Download Revealing effects of ocean acidification on the

Revealing effects of ocean acidification on the
calcified structures of marine invertebrates through
micro-computed tomography (micro-CT)
K. Keklikoglou1, E. Chatzinikolaou1#, S. Faulwetter1#, P. Grigoriou3#, N. Papageorgiou2#, A.
Queirós4 , S. Rühl4, H. Schmidbaur5
1
Hellenic Centre for Marine Research, Institute of Marine Biology, Biotechnology and
Aquaculture, Thalassocosmos, Gournes, 71500 Heraklion, Crete, Greece,
2
Hellenic Centre for Marine Research, Institute of Oceanography, Thalassocosmos, Gournes,
71500 Heraklion, Crete, Greece
3
Cretaquarium, Thalassocosmos, Gournes, 71500 Heraklion, Crete, Greece
4
Plymouth Marine Laboratory, Prospect Place, Plymouth, Devon PL1 3DH, United Kingdom
5
University of Vienna, Department of Integrative Zoology, Althanstraße 14, 1090 Vienna,
Austria
#
equal contribution
Aims
Climate change is currently one of the major threats for the world's oceans and organisms. Its
consequences include changes in subocean circulation, ocean warming and sea level rise, as
well as impacts on ice cover, fresh water run-off, salinity, oxygen levels and acidification
(Nelleman et al. 2008). The ocean is a sink of atmospheric CO 2, including up to 48% of that
emitted since the beginning of industrial times (Sabine et al. 2004). Dissolved CO 2 reacts with
seawater, leading to changes in ocean carbonate chemistry and lowering of seawater pH, i.e.
ocean acidification. Scientists, policy stakeholders and decision-makers are concerned that
ocean acidification will have significant consequences on marine organisms and may alter
species composition, disrupt marine food webs and ecosystems and affect fishing, tourism
and other maritime human activities (Royal Society 2005). Thus, the impacts of ocean
acidification are global and may drastically change marine life as we know it (Nelleman et al.
2008).
Most marine organisms are sensitive to changes in carbonate chemistry, and their responses
to the predicted changes can lead to profound ecological shifts in marine ecosystems (Kroeker
et al. 2013). Ocean acidification is expected to reduce biocalcification of the shells, bones and
skeletons that most marine organisms possess. Recent studies suggest that the pH reduction
in seawater could impair shell and skeletal production and maintenance of many calcifying
marine organisms (Kroeker et al. 2010; Courtney et al. 2013).
This study aims to examine the effects of ocean acidification on the structure and density of
the shell and other calcified structures of different marine invertebrates. Animals from three
different experiments were studied: a) adult gastropods from long-term acidification
experiments (>12 months), which were conducted as a part of the UK Ocean Acidification
Research Programme; b) juvenile gastropods hatched under acidified conditions during the
abovementioned long-term experiment; c) adult gastropods and mussels, as well as juvenile
cuttlefish from a short-term experiment (2 months), which were conducted under a project
funded by the Latsis Foundation.
The effects of acidification on the organisms' calcified tissues were assessed through microcomputed tomography, which allows relative density measurements of the calcified tissues
and thus facilitates an assessment of the impact of the low pH and elevated temperature
conditions. This study presents and discusses the first results of the experiment, however,
more extensive analyses are ongoing.
Method
Specimen selection
For the long-term experiment, the gastropod (whelk) Nucella lapillus was selected which
exerts strong influence in temperate rocky-shore ecosystems through top-down controls.
Nucella lapillus individuals were collected from the low intertidal and subtidal fringe of the
rocky-shore in Mount Batten, in the Plymouth Sound in January 2011. From these individuals,
the second generation of whelks was hatched after about one year in the tanks of the
experiment. The young whelks were analysed as well to assess the impact of the acidified
conditions on growth and development.
For the short-term experiment, three different species were selected which are characterised
by the presence of external or internal calcified structures: a) Nassarius nitidus (whelk); b)
Sepia officinalis (cuttlefish); c) Mytilus galloprovincialis (mussel). These species are very
abundant in the Mediterranean Sea and play an important role in the respective ecosystems.
The whelks were collected in April 2014 in the lagoons of Amvrakikos Bay (Western Greece),
the mussels in May 2014 in the harbour of Heraklion (Crete) and the cuttlefish were born in the
Cretaquarium in May 2014.
