Description: Although ocean warming and acidification are recognized as two major anthropogenic perturbations of today’s oceans we know very little about how marine phytoplankton may respond via evolutionary change. We tested for adaptation to ocean warming in combination with ocean acidification in the globally important phytoplankton species Emiliania huxleyi. Temperature adaptation occurred independently of ocean acidification levels. Growth rates were up to 16% higher in populations adapted for one year to warming when assayed at their upper thermal tolerance limit. Particulate inorganic (PIC) and organic (POC) carbon production was restored to values under present-day ocean conditions, owing to adaptive evolution, and were 101% and 55% higher under combined warming and acidification, respectively, than in non-adapted controls. Cells also evolved to a smaller size while they recovered their initial PIC:POC ratio even under elevated CO2. The observed changes in coccolithophore growth, calcite and biomass production, cell size and elemental composition demonstrate the importance of evolutionary processes for phytoplankton performance in a future ocean.

Description: Highlights • Calcification rates are stimulated by CO2 and HCO3− and inhibited by H+. • This novel substrate–inhibitor concept is tested with experimental data. • The concept enables us to reconcile conflicting results among laboratory studies. • We illustrate how this physiological concept can be included in ecological theory. • We apply the concept to discuss coccolithophore dispersal in the oceans. Abstract Coccolithophores are a group of unicellular phytoplankton species whose ability to calcify has a profound influence on biogeochemical element cycling. Calcification rates are controlled by a large variety of biotic and abiotic factors. Among these factors, carbonate chemistry has gained considerable attention during the last years as coccolithophores have been identified to be particularly sensitive to ocean acidification. Despite intense research in this area, a general concept harmonizing the numerous and sometimes (seemingly) contradictory responses of coccolithophores to changing carbonate chemistry is still lacking to date. Here, we present the “substrate–inhibitor concept” which describes the dependence of calcification rates on carbonate chemistry speciation. It is based on observations that calcification rate scales positively with bicarbonate (HCO3−), the primary substrate for calcification, and carbon dioxide (CO2), which can limit cell growth, whereas it is inhibited by protons (H+). This concept was implemented in a model equation, tested against experimental data, and then applied to understand and reconcile the diverging responses of coccolithophorid calcification rates to ocean acidification obtained in culture experiments. Furthermore, we (i) discuss how other important calcification-influencing factors (e.g. temperature and light) could be implemented in our concept and (ii) embed it in Hutchinson’s niche theory, thereby providing a framework for how carbonate chemistry-induced changes in calcification rates could be linked with changing coccolithophore abundance in the oceans. Our results suggest that the projected increase of H+ in the near future (next couple of thousand years), paralleled by only a minor increase of inorganic carbon substrate, could impede calcification rates if coccolithophores are unable to fully adapt. However, if calcium carbonate (CaCO3) sediment dissolution and terrestrial weathering begin to increase the oceans’ HCO3− and decrease its H+ concentrations in the far future (10–100 kyears), coccolithophores could find themselves in carbonate chemistry conditions which may be more favorable for calcification than they were before the Anthropocene.

