Microsensor studies on Padina from a natural CO2 seep: implications of morphology on acclimation to low pH

Low seawater pH can be harmful to many calcifying marine organisms, but the calcifying macroalgae Padina spp. flourish at natural submarine carbon dioxide seeps where seawater pH is low. We show that the microenvironment created by the rolled thallus margin of Padina australis facilitates supersaturation of CaCO3 and calcifi-cation via photosynthesis-induced elevated pH. Using microsensors to investigate oxygen and pH dynamics in the microenvironment of P. australis at a shallow CO2 seep, we found that, under saturating light, the pH inside the microenvironment (pHME) was higher than the external seawater (pHSW) at all pHSW levels investigated, and the difference (i.e., pHME-pHSW) increased with decreasing pHSW (0.9 units at pHSW 7.0). Gross photosynthesis (Pg) inside the microenvironment increased with decreasing pHSW, but algae from the control site reached a threshold at pH 6.5. Seep algae showed no pH threshold with respect to Pg within the pHSW range investigated. The external carbonic anhydrase (CA) inhibitor, acetazolamide, strongly inhibited Pg of P. australis at pHSW 8.2, but the effect was diminished under low pHSW (6.4-7.5), suggesting a greater dependence on membrane-bound CA for the dehydration of HCO3- ions during dissolved inorganic carbon uptake at the higher pHSW. In comparison, a calcifying green alga, Halimeda cuneata f. digitata, was not inhibited by AZ, suggesting efficient bicarbonate transport. The ability of P. australis to elevate pHME at the site of calcification and its strong dependence on CA may explain why it can thrive at low pHSW.

Proteomic and metabolomic responses of Pacific oyster Crassostrea gigas to elevated pCO2 exposure

The gradually increased atmospheric CO2 partial pressure (pCO2) has thrown the carbonate chemistry off balance and resulted in decreased seawater pH in marine ecosystem, termed ocean acidification (OA). Anthropogenic OA is postulated to affect the physiology of many marine calcifying organisms. However, the susceptibility and metabolic pathways of change in most calcifying animals are still far from being well understood. In this work, the effects of exposure to elevated pCO2 were characterized in gills and hepatopancreas of Crassostrea gigas using integrated proteomic and metabolomic approaches. Metabolic responses indicated that high CO2 exposure mainly caused disturbances in energy metabolism and osmotic regulation marked by differentially altered ATP, glucose, glycogen, amino acids and organic osmolytes in oysters, and the depletions of ATP in gills and the accumulations of ATP, glucose and glycogen in hepatopancreas accounted for the difference in energy distribution between these two tissues. Proteomic responses suggested that OA could not only affect energy and primary metabolisms, stress responses and calcium homeostasis in both tissues, but also influence the nucleotide metabolism in gills and cytoskeleton structure in hepatopancreas. This study demonstrated that the combination of proteomics and metabolomics could provide an insightful view into the effects of OA on oyster C. gigas. BIOLOGICAL SIGNIFICANCE: The gradually increased atmospheric CO2 partial pressure (pCO2) has thrown the carbonate chemistry off balance and resulted in decreased seawater pH in marine ecosystem, termed ocean acidification (OA). Anthropogenic OA is postulated to affect the physiology of many marine calcifying organisms. However, the susceptibility and metabolic pathways of change in most calcifying animals are still far from being understood. To our knowledge, few studies have focused on the responses induced by pCO2 at both protein and metabolite levels. The pacific oyster C. gigas, widely distributed throughout most of the world's oceans, is a model organism for marine environmental science. In the present study, an integrated metabolomic and proteomic approach was used to elucidate the effects of ocean acidification on Pacific oyster C. gigas, hopefully shedding light on the physiological responses of marine mollusk to the OA stress.

