The response of coral skeletal nano structure and hardness to ocean acidification conditions.

Ocean acidification typically reduces coral calcification rates and can fundamentally alter skeletal morphology. We use atomic force microscopy (AFM) and microindentation to determine how seawater pCO2 affects skeletal structure and Vickers hardness in a Porites lutea coral. At 400 µatm, the skeletal fasciculi are composed of tightly packed bundles of acicular crystals composed of quadrilateral nanograins, approximately 80-300 nm in dimensions. We interpret high adhesion at the nanograin edges as an organic coating. At 750 µatm the crystals are less regular in width and orientation and composed of either smaller/more rounded nanograins than observed at 400 µatm or of larger areas with little variation in adhesion. Coral aragonite may form via ion-by-ion attachment to the existing skeleton or via conversion of amorphous calcium carbonate precursors. Changes in nanoparticle morphology could reflect variations in the sizes of nanoparticles produced by each crystallization pathway or in the contributions of each pathway to biomineralization. We observe no significant variation in Vickers hardness between skeletons cultured at different seawater pCO2. Either the nanograin size does not affect skeletal hardness or the effect is offset by other changes in the skeleton, e.g. increases in skeletal organic material as reported in previous studies.

Ocean acidification, warming and feeding impacts on biomineralization pathways and shell material properties of Magallana gigas and Mytilus spp.

Molluscs are among the organisms affected by ocean acidification (OA), relying on carbon for shell biomineralization. Metabolic and environmental sourcing are two pathways potentially affected by OA, but the circumstances and patterns by which they are altered are poorly understood. From previous studies, mollusc shells grown under OA appear smaller in size, brittle and thinner, suggesting an important alteration in carbon sequestration. However, supplementary feeding experiments have shown promising results in offsetting the negative consequences of OA on shell growth. Our study compared carbon uptake by δ13C tracing and deposition into mantle tissue and shell layers in Magallana gigas and Mytilus species, two economically valuable and common species. After subjecting the species to 7.7 pH, +2 °C seawater, and enhanced feeding, both species maintain shell growth and metabolic pathways under OA without benefitting from extra feeding, thus, showing effective acclimation to rapid and short-term environmental change. Mytilus spp. increases metabolic carbon into the calcite and environmental sourcing of carbon into the shell aragonite in low pH and high temperature conditions. Low pH affects M. gigas mantle nitrogen isotopes maintaining growth. Calcite biomineralization pathway differs between the two species and suggests species-specific response to OA.

The impact of environmental acidification on the microstructure and mechanical integrity of marine invertebrate skeletons.

Ocean acidification (OA), from seawater uptake of anthropogenic CO2, has a suite of negative effects on the ability of marine invertebrates to produce and maintain their skeletons. Increased organism pCO2 causes hypercapnia, an energetically costly physiological stress. OA alters seawater carbonate chemistry, limiting the carbonate available to form the calcium carbonate (CaCO3) minerals used to build skeletons. The reduced saturation state of CaCO3 also causes corrosion of CaCO3 structures. Global change is also accelerating coastal acidification driven by land-run off (e.g. acid soil leachates, tannic acid). Building and maintaining marine biomaterials in the face of changing climate will depend on the balance between calcification and dissolution. Overall, in response to environmental acidification, many calcifiers produce less biomineral and so have smaller body size. Studies of skeleton development in echinoderms and molluscs across life stages show the stunting effect of OA. For corals, linear extension may be maintained, but at the expense of less dense biomineral. Conventional metrics used to quantify growth and calcification need to be augmented by characterisation of the changes to biomineral structure and mechanical integrity caused by environmental acidification. Scanning electron microscopy and microcomputed tomography of corals, tube worms and sea urchins exposed to experimental (laboratory) and natural (vents, coastal run off) acidification show a less dense biomineral with greater porosity and a larger void space. For bivalves, CaCO3 crystal deposition is more chaotic in response to both ocean and coastal acidification. Biomechanics tests reveal that these changes result in weaker, more fragile skeletons, compromising their vital protective roles. Vulnerabilities differ among taxa and depend on acidification level. Climate warming has the potential to ameliorate some of the negative effects of acidification but may also make matters worse. The integrative morphology-ecomechanics approach is key to understanding how marine biominerals will perform in the face of changing climate.

