CO2 induced seawater acidification impacts sea urchin larval development II: gene expression patterns in pluteus larvae.

Extensive use of fossil fuels is leading to increasing CO(2) concentrations in the atmosphere and causes changes in the carbonate chemistry of the oceans which represents a major sink for anthropogenic CO(2). As a result, the oceans' surface pH is expected to decrease by ca. 0.4 units by the year 2100, a major change with potentially negative consequences for some marine species. Because of their carbonate skeleton, sea urchins and their larval stages are regarded as likely to be one of the more sensitive taxa. In order to investigate sensitivity of pre-feeding (2 days post-fertilization) and feeding (4 and 7 days post-fertilization) pluteus larvae, we raised Strongylocentrotus purpuratus embryos in control (pH 8.1 and pCO(2) 41 Pa e.g. 399 µatm) and CO(2) acidified seawater with pH of 7.7 (pCO(2) 134 Pa e.g. 1318 µatm) and investigated growth, calcification and survival. At three time points (day 2, day 4 and day 7 post-fertilization), we measured the expression of 26 representative genes important for metabolism, calcification and ion regulation using RT-qPCR. After one week of development, we observed a significant difference in growth. Maximum differences in size were detected at day 4 (ca. 10% reduction in body length). A comparison of gene expression patterns using PCA and ANOSIM clearly distinguished between the different age groups (two-way ANOSIM: Global R=1) while acidification effects were less pronounced (Global R=0.518). Significant differences in gene expression patterns (ANOSIM R=0.938, SIMPER: 4.3% difference) were also detected at day 4 leading to the hypothesis that differences between CO(2) treatments could reflect patterns of expression seen in control experiments of a younger larva and thus a developmental artifact rather than a direct CO(2) effect. We found an up regulation of metabolic genes (between 10%and 20% in ATP-synthase, citrate synthase, pyruvate kinase and thiolase at day 4) and down regulation of calcification related genes (between 23% and 36% in msp130, SM30B, and SM50 at day 4). Ion regulation was mainly impacted by up regulation of Na(+)/K(+)-ATPase at day 4 (15%) and down regulation of NHE3 at day 4 (45%). We conclude that in studies in which a stressor induces an alteration in the speed of development, it is crucial to employ experimental designs with a high time resolution in order to correct for developmental artifacts. This helps prevent misinterpretation of stressor effects on organism physiology.

CO2 induced seawater acidification impacts sea urchin larval development I: elevated metabolic rates decrease scope for growth and induce developmental delay.

Anthropogenic CO(2) emissions are acidifying the world's oceans. A growing body of evidence is showing that ocean acidification impacts growth and developmental rates of marine invertebrates. Here we test the impact of elevated seawater pCO(2) (129 Pa, 1271 µatm) on early development, larval metabolic and feeding rates in a marine model organism, the sea urchin Strongylocentrotus purpuratus. Growth and development was assessed by measuring total body length, body rod length, postoral rod length and posterolateral rod length. Comparing these parameters between treatments suggests that larvae suffer from a developmental delay (by ca. 8%) rather than from the previously postulated reductions in size at comparable developmental stages. Further, we found maximum increases in respiration rates of +100% under elevated pCO(2), while body length corrected feeding rates did not differ between larvae from both treatments. Calculating scope for growth illustrates that larvae raised under high pCO(2) spent an average of 39 to 45% of the available energy for somatic growth, while control larvae could allocate between 78 and 80% of the available energy into growth processes. Our results highlight the importance of defining a standard frame of reference when comparing a given parameter between treatments, as observed differences can be easily due to comparison of different larval ages with their specific set of biological characters.

Trans-life cycle acclimation to experimental ocean acidification affects gastric pH homeostasis and larval recruitment in the sea star Asterias rubens.

