# Effects of increased pCO2 and temperature on the North Atlantic spring bloom. III. Dimethylsulfoniopropionate

P.A. Lee, A.R. Rudisill, A.R. Neeley,, J.M. Maucher,, David A. Hutchins, Y.

Feng, C.E. Hare, Karine Leblanc, J.M. Rose, S.W. Wilhelm, et al.

► To cite this version:

P.A. Lee, A.R. Rudisill, A.R. Neeley,, J.M. Maucher,, David A. Hutchins, et al.. Effects of increased pCO2 and temperature on the North Atlantic spring bloom. III. Dimethylsulfoniopropionate. Marine Ecology Progress Series, 2009, 388, pp.41-49. 10.3354/meps08135. hal-00700399

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# Effects of increased pCO₂ and temperature on the North Atlantic spring bloom. III. Dimethylsulfoniopropionate

Peter A. Lee<fnref n="1" />*, Jamie R. Rudisill<fnref n="1" />, Aimee R. Neeley<fnref n="1" />,<fnref n="7" />, Jennifer M. Maucher<fnref n="2" />, David A. Hutchins<fnref n="3" />,<fnref n="8" />, Yuanyuan Feng<fnref n="3" />,<fnref n="8" />, Clinton E. Hare<fnref n="3" />, Karine Leblanc<fnref n="3" />,<fnref n="9" />,<fnref n="10" />, Julie M. Rose<fnref n="3" />,<fnref n="11" />, Steven W. Wilhelm<fnref n="4" />, Janet M. Rowe<fnref n="4" />,<fnref n="5" />, Giacomo R. DiTullio<fnref n="1" />,<fnref n="6" />

**ABSTRACT:** The CLAW hypothesis argues that a negative feedback mechanism involving phytoplankton-derived dimethylsulfoniopropionate (DMSP) could mitigate increasing sea surface temperatures that result from global warming. DMSP is converted to the climatically active dimethylsulfide (DMS), which is transferred to the atmosphere and photochemically oxidized to sulfate aerosols, leading to increases in planetary albedo and cooling of the Earth's atmosphere. A shipboard incubation experiment was conducted to investigate the effects of increased temperature and pCO₂ on the algal community structure of the North Atlantic spring bloom and their subsequent impact on particulate and dissolved DMSP concentrations (DMSPₚ and DMSPₐ). Under 'greenhouse' conditions (elevated pCO₂; 690 ppm) and elevated temperature (ambient + 4°C), coccolithophorid and pelagophyte abundances were significantly higher than under control conditions (390 ppm CO₂ and ambient temperature). This shift in phytoplankton community structure also resulted in an increase in DMSPₚ concentrations and DMSPₐ:chl a ratios. There were also increases in DMSP-lyase activity and biomass-normalized DMSP-lyase activity under 'greenhouse' conditions. Concentrations of DMSPₐ decreased in the 'greenhouse' treatment relative to the control. This decline is thought to be partly due to changes in the microzooplankton community structure and decreased grazing pressure under 'greenhouse' conditions. The increases in DMSPₚ in the high temperature and greenhouse treatments support the CLAW hypothesis; the declines in DMSPₐ do not.

**KEY WORDS:** Particulate DMSP · Dissolved DMSP · Climate change · Global warming · Carbon dioxide · Temperature · Biogeochemistry

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## INTRODUCTION

The CLAW hypothesis (Charlson et al. 1987) postulates that changes in the Earth's radiation budget and any subsequent changes in oceanic conditions will be

mitigated through a feedback mechanism involving phytoplankton, the climatically active compound dimethylsulfide (DMS) and planetary albedo. In essence, the CLAW hypothesis proposes that higher sea surface temperatures (SST) will result in an enhanced DMS

<footnote n="1">¹Hollings Marine Laboratory, College of Charleston, 331 Fort Johnson Road, Charleston, South Carolina 29412, USA</footnote>

<footnote n="2">²Center for Coastal Environmental Health and Biomolecular Research, National Oceanic and Atmospheric Administration, 219 Fort Johnson Road, Charleston, South Carolina 29412, USA</footnote>

<footnote n="3">³College of Marine and Earth Studies, University of Delaware, 700 Pilottown Road, Lewes, Delaware 19958, USA</footnote>

<footnote n="4">⁴Department of Microbiology, University of Tennessee, 1414 West Cumberland Ave, Knoxville, Tennessee 37996, USA</footnote>

<footnote n="5">⁵Department of Plant Pathology, The University of Nebraska, 205 Morrison Center, Lincoln, Nebraska 68583, USA</footnote>

