Shotgun proteomics reveals physiological response to ocean acidification in Crassostrea gigas

Background. Ocean acidification as a result of increased anthropogenic CO2 emissions is occurring in marine and estuarine environments worldwide. The coastal ocean experiences additional daily and seasonal fluctuations in pH that can be lower than projected end of century open ocean pH reductions. Projected and current ocean acidification have wide-ranging effects on many aquatic organisms, however the exact mechanisms of the impacts of ocean acidification on many of these animals remains to be characterized. Methods. In order to assess the impact of ocean acidification on marine invertebrates, Pacific oysters (Crassostrea gigas) were exposed to one of four different pCO2 levels for four weeks: 400 µatm (pH 8.0), 800 µatm (pH 7.7), 1000 µatm (pH 7.6), or 2800 µatm (pH 7.3). At the end of 4 weeks a variety of physiological parameters were measured to assess the impacts of ocean acidification: tissue glycogen content and fatty acid profile, shell micromechanical properties, and response to acute heat shock. To determine the effects of ocean acidification on the underlying molecular physiology of oysters and their stress response, some of the oysters from 400 µatm and 2800 µatm were exposed to an additional mechanical stress and shotgun proteomics were done on oysters from high and low pCO2 and from with and without mechanical stress. Results. At the end of the four week exposure period, oysters in all four pCO2 environments deposited new shell, but growth rate was not different among the treatments. However, micromechanical properties of the new shell were compromised by elevated pCO2. Elevated pCO2 affected neither whole body fatty acid composition, nor glycogen content, nor mortality rate associated with acute heat shock. Shotgun proteomics revealed that several physiological pathways were significantly affected by ocean acidification, including antioxidant response, carbohydrate metabolism, and transcription and translation. Additionally, the proteomic response to a second stress differed with pCO2, with numerous processes significantly affected by mechanical stimulation at high versus low pCO2 (all proteomics data are available in the ProteomeXchange under the identifier PXD000835). Discussion. Oyster physiology is significantly altered by exposure to elevated pCO2, indicating changes in energy resource use. This is especially apparent in the assessment of the effects of pCO2 on the proteomic response to a second stress. The altered stress response illustrates that ocean acidification may impact how oysters respond to other changes in their environment. These data contribute to an integrative view of the effects of ocean acidification on oysters as well as physiological trade-offs during environmental stress.

Influence of sediment acidification and water flow on sediment acceptance and dispersal of juvenile soft-shell clams (Mya arenaria L.)

Although ocean acidification is expected to reduce carbonate saturation and yield negative impacts on open-ocean calcifying organisms in the near future, acidification in coastal ecosystems may already be affecting these organisms. Few studies have addressed the effects of sedimentary saturation state on benthic invertebrates. Here, we investigate whether sedimentary aragonite saturation (Omega aragonite) and proton concentration ([H+]) affect burrowing and dispersal rates of juvenile soft-shell clams (Mya arenaria) in a laboratory flume experiment. Two size classes of juvenile clams (0.5-1.5 mm and 1.51-2.5 mm) were subjected to a range of sediment Omega aragonite and [H+] conditions within the range of typical estuarine sediments (Omega aragonite 0.21-1.87; pH 6.8-7.8; [H+] 1.58 × 10**-8-1.51 × 10**- 7) by the addition of varying amounts of CO2, while overlying water pH was kept constant ~ 7.8 (Omega aragonite ~ 1.97). There was a significant positive relationship between the percent of juvenile clams burrowed in still water and Omega aragonite and a significant negative relationship between burrowing and [H+]. Clams were subsequently exposed to one of two different flow conditions (flume; 11 cm/s and 23 cm/s) and there was a significant negative relationship between Omega aragonite and dispersal, regardless of clam size class and flow speed. No apparent relationship was evident between dispersal and [H+]. The results of this study suggest that sediment acidification may play an important role in soft-shell clam recruitment and dispersal. When assessing the impacts of open-ocean and coastal acidification on infaunal organisms, future studies should address the effects of sediment acidification to adequately understand how calcifying organisms may be affected by shifting pH conditions.

