Seawater carbonate chemistry and elemental composition of purple sea urchin (Strongylocentrotus purpuratus) calcite in a laboratory experiment

Ocean acidification will likely have negative impacts on invertebrates producing skeletons composed of calcium carbonate. Skeletal solubility is partly controlled by the incorporation of "foreign" ions (e.g. magnesium) into the crystal lattice of these skeletal structures, a process that is sensitive to a variety of biological and environmental factors. Here we explore effects of life stage, oceanographic region of origin, and changes in the partial pressure of carbon dioxide in seawater (pCO2) on trace elemental composition in the purple sea urchin (Strongylocentrotus purpuratus). We show that, similar to other urchin taxa, adult purple sea urchins have the ability to precipitate skeleton composed of a range of biominerals spanning low- to high-Mg calcites. Mg / Ca and Sr / Ca ratios were substantially lower in adult spines compared to adult tests. On the other hand, trace elemental composition was invariant among adults collected from four oceanographically distinct regions spanning a range of carbonate chemistry conditions (Oregon, Northern California, Central California, and Southern California). Skeletons of newly settled juvenile urchins that originated from adults from the four regions exhibited intermediate Mg / Ca and Sr / Ca between adult spine and test endmembers, indicating that skeleton precipitated during early life stages is more soluble than adult spines and less soluble than adult tests. Mean skeletal Mg / Ca or Sr / Ca of juvenile skeleton did not vary with source region when larvae were reared under present-day, global-average seawater carbonate conditions (400 µatm; pHT = 8.02 ± 0.03 1 SD; Omega calcite = 3.3 ± 0.2 1 SD). However, when reared under elevated pCO2 (900 µatm; pHT = 7.73 ± 0.03; Omega calcite = 1.8 ± 0.1), skeletal Sr / Ca in juveniles exhibited increased variance across the four regions. Although larvae from the northern populations (Oregon, Northern California, Central California) did not exhibit differences in Mg or Sr incorporation under elevated pCO2 (Sr / Ca = 2.10 ± 0.06 mmol/mol; Mg / Ca = 67.4 ± 3.9 mmol/mol), juveniles of Southern California origin partitioned ~8% more Sr into their skeletons when exposed to higher pCO2 (Sr / Ca = 2.26 ± 0.08 vs. 2.09 ± 0.005 mmol/mol 1 SD). Together these results suggest that the diversity of carbonate minerologies present across different skeletal structures and life stages in purple sea urchins does not translate into an equivalent geochemical plasticity of response associated with geographic variation or temporal shifts in seawater properties. Rather, composition of S. purpuratus skeleton precipitated during both early and adult life history stages appears relatively robust to spatial gradients and predicted future changes in carbonate chemistry. An exception to this trend may arise during early life stages, where certain populations of purple sea urchins may alter skeletal mineral precipitation rates and composition beyond a given pCO2 threshold. This potential for geochemical plasticity during early development in contrast to adult stage geochemical resilience adds to the growing body of evidence that ocean acidification can have differing effects across organismal life stages.

Seawater carbonate chemistry and Strongylocentrotus purpuratus body length and gene expression pattern changes during experiments, 2011

Extensive use of fossil fuels is leading to increasing CO2 concentrations in the atmosphere and causes changes in the carbonate chemistry of the oceans which represents a major sink for anthropogenic CO2. 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 pCO2 41 Pa e.g. 399 µatm) and CO2 acidified seawater with pH of 7.7 (pCO2 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 CO2 treatments could reflect patterns of expression seen in control experiments of a younger larva and thus a developmental artifact rather than a direct CO2 effect. We found an up regulation of metabolic genes (between 10 to 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, 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.

