Introduction
The steady increase of carbon dioxide (CO2) due to anthropogenic
emission since the beginning of the industrial era has increased the
atmospheric concentration (Boyd et al., 2014). The ocean has a large carbon
sink capacity, and increasing atmospheric CO2 absorbed by the ocean is
changing the chemistry of the seawater, causing a decline in pH, termed
“ocean acidification” (OA; Boyd et al., 2014). OA has been shown to affect
various biological processes of diverse marine species (Doney et al., 2009;
Kroeker et al., 2010). For instance, OA can impact the biochemical and
elemental composition of organisms (Sato et al., 2003; Torstensson et al.,
2013), which can be transferred to higher trophic levels (Rossoll et al.,
2012). OA can also drive alterations in the community composition structure
of primary producers (Hare et al., 2007; Biswas et al., 2011; Schulz et al.,
2013). Strong CO2 effects may be particularly significant in marine
species that experience low natural fluctuations in CO2 (Riebesell,
2004). In contrast, coastal and brackish-water environments encounter wide
and frequent fluctuations in pCO2 (Hinga, 2002; Rossoll et al.,
2013) due to large fluxes of organic and inorganic carbon from river run-off
(Hinga, 2002), seasonal processes (Melzner et al., 2013) and upwelling of
CO2-enriched water (Feely et al., 2008), all of which lead to wider pH
variation in coastal systems compared to the open ocean (Hinga, 2002).
Consequently, it can be expected that coastal and brackish communities are
more tolerant of OA effects (Rossoll et al., 2013; Reusch and Boyd, 2013),
and adverse CO2 effects in terms of the biochemical composition of
primary producers and variations in community composition may be diminished.
Fatty acids (FAs) are the main components of lipids in cell membranes. In
particular, polyunsaturated fatty acids (PUFAs) have important physiological
roles in algae, which synthesise them in high amounts. Heterotrophs at higher
trophic levels cannot synthesise certain FAs de novo, especially PUFAs, and
have to acquire them from dietary sources (Izquierdo et al., 2001). Diverse
laboratory studies of monocultures showed that CO2 alters the FA profile
of individual algal species (Sato et al., 2003; Fiorini et al., 2010;
Torstensson et al., 2013; Bermúdez et al., 2015). A CO2-driven
change in algal food quality can be detrimental for grazers, as has been
shown in a laboratory study under elevated CO2 levels (Rossoll et al.,
2012). A strong decline of PUFAs in a diatom, grown at high CO2,
affected the FA composition of copepods grazing on them and severely impaired
their development and egg production rates. Furthermore, increasing seawater
CO2 can modify phytoplankton community composition by favouring certain
taxa of primary producers (Graeme et al., 2005). In particular, small-sized
cells benefit from high CO2 (Hare et al., 2007; Biswas et al., 2011;
Brussaard et al., 2013). This is ecologically relevant as taxonomic
phytoplankton groups have contrasting FA profiles (Galloway and Winder, 2015)
and a change in community structure can affect higher trophic levels. For
instance, a field study of two cladocerans with different phytoplankton
compositions as food sources showed decreased egg production, lipid reserves,
body size and abundance when fed with algae from an acidic lake (Locke and
Sprules, 2000).
The above observations suggest that changes in planktonic biochemical make-up
and associated shifts in community composition of primary producers as a
result of OA can affect the transfer of essential compounds to upper trophic
levels. Laboratory studies have shown that algae subjected to long-term high
CO2 levels can restore their physiological optima through adaptive
evolution (Lohbeck et al., 2012; Bermúdez et al., 2015) and that coastal
communities are resilient to OA-driven changes in community composition and
biomass (Nielsen et al., 2010; Rossoll et al., 2013). Therefore, it can be
expected that organisms in these areas are adapted to high CO2
fluctuations (Thomsen et al., 2010; Nielsen et al., 2010; Rossoll et al.,
2013), hampering any CO2-driven effects previously observed in plankton
communities (Locke and Sprules, 2000; Biswas et al., 2011).