<footnote n="6">⁶Grice Marine Laboratory, College of Charleston, 205 Fort Johnson Road, Charleston, South Carolina 29412, USA</footnote>

<footnote n="7">⁷Present address: National Aeronautics and Space Administration, Calibration and Validation Office, 1450 S. Rolling Road, Suite 4.111, Halethorpe, Maryland 21227, USA</footnote>

<footnote n="8">⁸Present address: Department of Biological Sciences, University of Southern California, 3616 Trousdale Parkway, Los Angeles, California 90089, USA</footnote>

<footnote n="9">⁹Present address: Aix-Marseille Université, CNRS, LOB-UMR 6535, Laboratoire d'Océanographie et de Biogéochimie, OSU/Centre d'Océanologie de Marseille, 163 Avenue de Luminy, 13288 Marseille Cedex 09, France</footnote>

<footnote n="10">¹⁰Present address: CNRS (CNRS/INSU), UMR 6535, Campus de Luminy, Case 901, 163 Avenue de Luminy, 13288 Marseille Cedex 09, France</footnote>

<footnote n="11">¹¹Present address: Biology Department, MS #32, Woods Hole Oceanographic Institution, 266 Woods Hole Road, Woods Hole, Massachusetts 02543, USA</footnote>
flux to the atmosphere, presumably due to an increased production of dimethylsulfoniopropionate (DMSP), the precursor to DMS, by phytoplankton. According to the CLAW hypothesis, this DMSP increase could occur either through an increase in phytoplankton biomass or a shift in community structure towards DMSP-producing species. It is assumed that any increases in DMSP concentrations will lead to increases in the highly volatile DMS. Enhanced vertical mixing rates (i.e. stronger winds), or decreased microbial DMS consumption, however, could also increase sea-to-air DMS fluxes without a concomitant rise in DMSP production (Simó & Pedrós-Alió 1999). Once in the atmosphere, DMS would be rapidly oxidized to sulfur dioxide, with a subsequent increase in the formation of sulfate aerosols, which are a significant source of cloud condensation nuclei in remote marine environments (Ayers & Cainey 2007). Consequently, cloud coverage and ultimately planetary albedo would increase, thereby altering the Earth's radiation budget and providing a negative feedback mechanism for the regulation of planetary climate (Vallina & Simó 2007).

Global models predict that by the end of this century concentrations of atmospheric carbon dioxide (pCO₂) will have risen from current levels of 380 to >700 ppm (IPCC 2007). These changes in atmospheric CO₂ will also impact the world's oceans with anticipated increases in SST of 1 to 4°C, decreases in ocean pH of 0.3 units, and major impacts on upper-ocean stratification (Sarmiento et al. 1998, Wolf-Gladrow et al. 1999, IPCC 2007). Furthermore, such changes will have dramatic effects on marine phytoplankton communities, in terms of both the cellular physiology of individual species and the taxonomic composition of the communities (Boyd & Doney 2002, Tortell et al. 2002, Hare et al. 2007b).

Currently, there is little direct information on the effects of climate-sensitive variables on algal community structure and the production of DMSP or DMS. While an earlier study was unable to find a link between long-term climate variations and DMS levels (Bates & Quinn 1997), more recent studies have suggested that climate-driven variations in SST and mixed layer depth may influence DMSP and DMS levels (Simó & Pedrós-Alió 1999, Wong et al. 2006). Even results obtained during a high-latitude mesocosm experiment have led to differing conclusions regarding the role of increasing pCO₂ on DMSP and DMS cycling. Wingenter et al. (2007) concluded that, although absolute DMS concentrations were not different when pCO₂ levels were doubled or tripled, time-integrated values of DMS were 26 and 18% higher, respectively. During the same experiment, Vogt et al. (2008) observed no differences in particulate DMSP (DMSPₚ), dissolved DMSP (DMSPₐ), total DMSP (DMSPt), or DMSP-lyase activity (DLA), leading them

to conclude that the system had ‘a certain resilience’ to changing pCO₂. These authors also reported that temporal differences in DMS levels were observed, but concluded that they were not statistically significant.

