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.

Grazing under experimental hypercapnia and elevated temperature does not affect the radula of a chiton (Mollusca, Polyplacophora, Lepidopleurida)

Chitons (class Polyplacophora) are benthic grazing molluscs with an eight-part aragonitic shell armature. The radula, a serial tooth ribbon that extends internally more than half the length of the body, is mineralised on the active feeding teeth with iron magnetite apparently as an adaptation to constant grazing on rocky substrates. As the anterior feeding teeth are eroded they are shed and replaced with a new row. The efficient mineralisation and function of the radula could hypothetically be affected by changing oceans in two ways: changes in seawater chemistry (pH and pCO2) may impact the biomineralisation pathway, potentially leading to a weaker or altered density of the feeding teeth; rising temperatures could increase activity levels in these ectothermic animals, and higher feeding rates could increase wear on the feeding teeth beyond the animals' ability to synthesise, mineralise, and replace radular rows. We therefore examined the effects of pH and temperature on growth and integrity in the radula of the chiton Leptochiton asellus. Our experiment implemented three temperature (10, 15, 20 °C) and two pCO2 treatments (400 µatm, pH 8.0; 2000 µatm, pH 7.5) for six treatment groups. Animals (n = 50) were acclimated to the treatment conditions for a period of 4 weeks. This is sufficient time for growth of ca. 7-9 new tooth rows or 20% turnover of the mineralised portion. There was no significant difference in the number of new (non-mineralised) teeth or total tooth row count in any treatment. Examination of the radulae via SEM revealed no differences in microwear or breakage on the feeding cusps correlating to treatment groups. The shell valves also showed no signs of dissolution. As a lineage, chitons have survived repeated shifts in Earth's climate through geological time, and at least their radulae may be robust to future perturbations.

Projected near-future CO2 levels increase activity and alter defensive behaviours in the tropical squid Idiosepius pygmaeus

Carbon dioxide (CO2) levels projected to occur in the oceans by the end of this century cause a range of behavioural effects in fish, but whether other highly active marine organisms, such as cephalopods, are similarly affected is unknown. We tested the effects of projected future CO2 levels (626 and 956 µatm) on the behaviour of male two-toned pygmy squid, Idiosepius pygmaeus. Exposure to elevated CO2 increased the number of active individuals by 19-25% and increased movement (number of line-crosses) by nearly 3 times compared to squid at present-day CO2. Squid vigilance and defensive behaviours were also altered by elevated CO2 with >80% of individuals choosing jet escape responses over defensive arm postures in response to a visual startle stimulus, compared with 50% choosing jet escape responses at control CO2. In addition, more escape responses were chosen over threat behaviours in body pattern displays at elevated CO2 and individuals were more than twice as likely to use ink as a defence strategy at 956 µatm CO2, compared with controls. Increased activity could lead to adverse effects on energy budgets as well as increasing visibility to predators. A tendency to respond to a stimulus with escape behaviours could increase survival, but may also be energetically costly and could potentially lead to more chases by predators compared with individuals that use defensive postures. These results demonstrate that projected future ocean acidification affects the behaviours of a tropical squid species.

Elevated CO2 levels do not affect the shell structure of the bivalve Arctica islandica from the western Baltic

Shells of the bivalve Arctica islandica are used to reconstruct paleo-environmental conditions (e.g. temperature) via biogeochemical proxies, i.e. biogenic components that are related closely to environmental parameters at the time of shell formation. Several studies have shown that proxies like element and isotope-ratios can be affected by shell growth and microstructure. Thus it is essential to evaluate the impact of changing environmental parameters such as high pCO2 and consequent changes in carbonate chemistry on shell properties to validate these biogeochemical proxies for a wider range of environmental conditions. Growth experiments with Arctica islandica from the Western Baltic Sea kept under different pCO2 levels (from 380 to 1120 µatm) indicate no affect of elevated pCO2 on shell growth or crystal microstructure, indicating that A. islandica shows an adaptation to a wider range of pCO2 levels than reported for other species. Accordingly, proxy information derived from A. islandica shells of this region contains no pCO2 related bias.