The goal of the present study was to determine whether an increase in
CO2 affects phytoplankton community composition and their FA profile
and if any effects are transferred to grazers of a natural plankton
community in a coastal/brackish environment. A set of offshore mesocosms,
which enclosed a natural plankton assemblage of the Baltic Sea, were used as
experimental units. The CO2 levels ranged from current to projected next
century values (Boyd et al., 2014, scenario A2). Algal FAs were measured from
total seston and from the copepods Acartia bifilosa and
Eurytemora affinis, which were the dominant zooplankton during the
experiment (Almén et al., 2016).
Material and methods
Experimental set-up and CO2 manipulation
Our study was conducted during an offshore CO2 mesocosm perturbation
experiment off the Tvärminne Zoological Station at the entrance to the
Gulf of Finland at 59∘51.5′ N, 23∘15.5′ E during late
spring 2012. We used six enclosures with a length of 17 m containing
∼ 55 m3 of natural sea water (Paul et al., 2015). The mesocosms
were set up and manipulated as described in detail by Paul et al. (2015) and
Riebesell et al. (2013). Carbon dioxide enrichment was achieved in two phases
through the addition of CO2-saturated seawater to four out of six
mesocosms. In phase 1, CO2 was added in five steps between day 1 and day
5 to achieve values from ambient levels
(∼ 240 µatm) and a fugacity of carbon dioxide (fCO2)
up to ∼ 1650 micro-atmospheres (µatm). In phase 2 on day 15,
CO2 was again added in the upper 7 m to compensate for pronounced
outgassing in the CO2-enriched mesocosms. As described by Paul et
al. (2015), dissolved inorganic carbon and total pH (on the total pH scale)
were taken every sampling day to determine the carbonate system and determine
fCO2 in the mesocosms. Samples for nutrients were collected and
analysed as described by Paul et al. (2015). Samples for phytoplankton counts
were taken every second day and for fatty acid concentrations every fourth
day using a depth-integrated water sampler (Hydrobios, Kiel, Germany), which
covered the upper 15 m of the water column. Integrated zooplankton net tows
were taken every seventh day as described by Almén et al. (2016).
Phytoplankton abundance and biomass calculation
Phytoplankton cell counts up to a cell size of 200 µm were carried
out from 50 mL water samples, fixed with alkaline Lugol's iodine (1 %
final concentration) using the Utermöhl's (1958) method with an inverted microscope (ZEISS Axiovert 100). At
200× magnification, cells larger than 12 µm were counted
across half of the chamber area, while smaller cells were counted at
400× magnification on two radial strips. The plankton was identified
to genus or species level according to Tomas (1997); Hoppenrath et al. (2009) and Kraberg et al. (2010).
Algal biovolume was calculated according to geometric shapes and converted to
cellular organic carbon using taxon-specific conversion equations for
phytoplankton (Menden-Deuer and Lessard, 2000).
Fatty acid composition
For analysis of seston fatty acid (FA), 500 mL of seawater was filtered by a
100 µm size pore net and samples were collected in a pre-combusted
(450 ∘C, 6 h) Whatman GF/F (∼ 0.7 µm nominal pore
size) filters. For zooplankton, gravid copepod females of Acartia bifilosa and Eurytemora affinis were picked up with sterile
tweezers under two stereomicroscopes (Nikon SMZ800, 25× magnification
and Leica 25× magnification) and placed in pre-weighted tin cups. All
samples were immediately stored at -80 ∘C until analysis. FAs were
measured by gas chromatography as fatty acid methyl esters (FAMEs) following
Breteler et al. (1999). Lipids were
extracted overnight from the filters using 3 mL of a solvent mixture
(dichloromethane : methanol : chloroform in 1:1:1 volume ratios). As an
internal standard, FAME C19:0 (Restek, Bad Homburg, Germany; c= 20 ng of
component per sample) was added, and a C23:0 FA standard
(c= 25.1 ng µL-1) was used as an esterification efficiency
control (usually 80–85 %). Water-soluble fractions were removed by
washing the samples with 2.25 mL of KCl solution (c= 1 mol L-1), and the
remainder dried by addition of NaSO4. The solvent was evaporated to
dryness in a rotary film evaporator (100–150 mbar), redissolved in
chloroform and transferred into a glass cocoon. The solvent was evaporated
again (10–30 mbar), and esterification was performed overnight using
200 µL 1 % H2SO4 (in CH3OH) and 100 µL
toluene at 50 ∘C. Phases were split using 300 µL 5 %
sodium chloride solution, and FAMEs were separated using n-Hexane,
transferred into a new cocoon, evaporated and 100 µL (final volume)
was added. All solvents used were gas chromatography (GC) grade. FAMEs were
analysed using a Thermo GC Ultra gas chromatograph equipped with a non-polar
column (RXI1-SIL-MS 0.32 µm, 30 m, company Restek) and Flame
ionisation detector. The column oven was initially set to 100 and heated to
220 ∘C at 2 ∘C min-1. The carrier gas was helium at a
constant flow of 2 mL min-1. The flame ionisation detector was set to
280 ∘C, with gas flows of 350, 35 and 30 mL min-1 for
synthetic air, hydrogen and helium respectively. A 1 µL aliquot of
the sample was injected. The system was calibrated with a 37-component
FAME-mix (Supelco, Germany) and chromatograms were analysed using Chrom-Card
Trace-Focus GC software and the fatty acids were clustered according to their
degree of saturation: saturated (SFA), monounsaturated (MUFA) and
polyunsaturated (PUFA).