The overarching objective of the North Atlantic spring bloom (NASB) expedition in 2005 was to investigate the impact of predicted increases in SST and pCO₂ on the algal community structure and biogeochemistry of the NASB, one of the most predictable and geographically extensive oceanic phytoplankton blooms (Sverdrup 1953, Lewis 1989, Savidge & Williams 2001). The area of the North Atlantic Ocean encompassed by the NASB is known to be an important source of DMS as result of the presence of small prymnesiophytes, such as the coccolithophorid *Emilia huxleyi* (Sieracki et al. 1993, Simó et al. 2002, Steinke et al. 2002). The presence of these calcifying phytoplankton species also makes this region a critical sink for anthropogenic CO₂ (Sabine et al. 2004). However, despite numerous intensive studies (Ducklow & Harris 1993, Sieracki et al. 1993, Savidge & Williams 2001), little is known about how this ecosystem will respond to the effects of climate change. Using a shipboard incubation system (Hutchins et al. 2003), seawater samples were manipulated to mimic SST and pCO₂ levels predicted for high-latitude oceanic regimes by the year 2100 (IPCC 2007).

This article is the third in a series of 3 companion papers that detail the findings made during the incubation experiment and focuses on the changes in DMSP concentrations and DLA that result from changes in algal community composition. The lead article (Feng et al. 2009, this volume) describes the impact of increasing temperature and pCO₂ on various biogeochemical parameters and the algal community composition, with particular emphasis on whole-community photosynthetic rates and the potential for decreased export of particulate inorganic carbon (PIC) relative to particulate organic carbon (POC) under ‘greenhouse’ conditions. The second article (Rose et al. 2009, this volume) describes the changes in and links between microzooplankton grazing dynamics and the algal community composition.

## MATERIALS AND METHODS

The experiment was conducted aboard the RV 'Seward Johnson' between June 20 and July 4, 2005 using seawater collected at 57.58°N, 15.32°W. Additional information regarding the experimental methodology can be found elsewhere (Feng et al. 2009). However, to give the reader an overview of the incubation system, experimental design and manipulation, a summary is also presented here. The experi-
ment was carried out using acid-washed, temperature-
controlled, continuous-culture incubation systems
('Ecostats'; Hutchins et al. 2003, Hare et al. 2005) that
allow simultaneous manipulation of pCO₂ and temper-
ature levels. Temperature control was achieved by
the use of a thermostatically controlled heat-exchange
cooling system coupled to in-line electric heaters.

A trace-metal-clean (TMC) towed-intake pumping
system (Bruland et al. 2005) was used to collect surface
seawater (~5 to 10 m) containing the intact bloom com-
munity, which was passed through an acid-washed
200 µm Nitex mesh (to remove mesozooplankton graz-
ers) and collected in an acid-cleaned 50 l mixing car-
boy. The incubation water was then dispensed into
TMC clear polycarbonate bottles (2.7 l) for incubation.
At the same location, TMC surface seawater was fil-
tered through 0.2 µm in-line filters and transferred to
acid-cleaned 50 l carboys for use as dilution media dur-
ing the continuous-dilution phase of the experiment.
The use of TMC techniques for water collection was
considered essential, as changes in ambient iron con-
centrations resulting from inadvertent contamination
in the bulk seawater collected for the experiment, in
any of the carboys used to store the seawater or in any
of the incubation bottles could have significantly
altered the phytoplankton community composition and
intracellular DMSP concentrations and introduced ran-
dom errors into the analyses (Sunda et al. 2002,
Hutchins et al. 2003, Hare et al. 2007a). As a result of
low *in situ* nutrient concentrations, nitrate and phos-
phate were added to both the initial incubation bottles
and dilution media to give final concentrations of 5 and
0.31 µmol l⁻¹, respectively. These additions yielded
final Si:N and Si:P ratios of 0.13 and 0.08, respectively,
reflecting the silicate limitation typical of late bloom
conditions during the NASB (Feng et al. 2009).

The incubation bottles were then subjected to 4 dif-
ferent treatments, with 6 replicates per treatment, as
follows: (1) 12°C and 390 ppm CO₂ (referred to as
'ambient'); (2) 12°C and 690 ppm CO₂ ('high CO₂'),
(3) 16°C and 390 ppm CO₂ ('high temperature'), and
(4) 16°C and 690 ppm CO₂ ('greenhouse'). The 2 pCO₂
levels were achieved by gently pumping HEPA-
filtered gas streams of either ambient air (with
390 ppm CO₂) or a commercially prepared air/CO₂
mixture containing 690 ppm CO₂. The light intensity
within the ecostats was set to 30% of the surface irra-
diance using spectrally corrected blue plastic and neu-
tral-density shade screen (Hutchins et al. 2003). This
value corresponded to the light intensity measured at
the depth where the incubation water was collected
and was selected so that the light level experienced by
phytoplankton in the ecostats would mimic the aver-
age light intensity experienced by cells in the 40 to
50 m mixed layer.

