addition

1438 of nutrients (Table 2). In calculating means and standard deviations, we used data from all paired

1439 incubations, whether or not grazing was statistically significant; we substituted “0” for negative “g”

1440 values in calculating means. In 2008, we did 21 comparative experiments, with and without the addition

1441 of 5 µM nitrogen as sodium nitrate. Mean phytoplankton growth rates (µ, d-1) in the control (no nutrient

1442 addition) and nutrient addition treatments were similar, however grazing coefficients (g, d-1) were

1443 significantly lower in the nutrient treatments (Table 2). In 2009, we did 5 comparative experiments in

1444 which the 5 µM nitrogen nutrient treatment was attained using ammonium chloride. The effects on

1445 phytoplankton growth and grazing were both non-significant (Table 2). In 2010, we did 15 experiments

1446 in which the nutrient treatment was addition of 5 µM nitrogen as sodium nitrate in combination with 0.3 5

1447 µM phosphate as sodium phosphate. The nutrient treatment significantly increased mean phytoplankton

1448 growth but not estimation of microzooplankton grazing (Table 2). Based on these results, we report

1449 microzooplankton grazing based upon the no addition dilution series. Olson and Strom (2002) also noted

1450 that nutrient enrichment can sometimes result in a decrease in phytoplankton growth rates in dilution

1451 experiments in the Bering Sea.

1452

1453 3.2. Phytoplankton growth, microzooplankton grazing, ingestion and production in the mixed layer

1454

1455 The mixed layer dilution experiments were with WSW samples collected at the depth of 55% of

1456 surface irradiance (PAR) and incubated under simulated in situ conditions. The sample depth varied from

1457 3 to 10 m (Table 1) and was always within the surface mixed layer. Water was prescreened with a 200

1458 µm Nitex mesh prior to set-up of the dilution series to remove mesozooplankton so large phytoplankton

1459 chains and colonies were not included in the incubations. The <200 µm fraction ranged from an average

1460 of 69% at the off shelf, 79-90% at the outer shelf stations, 41-98% at the middle shelf stations, 77% at the

1461 inner shelf stations and 74% at the Alaska Peninsula stations of the total chlorophyll (Table 3). Grazing

1462 coefficients were greater than “0” in 61% of the 59 dilution experiments conducted with mixed layer

1463 assemblages (Table 3). To avoid biasing the data against low growth and grazing rates, we included all

1464 coefficients, whether or not they were significant, in our estimates of average rates, however we did

1465 replace negative grazing rates with “0” in calculating means. Estimated average phytoplankton growth

1466 coefficients for most regions were in the range of 0.24-0.32 d-1 (Table 3). The average phytoplankton

1467 growth coefficient for the mid north Middle Shelf was low (Table 3) because of an experiment with a

1468 negative growth rate (µ=-0.80); without this one experiment, the average was 0.37 d-1. The average

1469 phytoplankton growth rate in the Alaska Peninsula region appeared to be higher than rates in the other

1470 areas, but differences among regions were not statistically significant (1-WAY ANOVA, p>0.05).

1471 Likewise, estimates of average microzooplankton grazing were relatively similar across regions, ranging

1472 from 0.16 to 0.34 d-1 (Table 3) and differences among regions were not statistically significant (1-WAY

1473 ANOVA , p>0.05). Correlations between µ or g and mixed layer water temperature, inorganic nutrients

1474 (phosphate, silicate, nitrate, ammonium) and estimated total PAR for incubations (data not shown) were

1475 not statistically significant (Pearson Product Moment Correlation; p>0.05). Both microzooplankton

1476 grazing and phytoplankton growth coefficients correlated negatively with total chlorophyll a estimated

1477 either as total chlorophylls from CTD bottles as part of core measurements or by the primary production

1478 study (Lomas et al., 2012). The regressions only accounted for a small amount of the variability (Figure

1479 3 A and B). Microzooplankton grazing (<200 µm fraction) was not correlated with the biomass of 20-200

1480 µm ciliate and dinoflagellate microzooplankton (p>0.05) (data not shown). Microzooplankton grazing

1481 was also not correlated with abundance of Phaeocystis cells (p>0.05) (data not shown).

1482 We estimated the proportion of phytoplankton growth grazed by phytoplankton as “g/µ” from the

1483 average “g” and “µ” for regions (Table 3). Estimates for individual incubations could not be made

1484 because of negative or zero coefficients. Estimated average proportion of phytoplankton growth in the

1485 <200 µm fraction grazed by phytoplankton appeared to vary among regions (Figure 2). In the off shelf

1486 and outer shelf regions, microzooplankton grazing consumed 67-78% of phytoplankton growth but on the

1487 middle shelf and inner shelf (with the exception of the south middle shelf) microzooplankton grazing

1488 coefficients were greater than phytoplankton growth rates in the mixed layer (Figure 2 and Table 3).

1489 Estimated phytoplankton carbon ingestion by microzooplankton was 11.0 µg carbon l-1 d-1 in the

1490 Alaska Peninsula region, 8.2 off shelf, 7.7 on the outer shelf, 4.7 on the middle shelf and 4.4 d-1 on the

1491 inner shelf, with differences among regions not statistically significant (1 Way ANOVA, p>0.05) (Figure

1492 4). Assuming that all microzooplankton were strictly heterotrophic and a gross growth efficiency of 35%,

1493 the estimated secondary production of microzooplankton in the mixed layer was 3.8 µg carbon l-1 d-1, 2.9,

1494 2.7, 1.6, and 1.5 for the Alaska Peninsula, off shelf, outer shelf, middle shelf and inner shelf regions,

1495 respectively.

1496

1497 3.3. Microzooplankton grazing, ingestion and production in the DCM

1498

1499 We conducted dilution grazing experiments with deep chlorophyll maximum assemblages at 6

1500 stations in 2010, four of these were paired with mixed layer incubations at the same station (Table 4).

1501 Irradiance levels and water temperatures were low in the DCM so we decided to incubate the DCM

1502 experiments in the dark either in a cold room (~4oC) or on ice (~0 oC), whichever provided conditions

1503 closer to in situ (Table 4). Grazing coefficients in the DCM ranged from ~0 to 0.70 d-1 and in 4 out of 6

1504 DCM incubations, the grazing coefficients were significant (p<0.05) (Table 4). It is interesting that in

1505 paired mixed layer and DCM incubations, the DCM grazing coefficients were similar in magnitude to the

1506 mixed layer coefficients (Table 4) however, because of the higher phytoplankton biomass in the DCM

1507 than the mixed layer estimated ingestion of phytoplankton carbon by microzooplankton in the DCM was

1508 higher than in the mixed layer (Figure 5). Assuming similar gross growth efficiencies for

1509 microzooplankton in the DCM and in the mixed layer, microzooplankton secondary production in the

1510 DCM on a per liter basis is ~4-fold higher than in the mixed layer.

1511

1512 3.4. Non-significant and “negative” grazing coefficients and Fv/Fm

1513

1514 In 39% of our dilution grazing experiments with mixed layer assemblages the grazing coefficients

1515 were not statistically significant (p>0.05) and in 3% we found negative grazing coefficients that were

1516 statistically significant. Estimation of microzooplankton grazing in dilution experiments is based on the

1517 assumption that phytoplankton growth rate is the same in diluted and undiluted treatments. To determine

1518 if dilution was having a negative effect on the physiology of phytoplankton, and potentially on their

1519 growth rates, we measured variable fluorescence (Fv/Fm) in both the undiluted (WSW) and diluted (20%

1520 WSW) treatments at the end of paired incubations with and without added nitrate (Table 1). Fv/Fm was

1521 significantly lower (t-test, p<0.05) in the diluted than undiluted treatments in 11 out of 14 incubations

1522 without added nutrients and in 7 out of 14 incubations with added nutrients (Table 5). Fv/Fm was

1523 significantly higher (p<0.05) in the diluted treatments with than without added nitrate in 3 of the 14

1524 experiments (data not shown). In only two experiments did this alleviate the negative effect of dilution on

1525 variable fluorescence of phytoplankton (Table 5). Low estimates of microzooplankton grazing coefficient

1526 were associated with experiments in which dilution had a relatively large negative effect of on Fv/Fm

1527 (Figure 6).