Experimental setup
Long-term experiment
The long term experiment was conducted in the Plymouth Marine Laboratory, UK, as part of
the UK Ocean Acidification Programme. Adult whelks were kept in 1m 3 mesocosm tanks (PML
Intertidal Mesocosm Acidification System/ PML-IMAS) (700 l of seawater and 300 l of
overlying atmosphere). Five experimental treatments were set up: three CO 2 concentration
treatments at ambient temperature (A), 380, 750 and 1000 ppm (corresponding to a pH of
8.08±0.08, 7.93±0.09 and 7.79±0.08, respectively); and two CO 2 concentration treatments
(380 and 750 ppm, corresponding to a pH of 8.0±0.09 and 7.90±0.1, respectively) at elevated
temperature (T) (ambient temperature plus 2°C). Treatments will hereafter be referred to as
380A, 380T, 750A, 750T and 1000A (expressing the combinations of CO 2 concentration and
temperature). Temperature was controlled by use of immersion heaters, the CO 2
concentration was achieved by bubbling a CO2 air mix into the water (a pre-mixed gas system
modified from Findlay et al. (2008)) was employed. The level of CO 2 was monitored and kept
constant with a closed path CO2 analyser (820, Li-Cor). Details on the setup can be found in
Queirós et al. (2015).
Short-term experiment
The short-term experiment was conducted in the Cretaquarium (Hellenic Centre for Marine
Research, Greece). Selected invertebrate species were kept in aquaria with two different pH
treatments and two different temperature levels for a period of three months (June-August
2014). An existing marine mesocosm system was used to ensure stable and controlled
conditions for the experiments. This system precluded sources of environmental conditions
variability other than the specifically manipulated variables (pH level, temperature).
Specialised heaters were used to regulate the temperature, whereas the lowered pH was
achieved through a bubbling system which added CO2 to the water. The lowered pH was kept
constant at 7.6 (i.e. 7.59± 0.01) and continuously monitored through an automated pH
controller system attached to the CO2 bottles. In addition, conditions in the tanks were
manually checked daily and, if needed, parameters were adjusted to keep the pH and
temperature constant.
Four tanks of 300 l each were used. Two of them contained seawater with ambient
temperature (22°C during the period of experiment), while in the other two the seawater was 3
degrees warmer, according to the Intergovernmental Panel on Climate Change (IPCC) models
for the temperature rise until the year 2100 in the eastern Mediterranean (IPCC 2013). The pH
was likewise adjusted according to the IPCC predictions and was regulated to 8.15 (ambient)
and 7.6 (future prediction). The following final setup was used (codes of tanks refer to the
combination of pH and temperature: 7 / 8 for acidified / ambient pH; A / W for ambient / warm
temperature):
 Tank 7-A: ambient seawater temperature (22°C), acidified conditions (pH: 7.6)
 Tank 8-A: ambient seawater temperature (22°C), normal pH (pH: 8.15)
 Tank 7-W: elevated (“warm”) seawater temperature (25°C), acidified conditions (pH: 7.6)
 Tank 8-W: elevated (“warm”) seawater temperature (25°C), normal pH (pH: 8.15)
Three small containers with perforated lids were submerged in each 300 l experimental tank.
The different species were kept separately in order to avoid stress from predation or
competition. Organisms were maintained following standard aquarist practices (regular
feeding ad libitum, cleaning of tanks). Oxygen, nitrate, nitrite and ammonium levels were kept
at a sufficient level by flow-through water renewal, always taking care that the temperature
and pH were kept constant as well.
Sampling for scanning and analyses
In the long-term experiments, four adult specimens of Nucella were selected from each of the
380A and 1000A treatments at exposure month 14. Juvenile Nucella were collected from two
age groups: three weeks and nine to ten weeks after hatching. Twenty-four individuals used in
the study (three from each age group and treatment, excluding the 1000 ppm treatment where
adults did not produce enough offspring).