AIM: Experimental simulation of near-future ocean acidification (OA) has been demonstrated to affect growth and development of echinoderm larval stages through energy allocation towards ion and pH compensatory processes. To date, it remains largely unknown how major pH regulatory systems and their energetics are affected by trans-generational exposure to near-future acidification levels. METHODS: Here, we used the common sea star Asterias rubens in a reciprocal transplant experiment comprising different combinations of OA scenarios, to study trans-generational plasticity using morphological and physiological endpoints. RESULTS: Acclimation of adults to pHT 7.2 (pCO2 3500 µatm) led to reductions in feeding rates, gonad weight and fecundity. No effects were evident at moderate acidification levels (pHT 7.4; pCO2 2000 µatm). Parental pre-acclimation to pHT 7.2 for 85 days reduced developmental rates even when larvae were raised under moderate and high pH conditions, whereas pre-acclimation to pHT 7.4 did not alter offspring performance. Microelectrode measurements and pharmacological inhibitor studies carried out on larval stages demonstrated that maintenance of alkaline gastric pH represents a substantial energy sink under acidified conditions that may contribute up to 30% to the total energy budget. CONCLUSION: Parental pre-acclimation to acidification levels that are beyond the pH that is encountered by this population in its natural habitat (eg, pHT 7.2) negatively affected larval size and development, potentially through reduced energy transfer. Maintenance of alkaline gastric pH and reductions in maternal energy reserves probably constitute the main factors for a reduced juvenile recruitment of this marine keystone species under simulated OA.

Intra-population variability of ocean acidification impacts on the physiology of Baltic blue mussels (Mytilus edulis): integrating tissue and organism response.

Increased maintenance costs at cellular, and consequently organism level, are thought to be involved in shaping the sensitivity of marine calcifiers to ocean acidification (OA). Yet, knowledge of the capacity of marine calcifiers to undergo metabolic adaptation is sparse. In Kiel Fjord, blue mussels thrive despite periodically high seawater PCO2, making this population interesting for studying metabolic adaptation under OA. Consequently, we conducted a multi-generation experiment and compared physiological responses of F1 mussels from 'tolerant' and 'sensitive' families exposed to OA for 1 year. Family classifications were based on larval survival; tolerant families settled at all PCO2 levels (700, 1120, 2400 µatm) while sensitive families did not settle at the highest PCO2 (≥99.8% mortality). We found similar filtration rates between family types at the control and intermediate PCO2 level. However, at 2400 µatm, filtration and metabolic scope of gill tissue decreased in tolerant families, indicating functional limitations at the tissue level. Routine metabolic rates (RMR) and summed tissue respiration (gill and outer mantle tissue) of tolerant families were increased at intermediate PCO2, indicating elevated cellular homeostatic costs in various tissues. By contrast, OA did not affect tissue and routine metabolism of sensitive families. However, tolerant mussels were characterised by lower RMR at control PCO2 than sensitive families, which had variable RMR. This might provide the energetic scope to cover increased energetic demands under OA, highlighting the importance of analysing intra-population variability. The mechanisms shaping such difference in RMR and scope, and thus species' adaptation potential, remain to be identified.

Calcium mobilisation following shell damage in the Pacific oyster, Crassostrea gigas.

Shell growth of oysters requires calcium uptake from the environment and transport to the area of shell formation. A shell regeneration assay in combination with radiolabelled calcium was used to investigate uptake and distribution of calcium to different tissues and hemolymph fractions in Pacific oysters, Crassostrea gigas (Bivalvia, Ostreoida). Oysters were notched at the shell margin and subsequently sampled for hemolymph and grading of shell regeneration during a two week experimental period. Half of the oysters were additionally exposed to (45)Ca and sampled for hemolymph and tissues. Total plasma calcium concentrations increased in notched oysters compared to controls on 1, 2 and 7days after notching. A decrease in plasma calcium levels was apparent on day 4, for both total and ionic calcium. The shell regeneration assay in the notched oysters resulted in a visible deposition of CaCO3 onto the regenerate from day 7 onwards. This was coinciding with an increased uptake of total calcium on days 11 and 14 as well as free, i.e. ionic and ligand-bound calcium, on day 14. At day 1, notching also increased calcium uptake into the mantle tissues, in areas above the notch and near the hinge. During the experiment, both the total hemocyte count and the number of granulocytes increased in notched compared to control oysters. The present study suggests that induced shell damage results in a dynamic regulation of the calcium uptake from the environment and the distribution of calcium within the body, starting directly after notching. Increases in both total calcium concentrations and uptake rates coincided with the visible depositions of CaCO3 on the regenerate shell. C. gigas was found to transport calcium mainly in the ionic form in the hemolymph, with only minor parts being bound to proteins or smaller ligands. Hemolymph measurement also revealed that C. gigas is able to regulate the extracellular concentrations of calcium and potassium. The changes in plasma calcium concentrations and speciation, concomitant with increases in granulocytes indicate that multiple calcium transport processes are activated after induced shell damage.

Resource allocation and extracellular acid-base status in the sea urchin Strongylocentrotus droebachiensis in response to CO2 induced seawater acidification.

