Introduction
In the 20th century, the atmospheric carbon dioxide (CO2) concentration
continued to increase as a result of fossil fuel combustion and other human
activities. It was in turn taken up by the ocean gradually through air–sea
exchange. Oceanic CO2 can hydrolyze to increase the concentration of
hydrogen ions (H+), which leads to the reduction of pH in the ocean by
0.1 units (Orr et al., 2005). Based on the amount of global CO2
emissions at present, the pH of the ocean is likely to drop by 0.3–0.4 units by the end
of the 21st century and by 0.7 units after 300 years. The current and
predicted levels of CO2 and acidity of seawater of nearshore, estuarine,
and higher-latitude habitats are expected to be even greater and
substantially more variable than those of the open ocean (Gruber et al.,
2012; Zhai et al., 2014). One alarming consequence is the rapid change in
seawater chemistry and decrease in ocean pH, which could have great impacts
on marine ecosystems and pose a threat to marine life (Frommel et al.,
2012; Kerr, 2010). Elevated CO2 concentrations can disturb the
acid-base regulation, blood circulation, respiration, and the nervous system
of marine organisms, leading to long-term effects such as reduced growth
rates and reproduction (Frommel et al., 2012). Other direct response to
ocean acidification (OA) have been found in the alteration of behaviour
(Dixson et al., 2010; Munday et al., 2009a), development (Frommel et al.,
2013), RNA/DNA ratio (Franke and Clemmesen, 2011), and otoliths (Checkley et
al., 2009; Maneja et al., 2013; Munday et al., 2011b) of marine fish larvae.
However, the emerging picture remains intriguingly complex. While the
majority of responses to high CO2 appear to be negative (Branch et al.,
2013) with highest sensitivities observed during the early life stages (ELS)
and in the calcifying invertebrates such as corals, bivalves, pteropods, and
echinoderms, there are substantial evidences for non-linear, neutral, or even
positive reaction to increasing CO2 conditions (Hurst et al., 2013;
Munday et al., 2011b; Murray et al., 2014). Moreover, marine fish exemplifies
this complexity. Decades of empirical data suggest that juvenile and adult
fish possess sufficient acid-base and osmoregulatory capabilities for the
toleration of very high metabolic and ambient CO2 levels
(> 2000 µatm) (Murray et al., 2014). Although fish possess the
ability of acid-base balance regulation, its physiological function will
certainly decline under such regulation for a long time from the perspective
of energetics, especially in its most fragile and sensitive early life stage
during its life history. In addition, in ELS of multiple taxa including fish,
elevated CO2 was shown to affect calcification of shells and skeletons
due to a drop in the carbonate availability (Riebesell, et al., 2010). Munday
et al. (2011a) observed no effect on spiny damselfish otolith calcification
at 850 µatm, while Munday et al. (2011b) and Checkley et al. (2009)
highlighted an otolith hypercalcification in white seabass
(Atractoscion nobilis) larvae exposed at 993 and 2558 µatm
pCO2 and in clownfish (Pomacentridae) larvae at
1721 µatm pCO2, respectively. In case of calcification
modulation, otolith morphology can be affected, which may have negative
repercussions on the behaviour and acoustic function of fish and decrease
their survival probabilities (Bignami et al., 2013; Réveillac et al.,
2015).
Marine medaka, Oryzias melastigma or Oryzias javanicus, is
one of the 14 species belonging to the genus Oryzias, which
distribute in estuarine waters from East to Southeast Asia (Koyama et al.,
2008). It has been proposed as a model species in marine environmental risk
assessments (Mu et al., 2014). However, few studies have addressed OA effects
on the ELS of marine medaka so far. The objective of this study was to
examine how CO2-driven OA affected the embryos and newly hatched larvae
of marine medaka after 21 days exposure through investigating the embryonic,
larval, and otolith development.
Materials and methods
Fish rearing
Marine medaka, O.melastigma, were provided by the Key Laboratory of
Coastal Ecological Environment of State Oceanic Administration. Fish were
maintained in an aquatic habitats system (Aquatic Habitats, USA) with a salinity
of 30 ± 2, temperature of 26 ± 1 ∘C, and a photoperiod
of 14 h:10 h (light:dark). All fish were fed with nauplii of
Artemia three times a day and synthetic food (New life spectrum
thera-A formula, Made in the Newlife International, Inc, USA) twice a day.
