Introduction
Increased concentrations of carbon dioxide (CO2) in the atmosphere is
changing the carbon chemistry of the world's oceans. CO2 dissolves in seawater, thereby decreasing ocean pH. Ocean acidification is
increasing fast and pH is expected to decrease by a further 0.14–0.43 pH
units during the coming century (IPCC, 2007). Acidification can cause
various problems to biochemical and physiological processes in aquatic
organisms. In addition to affecting calcification of calcareous organisms,
maintenance of acid-base equilibrium of body fluids may become more
difficult and have consequences, for example, on protein synthesis, metabolism
and volume control (Whiteley, 2011).
In a changing environment, populations can respond in three main ways:
through plastic responses of individuals, through genetic changes across
generations or through escaping in space or time by modification of
phenology. During rapid change, phenotypic plasticity, i.e. the ability of
an individual or a population to alter its physiological state, appearance
or behaviour in response to the environment, is of major importance
(West-Eberhard, 2003). Theory predicts that higher plasticity evolves in
extreme environments and that spatial heterogeneity and dispersal select for
higher plasticity (Chevin et al., 2013). One could, therefore, hypothesise
that organisms inhabiting a variable environment, such as the study area,
could be fairly plastic in their response to ocean acidification because
they have to cope with both seasonal and sudden changes in pH (Almén et
al., 2014; Lewis et al., 2013).
Proteomic studies suggest that oxidative stress is a common co-stress of
temperature and acidification (Tomanek, 2014). Increased production of
reactive oxygen species (ROS) may result in increased antioxidant and/or
repair costs as well as in reduced investment in reproduction or other
functions, such as immune defence. In addition, increased production of ROS
may lead to accumulation of oxidative damage and to acceleration
of senescence (Monaghan et al., 2009). There can also be a connection
between maternal oxidative balance and offspring quality. In birds, for
example, females allocate diverse antioxidants to the eggs that protect the
embryo from oxidative stress. This maternal effect has a positive effect on
offspring development and growth (Rubolini et al., 2006).
Copepods (zooplankton) are indispensable to the functioning of the whole
pelagic ecosystem and contribute significantly to many ecosystem services
(Bron et al., 2011). They provide, for example, food for early life stages
as well as some adults of many economically important fish species
(Steele, 1974; Cushing, 1990).
Previous results suggest that calanoid copepods have high buffering capacity
against projected ocean acidification for the year 2100 and beyond (Kurihara
and Ishimatsu, 2008; Weydmann et al., 2012; McConville et al., 2013; Vehmaa
et al., 2013), meaning that they are able to survive, grow, develop and
reproduce in lower pH (Reusch, 2014). However, there are also studies
showing negative impacts on moderate CO2 levels (Fitzer et al., 2012),
whereas most of the negative impacts have been discovered for extreme carbon
storage scenarios (Kurihara et al., 2004; Mayor et al., 2007; Weydmann et
al., 2012). Many studies have tested only one life stage, adult females, and
have, therefore, possibly underestimated the effects of ocean acidification on
copepods (Cripps et al., 2014a). There are indications that transgenerational
effects are the mechanism responsible for the high plasticity of copepod
reproduction against altered pH conditions (Vehmaa et al., 2012). This
maternal effect is most likely dependent on the condition of the mother,
the availability of food and the quality of her diet (Vehmaa et al., 2012;
Pedersen et al., 2014a). Paternal effects can also influence offspring
traits. The exposure of both parents to CO2 leads to fewer adverse effects
on egg production and hatching than exposure of only gravid copepod females
(Cripps et al., 2014b). Thor and Dupont (2015) also highlight the importance
of testing transgenerational effects. They found significantly lower copepod
egg production after two generations when exposed to 900 and 1500 compared to
400 µatm, but transgenerational effects alleviated the negative
CO2 response in 1500 µatm (Thor and Dupont, 2015).
We tested direct and indirect effects of ocean acidification (i.e. via food
quantity and quality) on the copepod Acartia sp. egg production rate
(EPR), egg-hatching success (EH), female body size measured as prosome
length (PL), as well as antioxidant capacity (ORAC). The study was conducted
in association with the KOSMOS (Kiel Off-Shore Mesocosms for Ocean
Simulations) project in the Baltic Sea (Paul et al., 2015). The study was
intended to cover the low-productivity late spring and early summer period,
i.e. the post-spring bloom period when pCO2 concentrations are at the
annual minimum. Over the annual cycle, pCO2 and pH vary substantially
at the study site as a result of biological activity and mixing/upwelling of
CO2-enriched deep water (Niemi, 1975; Omstedt et al., 2014). There are
also strong spatial gradients in seawater pCO2/pH, most prominently
between the surface layer and the CO2-rich deeper waters (Almén et
al., 2014). Thus, the copepods in the study area are likely to experience
strong changes in seawater carbonate chemistry, both seasonally and during
their diurnal migration. Total particulate carbon
(TPC < 55 µm) was used as the measure of food quantity.