The initial (starting) bloom community was grown in
batch culture mode (i.e. no dilution) with the amended
nutrients to avoid any lag-phase growth induced by
sampling and to allow the biomass to increase to pre-
vent immediate wash-out of the bloom community
upon dilution. On Day 4 of the experiment, the system
was switched to continuous culture mode at a dilution
rate of 0.5 d⁻¹, which corresponds to the 'typical'
whole-community growth rate for the area (Gaul et al.
1999). The continuous culture phase of the experiment
was carried out for an additional 11 d.

During the experiment, total phytoplankton biomass
(measured as chlorophyll *a* [chl *a*]) and carbonate sys-
tem parameters (dissolved inorganic carbon [DIC] and
pH) were monitored on a daily basis using samples col-
lected directly from the incubation bottles using a gas-
tight syringe. On a less frequent basis, samples were
collected directly from the incubation bottles for cell
counts (by microscopy and flow cytometry), DMSP<sub>t</sub>,
and DMSP<sub>d</sub>. Samples for DLA, and algal pigment com-
position, POC, and particulate organic nitrogen (PON)
were collected from the outflow. Full details of the ana-
lytical protocols used for chl *a*, DIC, pH, cell counts,
PON, and POC are presented in the companion papers
(Feng et al. 2009, Rose et al. 2009).

Samples for DMSP were collected and preserved
using the methodology of Kiene & Slezak (2006). Small
volumes of unfiltered seawater (≤20 ml) were pre-
served with 50% sulfuric acid (100 µl per 10 ml of sam-
ple) for the determination of DMSP. A second small
volume of each sample (≤20 ml) was gravity filtered
(Whatman GF/F), and the filtrate was preserved with
50% sulfuric acid for the determination of DMSP. All
DMSP samples were base-hydrolyzed and measured
using a cryogenic purge and trap system coupled to a
Hewlett-Packard 5890 Series II gas chromatograph fit-
ted with flame photometric detector (DiTullio & Smith
1995). DMSP, was calculated as the difference be-
tween DMSP and DMSP. A recent study (Kiene et al.
2007) has noted that DMSP can be underestimated
when colonial *Phaeocystis* sp. is present in samples
preserved using the Kiene & Slezak (2006) method.
Since colonial *Phaeocystis* was not observed in the
samples collected during the present study, the results
presented here are unlikely to have been significantly
affected by this problem.

DLA measurements were carried out following the
protocol of Steinke et al. (2000). Optimal conditions for
the lyase assay (25°C, pH 7, 500 mmol l⁻¹ sodium chlo-
ride) were determined using a strain of *Emiliania hux-
leyi* (CCMP 373) that is known to possess the DMSP-
lyase (Steinke et al. 1998).

Statistical analysis was conducted with the statisti-
cal software program R (www.r-project.org). A modi-
fied ANOVA test based on a percentile bootstrap
method (Wilcox 2003) was used to determine the significance of any observed differences among treatments on individual sampling days. Further details of the statistical analysis are described in Rose et al. (2009).

## RESULTS

Phytoplankton pigment analyses indicated that community structure changed significantly during the course of the experiment (Fig. 1). During the first 6 d of the experiment, the abundances of pelagophytes, as indicated by the concentrations of the pigment 19'-butanoyloxyfucoxanthin (19'-But; Fig. 1A), remained unchanged, while the abundances of haptophytes (presumably coccolithophorids based on microscopic analyses) appeared to decline, as indicated by the concentration of 19'-hexanoyloxyfucoxanthin (19'-Hex; Fig. 1B). At the same time, the abundances of diatoms, as indicated by the concentrations of fucoxanthin (Fuco; Fig. 1C), increased substantially in all treatments, with the smallest increases observed under greenhouse and high temperature conditions. On Day 6, the differences in Fuco concentrations observed between the control versus high temperature and control versus greenhouse were statistically significant (p < 0.05; Table 1).