Seawater carbonate chemistry, survival rate, shell mass growth, shell dissolution and locomotory speed of Limacina retroversa in a laboratory experiment

Anthropogenic carbon dioxide emissions induce ocean acidification, thereby reducing carbonate ion concentration, which may affect the ability of calcifying organisms to build shells. Pteropods, the main planktonic producers of aragonite in the worlds' oceans, may be particularly vulnerable to changes in sea water chemistry. The negative effects are expected to be most severe at high-latitudes, where natural carbonate ion concentrations are low. In this study we investigated the combined effects of ocean acidification and freshening on Limacina retroversa, the dominant pteropod in sub polar areas. Living L. retroversa, collected in Northern Norwegian Sea, were exposed to four different pH values ranging from the pre-industrial level to the forecasted end of century ocean acidification scenario. Since over the past half-century the Norwegian Sea has experienced a progressive freshening with time, each pH level was combined with a salinity gradient in two factorial, randomized experiments investigating shell degradation, swimming behavior and survival. In addition, to investigate shell degradation without any physiologic influence, one perturbation experiments using only shells of dead pteropods was performed. Lower pH reduced shell mass whereas shell dissolution increased with pCO2. Interestingly, shells of dead organisms had a higher degree of dissolution than shells of living individuals. Mortality of Limacina retroversa was strongly affected only when both pH and salinity reduced simultaneously. The combined effects of lower salinity and lower pH also affected negatively the ability of pteropods to swim upwards. Results suggest that the energy cost of maintaining ion balance and avoiding sinking (in low salinity scenario) combined with the extra energy cost necessary to counteract shell dissolution (in high pCO2 scenario), exceed the available energy budget of this organism causing the pteropods to change swimming behavior and begin to collapse. Since L. retroversa play an important role in the transport of carbonates to the deep oceans these findings have significant implications for the mechanisms influencing the inorganic carbon cycle in the sub-polar area.

GPS raw data (control points and ground control points) from the Liguria Region, Borghetto Santo Spirito, Italy

Monitoring the impact of sea storms on coastal areas is fundamental to study beach evolution and the vulnerability of low-lying coasts to erosion and flooding. Modelling wave runup on a beach is possible, but it requires accurate topographic data and model tuning, that can be done comparing observed and modeled runup. In this study we collected aerial photos using an Unmanned Aerial Vehicle after two different swells on the same study area. We merged the point cloud obtained with photogrammetry with multibeam data, in order to obtain a complete beach topography. Then, on each set of rectified and georeferenced UAV orthophotos, we identified the maximum wave runup for both events recognizing the wet area left by the waves. We then used our topography and numerical models to simulate the wave runup and compare the model results to observed values during the two events. Our results highlight the potential of the methodology presented, which integrates UAV platforms, photogrammetry and Geographic Information Systems to provide faster and cheaper information on beach topography and geomorphology compared with traditional techniques without losing in accuracy. We use the results obtained from this technique as a topographic base for a model that calculates runup for the two swells. The observed and modeled runups are consistent, and open new directions for future research.

Seawater carbonate chemistry, length and survival of Inland silverside, Menidia beryllina, during experiments, 2012

Absorption of anthropogenic carbon dioxide by the world's oceans is causing mankind's 'other CO2 problem', ocean acidification. Although this process will challenge marine organisms that synthesize calcareous exoskeletons or shells, it is unclear how it will affect internally calcifying organisms, such as marine fish. Adult fish tolerate short-term exposures to CO2 levels that exceed those predicted for the next 300 years (~2,000 ppm), but potential effects of increased CO2 on growth and survival during the early life stages of fish remain poorly understood. Here we show that the exposure of early life stages of a common estuarine fish (Menidia beryllina) to CO2 concentrations expected in the world's oceans later this century caused severely reduced survival and growth rates. When compared with present-day CO2 levels (~400 ppm), exposure of M. beryllina embryos to ~1,000 ppm until one week post-hatch reduced average survival and length by 74% and 18%, respectively. The egg stage was significantly more vulnerable to high CO2-induced mortality than the post-hatch larval stage. These findings challenge the belief that ocean acidification will not affect fish populations, because even small changes in early life survival can generate large fluctuations in adult-fish abundance.