One-tenth of the total amount of water in the system was automatically
renewed daily. To ensure developmental synchronization of embryos during
experiment, all eggs were collected within 3–5 h after initiation of
spawning, and fertilized and viable ones were selected under a dissecting
microscope.
Schematic illustration of the pH control system applied in
exposure experiment (For details refer to the text).
The experimental seawater (salinity of 30.7 ± 0.1) was prepared by
diluting sea salts (Instant Ocean, Aquarium Systems, USA) with deionized
water. The standard NBS pH was 8.2 ± 0.004.
Seawater manipulation and experimental design
The design of seawater pH control system was based on Riebesell et
al. (2010) with some modifications. Briefly, partial pressure of CO2
(pCO2) was adjusted by pH modulator (aquastar pH Modul?, IKS) with a
standard deviation of ±0.01. Three pH gradients, 8.2, 7.6, and 7.2 were
set according to the predicted levels upon CO2 emission at present,
after 100 and 300 years (Orr et al., 2005), respectively. The pH control
system consists of three parts, namely monitor, controller and aeration
(Fig. 1). The pH metre in water monitored the real-time pH changes during
the experiment. The controller associated with pH metre was also connected with
an electromagnetic valve, which opened or closed the electromagnetic valve based
on the feedback of the pH metre. The intake of electromagnetic valve connected to
a cylinder equipped with a high concentration of CO2 (0.1 %
CO2 : 99.9 % air-pCO2 of 1000 ppm), and its outtake connected
to silicone tube, drying tube, check valve and refiner which inserts into
seawater. The refiner was placed in the middle of the aquarium (10 L) bottom
to make the gas bubbled into water quickly and homogeneously. When the pH in
seawater was higher than the set value, the electromagnetic valve opened
automatically to pipe concentrated CO2 into the water until the
pH dropped to the set value, and then the valve closed. During the exposure
experiment, parameters including pH, inorganic carbon (DIC), temperature,
salinity, total alkalinity (TA) and dissolved oxygen (DO) were continuously
monitored and analyzed to ensure the stability of the pH control system.
For each pH treatment, 90 fertilized eggs were randomly assigned to three
tanks (three replicates) with 30 eggs per replicate. These tanks were
monitored daily for dead embryos, hatched larvae, and hatching time. Subsets
of hatched larvae per tank were then transferred to the alternative aquariums
with the same exposure conditions to start the larval exposure sub-experiment
(3 CO2 levels × 3 replicates). Larvae were monitored daily and
dead ones were removed until the termination of the experiment at 21 days
(approximately 1 week post-hatch). By the end of the experiments, the
surviving larvae were anaesthetized and photographed under a microscope (Leica DMI4000B)
for deformity analyses, and the otoliths were then removed and dry-stored in
well plates.
Determination of water quality parameters
The determination of pH, TA, and DIC referred to the methods of Dickson et
al. (2007). In brief, samples were collected into vials without obvious
bubbles by an overflow manner, and then fixed with 0.1 % saturated
HgCl2 solution. The pH was detected using combined electrode (Orion 8102
BN Ross) and high-precision pH metre (Thermo Orion 3-Star, USA) in
25∘ water bath within 2 h after sampling. The deviation was less
than 0.01. TA and DIC were detected by TA analyzer (Apollo AS-ALK2, USA) and
DIC analyzer (Apollo AS-C3, USA) with an accuracy of more than
±2 µmol kg-1, respectively. Salinity, temperature and DO
of seawater were detected by YSI-85 water quality monitor (YSI Inc, USA), and
the accuracy of each parameter was more than ±0.1, ±0.1∘ and
±2 % air saturation, respectively. Aragonite saturation (ΩAr) was calculated based on temperature, salinity, and measured TA and
DIC through CO2-SYS carbonate system software (Pelletier et al., 2011).
Other parameters adoption including dissociation constants of carbonic acid
and sulfuric acid, saturated solubility product of CaCO3 were consistent
with those internationally applied (Millero et al., 2006).