Calculated biomass after cell counts of the main plankton taxonomic
groups in the different CO2 treatments between days 1 and 29. Each
treatment is labelled with the average fCO2 level of the entire
experimental period (top).
Statistical analyses
The data were analysed with a nested mixed-effects ANOVA model
(LME) to determine
the differences in taxa biomass (µgC mL-1) and relative fatty
acid content (% in the seston and zooplankton) between the CO2
treatments (µatm fCO2), with fCO2, silicate, inorganic
nitrogen (nitrite + nitrate), phosphate, temperature and salinity as
fixed effects and sampling day and mesocosm position as nested random
variables (random distribution of CO2 treatments among the mesocosm).
Average mesocosm fCO2 was calculated for the total duration of the
sampling period plankton community composition (days 1 to 29) and for FA data
analysis (days 1 to 25 for seston FA and days -1 to 33 for zooplankton FA).
Linear regression models were used to determine the relation between PUFA and
phytoplankton biomass. The similarity in the structure of the plankton
community between the treatments was analysed using non-metric
multidimensional scaling (NMDS) with Bray distance, auto-transformation and 3
dimensions (k= 3). This analysis distributes the samples in an ordination
space according to the biomass of the different taxa in the community along
orthogonal principal components using non-Euclidean distances for ordination
space, which makes it more robust to the presence of zero values (Clarke,
1993). All statistical analyses were done using the R software environment
3.0.1 (R Development Core Team, 2013).
The
top panels show the mean of the calculated biomass of each plankton taxon in
(a) phase 1, between days 0 and 15 and (b) phase 2, between
days 15 and 29, in the CO2 gradient treatments. The bottom panels show
the relative biomass of the different plankton groups between
(c) phases 1 and (d) phase 2. The x axes show the
measured average fCO2 in each phase, error bars show standard error
in (a) and (b) (n= 5 for a; n= 5 for
b).
Results
Plankton community composition
The initial algal community consisted of post-bloom species dominated by
small-sized cells, with dinophyta being the most abundant phytoplankton group
in all mesocosm throughout the experiment followed by heterokontophyta,
euglenophyta, cholorophyta, cyanobacteria bigger than 5 µm (usually
filamentous) and small abundances of cryptophyta (Fig. 1). Microzooplankton
was present during the entire experimental period, particularly the
choanoflagellate Calliacantha natans (Fig. 1). The plankton
community was analysed from days 1 to 29, which comprised of two phases as
described by Paul et al. (2015). In phase 1 (from days 1 to 15),
phytoplankton biomass gradually increased until day 10 when a bloom started
and it reached a peak around day 15 in all treatments, while in phase 2 (from
days 17 to 29) the biomass began to decay from around day 19 up to day 29
(Fig. 1).