Temperature is the evil twin: Effects of increased temperature and ocean acidification on reproduction in a reef fish

Reproduction in many organisms can be disrupted by changes to the physical environment, such as those predicted to occur during climate change. Marine organisms face the dual climate change threats of increasing temperature and ocean acidification, yet no studies have examined the potential interactive effects of these stressors on reproduction in marine fishes. We used a long-term experiment to test the interactive effects of increased temperature and CO2 on the reproductive performance of the anemonefish, Amphiprion melanopus. Adult breeding pairs were kept for 10 months at three temperatures, 28.5°C (+0.0°C), 30.0°C (+1.5°C) and 31.5°C (+3.0°C), cross-factored with 3 CO2 levels, a current day control (417 µatm) and moderate (644 µatm) and high (1134 µatm) treatments consistent with the range of CO2 projections for the year 2100 under RCP8.5. We recorded each egg clutch produced during the breeding season, the number of eggs laid per clutch, average egg size, fertilization success, survival to hatching, hatchling length and yolk provisioning. Adult body condition, hepatosomatic index, gonadosomatic index, and plasma 17beta-estradiol concentrations were measured at the end of the breeding season to determine the effect of prolonged exposure to increased temperature and elevated CO2 on adults, and to examine potential physiological mechanisms for changes in reproduction. Temperature had by far the stronger influence on reproduction, with clear declines in reproduction occurring in the +1.5°C treatment and ceasing altogether in the +3.0°C treatment. In contrast, CO2 had a minimal effect on the majority of reproductive traits measured, but caused a decline in offspring quality in combination with elevated temperature. We detected no significant effect of temperature or CO2 on adult body condition or hepatosomatic index. Elevated temperature had a significant negative effect on plasma 17beta-estradiol concentrations, suggesting that declines in reproduction with increasing temperature were due to the thermal sensitivity of reproductive hormones rather than a reduction in energy available for reproduction. Our results show that elevated temperature exerts a stronger influence than high CO2 on reproduction in A. melanopus. Understanding how these two environmental variables interact to affect the reproductive performance of marine organisms will be important for predicting the future impacts of climate change.

Effects of increasing seawater carbon dioxide concentrations on chain formation of the diatom Asterionellopsis glacialis

Diatoms can occur as single cells or as chain-forming aggregates. These two strategies affect buoyancy, predator evasion, light absorption and nutrient uptake. Adjacent cells in chains establish connections through various processes that determine strength and flexibility of the bonds, and at distinct cellular locations defining colony structure. Chain length has been found to vary with temperature and nutrient availability as well as being positively correlated with growth rate. However, the potential effect of enhanced carbon dioxide (CO2) concentrations and consequent changes in seawater carbonate chemistry on chain formation is virtually unknown. Here we report on experiments with semi-continuous cultures of the freshly isolated diatom Asterionellopsis glacialis grown under increasing CO2 levels ranging from 320 to 3400 µatm. We show that the number of cells comprising a chain, and therefore chain length, increases with rising CO2 concentrations. We also demonstrate that while cell division rate changes with CO2 concentrations, carbon, nitrogen and phosphorus cellular quotas vary proportionally, evident by unchanged organic matter ratios. Finally, beyond the optimum CO2 concentration for growth, carbon allocation changes from cellular storage to increased exudation of dissolved organic carbon. The observed structural adjustment in colony size could enable growth at high CO2 levels, since longer, spiral-shaped chains are likely to create microclimates with higher pH during the light period. Moreover increased chain length of Asterionellopsis glacialis may influence buoyancy and, consequently, affect competitive fitness as well as sinking rates. This would potentially impact the delicate balance between the microbial loop and export of organic matter, with consequences for atmospheric carbon dioxide.

Temperature dependence of CO2-enhanced primary production in the European Arctic Ocean

The Arctic Ocean is warming at two to three times the global rate and is perceived to be a bellwether for ocean acidification. Increased CO2 concentrations are expected to have a fertilization effect on marine autotrophs, and higher temperatures should lead to increased rates of planktonic primary production. Yet, simultaneous assessment of warming and increased CO2 on primary production in the Arctic has not been conducted. Here we test the expectation that CO2-enhanced gross primary production (GPP) may be temperature dependent, using data from several oceanographic cruises and experiments from both spring and summer in the European sector of the Arctic Ocean. Results confirm that CO2 enhances GPP (by a factor of up to ten) over a range of 145-2,099?µatm; however, the greatest effects are observed only at lower temperatures and are constrained by nutrient and light availability to the spring period. The temperature dependence of CO2-enhanced primary production has significant implications for metabolic balance in a warmer, CO2-enriched Arctic Ocean in the future. In particular, it indicates that a twofold increase in primary production during the spring is likely in the Arctic.

