Introduction
Edible oysters belonging to the genus Magallana have a complex life
cycle, in which the free-swimming larvae attach onto a suitable hard
substrate and then metamorphose into sessile juveniles within a few hours (Medaković et al., 1997; Salvi and Mariottini, 2017). The oyster larval
shell is primarily made of aragonite, a denser and mechanically stronger form
of calcium carbonate (CaCO3), compared to calcite which is a stable
but mechanically brittle polymorphous CaCO3 (Lawn and Wilshaw, 1993;
Han et al., 1991). Upon metamorphosis, the fraction of calcite rapidly
increases and becomes the main component in the juvenile and adult oyster
shell (Medaković et al., 1997; Weiner and Addadi, 1997). The composition
of the mineral and its organic matrix define a wide array of composites that
relate to the mechanical strengths of the shell of each of these life stages
(Lee et al., 2011). Early life stages of marine invertebrates, oysters
included, are highly vulnerable to predators (Newell et al., 2007) and
environmental stressors (Thomsen et al., 2015) when compared to the adult
stages. Production of mechanically strong shells during larval and juvenile
stages is essential to the post-larval phase because shell integrity and
strength act as a protective barrier against shell-breaking and drilling
predators.
Oceans currently absorb about a third of anthropogenic carbon dioxide
(CO2), which dissolves in seawater, forming carbonic acid, and
increases the concentration of hydrogen ions, known as ocean acidification (OA).
A study showed that the calcification rate of marine organisms, including
oysters, is highly vulnerable to high carbon dioxide partial pressure
(pCO2; µatm) driven decreases in seawater pH (Feely et
al., 2009; De Bodt et al., 2010). If the early life stages of edible oysters
are vulnerable to near-future OA, then it could directly harm oyster survival
and aquaculture production. Evidence of the negative effects of OA is, for
example, the decline of spat production in oyster hatcheries on the west
coast of the USA due to poorly calcified larval shells consequence of
upwelled high pCO2 waters (Barton et al., 2012). Previous studies
on calcifying organisms suggest that OA not only reduces calcification rates,
but also increases dissolution of formed shells in very high pCO2
scenarios (Ries, 2011; Bednarsek et al., 2012). The decreased pH depletes
carbonate ions necessary for CaCO3 mineralization, as well as weakens
marine organisms physiologically by causing acidosis and impairing internal
pH homeostasis needed for optimal calcification (Dupont and Portner, 2013).
Recently, an increasing number of studies have captured the importance of the
mechanical properties of calcareous shells, the end product of calcification,
under OA scenarios (Dickinson et al., 2012; Ivanina et al., 2013; Li et al.,
2014; Fitzer et al., 2015; Collard et al., 2016; Teniswood et al., 2016;
Milano et al., 2016). For instance, it has been reported that the Pacific
oyster, Magallana gigas (previously Crassostrea gigas), and
the Eastern oyster, Crassostrea virginica, produced softer shells
with reduced mechanical strength under OA conditions (Beniash et al., 2010;
Dickinson et al., 2012). Despite these OA threats to oyster
calcification process, studies are yet to demonstrate the structural
organization of oyster shells under elevated CO2 conditions.
Importantly, modulating effects of OA on the inherent relationship between
shell structural and mechanical features is yet to be studied in detail.
This study is designed specifically to fill this gap in knowledge using the
ecologically and economically important edible oyster Magallana angulata (previously Crassostrea angulata) also as a model species.
Here, the quantitative relationship between microstructural and mechanical
properties was examined using the newly formed juvenile oyster shells.
Specifically, the effect of OA on this relationship was tested using three
levels of environmentally and climatically relevant high CO2 scenarios which induced decreased pH. As the calcitic foliated layer is the major
shell structure for mechanical support in oysters (Lee et al., 2008), we
examined its structural and mechanical properties by using a variety of
characterization and imaging techniques such as scanning electron microscopy
(SEM), electron back-scattered diffraction (EBSD) and nanoindentation tests. To
further evaluate the overall shell integrity, we quantified shell density and shell density–volume ratio relationships using high-resolution
micro-computed tomography scanning (micro-CT).
Scheme of the experimental system. Decreased pH conditions were
obtained through bubbling CO2-enriched air with the appropriate
CO2 concentration. Black and blue solid arrows indicate air and
CO2 flow respectively and orange arrows indicate
CO2-enriched air flow. The appropriate CO2 concentrations
in the injected CO2-enriched air flow were controlled by using a
dual variable area flow meter.
