Gene expression profiling in gills of the great spider crab Hyas araneus in respond to ocean acidification and warming, supplementary data

Hypercapnia and elevated temperatures resulting from climate change may have adverse consequences for many marine organisms. While diverse physiological and ecological effects have been identified, changes in those molecular mechanisms, which shape the physiological phenotype of a species and limit its capacity to compensate, remain poorly understood. Here, we use global gene expression profiling through RNA-Sequencing to study the transcriptional responses to ocean acidification and warming in gills of the boreal spider crab Hyas araneus exposed medium-term (10 weeks) to intermediate (1,120 µatm) and high (1,960 µatm) PCO2 at different temperatures (5°C and 10°C). The analyses reveal shifts in steady state gene expression from control to intermediate and from intermediate to high CO2 exposures. At 5°C acid-base, energy metabolism and stress response related genes were upregulated at intermediate PCO2, whereas high PCO2 induced a relative reduction in expression to levels closer to controls. A similar pattern was found at elevated temperature (10°C). There was a strong coordination between acid-base, metabolic and stress-related processes. Hemolymph parameters at intermediate PCO2 indicate enhanced capacity in acid-base compensation potentially supported by upregulation of a V-ATPase. The likely enhanced energy demand might be met by the upregulation of the electron transport system (ETS), but may lead to increased oxidative stress reflected in upregulated antioxidant defense transcripts. These mechanisms were attenuated by high PCO2, possibly as a result of limited acid-base compensation and metabolic down-regulation. Our findings indicate a PCO2 dependent threshold beyond which compensation by acclimation fails progressively. They also indicate a limited ability of this stenoecious crustacean to compensate for the effects of ocean acidification with and without concomitant warming.

Carbon sources in the North Sea evaluated by means of carbon isotope tracers

A multitracer approach is applied to assess the impact of boundary fluxes (e.g., benthic input from sedi- ments or lateral inputs from the coastline) on the acid-base buffering capacity, and overall biogeochemistry, of the North Sea. Analyses of both basin-wide observations in the North Sea and transects through tidal basins at the North-Frisian coastline, reveal that surface distributions of the d13C signature of dissolved inorganic carbon (DIC) are predominantly controlled by a balance between biological production and respiration. In particular, variability in metabolic DIC throughout stations in the well-mixed southern North Sea indi- cates the presence of an external carbon source, which is traced to the European continental coastline using naturally occurring radium isotopes (224Ra and 228Ra). 228Ra is also shown to be a highly effective tracer of North Sea total alkalinity (AT) compared to the more conventional use of salinity. Coastal inputs of meta- bolic DIC and AT are calculated on a basin-wide scale, and ratios of these inputs suggest denitrification as a primary metabolic pathway for their formation. The AT input paralleling the metabolic DIC release prevents a significant decline in pH as compared to aerobic (i.e., unbuffered) release of metabolic DIC. Finally, long- term pH trends mimic those of riverine nitrate loading, highlighting the importance of coastal AT production via denitrification in regulating pH in the southern North Sea.

Seawater carbonate chemistry and processes during experiments with crabs Chionoecetes tanneri and Cancer magister, 2007

Rising levels of atmospheric carbon dioxide could be curbed by large-scale sequestration of CO2 in the deep sea. Such a solution requires prior assessment of the impact of hypercapnic, acidic seawater on deep-sea fauna. Laboratory studies were conducted to assess the short-term hypercapnic tolerance of the deep-sea Tanner crab Chionoecetes tanneri, collected from 1000 m depth in Monterey Canyon off the coast of central California, USA. Hemolymph acid- base parameters were monitored over 24 h of exposure to seawater equilibrated with ~1% CO2 (seawater PCO2 ~6 torr or 0.8 kPa, pH 7.1), and compared with those of the shallow-living Dungeness crab Cancer magister. Short-term hypercapnia-induced acidosis in the hemolymph of Chionoecetes tanneri was almost uncompensated, with a net 24 h pH reduction of 0.32 units and a net bicarbonate accumulation of only 3 mM. Under simultaneous hypercapnia and hypoxia, short-term extracellular acidosis in Chionoecetes tanneri was completely uncompensated. In contrast, Cancer magister fully recovered its hemolymph pH over 24 h of hypercapnic exposure by net accumulation of 12 mM bicarbonate from the surrounding medium. The data support the hypothesis that deep-sea animals, which are adapted to a stable environment and exhibit reduced metabolic rates, lack the short-term acid-base regulatory capacity to cope with the acute hypercapnic stress that would accompany large-scale CO2 sequestration. Additionally, the data indicate that sequestration in oxygen-poor areas of the ocean would be even more detrimental to deep-sea fauna.