Seawater carbonate chemistry and its effects on properties and sinking velocity of aggregates of the coccolithophore Emiliania huxleyi, 2010

Coccolithophores play an important role in organic matter export due to their production of the mineral calcite that can act as ballast. Recent studies indicated that calcification in coccolithophores may be affected by changes in seawater carbonate chemistry. We investigated the influence of CO2 on the aggregation and sinking behaviour of the coccolithophore Emiliania huxleyi (PML B92/11) during a laboratory experiment. The coccolithophores were grown under low (~180 µatm), medium (~380 µatm), and high (~750 µatm) CO2 conditions. Aggregation of the cells was promoted using roller tables. Size and settling velocity of aggregates were determined during the incubation using video image analysis. Our results indicate that aggregate properties are sensitive to changes in the degree of ballasting, as evoked by ocean acidification. Average sinking velocity was highest for low CO2 aggregates (~1292 m d-1) that also had the highest particulate inorganic to particulate organic carbon (PIC/POC) ratio. Lowest PIC/POC ratios and lowest sinking velocity (~366 m d-1) at comparable sizes were observed for aggregates of the high CO2 treatment. Aggregates of the high CO2 treatment showed a 4-fold lower excess density (~4.2*10**-4 g cm**-3) when compared to aggregates from the medium and low CO2 treatments (~1.7 g*10**-3 cm**-3). We also observed that more aggregates formed in the high CO2 treatment, and that those aggregates contained more bacteria than aggregates in the medium and low CO2 treatment. If applicable to the future ocean, our findings suggest that a CO2 induced reduction of the calcite content of aggregates could weaken the deep export of organic matter in the ocean, particularly in areas dominated by coccolithophores.

Seawater carbonate chemistry and clownfish Amphiprion percula size and otholith development during experiments, 2011

Calcification in many invertebrate species is predicted to decline due to ocean acidification. The potential effects of elevated CO2 and reduced carbonate saturation state on other species, such as fish, are less well understood. Fish otoliths (earbones) are composed of aragonite, and thus, might be susceptible to either the reduced availability of carbonate ions in seawater at low pH, or to changes in extracellular concentrations of bicarbonate and carbonate ions caused by acid-base regulation in fish exposed to high pCO2. We reared larvae of the clownfish Amphiprion percula from hatching to settlement at three pHNBS and pCO2 levels (control: ~pH 8.15 and 404 µatm CO2; intermediate: pH 7.8 and 1050 µatm CO2; extreme: pH 7.6 and 1721 µatm CO2) to test the possible effects of ocean acidification on otolith development. There was no effect of the intermediate treatment (pH 7.8 and 1050 µatm CO2) on otolith size, shape, symmetry between left and right otoliths, or otolith elemental chemistry, compared with controls. However, in the more extreme treatment (pH 7.6 and 1721 µatm CO2) otolith area and maximum length were larger than controls, although no other traits were significantly affected. Our results support the hypothesis that pH regulation in the otolith endolymph can lead to increased precipitation of CaCO3 in otoliths of larval fish exposed to elevated CO2, as proposed by an earlier study, however, our results also show that sensitivity varies considerably among species. Importantly, our results suggest that otolith development in clownfishes is robust to even the more pessimistic changes in ocean chemistry predicted to occur by 2100.

Effects of ocean acidification on the calcification of otoliths of larval Atlantic cod Gadus morhua

The growth and development of the aragonitic CaCO3 otoliths of teleost fish could be vulnerable to processes resulting from ocean acidification. The potential effects of an increase in atmospheric CO2 on the calcification of the otoliths were investigated by rearing Atlantic cod Gadus morhua L. larvae in 3 pCO2 concentrations-control (370 µatm), medium (1800 µatm) and high (4200 µatm)-from March to May 2010. Increased otolith growth was observed in 7 to 46 d post hatch (dph) cod larvae at elevated pCO2 concentrations. The sagittae and lapilli were usually largest in the high pCO2 treatment followed by the medium and control treatments. The greatest difference in mean otolith surface area (normalized to fish length) was for sagittae at 11 dph, with medium and high treatments being 46 and 43% larger than the control group, respectively. There was no significant pCO2 effect on the shape of the otoliths nor were there any trends in the fluctuating asymmetry, defined as the difference between the right and left sides, in relation to the increase in otolith growth from elevated pCO2.

