Ocean acidification reduces mechanical properties of the Portuguese oyster shell with impaired microstructure: a hierarchical analysis

. The rapidly intensifying process of ocean acidification (OA) in coastal areas due to anthropogenic CO 2 is not only depleting carbonate ions necessary for calcification but also causing acidosis and disrupting internal pH homeostasis in several marine organisms. These negative consequences of OA on marine communities, particularly to shellfish oyster species, has been very well documented in recent studies, however, the consequences of these reduced or impaired calcification processes on the end-product, shells or skeletons, still remains one of the major research gaps. Shells produced 20 by marine organisms under OA are expected to be corroded with disorganized or impaired crystal orientation or microstructures with reduced mechanical property. To bridge this knowledge gap and to test the above hypothesis, we investigated the effect of OA on shell of the commercially important oyster species ( Crassostrea angulata ) at ecologically and climatically relevant OA levels (using pH 8.1, 7.8, 7.5, 7.2 as proxies). In decreased pH conditions, a drop of shell hardness and stiffness was revealed by nanoindentation tests, while an evident loosened internal 25 microstructure was detected by scanning electron microscopy (SEM). In contrary, the crystallographic orientation of oyster shell showed no significant difference with decreasing pH by Electron Back Scattered Diffraction (EBSD) analyses. These results indicate the loosened internal microstructure may be the cause of the OA induced reduction in shell hardness and stiffness. Micro-computed tomography analysis (Micro-CT) indicated that an overall “down-shifting” of mineral density in the shell with decreasing pH, which implied the loosened internal microstructure may run through 30 the shell, thus inevitably limiting the effectiveness of the shell defensive function. This study surfaces potential bottom-up deterioration induced by OA on oyster shells, especially in their early juvenile life stage. This knowledge is critical to forecast the survival and production of edible oysters in future ocean.

example, spat production in oyster hatcheries on the west coast of USA has started to decline, partially due to poorly calcified larval shells under upwelled high-CO2 waters (Barton et al., 2012). Previous studies on calcifying organisms, including oysters, suggest that OA not only reduces calcification rates, but also increases dissolution of formed shells (Ries, 2011;Bednarsek et al., 2012). The decreased pH depletes carbonate ions necessary for CaCO3 mineralization chemically, as well as weakens marine organisms physiologically by causing acidosis and impairing internal pH homeostasis needed for 55 optimal calcification (Dupont and Portner, 2013) . Recently, an increasing number of studies capture the importance of the mechanical properties of calcareous shell, the end-products of calcification, under OA scenario (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 and the Eastern oyster produced softer shells with reduced mechanical strength under OA condition (Beniash et al., 2010;Dickinson et al., 2012). Despite these OA threats to oyster 60 calcification process, studies are yet to demosntrate the hierarchical structural organization of oyster shells under elevated CO2 and OA conditions. Importantly, the modulating effect of OA on the inherent relationship between shell strctural and mechanical features is yet to be studied in detail.
This study is designed specifically to fill this gap in our current knowledge using the ecologically and economically important edible oyster (Crassostrea angulata) as model species. Here, the quantitative relationship between microstructural 65 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 levels of high-CO2 induced decreased pH. As the calcitic foliated layer is the major shell structure for mechanical support in oysters (Lee et al., 2008), we specifically examined its structural and mechanical properties by using variety of materials science techniques such as scanning electron microscopy (SEM), crystallography by electron backscatter diffraction (EBSD) and nanoindentation tests. 70 To further evaluate the overall structural integrity, we quantified shell mineral density, mineral density-volume ratio relationships using high-resolution micro-computed tomography scanning (Micro-CT).