Developmental toxicity
The numbers of embryos surviving to hatching were counted based on daily
inspection of the embryos in each treatment. Hatching rate data were summed
and converted to proportions of survival numbers out of 30 eggs per
replicate. After 8 days post fertilization, and 3 days before expected
hatching, the embryos were inspected at least twice a day and hatching numbers
were recorded. Heart rates were estimated by counting the number of heart
beats over a 30 s period (n=10) at day 8. The time when ≥ 50 %
of the embryos had hatched was recorded as the hatching time (Forsgren et
al., 2013). As observations of spawning and hatching were made at somewhat
irregular intervals over the course of the study, spawning and hatching times
were analyzed. The embryonic hatching time was calculated as the time elapsed
between spawning and hatching.
On day 21, thirty larvae (10 larvae per replicate) from each CO2
treatment were randomly selected and photographed for deformity analyses.
The deformity rate was calculated based on the proportions of abnormal
larvae numbers out of 10 eggs per replicate. Survival of larvae was based on
the number of newly-hatched larvae per replicate and the number remaining at
the termination of the experiment.
Otolith measurement
The measurement of marine medaka otolith was based on the method of Franke
and Clemmesen (2011). Briefly, the left and right otoliths were removed from
16 fish larvae randomly selected from each CO2 treatment. Each otolith
was observed and photographed under a microscope (Leica DMI4000B). Digital
pictures of otolith were taken at 1000 × magnification using the
microscope equipped with a Leica DFC420C Digital Camera. Otolith area
(µm2) was calculated through Image-Pro Plus 5.0 software after
calibration and gray-scale processing of photos.
Statistical analyses
Data analyses were performed using SPSS ver.16.0 (Chicago, IL) software. All
data were tested for normal distribution using the Kolmogorov–Smirnov test.
Non-normally distributed data were log transformed. The difference between
measured and nominal pH was analyzed by T-test. For heart rate, hatching
rate, hatching time, and deformity rate, one-way analysis of variance (ANOVA)
followed by Bonferroni post hoc tests were applied to test the differences
between and among groups. An independent sample test was used to compare the
difference of otolith areas between left and right sides in each treatment.
If there was a significant difference, one-way ANOVA was used to further
compare the difference between treatments for left and right sides,
respectively. If not, one-way ANOVA was performed after data combining of
left and right sides. Results were expressed as means ± standard
deviation (SD).
Results
Seawater chemical parameters
Measured pH in three treatments and different chemical parameters in
seawater were shown in Fig. 2 and Table 1, respectively. During the 21 days of
exposure, measured pH in pH 8.2, 7.6, and 7.2 groups were
8.22 ± 0.004, 7.63 ± 0.007, and 7.22 ± 0.002, respectively.
The fluctuation was less than 0.05 (Fig. 2), indicating the stability of the pH
control system.
Measured mean pHNBS of seawater in three
pH treatments during 21 d of exposure (n=3).
The hatching time, hatching rate, and heart rate of marine
medaka embryos exposed to three pH levels. (a) Hatching time; (b) hatching
rate; (c) heart rate.
Summary of chemical parameters in control and acidic seawater (n=3).
pHNBS∗
DIC
pCO2
CO2
HCO3-
CO32-
ΩAr
(µmol kg-1)
(µatm)
(µmol kg-1)
(µmol kg-1)
(µmol kg-1)
8.22 ± 0.004
2645.1 ± 28.5
495.9 ± 2.2
14.4 ± 0.1
2380.3 ± 10.1
280.4 ± 3.9
4.5 ± 0.06
7.63 ± 0.007
3014.2 ± 74.3
2372.6 ± 52.3
68.7 ± 1.4
2861.0 ± 20.7
84.5 ± 0.3
1.4 ± 0.006
7.22 ± 0.002
3202.7 ± 18.5
6165.7 ± 56.4
178.4 ± 1.8
2988.8 ± 9.3
35.5 ± 0.6
0.6 ± 0.01
∗ pH NBS: The fundamental definition of pH in terms of the hydrogen ion
activity; NBS: National Bureau of Standard.