Food quality was indicated by the carbon to nitrogen ratio of the same sized
fraction of seston (C : N < 55 µm) (Elser and Hasset,
1994; Sterner and Hessen, 1994). In addition, in order to separate
transgenerational plasticity (i.e. maternal and paternal effects) and the
effect of environment on copepod egg-hatching and development, we performed
an egg-transplant experiment. Half of the produced eggs were allowed to
develop in respective mesocosm water and the other half in water collected
outside the mesocosm bags.
Due to the high buffering capacity of Acartia sp., we hypothesised
that there are no fCO2-related differences in egg production rate, egg-hatching success and prosome length between the mesocosms. In addition, we
hypothesised that copepod eggs hatch and develop better in the same
environment in which they are produced, because transgenerational effects can
alleviate the negative effects of environmental change. Our third hypothesis
stated that low food quantity (TPC) and poor quality (high C : N) will
weaken the maternal effect by deteriorating the condition of the mother.
Finally, we tested whether mothers with higher ORAC produce
better quality offspring (EH) by calculating correlation coefficients between
the two variables.
Materials and methods
The study was performed in summer 2012 in the vicinity of Tvärminne
Zoological Station on the south-western coast of Finland. Six large mesocosms
were moored on site at the beginning of June. To enclose the natural plankton
community, the mesocosms were left open with only a 3 mm-sized mesh net
covering the top and the bottom during filling. After 4 days, the net was
removed and the top was pulled up 1.5 m above the water surface and closed
at the bottom (Riebesell et al., 2013; Paul et al., 2015). pH was ∼ 8
and fCO2 concentrations in the mesocosms prior to adjustment were
237 ± 9 µatm (average ± SD of daily measurements from
all bags). Four mesocosm were manipulated with CO2-enriched seawater,
during three consecutive days to reach fCO2 concentrations of
600–1650 µatm (Paul et al., 2015). Two untreated mesocosms were
used as controls. The water column was mixed at the beginning of the
experiment to avoid salinity stratification. Due to outgassing, CO2 was
also added on day 15 to the upper 7 m of the high-CO2 mesocosms to
maintain the treatment levels. No nutrients were added.
Sampling
Sampling took place once a week during the first 4 weeks of the experiment,
and once more at the end of the whole experiment (days 3, 10, 17, 24 and 45).
Mesozooplankton were sampled from all mesocosms by taking two hauls with a
300 µm net (17 cm diameter) from 17 m depth. The samples were
rinsed into containers with 4 L of seawater from respective mesocosm, taken
from 9 m depth with a water sampler (Limnos, Hydrobios). On the same day,
integrated water samples (0–17 m) were collected from all mesocosms and the
Baltic Sea, directly into 1.2 L Duran bottles that were closed without head
space. Water samples were kept in cool bags and zooplankton samples were
protected from light until transported to a temperature- and light-controlled
room at Tvärminne Zoological Station within 4 h. The light–dark cycle
in the room was 16 : 8 h and light intensity was 7 µmol
photons m-2 s-1 (LI-COR LI-1000). Temperature followed the in
situ temperature [9 ∘C (day 3), 11 ∘C (day 10),
15 ∘C (day 17), 16 ∘C (days 24 and 45)].
Measurements of egg production, egg-hatching success and prosome
length
Twenty adult Acartia sp. (17 females and 3 males) were picked with
pipettes from each sample using stereo microscopes and gently placed in
prefilled glass bottles with respective mesocosm water. The bottles were
closed without head space, to minimise CO2-outgassing during the
incubation. pH in the bottles was measured before closing and right after
opening them at the end of the incubation using an Ecosense pH 10
pH/temperature pen (Table S1 in the Supplement). The pen was calibrated with
standard buffer solutions (Certipur, Titripac pH 4.00, 7.00 and 10.00) every
second day. The bottles were incubated in temperature- and light-controlled
room in the conditions described above (Sect. 2.1) and mixed 3 times a day,
and their place on the shelf was changed randomly. After the incubation
(24.3 ± 2.3 h, average ± SD), the copepods and produced eggs
were filtered using 250 and 30 µm sieves respectively. The copepods
were counted and their viability checked before preserving them in
RNAlater (Sigma). RNAlater can affect size (Foley et al.,
2010), and the effect depends on the number of segments in the animal, i.e.
the more segments, the larger effect. Shrinkage is ∼ 15 % for
copepods (E. Gorokhova, Stockholm University, personal
communication, 2015). Prosome length of the preserved
female copepods was measured using a stereo microscope (Leica MZ12) and
ocular micrometre (total magnification 100×). As all the measured
copepods were adult females, we assume the shrinkage to be of a similar
proportion for all individuals, which means that our results are quite
conservative and comparable between mesocosms.