In the short-term experiment, three specimens of each species were collected from each
treatment after 2 months of exposure. Cuttlefish were collected earlier due to an increased
mortality rate, in a time span of three to five weeks after their introduction to the experimental
tanks. Specimens were anesthetised using a rising concentration of MgCl 2 starting at 1.5 %
and gradually reaching 3.5 % according to the European Directive 2010/63 EU. Then the
animals were placed into liquid nitrogen in order to achieve immediate freezing and to
preserve the shell surface biofilm and structure intact. Finally, the samples were stored
at -80°C until scanning was performed.
Scanning and data generation
All scans were performed with a Skyscan 1172 microtomograph at the Hellenic Center for
Marine Research (HCMR). Adult specimens of the long-term experiment were scanned at a
voltage of 100kV and 100μA with a copper and aluminium filter for a full rotation of 360° at the
highest camera resolution. Juvenile Nucella specimens were scanned at a voltage of 59kV
and 167μA with a custom made aluminium filter (two layers of aluminium foil) for a full rotation
of 360° at the highest camera resolution.
For the short-term experiment, specimens were scanned at a voltage of 70kV and 142μA with
an aluminium filter of 0.5mm and images were acquired at highest camera resolution. To
minimise the scanning duration, scans were performed for a half rotation of 180°. Initial tests
had shown that there was no significant loss of information or increase of artefacts compared
to a full rotation. Projection images were reconstructed into cross sections using SkyScan’s
NRecon software. All scanning, reconstruction parameters as well as the histogram values
were the same for each species in order to obtain comparable results.
For the assessment of acidification effects, only the surface of the shell in the cases of
gastropods and mussels and the cuttlebone in the case of cuttlefish were examined, since any
damage was more likely to occur on the shell surface. Furthermore, it was expected that the
growth zones of the shell/bone would be more affected by the acidification. Towards this end,
cross-sections were extracted from specific body regions. Specimens were rotated upright in
the software DataViewer (Bruker, Kontich), and ten slices were chosen of each individual of
the whelks (Nucella and Nassarius) and nine slices for each individual for the mussels and the
cuttlefish (Figure 1). This was done for all animals from all treatments.
From each slice (except for juvenile Nucella which were not analysed statistically), a 65 µm
deep layer of the shell surface was isolated (80 µm for the adult Nucella from the long term
experiment). The isolation of the shell surface was achieved using Image J for the animals in
the long-term experiment and Skyscan’s CTAnalyzer software (CTAn) for all animals in the
short-term experiment. The cross section images were loaded into CTAn and using the
custom processing plugin, 10 pixels of the shell surface in each slice were selected. Then, the
mean grayscale values of these defined areas were extracted using the binary selection
module. The range of the grayscale histogram was the same for specimens of the same
species (whelks:1-255; mussels:50-255; cuttlefish:15-255).
Figure 1: Selection of slices to be included in the analysis. Left: Nassarius and Nucella,
middle: Sepia, right: Mytilus. Distances were defined as percentages of the total length: Apex
1, End 1 and lip distal at 3% from the respective end, lip2 at 50% of the lip length, widest was
defined manually at the widest point. Replicates at each point are 1% of total length apart.
Analyses
Visual comparison of the scans
The selected slices were loaded into the DataViewer software. By applying the Colour 1
module in the histogram, slices were displayed with different colours which represent different
densities. Furthermore, 3D volume renderings of the scanned specimens were created using
the CTVox software to visually investigate changes in the shell morphology.
Statistical analyses
The mean grayscale values of the shell surface area was used as a proxy for relative shell
density in all statistical analyses.
For the assessment of the long-term acidification impact on adult Nucella, the overall relative
density of the shell surface (represented by 9 slices per specimen, 4 specimens per treatment)
and the distal part of the lip (1 slice per specimen) was compared between the control and the
low pH treatments using one-tailed t-tests. Data analysis was carried out in R. Grayscale
values are not yet available for juvenile Nucella as analyses are ongoing.
For the assessment of the short-term acidification impact on Nassarius, Mytilus and Sepia, the
overall relative density of the shell surface (represented by pooling 10 slices per specimen in
Nassarius and 9 slices per specimen in Mytilus and Sepia; 3 specimens per treatment) was
compared between the 4 treatments using a factorial ANOVA in order to examine the impact of
each of the two factors (pH and temperature) plus the potential impact of the interaction of
these factors. Non-parametric tests (Kruskal-Wallis) were applied when normality and/or equal
variance assumptions were not met. The same statistical methods were used for the analysis
of the separate (i.e. not pooled) shell/cuttlebone regions. Significant differences were indicated
for p-values <0.05. Data analysis was performed using MINITAB software (v. 13).