Effect of increasing sea water pCO2 on the northern Atlantic krill species Nyctiphanes couchii

Surprisingly little is known about potential effects of ocean acidification on krill of the Northern Hemisphere as ecologically very important food web component. Sub-adult individuals of the northern Atlantic krill species Nyctiphanes couchii (caught at Austevoll near Bergen, Norway, in January 2013) were exposed in the laboratory to four different levels of pCO2 (430, 800, 1,100, and 1,700 µatm) for 5 weeks in order to assess potential changes in a set of biological response variables. Survival decreased and the frequency of moulting-related deaths increased with increasing pCO2. Survival was considerably reduced at relatively high pCO2 of 1,700 µatm and tended to be negatively affected at 1,100 µatm pCO2. However, the experimental results show no significant effects of pCO2 on inter-moult period and growth at pCO2 levels below 1,100 µatm. No differences in length measurements of the carapace and uropod were observed across pCO2 levels, indicating no effect of changing carbonate chemistry on the morphology of those calciferous parts of the exoskeleton. The results suggest that sub-adult N. couchii may not suffer dramatically from predicted near-future changes in pCO2. However, potential detrimental effects on the moulting process and associated higher mortality at 1,100 µatm pCO2 cannot be excluded. Further experiments are needed in order to investigate whether early life stages of N. couchii show a different sensitivity to elevated sea water pCO2 and whether those results are transferable to other krill species of the Northern Hemisphere.

Predominance of heavily calcified coccolithophores at low CaCO3 saturation during winter in the Bay of Biscay

Coccolithophores are an important component of the Earth system, and, as calcifiers, their possible susceptibility to ocean acidification is of major concern. Laboratory studies at enhanced pCO2 levels have produced divergent results without overall consensus. However, it has been predicted from these studies that, although calcification may not be depressed in all species, acidification will produce "a transition in dominance from more to less heavily calcified coccolithophores" [Ridgwell A, et al., (2009) Biogeosciences 6:2611-2623]. A recent observational study [Beaufort L, et al., (2011) Nature 476:80-83] also suggested that coccolithophores are less calcified in more acidic conditions. We present the results of a large observational study of coccolithophore morphology in the Bay of Biscay. Samples were collected once a month for over a year, along a 1,000-km-long transect. Our data clearly show that there is a pronounced seasonality in the morphotypes of Emiliania huxleyi, the most abundant coccolithophore species. Whereas pH and CaCO3 saturation are lowest in winter, the E. huxleyi population shifts from <10% (summer) to >90% (winter) of the heavily calcified form. However, it is unlikely that the shifts in carbonate chemistry alone caused the morphotype shift. Our finding that the most heavily calcified morphotype dominates when conditions are most acidic is contrary to the earlier predictions and raises further questions about the fate of coccolithophores in a high-CO2 world.

Multiple stressor effects of near-future elevated seawater temperature and decreased pH on righting and escape behaviors of two common Antarctic gastropods

Warming seawater temperatures and ocean acidification on the coastal western Antarctic Peninsula pose unique challenges to stenothermal marine invertebrates. The present study examines prospective sub-lethal effects of elevated temperature, pCO2, and resultant decrease in seawater pH, on righting behavior and maximal escape speeds for two common gastropods, the limpet Nacella concinna (Strebel) and mesogastropod snail Margarella antarctica (Lamy). Replicate individuals held in individual containers were exposed to four combinations of seawater temperature (1.5 °C-current average, 3.5 °C-projected average by 2100) and pH (pH 8.0-current average, pH 7.8-projected average by 2100 as a result of elevated pCO2 levels) for a period of 6 weeks. Following this chronic exposure, righting behavior, determined for the limpets as proportion to right over 24 h and for snails as time to right, as well as maximum escape speed following contact with a sea star predator were measured. We found no significant differences in proportions of limpets displaying the capacity to right among the four temperature-pH treatments. However, there was a significant temperature-pH interaction effect for mean righting times in snails, indicating that the effect of pH on the time to right is dependent on temperature. We found no significant effects of temperature or pH on mean maximal escape speed in limpets. Additionally, we observed a significant temperature-pH interaction effect for mean maximal escape speed in snails. These interactive effects make it difficult to make clear predictions about how these environmental factors may impact behavioral responses.