In the egg transplant experiment, the collected eggs were divided for
hatching into two 50 mL petri dishes with different conditions; one dish was
filled with respective mesocosm water and the other with Baltic water. The pH
of the water was measured as above before the incubations and right after the
petri dishes were opened after the incubation (Table S1). The eggs were
counted before the petri dishes were completely filled and sealed without
head space using Parafilm. Egg hatching was followed by counting the number
of remaining eggs on the dish through the lid using a stereo microscope twice
a day. When the number of eggs had remained the same on two consecutive
counting times, the dishes were opened and the water containing the remaining
eggs and hatched nauplii was preserved with acid Lugol's solution. The
hatching incubation time varied between 63.9 and 137.6 h, depending on
incubation temperature. Acartia sp. nauplii stages were determined
and the number of nauplii and remaining copepod eggs counted using a stereo
microscope.
Some adults, copepodites, nauplii or eggs could have ended up in the
incubation bottles or petri dishes with the unfiltered incubation water. The
possible additional adults and their contribution to the EPR, eggs
copepod-1 d-1) were taken into account as EPR was calculated using
the number of eggs and adult Acartia sp. females found in the
incubation bottles after the 24 h incubation. When estimating the
egg-hatching success (EH, %), the total number of hatched
Acartia sp. nauplii and remaining eggs at the end of the hatching
incubation were compared with the number of eggs were counted before the
hatching incubation. If the total number exceeded the egg number prior to
hatching, the most developed nauplii (> N4) were considered to be carry-over individuals and were, therefore,
not considered in the estimation of EH. For an estimation of the nauplii
development rate, the development index (DI) was calculated (Knuckey et al.,
2005) accordingly.
DI=∑i=03(Ni×ni)∑i=03nim,
where Ni is the assigned stage value (0 for eggs, 1 for N1, 2 for
N2 and 3 for N3 and N4) and ni the number of individuals at
that stage. We assume all the Acartia sp. adults and nauplii to be
species A. bifilosa. However, because another Acartia
species, A. tonsa occurs in the area in late summer too (Katajisto
et al., 1998), we cannot be completely sure that we only had one species in
the experiments.
Antioxidant capacity
For ORAC samples ∼ 25 live female Acartia sp. were picked from
every zooplankton sample onto a piece of plankton net in the temperature- and
light-controlled room on days 3, 10, 17 and 31. The net containing the
copepods was folded and stored in Eppendorf tubes at -80 ∘C. The
samples were homogenised in 150 µL Tris-EDTA buffer containing
1 % sarcosyl. The antioxidative capacity was assayed as ORAC (Ou et al.,
2001). As a source of peroxyl radicals, 2, 2-azobis (2-amidinopropane)
dihydrochloride (AAPH) (152.66 mM) was used and fluorescein was used as a
fluorescent probe (106 nM). We used trolox (218 µM, Sigma-Aldrich)
as a standard and the assay was performed on a 96-well microplate and to each
well, 20 µL sample, 30 µL AAPH and 150 µL
fluorescein were added. ORAC values were normalised to protein and expressed
as mg Trolox eq. mg protein-1. Protein concentration was measured with
NanoOrange® (Life Technologies).
C : N and TPC
Samples for TPC and C : N were collected onto GF/F filters (Whatman,
nominal pore size 0.7 µm) using gentle vacuum filtration
(< 200 mbar), then stored in glass petri dishes at
-20 ∘C. GF/F filters and petri dishes were combusted at
450 ∘C for 6 h before use. Gauze prefilters were used to separate
the size fraction < 55 µm. Filters were not acidified to
remove inorganic carbon; therefore total particulate carbon was used. C and N
concentrations were determined on an elemental analyser (EuroEA) following
Sharp (1974), coupled by a Conflo II to a Finnigan DeltaPlus mass
spectrometer and were used to calculate C : N ratios in mol : mol. For
further details on sampling and analyses, please refer to Paul et al. (2015).
