Impacts of seawater acidification on mantle gene expression patterns of the Baltic Sea blue mussel: implications for shell formation and energy metabolism, link to supplementary material

Marine organisms have to cope with increasing CO2 partial pressures and decreasing pH in the oceans. We elucidated the impacts of an 8-week acclimation period to four seawater pCO2 treatments (39, 113, 243 and 405 Pa/385, 1,120, 2,400 and 4,000 µatm) on mantle gene expression patterns in the blue mussel Mytilus edulis from the Baltic Sea. Based on the M. edulis mantle tissue transcriptome, the expression of several genes involved in metabolism, calcification and stress responses was assessed in the outer (marginal and pallial zone) and the inner mantle tissues (central zone) using quantitative real-time PCR. The expression of genes involved in energy and protein metabolism (F-ATPase, hexokinase and elongation factor alpha) was strongly affected by acclimation to moderately elevated CO2 partial pressures. Expression of a chitinase, potentially important for the calcification process, was strongly depressed (maximum ninefold), correlating with a linear decrease in shell growth observed in the experimental animals. Interestingly, shell matrix protein candidate genes were less affected by CO2 in both tissues. A compensatory process toward enhanced shell protection is indicated by a massive increase in the expression of tyrosinase, a gene involved in periostracum formation (maximum 220-fold). Using correlation matrices and a force-directed layout network graph, we were able to uncover possible underlying regulatory networks and the connections between different pathways, thereby providing a molecular basis of observed changes in animal physiology in response to ocean acidification.

Seawater carbonate chemistry and Emiliania huxleyi biological processes during experiments, 2010

The calcifying phytoplankton species, coccolithophores, have their calcified coccoliths around the cells, however, their physiological roles are still unknown. Here, we hypothesized that the coccoliths may play a certain role in reducing solar UV radiation (UVR, 280-400 nm) and protect the cells from being harmed. Cells of Emiliania huxleyi with different thicknesses of the coccoliths were obtained by culturing them at different levels of dissolved inorganic carbon and their photophysiological responses to UVR were investigated. Although increased dissolved inorganic carbon decreased the specific growth rate, the increased coccolith thickness significantly ameliorated the photoinhibition of PSII photochemical efficiency caused by UVR. Increase by 91% in the coccolith thickness led to 35% increase of the PSII yield and 22% decrease of the photoinhibition of the effective quantum yield by UVR. The coccolith cover reduced more UVA (320-400 nm) than UVB (280-315 nm), leading to less inhibition per energy at the UV-A band.

Seawater carbonate chemistry, photosynthesis and calcification rate during experiments with coral Acropora muricata, 2011

The effect of decreasing aragonite saturation state (Omega Arag) of seawater (elevated pCO2) on calcification rates of Acropora muricata was studied using nubbins prepared from parent colonies located at two sites of La Saline reef (La Réunion Island, western Indian Ocean): a back-reef site (BR) affected by nutrient-enriched groundwater discharge (mainly nitrate), and a reef flat site (RF) with low terrigenous inputs. Protein and chlorophyll a content of the nubbins, as well as zooxanthellae abundance, were lower at RF than BR. Nubbins were incubated at ~27°C over 2 h under sunlight, in filtered seawater manipulated to get differing initial pCO2 (1,440-340 µatm), Omega Arag (1.4-4.0), and dissolved inorganic carbon (DIC) concentrations (2,100-1,850 µmol kg-1). Increasing DIC concentrations at constant total alkalinity (AT) resulted in a decrease in Omega Arag and an increase in pCO2. AT at the beginning of the incubations was kept at a natural level of 2,193 +- 6 µmol kg-1 (mean +- SD). Net photosynthesis (NP) and calcification were calculated from changes in pH and AT during the incubations. Calcification decrease in response to doubling pCO2 relative to preindustrial level was 22% for RF nubbins. When normalized to surface area of the nubbins, (1) NP and calcification were higher at BR than RF, (2) NP increased in high pCO2 treatments at BR compared to low pCO2 treatments, and (3) calcification was not related to Omega Arag at BR. When normalized to NP, calcification was linearly related to Omega Arag at both sites, and the slopes of the relationships were not significantly different. The increase in NP at BR in the high pCO2 treatments may have increased calcification and thus masked the negative effect of low Omega Arag on calcification. Removing the effect of NP variations at BR showed that calcification declined in a similar manner with decreased Omega Arag (increased pCO2) whatever the nutrient loading.