Materials and methods
Experimental design
Sexually matured adult oysters of the Portuguese oyster, Magallana angulata, were collected from the coastal area in Fujian, China
(26∘05′53.36′′ N 119∘47′45.81′′ W), in the South
China Sea on 29 July 2014. The adults were transported to the laboratory at
the Swire Institute of Marine Science, University of Hong Kong. They were
left to acclimatize in flow-through tanks in natural seawater at ambient
conditions (31 salinity, 29 ∘C and pH(NBS) 8.1) for a
week. They were fed with a mixed algae diet (Isochrysis galbana and Chaetoceros gracilis). Sperm and eggs were obtained
from more than 10 males and 10 females using the “strip spawning” method
(Dineshram et al., 2013), and cultured under ambient conditions. A period of
24 h post-fertilization, embryos developed into D-shaped veliger larvae.
Four environmentally and climatically relevant pH levels (the control: pH
8.1; the low treatments: pH 7.8, 7.5 and 7.2) were selected as proxies to
investigate the effect of CO2-driven OA on oyster shells. According
to IPCC projections, the average pH of oceans (currently pH 8.1) is expected
to drop to pH 7.8 and 7.5 by the year 2100 and 2300, respectively (Feely et
al., 2009). Lowest pH treatment was included in this study to understand the
impact of extreme environmental conditions in the coastal habitats of
M. angulata (pH 7.2), which manifest naturally fluctuating pH levels up to
-0.8 units due to river runoff and microbial respiration (Duarte et al.,
2013; Thiyagarajan and Ko, 2012). Treatment levels of pH were maintained by
bubbling filtered natural seawater with air enriched with CO2 at
the required concentrations using gas flow meters/controllers (Cole-Parmer,
USA) (Fig. 1). Oyster larvae were raised from the D-shaped veliger stage to
the juvenile stage under the four pH levels with four biologically
independent replicates tanks for each treatment. D-shaped larvae (10 larvae mL-1, 50 L replicate tanks, 1 µm FSW, 31 salinity, at
29 ∘C ± 2 ∘C) were reared until the pediveliger stage
following methods described by Dineshram et al. (2013). After about 2 to
3 weeks, larvae attained competency for attachment and metamorphosis. Larvae
were transferred from each 50 L replicate tank to 1 L replicate tanks
containing plastic substrates coated with 7-day-old natural biofilms.
Attachment and metamorphosis took place within 24 h. Attached oysters were
reared in 1 L replicate tanks with the same pH level as before attachment
for 35 days until collection. Larvae and juveniles were fed twice a day using
a mixture of live I. galbana and C. gracilis (5–10×106 cells mL-1, 1:1 ratio). Seawater
pH (NBS scale) and temperature were measured using a Metter-Toledo (SG2)
probe, and salinity was measured using a refractometer (ATAGO, S/Mill0E;
Japan). The probe was calibrated using NIST buffers (pH = 4.01, 7.00, and
9.21; Mettler Toledo, Gmbh Analytical CH8603 Schwerzenbach, Switzerland). In
each culture, tanks levels of pH, temperature and salinity were measured
daily. Daily measurements were firstly averaged within and among days per
each replicate tank. Samples of seawater (50 mL) from each culture tank were
collected every 4 days and poisoned with 10 µL of 250 mM mercuric
chloride for total alkalinity (TA) analysis using the Alkalinity Titrator
(AC-A2, Apollo SciTech's Inc., US). The TA measurement was standardized with
a certified seawater reference material (Batch 106,
Andrew G. Dickson, Scripps Institution of Oceanography,
USA). The carbonate system parameters, i.e. carbon dioxide partial pressure
(pCO2; µatm), carbonate ion concentration
(CO32-; µmol kg-1), calcite and aragonite
saturation state (ΩCa, ΩAr), were calculated
from pH, salinity, temperature and TA measured from each replicated tank (n=4), using the CO2SYS software program (Pierrot et al., 2006) with
equilibrium constants K1, K2 and KSO4 (Mehrbach et al.,
1973; Dickson and Millero, 1987). The treatment level (mean ± SD;
Table 1) was calculated using averages of the replicate culture tanks within
each treatment (n=4). On the 35th day post-metamorphosis, juveniles were
collected and preserved in 75 % ethanol for the following analyses (Chan
et al., 2012).
Seawater physico-chemical parameters in the experimental system.