Selectively bred oysters can alter their biomineralization pathways, promoting resilience to environmental acidification.

Commercial shellfish aquaculture is vulnerable to the impacts of ocean acidification driven by increasing carbon dioxide (CO2 ) absorption by the ocean as well as to coastal acidification driven by land run off and rising sea level. These drivers of environmental acidification have deleterious effects on biomineralization. We investigated shell biomineralization of selectively bred and wild-type families of the Sydney rock oyster Saccostrea glomerata in a study of oysters being farmed in estuaries at aquaculture leases differing in environmental acidification. The contrasting estuarine pH regimes enabled us to determine the mechanisms of shell growth and the vulnerability of this species to contemporary environmental acidification. Determination of the source of carbon, the mechanism of carbon uptake and use of carbon in biomineral formation are key to understanding the vulnerability of shellfish aquaculture to contemporary and future environmental acidification. We, therefore, characterized the crystallography and carbon uptake in the shells of S. glomerata, resident in habitats subjected to coastal acidification, using high-resolution electron backscatter diffraction and carbon isotope analyses (as δ13 C). We show that oyster families selectively bred for fast growth and families selected for disease resistance can alter their mechanisms of calcite crystal biomineralization, promoting resilience to acidification. The responses of S. glomerata to acidification in their estuarine habitat provide key insights into mechanisms of mollusc shell growth under future climate change conditions. Importantly, we show that selective breeding in oysters is likely to be an important global mitigation strategy for sustainable shellfish aquaculture to withstand future climate-driven change to habitat acidification.

Ocean acidification affects calcareous tube growth in adults and reared offspring of serpulid polychaetes.

The energetically costly transition from free-swimming larvae to a benthic life stage and maintenance of a calcareous structure can make calcifying marine invertebrates vulnerable to ocean acidification. The first goal of this study was to evaluate the impact of ocean acidification on calcified tube growth for two Serpulidae polychaete worms. Spirorbis sp. and Spirobranchus triqueter were collected at 11 m depth from the northwest Mediterranean Sea and maintained for 30 and 90 days at three mean pHT levels (total scale): 8.1 (ambient), 7.7 and 7.4. Moderately decreased tube elongation rates were observed in both species at pHT 7.7 while severe reductions occurred at pHT 7.4. There was visual evidence of dissolution and tubes were more fragile at lower pH but fragility was not attributed to changes in fracture toughness. Instead, it appeared to be due to the presence of larger alveoli covered in a thinner calcareous layer. The second objective of this study was to test for effects on S. triqueter offspring development. Spawning was induced, and offspring were reared in the same pH conditions that the parents experienced. Trochophore size was reduced at the lowest pH level but settlement success was similar across pH conditions. Post-settlement tube growth was most affected. At 38 days post-settlement, juvenile tubes at pHT 7.7 and 7.4 were half the size of those at pHT 8.1. The results suggest future carbonate chemistry will negatively affect the initiation and persistence of both biofouling and epiphytic polychaete tube worms.

Coastal acidification impacts on shell mineral structure of bivalve mollusks.

Ocean acidification is occurring globally through increasing CO 2 absorption into the oceans creating particular concern for calcifying species. In addition to ocean acidification, near shore marine habitats are exposed to the deleterious effects of runoff from acid sulfate soils which also decreases environmental pH. This coastal acidification is being exacerbated by climate change-driven sea-level rise and catchment-driven flooding. In response to reduction in habitat pH by ocean and coastal acidification, mollusks are predicted to produce thinner shells of lower structural integrity and reduced mechanical properties threatening mollusk aquaculture. Here, we present the first study to examine oyster biomineralization under acid sulfate soil acidification in a region where growth of commercial bivalve species has declined in recent decades. Examination of the crystallography of the shells of the Sydney rock oyster, Saccostrea glomerata, by electron back scatter diffraction analyses revealed that the signal of environmental acidification is evident in the structure of the biomineral. Saccostrea glomerata, shows phenotypic plasticity, as evident in the disruption of crystallographic control over biomineralization in populations living in coastal acidification sites. Our results indicate that reduced sizes of these oysters for commercial sale may be due to the limited capacity of oysters to biomineralize under acidification conditions. As the impact of this catchment source acidification will continue to be exacerbated by climate change with likely effects on coastal aquaculture in many places across the globe, management strategies will be required to maintain the sustainable culture of these key resources.