In the second half of the experiment, a dramatic shift in community structure was observed. Diatom abundances declined in all treatments, while increases were seen in the relative abundances of pelagophytes and coccolithophorids under both high temperature and greenhouse conditions. On Day 14, levels of Fuco in the greenhouse treatment were significantly higher than in the high CO₂ treatment, but not higher than in the ambient control (Table 1, Fig. 1C). 19'-But concentrations in the greenhouse and high temperature treatments were significantly

<figure><img src="image_2.png" /><figcaption>Fig. 1. Average values of (A) 19'-butanoyloxyfucoxanthin (19'-But), (B) 19'-hexanoyloxyfucoxanthin (19'-Hex) and (C) fucoxanthin (Fuco) during the course of the experiment. Error bars represent the 90% confidence interval. See 'Materials and methods' for treatment details</figcaption></figure>

<table><caption>Table 1. Summary of the statistical analysis for 19'-butanoyloxyfucoxanthin, 19'-hexanoyloxyfucoxanthin and Fucoxanthin on Days 6 and 14 of the 14 d experiment. *p < 0.05 between the treatments; NS: no significant difference between the treatments. See 'Materials and methods' for treatment details</caption><thead><tr><th></th><th>High CO<sub>2</sub></th><th>Ambient vs. High temp.</th><th>Greenhouse</th><th>High CO<sub>2</sub> vs. High temp.</th><th>High temp. vs. Greenhouse</th></tr></thead><tbody><tr><td><strong>19'-butanoyloxyfucoxanthin</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Day 6</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td></tr><tr><td>Day 14</td><td>NS</td><td>*</td><td>*</td><td>NS</td><td>*</td></tr><tr><td><strong>19'-hexanoyloxyfucoxanthin</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Day 6</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td></tr><tr><td>Day 14</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td></tr><tr><td><strong>Fucoxanthin</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Day 6</td><td>NS</td><td>*</td><td>*</td><td>NS</td><td>NS</td></tr><tr><td>Day 14</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>*</td></tr></tbody></table>
<table><caption>Table 2. Summary of the averages (±1 SD; n = number of replicates) of the major pigments normalized to the sum of all fucoxanthin (Fuco) derivatives and sulfur parameters (particulate and total DSMP) normalized to the sum of the pigments for the major DMSP-producing taxa (19'-hexanoyloxyfucoxanthin [Hex] and butanoyloxyfucoxanthin [But]) as measured at the start of the experiment (initial) and on Day 14 (end of the experiment) in each of the treatments. See 'Materials and methods' for treatment details</caption><thead><tr><th>Treatment:</th><th>Initial</th><th>Ambient</th><th>High temp.</th><th>High CO<sub>2</sub></th><th>Greenhouse</th></tr></thead><tbody><tr><td><strong>Pigment ratios</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>But:ΣFucos</td><td>0.180 ± 0.003 (n = 3)</td><td>0.17 ± 0.05 (n = 6)</td><td>0.28 ± 0.13 (n = 6)</td><td>0.19 ± 0.07 (n = 6)</td><td>0.32 ± 0.13 (n = 6)</td></tr><tr><td>Fuco:ΣFucos</td><td>0.139 ± 0.001 (n = 3)</td><td>0.31 ± 0.01 (n = 6)</td><td>0.30 ± 0.11 (n = 6)</td><td>0.40 ± 0.11 (n = 6)</td><td>0.25 ± 0.04 (n = 6)</td></tr><tr><td>Hex:ΣFucos</td><td>0.680 ± 0.003 (n = 3)</td><td>0.52 ± 0.13 (n = 6)</td><td>0.43 ± 0.23 (n = 6)</td><td>0.42 ± 0.16 (n = 6)</td><td>0.43 ± 0.16 (n = 6)</td></tr><tr><td><strong>Sulfur ratios (nmol ng<sup>-1</sup>)</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>DMSP<sub>t</sub>:Hex + But</td><td>0.29 ± 0.03 (n = 3)</td><td>0.31 ± 0.05 (n = 6)</td><td>0.25 ± 0.04 (n = 6)</td><td>0.27 ± 0.06 (n = 5)</td><td>0.24 ± 0.08 (n = 6)</td></tr><tr><td>DMSP<sub>p</sub>:Hex + But</td><td>0.25 ± 0.03 (n = 3)</td><td>0.20 ± 0.04 (n = 6)</td><td>0.23 ± 0.03 (n = 6)</td><td>0.20 ± 0.06 (n = 5)</td><td>0.23 ± 0.07 (n = 6)</td></tr></tbody></table>

greater than those observed under ambient conditions. Similarly, 19'-But was significantly higher in the greenhouse treatment than in the high CO₂ treatment (Table 1, Fig. 1A). In the case of 19'-Hex, no significant differences were observed at the end of the experiment despite large increases in the 19'-Hex concentrations in the greenhouse treatment (Table 1, Fig. 1B).