Statistics
The effect of acidification and food quantity and quality on Acartia sp. EPR, PL, ORAC and nauplii development index (DI) was tested using linear
mixed effect models (LMM) with restricted likelihood (REML) approximation
from the nlme-package (Pinheiro et al., 2014), where EPR, PL or ORAC were
used as response variables, fCO2, TPC (< 55 µm) and
C : N as fixed explanatory variables and repeated measure of the mesocosms
over time as a random factor (Table 1). Due to the binomial nature of the
data, the effect of fCO2, TPC (< 55 µm) and C : N
on EH was tested with a generalized linear mixed model (GLMM) with Laplace
likelihood approximation, binomial error structure and logit link function
from the lme4-package (Bates et al., 2014) (Table 1). The average of
fCO2, TPC (< 55 µm) and C : N measurements from
each mesocosm within 3 days before the zooplankton sampling were used as
explanatory variables for EPR, ORAC and EH, because 2–3 days are considered
to be an appropriate acclimatisation period for A. bifilosa (Yoon et
al., 1998; Koski and Kuosa, 1999). For PL, the average of all fCO2,
TPC (< 55 µm) and C : N measurements from the start of
the mesocosm experiment were used since PL reflects the environmental
conditions of the whole lifespan of the animal. In addition, day 3 was
excluded in the LMM testing the PL (Table 1), since 3 days is too short a
period for detecting differences in copepod size. Egg to adult generation
time for A. bifilosa at 17 ∘C is approximately 16 days of
which ∼ 7.5 d taken by nauplii stages and ∼ 8.5 days by
copepodite stages (Yoon et al., 1998). Collinearity between all explanatory
variables was checked. Temperature was not considered in the models, because
it changed similarly in all the bags (Paul et al., 2015). The model
simplifications were done manually in a backward stepwise manner by removing
the non-significant effects and by using Akaike's information criterion
(AIC). We report t or z statistics (EH) of the retained fixed effects. To
separate the effect of the hatching environment from the maternal
environment, EH and DI were divided by the corresponding values measured in
the Baltic Sea water. The ratio of Mesocosm EH (or DI) / Baltic EH (or
DI) > 1 indicates that eggs hatch or develop better in the
maternal conditions (Mesocosm water), whereas the ratio < 1
indicates that eggs hatch or develop better in the Baltic Sea water. The
effect of maternal environment (fCO2, TPC (< 55 µm)
and C : N) on the ratio was tested with LMM, where the ratio of Mesocosm
EH / Baltic EH and Mesocosm DI / Baltic DI were used as response
variables; fCO2, TPC (< 55 µm) and C : N as fixed
explanatory variables; and repeated measure of the mesocosms over time as a
random factor. The model simplifications were made as above.
The structure of the full LMM or GLMM models that were used to test
the effects of ocean acidification, food quantity and food quality on copepod
EPR, EH, PL, ORAC, the ratio of EH mesocosm / EH Baltic and
the ratio of nauplii DI mesocosm / DI Baltic. The
sampling days that were included in each of the models are listed. Repeated
measures of the same mesocosm bags were used as a random effect in all the models,
because copepods that come from the same bags are more alike than copepods
from different bags.
Response variable
Fixed effects
Effect tested
Days included in the model
3
10
17
24
31
45
EPR (LMM)
fCO2
Ocean acidification
X
X
X
X
X
TPC (< 55 µm)
Food quantity
C : N (< 55 µm)
Food quality
EH (GLMM)
fCO2
Ocean acidification
X
X
X
X
TPC (< 55 µm)
Food quantity
C : N (< 55 µm)
Food quality
PL (LMM)
fCO2
Ocean acidification
X
X
X
X
TPC (< 55 µm)
Food quantity
C : N (< 55 µm)
Food quality
ORAC (LMM)
fCO2
Ocean acidification
X
X
X
X
TPC (< 55 µm)
Food quantity
C : N (< 55 µm)
Food quality
EH MC/Baltic (LMM)
fCO2
Ocean acidification
X
X
X
X
TPC (< 55 µm)
Food quantity
C : N (< 55 µm)
Food quality
DI MC/Baltic (LMM)
fCO2
Ocean acidification
X
X
X
X
TPC (< 55 µm)
Food quantity
C : N (< 55 µm)
Food quality
To test whether maternal ORAC correlates with egg-hatching success, Spearman
rank correlation tests were used. Data from days 3, 10 and 17 were included
in the test (n=17, EH result for MC (mesocosm) 6 in day 3 is missing) because those are the days when both ORAC
and EH were measured.
All the statistical analyses were performed using software R 3.0.2 (R Core
Team, 2013), and the significance level was 0.05.
Results
Egg production, prosome length, antioxidant capacity and egg-hatching
success
Acartia sp. EPR increased in all mesocosms between
day 3 and day 10, but decreased after that, reaching very low rates (1–2
eggs copepod-1 d-1) on days 24 and 45 (Fig. 1a). Neither food
quantity (TPC, < 55 µm), food quality (C : N,
< 55 µm), nor ocean acidification (fCO2) had a
statistically significant effect on copepod egg production (Table 2), even
though there seemed to be variations in those parameters between the mesocoms
(Table 3).
T-statistics of the retained fixed effects in the linear mixed
effect models testing the effects of TPC (< 55 µm), C : N and
fCO2 on EPR, female PL
and female ORAC. Repeated measures of same mesocosm
bags were used as a random effect in all the models, because copepods that
come from the same bags are more alike than copepods from different bags.