Seawater carbonate chemistry and community metabolism during an experiment with a coral reef, 2003

The effect of elevated pCO2 on the metabolism of a coral reef community dominated by macroalgae has been investigated utilizing the large 2650 m3 coral reef mesocosm at the Biosphere-2 facility near Tucson, Arizona. The carbonate chemistry of the water was manipulated to simulate present-day and a doubled CO2 future condition. Each experiment consisted of a 1-2 month preconditioning period followed by a 7-9 day observational period. The pCO2 was 404 ± 63 ?atm during the present-day pCO2 experiment and 658 ± 59 ?atm during the elevated pCO2 experiment. Nutrient levels were low and typical of natural reefs waters (NO3? 0.5-0.9 ?M, NH4+ 0.4 ?M, PO43? 0.07-0.09 ?M). The temperature and salinity of the water were held constant at 26.5 ± 0.2°C and 34.4 ± 0.2 ppt. Photosynthetically available irradiance was 10 ± 2 during the present-day experiment and 7.4 ± 0.5 mol photons m?2 d?1 during the elevated pCO2 experiment. The primary producer biomass in the mesocosm was dominated by four species of macroalgae; Haptilon cubense, Amphiroa fragillisima, Gelidiopsis intricata and Chondria dasyphylla. Algal biomass was 10.4 mol C m?2 during the present-day and 8.7 mol C m?2 and during the elevated pCO2 experiments. As previously observed, the increase in pCO2 resulted in a decrease in calcification from 0.041 ± 0.007 to 0.006 ± 0.003 mol CaCO3 m?2 d?1. Net community production (NCP) and dark respiration did not change in response to elevated pCO2. Light respiration measured by a new radiocarbon isotope dilution method exceeded dark respiration by a factor of 1.2 ± 0.3 to 2.1 ± 0.4 on a daily basis and by 2.2 ± 0.6 to 3.9 ± 0.8 on an hourly basis. The 1.8-fold increase with increasing pCO2 indicates that the enhanced respiration in the light was not due to photorespiration. Gross production (GPP) computed as the sum of NCP plus daily respiration (light + dark) increased significantly (0.24 ± 0.03 vs. 0.32 ± 0.04 mol C m?2 d?1). However, the conventional calculation of GPP based on the assumption that respiration in the light proceeds at the same rate as the dark underestimated the true rate of GPP by 41-100% and completely missed the increased rate of carbon cycling due to elevated pCO2. We conclude that under natural, undisturbed, nutrient-limited conditions elevated CO2 depresses calcification, stimulates the rate of turnover of organic carbon, particularly in the light, but has no effect on net organic production. The hypothesis that an increase pCO2 would produce an increase in net production that would counterbalance the effect of decreasing saturation state on calcification is not supported by these data.

Seawater carbonate chemistry and shell length of Mediterranean pteropod Cavolinia inflexa larvae during experiments

Larvae of the Mediterranean pteropod Cavolinia inflexa were maintained at controlled pHT values of 8.1, 7.82 and 7.51, equivalent respectively to pCO2 levels of 380, 857 and 1713 µatm. At pHT 7.82 larvae exhibited malformations and lower shell growth, compared to the control condition. At pHT 7.51 the larvae did not make shells but were viable and showed a normal development. However, smaller shells or no shells will have both ecological (food web) and biogeochemical (export of carbon and carbonate) consequences. These results confirm that 1pteropods, as well as the species dependent upon them as a food resource, will be severely impacted by ocean acidification.