Seawater carbonate chemistry and growth rate during experiments with phylotypes of Symbiodinium (Dinophyceae), 2011

We investigated the effect of elevated partial pressure of CO2 (pCO2) on the photosynthesis and growth of four phylotypes (ITS2 types A1, A13, A2, and B1) from the genus Symbiodinium, a diverse dinoflagellate group that is important, both free-living and in symbiosis, for the viability of cnidarians and is thus a potentially important model dinoflagellate group. The response of Symbiodinium to an elevated pCO2 was phylotype-specific. Phylotypes A1 and B1 were largely unaffected by a doubling in pCO2 in contrast, the growth rate of A13 and the photosynthetic capacity of A2 both increased by ~ 60%. In no case was there an effect of ocean acidification (OA) upon respiration (dark- or light-dependent) for any of the phylotypes examined. Our observations suggest that OA might preferentially select among free-living populations of Symbiodinium, with implications for future symbioses that rely on algal acquisition from the environment (i.e., horizontal transmission). Furthermore, the carbon environment within the host could differentially affect the physiology of different Symbiodinium phylotypes. The range of responses we observed also highlights that the choice of species is an important consideration in OA research and that further investigation across phylogenetic diversity, for both the direction of effect and the underlying mechanism(s) involved, is warranted.

Seawater carbonate chemistry and survival of mollusks: Mercenaria mercenaria, Argopecten irradians, and Crassostrea virginica during experiments, 2009

We present experiments that examined the metamorphosis, growth, and survivorship of larvae from three species of commercially and ecologically valuable shellfish (Mercenaria mercenaria, Argopecten irradians, and Crassostrea virginica) at the levels of CO2 projected to occur during the 21st century and beyond. Under CO2 concentrations estimated to occur later this century (~66 Pa, 650 ppm), M. mercenaria and A. irradians larvae exhibited dramatic declines (>50%) in survivorship as well as significantly delayed metamorphosis and significantly smaller sizes. Although C. virginica larvae also experienced lowered growth and delayed metamorphosis at ~66 Pa CO2, their survival was only diminished at ~152 Pa CO2. The extreme sensitivity of larval stages of shellfish to enhanced levels of CO2 indicates that current and future increases in pelagic CO2 concentrations may deplete or alter the composition of shellfish populations in coastal ecosystems.

Seawater carbonate chemistry, nutrients, and Calcidiscus leptoporus (strain RCC1135) growth rate and calcification rate during experiments, 2012

The coccolithophore Calcidiscus leptoporus was grown in batch culture under nitrogen (N) as well as phosphorus (P) limitation. Growth rate, particulate inorganic carbon (PIC), particulate organic carbon (POC), particulate organic nitrogen (PON), and particulate organic phosphorus (POP) production were determined and coccolith morphology was analysed. While PON production decreased by 70% under N-limitation and POP production decreased by 65% under P-limitation, growth rate decreased by 33% under N- as well as P-limitation. POC as well as PIC production (calcification rate) increased by 27% relative to the control under P-limitation, and did not change under N-limitation. Coccolith morphology did not change in response to either P or N limitation. While these findings, supported by a literature survey, suggest that coccolith morphogenesis is not hampered by either P or N limitation, calcification rate might be. The latter conclusion is in apparent contradiction to our data. We discuss the reasons for this inference.