1528

1529 4. DISCUSSION

1530

1531 During summers of 2008-2010, all “cold years” in the eastern Bering Sea (Stabeno et al., 2012b),

1532 the over-all average mixed layer microzooplankton grazing coefficient was 0.26 d-1, which is quite similar

1533 to the average coefficient of 0.29 d-1 observed on the SE shelf by Olson and Strom during summer 1999,

1534 another cold year (Table 6). In 2004, a “warm year” (Ladd and Stabeno, 2012), microzooplankton

1535 grazing was low on the SE Shelf (Strom and Fredrickson, 2008)(Table 6). The low grazing in 2004 was

1536 probably a response of microzooplankton to poor food quality caused by phytoplankton nutrient

1537 limitation due to intense stratification (Strom and Fredrickson, 2008) however “warm” years are not

1538 always associated with high stratification on the Bering Sea shelf (Ladd and Stabeno, 2012), so it is

1539 unlikely that summer microzooplankton grazing is predictable from temperature alone. During 2004,

1540 phytoplankton growth rates responded strongly to addition of nutrients (Strom and Fredrickson, 2008) but

1541 we only observed increases in phytoplankton growth in response to nutrient additions in one year, 2010

1542 (Table 2). Contrary to expectations, nutrient addition sometimes resulted in lower estimates of

1543 phytoplankton growth and sometimes microzooplankton grazing. Possible inhibition of growth and

1544 grazing due to nutrient addition has been previously reported (Gifford, 1988; Olson and Strom, 2002).

1545 Average microzooplankton grazing coefficients during spring sea ice conditions are <50% of

1546 summer grazing coefficients (Table 6). The lower grazing coefficients in spring are probably due to a

1547 combination of factors including lower ratios of microzooplankton biomass to phytoplankton biomass in

1548 spring than in summer (Sherr et al., submitted; Stoecker et al., this volume), differences in size

1549 distribution and species composition of phytoplankton (Lomas et al., 2012) and lower water temperatures

1550 in spring.

1551 It is interesting that both the growth rate of phytoplankton (µ) and microzooplankton grazing (g)

1552 were correlated negatively with total chlorophyll a in the mixed layer. Negative or no correlation of

1553 growth and grazing coefficients with chlorophyll have also been noted by Olson and Strom (2002), Strom

1554 et al. (2007), and Calbet et al. (2011) in northern seas in summer. This is consistent with domination of

1555 the phytoplankton by < 5 µm cells, and a rate, rather than biomass, controlled production system

1556 dependent on nutrient recycling (Lomas et al., 2012). Based on primary production measurements,

1557 integrated phytoplankton growth rates (µ) averaged 0.42 d-1 (SD, 0.17) (Lomas et al., 2012), which is

1558 within the wide range of phytoplankton growth rates we estimated in mixed layer dilution experiments

1559 (Table 6). In the north middle and inner domains, the biomass of microzooplankton to phytoplankton was

1560 high in summer (Stoecker et al., this issue), consistent with the high ratios of microzooplankton grazing to

1561 phytoplankton growth. A confounding factor in estimating phytoplankton growth and microzooplankton

1562 grazing from chlorophyll in dilution experiments on the Bering Sea shelf is the high biomass of plastidic

1563 ciliates in summer (Stoecker et al., this issue). The incorporation of phytoplankton chloroplasts into

1564 ciliates may result in underestimation of microzooplankton grazing and an overestimate of

1565 “phytoplankton” biomass.

1566 For the 2008 summer cruise, the spatial pattern of mixed layer phytoplankton growth and

1567 microzooplankton grazing (Figure 7) can be compared with the spatial distribution of chl a, variable

1568 fluorescence and phytoplankton composition from HPLC (Goes et al., this issue). The high phytoplankton

1569 growth (µ, d-1) and moderate microzooplankton grazing (g, d-1) were measured on the north middle shelf,

1570 where diatom and cryptophytes patches, probably remnants of spring ice associated blooms, were

1571 observed. Elevated phytoplankton growth and microzooplankton grazing were observed on the middle

1572 shelf near 60oN, at the border between the St. Matthews and North Inner Shelf regions. This area was

1573 characterized by relatively low surface chlorophyll a and moderate variable fluorescence. Diatom patches

1574 were to the east and haptophytes to the west of 170OW in this area. Phytoplankton growth and

1575 microzooplankton grazing were also relatively high on parts of the north outer shelf and off shelf north

1576 regions on the inner edge of the greenbelt. The greenbelt was characterized by low to moderate surface

1577 chlorophyll a, moderate variable fluorescence and dominance of phytoplankton biomass by cryptophytes

1578 and haptophytes. The Pribilof Island area was a hot spot for phytoplankton growth, with patches of both

1579 diatoms and haptophytes present, but microzooplankton grazing was moderate in this region. In contrast,

1580 the southeastern shelf and Peninsula area tended to have lower phytoplankton growth and

1581 microzooplankton grazing coefficients, although small flagellates, including cryptophytes and

1582 haptophytes, dominated (Goes et al., this issue).

1583 In 2004, weak trophic coupling of phytoplankton growth to microzooplankton grazing was

1584 observed (Strom and Fredrickson, 2008), but strong coupling was observed in 1999 (Olson and Strom,

1585 2002) and in summers of 2008, 2009, and 2010 (Table 6). On the shelf, microzooplankton grazing

1586 coefficients (g) often exceeded phytoplankton growth coefficients (µ) in the mixed layer, but g/µ was <1

1587 in Alaska Peninsula, Off Shelf and Outer Shelf waters in summer (Table 3). On average, grazing is

1588 equivalent to phytoplankton growth in the sea (Banse, 1992), with ratios of g to µ exceeding 1 often

1589 found during the demise of blooms. For example, in the more southerly Gulf of Alaska, the ratio of

1590 microzooplankton grazing to phytoplankton growth on the middle and inner shelf reaches a maximum in

1591 summer with g/µ > 1.0 whereas in summer the ratio is lower on the outer shelf (Strom et al., 2007),

1592 similar to our observations in the eastern Bering Sea. Another example is the Sea of Okhotsk, Liu et al.

1593 (2009) found that microzooplankton grazing (g) was about 3X higher than phytoplankton growth (µ) in

1594 late summer in nutrient-depleted shelf waters whereas in the higher nutrient shelf break and strait waters,

1595 g/µ estimates were <0.5. Ratios of microzooplankton grazing to phytoplankton growth in excess of 1

1596 may be a general, although transient, feature of highly stratified boreal and arctic shelf ecosystems in

1597 summer.