Response variable
Fixed effect
Estimate
DF
t
p value
EPR
TPC < 55 µm
0.21 ± 0.14
23
1.54
0.137
PL
fCO2
–0.000027 ± 0.000011
16
–2.39
0.030
TPC < 55 µm
–0.0037 ± 0.0017
16
–2.21
0.042
ORAC
TPC < 55 µm
–0.0045 ± 0.0021
22
–2.17
0.041
Development of Acartia bifilosa (a) egg
production, (b)
prosome length (average ± s.e.), (c) antioxidant capacity and (d) egg-hatching success in the mesocosms and (e) egg-hatching success in Baltic
water when eggs are produced in mesocosms in the course of the study. The
fCO2 (µatm) values represent the average in days 1–43
(Paul et al., 2015).
![]()
Ranges of fCO2, TPC < 55 µm and
C : N < 55 µm that were used as explanatory variables
in the full LMM and GLMM models. Three-day averages (measured within the latest
three days of the sampling day) were used for testing the effects of the
explanatory variables on copepod EPR, ORAC and EH, whereas average of all measurements
since the start of the experiments until the sampling day were used when
testing the effects of the explanatory variables on copepod size (PL).
Variations in fCO2, TPC < 55 µm, and
C : N < 55 µm in the course
of the study are presented in Paul et al. (2015).
fCO2 (µatm)
TPC < 55 µm
C : N < 55 µm
3-D average
Average since
3-D average
Average since
3-D average
Average since
Day 1
Day 1
Day 1
MC 1
267–477
267–365
15.1–31.6
21.4–31.6
5.51–8.43
7.26–8.03
MC 3
745–1201
884–1121
17.4–29.7
20.4–29.7
6.94–8.36
7.79–8.20
MC 5
275–481
274–368
15.8–24.5
19.2–24.8
7.24–8.57
7.24–7.59
MC 6
663–991
683–896
16.5–34.3
21.0–34.3
7.14–8.25
7.60–7.81
MC 7
390–565
390–497
17.5–30.0
21.4–29.9
6.92–8.25
7.43–7.74
MC 8
874–1525
1117–1413
17.4–26.3
21.6–26.3
7.16–8.53
7.59–7.93
PL of Acartia sp. females increased during the
first week of the study; however there seemed to be some differences between
the mesocosms already on day 3, which was not included in the analysis
(Fig. 1b). From day 10 onwards, the smallest A. bifilosa adults were
found in the mesocosm with the highest fCO2 concentration (Fig. 1b).
fCO2, but also TPC (< 55 µm) had a statistically
significant negative impact on copepod body size (Table 2).
ORAC of the female copepods increased from day 3 to day 10 in all mesocosms
(Fig. 1c). Interestingly, on day 3 ORAC was highest in the three mesocosms
and had the highest fCO2 treatment, whereas on day 31 the situation
was reversed and ORAC was lowest in the three mesocosms with the highest
fCO2 (Fig. 1c). Despite this, only TPC (< 55 µm)
had a statistically significant effect on ORAC, which decreases with
increasing TPC (Table 2).
The overall EH was high throughout the study: over 80 % of the
Acartia sp. eggs hatched. As seen for EPR, PL and ORAC, EH also
increased from day 3 to day 10 in all mesocosms (Fig. 1d). Variance in the EH
between the four samplings was highest in the mesocosms with highest
fCO2, whereas EH varied the least and remained > 90 %
in both control mesocosms (MC 1, MC 5). In spite of this, only TPC
(< 55 µm) had a statistically significant negative effect
on EH (Table 4). Eggs that were produced in MCs 3, 5, 6 and 7 had fairly
similar hatching success in Baltic water, whereas the hatching success of
eggs that were produced in MCs 1 (control) and 8 (the highest fCO2)
was alternately either lower or higher than in the other MCs (Fig. 1e).
Z statistics of the retained fixed effects in the GLMM testing the
effect of fCO2, TPC (< 55 µm) and C : N on EH. Repeated measures of the same mesocosm bags were used as a
random effect in the model, because copepods that come from the same bags
are more alike than copepods from different bags.
Response
Fixed effect
Estimate
z
p value
variable
EH
fCO2
–0.00062 ± 0.00032
1.94
0.052
TPC < 55 µm
–0.09557 ± 0.02505
3.82
< 0.001
Egg-hatching and nauplii development in mesocosm vs. Baltic Sea
conditions
Neither the maternal food quantity (TPC) nor the quality (C : N) had a
statistically significant effect on offspring quality (EH and DI) in the egg
transplant experiment (Table 5). The fCO2 was the only detected
variable in the maternal environment that influenced the ratio of EH and DI
between mesocosm and Baltic conditions.
T statistics of the retained fixed effects in the LMMs testing the
effect of fCO2, TPC (< 55 µm) and C : N on ratio
of EH mesocosm / EH Baltic and nauplii development
index (DI) mesocosm / DI Baltic. Ratio > 1: higher EH or DI in
the mesocosm water (maternal environment) than in the Baltic Sea water,
ratio < 1: lower EH or DI in the mesocosm water (maternal
environment) than in the Baltic Sea water. Repeated measures of same
mesocosm bags were used as a random effect in both models, because copepods
that come from the same bags are more alike than copepods from different
bags.