The more abundant taxa did not show differences in abundance between the
CO2 treatments on both phases (Fig. 2a, b). However, the biomass of some
of the less abundant groups was affected by CO2 within the different
phases. In phase 1, the nested mixed effects model analysis of the algal
biomass showed that chlorophyta decrease significantly towards high CO2
levels (Fig. 2a; LME, F= 7.27, p= 0.01, df= 20). Nevertheless, there
was a difference in the relative biomass of the more abundant plankton groups
between phases 1 and 2, with a decrease in dinophyta (37.2 ± 3.2 to
28.3 ± 2.9 %) and heterokontophyta (19.1 ± 2.2 % to
14 ± 2.6) from phase 1 (Fig. 2c) to phase 2 (Fig. 2d), and an increase
of euglenophyta (7.5 ± 1.4 % to 21 ± 2.7) and chlorophyta
(14.0 ± 1.5 % to 19.1 ± 2.4) in the same period. An NMDS
analysis of the entire phytoplankton community showed a rather homogeneous
community composition between the different CO2 treatments but variation
among sampling days, especially on day 7, when the diatom Melosira varians was abundant during that particular sampling day (Fig. S1 in the
Supplement).
(a) Relative polyunsaturated (PUFAs), monounsaturated
(MUFAs) and saturated (SFAs) fatty acids content in the seston as a function
of fCO2 between days 1 and 29. The x axes show the mean
fCO2 measured during the sampling period, bars shows standard error.
(b) Relative PUFA composition of the seston showed over time in the
876, 1012 and 1314 µatm fCO2 levels (high CO2
treatments) and the 362, 403 and 590 µatm fCO2 levels (low
CO2 treatments). Horizontal dashed line indicates the position of the
overall mean PUFA value.
Seston fatty acid composition
The PUFAs represented on average ∼ 26 ± 4, MUFAs
∼ 22 ± 3 and SFAs ∼ 52 ± 4 % of the total FA
content in the seston over the entire experimental period. The LME
analysis of relative PUFA content data showed no significant difference among
the CO2 treatments (LME, F45= 0.0, p > 0.05;Fig. 3a
PUFA). Neither did the MUFAs and SFAs show any significant change in
abundance in relation with CO2 (LME, F45= 0.0, p= 0.8 and
F45= 0.06, p= 0.79; Fig. 3a shows MUFA and SFA). However, the FA
composition of the seston showed that the relative PUFA content significantly
decreased over time in all mesocosms (linear regression, R2= 0.52,
t= -7.64, p < 0.0001, n= 22; Fig. 3b shows high CO2
treatments and low CO2 treatments), while the MUFA and SFA increased,
although the relation of both with time is weak (linear regression, R2=
0.12, t= 2.88, p= 0.005 and R2= 0.15, t= 3.26, p= 0.001,
n= 22; Fig. S2). Regarding specific PUFAs, 18:2n6c showed a significant
correlation with CO2 and Si, 16:3n4 with CO2, P and Si and 18:3n6
with CO2 and N (Fig. S3).
(a) Relation between sestonic relative polyunsaturated
fatty acids (PUFAs) with heterokontophyta (PUFA, heterokontophyta) and
dinophyta (PUFA, dinophyta) biomass. (b) Relation between relative
sestonic PUFA content with silicate and phosphate abundance in the
mesocosms.
Nevertheless, PUFAs showed a positive relation with heterokontophyta (linear
regression, R2=0.58, p < 0.001) and dinophyta (linear
regression, R2=0.41, p < 0.001) biomass (Fig. 4a), and with
an abundance of silicate (LME, F= 22.8, p < 0.001, df= 35)
and phosphate (LME, F= 9.3, p < 0.01, df= 35) in the
mesocosms (Fig. 4b). The PUFAs 18:2n6c and 18:3n3 showed a positive effect of
silicate, while 20:5n3c and 22:6n3c showed a significant effect of silicate
and phosphate (Fig. S4).
Copepod fatty acids
The overall PUFA content represented ∼ 12 % (311 ± 175 ng FA
mg dry wt-1) of the total FA of the copepod A. bifilosa and in
E. affinis it was ∼ 16 % (433 ± 597 ng FA mg dry
wt-1).