Seawater carbonate chemistry and effects of CO2 and their modulation by light in the life-cycle stages of the coccolithophore Emiliania huxleyi strains RCC 1216 and 1217 during experiments, 2012

The effects of ocean acidification on the life-cycle stages of the coccolithophore Emiliania huxleyi and their by light were examined. Calcifying diploid and noncalcifying haploid cells (Roscoff culture collection 1216 and 1217) were acclimated to present-day and elevated CO2 partial pressures (PCO2; 38.5 vs. 101.3 Pa, ., 380 vs. 1000 matm) under low and high light (50 vs. 300 mmol photons m-2 s-1). Growth rates as well as quotas and production rates of C and N were measured. Sources of inorganic C for biomass buildup were using a 14C disequilibrium assay. Photosynthetic O2 evolution was measured as a function of dissolved inorganic C and light by means of membrane-inlet mass spectrometry. The diploid stage responded to elevated PCO2 by shunting resources from the production of particulate inorganic C toward organic C yet keeping the production of total particulate C constant. As the effect of ocean acidification was stronger under low light, the diploid stage might be less affected by increased acidity when energy availability is high. The haploid stage maintained elemental composition and production rates under elevated PCO2. Although both life-cycle stages involve different ways of dealing with elevated PCO2, the responses were generally modulated by energy availability, being typically most pronounced under low light. Additionally, PCO2 responses resembled those induced by high irradiances, indicating that ocean acidification affects the interplay between energy-generating processes (photosynthetic light reactions) and processes competing for energy (biomass buildup and calcification). A conceptual model is put forward explaining why the magnitude of single responses is determined by energy availability.

Seawater carbonate chemistry and biological processes of Emiliania huxleyi (strains RCC1256 and NZEH) during experiments, 2011

With respect to their sensitivity to ocean acidification, calcifiers such as the coccolithophore Emiliania huxleyi have received special attention, as the process of calcification seems to be particularly sensitive to changes in the marine carbonate system. For E. huxleyi, apparently conflicting results regarding its sensitivity to ocean acidification have been published (Iglesias-Rodriguez et al., 2008a; Riebesell et al., 2000). As possible causes for discrepancies, intra-specific variability and different effects of CO2 manipulation methods, i.e. the manipulation of total alkalinity (TA) or total dissolved inorganic carbon (DIC), have been discussed. While Langer et al. (2009) demonstrate a high degree of intra-specific variability between strains of E. huxleyi, the question whether different CO2 manipulation methods influence the cellular responses has not been resolved yet. In this study, closed TA as well as open and closed DIC manipulation methods were compared with respect to E. huxleyi's CO2-dependence in growth rate, POC- and PIC-production. The differences in the carbonate chemistry between TA and DIC manipulations were shown not to cause any differences in response patterns, while the latter differed between open and closed DIC manipulation. The two strains investigated showed different sensitivities to acidification of seawater, RCC1256 being more negatively affected in growth rates and PIC production than NZEH.

Seawater carbonate chemistry and reproduction of a coral reef fish in a laboratory experiment

Ocean acidification is predicted to negatively impact the reproduction of many marine species, either by reducing fertilization success or diverting energy from reproductive effort. While recent studies have demonstrated how ocean acidification will affect larval and juvenile fishes, little is known about how increasing partial pressure of carbon dioxide (pCO2) and decreasing pH might affect reproduction in adult fishes. We investigated the effects of near-future levels of pCO2 on the reproductive performance of the cinnamon anemonefish, Amphiprion melanopus, from the Great Barrier Reef, Australia. Breeding pairs were held under three CO2 treatments [Current-day Control (430 µatm), Moderate (584 µatm) and High (1032 µatm)] for a 9-month period that included the summer breeding season. Unexpectedly, increased CO2 dramatically stimulated breeding activity in this species of fish. Over twice as many pairs bred in the Moderate (67% of pairs) and High (55%) compared to the Control (27%) CO2 treatment. Pairs in the High CO2 group produced double the number of clutches per pair and 67% more eggs per clutch compared to the Moderate and Control groups. As a result, reproductive output in the High group was 82% higher than that in the Control group and 50% higher than that in the Moderate group. Despite the increase in reproductive activity, there was no difference in adult body condition among the three treatment groups. There was no significant difference in hatchling length between the treatment groups, but larvae from the High CO2 group had smaller yolks than Controls. This study provides the first evidence of the potential effects of ocean acidification on key reproductive attributes of marine fishes and, contrary to expectations, demonstrates an initially stimulatory (hormetic) effect in response to increased pCO2. However, any long-term consequences of increased reproductive effort on individuals or populations remain to be determined.

