Seawater carbonate chemistry and biological processes during experiments with postlarvae of barnacle of Semibalanus balanoides and Elminius modestus, 2010

Ocean acidification and global warming are occurring concomitantly, yet few studies have investigated how organisms will respond to increases in both temperature and CO2. Intertidal microcosms were used to examine growth, shell mineralogy and survival of two intertidal barnacle post-larvae, Semibalanus balanoides and Elminius modestus, at two temperatures (14 and 19°C) and two CO2 concentrations (380 and 1,000 ppm), fed with a mixed diatom-flagellate diet at 15,000 cells ml-1 with flow rate of 10 ml-1 min-1. Control growth rates, using operculum diameter, were 14 ± 8 µm day-1 and 6 ± 2 µm day-1 for S. balanoides and E. modestus, respectively. Subtle, but significant decreases in E. modestus growth rate were observed in high CO2 but there were no impacts on shell calcium content and survival by either elevated temperature or CO2. S. balanoides exhibited no clear alterations in growth rate but did show a large reduction in shell calcium content and survival under elevated temperature and CO2. These results suggest that a decrease by 0.4 pH(NBS) units alone would not be sufficient to directly impact the survival of barnacles during the first month post-settlement. However, in conjunction with a 4-5°C increase in temperature, it appears that significant changes to the biology of these organisms will ensue.

Seawater carbonate chemistry and biological processes of Amphisorus hemprichii, Amphisorus kudakajimensis and Calcarina gaudichaudii during experiments, 2011

Ocean acidification, which like global warming is an outcome of anthropogenic CO2emissions, severely impacts marine calcifying organisms, especially those living in coral reef ecosystems. However, knowledge about the responses of reef calcifiers to ocean acidification is quite limited, although coral responses are known to be generally negative. In a culture experiment with two algal symbiont-bearing, reef-dwelling foraminifers, Amphisorus kudakajimensis and Calcarina gaudichaudii, in seawater under five different pCO2 conditions, 245, 375, 588, 763 and 907 µatm, maintained with a precise pCO2-controlling technique, net calcification of A. kudakajimensis was reduced under higher pCO2, whereas calcification of C. gaudichaudii generally increased with increased pCO2. In another culture experiment conducted in seawater in which bicarbonate ion concentrations were varied under a constant carbonate ion concentration, calcification was not significantly different between treatments in Amphisorus hemprichii, a species closely related to A. kudakajimensis, or in C. gaudichaudii. From these results, we concluded that carbonate ion and CO2 were the carbonate species that most affected growth ofAmphisorus and Calcarina, respectively. The opposite responses of these two foraminifer genera probably reflect different sensitivities to these carbonate species, which may be due to their different symbiotic algae.

Seawater carbonate chemistry and hydrogen ions and carbonate chemistry in calcifying fluid of coral Astrangia poculata during experiments, 2011

A generalized physicochemical model of the response of marine organisms' calcifying fluids to CO2-induced ocean acidification is proposed. The model is based upon the hypothesis that some marine calcifiers induce calcification by elevating pH, and thus Omega aragonite, of their calcifying fluid by removing protons (H+). The model is explored through two end-member scenarios: one in which a fixed number of H+ is removed from their calcifying fluid, regardless of atmospheric pCO2, and another in which a fixed external-internal proton ratio ([H+]E/[H+]I) is maintained. The model is able to generate the full range of calcification response patterns observed in prior ocean acidification experiments and is consistent with the assertion that organisms' calcification response to ocean acidification is more negative for marine calcifiers that exert weaker control over their calcifying fluid pH. The model is empirically evaluated for the temperate scleractinian coral Astrangia poculata with in situ pH microelectrode measurements of the coral's calcifying fluid under control and acidified conditions. These measurements reveal that (1) the pH of the coral's calcifying fluid is substantially elevated relative to its external seawater under both control and acidified conditions, (2) the coral's [H+]E/[H+]I remains constant under control and acidified conditions, and (3) the coral removes fewer H+ from its calcifying fluid under acidified conditions than under control conditions. Thus, the carbonate system dynamics of A. poculata's calcifying fluid appear to be most consistent with the fixed [H+]E/[H+]I end-member scenario. Similar microelectrode experiments performed on additional taxa are required to assess the model's general applicability.