Experimental animal and design
Sexually matured adult oysters of the Portuguese oyster species, Crassostrea angulata, were collected from the coastal area 75 flow-through tanks in natural seawater at ambient conditions (31 psu salinity, 29 o 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 by the "strip spawning" method , and cultured under ambient conditions. After 80 24 h postfertilization, embryos developed into D-shaped veliger larvae. Larvae were subjected to pH perturbation to study the effects of ocean acidification (OA) process on oyster shell structure and mechanical features in the early stages of development.
Four environmentally and climatically relevant pH levels (pH 8.1, 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 85 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). The very low pH 7.2 treatment was included in this study to understand the impact of extreme environmental conditions in the coastal habitats of C. angulata, where seawater pH naturally fluctuates and may decrease by as much as 0.8 units due to river runoff and microbial respiration (Duarte et al., 2013;Thiyagarajan and Ko, 2012). Treatment pH levels were maintained by bubbling filtered natural seawater with air enriched with CO2 at the required concentrations using gas flow meters/controllers (Cole-90 Parmer, USA). 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. Briefly, D-shaped larvae (10 larvae/mL, 50L replicate tanks, 1 μm FSW, 31 psu salinity, at 29 o C ± 2 o C) were reared until the pediveliger stage following previously described methods . After about 2 to 3 weeks, larvae attained competency for attachment and metamorphosis.
They were transferred from each 50 L replicate tanks to 1 L replicate tanks containing plastic substrates coated with 7-day-95 old natural biofilms. Attachment and metamorphosis took place within 24 h, and attached oyster were reared in 1 L replicate tanks with the same pH level before attachment for 35 days until shell collection for subsequent analysis. Larvae and juveniles were fed twice a day using mixture of live Isochrysis galbana and Chaetoceros gracilis (5-10×10 6 cells/mL, 1:1 ratio). Seawater pH (NBS scale) and the temperature were measured using a Metter-Toledo (SG2) probe and salinity with a refractometer (ATAGO, S/Mill0E; Japan). The probe was calibrated using NIST buffers (pH =4.01, 7.00, and 9.21; Mettler 100 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.

Shell microstructure analysis
The sessile juvenile oyster permanently cements the left valve of its shell to 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; 115 Hitachi S-3400N VP SEM, Hitachi, Japan). To examine sectional surface microstructures, 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 bill region, formed most 120 recently, was not included in this study because it is too 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 seconds 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 buildup, and the sectioned surfaces were sputter-coated with 50-nm thick gold−palladium alloy. The shell microstructures were 125 examined under 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. 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 of greater than 0.001 µm 2 . Three to four specimens from each treatment were randomly selected and examined (n = 3~4). All data was tested for normality of 130 residuals, normality, and homogeneity of variance before analysing by ANOVA. Student-Newman-Keuls test was used to compare the means following one-way ANOVA.

Shell crystallography analysis
Shell crystallographic orientation was analysed by Electron Backscatter Diffraction (EBSD). Shells were prepared according to the above method, but without etching. The shell surfaces were ultra-polished for 4 min using cloths with 1 μm and 0.3 135 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 were 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 backscatter 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 140 clean-up procedures to display grains with a confidence index (CI) 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.

Shell mechanical properties analysis 145
After SEM and EBSD analysis, the resin blocks were re-polished for 5 min using grit papers (P2500 and P4000) and for another 5min 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 150 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 loadingunloading curve using the Oliver-Pharr model (Doerner and Nix, 1986;Oliver and Pharr, 1992). Five to six specimens of 155 each treatment were randomly selected for nanoindentation tests. Measurements were firstly averaged within per specimen and then per replicate tank. Finally, three to four replicate values per treatment were compared (n = 3~4). All data was tested for normality of residuals, normality, and homogeneity of variance before analysing by ANOVA. Student-Newman-Keuls test was used to compare the means following one-way ANOVA.

Shell mineral density analysis 160
The three-dimensional shell density maps, the overall mineral density and the mineral 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 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 165 Celenk, 2012). The 3D digital data was converted from ~1000 2D 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 g/cm 3 , 0.5 to 1 g/cm 3 , and >1.5 g/cm 3 , and density of the treatment groups were compared with the controls by one-way ANOVAs. 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. 170 For all other datasets, Student-Newman-Keuls test was used to compare the means following one-way ANOVA. Otherwise, Dunn's test was used after Kruskal-Wallis test. Linear regressions (Volume ratio (%) = b× mineral density (g/cm 3 ) +a) was utilized to determine the relationships between mineral density and 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 175 mineral 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 (Team, 2013).