Effects of ocean acidification on respiration, growth and C:N ratio of arctic Hyas araneus larvae

Early life stages of marine crustaceans respond sensitively to elevated seawater PCO2. However, the underlying physiological mechanisms have not been studied well. We therefore investigated the effects of elevated seawater PCO2 on oxygen consumption, dry weight, elemental composition, median developmental time (MDT) and mortality in zoea I larvae of the spider crab Hyas araneus (Svalbard 79°N/11°E; collection, May 2009; hatch, December 2009). At the time of moulting, oxygen consumption rate had reached a steady state level under control conditions. In contrast, elevated seawater PCO2 caused the metabolic rate to rise continuously leading to a maximum 1.5-fold increase beyond control level a few days before moulting into the second stage (zoea II), followed by a pronounced decrease. Dry weight of larvae reared under high CO2 conditions was lower than in control larvae at the beginning of the moult cycle, yet this difference had disappeared at the time of moulting. MDT of zoea I varied between 45 ± 1 days under control conditions and 42 ± 2 days under the highest seawater CO2 concentration. The present study indicates that larval development under elevated seawater PCO2 levels results in higher metabolic costs during premoulting events in zoea I. However, H. araneus zoea I larvae seem to be able to compensate for higher metabolic costs as larval MDT and survival was not affected by elevated PCO2 levels.

Seawater carbonate chemistry, clearance rates, respiration rates, condition index and cellular turnover (RNA: DNA) of Juvenile King Scallop, Pecten maximus in a laboratory experiment

The decline in ocean water pH and changes in carbonate saturation states through anthropogenically mediated increases in atmospheric CO2 levels may pose a hazard to marine organisms. This may be particularly acute for those species reliant on calcareous structures like shells and exoskeletons. This is of particular concern in the case of valuable commercially exploited species such as the king scallop, Pecten maximus. In this study we investigated the effects on oxygen consumption, clearance rates and cellular turnover in juvenile P. maximus following 3 months laboratory exposure to four pCO2 treatments (290, 380, 750 and 1140 µatm). None of the exposure levels were found to have significant effect on the clearance rates, respiration rates, condition index or cellular turnover (RNA: DNA) of individuals. While it is clear that some life stages of marine bivalves appear susceptible to future levels of ocean acidification, particularly under food limiting conditions, the results from this study suggest that where food is in abundance, bivalves like juvenile P. maximus may display a tolerance to limited changes in seawater chemistry.

Seawater carbonate chemistry and sea urchin larval size in a laboratory experiment

Ocean acidification results from an increase in the concentrations of atmospheric carbon dioxide (CO2) impacts on marine calcifying species, which is predicted to become more pronounced in the future. By the end of this century, atmospheric pCO2 levels will have doubled relative to the pre-industrial levels of 280 ppm. However, the effects of pre-industrial pCO2 levels on marine organisms remain largely unknown. In this study, we investigated the effects of pre-industrial pCO2 conditions on the size of the pluteus larvae of sea urchins, which are known to be vulnerable to ocean acidification. The larval size of Hemicentrotus pulcherrimus significantly increased when reared at pre-industrial pCO2 level relative to the present one, and the size of Anthocidaris crassispina larvae decreased as the pCO2 levels increased from the pre-industrial level to the near future ones after 3 days' exposure. In this study, it is suggested that echinoid larvae responded to pre-industrial pCO2 levels. Ocean acidification may be affecting some sensitive marine calcifiers even at the present pCO2 level.

Size matters: plasticity in metabolic scaling shows body-size may modulate responses to climate change

Variability in metabolic scaling in animals, the relationship between metabolic rate ( R) and body mass ( M), has been a source of debate and controversy for decades. R is proportional to Mb, the precise value of b much debated, but historically considered equal in all organisms. Recent metabolic theory, however, predicts b to vary among species with ecology and metabolic level, and may also vary within species under different abiotic conditions. Under climate change, most species will experience increased temperatures, and marine organisms will experience the additional stressor of decreased seawater pH ('ocean acidification'). Responses to these environmental changes are modulated by myriad species-specific factors. Body-size is a fundamental biological parameter, but its modulating role is relatively unexplored. Here, we show that changes to metabolic scaling reveal asymmetric responses to stressors across body-size ranges; b is systematically decreased under increasing temperature in three grazing molluscs, indicating smaller individuals were more responsive to warming. Larger individuals were, however, more responsive to reduced seawater pH in low temperatures. These alterations to the allometry of metabolism highlight abiotic control of metabolic scaling, and indicate that responses to climate warming and ocean acidification may be modulated by body-size.