1598 Large copepods, such as Neocalanus spp., Calanus glacialis, and Metridia longa, have a strong

1599 prey preference for microzooplankton, but are largely absent from the middle and inner shelf waters in

1600 summer (Vidal and Smith, 1986; Gifford, 1993; Campbell et al., 2009; Hunt et al., 2008). A reduction in

1601 top down control of microzooplankton on the eastern Bering Sea shelf may be partially responsible for the

1602 high biomass of microzooplankton (Stoecker et al., this issue) and their high grazing impact on the Bering

1603 Sea shelf in summer. A similar phenomenon occurs on the Gulf of Alaska shelf, where summer

1604 populations of large copepods are low and large cell-size microzooplankton are very abundant due to

1605 diminished top down control (Strom et al., 2007).

1606 Although the grazing impact of microzooplankton on phytoplankton growth (g/µ) was highest in

1607 middle and inner shelf waters, estimated microzooplankton ingestion of phytoplankton carbon and

1608 secondary production of microzooplankton was highest in the Alaska Peninsula, off shelf and outer shelf

1609 waters. This was due to higher chlorophyll levels than on the middle and inner shelf. However,

1610 microzooplankton (20-200 µm) biomass in the off shelf and outer shelf regions was about half that in the

1611 inner and middle domains (Stoecker et al., this issue). One reason for this discrepancy may be that grazing

1612 by nanozooplankton (which are not included in the microzooplankton biomass estimates) makes a larger

1613 contribution to grazing in the higher chlorophyll regions. Nanozooplankton have been shown to be more

1614 abundant in frontal areas on the outer shelf and at the shelf break than in lower chlorophyll inner and

1615 middle domains in summer (Flint et al., 2002). The relatively low biomass of 20-200 µm

1616 microzooplankton (ciliates and large dinoflagellates) and high biomass of nanozooplankton in Peninsula,

1617 off shelf and outer shelf waters suggests a trophic cascade in which top down control of large

1618 microzooplankton by crustacean zooplankton releases nanozooplankton from grazing control.

1619 Conversely, large microzooplankton are probably relatively more abundant on the shelf in summer due to

1620 relaxation in top down control by copepods which are mostly absent from the shelf in summer (Vidal and

1621 Smith, 1986; Hunt et al., 2008).

1622 During the summer, the DCM in the northern domain can be very well developed with > 10 µg

1623 chlorophyll a l-1 at some stations (Lomas et al., 2012). Thus, the processes within the DCM may be very

1624 important to carbon flux and trophic transfer. Microzooplankton biomass (Stoecker et al., this issue) and

1625 grazing coefficients in the DCM were usually similar to those in the mixed layer above. The DCM

1626 samples ranged in depth from 26 to 35 m (Table 4), which was near or below the average depth of the 1%

1627 PAR isolume, 30 m (Lomas et al., 2012). If we assume that phytoplankton growth was usually low at

1628 these depths, it is clear that the ratio of grazing to phytoplankton growth must have been very high.

1629 Microzooplankton grazing is often important in erosion of “plankton patches” (Menden-Deuer and

1630 Fredrickson, 2010). Although grazing coefficients were roughly similar in the mixed layer and

1631 corresponding DCM, the ingestion rates in the DCM were 3-30X higher than in the mixed layer (Figure

1632 5). If we assume that microzooplankton growth efficiency was similar in the mixed layer and DCM, the

1633 DCM may be an important site of microzooplankton production during summer, particularly on the

1634 northern shelf. The lack of accumulation of microzooplankton biomass in the DCM suggests that

1635 predation pressure on microzooplankton is high and thus that these layers may be important in trophic

1636 transfer to higher trophic levels. The impact of microzooplankton grazing on phytoplankton and export

1637 fluxes can be under-estimated if grazing near the base of the euphotic zone is not included (Landry et al.,

1638 2011).

1639 The decrease in variable fluorescence during the incubations from some stations suggest that

1640 dilution had a negative impact on phytoplankton photosynthetic physiology and hence potentially growth

1641 rate. This effect was not simply due to N limitation alone in the diluted treatments; it occurred in control

1642 and, in most cases, the paired nitrate amended dilutions. One of the assumptions of the dilution method is

1643 that dilution does not change the growth rate of phytoplankton, and this assumption likely was violated in

1644 at least some dilution experiments. A decrease in phytoplankton growth rate with dilution would result in

1645 an underestimation of the grazing coefficients. This may partially account for the low or non-significant

1646 grazing coefficients. Low grazing rates of microzooplankton for the biomass of microzooplankton have

1647 previously been reported in the SE Bering Sea during intense summer stratification and nutrient limitation

1648 of phytoplankton (Strom and Fredrickson, 2008). Non-significant microzooplankton grazing coefficients

1649 have also been reported during the spring at both non-bloom and diatom bloom ice edge stations (Sherr et

1650 al., submitted). In addition to non-significant grazing coefficients, statistically significant negative rates

1651 occur. Negative dilution grazing results are usually not reported (Dolan and McKeon, 2005), or the

1652 negative coefficients treated as “0” (Strom and Fredrickson, 2008). Negative rates can only occur when

1653 the growth rate of phytoplankton, µ, is lower in the diluted treatment and than in the whole seawater. In

1654 the polar and subpolar ecosystems, non-significant and negative results in dilution experiments are

1655 common, particularly during Phaeocystis blooms (Calbet et al., 2011; Caron et al., 2000).

1656 In culturing phytoplankton, a “lag” phase typically occurs after transfer of cells to new media,

1657 this has been ascribed to the time it takes cells to “ramp up” to better growth conditions, the “shock” of

1658 transfer and to lack of “conditioning factors” in the media. Perhaps something similar happens when

1659 phytoplankton are diluted with filtered seawater. Another possibility is that the mechanical stress

1660 involved in passage through filters results in release of “toxic’ or “inhibitory” compounds into the filtered

1661 seawater used to make the dilutions. Strom and Fredrickson (2008) noted that release of diatom extracts

1662 from filters used to prepare filtered seawater may have inhibited growth of phytoplankton. During certain

1663 growth phases, Phaeocystis pouchetti and many bloom forming diatoms produce cytotoxic aldehydes that

1664 are inhibitory to phytoplankton growth, including their own (Hansen and Eilertsen, 2007; Paul et al.,

1665 2009). Mechanical stress can trigger the release of these compounds from cells (Hansen & Eilertsen,

1666 2007). We hypothesize that decreases in variable fluorescence and low or negative phytoplankton growth

1667 rates in diluted treatments could be due to presence of toxic aldehydes released from phytoplankton

1668 during preparation of filtered seawater. Preliminary results indicate that with some diatom and

1669 Phaeocystis blooms, treatment of filtered seawater with activated carbon to remove organic material prior

1670 to its use in dilution can reverse the negative effects on both “u” and “g” (Stoecker and Nejstgaard ,

1671 unpubl. data). However, contrary to expectation, grazing coefficients were not negatively correlated with

1672 abundance of Phaeocystis pouchetti cells in the Bering Sea. Production of inhibitory compounds by

1673 Phaeocystis varies with life form and bloom stage (Nejstgaard et al., 2007), thus not all Phaeocystis cells

1674 will have the same impact on water chemistry. Furthermore, diatoms are also a source of cytotoxic

1675 aldehydes and are bloom dominants in the Bering Sea (Flint et al., 2002; Lomas et al., 2012). A simple

1676 relationship between one of these factors (Phaeocystis cells) and apparent low grazing coefficients is

1677 unlikely due to confounding factors.