Embryonic development
Three replicates produced a total of 90 eggs in each CO2 treatment. The
hatching times were extended with decreasing pH level, but there was no
significant difference among the three pH treatments (F2, 6=5.8, p=0.066) (Fig. 3a). On average, 83 % of eggs in three pH treatments
survived to hatch, and the hatching rate of eggs was not significantly
different among the three pH treatments (F2, 6=1.1, p=0.4)
(Fig. 3b). For the heart rates of embryos, pH 7.6 and 7.2 groups were not
significantly different from those in the control group (F2,28=1.7, p=0.7) (Fig. 3c).
The deformity and survival rates of larvae exposed to three
pH levels. (a) Deformity rate; (b) survival rate. (a) indicates that the
value in pH 7.2 or in pH 7.6 differs significantly from that in the control
(pH 8.2), and (b) indicates that the value in pH 7.2 differs significantly
from that in pH 7.6.
Morphological changes of medaka larvae exposed to three
pH levels. 1–2: Control-nNormal (pH 8.2); 3–4: pH 7.6
treatment; 5–6: pH 7.2 treatment. SD: Spinal deformities; CF:
Craniofacial deformities; PE: Pericardial edema; SH: Stretched heart.
The effects of different pH levels on the otolith area of
marine medaka larvae after 21 days of exposure. (a) indicates that the value
in pH 7.2 or in pH 7.6 differs significantly from that in the control
(pH 8.2).
Larval development
Three replicates produced a total of 66–75 newly hatched larvae in each
CO2 treatment level. By the end of the experiment, larvae survival rate was
highly variable but did not differ significantly between the control and
acidified water groups (F2, 6=0.3, p=0.7) (Fig. 4b).
However, the two lower pH treatments (pH 7.6 and pH 7.2) can both cause
spinal deformities, craniofacial deformities, stretched heart and pericardial
edema of marine medaka larvae (Fig. 5). Furthermore, in pH 7.2 treatment,
the deformity rate was significantly higher than that of control group
(F1, 4=32, p=0.005) (Fig. 4a).
Otolith development of larvae
The effects of different pH treatments on otolith size of marine medaka
larvae were shown in Fig. 6. There was no statistically significant
difference between the areas of left and right sides in each pH treatment
(pH 8.2: F1, 59=0.092, p=0.76; pH 7.6: F1, 67=0.045, p=0.83; pH 7.2: F1, 68=0.005, p=0.95, respectively)
(Fig. 6a). In pH 7.6 treatment, the average areas of left and right sides
were significantly smaller than those of the control treatment (F1, 128=8.8, p=0.013) (Fig. 6b).
Discussions
Assessment of species sensitivity or tolerance to CO2-driven
acidification in marine environment is critical to evaluate the impacts of OA
on marine biodiversity and ecosystem function (Fabry et al.,
2008; Melzner et al.,
2009). A number of studies found that
CO2-driven acidification had obvious influences on ELS of many marine
invertebrates, especially calcified organisms including coral (Doropoulos et
al., 2012; Fabricius et al., 2011), coccolithophores (Berry et al., 2002),
and mollusk (Kroeker et al., 2013; Thomsen et al., 2013; Waldbusser et al.,
2011). OA was predicted to potentially affect individual behaviour such as
development, growth, survival, and swimming particularly during the early life
stage of marine organisms (Munday et al., 2008). In our experiments, the
duration of embryonic stage, egg survival and embryonic heart rate of marine
medaka were unaffected by acidification water with pH 7.6 and pH 7.2. There
was a slight increase in the embryonic duration of the eggs, but the size
effect was not different among the three pH treatments. Overall, these
results suggest that the egg stage of marine medaka is relatively tolerant to
elevated CO2 and low pH level, which were consistent with the results
reported by other studies on a diverse set of marine fishes. For instance,
Munday et al. (2009b) found
the survival to hatch of orange clownfish (Amphiprion percula) from
the Great Barrier Reef, Australia, to be nonresponsive to pCO2 levels