The FAs did not show a CO2-related effect in A. bifilosa (LME,
F= 0.62, p= 0.4374, df= 26; Fig. 5a) or E. affinis
(F= 0.13, p= 0.71, df= 26; Fig. 5b). Nevertheless, the relative PUFA
content of A. bifilosa and E. affinis showed a significant
decrease over time in all high and low CO2 treatments (linear
regression, A. bifilosa; R2= 0.22, t= -3.288, p= 0.002
E. affinis; R2= 0.47, t= -5.51, p < 0.0001;
Fig. 5c) but no specific PUFA in A. bifilosa (Fig. S5) or E. affinis did (Fig. S6). However there was a decrease in MUFA and an increase
in SFA in both copepod species (Fig. S7). Furthermore, the relative FA
content in E. affinis varied over time following the changes in the
seston FA. This relation was significant but weak for PUFA, MUFA and SFA
(Fig. S8), while in A. bifilosa this change appeared only in MUFA
(Fig. S8).
Panels (a) and (b) show the relative polyunsaturated
(PUFA), monounsaturated (MUFA) and saturated (SFA) fatty acid content in the
copepods Acartia bifilosa and Eurytemora affinis,
respectively, under the fCO2 gradient treatments between days 1 and
29. The x axes show the mean fCO2 measured during the sampling
period, bars shows standard error. (c) Relative PUFA composition of
Acartia bifilosa (Ac) and Eurytemora affinis (Eu) over time
in the 876, 1012 and 1314 µatm fCO2 levels (high CO2
treatments) and the 362, 403 and 590 µatm fCO2 levels (low
CO2 treatments). Horizontal dashed line indicates the position of the
overall mean PUFA value.
Discussion
Community composition
The plankton community composition in the present experiment changed over
time and showed few differences in relation to the different CO2
treatments. The observed absence of a strong CO2 effect on the community
composition in the present study is in line with the observations in the
western Baltic Sea (Thomsen et al., 2010; Nielsen et al., 2010; Rossoll et
al., 2013). In these studies, the authors suggested that the plankton
community is adapted to OA due to the recurrent large seasonal and daily
variance of pH and CO2 experienced by the communities in this productive
low-salinity region (Thomsen et al., 2010; Nielsen et al., 2010; Rossoll et
al., 2013; Almén et al., 2014). Our study region, a coastal zone in the
western Gulf of Finland in the northern Baltic Sea, is a brackish environment
with low salinity (∼ 5.7 ‰) and has a high fresh water run-off
(∼ 111 km3 yr-1; Savchuk, 2005) and a strong inter- and
intra-seasonal pH variability, sometimes reaching extreme values of 9.2 and
7.4 with an average of 8.1 (Brutemark et al., 2011). Therefore, it seems that
the plankton community in our study area, which experiences high natural pH
fluctuations, is composed of species and genotypes that are less pH/CO2
sensitive (Nielsen et al., 2010; Lohbeck et al., 2012; Melzner et al., 2013;
Rossoll et al., 2013), which allows them to cope with the CO2 range
applied in the current field experiment.
Chlorophytes were the only group that showed a significant response to the
CO2 treatment, although their contribution to total biomass was low.
Chlorophytes decreased at elevated fCO2, which is in contrast to
laboratory studies showing that several species in this group benefit from
high CO2 and can increase their growth rates (Tsuzuki et al., 1990; Yang
and Gao, 2003).
Seston FAs
The relative PUFA content of seston showed a significant decrease over time,
which can be attributed to nutrient depletion in the mesocosms, particularly
silicate and phosphate concentrations, which caused a decrease in dinophyta
and heterokontophyta abundances. These two groups of microalgae have been
identified as rich in PUFA content (Galloway and Winder, 2015) and their
decrease in the mesocosms explains the concomitant decrease in PUFA. Silicate
is required by heterokontophyta for the formation of new frustules during
cell division and, when limited, cell division stops (Flynn,
2000). Phosphorus is required for the
production of PUFA-rich membrane phospholipids during cell division and
growth (Guschina and Harwood, 2009). Nutrient limitation, which causes
reduced cell division rates, results in a lower production of phospholipid
and increased production of storage lipid, primarily triacylglycerols
(Guschina and Harwood, 2009). Triacylglycerols are rich in SFA and MUFA;
therefore the increase in triacylglycerols with nutrient limitation typically
resulted in decreased proportions of PUFA in most algae (Guschina and
Harwood, 2009). This is consistent with our observations in the mesocosms,
where the relative PUFA content of seston followed the phosphate
concentration. From this perspective, one may expect that any CO2 effect
in algal PUFA will occur when cells are actively growing, since nutrient
limitation (silicate and phosphorus) will have more profound consequences in
the physiology of the cell than an excess of CO2.