Deceased pH alters shell surface and internal microstructure 180
As shown by the SEM, decreased pH altered both shell topography (Fig. 1) and internal microstructure (Fig. 2). Mineral dissolution or erosion was prominent on the outer surface layers of shells under decreased pH. The shells of juveniles raised at pH 7.8 (Fig. 1b, f) and pH 7.5 (Fig. 1c, g) showed signs of erosion or physical damage when compared to the controls (Fig. 1a, 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. 1d, h). Though the overall calcitic foliated laminas alignment 185 were retained, those in the shells of untreated juveniles (controls) were compactly arranged and well-ordered with minimal gaps between layers (Fig. 2c, e). In contrast, the foliated layers in shells under all three decreased pH treatments were less tightly packed and irregularly arranged (Fig. 2g, i, k, m, o and q). The area porosity of foliated layers was significantly increased by decreased pH treatments, 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. 2r, s). 190

Decreased pH does not affect the crystallographic orientation of foliated layer
Electron backscatter diffraction (EBSD) intensity mapping analysis showed diffraction patterns for both calcite and aragonite crystals of older hinge regions in the juvenile shells (Fig. 3). From crystallographic orientation maps, 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. 3.i and ii). The 195 spread of data points in the pole figures (Fig. 3.ii) confirmed the identical preferred crystallographic orientation of foliated layers, resulting in the extent of the variation in crystal orientation of 40 degree regardless of pH treatments, which corresponded to the colours in the orientation maps (Fig. 3.i). The aragonite crystal units appeared to be similarly distributed ( Fig. 3.iii), but notably, there was an absence of aragonite in the shells formed under pH 7.2 ( Fig. 3.iv). Though decreased pH had a restricted effect on the marginal foliated laminas closed to interior of oyster shell, the overall crystallographic 200 orientation of main shell regions was not affected.