Response variable
Fixed effect
Estimate
DF
t
p value
EH mesocosm / EH Baltic
fCO2
–0.000061 ± 0.000028
16
–2.20
0.043
DI mesocosm / DI Baltic
fCO2
–0.000145 ± 0.000067
16
–2.15
0.047
Egg-hatching success for eggs hatching in the mesocosm water differed from
eggs hatching in the Baltic water. On days 3 and 10, hatching success was
higher in the mesocosm water for the control (MC 1, MC 5) and for
low-fCO2-treatment bags (MC 7, MC 6), whereas eggs produced in
high-fCO2-treatment bags (MC 3, MC 8) showed higher hatching in the
Baltic water (Fig. 2a). Thus, there may be a threshold fCO2 for
hatching success at high fCO2. However, on days 17 and 24 the
fCO2 treatment did not have a clear effect on hatching success.
Nevertheless, fCO2 had a statistically significant negative effect on
the ratio of EH mesocosm / Baltic, meaning that egg hatching was higher
in the Baltic water than in the maternal environment when the maternal
environment had a high fCO2 (Table 5). When the maternal environment had
low fCO2, the situation was reversed. The level of fCO2 also
had a significant negative effect on the DI mesocosm / Baltic ratio
(Fig. 2b; Table 5).
Development of the ratio of (a) EH
mesocosm / EH Baltic and (b) nauplii development index (DI) mesocosm / DI Baltic during
the study. Ratio > 1: higher EH or DI in the mesocosm water
(maternal environment) than in the Baltic Sea water, ratio < 1:
lower EH or DI in the mesocosm water (maternal environment) than in the
Baltic Sea water. Note that because of different development times, the DI
values are not comparable between the days. The fCO2 (µatm) values represent the average in days 1–43 (Paul et al., 2015).
![]()
Correlations between antioxidant capacity and offspring quality
Copepod ORAC was correlated significantly with copepod
egg-hatching success. The relationship between the two variables is positive
and stronger for eggs developing in the mesocosm water
(ρ = 0.75,
p < 0.001) than for eggs developing in the Baltic water
(ρ = 0.62, p = 0.007) (Fig. 3).
Correlations of copepod EH with maternal
ORAC.
![]()
Discussion
In this study, conducted in semi-natural mesocosm environments, reproduction
of the Acartia sp. copepod showed high phenotypic buffering against
acidification, i.e. the species was able to maintain similar egg production
rates and high egg-hatching success in all fCO2 conditions.
Nevertheless, we found a significant negative effect of ocean acidification
on adult female size. Even more interestingly, we found signs of a possible
threshold at high fCO2 for offspring development, above which adaptive
maternal effects cannot alleviate the negative effects of acidification on
egg hatching and nauplii development (Fig. 2). However, we did not find
support for the third hypothesis that lower TPC and
higher C : N would weaken the maternal effect by deteriorating
the condition of the mother. Conversely, higher TPC
< 55 µm correlated negatively with egg-hatching success,
adult female size and antioxidant capacity, whereas C : N ratio did not
correlate with any of the measured variables significantly. Copepods were
possibly food limited in all the mesocosms, especially after day 17 due to a
sharp decline in Chl a concentrations and in phytoplankton community size
structure (Paul et al., 2015). Dominance of picophytoplankton that are too
small to be consumed by copepods could be the reason for the observed
negative effects of food quantity and may also have masked the food
quality effect. Also, after day 17 egg production rate was so low that it was
practically impossible to find differences in egg production between the
mesocosms. Finally, we found a positive correlation between maternal
antioxidant capacity and egg-hatching success, suggesting that the female
antioxidant defence might also protect the embryo from oxidative stress.
The fact that Acartia sp. egg production and egg hatching were
unaffected by high fCO2 but the egg transplant experiment revealed
that development was slower for nauplii at high CO2 supports the
importance of looking beyond egg production and egg hatching, which is also
pointed out by Pedersen et al. (2014b). They concluded that the first
endogenously feeding nauplii stages of Calanus finmarchicus are more
sensitive to CO2-induced acidification than eggs or later nauplii stages
(Pedersen et al., 2014b). Longer developmental times in high
CO2 / low pH have been observed in crustaceans, echinoderms and
molluscs (Cripps et al., 2014a and references therein). Weydmann et
al. (2012) also reported a significant developmental delay for
Calanus glacialis eggs when exposed to highly acidified conditions.
Pedersen et al. (2014a) observed that development of C4 copepodites of
C. finmarchicus was delayed by 8.9 days in high-CO2 treatments
in comparison to the control condition, when also the previous generation had
been exposed to the same conditions.