Results
Visual comparison of the scans
The visual comparison of shell densities represented through colour codes in DataViewer only
revealed obvious differences between the treatments 7-A and 8-A for Nassarius nitidus in all
shell areas, indicating a lower shell density in the specimens treated under acidified
conditions at ambient temperature (Figure 2C). Differences were also found between adult
Nucella specimens from treatments 380A and 1000A (Figure 2A). In juvenile Nucella
specimens, differences between acidified and non-acidified conditions were only visible in the
older age group (9-10 weeks, Figure 2B). In the Sepia specimens, density differences were
difficult to observe in the thin structures. However, the morphology of the cuttlebone seems to
undergo changes in animals from acidified conditions). Here, the division of the chambers
(lamellae) which constitute the cuttlebone was more pronounced than in those from the control
treatments, with additional denser inclusions (Figure 3). No density differences could be
detected in any of the mussel specimens.
The 3D visualisation of the specimens revealed changes in the shell morphology of all whelks
(adult and juvenile Nucella, Nassarius) (Figure 4). Shell deformations included corrosion (loss
of the characteristic whorls), cracks and thinning of the shells, as well as perforations.
Figure 2: The apex, the widest point, the lip and the lip distal of A) adult and B) juvenile
Nucella and C) Nassarius in acidified and normal conditions. The warm colours indicate low
density, while the cold colours indicate high density.
Figure 3: Slices from the widest part of Sepia officinalis in A) acidified and B) normal
conditions. The warm colours indicate low density, while the cold colours indicate high density.
Arrows indicate the higher material density of the lamellae and cuttlebone in acidified
conditions compared to normal conditions.
Figure 4: 3D images of A) adult and B) juvenile Nucella and C) Nassarius. The left column
shows the specimens from the control treatment and the right column shows the specimens
from the acidified treatment. Arrows show specific shell deformations which include corrosion,
cracks and thinning of shells.
Statistical analyses
Long-term experiment
The statistical analysis of the shell surface revealed differences between adult Nucella
specimens from acidified and control conditions. Animals exposed to acidified conditions
exhibited a 20–30% decrease in shell density in the distal lip area (t6 = -1.80 and P < 0.10)
and in the overall shell surface (t6 = -2.32 and P < 0.05).
Short-term experiment
The mean grayscale values (i.e. a value that represents shell density) for the pooled shell
regions of Nassarius nitidus, differed significantly between the ambient acidified treatment
(tank 7-A) and all other treatments (Fig. 5). The shell of N. nitidus in 7-A tank had a
significantly lower mean density in comparison to all other tanks (Kruskal-Wallis, p<0.001).
However, no acidification impact was detected regarding the density of the pooled shell or
cuttlebone regions for the other two species (Mytilus galloprovincialis and Sepia officinalis)
(Kruskal-Wallis, p=0.588; p=0.309 respectively).
Figure 5: Mean grayscale values (±StDev) for the pooled shell regions of Mytilus
galloprovincialis, Sepia officinalis and Nassarius nitidus in four different treatments. The red
colour in the triangular tables indicates the significant differences (p<0.05) between the
different treatments as detected by the Kruskal-Wallis test.