Biomineral shell formation under ocean acidification: a shift from order to chaos.

Biomineral production in marine organisms employs transient phases of amorphous calcium carbonate (ACC) in the construction of crystalline shells. Increasing seawater pCO2 leads to ocean acidification (OA) with a reduction in oceanic carbonate concentration which could have a negative impact on shell formation and therefore survival. We demonstrate significant changes in the hydrated and dehydrated forms of ACC in the aragonite and calcite layers of Mytilus edulis shells cultured under acidification conditions (1000 µatm pCO2) compared to present day conditions (380 µatm pCO2). In OA conditions, Mytilus edulis has more ACC at crystalisation sites. Here, we use the high-spatial resolution of synchrotron X-ray Photo Emission Electron Microscopy (XPEEM) combined with X-ray Absorption Spectroscopy (XAS) to investigate the influence of OA on the ACC formation in the shells of adult Mytilus edulis. Electron Backscatter Diffraction (EBSD) confirms that OA reduces crystallographic control of shell formation. The results demonstrate that OA induces more ACC formation and less crystallographic control in mussels suggesting that ACC is used as a repair mechanism to combat shell damage under OA. However, the resultant reduced crystallographic control in mussels raises concerns for shell protective function under predation and changing environments.

Ocean acidification and temperature increase impact mussel shell shape and thickness: problematic for protection?

Ocean acidification threatens organisms that produce calcium carbonate shells by potentially generating an under-saturated carbonate environment. Resultant reduced calcification and growth, and subsequent dissolution of exoskeletons, would raise concerns over the ability of the shell to provide protection for the marine organism under ocean acidification and increased temperatures. We examined the impact of combined ocean acidification and temperature increase on shell formation of the economically important edible mussel Mytilus edulis. Shell growth and thickness along with a shell thickness index and shape analysis were determined. The ability of M. edulis to produce a functional protective shell after 9 months of experimental culture under ocean acidification and increasing temperatures (380, 550, 750, 1000 µatm pCO 2, and 750, 1000 µatm pCO 2 + 2°C) was assessed. Mussel shells grown under ocean acidification conditions displayed significant reductions in shell aragonite thickness, shell thickness index, and changes to shell shape (750, 1000 µatm pCO 2) compared to those shells grown under ambient conditions (380 µatm pCO 2). Ocean acidification resulted in rounder, flatter mussel shells with thinner aragonite layers likely to be more vulnerable to fracture under changing environments and predation. The changes in shape presented here could present a compensatory mechanism to enhance protection against predators and changing environments under ocean acidification when mussels are unable to grow thicker shells. Here, we present the first assessment of mussel shell shape to determine implications for functional protection under ocean acidification.

Ocean acidification alters the material properties of Mytilus edulis shells.

Ocean acidification (OA) and the resultant changing carbonate saturation states is threatening the formation of calcium carbonate shells and exoskeletons of marine organisms. The production of biominerals in such organisms relies on the availability of carbonate and the ability of the organism to biomineralize in changing environments. To understand how biomineralizers will respond to OA the common blue mussel, Mytilus edulis, was cultured at projected levels of pCO2 (380, 550, 750, 1000 µatm) and increased temperatures (ambient, ambient plus 2°C). Nanoindentation (a single mussel shell) and microhardness testing were used to assess the material properties of the shells. Young's modulus (E), hardness (H) and toughness (KIC) were measured in mussel shells grown in multiple stressor conditions. OA caused mussels to produce shell calcite that is stiffer (higher modulus of elasticity) and harder than shells grown in control conditions. The outer shell (calcite) is more brittle in OA conditions while the inner shell (aragonite) is softer and less stiff in shells grown under OA conditions. Combining increasing ocean pCO2 and temperatures as projected for future global ocean appears to reduce the impact of increasing pCO2 on the material properties of the mussel shell. OA may cause changes in shell material properties that could prove problematic under predation scenarios for the mussels; however, this may be partially mitigated by increasing temperature.

Ocean acidification reduces the crystallographic control in juvenile mussel shells.