Seawater carbonate chemistry and protein sports of barnicle Balanus amphitrite during experiments, 2011

The majority of benthic marine invertebrates have a complex life cycle, during which the pelagic larvae select a suitable substrate, attach to it, and then metamorphose into benthic adults. Anthropogenic ocean acidification (OA) is postulated to affect larval metamorphic success through an altered protein expression pattern (proteome structure) and post-translational modifications. To test this hypothesis, larvae of an economically and ecologically important barnacle species Balanus amphitrite, were cultured from nauplius to the cyprid stage in the present (control) and in the projected elevated concentrations of CO2 for the year 2100 (the OA treatment). Cyprid response to OA was analyzed at the total proteome level as well as two protein post-translational modification (phosphorylation and glycosylation) levels using a 2-DE based proteomic approach. The cyprid proteome showed OA-driven changes. Proteins that were differentially up or down regulated by OA come from three major groups, namely those related to energy-metabolism, respiration, and molecular chaperones, illustrating a potential strategy that the barnacle larvae may employ to tolerate OA stress. The differentially expressed proteins were tentatively identified as OA-responsive, effectively creating unique protein expression signatures for OA scenario of 2100. This study showed the promise of using a sentinel and non-model species to examine the impact of OA at the proteome level.

Seawater carbonate chemistry and impacts of calcium and carbonate ion concentration on Mg and Sr incorporation in cultured benthic foraminifera (Ammonia tepida and Heterostegina depressa), 2011

Laboratory culture experiments were conducted to determine effects of seawater carbonate ion concentration ([CO32-]), and thereby calcite saturation state, on Mg and Sr incorporation into calcite of two species of shallow-water benthic foraminifera: Ammonia tepida and Heterostegina depressa. Impact on Mg and Sr incorporation by increased seawater [CO32-] and thereby higher calcite saturation state, is absent in either species. Comparison to results from a similar culturing experiment, in which calcite saturation state was varied as a function of [Ca2+], reveals that saturation state affects incorporation of Mg and Sr through calcium- rather than carbonate availability. The similarity in response by both species is surprising since the average Mg/Ca ratio is ~ 70 times higher in H. depressa than in A. tepida. Furthermore, these results suggest that the ions involved in biomineralization (i.e. Ca2+ and DIC) are processed by separate cellular transport mechanisms. The similar response of Mg and Sr incorporation in this study suggests that only differences in the Ca2+ transport mechanism affect divalent cation partitioning.

Combined effects of solar UV radiation and CO2-induced seawater acidification on photosynthetic carbon fixation of phytoplankton assemblages in the South China Sea, 2010

We carried out short term pCO2/pH perturbation experiments in the coastal waters of the South China Sea to evaluate the combined effects of seawater acidification (low pH/high pCO2) and solar UV radiation (UVR, 280-400 nm) on photosynthetic carbon fixation of phytoplankton assemblages. Under photosynthetically active radiation (PAR) alone treatments, reduced pCO2 (190 ppmv) with increased pH resulted in a significant decrease in the photosynthetic carbon fixation rate (about 23%), while enriched pCO2 (700 ppmv) with lowered pH had no significant effect on the photosynthetic performance compared to the ambient level. The apparent photosynthetic efficiency decreased under the reduced pCO2 level, probably due to C-limitation as well as energy being diverged for up-regulation of carbon concentrating mechanisms (CCMs). In the presence of UVR, both UV-A and UV-B caused photosynthetic inhibition, though UV-A appeared to enhance the photosynthetic efficiency under lower PAR levels. UV-B caused less inhibition of photosynthesis under the reduced pCO2 level, probably because of its contribution to the inorganic carbon (Ci)-acquisition processes. Under the seawater acidification conditions (enriched pCO2), both UV-A and UV-B reduced the photosynthetic carbon fixation to higher extents compared to the ambient pCO2 conditions. We conclude that solar UV and seawater acidification could synergistically inhibit photosynthesis.