Diverse coral communities in naturally acidified waters of a Western Pacific reef

Anthropogenic carbon dioxide emissions are acidifying the oceans, reducing the concentration of carbonate ions ([CO32-) that calcifying organisms need to build and cement coral reefs. To date, studies of a handful of naturally acidified reef systems reveal depauperate communities, sometimes with reduced coral cover and calcification rates, consistent with results of laboratory-based studies. Here we report the existence of highly diverse, coral-dominated reef communities under chronically low pH and aragonite saturation state (Omega ar). Biological and hydrographic processes change the chemistry of the seawater moving across the barrier reefs and into Palau's Rock Island bays, where levels of acidification approach those projected for the western tropical Pacific open ocean by 2100. Nevertheless, coral diversity, cover, and calcification rates are maintained across this natural acidification gradient. Identifying the combination of biological and environmental factors that enable these communities to persist could provide important insights into the future of coral reefs under anthropogenic acidification.

Seasonality affects macroalgal community response to increases in pCO2

Ocean acidification is expected to alter marine systems, but there is uncertainty about its effects due to the logistical difficulties of testing its large-scale and long-term effects. Responses of biological communities to increases in carbon dioxide can be assessed at CO2 seeps that cause chronic exposure to lower seawater pH over localised areas of seabed. Shifts in macroalgal communities have been described at temperate and tropical pCO2 seeps, but temporal and spatial replication of these observations is needed to strengthen confidence our predictions, especially because very few studies have been replicated between seasons. Here we describe the seawater chemistry and seasonal variability of macroalgal communities at CO2 seeps off Methana (Aegean Sea). Monitoring from 2011 to 2013 showed that seawater pH decreased to levels predicted for the end of this century at the seep site with no confounding gradients in Total Alkalinity, salinity, temperature or wave exposure. Most nutrient levels were similar along the pH gradient; silicate increased significantly with decreasing pH, but it was not limiting for algal growth at all sites. Metal concentrations in seaweed tissues varied between sites but did not consistently increase with pCO2. Our data on the flora are consistent with results from laboratory experiments and observations at Mediterranean CO2 seep sites in that benthic communities decreased in calcifying algal cover and increased in brown algal cover with increasing pCO2. This differs from the typical macroalgal community response to stress, which is a decrease in perennial brown algae and proliferation of opportunistic green algae. Cystoseira corniculata was more abundant in autumn and Sargassum vulgare in spring, whereas the articulated coralline alga Jania rubens was more abundant at reference sites in autumn. Diversity decreased with increasing CO2 regardless of season. Our results show that benthic community responses to ocean acidification are strongly affected by season.

Phytoplankton responses and associated carbon cycling during shipboard carbonate chemistry manipulation experiments conducted around Northwest European shelf seas

The ongoing oceanic uptake of anthropogenic carbon dioxide (CO2) is significantly altering the carbonate chemistry of seawater, a phenomenon referred to as ocean acidification. Experimental manipulations have been increasingly used to gauge how continued ocean acidification will potentially impact marine ecosystems and their associated biogeochemical cycles in the future; however, results amongst studies, particularly when performed on natural communities, are highly variable, which may reflect community/environment-specific responses or inconsistencies in experimental approach. To investigate the potential for identification of more generic responses and greater experimentally reproducibility, we devised and implemented a series (n = 8) of short-term (2-4 days) multi-level (>=4 conditions) carbonate chemistry/nutrient manipulation experiments on a range of natural microbial communities sampled in Northwest European shelf seas. Carbonate chemistry manipulations and resulting biological responses were found to be highly reproducible within individual experiments and to a lesser extent between geographically separated experiments. Statistically robust reproducible physiological responses of phytoplankton to increasing pCO2, characterised by a suppression of net growth for small-sized cells (<10 µm), were observed in the majority of the experiments, irrespective of natural or manipulated nutrient status. Remaining between-experiment variability was potentially linked to initial community structure and/or other site-specific environmental factors. Analysis of carbon cycling within the experiments revealed the expected increased sensitivity of carbonate chemistry to biological processes at higher pCO2 and hence lower buffer capacity. The results thus emphasise how biogeochemical feedbacks may be altered in the future ocean.