Treatments/parameter
Control
pH 7.8
pH 7.5
pH 7.2
pH
8.14±0.04
7.88±0.02
7.46±0.01
7.23±0.01
Temperature (∘C)
27.04±0.14
27.02±0.08
24.35±0.12
27.50±0.08
Salinity (psu)
31±0.5
31±0.5
31±0.5
31±0.5
TA (µequiv kg-1)
2053.77±46.51
2032.63±25.60
2061.50±4.56
2091.37±39.37
pCO2 (µatm)*
352.93±11.04
861.37±130.34
1997.23±124.42
4091.73±447.85
CO32- (µmol kg-1)*
175.66±24.96
97.92±16.38
48.82±6.07
26.59±4.72
ΩCa*
4.59±0.25
2.43±0.41
1.21±0.15
0.66±0.12
ΩAr*
3.01±0.18
1.59±0.28
0.79±0.10
0.43±0.08
Data are mean ± SD of the replicate culture tanks (n=4) for the seawater
physico-chemical parameters measured or calculated during the duration of the
experiment: pH (National Bureau of Standards scale), temperature
(∘C), salinity (psu), TA (µequiv kg-1), carbon dioxide
partial pressure (pCO2; µatm), carbonate ion
concentration (CO32-; µmol kg-1), calcite
saturation state (ΩCa), and aragonite saturation state
(ΩAr). Values were first averaged within and among days per
each of the replicate culture tanks. Afterwards, the treatment mean was
computed. * Parameters were calculated using the CO2SYS software program
(Pierrot et al., 2006) with equilibrium constants K1, K2 and
KSO4 (Mehrbach et al., 1973; Dickson and Millero, 1987).
Shell microstructure analysis
The sessile juvenile oyster permanently cements the left valve of its shell
to the substratum, whereas its right valve provides protection from predators and
the environment. In this study, only the right valve was used in the shell
analysis. The surface topography of the intact shell was examined under
variable pressure at 30 kV using a scanning electron microscope (SEM; Hitachi
S-3400N VP SEM, Hitachi, Japan). To examine sectional surface microstructures
(MacDonald et al., 2010), shells were embedded in epoxy resin (EpoxyCure,
Buehler) and sliced along the dorsal-ventral axis using a diamond trim saw
blade. This allows for a more controlled comparison between the hinge region
and the middle region of the shell. The hinge region (hereafter also referred
to as “older shell”) is the part of the shell that is deposited first by
the juvenile oyster, whereas the middle region (hereafter also referred to as
“younger shell”) is the part of the shell that is deposited more recently.
The edge region (Galtsoff, 1964), formed most recently, was not included in
this study because it is too thin and fragile to handle. The sectioned
surfaces were polished for 2 to 5 min using grit papers (P320, P800, P1200,
P2500, and P4000) and etched for 20 s using 1 % acetic acid, and then
washed with distilled water and air-dried. The sectioned resin blocks were
mounted on aluminium stubs using carbon adhesive tape with the polished side
up. The area surrounding the specimen was painted with silver to reduce
charge build-up, and the sectioned surfaces were sputter-coated with 50 nm
thick gold–palladium alloy. The shell microstructure was examined under SEM
with an accelerating voltage of 5 kV using a LEO 1530 Gemini FSEM (Zeiss,
Germany). The cross-sectional porosity of foliated laminated structure was
calculated using ImageJ software by standardizing and converting an SEM image
to thresholding where the non-diffracted regions of SEM images were defined
as pores. The pore area was then calculated by using the ImageJ “Analyse
Particles” feature due to the divergence in the size of pores. The pores
area was sized with a confidence area greater than 0.001 µm2.
Three to four specimens from each treatment were randomly selected and
examined (n=3–4). All data were tested for normality of residuals,
normality and homogeneity of variance before conducting analysis of variance (ANOVA).
The Student–Newman–Keuls test was used to compare the means following one-way
ANOVA.
Shell crystallographic orientation analysis
Shell crystallographic orientation was analysed by electron back-scattered
diffraction (EBSD). Shells were prepared according to the above method, minus
etching. The shell surfaces were ultra-polished for 4 min using cloths with
1 and 0.3 µm Alpha alumina powders and for 2 min using colloidal
silica. In order to investigate both larva aragonite and juvenile calcite
composition, an area throughout the sectional surface of the older hinge
regions was selected. The EBSD analyses were carried out under low vacuum
mode (∼50 Pa) with a beam voltage of 20 kV using an FEI Quanta 200F
with the stage tilted at 70∘ to examine back-scattered Kikuchi patterns
(Perez-Huerta and Cusack, 2009). Diffraction intensity, phase and
crystallographic orientation maps were produced using the OIM Analysis 6.2
software. Data was partitioned through two clean-up procedures to display
grains with a confidence index greater than 0.1. Pole figures were used
to illustrate the spread of crystallographic orientation (Perez-Huerta and
Cusack, 2009). The colours in the crystallographic orientation maps and pole
figures were used to quantify the crystallographic orientation. Two randomly
selected specimens were examined per treatment.