Time series of seawater carbonate chemistry calculated throughout incubation periods of Boreogadus saida and Gadus morhua during exposure to different CO2 and temperature conditions

Oceans are experiencing increasing acidification in parallel to a distinct warming trend in consequence of ongoing climate change. Rising seawater temperatures are mediating a northward shift in distribution of Atlantic cod (Gadus morhua), into the habitat of polar cod (Boreogadus saida), that is associated with retreating cold water masses. This study investigates the competitive strength of the co-occurring gadoids under ocean acidification and warming (OAW) scenarios. Therefore, we incubated specimens of both species in individual tanks for 4 months, under different control and projected temperatures (polar cod: 0, 3, 6, 8 °C, Atlantic cod: 3, 8, 12, 16 °C) and PCO2 conditions (390 and 1170 µatm) and monitored growth, feed consumption and standard metabolic rate. Our results revealed distinct temperature effects on both species. While hypercapnia by itself had no effect, combined drivers caused nonsignificant trends. The feed conversion efficiency of normocapnic polar cod was highest at 0 °C, while optimum growth performance was attained at 6 °C; the long-term upper thermal tolerance limit was reached at 8 °C. OAW caused only slight impairments in growth performance. Under normocapnic conditions, Atlantic cod consumed progressively increasing amounts of feed than individuals under hypercapnia despite maintaining similar growth rates during warming. The low feed conversion efficiency at 3 °C may relate to the lower thermal limit of Atlantic cod. In conclusion, Atlantic cod displayed increased performance in the warming Arctic such that the competitive strength of polar cod is expected to decrease under future OAW conditions.

Seawater carbonate chemistry and conditions of Mytilus edulis extracellular body fluids and shell composition in a pH-treatment experiment: Acid-base status, trace elements and delta11B, 2012

Mytilus edulis were cultured for 3 months under six different seawater pCO2 levels ranging from 380 to 4000 µatm. Specimen were taken from Kiel Fjord (Western Baltic Sea, Germany) which is a habitat with high and variable seawater pCO2 and related shifts in carbonate system speciation (e.g., low pH and low CaCO3 saturation state). Hemolymph (HL) and extrapallial fluid (EPF) samples were analyzed for pH and total dissolved inorganic carbon (CT) to calculate pCO2 and [HCO3]. A second experiment was conducted for 2 months with three different pCO2 levels (380, 1400 and 4000 µatm). Boron isotopes (delta11B) were investigated by LA-MC-ICP-MS (Laser Ablation-Multicollector-Inductively Coupled Plasma-Mass Spectrometry) in shell portions precipitated during experimental treatment time. Additionally, elemental ratios (B/Ca, Mg/Ca and Sr/Ca) in the EPF of specimen from the second experiment were measured via ICP-OES (Inductively Coupled Plasma-Optical Emission Spectrometry). Extracellular pH was not significantly different in HL and EPF but systematically lower than ambient water pH. This is due to high extracellular pCO2 values, a prerequisite for metabolic CO2 excretion. No accumulation of extracellular [HCO3] was measured. Elemental ratios (B/Ca, Mg/Ca and Sr/Ca) in the EPF increased slightly with pH which is in accordance with increasing growth and calcification rates at higher seawater pH values. Boron isotope ratios were highly variable between different individuals but also within single shells. This corresponds to a high individual variability in fluid B/Ca ratios and may be due to high boron concentrations in the organic parts of the shell. The mean delta11B value shows no trend with pH but appears to represent internal pH (EPF) rather than ambient water pH.

Seawater carbonate chemistry and calcification rate during mesocosm experiments with coral Montipora capitata, 2008

A long-term (10 months) controlled experiment was conducted to test the impact of increased partial pressure of carbon dioxide (pCO2) on common calcifying coral reef organisms. The experiment was conducted in replicate continuous flow coral reef mesocosms flushed with unfiltered sea water from Kaneohe Bay, Oahu, Hawaii. Mesocosms were located in full sunlight and experienced diurnal and seasonal fluctuations in temperature and sea water chemistry characteristic of the adjacent reef flat. Treatment mesocosms were manipulated to simulate an increase in pCO2 to levels expected in this century [midday pCO2 levels exceeding control mesocosms by 365 ± 130 µatm (mean ± sd)]. Acidification had a profound impact on the development and growth of crustose coralline algae (CCA) populations. During the experiment, CCA developed 25% cover in the control mesocosms and only 4% in the acidified mesocosms, representing an 86% relative reduction. Free-living associations of CCA known as rhodoliths living in the control mesocosms grew at a rate of 0.6 g buoyant weight per year while those in the acidified experimental treatment decreased in weight at a rate of 0.9 g buoyant weight per year, representing a 250% difference. CCA play an important role in the growth and stabilization of carbonate reefs, so future changes of this magnitude could greatly impact coral reefs throughout the world. Coral calcification decreased between 15% and 20% under acidified conditions. Linear extension decreased by 14% under acidified conditions in one experiment. Larvae of the coral Pocillopora damicornis were able to recruit under the acidified conditions. In addition, there was no significant difference in production of gametes by the coral Montipora capitata after 6 months of exposure to the treatments.