Anthropogenic CO(2) emission will lead to an increase in seawater pCO(2) of up to 80-100 Pa (800-1000 µatm) within this century and to an acidification of the oceans. Green sea urchins (Strongylocentrotus droebachiensis) occurring in Kattegat experience seasonal hypercapnic and hypoxic conditions already today. Thus, anthropogenic CO(2) emissions will add up to existing values and will lead to even higher pCO(2) values >200 Pa (>2000 µatm). To estimate the green sea urchins' potential to acclimate to acidified seawater, we calculated an energy budget and determined the extracellular acid base status of adult S. droebachiensis exposed to moderately (102-145 Pa, 1007-1431 µatm) and highly (284-385 Pa, 2800-3800 µatm) elevated seawater pCO(2) for 10 and 45 days. A 45-day exposure to elevated pCO(2) resulted in a shift in energy budgets, leading to reduced somatic and reproductive growth. Metabolic rates were not significantly affected, but ammonium excretion increased in response to elevated pCO(2). This led to decreased O:N ratios. These findings suggest that protein metabolism is possibly enhanced under elevated pCO(2) in order to support ion homeostasis by increasing net acid extrusion. The perivisceral coelomic fluid acid-base status revealed that S. droebachiensis is able to fully (intermediate pCO(2)) or partially (high pCO(2)) compensate extracellular pH (pH(e)) changes by accumulation of bicarbonate (maximum increases 2.5mM), albeit at a slower rate than typically observed in other taxa (10-day duration for full pH(e) compensation). At intermediate pCO(2), sea urchins were able to maintain fully compensated pH(e) for 45 days. Sea urchins from the higher pCO(2) treatment could be divided into two groups following medium-term acclimation: one group of experimental animals (29%) contained remnants of food in their digestive system and maintained partially compensated pH(e) (+2.3mM HCO(3)(-)), while the other group (71%) exhibited an empty digestive system and a severe metabolic acidosis (-0.5 pH units, -2.4mM HCO(3)(-)). There was no difference in mortality between the three pCO(2) treatments. The results of this study suggest that S. droebachiensis occurring in the Kattegat might be pre-adapted to hypercapnia due to natural variability in pCO(2) in its habitat. We show for the first time that some echinoderm species can actively compensate extracellular pH. Seawater pCO(2) values of >200 Pa, which will occur in the Kattegat within this century during seasonal hypoxic events, can possibly only be endured for a short time period of a few weeks. Increases in anthropogenic CO(2) emissions and leakages from potential sub-seabed CO(2) storage (CCS) sites thus impose a threat to the ecologically and economically important species S. droebachiensis.

Acid-base regulatory ability of the cephalopod (Sepia officinalis) in response to environmental hypercapnia.

Acidification of ocean surface waters by anthropogenic carbon dioxide (CO(2)) emissions is a currently developing scenario that warrants a broadening of research foci in the study of acid-base physiology. Recent studies working with environmentally relevant CO(2) levels, indicate that some echinoderms and molluscs reduce metabolic rates, soft tissue growth and calcification during hypercapnic exposure. In contrast to all prior invertebrate species studied so far, growth trials with the cuttlefish Sepia officinalis found no indication of reduced growth or calcification performance during long-term exposure to 0.6 kPa CO(2). It is hypothesized that the differing sensitivities to elevated seawater pCO(2) could be explained by taxa specific differences in acid-base regulatory capacity. In this study, we examined the acid-base regulatory ability of S. officinalis in vivo, using a specially modified cannulation technique as well as (31)P NMR spectroscopy. During acute exposure to 0.6 kPa CO(2), S. officinalis rapidly increased its blood [HCO(3)(-)] to 10.4 mM through active ion-transport processes, and partially compensated the hypercapnia induced respiratory acidosis. A minor decrease in intracellular pH (pH(i)) and stable intracellular phosphagen levels indicated efficient pH(i) regulation. We conclude that S. officinalis is not only an efficient acid-base regulator, but is also able to do so without disturbing metabolic equilibria in characteristic tissues or compromising aerobic capacities. The cuttlefish did not exhibit acute intolerance to hypercapnia that has been hypothesized for more active cephalopod species (squid). Even though blood pH (pHe) remained 0.18 pH units below control values, arterial O(2) saturation was not compromised in S. officinalis because of the comparatively lower pH sensitivity of oxygen binding to its blood pigment. This raises questions concerning the potentially broad range of sensitivity to changes in acid-base status amongst invertebrates, as well as to the underlying mechanistic origins. Further studies are needed to better characterize the connection between acid-base status and animal fitness in various marine species.