1678 Although the overestimation of microzooplankton grazing by the dilution technique may occur

1679 under some circumstances (Dolan et al., 2000; Dolan and McKeon, 2005), it is possible that dilution

1680 experiments underestimate microzooplankton grazing during summer in the Bering Sea. Whether or not

1681 grazing is underestimated, it is clear that microzooplankton grazing consumes most of phytoplankton

1682 production and that secondary production by microzooplankton is important. Most phytoplankton

1683 production is by < 5 µm phytoplankton and thus passes through the microzooplankton link before it is

1684 available to crustacean zooplankton in summer. On the northern shelf, the DCM may often be a site of

1685 enhanced trophic transfer. Microzooplankton can comprise 49% or more of the food available to larger

1686 zooplankton on the Bering Sea shelf in summer (Stoecker et al., this volume).

1687

1688 Acknowledgements

1689

1690 The authors thank the captain and crew of the USCG Healy, R/V Knorr and R/V T.G. Thompson for their

1691 assistance during the cruises. We thank Michael Lomas for use of his fluorometer, Dean Stockwell and

1692 Michael Lomas for chlorophyll data, Carol Feierabend for enumerating Phaeocystis cells, Helga Gomes

1693 for help with the variable fluorescence measurements and Kristin Blattner for excellent technical

1694 assistance in 2008 and 2009. This research project was BSIERP B55. DKS and AW were supported by

1695 BSIERP project #55. JIG was supported by funding from NASA. This is BEST-BSIERP Bering Sea

1696 Project publication number XX and UMCES contribution number XX.

1697

1698 Reference

1699

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1702 the sea. Plenum Press, New York, pp. 409-440.

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1704 Calbet, A., Saiz, E., 2005. The ciliate-copepod link in marine ecosystems. Aquat. Microb. Ecol. 38, 157-

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1707 Calbet, A., Saiz, E., Almeda, R., Movilla, J.I., Alcarez, M., 2011. Low microzooplankton grazing rates in

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1711 Campbell, R.G., Sherr, E.B., Ashjian, C.J., Plourde, S., Sherr, B.F., Hill, V., Stockwell , D.A., 2009.

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1723

1724 Dolan, J.R., Gallegos, C.L., Moigis, A., 2000. Dilution effects on microzooplankton in dilution grazing

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1743 Hunt, G.L., Jr., Stabeno, P., Strom, S., Napp, J.M., 2008. Patterns of spatial and temporal variation in the

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1747 Hunt, G.L ., Jr., Stabeno, P., Walters, G., Sinclair, E., Brodeur, R.D., Napp, J.M., Bond, N.A., 2002.

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1751 Ladd, C., Stabeno, P.J., 2012. Stratification on the Eastern Bering Sea shelf revisited. Deep Sea Res. II

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1754 Landry, M.L., 1993. Estimating rates of growth and grazing mortality of phytoplankton by the dilution

1755 method, in Kemp, P.F., et al. (Eds) Handbook of Methods in Aquatic Microbial Ecology. Lewis

1756 Publishers, Boca Raton, pp. 715-722.

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1758 Landry, M.L., Calbet, A., 2004. Microzooplankton production in the oceans. ICES J. Mar. Sci. 61, 501-

1759 507.

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1761 Landry, M.R., Brown, S.L,, Yoshimi, M.R., Selph, K.E., Bidigare, R.R., Yang, E.J., Simmons, M.P.,

1762 2008. Depth-stratified phytoplankton dynamics in Cyclone Opal, a subtropical mesoscale eddy. Deep-Sea

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1765 Landry, M.R., Selph, K.E., Yang, E.J., 2011. Decoupled phytoplankton growth and microzooplankton

1766 grazing in the deep euphotic zone of the eastern equatorial Pacific. Mar. Ecol. Prog. Ser. 421, 13-24.

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1768 Levinsen, H., Nielsen, T.G. 2002. The trophic role of marine pelagic ciliates and heterotrophic

1769 dinoflagellates in Arctic and temperate coastal ecosystems: a cross-latitude comparison. Limnol.

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1772 Liu, H., Suzuki, K., Nishioka, J., Sohrin, R., Nakatsuka, T. ,2009. Phytoplankton growth and

1773 microzooplankton grazing in the Sea of Okhotsk during late summer of 2006. Deep-Sea Res. I 56, 561-

1774 570.

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1776 Liu, H., Suzuki ,K., Saino, T., 2002. Phytoplankton growth and microzooplankton grazing in the subarctic

1777 Pacific Ocean and the Bering Sea during summer 1999. Deep Sea Res. I 49, 363-375.

1778

1779 Lomas, M.W., Moran, S.B., Casey, J.R., Bell, D.W., Tiahlo, M., Whitefield, J., Kelly, R.P., Mathis, J.T.,

1780 Cokelet, E.D., 2012. Spatial and seasonal variability of primary production on the Eastern Bering Sea

1781 shelf. Deep-Sea Res. II 65-70, 126-140.

1782

1783 Menden-Deurer, S., Fredrickson, K., 2010. Structure-dependent, protistan grazing and its implication for

1784 the formation, maintenance and decline of plankton patches. Mar. Ecol. Prog. Ser. 420, 57-71.

1785

1786 Merico, A., Tyrrell, T., Lessard, E.J., Oguz, T., Stabeno ,P.J., Zeeman, S.I., Whitledge, T.E., 2004.

1787 Modelling phytoplankton succession on the Bering Sea shelf: role of climate influences and trophic

1788 intractions in generating Emiliamia huxleyi blooms 1997-2000. Deep Sea Res. I 51, 1803-1826.

1789

1790 Moran, S.B., Lomas, M.W., Kelly, R.P., Gradinger, R., Iken, K., Mathis, J.T., 2012. Seasonal succession

1791 of net primary productivity, particulate organic carbon export and autotrophic community composition in

1792 the eastern Bering Sea. 2012. Deep Sea Res.II 65-70, 84-97.

1793

1794 Mordy, C.W., Cokelet, E.D., Ladd, C., Menzia, F.A., Proctor, P., Stabeno, P.J., Wisegarver, E., 2012. Net

1795 community production on the middle shelf of the eastern Bering Sea. Deep Sea Res. II 65-70, 110-125.

1796

1797 Nejstgaard, J.C., Tang, K.W., Steinke, M., Dutz, J., Koski, M., Antajan, E., Long, J.D. , 2007.

1798 Zooplankton grazing on Phaeocystis: a quantitative review and future challenges. Biogeochemistry 83,

1799 147-172.

1800

1801 Olson, M.B., Strom, S.L., 2002. Phytoplankton growth, microzooplankton herbivory and community

1802 structure in the southeast Bering Sea: insight into the formation and temporal persistence of an Emiliania

1803 huxleyi bloom. Deep-Sea Res. II 49, 5969-5990.

1804

1805 Paul, C., Barofsky, A., Vidoudez, C., Pohnert, G., 2009. Diatom exudates influence metabolism and cell

1806 growth of co-cultured diatom species. Mar. Ecol. Prog. Ser. 389, 61-70.