The absence of a PUFA response to CO2 is countered by a report of an
Arctic plankton community showing an increase of PUFA at high CO2 levels
during part of a mesocosm experiment experiencing nutrient additions (Leu et
al., 2013). This was attributed to a change in the plankton community
composition due to a rise in abundance of dinoflagellates at high CO2
(Leu et al., 2013). Our results show a decrease in PUFA due to a decline in
dinoflagellates. The different PUFA trends between these experiments can be
attributed to the specific plankton community composition and their related
FA profiles alongside limited phosphate and silicate in our study, which
causes a reduction of the biomass of some PUFA-rich taxa. Species composition
of a natural plankton assemblage determines its food quality properties as
distinct algal taxonomic groups have different FA composition profiles
(Galloway and Winder, 2015). A CO2-driven change in the Arctic plankton
community composition (Leu et al., 2013) promoted the presence of species
rich in PUFA. In our study the absence of a CO2 response in taxa
composition and the apparent influence of phosphate and silicate limitation
in the algal FA composition resulted in a rather homogeneous PUFA
concentration between CO2 treatments.
Copepod fatty acids
Our results showed that the PUFA concentration of the dominating copepod
species, A. bifilosa and E. affinis did not vary between
the different CO2 treatments. However, the PUFAs decrease in both
copepods over the experimental period reflects the decline in the PUFA
content of the seston. This observation is consistent with other studies
showing that copepods strongly rely on their diet as a source of FA and that
their composition, especially PUFA, mirrors the algae they graze on (Ishida
et al., 1998; Caramujo et al., 2007; Rossoll et al., 2012).
Several studies have shown a limited direct effect on CO2 in the copepod
FA of some species, like the genus Acartia, which is rather
insensitive to projected high CO2 exposure up to 5000 µatm
CO2 (Kurihara et al., 2004; Kurihara and Ishimatsu, 2008). Copepods
experience widely varying pH conditions on a daily basis during their
vertical migration, shown in the same area as the current study (Almén et
al., 2014), which may explain their tolerance to pH variations. Several
studies have demonstrated that food quality of the available prey in terms of
PUFA content can affect egg production, hatching success and nauplii survival
in copepods (Jónasdóttir, 1994; Jónasdóttir et al., 2009;
Caramujo et al., 2007). Indirect adverse CO2 effects through the diet of
primary consumers have been reported in laboratory and field experiments
(Rossoll et al., 2012; Locke and Sprules, 2000). However, the absence of a
CO2-driven change in the community composition of primary producers and
the homogeneous algal FA composition due to phosphate and silicate
limitations masked any noticeable CO2-related effects in the algae FA
profile which could have affected the copepods during our experiment.
Conclusions
Considering that the Baltic Sea is a coastal sea with a natural frequent and
wide pH variability (Omstedt et al., 2009), it can be expected that the
effects of OA on plankton communities will be rather small within the range
of predicted values for this century (Havenhand, 2012). A reduced OA
sensitivity in systems experiencing high CO2 fluctuations is supported
by our results and other studies using communities from the Baltic (Thomsen
et al., 2010; Nielsen et al., 2010; Rossoll et al., 2013). However, in
coastal upwelling areas undergoing an increase in hypoxic events, it is
likely that elevated CO2 values presently experienced by coastal
organisms and projected by the end of the century (Melzner et al., 2013) will
be more recurrent in the future (Feely et al., 2004), with a potential to
affect various properties of plankton communities.
Nonetheless, it is clear that the plankton community response to OA and
concomitant effects on its food quality for higher trophic levels will
strongly depend on the sensitivity of primary producers and on how OA affects
the species composition of plankton assemblages (Leu et al., 2013; Rossoll et
al., 2013). This result is important as any change in primary producers in
terms of FAs, in particular essential biomolecules such as PUFAs, may scale
up in food webs since FAs are incorporated into the lipids of larval fish
(Fraser et al., 1989; Izquierdo et al., 2001). Considering that fish is a
critical natural resource (FAO, 2010), adverse OA effects on food quality can
reach human populations, who rely on fisheries as an important food source
(Sargent et al., 1997; Arts et al., 2001).