Euechinoidea and Cidaroidea respond differently to ocean acidification

The impact of the chemical changes in the ocean waters due to the increasing atmospheric CO2 depends on the ability of an organism to control extracellular pH. Among sea urchins, this seems specific to the Euechinoidea, sea urchins except Cidaroidea. However, Cidaroidea survived two ocean acidification periods: the Permian-Trias and the Cretaceous-Tertiary crises. We investigated the response of these two sea urchin groups to reduced seawater pH with the tropical cidaroid Eucidaris tribuloides, the sympatric euechinoid Tripneustes ventricosus and the temperate euechinoid Paracentrotus lividus. Both euechinoid showed a compensation of the coelomic fluid pH due to increased buffer capacity. This was linked to an increased concentration of DIC in the coelomic fluid and thus of bicarbonate ions (most probably originating from the surrounding seawater as isotopic signature of the carbon -delta 13C- was similar). On the other hand, the cidaroid showed no changes within the coelomic fluid. Moreover, the delta 13C of the coelomic fluid did not match that of the seawater and was not significantly different between the urchins from the different treatments. Feeding rate was not affected in any species. While euechinoids are able to regulate their extracellular acid-base balance, many questions are still unanswered on the costs of this capacity. On the contrary, cidaroids do not seem affected by a reduced seawater pH. Further investigations need to be undertaken to cover more species and physiological and metabolic parameters in order to determine if energy trade-offs occur and how this mechanism of compensation is distributed among sea urchins.

(Supplement) Groundwater quality in Lahore Pakistan: emphasis on Arsenic and Fluoride levels

The dataset provides detailed information on the study that was conducted in Lahore's 7 major towns. The sample was taken from 472 tubewells and analyzed for major cations and anions using APHA 2012 techniques as explained herein. Besides, E.coli determination was done to check for microbial contamination. The data includes results from PHREEQC modeling of As(III)/ As(V) species and saturation indices as well as Aquachem's computed hydrochemical water facies. The WHO (2011) and EPA standards included in Aquachem identified the parameters that where in violation. Bicarbonates dominated the groundwater types with 50.21% of the samples exceeding the EPA maximum permissible limit of 250 mg/L in drinking water. Similarly, 30.51% of the samples had TDS values greater than 500 mg/L while 85.38 % of the samples exceed 10 µg/L threshold limit value of arsenic. Also, instances of high magnesium hazard values were observed which requires constant assessment if the groundwater is used for irrigation. Higher than 50% MH values are detrimental to crops which may reduce the expected yields. The membrane filtration technique using m-Endo Agar indicated that 3.59% samples had TNC (too numerous to count) values for E.coli while 5.06% showed values higher than 0 cfu/ 100 ml acceptable value in drinking water. Any traces of E-coli in a groundwater sample indicate recent fecal contamination. Such outcomes signify presence of enteric pathogens. If the groundwater is not properly dosed with disinfectants it may cause harm to human health. It is concluded that more studies are needed and proper groundwater management implement to safeguard the lives of communities that depend solely on groundwater in the city.

Effects of ocean acidification on sponge communities

The effects of ocean acidification on lower invertebrates such as sponges may be pronounced because of their low capacity for acid-base regulation. However, so far, most studies have focused on calcifiers. We present the first study of the effects of ocean acidification on the Porifera. Sponge species composition and cover along pH gradients at CO2 vents off Ischia (Tyrrhenian Sea, Italy) was measured at sites with normal pH (8.1-8.2), lowered pH (mean 7.8-7.9, min 7.4-7.5) and extremely low pH (6.6). There was a strong correlation between pH and both sponge cover and species composition. Crambe crambe was the only species present in any abundance in the areas with mean pH 6.6, seven species were present at mean pH 7.8-7.9 and four species (Phorbas tenacior, Petrosia ficiformis, Chondrilla nucula and Hemimycale columella) were restricted to sites with normal pH. Sponge percentage cover decreased significantly from normal to acidified sites. No significant effect of increasing CO2 levels and decreasing pH was found on spicule form in Crambe crambe. This study indicates that increasing CO2 concentrations will likely affect sponge community composition as some demosponge species appear to be more vulnerable than others. Further research into the mechanisms by which acidification affects sponges would be useful in predicting likely effects on sessile marine communities.