Assessing the physiological responses of the gastropod Crepidula fornicata to predicted ocean acidification and warming

Organisms inhabiting coastal waters naturally experience diel and seasonal physico-chemical variations. According to various assumptions, coastal species are either considered to be highly tolerant to environmental changes or, conversely, living at the thresholds of their physiological performance. Therefore, these species are either more resistant or more sensitive, respectively, to ocean acidification and warming. Here, we focused on Crepidula fornicata, an invasive gastropod that colonized bays and estuaries on northwestern European coasts during the 20th century. Small (<3 cm in length) and large (>4.5 cm in length), sexually mature individuals of C. fornicata were raised for 6 months in three different pCO2 conditions (390 µatm, 750 µatm, and 1400 µatm) at four successive temperature levels (10°C, 13°C, 16°C, and 19°C). At each temperature level and in each pCO2 condition, we assessed the physiological rates of respiration, ammonia excretion, filtration and calcification on small and large individuals. Results show that, in general, temperature positively influenced respiration, excretion and filtration rates in both small and large individuals. Conversely, increasing pCO2 negatively affected calcification rates, leading to net dissolution in the most drastic pCO2 condition (1400 µatm) but did not affect the other physiological rates. Overall, our results indicate that C. fornicata can tolerate ocean acidification, particularly in the intermediate pCO2 scenario. Moreover, in this eurythermal species, moderate warming may play a buffering role in the future responses of organisms to ocean acidification.

Effects of ocean acidification on Posidonia oceanica epiphytic community and shoot productivity

1. Biological interactions can alter predictions that are based on single-species physiological response. It is known that leaf segments of the seagrass Posidonia oceanica will increase photosynthesis with lowered pH, but it is not clear whether the outcome will be altered when the whole plant and its epiphyte community, with different respiratory and photosynthetic demands, are included. In addition, the effects on the Posidonia epiphyte community have rarely been tested under controlled conditions, at near-future pH levels. 2. In order to better evaluate the effects of pH levels as projected for the upcoming decades on seagrass meadows, shoots of P. oceanica with their associated epiphytes were exposed in the laboratory to three pH levels (ambient: 8.1, 7.7 and 7.3, on the total scale) for 4 weeks. Net productivity, respiration, net calcification and leaf fluorescence were measured on several occasions. At the end of the study, epiphyte community abundance and composition, calcareous mass and crustose coralline algae growth were determined. Finally, photosynthesis vs. irradiance curves (PE) was produced from segments of secondary leaves cleaned of epiphytes and pigments extracted. 3. Posidonia leaf fluorescence and chlorophyll concentrations did not differ between pH treatments. Net productivity of entire shoots and epiphyte-free secondary leaves increased significantly at the lowest pH level yet limited or no stimulation in productivity was observed at the intermediate pH treatment. Under both pH treatments, significant decreases in epiphytic cover were observed, mostly due to the reduction of crustose coralline algae. The loss of the dominant epiphyte producer yet similar photosynthetic response for epiphyte-free secondary leaves and shoots suggests a minimal contribution of epiphytes to shoot productivity under experimental conditions. 4. Synthesis. Observed responses indicate that under future ocean acidification conditions foreseen in the next century an increase in Posidonia productivity is not likely despite the partial loss of epiphytic coralline algae which are competitors for light. A decline in epiphytic cover could, however, reduce the feeding capacity of the meadow for invertebrates. In situ long-term experiments that consider both acidification and warming scenarios are needed to improve ecosystem-level predictions.