Scanning electron micrographs of 35-day-old juvenile
Magallana angulata shells cultured at ambient or control pH
8.1 (a, e), treatment pH 7.8 (b, f), pH 7.5 (c, g)
and pH 7.2 (d, h) were compared. Panels (a–d) show the low magnification tomography of the juvenile shells. Panels (e–h) show the enlarged view of
the crystallite units (top view). (e) The prism units were arranged
in compact prismatic structures at pH 8.1. (f) Prismatic arrangement
was partially lost at pH 7.8. (g) A rough surface was observed,
demonstrating a much lower level of organization at pH 7.5. (h) A
smooth surface was observed with no prismatic arrangement due to dissolution
by environmental seawater.
Shell mechanical properties analysis
After SEM and EBSD analysis, the resin blocks were re-polished for 5 min
using grit papers (P2500 and P4000) and for another 5 min using cloth with
colloidal silica to remove the gold–palladium coating and etched shell
surface. The mechanical properties of the polished longitudinal cross
sections were determined by measuring the hardness (H) and stiffness (E)
using load and displacement sensing nanoindentation tests (Perez-Huerta et
al., 2007). Hardness and stiffness of foliated layers were measured in the
older hinge and younger middle regions of the specimens used in the SEM
analysis. The nanoindentation tests were carried out from the interior to the
exterior shell in these regions at ambient temperature with a Hysitron
TriboIndenter TI 900 (TI 900, Hysitron, MN, USA) equipped with a Berkovich
indenter (with a half-angle of 63.5∘). Indentations were made in each
specimen using a 6–11 indent-per-row pattern and a maximum load of
2000 µN with valid contact depth of 16 to 184 nm. The hardness and
stiffness from each indentation were obtained from the loading–unloading
curve using the Oliver–Pharr model (Doerner and Nix, 1986; Oliver and Pharr,
1992). Five to six specimens of each treatment were randomly selected for
nanoindentation tests (one to two specimens per replicate tanks). Measurements
per replicate tanks were calculated by firstly averaging the values among
indentations per specimen, and then among specimens per replicates.
Afterwards, the effect of decreased pH on the hardness and stiffness of
juvenile oyster shells was compared by three to four replicate measurements
(n=3–4). All data were tested for normality of residuals, normality, and
homogeneity of variance before conducting ANOVA. The Student–Newman–Keuls test
was used to compare the means following one-way ANOVA.
Microstructures were observed in the cross-sectional shell surfaces
of 35-day-old juvenile Magallana angulata. Scanning electron
micrographs were taken near the older hinge region (b, c, f, g, j, k, n, o) and the younger middle region (d, e, h, i, l, m, p and q).
Panel (a) shows the scanning electron micrograph of the full shell cross-sectional
surface. Second row: the prismatic layer (b, d) and
tightly packed foliated structure (c, e) at pH 8.1. Third row: the
prismatic layer (f, h) and the foliated structure with more and
bigger pores (g, i) at pH 7.8 compared with at pH 8.1. Fourth row:
the incomplete prismatic layer (j, l) and more porous foliated
structure (k, m) at pH 7.5 compared with at pH 8.1. Fifth row: the
prismatic layer was not detectable (n, p) with porous foliated
structure (o, q) at pH 7.2. The porosity of foliated layers at the
older (r) and younger regions (s) of the shell reared under
control and low pH treatments. The mean values are presented in the bar chart
(mean ± SD, n=3–4). Annotations: P is the prismatic layer; F is the foliated
layer.
Shell density analysis
The three-dimensional shell density maps, the overall shell density and the
density–volume ratio relationships were obtained using a high-resolution
micro-CT scanning system (SkyScan 1076, Skyscan, Kontich, Belgium) with a
spatial resolution of 9 µm. Individual shells were placed in a
small plastic container held securely in the chamber of the micro-CT scanner.
Shell densities and the corresponding volume ratios of partial density were
calculated by relative comparison using standardized phantoms used for bone
density measurement in the analytical software CT-Analyser v 1.14.4.1
(SkyScan) (Celenk and Celenk, 2012). The three-dimensional digital data were converted from
∼1000 two-dimensional layers using reconstruction software CT-Volume v
2.2.1.0 (SkyScan). Three randomly selected specimens were used per treatment
(n=3). The volume ratio with partial density ranges of 0 to 0.5, 0.5 to
1 and > 1.5 g cm-3, and density of the treatment groups
were compared with the controls by following one-way ANOVA. For the datasets that did
not meet the requirement of variance homogeneity, i.e. the volume ratio with
a partial density range of 1 to 1.5 g cm-3, Kruskal–Wallis tests were
used to compare the effect of pH on these shell properties. For all other
datasets, the Student–Newman–Keuls test was used to compare the means by following
one-way ANOVA. Otherwise, Dunn's test was used after the Kruskal–Wallis test.