Description: Biomineralization processes in bivalve molluscs are still poorly understood. Here we provide an analysis of specifically expressed sequences from a mantle transcriptome of the blue mussel, Mytilus edulis. We then developed a novel, integrative shell injury assay to test, whether biomineralization candidate genes highly expressed in marginal and pallial mantle could be induced in central mantle tissue underlying the damaged shell areas. This experimental approach makes it possible to identify gene products that control the chemical micro-environment during calcification as well as organic matrix components. This is unlike existing methodological approaches that work retroactively to characterize calcification relevant molecules and are just able to examine organic matrix components that are present in completed shells. In our assay an orthogonal array of nine 1 mm holes was drilled into the left valve, and mussels were suspended in net cages for 20, 29 and 36 days to regenerate. Structural observations using stereo-microscopy, SEM and Raman spectroscopy revealed organic sheet synthesis (day 20) as the first step of shell-repair followed by the deposition of calcite crystals (days 20 and 29) and aragonite tablets (day 36). The regeneration period was characterized by time-dependent shifts in gene expression in left central mantle tissue underlying the injured shell, (i) increased expression of two tyrosinase isoforms (TYR3: 29-fold and TYR6: 5-fold) at day 20 with a decline thereafter, (ii) an increase in expression of a gene encoding a nacrein-like protein (max. 100-fold) on day 29. The expression of an acidic Asp-Ser-rich protein was enhanced during the entire regeneration process. This proof-of-principle study demonstrates that genes that are specifically expressed in pallial and marginal mantle tissue can be induced (4 out of 10 genes) in central mantle following experimental injury of the overlying shell. Our findings suggest that regeneration assays can be used systematically to better characterize gene products that are essential for distinct phases of the shell formation process, particularly those that are not incorporated into the organic shell matrix.

Description: The cellular mechanisms of calcification in sea urchin larvae are still not well understood. Primary mesenchyme cells within the larval body cavity form a syncytium to secrete CaCO3 spicules from intracellular amorphous CaCO3 (ACC) stores. We studied the role of Na+K+2Cl− cotransporter (NKCC) in intracellular ACC accumulation and larval spicule formation of Strongylocentrotus droebachiensis. First, we incubated growing larvae with three different loop diuretics (azosemide, bumetanide, and furosemide) and established concentration-response curves. All loop diuretics were able to inhibit calcification already at concentrations that specifically inhibit NKCC. Calcification was most effectively inhibited by azosemide (IC50 = 6.5 μM), while larval mortality and swimming ability were not negatively impacted by the treatment. The inhibition by bumetanide (IC50 = 26.4 μM) and furosemide (IC50 = 315.4 μM) resembled the pharmacological fingerprint of the mammalian NKCC1 isoform. We further examined the effect of azosemide on the maintenance of cytoplasmic cords and on the occurrence of calcification vesicles using fluorescent dyes (calcein, FM1-43). Fifty micromolars of azosemide inhibited the maintenance of cytoplasmic cords and resulted in increased calcein fluorescence within calcification vesicles. The expression of NKCC in S. droebachiensis was verified by PCR and Western blot with a specific NKCC antibody. In summary, the pharmacological profile of loop diuretics and their specific effects on calcification in sea urchin larvae suggest that they act by inhibition of NKCC via repression of cytoplasmic cord formation and maintenance.

Description: Anthropogenic CO2 emissions lead to chronically elevated seawater CO2 partial pressures (hypercapnia). The induced ocean acidification will very likely be a relevant factor shaping future marine environments. CO2 exposure concomitantly challenges the animal's capacity of acid-base and ionic regulation as well as the ability to maintain energy metabolism and calcification. Under conditions of acute hypercapnia, numerous studies have revealed a broad range of tolerance levels displayed by various marine taxa. Thus, it is well known that, in contrast to many marine invertebrates, most teleost fish are able to fully compensate acid-base disturbances in short-term experiments (hours to several days). in order to determine whether marine fish are able to preserve aerobic scope following long-term incubation to elevated CO2, we exposed two groups of Atlantic Cod for 4 and 12 months to 0.3 and 0.6 kPa P-CO2, respectively. Measurements of standard and active metabolic rates, critical swimming speeds and aerobic scope of long-term incubated cod showed no deviations from control values, indicating that locomotory performance is not compromised by the different levels of chronic hypercapnia. While the maintenance of high activity levels is supported by a 2-fold increased Na+/K+-ATPase protein expression and 2-fold elevated Na+/K+-ATPase activity in the 12 month incubated fish (0.6 kPa P-CO2), no such elevation in Na+/K+-ATPase activity could be observed in the group treated with 0.3 kPa P-CO2. Owing to the relevance of Na+/K+-ATPase as a general indicator for ion regulatory capacity, these results point at an adjustment of enzymatic activity to cope with the CO2 induced acid-base load at 0.6 kPa P-CO2 while under milder hypercapnic conditions the 'standard' Na+/K+-ATPase capacity might still be sufficient to maintain acid-base status. (c) 2009 Elsevier B.V. All rights reserved.