Seawater carbonate chemistry and biological processes of mussel Crassostrea gigas during experiments, 2011

Climate change with increasing temperature and ocean acidification (OA) poses risks for marine ecosystems. According to Pörtner and Farrell [1], synergistic effects of elevated temperature and CO2-induced OA on energy metabolism will narrow the thermal tolerance window of marine ectothermal animals. To test this hypothesis, we investigated the effect of an acute temperature rise on energy metabolism of the oyster, Crassostrea gigas chronically exposed to elevated CO2 levels (partial pressure of CO2 in the seawater ~0.15 kPa, seawater pH ~ 7.7). Within one month of incubation at elevated PCO2 and 15 °C hemolymph pH fell (pHe = 7.1 ± 0.2 (CO2-group) vs. 7.6 ± 0.1 (control)) and PeCO2 values in hemolymph increased (0.5 ± 0.2 kPa (CO2-group) vs. 0.2 ± 0.04 kPa (control)). Slightly but significantly elevated bicarbonate concentrations in the hemolymph of CO2-incubated oysters ([HCO-3]e = 1.8 ± 0.3 mM (CO2-group) vs. 1.3 ± 0.1 mM (control)) indicate only minimal regulation of extracellular acid-base status. At the acclimation temperature of 15 °C the OA-induced decrease in pHe did not lead to metabolic depression in oysters as standard metabolism rates (SMR) of CO2-exposed oysters were similar to controls. Upon acute warming SMR rose in both groups, but displayed a stronger increase in the CO2-incubated group. Investigation in isolated gill cells revealed a similar temperature-dependence of respiration between groups. Furthermore, the fraction of cellular energy demand for ion regulation via Na+/K+-ATPase was not affected by chronic hypercapnia or temperature. Metabolic profiling using 1H-NMR spectroscopy revealed substantial changes in some tissues following OA exposure at 15 °C. In mantle tissue alanine and ATP levels decreased significantly whereas an increase in succinate levels was observed in gill tissue. These findings suggest shifts in metabolic pathways following OA-exposure. Our study confirms that OA affects energy metabolism in oysters and suggests that climate change may affect populations of sessile coastal invertebrates such as mollusks

Regulation of intracellular pH in cnidarians: response to acidosis in Anemonia viridis

The regulation of intracellular pH (pHi) is a fundamental aspect of cell physiology that has received little attention in studies of the phylum Cnidaria, which includes ecologically important sea anemones and reef-building corals. Like all organisms, cnidarians must maintain pH homeostasis to counterbalance reductions in pHi, which can arise because of changes in either intrinsic or extrinsic parameters. Corals and sea anemones face natural daily changes in internal fluids, where the extracellular pH can range from 8.9 during the day to 7.4 at night. Furthermore, cnidarians are likely to experience future CO2-driven declines in seawater pH, a process known as ocean acidification. Here, we carried out the first mechanistic investigation to determine how cnidarian pHi regulation responds to decreases in extracellular and intracellular pH. Using the anemone Anemonia viridis, we employed confocal live cell imaging and a pH-sensitive dye to track the dynamics of pHi after intracellular acidosis induced by acute exposure to decreases in seawater pH and NH4Cl prepulses. The investigation was conducted on cells that contained intracellular symbiotic algae (Symbiodinium sp.) and on symbiont-free endoderm cells. Experiments using inhibitors and Na-free seawater indicate a potential role of Na/H plasma membrane exchangers (NHEs) in mediating pHi recovery following intracellular acidosis in both cell types. We also measured the buffering capacity of cells, and obtained values between 20.8 and 43.8 mM per pH unit, which are comparable to those in other invertebrates. Our findings provide the first steps towards a better understanding of acid-base regulation in these basal metazoans, for which information on cell physiology is extremely limited.