Experiment: Acidified seawater impacts sea urchin larvae pH regulatory systems relevant for calcification

Calcifying echinoid larvae respond to changes in seawater carbonate chemistry with reduced growth and developmental delay. To date, no information exists on how ocean acidification acts on pH homeostasis in echinoderm larvae. Understanding acid-base regulatory capacities is important because intracellular formation and maintenance of the calcium carbonate skeleton is dependent on pH homeostasis. Using H(+)-selective microelectrodes and the pH-sensitive fluorescent dye BCECF, we conducted in vivo measurements of extracellular and intracellular pH (pH(e) and pH(i)) in echinoderm larvae. We exposed pluteus larvae to a range of seawater CO(2) conditions and demonstrated that the extracellular compartment surrounding the calcifying primary mesenchyme cells (PMCs) conforms to the surrounding seawater with respect to pH during exposure to elevated seawater pCO(2). Using FITC dextran conjugates, we demonstrate that sea urchin larvae have a leaky integument. PMCs and spicules are therefore directly exposed to strong changes in pH(e) whenever seawater pH changes. However, measurements of pH(i) demonstrated that PMCs are able to fully compensate an induced intracellular acidosis. This was highly dependent on Na(+) and HCO(3)(-), suggesting a bicarbonate buffer mechanism involving secondary active Na(+)-dependent membrane transport proteins. We suggest that, under ocean acidification, maintained pH(i) enables calcification to proceed despite decreased pH(e). However, this probably causes enhanced costs. Increased costs for calcification or cellular homeostasis can be one of the main factors leading to modifications in energy partitioning, which then impacts growth and, ultimately, results in increased mortality of echinoid larvae during the pelagic life stage.

Seawater carbonate chemistry and calcification at Anse des Cuivres, NW Mediterranean, 2004

The community metabolism of a shallow infralittoral ecosystem dominated by the calcareous macroalgae Corallina elongata was investigated in Marseilles (NW Mediterranean), by monitoring hourly changes of seawater pH and total alkalinity over 6 d in February 2000. Fair weather conditions prevailed over the study period as indicated by oceanographic (temperature, salinity, and current velocity and direction) and meteorological variables, which validated the standing water hypothesis. This temperate ecosystem exhibited high community gross primary production (GPP = 519 ± 106 mmol C m-2 d-1, n = 6) and also supported high rates of community respiration (R). As a result, the system was slightly autotrophic (net community production, NCP = 20 mmol C m-2 d-1), with a GPP/R ratio of 1.06. NCP exhibited circadian variations with 2- to 3-fold changes in community respiration, both in the light and in the dark. Rates of net community calcification also exhibited circadian variations, with positive rates (up to 24 mmol CaCO3 m-2 h-1) for irradiance values >300 W m-2 (about 1380 µmol photon m-2 s-1). Below this irradiance threshold, net community dissolution prevailed. Daily net calcification (G) was on average 8 mmol CaCO3 m-2 d-1. CO2 fluxes generated by primary production, respiration, and calcification suggest that the study site was a potential atmospheric CO2 sink of 15 mmol CO2 m-2 d-1 at the time of measurement.