We expected maternal effects to be most obvious in a high-stress situation
(high-fCO2 treatments), as seen for three-spined sticklebacks in a
study testing the effects of global warming (Shama et al., 2014). Instead,
egg hatching was higher and nauplii development faster in the maternal
environment than in the Baltic water, when the maternal environment had low
fCO2 (low stress). In the high-fCO2 maternal environment the
opposite response was observed, thus indicating that maternal effects are in
fact weak and cannot compensate for the higher fCO2 levels that
correspond to near-future levels or that the eggs are damaged by the high-fCO2. This suggests that Acartia sp. and its reproduction are
after all somewhat sensitive to ocean acidification. However, the effects
were not as clear over the following weeks as at the beginning of the study,
which may be due to an overall low egg number and large variation in hatching
after day 17 or to acclimation of the copepods to the treatment
conditions. In addition, the maternal effects seemed to weaken over time.
This could be due to the weakening condition of the mothers. In the absence of
fish predators, zooplankton density, especially Bosmina sp.
(cladocerans), increased strongly in the mesocosms (Lischka et al., 2015).
Senescence and food limitation were thus plausible problems for copepods and
a likely cause of weakening maternal provisioning. In addition, conditions in
the Baltic Sea changed after day 17 due to an upwelling event, which caused
an increase in fCO2 and decrease in pH (Paul et al., 2015). This might
have made the Baltic conditions less favourable for copepod egg development
and evened out the differences between high-fCO2 mesocosms and the
Baltic conditions.
A few studies have highlighted the importance of testing for
transgenerational effects to avoid over- or underestimation of the effects of
ocean acidification on copepods. Thor and Dupont (2015) found decreasing egg
hatching of Pseudocalanus acuspes with increasing pCO2. In
addition, transgenerational effects alleviated the negative effects on egg
production and hatching of the second generation when the mothers had been
acclimatised to the same treatment. Also, a reciprocal transplant experiment
showed that the effect was reversible and an expression of phenotypic
plasticity (Thor and Dupont, 2015). Contrary to the current study, Pedersen
et al. (2014a) found no effect of the CO2 environment on egg hatching or
development of prefeeding nauplii stages N1 and N2 in their multigenerational study using C. finmarchicus. However, the development time of larger nauplii and copepodite
stages was increased by pCO2, although the development delay was not
detected in the following generation (Pedersen et al., 2014a). Vehmaa et
al. (2012) studied combined effects of ocean acidification and warming and
found indications that negative effects on Acartia sp. reproductive
success can partly be combated with maternal effects. The used pH treatments
(-0.4 from ambient) were at the same level as the low-fCO2 treatments in this study (MC 6, MC 7), which makes the results of
the two studies consistent.
The measurements of female copepod antioxidant capacity were done in order to
provide possible additional information of the maternal provisioning of the
offspring. A preferable practice in oxidative stress studies is to measure
several of the four components consisting of free radical production,
antioxidant defences, oxidative damage and repair mechanisms (Monaghan et
al., 2009). In the current study we only have the estimate for the defences,
ORAC measurements, which makes our conclusions
slightly more uncertain. However, an earlier study with the same species has
indicated that, at intermediate stress levels, an upregulation of the
antioxidant system enhances protection against oxidative damage, but at
higher stress, the pro-oxidants may exceed the capacity of the antioxidant
system and lead to oxidative damage (Vehmaa et al., 2013). In this study,
upregulated antioxidant defence seemed to have a positive effect on offspring
quality, as indicated by the positive correlation between female ORAC and egg-hatching success. Higher ORAC in the two highest fCO2 mesocosms at the beginning of the study could be a sign of an upregulated antioxidant system
in a sudden stressful situation, whereas the lowest ORAC in the high-fCO2 treatments at day 31 (Fig. 1c) could be caused by prolonged
stress and exhausted antioxidant defence. The change from positive to
negative effect in the course of the study could explain why fCO2 did
not show a significant correlation with ORAC, whereas food quantity (TPC
< 55 µm) did.
Ismar et al. (2008) showed that Acartia spp. development can be
either slow or altered by certain algal groups causing death before the first
copepodite or reproductive stage. A non-optimal diet could explain why higher
food quantity would cause smaller adult female size, lower egg-hatching
success or lower antioxidant capacity. Skeletonema-diatoms had
fairly high abundance in the mesocosms during the first days of the
experiment when egg-hatching success was lowest in every mesocosm, but then
declined rapidly. Diatom-dominated phytoplankton composition has been shown
to cause low copepod egg-hatching success in the field (Miralto et al.,
1999). Another quality aspect is the size and shape of the food, which may
make it difficult to ingest or assimilate. From day 16 onwards, over 50 %
of chlorophyll a was in picophytoplankton (< 2 µm) (Paul
et al., 2015), which is too small for Acartia consumption (Rollwagen
Bollens and Penry, 2003). Since we did not study what the copepods preyed
upon, we can only speculate on diet quantity and quality. Satiated food
conditions can strengthen the maternal or transgenerational effects. The
transgenerational effects were of minor importance for hatching success in
C. finmarchicus when exposed to long-term high-CO2 and
food-limited conditions (Pedersen et al., 2014a). Long-term stress and food
limitation could thus also be a reason for weakening maternal effects in
the current study.