Juvenile sea stars exposed to acidification decrease feeding and growth with no acclimation potential

Ocean acidification has the potential to affect growth and calcification of benthic marine invertebrates, particularly during their early life history. We exposed field-collected juveniles of Asterias rubens from Kiel Fjord (western Baltic Sea) to 3 seawater CO2 partial pressure (pCO2) levels (ranging from around 650 to 3500 µatm) in a long-term (39 wk) and a short-term (6 wk) experiment. In both experiments, survival and calcification were not affected by elevated pCO2. However, feeding rates decreased strongly with increasing pCO2, while aerobic metabolism and NH4+ excretion were not significantly affected by CO2 exposure. Consequently, high pCO2 reduced the scope for growth in A. rubens. Growth rates decreased substantially with increasing pCO2 and were reduced even at pCO2 levels occurring in the habitat today (e.g. during upwelling events). Sea stars were not able to acclimate to higher pCO2, and growth performance did not recover during the long-term experiment. Therefore, the top-down control exerted by this keystone species may be diminished during periods of high environmental pCO2 that already occur occasionally and will be even higher in the future. However, some individuals were able to grow at high rates even at high pCO2, indicating potential for rapid adaption. The selection of adapted specimens of A. rubens in this seasonally acidified habitat may lead to higher CO2 tolerance in adult sea stars of this population compared to the juvenile stage. Future studies need to address the synergistic effects of multiple stressors such as acidification, warming and reduced salinity, which will simultaneously impact the performance of sea stars in this habitat.

Conflicting effects of predator cue and ocean acidification on the mussel Mytilus coruscus byssus production

Understanding the impact of ocean acidification and warming on communities and ecosystems is a researcher priority. This can only be achieved through a combination of experimental and field approaches that would allow developing a mechanistic understanding of impacts across level of biological organizations. Surprisingly, most published studies are still focusing on single species responses with little consideration for interspecific interactions. In this study, the impacts of a 3 days exposure to three parameters (temperature, pH, and presence/absence of the predator cue of the crab Charybdis japonica) and their interactions on an ecologically important endpoint were evaluated: the byssus production of the mussel Mytilus coruscus. Tested temperatures (25°C and 30°C) were within the present range of natural variability whereas pH (8.1, 7.7, and 7.4) covered present as well as near-future natural variability. As expected, the presence of the crab cue induced an antipredator response in Mytilus coruscus (significant 10% increase in byssus secretion rate, 22% increase in frequency of shed byssus, and 30% longer byssus). Decreased pH but not temperature had a significant negative impact on the same endpoints (up to a 17% decrease in byssus secretion rate, 40% decrease in frequency of shed byssus, and 10% shorter byssus at pH 7.3 as compared with pH 8.1) with no significant interactions between the three tested parameters. In this study, it has been hypothesized that pH and predator cue have different modes of action and lead to conflicting functional responses (escape response versus stronger attachment). Functional consequences for ecosystem dynamics still need to be investigated.

Sponge bioerosion accelerated by ocean acidification across species and latitudes?

In many marine biogeographic realms, bioeroding sponges dominate the internal bioerosion of calcareous substrates such as mollusc beds and coral reef framework. They biochemically dissolve part of the carbonate and liberate so-called sponge chips, a process that is expected to be facilitated and accelerated in a more acidic environment inherent to the present global change. The bioerosion capacity of the demosponge Cliona celata Grant, 1826 in subfossil oyster shells was assessed via alkalinity anomaly technique based on 4 days of experimental exposure to three different levels of carbon dioxide partial pressure (pCO2) at ambient temperature in the cold-temperate waters of Helgoland Island, North Sea. The rate of chemical bioerosion at present-day pCO2 was quantified with 0.08-0.1 kg/m**2/year. Chemical bioerosion was positively correlated with increasing pCO2, with rates more than doubling at carbon dioxide levels predicted for the end of the twenty-first century, clearly confirming that C. celata bioerosion can be expected to be enhanced with progressing ocean acidification (OA). Together with previously published experimental evidence, the present results suggest that OA accelerates sponge bioerosion (1) across latitudes and biogeographic areas, (2) independent of sponge growth form, and (3) for species with or without photosymbionts alike. A general increase in sponge bioerosion with advancing OA can be expected to have a significant impact on global carbonate (re)cycling and may result in widespread negative effects, e.g. on the stability of wild and farmed shellfish populations, as well as calcareous framework builders in tropical and cold-water coral reef ecosystems.