An overall "down-shifting" of shell density with decreased pH
Three-dimensional (3D) shell density maps, the overall shell density and mineral density-volume ratio relationships by micro-computed tomography (Micro-CT) showed an overall "down-shifting" of mineral density in the shell with 210 decreasing pH (Fig. 5). The shell mineral density was significantly reduced by decreased pH (Fig. 5e) (F (3,8) = 5.318, p = 0.026) which may be due to the altered mineral density-volume ratio relationships (Fig. 5f). Volume ratios were decreased with the increased mineral density in all pH treatments (ANCOVA; mineral density, F (1,263) = 1253.14, p < 0.001). There was an interaction between pH and mineral density (ANCOVA; pH ×mineral density, F (3,263) = 4.994, p = 0.002), indicating that the effect of pH on the mineral density-volume ratio was different. The lower scaling of consumptions at pH 7.8 (mean 215 exponent -0.063), pH 7.5 (mean exponent -0.065), pH 7.2 (mean exponent -0.062) versus the control pH level of 8.1 (mean exponent -0.052), indicating the volume ratio of denser shell was reduced with decreased pH while the volume ratio of less denser shell was increased correspondingly (Fig. 5f). 3D shell density map (Fig. 5 a-d) reinforced the effect of decreased pH on the mineral density-volume ratio relationships. In the controls, shells were produced with denser minerals compared to shells in decreased pH (Fig. 5a). Shells in pH 7.8, pH.7.5 and pH 7.2 had larger proportions of lower mineral density regions 220 or "pores" (Fig. 5b-d). These pores were observed in the 3D density maps as density values below the detection threshold ( Fig. 5a-d). With classifying the shell volumes into four density categories, i.e., < 0.5 g/cm 3 , 0.5-1 g/cm 3 , 1-1.5 g/cm 3 and > 1.5 g/cm 3 , the proportions of high (>1.5 g/cm 3 ) and low (< 0.5 g/cm 3 ) shell mineral density areas were significantly affected by decreasing pH (Fig. 5a-d). The volume ratios of high density areas were significantly reduced in all three decreased pH treatments (pH 7.8, pH 7.5 and pH 7.2) when compared to the controls (F (3,8) = 4.856, p = 0.033). Meanwhile, the volume 225 ratios of low density areas (< 0.5 g/cm 3 ) significantly increased in decreased pH treatments (pH 7.8, pH 7.5 and pH 7.2) when compared to the controls (F (3,8) = 6.945, p = 0.013). There were no significant differences in the volume ratios of the middle mineral 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.  5a-d). This study provided new compiling 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 conditions of elevated CO2 driven decreased pH. The reveled structural information and subsequent analysis of mechanical features in this study provided an important experimental basis for developing predictive models to forecast the impact of ocean acidification process on marine calcifying organisms. The rate of calcareous shell formation of many marine 235 organisms is expected to be significantly reduced in near-future oceans with a reduced pH of 7.8 due to ocean acidification (OA) process (Ries, 2011;Bednarsek et al., 2012). We have also observed a similar trend on calcification process in the Crassostrea angulata because decreased pH due to OA is not only depleting carbonate ions necessary for CaCO3 mineralization, but also metabolically weakening marine organisms through the altered physiological processes, i.e. acidosis (Dupont and Portner, 2013). Importantly, this study provided a strong evidence to support the argument that shells produced 240 by oysters under OA are corroded with disorganized or impaired crystal orientation or microstructures with reduced mechanical properties. The possible mechanisms and consequences underlying such a 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 materials, mechanical properties at macroscale is generally depends on composition of 245 material component and materials micro-structural features (Rodriguez-Navarro et al., 2002). In this study, oyster shell material is composed of two inorganic CaCO3 compounds, calcite and aragonite. Oysters begin their life (larvae) with aragonite-based shell, but it is completely replaced by calcite in adults though juvenile shells may retain a tiny portion of aragonite. Calcite is a relatively less soluble form of CaCO3 to decreased environmental pH when compared to aragonite.
This chemical feature of calcite may have made feasible for the juvenile oysters to successfully mineralize and retain the 250 laminated calcareous structure even under undersaturated CaCO3 saturation levels, e.g. decreased pH 7.4 (Fig. 2).
Like a previously described oyster shell microstructure, the materials used in this study composed of structurally organized layers. The bulk of the microstructure is characterized by the foliated layer with laminated lamellar structure of crystal units. 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 gap or pore" size between laminated layers within the 255 folia. The decreased pH significantly increased size and quantity of the pore in the folia layer. The presence of such a loosened laminated folia with pores or gaps was an obvious impairment of decreased pH. This micro-structural impairment was observed even under the near-future level of decreased pH 7.8, where the porosity was increased by 10 folds (Fig. 2r).
On the other hand, the preferred orientation of crystal units within the folia layer showed no difference in all decreased pH treatments, with c-axis of calcite units approximately perpendicular to the outer and inner shell surface. Nevertheless, 260 hardness and stiffness of the folia layer were significantly reduced under decreased pH, possibly due to the impaired microstructure with significantly higher pore size and numbers.
Furthermore, we have measured the impacts of decreased pH on whole shell mineral density and thus on "pores or gaps" in foliated layers using micro-CT analysis. Notably, higher density mineral volume has started reducing with decreasing pH. This result supports our finding on the effect of decreased pH on microscale structure and mechanical 265 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 depend on stiffness parameter of the shell. Here, we have found that both the hardness and stiffness of the folia layer has started to reduce with decreasing pH, which may have triggered shell failure phenomenon under stimulated external attack. Under the condition of same external forces, the folia layer with lower Biogeosciences Discuss., https://doi. org/10.5194/bg-2018-204 Manuscript under review for journal Biogeosciences Discussion started: 29 May 2018 c Author(s) 2018. CC BY 4.0 License. vulnerable to predatory attack even though the preferred orientation of the brittle material (i.e. calcite) is unaffected (Kemeny and Cook, 1986). In addition, the overall "down-shifting" of mineral density detected by Micro-CT analysis indicates the loosed internal microstructure may run through the juvenile shell with decreasing pH, thus the above conclusion may be applicable for the entire oyster shell. In other words, the juvenile oyster shell with impaired microstructural features is more prone to predator attack under near-future level of decreased pH due to OA processes. 275