We found body size (prosome length) to be negatively affected by high
CO2. The result seems to be mostly driven by the mesocosm with the
highest fCO2 (MC 8), where the adult Acartia sp. copepods
were smallest on all the four sampling times that were included in the
analysis (days 10, 17, 24 and 45) (Fig. 1b). It takes ∼ 8.5 days for a
sixth-stage nauplius of A. bifilosa to develop through the five
copepodite stages and reach adulthood at 17 ∘C (Yoon et al., 1998).
According to the Bělehrádek's temperature function it takes 12–15
days for VI nauplii to reach adulthood at 9–11 ∘C
(Bělehrádek, 1935; McLaren, 1966). The constants used in the equation
(α=1008, a=-8.701) were the same as used in Dzierzbicka-Glowacka et
al. (2009) for the Baltic Sea Acartia spp. It is thus possible that
the copepods could have developed through several stages causing the
differences in prosome length between the treatments on day 10. Lowered pH
may have increased copepods' energy requirements and if energy is reallocated
towards maintaining homeostasis, their somatic growth can be reduced.
Pedersen et al. (2014a) found C. finmarchicus body size to be
inversely related to pCO2. They also found a higher respiration rate
under more acidified conditions and claimed that increased energy
expenditure via rising respiration and consecutive decreasing growth and
reproduction could lower the energy transfer to higher trophic levels, thus hampering the productivity of the whole ecosystem (Pedersen et al., 2014a).
This is especially alarming when considering the projected climate warming,
since copepod size is negatively correlated with temperature (Foster et al.,
2011). In addition to temperature, food quantity and quality can affect the
copepod body size (Hart and Bychek, 2011) and create surprising combined
effects with acidification. Garzke et al. (2016) reported an indirect
positive effect of pCO2 on copepod body size, which was explained by
higher food availability when acidification acted as a fertiliser for
phytoplankton. Temperature and food also interact because temperature affects
the respiration and metabolism, thus the satisfying diet depends on
temperature (Boersma et al., 2016). If high-CO2 treatment (MC 8) caused
a developmental delay in maturation, as could be interpreted from the prosome
length results (Fig. 1b), the maturation would have occurred at a different
temperature than in other mesocosms and possibly in non-optimal food
conditions. Anyway, higher food quantity and quality would be expected to
increase copepod size, contrary to our results. It is, therefore, possible that
the used food quantity (TPC < 55 µm) and quality
estimates (C : N < 55 µm) do not fully describe the diet
that Acartia sp. was consuming in the mesocosms.
Adult copepods have in general shown robustness against acidification (Mayor
et al., 2012; McConville et al., 2013), whereas eggs and nauplii appear to be
more sensitive (Cripps et al., 2014b; Fitzer et al., 2012). In addition,
there seem to be notable differences in sensitivity between species. Nauplii
production, adult female fatty acid content and ORAC
of Eurytemora affinis were not affected by fCO2 in the
current mesocosm campaign (Almén et al., 2016). Similarly, Lewis et
al. (2013) found differences in ocean acidification sensitivity between the
species Oithona similis and Calanus spp. (C. glacialis and C. hyperboreus). They argued that O. similis is more sensitive to future ocean acidification than Calanus spp., because O. similis remains in the surface waters whereas
Calanus spp. migrates vertically and encounters wider pCO2
ranges daily than O. similis (Lewis et al., 2013). The same applies
to Acartia sp. and E. affinis in our study area. Although
Acartia spp. is exposed to natural variability in pH environment due
to daily variations as well as due to staying at greater depths during the
day (low pH in deep water), it does not reside as deep as E. affinis (Almén et al., 2014) and may, therefore, show higher sensitivity
than E. affinis during the current mesocosm campaign (Almén et
al., 2016).
The results obtained for Acartia sp. reproduction in the current
study seem to contradict the results obtained for the Acartia sp.
abundance determined in the mesocosms. Although our results indicate that
Acartia sp. reproduction is in fact sensitive to ocean
acidification, no fCO2 effect was found for the abundance of this
species (Lischka et al., 2015). It is possible that 45 days was not long
enough to detect small negative effects of CO2 on copepod size, egg
hatching and nauplii development, to be reflected in copepod abundance. In
addition, especially at the beginning of the study, Acartia eggs in
the mesocosms might have ended up in the sediment trap before hatching due to
slow development at low temperatures, which might have made it difficult to
detect differences in Acartia abundance between the mesocosms. On a
longer timescale, small acidification-induced delays in offspring
development could translate into negative effects for the copepod population
and on energy transfer within the pelagic food web. In addition,
warming will probably enhance the sensitivity of the species towards ocean
acidification (Vehmaa et al., 2012, 2013).