Microenvironmental changes support evidence of photosynthesis and calcification inhibition in Halimeda under ocean acidification and warming

The effects of elevated CO2 and temperature on photosynthesis and calcification of two important calcifying reef algae (Halimeda macroloba and Halimeda cylindracea) were investigated with O2 microsensors and chlorophyll a fluorometry through a combination of two pCO2 (400 and 1,200 µatm) and two temperature treatments (28 and 32 °C) equivalent to the present and predicted conditions during the 2100 austral summer. Combined exposure to pCO2 and elevated temperature impaired calcification and photosynthesis in the two Halimeda species due to changes in the microenvironment around the algal segments and a reduction in physiological performance. There were no significant changes in controls over the 5-week experiment, but there was a 50-70 % decrease in photochemical efficiency (maximum quantum yield), a 70-80 % decrease in O2 production and a threefold reduction in calcification rate in the elevated CO2 and high temperature treatment. Calcification in these species is closely coupled with photosynthesis, such that a decrease in photosynthetic efficiency leads to a decrease in calcification. Although pH seems to be the main factor affecting Halimeda species, heat stress also has an impact on their photosystem II photochemical efficiency. There was a strong combined effect of elevated CO2 and temperature in both species, where exposure to elevated CO2 or temperature alone decreased photosynthesis and calcification, but exposure to both elevated CO2 and temperature caused a greater decline in photosynthesis and calcification than in each stress individually. Our study shows that ocean acidification and ocean warming are drivers of calcification and photosynthesis inhibition in Halimeda. Predicted climate change scenarios for 2100 would therefore severely affect the fitness of Halimeda, which can result in a strongly reduced production of carbonate sediments on coral reefs under such changed climate conditions.

Temperature modulates coccolithophorid sensitivity of growth, photosynthesis and calcification to increasing seawater pCO2

Increasing atmospheric CO2 concentrations are expected to impact pelagic ecosystem functioning in the near future by driving ocean warming and acidification. While numerous studies have investigated impacts of rising temperature and seawater acidification on planktonic organisms separately, little is presently known on their combined effects. To test for possible synergistic effects we exposed two coccolithophore species, Emiliania huxleyi and Gephyrocapsa oceanica, to a CO2 gradient ranging from ~0.5-250 µmol/kg (i.e. ~20-6000 µatm pCO2) at three different temperatures (i.e. 10, 15, 20°C for E. huxleyi and 15, 20, 25°C for G. oceanica). Both species showed CO2-dependent optimum-curve responses for growth, photosynthesis and calcification rates at all temperatures. Increased temperature generally enhanced growth and production rates and modified sensitivities of metabolic processes to increasing CO2. CO2 optimum concentrations for growth, calcification, and organic carbon fixation rates were only marginally influenced from low to intermediate temperatures. However, there was a clear optimum shift towards higher CO2 concentrations from intermediate to high temperatures in both species. Our results demonstrate that the CO2 concentration where optimum growth, calcification and carbon fixation rates occur is modulated by temperature. Thus, the response of a coccolithophore strain to ocean acidification at a given temperature can be negative, neutral or positive depending on that strain's temperature optimum. This emphasizes that the cellular responses of coccolithophores to ocean acidification can only be judged accurately when interpreted in the proper eco-physiological context of a given strain or species. Addressing the synergistic effects of changing carbonate chemistry and temperature is an essential step when assessing the success of coccolithophores in the future ocean.