Description: Anthropogenic CO2 emissions will lead to an increased average ocean pCO2 of potentially 1900 ppm (ca. 0.2 kPa) by the year 2300 (Caldeira and Wickett 2003). Associated shifts in carbonate system speciation will cause ocean pH to fall by a maximum of 0.5–0.8 units. Most existing studies suggest that organisms/taxa with high standard metabolic rates, highly sophisticated convection systems and efficient gas exchange organs are coping best with elevated ocean pCO2. Typically, these taxa (decapod crustaceans, and cephalopods) are able to rapidly compensate extracellular pH by accumulating large amounts of bicarbonate. The underlying molecular machinery for this accumulatory response is still largely unknown for most marine invertebrate taxa. Thus, we will briefly review (a) acid-base regulatory responses in a range of marine invertebrates (bivalves, echinoderms, crustacea, and cephalopoda) and then (b) highlight and discuss their main ion-regulatory organs and potential acid-base regulatory proteins. In addition, we will present results from ongoing gene expression studies that specifically target transcripts relevant for ion- and acid-base regulation in the gill of the cephalopod Sepia officinalis, the crustacean Carcinus maenas and pluteus larvae of the sea urchin Strongylocentrotus purpuratus in response to environmental hypercapnia.

Description: Experimental ocean acidification leads to a shift in resource allocation and to an increased [HCO3-] within the perivisceral coelomic fluid (PCF) in the Baltic green sea urchin Strongylocentrotus droebachiensis. We investigated putative mechanisms of this pH compensation reaction by evaluating epithelial barrier function and the magnitude of skeleton (stereom) dissolution. In addition, we measured ossicle growth and skeletal stability. Ussing chamber measurements revealed that the intestine formed a barrier for HCO3- and was selective for cation diffusion. In contrast, the peritoneal epithelium was leaky and only formed a barrier for macromolecules. The ossicles of 6 week high CO2-acclimatised sea urchins revealed minor carbonate dissolution, reduced growth but unchanged stability. On the other hand, spines dissolved more severely and were more fragile following acclimatisation to high CO2. Our results indicate that epithelia lining the PCF space contribute to its acid–base regulation. The intestine prevents HCO3- diffusion and thus buffer leakage. In contrast, the leaky peritoneal epithelium allows buffer generation via carbonate dissolution from the surrounding skeletal ossicles. Long-term extracellular acid–base balance must be mediated by active processes, as sea urchins can maintain relatively high extracellular [HCO3-]. The intestinal epithelia are good candidate tissues for this active net import of HCO3- into the PCF. Spines appear to be more vulnerable to ocean acidification which might significantly impact resistance to predation pressure and thus influence fitness of this keystone species.

Description: Ocean acidification and associated changes in seawater carbonate chemistry negatively influence calcification processes and depress metabolism in many calcifying marine invertebrates. We present data on the cephalopod mollusc Sepia officinalis, an invertebrate that is capable of not only maintaining calcification, but also growth rates and metabolism when exposed to elevated partial pressures of carbon dioxide (pCO2). During a 6 wk period, juvenile S. officinalis maintained calcification under ~4000 and ~6000 ppm CO2, and grew at the same rate with the same gross growth efficiency as did control animals. They gained approximately 4% body mass daily and increased the mass of their calcified cuttlebone by over 500%. We conclude that active cephalopods possess a certain level of pre-adaptation to long-term increments in carbon dioxide levels. Our general understanding of the mechanistic processes that limit calcification must improve before we can begin to predict what effects future ocean acidification will have on calcifying marine invertebrates.