Linear regressions (volume ratio (%) =b× density (g cm-3)
+ a) were utilized to determine the relationships between shell density and
the corresponding volume ratio; a is the y intercept and b is the scaling
exponent of consumption. To compare slopes of the resulting linear models,
analysis of covariance (ANCOVA) was performed by using log10 transformed
volume ratio as the dependent variable, pH levels as the independent
variable, and shell density range as covariates. All data met the homogeneity
of variance and normality assumptions of parametric tests. ANCOVA were
implemented in R 3.3.2 using the statistical package Linear and Nonlinear
Mixed Effects Models (R Core Team, 2013).
Results
Shell surface and internal microstructure
As shown by the SEM, decreased pH altered both shell topography (Fig. 2) and
internal microstructure (Fig. 3). Mineral dissolution was prominent on the
outer surface layers of shells under decreased pH. The shells of juveniles
raised at pH 7.8 (Fig. 2b, f) and pH 7.5 (Fig. 2c, g) showed signs of
dissolution or physical damage when compared to the controls (pH 8.1)
(Fig. 2a, e). At the lowest pH of 7.2 with undersaturated calcite conditions,
the outer prismatic layer was completely absent at the older hinge and
younger middle regions of the shell (Fig. 2d, h). Though the overall calcitic
foliated laminas' alignment was retained, those in the shells of controls
(pH 8.1) were compactly arranged and well-ordered with minimal gaps between
layers (Fig. 3c, e). In contrast, the foliated layers in shells under all
three decreased pH treatments presented a more porous alignment in that the
foliated laminas were less tightly packed and irregularly arranged (Fig. 3g,
i, k, m, o and q). The area porosity of foliated layers increased
significantly with decreasing pH levels, regardless of older and younger
shell (older region: F(3,11)=3.683, p=0.045; younger region:
F(3,11)=7.480, p=0.005; Fig. 3r, s).
Electron back-scattered diffraction analyses of shells grown for 35
days at control pH 8.1 (a), treatment pH 7.8 (b),
pH 7.5 (c) and pH 7.2 (d). Crystallographic orientation
maps (left column) of calcite crystals in reference to the
0001 plane and aragonite crystals in
reference to the 001 plane. Crystallographic
planes of calcite are colour-coded according to the normal crystallographic
direction shown in the colour key (Perez-Huerta and Cusack, 2009). Pole
figures for calcite (centre column) corresponding to the crystallographic orientation
maps with the same colour key. The right column shows the phase maps of calcite exhibited in red
and aragonite in green. The white arrow denotes a change in colour of 5 to 10 marginal
foliated laminates. Scale bar = 45 µm.
Crystallographic orientation
Electron back-scattered diffraction (EBSD) intensity mapping analysis showed
diffraction patterns for both calcite and aragonite crystals of older hinge
regions in the juvenile shells (Fig. 4). The crystallographic orientation
maps (Fig. 4 left column) showed changes in crystallographic orientation from the
control (pH 8.1) to low pH conditions (pH 7.8, 7.5 and 7.2) as represented by
colour change corresponding to the colour key (Fitzer et al., 2014). The
spread of data points in pole figures (Fig. 4 centre column) highlighted the variation in crystallographic orientation
between the juvenile oysters under the low pH and the control conditions.
Though the foliated layers of shells under decreased pH showed colour
variations within a limited area (∼5–10 foliated laminas) close to the
interior, the majority of calcite crystal units showed uniform orientation,
the same as those in the control (Fig. 4 left and centre columns). It was confirmed by the pole figures that the preferred
crystallographic orientation of foliated layers was identical, resulting in
the extent of the variation in crystal orientation of 40∘ regardless
of pH treatments, corresponding to the colours in the orientation maps
(Fig. 4 left column). But notably, there was an
absence of aragonite in the shells formed under pH 7.2
(Fig. 4 right column). Although the aragonitic
crystals are not present in the most extreme treatment (pH 7.2), the overall
crystallographic orientation of the calcitic fraction did not change between
treatments.
Shell hardness and stiffness
Shell hardness was significantly reduced as treatment pH decreased, relative
to control (older region: F(3,11)=21.987, p < 0.001;
younger region: F(3,11)=4.135, p=0.034). Similarly, shells at pH
7.5 and 7.2 had reduced stiffness compared to the controls (Fig. 5c, d)
(older region: F(3,11)=4.525, p=0.027; younger region: F(3,11)=7.369, p=0.006). The reduced mechanical features due to decreased pH
were observed in both the older hinge regions and younger middle regions
(Fig. 5).