Effect of ocean acidification on shell microstructure and crystallography
The outermost prismatic layers of the older hinge and younger middle regions was completely disappeared when juvenile oysters exposed to the extreme, but still environmentally and climatically relevant, the decreased pH of 7.2 with calcite undersaturation (Ωcal ≈ 0.66) ( Fig. 1h and Fig. 2n, p). This may be because of the corrosive effect of the calciteundersaturated seawater in the environment (Bednarsek et al., 2012). Similar impacts were observed in the juvenile scallop 280 (pH 7.8 and pH 7.5), Argopecten irradians (Talmage and Gobler, 2010), juvenile hard-shell clams (pH 7.7), Mercenaria mercenaria  and the rock oyster (pH 7.8 and pH 7.6), Saccostrea glomerata (Watson et al., 2009).
The juvenile oysters exposed to decreased pH exhibited loosened microstructure in foliated layers (Fig. 2). Firstly, it 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, the dissolution of the newly formed minerals of 285 the inner surface in the decreased pH conditions may be another probable reason (Melzner et al., 2011). Based on the calcification mechanism of mollusc, undersaturated calcite conditions may be in contact with the inner shell surface (Addadi et al., 2006;Thomsen et al., 2010), where the newly formed minerals grow as the structural building blocks for foliated layers, in the decreased pH conditions. Thus, the newly formed minerals may still be prone to dissolution. When the dissolution rate is faster than the calcification rate, organisms may tend to produce loosened microstructure of foliated layers. 290 Similarly, mussel shells grown in decreased pH conditions (pH 7.65) showed inner shell surface dissolution (Melzner et al., 2011) and impaired shell microstructure (Hahn et al., 2012), which were consistent with the results in this study. The crystallography of marine shell is the other important proxy to environmental stressors (Milano et al., 2017). Compared to calcite, aragonite occupies much less amount of oyster shells and is more soluble under decreased pH conditions (Fitzer et al., 2014). Therefore, because of the same reasons discussed above, an absent of aragonite in the older hinge regions at pH 7.2 295 ( Fig. 3.iv) is observed in this study. A similar absence of aragonite also was reported in mussel shells in high pCO2 (1000 µatm) conditions (Fitzer et al., 2014). Nevertheless, the aragonitic portion in the adult oyster shell is insignificant and it plays no role in determining the ultimate mechanical properties of the calcite predominant adult shells.

Ecological implications and conclusion
Although previous studies showed that early larval life stages of several edible oyster species were relatively physiologically 300 tolerant to the near-future pH 7.8 due to OA Ko et al., 2013;Ko et al., 2014), this study shows that they are still vulnerable due to the softer and less stiff shells in decreased pH conditions. 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 acidified seawater at pH 7.8 (Welladsen et al., 2010). Decreasing shell hardness in decreased pH conditions was also observed in the California mussel (pH 7.95 and pH 7.75), Mytilus 305 californianus (Gaylord et al., 2011), the hard clam (pH 7.7), Mercenaria mercenaria Ivanina et al., 2013), and the serpulid tubeworm (pH 7.8), Hydroides elegans (Li et al., 2014). However, the effects of increased pCO2 on shell mechanical properties are species-specific. Near-future decreased 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). Indeed, juvenile oysters of C. gigas significantly increased their shell strength and size as a compensatory adaptive response to the 310 high pCO2 condition (i.e., pCO2 1000 µatm) (Wright et al., 2014), and the blue mussel, Mytilus edulis, produced a stiffer and harder calcite layer under in increased pCO2 condition (i.e., pCO2 1000 µatm) (Fitzer et al., 2015).
The long-term survival strategy of oysters with mechanical softer and less stiff shells as yet to be studied. However, as shown in a recent study (Sanford et al., 2014), it appears that the mechanically weaker shell will result in compromised defence ability, thus lead to potential experience of increased predation which may act as a strong selective pressure for 315 oysters in the decreased pH condition. Moreover, results from a recent study suggest that oysters with reduced and impaired calcification mechanisms have lower capability to repair their shells once natural damage occurs (Coleman et al., 2014). This hierarchical study revealed that the OA-induced decreased pH conditions may cause a bottom-up deterioration on oyster shells, 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 to the coastal oceanographic 320 biophysical models to accurately project the survival of oysters in near-future coastal oceans.