Seawater carbonate chemistry and biological processes of oysters Crassostrea virginica during experiments, 2010

Estuarine organisms are exposed to periodic strong fluctuations in seawater pH driven by biological carbon dioxide (CO2) production, which may in the future be further exacerbated by the ocean acidification associated with the global rise in CO2. Calcium carbonate-producing marine species such as mollusks are expected to be vulnerable to acidification of estuarine waters, since elevated CO2 concentration and lower pH lead to a decrease in the degree of saturation of water with respect to calcium carbonate, potentially affecting biomineralization. Our study demonstrates that the increase in CO2 partial pressure (pCO2) in seawater and associated decrease in pH within the environmentally relevant range for estuaries have negative effects on physiology, rates of shell deposition and mechanical properties of the shells of eastern oysters Crassostrea virginica (Gmelin). High CO2 levels (pH ~7.5, pCO2 ~3500 µatm) caused significant increases in juvenile mortality rates and inhibited both shell and soft-body growth compared to the control conditions (pH ~8.2, pCO2 ~380 µatm). Furthermore, elevated CO2 concentrations resulted in higher standard metabolic rates in oyster juveniles, likely due to the higher energy cost of homeostasis. The high CO2 conditions also led to changes in the ultrastructure and mechanical properties of shells, including increased thickness of the calcite laths within the hypostracum and reduced hardness and fracture toughness of the shells, indicating that elevated CO2 levels have negative effects on the biomineralization process. These data strongly suggest that the rise in CO2 can impact physiology and biomineralization in marine calcifiers such as eastern oysters, threatening their survival and potentially leading to profound ecological and economic impacts in estuarine ecosystems.

Tolerance of juvenile barnacles (Amphibalanus improvisus) to warming and elevated pCO2

We investigated the impacts of warming and elevated pCO2 on newly settled Amphibalanus improvisus from Kiel Fjord, an estuarine ecosystem characterized by significant natural pCO2 variability. In two experiments, juvenile barnacles were maintained at two temperature and three pCO2 levels (20/24°C, 700-2.140 µatm) for 8 weeks in a batch culture and at four pCO2 levels (20°C, 620-2.870 µatm) for 12 weeks in a water flow-through system. Warming as well as elevated pCO2 hardly affected growth or the condition index of barnacles, although some factor combinations led to temporal significances in enhanced or reduced growth with an increase in pCO2. While warming increased the shell strength of A. improvisus individuals, elevated pCO2 had only weak effects. We demonstrate a strong tolerance of juvenile A. improvisus to mean acidification levels of about 1,000 µatm pCO2 as is already naturally experienced by the investigated barnacle population.

Seawater carbonate chemistry and Ruditapes decussatus biological processes during experiments, 2011

We investigated the effects of ocean acidification on juvenile clams Ruditapes decussatus (average shell length 10.24 mm) in a controlled CO2 perturbation experiment. The carbonate chemistry of seawater was manipulated by diffusing pure CO2, to attain two reduced pH levels (by -0.4 and -0.7 pH units), which were compared to unmanipulated seawater. After 75 days we found no differences among pH treatments in terms of net calcification, size or weight of the clams. The naturally elevated total alkalinity of local seawater probably contributed to buffer the effects of increased pCO2 and reduced pH. Marine organisms may, therefore, show diverse responses to ocean acidification at local scales, particularly in coastal, estuarine and transitional waters, where the physical-chemical characteristics of seawater are most variable. Mortality was significantly reduced in the acidified treatments. This trend was probably related to the occurrence of spontaneous spawning events in the control and intermediate acidification treatments. Spawning, which was unexpected due to the small size of the clams, was not observed for the pH -0.7 treatment, suggesting that the increased survival under acidified conditions may have been associated with a delay in the reproductive cycle of the clams. Future research about the impacts of ocean acidification on marine biodiversity should be extended to other types of biological and ecological processes, apart from biological calcification.