Shell mechanical properties in terms of hardness (a, b) and
stiffness (c, d) with longer and shorter exposures in older hinge
regions (a, c) and younger middle regions (b, d) in
cross-sectional shell surfaces of Magallana angulata were compared.
Data of mechanical properties are presented as mean ± SD of three to
four replicates (n=3 or 4).
Effects of low pH on the shell density map (a–d), overall
density (e), and shell density–volume ratio
relationships (f) for the four experimental pH treatment groups were
examined by micro-CT of shells of Magallana angulata. Three-dimensional reconstructions represent the density distribution of the shells
produced in ambient or control pH 8.1 (a), treatment
pH 7.8 (b), pH 7.5 (c) and pH 7.2 (d). The volume
ratios of density categories of < 0.5, 0.5–1.0, 1.0–1.5, and
> 1.5 g cm-3 were quantified. (e) The overall
density was presented as mean ± SD of three replicates (n=3).
(f) shell density–volume ratio relationships for the four
experimental pH treatment groups of C. angulata. Regression lines
for the three low pH treatments closely overlap and are partly obscured.
Shell density
Three-dimensional shell density maps (Fig. 6a–d), the overall shell
density and the relationship of shell density–volume ratio by
micro-computed tomography (micro-CT) showed an overall decrease of shell
density with decreasing pH (Fig. 6e) (F(3,8)=5.318, p=0.026). A
similar decrease is visible in the linear regressions (volume ratio (%) =b× density (g cm-3) +a) in Fig. 6f. Volume ratios were
decreased with the increased shell density in all pH treatments (ANCOVA;
shell density, F(1,263)=1253.14, p < 0.001). There was an
interaction between pH and shell density (ANCOVA; pH × density, F(3,263)=4.994, p=0.002), indicating that the effect of pH on the
density–volume ratio relationship was different. The lower scaling of
consumptions at pH 7.8 (mean exponent -0.063), pH 7.5 (mean exponent
-0.065) and pH 7.2 (mean exponent -0.062) versus the control pH level of 8.1
(mean exponent -0.052) indicates that the volume ratio of denser shell was
reduced with decreased pH while the volume ratio of less dense shell was
increased correspondingly (Fig. 6f). The three-dimensional shell density map (Fig. 6a–d)
reinforces the effect of decreased pH on the density–volume ratio
relationships. In the controls, shells were produced with denser minerals
compared to shells in decreased pH (Fig. 6a). Shells at pH 7.8, pH 7.5 and pH
7.2 had larger proportions of lower shell density regions or “pores”
(Fig. 6b–d). These pores were observed in the three-dimensional density maps as density
values below the detection threshold (Fig. 6a–d). Classifying the shell
volumes into four density categories, i.e. < 0.5, 0.5–1, 1–1.5
and > 1.5 g cm-3, showed that the proportions of high
(> 1.5 g cm-3) and low (< 0.5 g cm-3)
shell density areas were significantly different between pH treatments
(Fig. 6a–d). The volume ratios of high density areas were significantly
reduced in each pH treatment (pH 7.8, pH 7.5 and pH 7.2) when compared to the
controls (pH 8.1) (F(3,8)=4.856, p=0.033). Meanwhile, the
volume ratios of low density areas (< 0.5 g cm-3)
significantly increased in each of the lower pH treatments (pH 7.8, pH 7.5
and pH 7.2) compared to the controls (F (3,8)=6.945, p=0.013).
There were no significant differences in the volume ratios of the middle
densities (0.5–1 g cm-3: χ(2)2=5.615, p=0.132;
1–1.5 g cm-3: F(3,8)=3.713, p=0.061) among treatments
(Fig. 6a–d).
Discussion
This study provided new compelling information of structure–property
relationships in calcareous shells of commercially important oyster species
at different spatial scales and under a variety of environmentally and
climatically relevant OA scenarios (i.e. different levels of decreased pH via
pCO2 increase). The revealed structural information and
subsequent analysis of mechanical features in this study provided an
important experimental basis for developing models to forecast the impact of
ocean acidification on marine calcifying organisms. The rate of calcification
of many marine organisms is expected to be significantly reduced in
near-future oceans with a reduced pH of 7.8 due to OA (Ries, 2011; Bednarsek
et al., 2012; Duquette et al., 2017; Chatzinikolaou et al., 2017). This study
observed the same calcification trend in Magallana angulata
because OA not only depletes carbonate ions necessary for CaCO3
mineralization, but it also metabolically weakens marine organisms
through altered physiological processes, i.e. acidosis (Dupont and
Portner, 2013). Importantly, this study provides strong evidence to support
the argument that shells produced by oysters under OA suffer from dissolution
with disorganized or impaired crystal orientation and microstructures, and
reduced mechanical properties. The possible mechanisms and consequences
underlying such negative effects of decreased pH on mechanics of shell
structure are discussed in the following sections.