Calcification of the Arctic coralline red algae Lithothamnion glaciale in response to elevated CO2

Rising atmospheric CO2 concentrations could cause a calcium carbonate subsaturation of Arctic surface waters in the next 20 yr, making these waters corrosive for calcareous organisms. It is presently unknown what effects this will have on Arctic calcifying organisms and the ecosystems of which they are integral components. So far, acidification effects on crustose coralline red algae (CCA) have only been studied in tropical and Mediterranean species. In this work, we investigated calcification rates of the CCA Lithothamnion glaciale collected in northwest Svalbard in laboratory experiments under future atmospheric CO2 concentrations. The algae were exposed to simulated Arctic summer and winter light conditions in 2 separate experiments at optimum growth temperatures. We found a significant negative effect of increased CO2 levels on the net calcification rates of L. glaciale in both experiments. Annual mean net dissolution of L. glaciale was estimated to start at an aragonite saturation state between 1.1 and 0.9 which is projected to occur in parts of the Arctic surface ocean between 2030 and 2050 if emissions follow 'business as usual' scenarios (SRES A2; IPCC 2007). The massive skeleton of CCA, which consist of more than 80% calcium carbonate, is considered crucial to withstanding natural stresses such as water movement, overgrowth or grazing. The observed strong negative response of this Arctic CCA to increased CO2 levels suggests severe threats of the projected ocean acidification for an important habitat provider in the Arctic coastal ocean.

Experiment: Dissecting the impact of CO2 and pH on the mechanisms of photosynthesis and calcification in the coccolithophore Emiliania huxleyi

Coccolithophores are important calcifying phytoplankton predicted to be impacted by changes in ocean carbonate chemistry caused by the absorption of anthropogenic CO2. However, it is difficult to disentangle the effects of the simultaneously changing carbonate system parameters (CO2, bicarbonate, carbonate and protons) on the physiological responses to elevated CO2. Here, we adopted a multifactorial approach at constant pH or CO2 whilst varying dissolved inorganic carbon (DIC) to determine physiological and transcriptional responses to individual carbonate system parameters. We show that Emiliania huxleyi is sensitive to low CO2 (growth and photosynthesis) and low bicarbonate (calcification) as well as low pH beyond a limited tolerance range, but is much less sensitive to elevated CO2 and bicarbonate. Multiple up-regulated genes at low DIC bear the hallmarks of a carbon-concentrating mechanism (CCM) that is responsive to CO2 and bicarbonate but not to pH. Emiliania huxleyi appears to have evolved mechanisms to respond to limiting rather than elevated CO2. Calcification does not function as a CCM, but is inhibited at low DIC to allow the redistribution of DIC from calcification to photosynthesis. The presented data provides a significant step in understanding how E. huxleyi will respond to changing carbonate chemistry at a cellular level

Impact of medium-term exposure to elevated pCO2 levels on the physiological energetics of the mussel Mytilus chilensis

This study evaluated the impact of medium-term exposure to elevated pCO2 levels (750-1200 ppm) on the physiological processes of juvenile Mytilus chilensis mussels over a period of 70 d in a mesocosm system. Three equilibration tanks filled with filtered seawater were adjusted to three pCO2 levels: 380 (control), 750 and 1200 ppm by bubbling air or an air-CO2 mixture through the water. For the control, atmospheric air (with aprox. 380 ppm CO2) was bubbled into the tank; for the 750 and 1200 ppm treatments, dry air and pure CO2 were blended to each target concentration using mass flow controllers for air and CO2. No impact on feeding activity was observed at the beginning of the experiment, but a significant reduction in clearance rate was observed after 35 d of exposure to highly acidified seawater. Absorption rate and absorption efficiency were reduced at high pCO2 levels. In addition, oxygen uptake fell significantly under these conditions, indicating a metabolic depression. These physiological responses of the mussels resulted in a significant reduction of energy available for growth (scope for growth) with important consequences for the aquaculture of this species during medium-term exposure to acid conditions. The results of this study clearly indicate that high pCO2 levels in the seawater have a negative effect on the health of M. chilensis. Therefore, the predicted acidification of seawater associated with global climate change could be harmful to this ecologically and commercially important mussel.