However the analysis of the separate shell regions (i.e. not pooled) revealed differences
between organisms in different treatments (Fig. 6). In Sepia officinalis, the factorial ANOVA
revealed a significant interaction between temperature and pH (p=0.016) only regarding the
widest part of the cuttlebone. More specifically, significant differences were found in the grey
scale values a) between the organisms in the acidified (tank 7-A) and the control (tank 8-A)
treatments for the low temperature (p<0.001), and b) between the organisms in the high (tank
7-W) and low temperature (tank 7-A) in the acidified treatment (p<0.001). In Nassarius nitidus
significant interactions were found in all the studied parts of the shell (apex, widest, lip and lip
distal). More specifically, for the apex of the Nassarius shell (p<0.001) there were significant
differences in the grey scale values a) between the organisms in the acidified (tank 7-A) and
the control (tank 8-A) treatments for the lower temperature (p<0.001), and b) between the
organisms in the high (tank 7-W) and low temperature (tank 7-A) in the acidified treatment
(p<0.001). For the widest part of the Nassarius shell (p=0.038) there were significant
differences in the grey scale values between a) the organisms in the acidified (tank 7-A) and
the control (tank 8-A) treatments for the lower temperature (p=0.007), and b) between the
organisms in the high (tank 8-W) and low temperature (tank 8-A) in the control pH treatment
(p=0.035). The grey scale values of the shell lip of Nassarius (p=0.007) were significantly
different between a) the organisms in the acidified (tank 7-A) and the control (tank 8-A)
treatments for the lower temperature (p=0.006), and b) between the organisms in the high
(tank 8-W) and low temperature (tank 8-A) in the control pH treatment (p=0.009). The lip distal
part of the Nassarius shell had significantly different grey scale values (p=0.028) between a)
the organisms in the acidified (tank 7-A) and the control (tank 8-A) treatments for the lower
temperature (p=0.004), and b) between the organisms in the high (tank 7-W) and low
temperature (tank 7-A) in the acidified treatment (p=0.021).
In the shell of Mytilus galloprovincialis only the apex part indicated a significant interaction
between temperature and pH (p=0.018). More specifically, there were significant difference in
the grey scale values between the organisms in the high (tank 7-W) and low temperature (tank
7-A) in the acidified treatment (p=0.003).
Figure 6: Mean grey scale values (±StDev) for the widest part, the apex, the end/lip and the
distal lip (i.e. not pooled shell regions) of a) Mytilus galloprovincialis, b) Sepia officinalis and c)
Nassarius nitidus in four different treatments.Colours as in Figure 5.
Discussion and conclusions
Generally, there is very strong evidence of marine organisms being affected by ocean
acidification. Changes in shell morphology will always result in a change in function/protection
and thus a change in ecosystem functioning (Doney et al. 2009). Micro-CT images helped to
visualise the effect of ocean acidification mostly in whelks which exhibited damaged and
thinner shells.
Micro-CT analysis revealed that whelks had a reduced shell-surface density under ambient
acidified conditions when compared to whelks from control treatments. The shell degradation
on whelks as a result of acidification impact has been reported in several studies (e.g. Lischka
et al. 2010; Melatunan et al. 2013; Queiros et al. 2015). As Melatunan et al. (2013) indicate,
either the dissolution of calcium carbonate structures which are exposed to acidified
seawaters, and/or at the same time the insufficient deposition rate of calcium carbonate could
cause a reduction in shell thickness.
The shell-surface density of whelks in ambient acidified conditions was significantly lower
comparing to whelks from the warm acidified treatment. Similarly, Melatunan et al. (2013)
reported that gastropods in high temperature acidified treatment had thicker shells than the
ones from the cooler acidified treatment. Melatunan et al. (2013) assumed that the elevated
temperature may increase the calcite and aragonite saturation.
Cuttlefish had a significantly higher density in the widest part of the cuttlebone under acidified
conditions than in control conditions. This fact could be related to an increase in the
calcification rate when pH is lower as has been reported by Gutowska et al. (2008). According
to these authors, cuttlefish may have the ability to acclimatise more efficiently in acidified
conditions, thus being able to continue their shell growth and calcification. However, an
increase in the density of the cuttlebone may decrease the buoyancy of the cuttlefish resulting
swimming difficulties (Gutowska et al. 2010).
No significant effect of acidification alone was evident in the surface-shell density of mussels.
However, a synergistic effect of both temperature and pH was revealed, where the apex of the
shell had a lower density in the ambient acidified treatment than in the warm acidified
treatment. These findings agree with the results of Kroeker et al. (2014) who indicated thicker
shells in elevated temperatures suggesting that mussels may be more vulnerable to ocean
acidification impact in lower temperatures.
Although further study is necessary to understand the impact of ocean acidification on
organisms, the present study succeeded to demonstrate the utility of micro-CT when applied
as a tool for the assessment of the ocean acidification impact.
Acknowledgements
The project was funded by John S. Latsis Public Benefit Foundation and supported by the EU
FP7 project MARBIGEN (FP7- REGPOT-2010-1). Dr. Christos Arvanitidis (HCMR) is thanked
for his support during various stages of the project.
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