Seawater carbonate chemistry, nutrient uptake and biological processes of coral Stylophora pistillata during experiments, 2011

The effects of ocean acidification and elevated seawater temperature on coral calcification and photosynthesis have been extensively investigated over the last two decades, whereas they are still unknown on nutrient uptake, despite their importance for coral energetics. We therefore studied the separate and combined impacts of increases in temperature and pCO2 on phosphate, ammonium, and nitrate uptake rates by the scleractinian coral S. pistillata. Three experiments were performed, during 10 days i) at three pHT conditions (8.1, 7.8, and 7.5) and normal temperature (26°C), ii) at three temperature conditions (26°, 29°C, and 33°C) and normal pHT(8.1), and iii) at three pHT conditions (8.1, 7.8, and 7.5) and elevated temperature (33°C). After 10 days of incubation, corals had not bleached, as protein, chlorophyll, and zooxanthellae contents were the same in all treatments. However, photosynthetic rates significantly decreased at 33°C, and were further reduced for the pHT 7.5. The photosynthetic efficiency of PSII was only decreased by elevated temperature. Nutrient uptake rates were not affected by a change in pH alone. Conversely, elevated temperature (33°C) alone induced an increase in phosphate uptake but a severe decrease in nitrate and ammonium uptake rates, even leading to a release of nitrogen into seawater. Combination of high temperature (33°C) and low pHT(7.5) resulted in a significant decrease in phosphate and nitrate uptake rates compared to control corals (26°C, pHT = 8.1). These results indicate that both inorganic nitrogen and phosphorus metabolism may be negatively affected by the cumulative effects of ocean warming and acidification.

Seawater carbonate chemistry and biological parameters of Sepia officinalis during experiments, 2009

Low pO2 values have been measured in the perivitelline fluids (PVF) of marine animal eggs on several occasions, especially towards the end of development, when embryonic oxygen consumption is at its peak and the egg case acts as a massive barrier to diffusion. Several authors have therefore suggested that oxygen availability is the key factor leading to hatching. However, there have been no measurements of PVF pCO2 so far. This is surprising, as elevated pCO2 could also constitute a major abiotic stressor for the developing embryo. As a first attempt to fill this gap in knowledge, we measured pO2, pCO2 and pH in the PVF of late cephalopod (Sepia officinalis) eggs. We found linear relationships between embryo wet mass and pO2, pCO2 and pH. pO2 declined from >12 kPa to less than 5 kPa, while pCO2 increased from 0.13 to 0.41 kPa. In the absence of active accumulation of bicarbonate in the PVF, pH decreased from 7.7 to 7.2. Our study supports the idea that oxygen becomes limiting in cephalopod eggs towards the end of development; however, pCO2 and pH shift to levels that have caused significant physiological disturbances in other marine ectothermic animals. Future research needs to address the physiological adaptations that enable the embryo to cope with the adverse abiotic conditions in their egg environment.

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.

Impact of ocean acidification and warming on the larval development of the spider crab Hyas araneus from different latitudes (54° vs 79°N), 2010

The combined impacts of future scenarios of ocean acidification and global warming on the larvae of a cold-eurythermal spider crab, Hyas araneus L., were investigated in one of its southernmost populations (living around Helgoland, southern North Sea, 54°N) and one of the northernmost populations (Svalbard, North Atlantic, 79°N). Larvae were exposed at temperatures of 3, 9 and 15°C to present day normocapnia (380 ppm CO2) and to CO2 conditions expected for the near or medium-term future (710 ppm by 2100 and 3000 ppm CO2 by 2300 and beyond). Larval development time and biochemical composition were studied in the larval stages Zoea I, II, and Megalopa. Permanent differences in instar duration between both populations were detected in all stages, likely as a result of evolutionary temperature adaptation. With the exception of Zoea II at 3°C and under all CO2 conditions, development in all instars from Svalbard was delayed compared to those from Helgoland, under all conditions. Most prominently, development was much longer and fewer specimens morphosed to the first crab instar in the Megalopa from Svalbard than from Helgoland. Enhanced CO2 levels (710 and particularly 3000 ppm), caused extended duration of larval development and reduced larval growth (measured as dry mass) and fitness (decreasing C/N ratio, a proxy of the lipid content). Such effects were strongest in the zoeal stages in Svalbard larvae, and during the Megalopa instar in Helgoland larvae.