Effect of ocean acidification on shell mechanical features: a
hierarchical analysis
In any given biologically formed material, mechanical properties at
the macroscale generally depend on the composition of material components and
materials' microstructural features (Rodriguez-Navarro et al., 2002; Meng et
al., 2018). In this study, oyster shell material is composed of two calcium
carbonate polymorphs, calcite and aragonite. Oysters begin their life
(larvae) with aragonite-based shells, and these are completely replaced by calcite
in adult shells, though juvenile shells may retain a tiny portion of aragonite
(Weiss et al., 2002). Calcite is relatively less soluble in seawater compared
to aragonite regardless of environmental pH. Calcite is a relatively less
soluble form of CaCO3 in conditions with decreased pH when compared to aragonite (Lippmann, 1973). This
chemical feature of calcite may have made it feasible for the juvenile
oysters to successfully mineralize and retain a laminated calcareous
structure, even under undersaturated CaCO3 saturation levels, e.g.
decreased pH 7.4 (Fig. 3).
Like the previously described oyster shell microstructure (Dauphin and Denis,
2000; MacDonald et al., 2010), the materials used in this study are composed
of structurally organized layers. The bulk of the microstructure is
characterized by foliated layers of crystal units organized in lamellae. In
order to understand the modulating effect of environmental pH on the
relationship between the shell structural and mechanical features, we have
quantified the space or pore size between laminated layers within the
folia. The decreased pH significantly increased size and quantity of the
pores in the folia layer. The presence of such laminated folia with pores or gaps
was an obvious consequence of decreased pH. However, the larvae were still
capable of producing a new foliated layer under these treatments whilst at
undersaturation (at pH 7.2). Although the juvenile shells show signs of
physical dissolution, the EBSD and porosity data suggest that the
microstructure growth is impaired initially. This microstructural impairment
was observed even under the near-future level of decreased pH 7.8, where the
porosity was increased 10-fold (Fig. 3r). On the other hand, the
preferred orientation of crystal units within the folia layer showed no
difference in all low pH treatments, with the c axis of calcite units
approximately perpendicular to the outer and inner shell surface. Thus, the
significantly reduced hardness and stiffness of the foliated layer under
decreased pH might be due to the impaired microstructure with significantly
higher pore size and numbers.
Furthermore, we measured the impacts of decreased pH on whole shell density
and thus on pores or gaps in foliated layers using micro-CT analysis.
Notably, higher density shell volume reduces with decreasing pH. This result
supports our finding on the effect of decreased pH on microscale structure
and mechanical features in the folia. Calcite shell materials are brittle in
nature, like egg shells or ceramics; therefore their resistance to
deformation (or breaking force) is largely dependent on the stiffness parameter of
the shell (Lawn and Wilshaw, 1993). Here, we found that both hardness and
stiffness of the folia layer reduce with decreasing pH, which may have
triggered shell fracture under simulated external attack. A folia layer with
lower stiffness and hardness resulting from a porous laminated microstructure
is expected to be highly vulnerable to predatory attack, even though the
preferred orientation remains unaffected (Kemeny and Cook, 1986). In
addition, the overall decrease of shell density detected by micro-CT analysis
indicates the porous internal microstructure may occur throughout the
juvenile shell. In other words, the juvenile oyster shell with impaired
microstructural features is more prone to predator attack under the near-future
level of decreased pH due to OA processes.
Effect of ocean acidification on shell microstructure and
crystallography
The outermost prismatic layers of the older hinge and younger middle regions
had completely disappeared when juvenile oysters were exposed to the extreme
scenarios (pH of 7.2 and calcite undersaturation Ωcal≈0.66) (Figs. 2h and 3n, p). Undersaturated waters, with regards to calcite
(Ωcal < 1), result in the dissolution of calcitic materials
(Bednarsek et al., 2012; Lippmann, 1973). Similar impacts were observed in
Argopecten irradians (pH 7.8 and pH 7.5) (Talmage and Gobler, 2010),
Mercenaria mercenaria (pH 7.7) (Dickinson et al., 2013) and
Saccostrea glomerata (pH 7.8 and pH 7.6) (Watson et al., 2009).
The juvenile oysters exposed to decreased pH exhibited a porous microstructure
in foliated layers (Fig. 3). Firstly, this may be due to the decreased
calcification rate resulted from the metabolic depression and/or energy
shortage in the decreased pH conditions (Gobler and Talmage, 2014; Lannig et
al., 2010). Secondly, it could be due to the dissolution of newly formed
minerals of the inner surface in the lower pH conditions (Melzner et al.,
2011). Marine invertebrates' calcification has highly controlled mechanisms
and remains to be explored by further studies (Krause-Nehring et al., 2011).