to 1020 ppm (pH 7.8). Similarly, Franke and Clemmesen (2012) found no
significant effect of elevated pCO2 levels from 460 to 4635 ppm
(corresponding to pH 8.08–pH 7.05) on survival to hatch of Atlantic
herring from the western Baltic Sea. In the study of Frommel et al. (2013),
the survival of embryos of Atlantic cod from the Bornholm Basin of the
western Baltic Sea was not altered at pCO2 levels up to 4000 ppm
(pH 7.2). Hurst et al. (2013) also reported no effect on embryo survival of
walleye pollock (Theragra chalcogramma), common in the temperate
eastern North Pacific, at pCO2 levels up to 1933 ppm (pH 7.4). In
other cases, however, a strong effect of CO2 was evident on the
embryo survival of summer flounder (Paralichthys dentatus), an
ecologically and economically important flatfish of the inshore and nearshore
waters of the mid-Atlantic Bight (Chambers et al., 2013). The relative
survival of summer flounder embryos was reduced to 48 % when maintained
at 1808 ppm pCO2 (pH 7.5) and to 16 % when maintained at 4714
ppm pCO2 (pH 7.1). Baumann et al. (2012) also reported a 74 %
reduction in survival of embryos and young larvae of inland silverside,
Menidia beryllina, native to estuaries of the US Atlantic coast,
when maintained at 1100 ppm pCO2 compared to those held at 410 ppm
pCO2. All of these studies varied in the number of parents used, the
time lapse between egg fertilization and initiation of CO2 treatment,
and how and when survival was scored. For example, the CO2 treatments of
inland silverside by Baumann et al. (2012) began at approximately 24 h
post-fertilization, and the survival was scored at approximately 1 week
post-hatching. The different approaches used in previous studies may preclude
a fair cross-study comparison (Chambers et al., 2014); however, the overall
present of effect of elevated CO2 environments on embryo survival is in
contrast to the findings here. Habitats occupied of species, particularly in
the ELS, may play a role in their sensitivities. It is counter to
expectations and requires further attention that species in their ELS are
found in estuarine (marine medaka) and inner shelf (summer flounder)
habitats, both with relatively high ambient CO2 levels, but exhibit
different sensitivities to experimentally elevated-CO2 levels.
An unexpected result of our study was that elevated levels of CO2
affected larval development abnormalities, and the average deformity rate of
marine medaka larvae (approximately 1 week post-hatch) increased
significantly by 16 % as CO2 increased from control level (pH 8.2)
to high-CO2 level (pH 7.2). Although CO2-induced acidification up
to the high-CO2 level (pH 7.2) had no noticeable effect on larval
survival by the end of the experiments (21 days), the larval development
abnormalities may ultimately influence the later life consequences and
therefore further reduce the productivity of fish stock in future acidified
oceans. Chambers et al. (2014) found no reduction in survival with CO2
for larvae during the first 4 weeks of larval life (experiment ended at 28 days post-hatching (dph)), however, the sizes, shapes, and developmental status
of larvae showed initially longer and faster growing when reared at pH 7.5
and pH 7.1 levels, and the tissue damage was evident in larvae as early as
7 dph from both elevated-CO2 levels. At present, it is unknown how
increasing CO2 levels affect development and survival in fish ELS. Even
if fish embryos and early larvae are capable of physiological adaptation to
increased CO2 somehow, this would incur further metabolic costs and thus
reduce energy available for tissue synthesis or post-hatch survival on
diminished yolk reserves. As some fish eggs, including those of O. melastigma, seem to be tolerant to low-pH conditions, the high levels of
CO2 or associated changes in carbonate chemistry may be more important
to larval-fish development than hydrogen ion concentrations. (Baumann et al.,
2011; Ishimatsu et al., 2008).