1807

1808 Sambrotto, R.N., Mordy , C., Zeeman, S.I., Stabeno, P.J., Macklin, S.A., 2008. Physical forcing and

1809 nutrient conditions associated with patterns of Chl a and phytoplankton productivity in the southeastern

1810 Bering Sea during summer. Deep-Sea Res. II 55, 1745-1760.

1811

1812 Sherr, E.B., Sherr, B.F., Hartz, A.J., 2009. Microzooplankton grazing impact in the western Artic Ocean.

1813 Deep-Sea Res. II 56, 1264-1273.

1814

1815 Sherr, E.B., Sherr, B.F., Ross, C., Submitted. Microzooplankton grazing impact in the Bering Sea during

1816 spring sea ice conditions. Deep Sea Res.

1817

1818 Stabeno, P.J., Farley, E.V., Jr., Kachel, N.B., Moore, S., Mordy, C.W., Napp, J.M., Overland, J.E.,

1819 Pinchuk, A.I., Sigler, M.F., 2012a. A comparison of the physics of the northern and southern shelves of

1820 the eastern Bering Sea and some implications for the ecosystem. Deep Sea Res. II 65-70, 14-30.

1821

1822 Stabeno, P.J., Kachel, N.B., Moore, S.E., Napp, J.M., Sigler, M., Yamaguchi, A., Zerbini, A.N., 2012b.

1823 Comparison of warm and cold years on the southeastern Bering Sea shelf and some implications for the

1824 ecosystem. Deep Sea Res. II 65-70, 31-45.

1825

1826 Stockwell, D.A., Whitledge, T.E., Zeeman, S.I., Coyle, K.O., Napp, J.M., Brodeur, R.D., Pinchuk ,A.I.,

1827 Hunt, G.L., 2001. Anomalous conditions in the south-eastern Bering Sea, 1997: nutrients, phytoplankton

1828 and zooplankton. Fisheries Oceanography 10, 99-116.

1829

1830 Stoecker, D.K., Weigel, A., Stockwell, D., Lomas, M., (this issue) Microzooplankton: Abundance,

1831 biomass and contribution to chlorophyll in the Eastern Bering Sea in summer. Deep Sea Res.

1832

1833 Strom, S.L., Fredrickson, K.A., 2008. Intense stratification leads to phytoplankton nutrient limitation and

1834 reduced microzooplankton grazing in the southeastern Bering Sea. Deep Sea Res. II 55, 1761-1774.

1835

1836 Strom, S.L., Macri, E.L., Olson, M.B., 2007. Microzooplankton grazing in the coastal Gulf of Alaska:

1837 variations in top-down control of phytoplankton. Limnol. Oceanogr. 52, 1480-1494.

1838

1839 Strom S.L., Olson, M.B., Macri, E.L., Mordy, C.W., 2006. Cross-shelf gradients in phytoplankton

1840 community structure, nutrient utilization, and growth rate in the coastal Gulf of Alaska. Mar. Ecol. Prog.

1841 Ser. 328, 75-92.

1842

1843 Sukhanova , I.N., Semina ,H.J., Venttsel, M.V., 1999. Spatial and temporal variability of phytoplankton

1844 in the Bering Sea, in Loughlin, T.R., Ohtani, K. (Eds) Dynamics of the Bering Sea. University of Alaska

1845 Sea Grant, Fairbanks, pp 453-483.

1846

1847 Vidal, J., Smith, S., 1986. Biomass, growth, and development of populations of herbivorous zooplankton

1848 in the southeastern Bering Sea during spring. Deep-Sea Res. 33, 523-556.

1849

1850

1851

1852

1853

1854

1855

1856

1857

1858

1859

1860

1861

1862

1863

1864

1865

1866 Figure Legends

1867

1868 Fig. 1. Stations where r dilution experiments were performed in the eastern Bering Sea during summers

1869 2008, 2009, and 2010. Refer to Table 1 for list of stations, regions and experimental parameters;

1870 experiments were not performed at all stations in all years.

1871

1872 Fig. 2. Estimated fraction of phytoplankton daily growth consumed by microzooplankton grazing (g/u) in

1873 the mixed layer, Eastern Bering Sea. Means (SD) of regions within larger domains. AK PEN=Alaska

1874 Peninsula region; OFF=Off Shelf; OUTER=Outer Shelf; MIDDLE=Middle Shelf; INNER= Inner Shelf

1875 or Coastal. Data for summers 2008,2009, and 2010.

1876

1877 Fig. 3. Microzooplankton grazing (g, d-1) (A) and phytoplankton growth (u, d-1) (B) vs. chlorophyll a,

1878 Eastern Bering Sea. Data for summers 2008-2010.

1879

1880 Fig. 4. Estimated microzooplankton ingestion of phytoplankton carbon in the mixed layer. Means (SD) of

1881 stations within a region, Eastern Bering Sea. AK PEN=Alaska Peninsula region; OFF=Off Shelf;

1882 OUTER=Outer Shelf; MIDDLE=Middle Shelf; INNER= Inner Shelf or Coastal. Data for summers 2008

1883 2009 and 2010.

1884

1885 Fig. 5. Comparison of estimated microzooplankton ingestion of phytoplankton carbon, paired mixed

1886 layer and DCM experiments, Eastern Bering Sea, summer 2010.

1887

1888 Fig. 6. Estimated microzooplankton grazing coefficients (g, d-1) vs. the difference in variable

1889 fluorescence (Fv/Fm) between WSW and diluted treatments in an experiment. Low grazing was

1890 associated with decreased Fv/Fm in the diluted compared to the whole seawater treatments.

1891

1892 Fig. 7. Spatial distribution of phytoplankton growth (µ, d-1) (upper panel) and microzooplankton grazing

1893 (g, d-1)(lower panel) in the eastern Bering Sea, summer 2008.

1894

1895

1896

1897

1898

1899

1900

1901

1902

1903

1904

1905

1906 Fig. 1

1907

1908

1909

1910

1911

1912

1913

1914

1915

1916

1917

1918 Fig. 2

1919

1.8 1920

1.6 1921 1.4 1922 1.2

1.0 1923 SD

+ µ 0.8 1924 g/

0.6 1925 0.4 1926 0.2 1927 0.0 AK PEN OFF OUTER MIDDLE INNER 1928

1929

1930

1931

1932

1933

1934

1935

1936

1937

1938

1939

1940 Fig. 3

1941 A.

1.8

1.6 R2=0.072, p<0.05 1.4

1.2

1.0 -1 0.8 g, d g, 0.6

0.4

0.2

0.0

0.0 0.5 1.0 1.5 2.0 2.5 3.0 -1 1942 Chl. α, µg l

1943 B.

1.0

0.8

0.6

0.4

0.2 -1 0.0 , d µ -0.2

-0.4

-0.6 R2=0.236, p<0.001 -0.8

-1.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 -1 1944 Chl a, µg l

1945

1946

1947 Fig. 4

-1 20 d -1

15 g Carbon l

µ 10

0 AK PEN OFF OUTER MIDDLE INNER

1948

1949

1950

1951

1952

1953

1954

1955 Fig. 5

1956

40 ) -1 d -1 Mixed Layer DCM

g C l g 30 µ

10 Microzooplankton Ingestion ( Ingestion Microzooplankton 0 SL-11 BN-3 70m-25 ML-13 Station

1957

1958

1959

1960

1961

1962

1963

1964

1965

1966

1967 Fig. 6

1968

0.8

0.7 r12=0.579, p=0.030

0.6

0.5

0.4 g d-1

0.3

0.2

0.1

0.0 -0.04 -0.02 0.00 0.02 0.04 0.06 0.08 0.10 0.12

Fv/FmWSW-Fv/Fm20%WSW

1969

1970

1971

1972

1973

1974

1975 Fig. 7

1976

1977

1978

1979

1980

1981

Table 1. Dilution Experiments, Eastern Bering Sea, Summers 2008, 2009, 2010.