Animals are capable of actively increasing the site of calcification by
pumping protons out of the calcification site, thereby enabling calcium
carbonate precipitation (Ries et al., 2010; Toyofuku et al., 2017).
Supersaturated calcite conditions of oysters were found restricted to the
shell edge including the outer mantle and the first intracellular nucleation
site (Mount et al., 2004). Undersaturated calcite conditions may be
maintained elsewhere in contact with the inner shell surface (Addadi et al.,
2006; Thomsen et al., 2010). Therefore, in low pH conditions due to OA, these
inner areas of newly formed minerals, which are precipitated as structural
building blocks for the prismatic and foliated layers, may still be prone to
dissolution. When the shell dissolution rate is faster than the
mineralization rate, organisms tend to produce thinner and lighter (less
dense) shells, resulting in an impaired shell microstructure. This may explain
the multiple negative effects of reduced pH in our results, including porous
and less dense foliated layers. Similarly, mussel shells grown in lower pH
conditions (pH 7.65) showed inner shell surface dissolution (Melzner et al.,
2011) and an impaired shell microstructure (Hahn et al., 2012), which is
consistent with the results in this study. The crystallography of marine
shells is the other important proxy for environmental stressors (Milano et al.,
2017). The crystallographic orientation maps of foliated layers showed a
preferred crystallographic orientation with ∼40∘ of variation
regardless of pH treatments, which concurs with the results of field samples
(Checa et al., 2018). Compared to calcite, aragonite represents a small
fraction of the oyster shells and is more soluble under decreased pH
conditions (Fitzer et al., 2014), which could explain the absence of
aragonite in the older hinge regions at pH 7.2 (Fig. 4 right column) is observed in this study. A similar absence of aragonite
was also reported in mussel shells in high pCO2
(1000 µatm) conditions (Fitzer et al., 2014). Nevertheless,
aragonite dissolution may be very relevant for oyster shells during
early life stages but not so much for adult shells. Therefore, it plays
an insignificant role in shell mechanical properties of the calcite-predominant
adult shells and thus the adult oyster survival.
Ecological implications and conclusion
Although previous studies showed that early larval life stages of several
edible oyster species were relatively physiologically tolerant of near-future
OA conditions (Dineshram et al., 2013; Ko et al., 2013, 2014; Thiyagarajan
and Ko, 2012), this study shows that they are still vulnerable due to the
effects decreased pH has on shell characteristics, like porosity, hardness
and stiffness. A similar negative impact of OA on shell mechanical properties
was reported in various marine calcifiers. For example, the pearl oyster,
Pinctada fucata, produced a 25.9 % weaker shell after exposure
to seawater at pH 7.8 (Welladsen et al., 2010). Decreasing shell mechanical
properties in decreased pH conditions were also observed in Mytilus californianus (Gaylord et al., 2011), which produced 20% weaker shells
under pH 7.8, Mercenaria mercenaria (Dickinson et al., 2013; Ivanina
et al., 2013), which produced approximately 60 % softer shells under pH
7.77 (16 salinity) and Hydroides elegans (Li et al., 2014), which
produced 80 % softer tubes under pH 7.8. However,
the effects of increased carbon dioxide partial pressure (pCO2) on
shell mechanical properties are species-specific. Near-future conditions (pH
7.8) did not affect shell hardness in the sea urchin Paracentrotus lividus (Collard et al., 2016) or in the barnacle Amphibalanus amphitrite (McDonald et al., 2009). Furthermore, juvenile oysters of
C. gigas significantly increased their shell strength and size as a
compensatory adaptive response to low pH conditions (i.e. pH 7.8) (Wright et
al., 2014), and the blue mussel, Mytilus edulis, produced a stiffer
and harder calcite layer under increased pCO2 conditions (i.e. pH
7.3) (Fitzer et al., 2015).
The long-term survival strategy of oysters with mechanically softer shells is
yet to be studied. However, as shown in a recent study (Sanford et al.,
2014), it appears that weaker shell structures will result in compromised
defence abilities. Moreover, results from a recent study suggest that oysters
with reduced and impaired calcification mechanisms have lower repair
capabilities (Coleman et al., 2014). This hierarchical study revealed that
the OA conditions may cause a deterioration of oyster shells and thus pose a
serious threat to oyster survival and the health of coastal oyster reef
structures in the near-future ocean. This biological effect of OA on shell
structures and mechanical features should be incorporated in coastal
oceanographic biophysical models to accurately project the survival of
oysters in near-future coastal oceans which could be used for commercial
shellfisheries to plan for sustainable growth under climate-change-induced
acidification.