Evolution of extreme stomach pH in bilateria inferred from gastric alkalization mechanisms in basal deuterostomes

The stomachs of most vertebrates operate at an acidic pH of 2 generated by the gastric H+/K+-ATPase located in parietal cells. The acidic pH in stomachs of vertebrates is believed to aid digestion and to protect against environmental pathogens. Little attention has been placed on whether acidic gastric pH regulation is a vertebrate character or a deuterostome ancestral trait. Here, we report alkaline conditions up to pH 10.5 in the larval digestive systems of ambulacraria (echinoderm + hemichordate), the closest relative of the chordate. Microelectrode measurements in combination with specific inhibitors for acid-base transporters and ion pumps demonstrated that the gastric alkalization machinery in sea urchin larvae is mainly based on direct H+ secretion from the stomach lumen and involves a conserved set of ion pumps and transporters. Hemichordate larvae additionally utilized HCO3- transport pathways to generate even more alkaline digestive conditions. Molecular analyses in combination with acidification experiments supported these findings and identified genes coding for ion pumps energizing gastric alkalization. Given that insect larval guts were also reported to be alkaline, our discovery raises the hypothesis that the bilaterian ancestor utilized alkaline digestive system while the vertebrate lineage has evolved a strategy to strongly acidify their stomachs.

Seawater carbonate chemistry during experiments with Patella vulgata, 2010

The effect of short-term (5 days) exposure to CO2-acidified seawater (year 2100 predicted values, ocean pH = 7.6) on key aspects of the function of the intertidal common limpet Patella vulgata (Gastropoda: Patellidae) was investigated. Changes in extracellular acid-base balance were almost completely compensated by an increase in bicarbonate ions. A concomitant increase in haemolymph Ca2+ and visible shell dissolution implicated passive shell dissolution as the bicarbonate source. Analysis of the radula using SEM revealed that individuals from the hypercapnic treatment showed an increase in the number of damaged teeth and the extent to which such teeth were damaged compared with controls. As radula teeth are composed mainly of chitin, acid dissolution seems unlikely, and so the proximate cause of damage is unknown. There was no hypercapnia-related change in metabolism (O2 uptake) or feeding rate, also discounting the possibility that teeth damage was a result of a CO2-related increase in grazing. We conclude that although the limpet appears to have the physiological capacity to maintain its extracellular acid-base balance, metabolism and feeding rate over a 5 days exposure to acidified seawater, radular damage somehow incurred during this time could still compromise feeding in the longer term, in turn decreasing the top-down ecosystem control that P. vulgata exerts over rocky shore environments.

Interactive effects of salinity and elevated CO2 levels on juvenile eastern oysters, Crassostrea virginica

Rising levels of atmospheric CO2 lead to acidification of the ocean and alter seawater carbonate chemistry, which can negatively impact calcifying organisms, including mollusks. In estuaries, exposure to elevated CO2 levels often co-occurs with other stressors, such as reduced salinity, which enhances the acidification trend, affects ion and acid-base regulation of estuarine calcifiers and modifies their response to ocean acidification. We studied the interactive effects of salinity and partial pressure of CO2 (PCO2) on biomineralization and energy homeostasis in juveniles of the eastern oyster, Crassostrea virginica, a common estuarine bivalve. Juveniles were exposed for 11 weeks to one of two environmentally relevant salinities (30 or 15 PSU) either at current atmospheric PCO2 (400 µatm, normocapnia) or PCO2 projected by moderate IPCC scenarios for the year 2100 (700-800 µatm, hypercapnia). Exposure of the juvenile oysters to elevated PCO2 and/or low salinity led to a significant increase in mortality, reduction of tissue energy stores (glycogen and lipid) and negative soft tissue growth, indicating energy deficiency. Interestingly, tissue ATP levels were not affected by exposure to changing salinity and PCO2, suggesting that juvenile oysters maintain their cellular energy status at the expense of lipid and glycogen stores. At the same time, no compensatory upregulation of carbonic anhydrase activity was found under the conditions of low salinity and high PCO2. Metabolic profiling using magnetic resonance spectroscopy revealed altered metabolite status following low salinity exposure; specifically, acetate levels were lower in hypercapnic than in normocapnic individuals at low salinity. Combined exposure to hypercapnia and low salinity negatively affected mechanical properties of shells of the juveniles, resulting in reduced hardness and fracture resistance. Thus, our data suggest that the combined effects of elevated PCO2 and fluctuating salinity may jeopardize the survival of eastern oysters because of weakening of their shells and increased energy consumption.