Abundance and Biomass from heterotrophic bacteria, Synechococcus, Prochlorococcus and Virus in the eastern Mediteranean Sea in October 2008 during SES_GR2

The HCMR_SES_LAGRANGIAN_GR2_ MICROBIAL PARAMETERS dataset is based on samples collected in the framework of the project SESAME, in the North Aegean Sea during October 2008. The objectives were to measure the standing stocks and calculate the production of the microbial compartment of the food web, describe the vertical distribution pattern and characterize its structure and function through the water column as influenced by the BSW. Heterotrophic bacteria, Synechococcus, Prochlorococcus and Virus abundance: Subsamples for virus, heterotrophic bacteria and cyanobacteria (Synechococcus spp. and Prochlorococcus spp.) counting were analyzed using a FACSCalibur (Becton Dickinson) flow cytometer equipped with a standard laser (488 nm) and filter set and using deionized water as sheath fluid. Fluorescent beads with a diameter of 0.97 µm (Polysciences) were added to each sample as an internal standard, and all parameters were normalized to the beads and expressed as relative units. SYBRGreen I stain (Molecular Probe) was used to stain viral and heterotrophic bacterial DNA. Viruses were counted according to (Brussaard 1984). In order to avoid bulk consentrations of viruses samples we dilluted to Tris-EDTA (pH=8,0) buffer to a final sollution of 1/5 to 1/100. Total abundance and nucleid content classes were calculated using the Paint-A-Gate software (Becton Dickinson). Heterotrophic Nanoflagellate abundance: Subsamples (30-150 ml) were concentrated on 25mm black polycarbonate filters of porosity 0.6?m and stained with DAPI for 10 min (Porter and Feig 1980). Under epifluorescence microscopy heterotrophic nanoflagellates (HNAN) were distinguished using UV and blue excitation and enumerated. Nanoflagellates were classified in size categories and the biovolume was calculated. Ciliate abundance: For ciliate identification and enumeration, 100-3000 ml samples were left for 24h-4d for sedimentation and then observed under an inverted microscope. Ciliates were counted, distinguished into size-classes and major taxonomic groups and identified down to genus or species level where possible (Pitta et al. 2005). Heterotrophic bacteria, Synechococcus, Prochlorococcus bacteria: Subsamples for virus, heterotrophic bacteria and cyanobacteria (Synechococcus spp. and Prochlorococcus spp.) counting were analyzed using a FACSCalibur (Becton Dickinson) flow cytometer equipped with a standard laser (488 nm) and filter set and using deionized water as sheath fluid. Fluorescent beads with a diameter of 0.97 µm (Polysciences) were added to each sample as an internal standard, and all parameters were normalized to the beads and expressed as relative units. SYBRGreen I stain (Molecular Probe) was used to stain viral and heterotrophic bacterial DNA. Viruses were counted according to (Brussaard 1984). In order to avoid bulk consentrations of viruses samples we dilluted to Tris-EDTA (pH=8,0) buffer to a final sollution of 1/5 to 1/100. Total abundance and nucleid content classes were calculated using the Paint-A-Gate software (Becton Dickinson). Abundance data were converted into C biomass using 250 fgC cell-1 (Kana & Glibert 1987) for Synechococcus, 50 fgC cell-1 (Campbell et al. 1994) for Prochlorococcus and 20fgC cell-1 (Lee & Fuhrman 1987) for heterotrophic bacteria. Heterotrophic Nanoflagellate biomass: Subsamples (30-150 ml) were concentrated on 25mm black polycarbonate filters of porosity 0.6µm and stained with DAPI for 10 min (Porter and Feig 1980). Under epifluorescence microscopy heterotrophic nanoflagellates (HNAN) were distinguished using UV and blue excitation and enumerated. Nanoflagellates were classified in size categories and the biovolume was calculated. Abundance data were converted into C biomass using 183 fgC µm**3 (Caron et al. 1995). Ciliate biomass: For ciliate identification and enumeration, 100-3000 ml samples were left for 24h-4d for sedimentation and then observed under an inverted microscope. Ciliates were counted, distinguished into size-classes and major taxonomic groups and identified down to genus or species level where possible (Pitta et al. 2005). Ciliate cell sizes were measured and converted into cell volumes using appropriate geometric formulae using image analysis. For biomass estimation, the conversion factor 190 fgC µm**3 was used (Putt and Stoecker 1989).