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 biological processes of sand dollars Dendraster excentricus during experiments, 2011

Reduction in global ocean pH due to the uptake of increased atmospheric CO2 is expected to negatively affect calcifying organisms, including the planktonic larval stages of many marine invertebrates. Planktonic larvae play crucial roles in the benthic-pelagic life cycle of marine organisms by connecting and sustaining existing populations and colonizing new habitats. Calcified larvae are typically denser than seawater and rely on swimming to navigate vertically structured water columns. Larval sand dollars Dendraster excentricus have calcified skeletal rods supporting their bodies, and propel themselves with ciliated bands looped around projections called arms. Ciliated bands are also used in food capture, and filtration rate is correlated with band length. As a result, swimming and feeding performance are highly sensitive to morphological changes. When reared at an elevated PCO2 level (1000 ppm), larval sand dollars developed significantly narrower bodies at four and six-arm stages. Morphological changes also varied between four observed maternal lineages, suggesting within-population variation in sensitivity to changes in PCO2 level. Despite these morphological changes, PCO2 concentration alone had no significant effect on swimming speeds. However, acidified larvae had significantly smaller larval stomachs and bodies, suggesting reduced feeding performance. Adjustments to larval morphologies in response to ocean acidification may prioritize swimming over feeding, implying that negative consequences of ocean acidification are carried over to later developmental stages.

Seawater carbonate chemistry and combined physiological effects of CO2 and light on the N2-fixing cyanobacterium Trichodesmium IMS101 during experiments, 2010

Recent studies on the diazotrophic cyanobacterium Trichodesmium erythraeum(IMS101) showed that increasing CO2 partial pressure (pCO2) enhances N2 fixation and growth. Significant uncertainties remain as to the degree of the sensitivity to pCO2, its modification by other environmental factors, and underlying processes causing these responses. To address these questions, we examined the responses ofTrichodesmium IMS101 grown under a matrix of low and high levels of pCO2 (150 and 900 µatm) and irradiance (50 and 200 µmol photons m-2 s-1). Growth rates as well as cellular carbon and nitrogen contents increased with increasing pCO2 and light levels in the cultures. The pCO2-dependent stimulation in organic carbon and nitrogen production was highest under low light. High pCO2 stimulated rates of N2fixation and prolonged the duration, while high light affected maximum rates only. Gross photosynthesis increased with light but did not change with pCO2. HCO3- was identified as the predominant carbon source taken up in all treatments. Inorganic carbon uptake increased with light, but only gross CO2 uptake was enhanced under high pCO2. A comparison between carbon fluxes in vivo and those derived from 13C fractionation indicates high internal carbon cycling, especially in the low-pCO2treatment under high light. Light-dependent oxygen uptake was only detected underlow pCO2 combined with high light or when low-light-acclimated cells were exposed to high light, indicating that the Mehler reaction functions also as a photoprotective mechanism in Trichodesmium. Our data confirm the pronounced pCO2 effect on N2fixation and growth in Trichodesmium and further show a strong modulation of these effects by light intensity. We attribute these responses to changes in the allocation of photosynthetic energy between carbon acquisition and the assimilation of carbon and nitrogen under elevated pCO2. These findings are supported by a complementarystudy looking at photosynthetic fluorescence parameters of photosystem II, photosynthetic unit stoichiometry (photosystem I:photosystem II), and pool sizes of key proteins in carbon and nitrogen acquisition.