Global climate change threatens the oceans as anthropogenic carbon dioxide causes ocean acidification and reduced carbonate saturation. Future projections indicate under saturation of aragonite, and potentially calcite, in the oceans by 2100. Calcifying organisms are those most at risk from such ocean acidification, as carbonate is vital in the biomineralisation of their calcium carbonate protective shells. This study highlights the importance of multi-generational studies to investigate how marine organisms can potentially adapt to future projected global climate change. Mytilus edulis is an economically important marine calcifier vulnerable to decreasing carbonate saturation as their shells comprise two calcium carbonate polymorphs: aragonite and calcite. M. edulis specimens were cultured under current and projected pCO2 (380, 550, 750 and 1000µatm), following 6months of experimental culture, adults produced second generation juvenile mussels. Juvenile mussel shells were examined for structural and crystallographic orientation of aragonite and calcite. At 1000µatm pCO2, juvenile mussels spawned and grown under this high pCO2 do not produce aragonite which is more vulnerable to carbonate under-saturation than calcite. Calcite and aragonite were produced at 380, 550 and 750µatm pCO2. Electron back scatter diffraction analyses reveal less constraint in crystallographic orientation with increased pCO2. Shell formation is maintained, although the nacre crystals appear corroded and crystals are not so closely layered together. The differences in ultrastructure and crystallography in shells formed by juveniles spawned from adults in high pCO2 conditions may prove instrumental in their ability to survive ocean acidification.

Ocean acidification impacts mussel control on biomineralisation.

Ocean acidification is altering the oceanic carbonate saturation state and threatening the survival of marine calcifying organisms. Production of their calcium carbonate exoskeletons is dependent not only on the environmental seawater carbonate chemistry but also the ability to produce biominerals through proteins. We present shell growth and structural responses by the economically important marine calcifier Mytilus edulis to ocean acidification scenarios (380, 550, 750, 1000 µatm pCO2). After six months of incubation at 750 µatm pCO2, reduced carbonic anhydrase protein activity and shell growth occurs in M. edulis. Beyond that, at 1000 µatm pCO2, biomineralisation continued but with compensated metabolism of proteins and increased calcite growth. Mussel growth occurs at a cost to the structural integrity of the shell due to structural disorientation of calcite crystals. This loss of structural integrity could impact mussel shell strength and reduce protection from predators and changing environments.

Response of copepods to elevated pCO2 and environmental copper as co-stressors--a multigenerational study.

We examined the impacts of ocean acidification and copper as co-stressors on the reproduction and population level responses of the benthic copepod Tisbe battagliai across two generations. Naupliar production, growth, and cuticle elemental composition were determined for four pH values: 8.06 (control); 7.95; 7.82; 7.67, with copper addition to concentrations equivalent to those in benthic pore waters. An additive synergistic effect was observed; the decline in naupliar production was greater with added copper at decreasing pH than for decreasing pH alone. Naupliar production modelled for the two generations revealed a negative synergistic impact between ocean acidification and environmentally relevant copper concentrations. Conversely, copper addition enhanced copepod growth, with larger copepods produced at each pH compared to the impact of pH alone. Copepod digests revealed significantly reduced cuticle concentrations of sulphur, phosphorus and calcium under decreasing pH; further, copper uptake increased to toxic levels that lead to reduced naupliar production. These data suggest that ocean acidification will enhance copper bioavailability, resulting in larger, but less fecund individuals that may have an overall detrimental outcome for copepod populations.

Impacts of boat paint chips on the distribution and availability of copper in an English ria.

Discarded paint chips collected from a leisure boat maintenance facility on the Kingsbridge estuary, SW England, have been fractionated to <63 microm and chemically characterised. At about 16% by weight, Cu was the most abundant metallic component, reflecting its biocidal application in antifouling paint. Bioavailability of Cu in the chips, determined by protein digestion, was about 4%, and sea water leachability was about 8%. Copper concentrations in fractionated intertidal sediment from the estuary were highly variable (<10-460 microg g(-1)). Specifically, greatest concentrations and greatest variability among replicates were found in samples collected near boat maintenance facilities. Bioavailability of Cu in sediment averaged 7% but was also variable. We attribute Cu "hot spots" to heterogeneous contamination of local sediment by small quantities of paint chips. Contamination may arise directly, from relatively inert particulates, or indirectly, via release of Cu from chips to interstitial waters and its subsequent adsorption to local sediment.