Since it is unclear how environmental factors affect the specific pigment:chl a ratio in different phytoplankton groups, the relative contribution of pelagophytes, diatoms, and coccolithophorids to the total algal biomass was estimated by normalizing their diagnostic pigments to the sum of all fucoxanthin derivatives (ΣFuco), respectively (Table 2). Based on pigment analyses, algal chl a was dominated initially by coccolithophorids (53%), with contributions from diatoms (18%), dinoflagellates (15%), and pelagophytes (14%). However, by Day 14, dramatic differences in the community structure were evident that were apparently driven by experimental conditions in each treatment. A significant increase (55%) in the percentage of pelagophytes was observed in the 2 high temperature treatments relative to the 2 low temperature treatments. In contrast, the percentage of diatoms decreased by 27% in the high temperature treatments compared to the lower temperature treatments. Although the highest accessory pigment concentration (19'-Hex) for all treatments was observed for the coccolithophorid population, no significant change was observed in the relative percentage of coccolithophorid to the total algal community. It is worth noting that, while dinoflagellates made a small contribution to the initial phytoplankton community, there was a marked decline in dinoflagellate abundances, based on peridinin levels, in all treatments during the experiment (data not shown).

During the early stages of the experiment, concentrations of DMSP<sub>d</sub>, DMSP<sub>p</sub>, and DMSP<sub>p:chl a</sub> all declined (Fig. 2), most likely in response to the decline in

<figure><img src="image_3.png" /><figcaption>Fig. 2. Average values of (A) dissolved dimethylsulfoniopropaneate (DMSP<sub>d</sub>), (B) particulate DMSP (DMSP<sub>p</sub>) and (C) DMSP<sub>p:chl a</sub> ratios during the course of the experiment. Error bars represent the 90% confidence interval. See 'Materials and methods' for treatment details</figcaption></figure>
<table><caption>Table 3. Summary of the statistical analysis for DMSP<sub>d</sub> (dissolved), DMSP<sub>p</sub> (particulate), and DMSP<sub>p:chl a</sub> on Days 8 and 14, and DMSP-lyase activity (DLA) and DLA:chl a on Day 13 of the 14 d experiment. *p < 0.05 between treatments; NS: no significant difference between treatments. See ‘Materials and methods’ for treatment details</caption><thead><tr><th rowspan="2"></th><th colspan="3">Ambient vs.</th><th colspan="3">High CO<sub>2</sub> vs.</th></tr><tr><th>High CO<sub>2</sub></th><th>High temp.</th><th>Greenhouse</th><th>High temp.</th><th>Greenhouse</th><th>High temp. vs. Greenhouse</th></tr></thead><tbody><tr><td><strong>DMSP<sub>d</sub></strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Day 8</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td></tr><tr><td>Day 14</td><td>NS</td><td>*</td><td>*</td><td>NS</td><td>NS</td><td>NS</td></tr><tr><td><strong>DMSP<sub>p</sub></strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Day 8</td><td>NS</td><td>*</td><td>*</td><td>*</td><td>*</td><td>*</td></tr><tr><td>Day 14</td><td>NS</td><td>*</td><td>*</td><td>NS</td><td>*</td><td>*</td></tr><tr><td><strong>DMSP<sub>p:chl a</sub></strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Day 8</td><td>NS</td><td>*</td><td>*</td><td>*</td><td>*</td><td>NS</td></tr><tr><td>Day 14</td><td>NS</td><td>NS</td><td>*</td><td>NS</td><td>*</td><td>NS</td></tr><tr><td><strong>DLA</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Day 13</td><td>NS</td><td>NS</td><td>*</td><td>NS</td><td>*</td><td>NS</td></tr><tr><td><strong>DLA:chl a</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Day 13</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td></tr></tbody></table>

coccolithophorid abundances and the increase in diatom abundances. However, starting on Day 5, small increases in DMSP<sub>p</sub> and DMSP<sub>p:chl a</sub> were observed in the high temperature and greenhouse treatments relative to the ambient control and high CO<sub>2</sub> treatment.

On Day 8, DMSP<sub>p</sub> levels in the high temperature and greenhouse treatments were significantly higher (p < 0.05; Fig. 2B, Table 3) than in the high CO<sub>2</sub> treatment and ambient control. By the end of the experiment, DMSP<sub>p</sub> concentrations in the greenhouse treatment were significantly higher (2- to 4-fold greater; p < 0.05; Fig. 2B) than the final values measured in the other treatments. In the case of DMSP<sub>p:chl a</sub> ratios, the values for the high temperature and greenhouse treatments were also significantly higher (p < 0.05) on Days 8 and 14, and by the end of the experiment were 50 to 60% greater than under the ambient conditions (Fig. 2C).