Water Water bc Regiona Date Station bc Regiona Date Station Depth (m) Temp. Depth (m) Temp.

(oC) (oC)

1 07/04/08 UP-3 5 5.39 N 8 07/05/10 ML-13 35 -1.07 B

1 06/15/09 UAP-7 3 6.08 8 06/27/10 SB-5 4 5.44 N+P

1 06/16/09 UAP-3 3 6.49 8 07/03/10 MN-16 7 5.36 B

1 06/18/10 UAP-5 5 5.02 8 07/03/10 MN-18 26 1.65 N

1 06/19/10 UAP-2 3 4.19 N+P 9 07/10/08 C-55 4 5.41 N

2 06/17/09 CN-2 3 3.89 9 07/23/08 MN-6 5 6.13 N

3 07/05/08 CN-6 7 5.12 9 07/28/08 70m-36 5 6.75

3 06/20/09 CNN-6 32 4.33 9 07/06/09 XB2-4 4 3.92

3 06/20/10 CN 8 7 3.24 N+P 9 07/09/09 70m41 4 4.15 N+P

3 06/23/10 CNN 4 5 2.86 N+P 9 06/30/10 70m-40 5 4.59 N+P

4 06/21/10 CN 17 3 5.58 9 07/02/10 MN-9 5 5.08 CF, B

4 06/18/09 CN-12 5 6.44 9 07/09/10 70m-50 36 -1.32 N+P

4 06/24/09 SB-1 5 6.16 9 07/10/10 70m-39 5 5.18 N

5 06/25/10 NP 9 6 3.77 N+P 10 07/12/08 SL-8 4 5.94 N

5 07/19/08 NP-11 3 4.26 N 10 07/26/08 SL-14 6 6.57 N

6 07/14/08 NP-7 7 5.21 N 10 07/27/08 70m-53 7 6.77

6 06/22/09 NP-7 7 5.25 N* 10 07/08/09 70m58 10 3.30 N+P

6 07/10/09 70m-25 6 4.67 10 07/06/10 ML-3 7 5.21

6 07/11/10 70m-25 3 6.53 10 07/07/10 SL 11 6 5.68 B

6 07/11/10 70m-25 28 -0.30 B 10 07/07/10 SL 11 33 -1.20

7 06/24/10 NP 1 4 3.62 N+P 10 07/08/10 BN-3 5 5.69 C

7 07/01/10 MN-1 5 3.52 N+P 10 07/08/10 BN-3 30 -1.22

8 07/18/08 NP-14 5 7.41 N 10 07/09/10 70m-51 6 6.04

8 07/15/08 NP-14 4 7.32 N 10 07/06/10 ML-3 7 5.21 N

8 07/20/08 LS1-6 5 7.47 N 11 07/09/08 MN-3 3 3.31 N

8 07/22/08 P14-2 7 7.07 N 11 07/11/08 W-4b 4 4.53

8 07/24/08 MN-12 7 6.24 N 11 07/01/09 MN-3 2 2.27 N*

8 07/25/08 MN-19 9 7.90 N 15 06/23/09 NP-15 4 6.40 N*

8 07/03/09 MN-19 10 6.03 15 06/26/09 P14-7 3 5.86 N+P

8 06/29/09 XB16 3 6.49 N* 15 06/28/10 P14N-10 10 5.77 N+P

8 07/04/09 X-6 2 5.50 N* 15 06/29/10 TR3 5 6.22 N

8 07/05/09 XB2-12 2 6.34 15 07/21/08 P14-4.5 7 7.72

8 06/26/10 TD2 4 5.86 N+P 15 06/25/09 SB-7 5 6.35

8 06/27/10 SB-5 4 5.44 N+P 16 06/19/09 CN-20 7 6.31 N

8 07/03/10 MN-16 7 5.36 N+P 16 07/06/08 PIT-1 6 7.09 N

8 07/03/10 MN-18 26 1.65 B 16 07/07/08 sta21 5 6.92

8 07/04/10 TM4 35 1.67 B

8 07/05/10 ML-13 3 5.97

1982

1983

1984

1985

1986

1987

1988

1989

1990

1991

1992

1993

1994 Table 2. Effect of nutrient additions (+nuts) on phytoplankton growth (µ) and microzooplankton grazing

1995 (µ) in dilution experiments, mixed layer, summer, Eastern Bering Sea 2008, 2009, 2010. Mean (SD).

1996

Year Number of Control + Nutsa Control Experiments vs. Nuts b 2008 21 µ 0.31(0.178) 0.31(0.204) ns g 0.24(0.237) 0.11(0.224) * 2009 5 µ 0.17(0.178) 0.18(0.159) ns g 0.12(0.114) 0.08(0.113) ns 2010 15 µ 0.28(0.157) 0.36(0.165) ** g 0.21(0.125) 0.21(0.138) ns 1997 a. 2008: 5 µM N as sodium nitrate; 2009: 5 µM N as NH4Cl; 2010: 5 µM N as sodium nitrate + 0.3 µM P as sodium 1998 phosphate. 1999 b. Repeated Measures ANOVA: ns=non-significant, *p<0.05, **p<0.01. 2000

2001

2002

2003

2004

2005

2006

2007

2008

2009

2010

2011

2012

2013 Table 3. Growth coefficients of phytoplankton (µ, d-1) and microzooplankton grazing coefficients (g, d-1),

2014 and proproportion phytoplankton daily growth grazed by microzooplankton (g/µ). Mixed Layer data for

2015 2008, 2009, 2010 combined by region. Mean (SD). For g and u, mean of all incubations without added

2016 nutrients, but “0” used for negative g. *=Number of experiments in which g was statistically significant

2017 (p<0.05). C=without outlier

2018

Region Chl a <200 % Chl Incubation µ g g/u µm (µg l-1) a PAR <200 (µE s-1 m-2) µm Off Shelf Southeast (n=3, 0.77(0.44) nd 742(300.1) 0.24(0.16) 0.16 (0.16) .67 1*) North(n=6, 4*) 0.60(0.54) 69(32) 278(119.3) 0.19(0.30) 0.46(0.57) .77c 0.30(0.12)c 0.23(0.09)c Outer Shelf South (n=3, 2*) 0.79(0.22) 90(14) 210(151.6) 0.32(0.20) 0.25(0.15) .78 North(n=14, 9*) 0.89(0.64) 79(14) 328(141.5) 0.25(0.24) 0.19(0.12) .76 Middle Shelf South (n=3, 2*) 0.38(0.22) 98(2) 517(146) 0.27(0.11) 0.24(0.06) .89 Mid-North(n=4, 0.40(0.35) 41(49) 241(41.0) 0.08(0.62) 0.19(0.20) 1.56 3*) St. Matthew 0.52(0.52) 62(1) 344(184.5) 0.25(0.15) 0.28(0.22) 1.17 (n=8, 6*) North(n=8, 4*) 0.33(0.41) 97(25) 440(188.4) 0.24(0.21) 0.34(0.28) 1.42 Inner Shelf (South, Mid- 0.47(0.28) 77(50) 263(116.2) 0.19(0.14) 0.22(0.25) 1.16 North & North combined)(n=6, 3*) AK Peninsula 0.71(0.47) 74(30) 334 0.45(0.23) 0.27(0.22) .60 (n=4, 2*) 2019

2020

2021

2022

2023

2024 Table 4. Deep chlorophyll maximum (DCM) Dilution Experiments, Middle Shelf, Summer 2010. For

2025 experiments with a paired Mixed Layer (ML) experiment, both presented. Means (SD).