The pH drop driven by CO2 can change concentrations of bicarbonate and
non-bicarbonate ions during which elevated CO2 affects saturation states
of calcium ions carbonate polymorphs (Munday et al., 2008). Otoliths are bony
structures of fish to sense orientation and acceleration and consist of
aragonite-protein bilayers, which document fish age and growth (Checkley et
al., 2009). Its formation starts during embryonic development, and any
alteration of otolith size or shape is important for physical performance and
individual adaptability of fish. Therefore, any substantial change in the
size, shape, or symmetry of otoliths could have serious implications for
individual performance and survival (Munday et al., 2008, 2011a). In this
study, we found no significant difference existing between the left and right
sides of marine medaka larval otolith under the same pH level. However,
otolith area of larval fish exposed to the intermediate-CO2 level
(pH 7.6) was smaller than that of control. Results suggested that there was
no significant pCO2 effect on otolith symmetry of marine medaka,
defined as the difference between the right and left sides. However, the
otolith area was significantly affected. The trend of reduction in otolith
area of marine medaka larvae exposed to elevated CO2 environments found
here has not been reported in most previous studies focusing on other marine
fishes. For instance, Checkley et al. (2009) found that otolith area of white
seabass (Atractoscion nobilis) larvae increased by 7–9
and 10–14 % after exposure to 993 and 2558 ppm CO2,
respectively. Munday et al. (2011b) found that the size, shape, and symmetry
of otoliths in larval clownfish was unaffected by exposure to simulated levels
of OA (pH 7.8 and 1050 µatm CO2); however, in a more extreme
treatment (pH 7.6 and 1721 µatm CO2) otolith area and maximum
length were larger than those of control otoliths. Maneja et al. (2013) found
that elevated CO2 had no significant effect on the shape of the otoliths
nor was there any trend in the fluctuating asymmetry, while increased otolith
growth was observed in 7 to 46 d post hatch cod larvae in two pCO2
treatments of 1800 and 4200 µatm. In contrast, Munday et
al. (2011a) did not detect any effect of elevated CO2 on otolith size of
juvenile spiny damselfish, Acanthochromis polyacanthus, which were
reared for 3 weeks in treatments up to 841 µatm CO2. Our
results seemed to support the hypothesis that otoliths of larvae reared in
seawater with elevated CO2 would grow more slowly than they do in
seawater with normal CO2. The reduction of otolith area was likely
associated with reduced CaCO3 saturation which slowed down its
formation. We do not know whether smaller otoliths have a deleterious effect,
although we do know that asymmetry between otoliths can be harmful (Checkley
et al., 2009).The difference between our results and other studies may be
related to: (1) different pCO2 levels; (2) different life histories;
or (3) different exposure duration (Munday et al., 2011a). However, another
interesting result from the present study was that the otolith area of marine
medaka larvae under the extreme CO2 level (pH 7.2) tended to increase
instead of continuously reduce. We should not ignore its own acid-base
regulation ability that increased the available amount of carbonate by
compensation mechanism for otolith to intensify the calcification process
under such acidic condition (Checkley et al., 2009). Calcium incorporation
into the otolith was modulated by the seawater pH. This questions the
stability of the element:Ca ratio under environmental hypercapnia. During
the biomineralization of the otolith, chemical elements such as metals and
metalloids are supposed to substitute for calcium (Réveillac et al.,
2015). The changes of pH and seawater chemistry caused by increased CO2
can modify the speciation of metals and their subsequent bioavailability to
organisms (Millero et al., 2006). The physiological response of fish to
hypercapnia might in turn stimulate processes to compensate for acidosis
based on the key role of ion transporters. In the present study, OA may interfere
with trace element uptake and body concentrations and therefore could affect
otolith growth and microchemical constituent. Further studies are thus needed
to investigate the possibility that OA impacts on the trace metals
properties, molecular-binding affinities and incorporation pathway into the
otolith.
In conclusion, this study demonstrated that, under projected near-future
pCO2 levels, the ELS of marine medaka exhibited a dramatic increase of
larval developmental deformity and otolith calcification while their
survival was not affected. Importantly, the observed CO2-induced
abnormal development of larvae might have predictably negative consequences
on the recruitment of fish population, the effects of which on later life
history and phenotype of subsequent generations should be concerned. As the
otolith is an essential tool used in reconstructing fish life history in
terms of age, somatic growth and attended habitats, further studies should
investigate the process of otolith biomineralization. Finally, we emphasize
that there is considerable variation among species in their sensitivities to
elevated CO2 and reduced pH. Determining the traits that render some
species more susceptible than others will be helpful and valuable in
predicting the long-term and ecological effects of OA.