Conversely, DMSP<sub>d</sub> concentrations in the high temperature and greenhouse treatments declined after Day 5 relative to the controls and high CO<sub>2</sub> treatments. By the end of the experiment on Day 14, DMSP<sub>d</sub> levels were significantly lower (p < 0.05) in both the high temperature and greenhouse treatments than under ambient conditions (Fig. 2A). The results obtained for DLA (Fig. 3A, Table 3) show a remarkably similar pattern to those obtained for DMSP<sub>p</sub>, with considerably higher DLA in the greenhouse treatment. At the end of the experiment, DLA rates were ca. 2.5-fold greater in the greenhouse treatment relative to the ambient treatment (p < 0.05; Table 3). DLA rates were also significantly different between the high CO<sub>2</sub> and greenhouse treatments. Although the DLA:chl a ratios (Fig. 3B, Table 3) tended to follow the same pattern as DLA, the differences between the greenhouse conditions and the other treatment conditions were not as marked and none were significant.

<figure><img src="image_4.png" /><figcaption>Fig. 3. Average values of (A) DMSP-lyase activity (DLA) and (B) DLA:chl a ratios at the start of the experiment (Initial) and in the 4 treatments on Day 13 of the 14 d experiment. Error bars represent the 90% confidence interval. See ‘Materials and methods’ for treatment details</figcaption></figure>
DISCUSSION

The results obtained for DMSPₚ during the experi-
ment were consistent with the expected changes pre-
dicted by the CLAW hypothesis. There was both an
increase in overall DMSPₚ concentrations and a shift in
phytoplankton community structure toward species
with a higher cellular DMSP content under green-
house conditions. Furthermore, the pigment-normalized
DMSP numbers did not vary greatly between treat-
ments, suggesting that the intracellular DMSP content
of the component of the phytoplankton community
responsible for DMSP production (the coccolitho-
phorids and pelagophytes) was not affected by the
changing conditions and confirms that the observed
changes in DMSPₚ and biomass-normalized DMSPₚ
resulted from increases in coccolithophorid and pelago-
phyte abundances.

Moreover, the results show that changes in DMSP<sub>p</sub>
(Fig. 2B to D) and in DLA (Fig. 3A,B) were driven pri-
marily by changes in temperature, with increasing
pCO<sub>2</sub> (alone) having no effect at ambient temperature.
Although DMSP<sub>p</sub> and DLA levels were higher under
greenhouse conditions than under elevated tempera-
ture alone by the end of the experiment, none of the
observed interactive changes were statistically signifi-
cant. This observation is perhaps not surprising given
that increasing pCO<sub>2</sub> does not increase intracellular
DMSP (it only rises under CO<sub>2</sub> limitation; Sunda et al.
2002), but can influence DMSP<sub>p</sub> levels by influencing
the taxonomic composition of the phytoplankton com-
munities (Tortell et al. 2002, Hare et al. 2007b). Despite
a number of methodological differences between the
present study and that of Vogt et al. (2008), the finding
that increasing pCO<sub>2</sub> alone had little effect on DMSP<sub>p</sub>
and DLA levels is remarkably consistent.

Intriguingly, the results also revealed that, under
greenhouse conditions, there was a decrease in
DMSP<sub>d</sub> concentrations, which contradicts the CLAW
hypothesis. This decline in DMSP<sub>d</sub> was likely associ-
ated with significant changes in microzooplankton
grazing pressure, species composition, and species
abundance at the end of the experiment (Rose et al.
2009). These changes in the microzooplankton com-
munity may themselves be a result of the observed
changes in the phytoplankton community, DMSP<sub>p</sub>, and
DLA. It is also worth noting that the same grazing/
DMSP<sub>p</sub>/DLA/DMSP<sub>d</sub> trends were observed in the high
temperature treatment, which was dominated ulti-
mately by pelagophytes.

Microzooplankton grazers have been shown to pref-
erentially avoid prey with high DLA. The mixing of
intracellular DMSP and the DMSP-lyase enzyme dur-
ing prey ingestion results in the formation of DMS and
acrylate, with the latter thought to render the prey

unpalatable to the predator (Wolfe & Steinke 1996,
Wolfe et al. 1997), or the presence of the former acting
as a ‘don’t eat me’ signal to deter grazing (Strom et al.
2003). A study has suggested that this mechanism may
have been partially responsible for the development
and persistence of large, anomalous coccolithophorid
blooms in the Bering Sea (Olson & Strom 2002). In the
current study, heavy grazing pressure in the green-
house treatment by the initial microzooplankton com-
munity may have allowed the coccolithophores and
pelagophytes to become the dominant taxa. As the
phytoplankton community became less and less palat-
able for the grazers, less grazing took place, resulting
in a decrease in the amount of DMSP released through
feeding, which was reflected in the decrease in DMSP<sub>d</sub>
levels.