2026

Domain Station Sample Temp. (oC) Avg. Chlorophyll µ g p-value - (region) Depth Incub. a (t0) (µg l Irradiance 1) (m) In Incubation µ E s-1 m-2 situ

Middle SL-11 6-ML 5.6 ~6 347 0.30 0.16 0.95 <0.001 (10) 33- -1.2 4 Dark 1.08 -1.03 0.70 <0.001 DCM

Middle BN-3 5-ML 5.7 ~6 524 0.10 0.29 0.31 <0.01 (10) 30- -1.2 0 Dark 0.85 0.03 0.21 <0.01 DCM

Middle(6) 70m-25 3-ML 6.5 ~6 262 0.16 0.39 0.40 <0.05 28- -0.3 4 Dark 1.84 -0.03 0.24 ns DCM

Outer (8) ML-13 3-ML 6.0 ~6 330 0.11 0.34 0.08 ns 35- -1.1 4 Dark 5.36 0.04 0.05 ns DCM

Outer (8) MN-18 26- 1.6 ~4 Dark 1.21 0.03 0.18 <0.1 DCM

Outer (8) TM-4 35- 1.7 4 Dark 0.65 0.08 0.15 <0.05 DCM

2027

2028

2029

2030

2031

2032 Table 5. Variable Fluorescence (Fv/Fm) at the end of mixed layer dilution experiments in undiluted

2033 (WSW) and diluted (20% WSW) treatments without and with the addition of 0.05 µM N as sodium

2034 nitrate. All experiments conducted in summer 2008. N=2 or 3.

Station Mean (SD), No nutrient addition T-test Mean(SD), Nutrient addition T-test WSW 20%WSW p WSW 20%WSW p C-55 0.345(0.0071) 0.357(0.0896) ns 0.348(0.0318) 0.353(0.4596) ns W-4b 0.237(0.0379) 0.263(0.0752) ns 0.245(0.0283) 0.283(0.0318) ns SL-8 0.350(0.0433) 0.327(0.0431) ns 0.353(0.0404) 0.426(0.0871) ns NP-7 0.330(0.0100) 0.232(0.0126) p<0.05 0.310(0.0100) 0.357(0.0416) ns NP-14 0.183(0.1626) 0.198(0.0775) ns 0.317(0.0635) 0.270(0.0200) ns NP-14 0.263(0.0115) 0.220(0.0200) p<0.05 0.268(0.0104) 0.267(0.0189) ns NP-11 0.341(0.0136) 0.315(0.0391) ns 0.362(0.0076) 0.300(0.0250) p<0.05 LS1-6 0.343(0.0152) 0.278(0.0202) p<0.05 0.345(0.0087) 0.265(0.0071) p<0.05 P14-4.5 0.253(0.0144) 0.158(0.0126) p<0.05 0.268(0.0293) 0.183(0.0231) p<0.05 P14-2 0.298(0.0126) 0.233(0.0252) p<0.05 0.313(0.0058) 0.245(0.0229) p<0.05 MN-6 0.280(0.0173) 0.227(0.0058) p<0.05 0.307(0.0115) 0.247(0.0058) p<0.05 MN-12 0.245(0.0071) 0.153(0.0153) p<0.05 0.273(0.0289) 0.150(0.0141) p<0.05 MN-19 0.250(0.0173) 0.183(0.0289) p<0.05 0.240(0.0265) 0.163(0.0321) p<0.05 SL-14 0.263(0.0306) 0.210(0.0346) ns 0.283(0.0643) 0.233(0.0106) ns 2035

2036

2037

2038

2039

2040

2041

2042

2043

2044

2045

2046 Table 6. Comparison of hydrographic conditions, phytoplankton growth coefficients and

2047 microzooplankton grazing coefficients from mixed layer dilution experiments conducted in the Bering

2048 Sea. Years classified as warm or cold, with high or low stratification index (Ladd and Stabeno, 2012;

2049 Stabeno et al., 2012b). Phytoplankton growth rates (µ) and MZ grazing rates (g) per day. Average with

2050 SD in parentheses, or range.

2051

Cruise dates and Water , oC Growth Grazing ref hydrographic conditions in SE Bering Sea July 99: south Bering 5.3-7.7 0.2-0.6 0.1-0.4 Liu et al., 2002 Sea (off shelf and outer 0.41 0.27 shelf) (cold year) (n=5) (n=5) July-August 99; SE 5.8-8.4 -0.7-.0.6 0.1-0.5 Olson and Strom, 2002 Bering Sea (cold year 0.33 0.29 with low stratification (n=13) (n=13) index) July-August 04: SE 9.3-13.4 0.0-1.0 0.0-0.27 Strom and Fredrickson, Bering Sea (warm year 0.35 (n=18) 0.13 (n=18) 2008 with high stratification index) April-May, 2008, 2009, -0.3(1.4) 0.17(0.14) 0.08(0.12) Sherr et al., submitted 2010-Non bloom, n=17 n=17 Eastern Bering Sea, ice edge (cold years) April-May, 2008, 2009, 0.8(1.7) 0.21(0.12) 0.09(0.08) Sherr et al., submitted 2010-Bloom, Eastern n=21 n=21 Bering Sea, ice edge (cold years) June-July, 2008, 2009, 2.3-7.9 -0.8-0.8 0.0-0.9 This study 2010-Eastern Bering Sea 0.26 (n=61) 0.26 (n=61) (cold years; low stratification index in 2008, stratification classification for 2009 & 2010 not yet available) 2052

2053

2054

2055

2056 Conclusions

2057 There are regional (domain) differences in microzooplankton coupling to phytoplankton in the 2058 eastern Bering Sea in summer (Chapters 1 and 2). In stratified shelf waters, microzooplankton (defined as 2059 < 200 µm, includes nanozooplankton) consumed most of primary production (Chapter 2). 2060 Microzooplankton biomass (20-200 µm) often exceeded phytoplankton biomass in the mixed layer 2061 (Chapter 1). Large and small crustacean zooplankton do not efficiently feed on small (<10 µm) 2062 phytoplankton (reviewed in Calbet and Saiz, 2005), and often most of the biomass of > 10 cells was 20- 2063 200 µm microzooplankton, not large phytoplankton such as diatoms. These data indicate that if 2064 zooplankton are present in stratified shelf waters in summer, they must be largely dependent on 2065 microzooplankton as food. In less stratified waters near the shelf break, in the “green belt” and in the 2066 Pribilof Island Domain, the ratio of microzooplankton biomass (20-200 µm) to phytoplankton biomass 2067 was lower but microzooplankton (<200 µm fraction) still consumed about 67-78% of daily phytoplankton 2068 production (Chapters 1 and 2). This indicates an important role for combined nanozooplankton and 2069 microzooplankton in carbon cycling and nutrient regeneration in these higher chlorophyll waters.