Another potential factor that could influence DMSP
release and degradation is viral infection and lysis
(Malin et al. 1992). However, results obtained during
subsequent studies have not provided clear-cut evi-
dence of this possibility. In experiments using field
samples, the results have been ambiguous as to the
effect of viruses on DMSP or have shown no correla-
tion between viruses and concentrations of DMSP and
DMS (Bratbak et al. 1995, Wilson et al. 1998, 2002).
Conversely, various studies using cultures have found
that viruses can have a significant impact on the
release of intracellular DMSP and can suppress the for-
mation of DMS through the cleavage of DMSP by
DMSP-lyase (Hill et al. 1998, Evans et al. 2007). In the
present study, viruses were unlikely to have been a
significant factor for 2 reasons. First, *in situ* water col-
umn samples collected during the cruise in the NASB
region showed no discernable pattern of viral produc-
tion (Rowe et al. 2008), and samples from the ecostat
experiment showed no relationship between the treat-
ments and viral production (Rowe & Wilhelm unpubl.
data). Second, DMSP<sub>d</sub> was lower in all treatments at
the end of the experiment rather than higher, as would
be expected if viral infection resulted in cell lysis.

Unfortunately, the ecostat design precludes the
accurate measurement of DMS, as active bubbling of
the incubation bottles with the gas mixtures stripped
the DMS from the growth media. Consequently, it is
not known if the declines in DMSP<sub>d</sub> resulted from a
decrease in the release of DMSP<sub>p</sub> or an increase in
microbial consumption of DMSP<sub>d</sub> (or a combination of
both). Thus, whether or not changes in the phytoplank-
ton, microbial, or microzooplankton communities re-
sulted in changes in DMS concentrations would be
conjecture. A decline in microzooplankton abun-
dances and grazing rates could result in a decline in
the production of DMS. This scenario would be con-
trary to previous findings that suggest oceanic DMS
will increase as a result of global warming, either
through stratification and temperature effects (Simó & Pedrós-Alió 1999, Wong et al. 2006) or increasing pCO₂ (Wingenter et al. 2007, Vogt et al. 2008).

Whilst the findings of this experiment are consistent with the CLAW hypothesis in that oceanic changes associated with global warming will likely result in increased levels of DMSP, the findings do not fully support the notion that this change in DMSP will also result in an increase in DMS levels that would lead to an increase in cloud albedo and a negative feedback mechanism on global warming. Furthermore, a number of uncertainties also remain. The responses observed during the relatively short duration of the experiment may be the result of acclimation to the experimental conditions rather than an adaptive response to 100 yr of global warming. For example, although grazing pressure was suppressed under greenhouse conditions during the experiment, will an oceanic microzooplankton community develop that is fully capable of consuming the relatively unpalatable coccolithophorids? Conversely, if no such grazing community develops, will the DMSP content of the cells still be released in the surface waters due to cell lysis when the bloom becomes senescent? A reduction in POC export due to global warming (Laws et al. 2000, Bopp et al. 2001) could result in the retention of DMSP near the surface, where it can be converted to DMS and is available for transfer to the atmosphere. Finally, how will bacterial utilization of DMSP, either through uptake or degradation to DMS and other products, be influenced by increasing temperature and changes in other climate-sensitive variables?

The results of the present study along with those of its companion studies (Feng et al. 2009, Rose et al. 2009) highlight the complex manner in which marine ecosystems may respond to global warming. Physiological (bottom-up) responses to changes in pCO₂ and temperature by the phytoplankton community (Feng et al. 2009) led indirectly to changes in the top-down pressure exerted by microzooplankton grazers (Rose et al. 2009). The impact of increasing pCO₂ and temperature on DMSP observed during this experiment is the net response of those changes and shows that further work examining these complex interactions is required.

*Acknowledgements*. The authors thank the captain and crew of the RV 'Seward Johnson' and fellow cruise participants for their support and many fruitful discussions. The authors also gratefully acknowledge the efforts of several anonymous reviewers whose comments greatly improved this manuscript. The present study was made possible through funding from the Biological Oceanography and Office of Polar Programs of the U.S. National Science Foundation (OCE-0423418 [0741412], OCE-0722337 and ANT-0338111 [0741411]) to D.A.H., ANT-0528715 to J.M.R., OCE 0452409 to S.W.W., and OCE-0422890 to G.R.D.).

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