2070 In the northern middle domain there was a well developed deep chlorophyll maximum (DCM) 2071 (Chapter 1). Although microzooplankton grazing coefficients were not elevated in the DCM, 2072 microzooplankton ingestion of phytoplankton carbon was higher than in the mixed layer due to the high 2073 phytoplankton biomass (Chapter 2). These observations suggest that the DCM may be an important site 2074 of trophic transfer of phytoplankton carbon to microzooplankton, and in turn to zooplankton, on the 2075 northern shelf in summer.

2076 Mixotrophic ciliates were a dominant component of the 20-200 µm fraction of the plankton in 2077 stratified shelf waters, particularly on the northern shelf (Chapter 1). Mixotrophic ciliates are members of 2078 the microzooplankton because they ingest phytoplankton, but they are also functionally members of the 2079 phytoplankton due to their chlorophyll content and photosynthetic activities. The dominance of the ciliate 2080 microzooplankton by mixotrophs may be a common feature of high latitude, stratified seas in summer. 2081 The mixotrophic ciliates, because of their relatively large cell sizes, were probably important as food for 2082 crustacean zooplankton in the mixed layer (Dutz and Peters, 2008). Due to photosynthesis, mixotrophic 2083 ciliates can have higher gross growth efficiency than strict heterotrophs (Stoecker et al., 2009). In the 2084 eastern Bering Sea shelf in summer, this may have had a positive impact on food web transfer efficiency 2085 to crustacean zooplankton.

2086 Several factors complicate comparison of the role of microzooplankton in summers of “warm” 2087 versus “cold” years in eastern Bering Sea. One is that summer stratification and episodic mixing influence

2088 phytoplankton biomass and coupling of microzooplankton grazing to phytoplankton growth. Ladd and 2089 Stabeno (2012) show that in the existing data set, stratification was not significantly correlated with 2090 “warm” or “cold” years. Thus, it is probably not possible to predict the role of microzooplankton in 2091 Bering Sea food webs based on temperature classifications alone. Another important factor in comparing 2092 years is differences in stations sampled and cruise timing in different years (Chapter 2). If domain is 2093 considered, there are no clear differences in microzooplankton biomass among “warm” and “cold” years 2094 (Chapter 1).

2095 Project Connections

2096 I liked working on the larger Bering Sea Project and enjoyed learning about and being exposed to 2097 the higher “vertebrate” trophic levels; this was new to me because on previous cruises that I have 2098 participated in as a plankton ecologist there were no vertebrate ecologists.

2099 I was disappointed that I was not able to link my results more strongly to “bottom up” and “top 2100 down” results. It would have helped to have more data available on phytoplankton assemblages, not just 2101 chlorophyll, from the larger project. Food species do matter, even to microzooplankton. I think the 2102 general species composition of blooms makes a big difference to trophic transfer to microzooplankton and 2103 higher trophic levels.

2104 Discussions and collaborations with the phytoplankton investigators, especially M. Lomas and D. 2105 Stockwell, were very useful. Collaboration with J. Goes, a guest on the 2008 cruise, helped tremendously 2106 in interpretation of the results from the dilution experiments. Our collaboration on dilution experiments 2107 resulted in general insight into the application of this technique. I also wish there had been research on 2108 the summer cruises on mesozooplankton in general (not just krill) to which to link results on 2109 microzooplankton. Specifically it would have been good to have data on zooplankton populations and 2110 zooplankton grazing --- I had to resort to the “general” literature, not data specific to the Bering Sea to 2111 link my results to higher trophic levels. One question is whether microzooplankton are abundant on the 2112 shelf in summer because there are not enough mesozooplankton to exert significant top down control on 2113 them.

2114 Management or policy implications

2115 The effects of global warming, ocean acidification and climate variability (changes in storm 2116 frequency, mixing events, stratification as well as timing of the spring bloom) will impact marine food 2117 webs through changes not just in “chlorophyll” but in phytoplankton and microzooplankton composition. 2118 This along with photosynthetic biomass and size fraction is the “frontline” for biological, bottom up

2119 effects. Phytoplankton composition, at least “rough” taxonomic composition of biomass, should be 2120 included in long-term monitoring. The dominance of certain species (for example Phaeocystis pouchetti 2121 and Emiliania huxleyi) may largely determine what type of planktonic food web develops. Likewise the 2122 the species and size distribution of microzooplankton matter since they can be an important prey for 2123 crustacean zooplankton that support pelagic fisheries. In low chlorophyll waters dominated by small cell 2124 size phytoplankton, such as observed on the Bering Sea Shelf in summer, plastidic ciliates appear to be a 2125 particularly important link between primary production and crustacean zooplankton. Although plastidic 2126 ciliates are common in temperate as well as arctic waters during summer stratification, they appear to 2127 occur on the Bering Sea shelf (and in other summer stratified arctic and sub-arctic seas) at higher biomass 2128 than in temperate waters. It is unclear if climate change will influence the magnitude and role of this 2129 “trophic link” under summer, low chlorophyll conditions.

2130 Publications

2131 Stoecker, D.K., Weigel, A., Goes, J.I. submitted (a) Microzooplankton grazing in the eastern Bering Sea 2132 in summer. Deep Sea Res. II, Special Bering Sea Issue.

2133 Stoecker, D.K., Weigel, A., Stockwell, D.A., Lomas, M.W. submitted (b) Microzooplankton: Abundance, 2134 biomass and contribution to chlorophyll in the eastern Bering Sea in summer. Deep Sea Res. II, Special 2135 Bering Sea Issue.

2136 Poster and Oral Presentations

2137 ASLO/AGU, Feb 2010, Portland Oregon, Oral presentation: Summer importance of microzooplankton on 2138 Bering Sea Shelf. (co-authors Blattner, Stockwell)

2139 Invited Speaker, 5th International Zooplankton Production Symposium, Population Connections, 2140 Community Dynamics and Climate Variability. March 2011, Pucón, Chile. Acquired phototrophy in 2141 ciliates: Does it boost trophic transfer to mesozooplankton? (co-authors Blattner, Weigel and Stockwell)

2142 Outreach

2143 Diane Stoecker and Kristen Blattner participated in outreach during the summer cruise 2008 by 2144 preparing presentations and talking with oceanography summer camp students, teachers and citizens of 2145 St. George Island. Diane Stoecker participated in an outreach webinar during the summer cruise. Diane 2146 Stoecker will use material from the cruise in teaching a graduate course in Biological Oceanography at 2147 University of Maryland. Diane Stoecker has digital micrographs of Bering Sea plankton (obtained in 2148 collaboration with John Casey) and digital photographs of research activities from the cruise which were

2149 available to other researchers on the cruise and which are available to the BSIERP program. Diane 2150 Stoecker used material from the 2008 cruise in teaching a graduate course in Biological Oceanography at 2151 University of Maryland.

2152

2153 Acknowledgements

2154 Refer to acknowledgements in Chapters 1 and 2.

2155 Literature cited

2156 Refer to references in Chapters 1 and 2.

2157