Seawater carbonate chemistry and biological processes of Emiliania huxleyi (PML B92/11) during experiments, 2011

The present study investigates the combined effect of phosphorous limitation, elevated partial pressure of CO2 (pCO2) and temperature on a calcifying strain of Emiliania huxleyi (PML B92/11) by means of a fully controlled continuous culture facility. Two levels of phosphorous limitation were consecutively applied by renewal of culture media (N:P = 26) at dilution rates (D) of 0.3 d- and 0.1 d-1. CO2 and temperature conditions were 300, 550 and 900 µatm pCO2 at 14 °C and 900 µatm pCO2 at 18 °C. In general, the steady state cell density and particulate organic carbon (POC) production increased with pCO2, yielding significantly higher concentrations in cultures grown at 900 µatm pCO2 compared to 300 and 550 µatm pCO2. At 900 µatm pCO2, elevation of temperature as expected for a greenhouse ocean, further increased cell densities and POC concentrations. In contrast to POC concentration, C-quotas (pmol C cell-1) were similar at D = 0.3 d-1 in all cultures. At D = 0.1 d-1, a reduction of C-quotas by up to 15% was observed in the 900 µatm pCO2 at 18 °C culture. As a result of growth rate reduction, POC:PON:POP ratios deviated strongly from the Redfield ratio, primarily due to an increase in POC. Ratios of particulate inorganic and organic carbon (PIC:POC) ranged from 0.14 to 0.18 at D = 0.3 d-1, and from 0.11 to 0.17 at D = 0.1 d-1, with variations primarily induced by the changes in POC. At D = 0.1 d-1, cell volume was reduced by up to 22% in cultures grown at 900 µatm pCO2. Our results indicate that changes in pCO2, temperature and phosphorus supply affect cell density, POC concentration and size of E. huxleyi (PML B92/11) to varying degrees, and will likely impact bloom development as well as biogeochemical cycling in a greenhouse ocean.

Egg and early larval stages of Baltic cod, Gadus morhua duirng ocean acidification experiments, 2012

The accumulation of carbon dioxide in the atmosphere will lower the pH in ocean waters, a process termed ocean acidification (OA). Despite its potentially detrimental effects on calcifying organisms, experimental studies on the possible impacts on fish remain scarce. While adults will most likely remain relatively unaffected by changes in seawater pH, early life-history stages are potentially more sensitive, due to the lack of gills with specialized ion-regulatory mechanisms. We tested the effects of OA on growth and development of embryos and larvae of eastern Baltic cod, the commercially most important fish stock in the Baltic Sea. Cod were reared from newly fertilized eggs to early non-feeding larvae in 5 different experiments looking at a range of response variables to OA, as well as the combined effect of CO2 and temperature. No effect on hatching, survival, development, and otolith size was found at any stage in the development of Baltic cod. Field data show that in the Bornholm Basin, the main spawning site of eastern Baltic cod, in situ levels of pCO2are already at levels of 1,100 µatm with a pH of 7.2, mainly due to high eutrophication supporting microbial activity and permanent stratification with little water exchange. Our data show that the eggs and early larval stages of Baltic cod seem to be robust to even high levels of OA (3,200 µatm), indicating an adaptational response to CO2.

Interactive effects of ocean acidification and neighboring corals on the growth of Pocillopora verrucosa

The physical and chemical environment around corals, as well as their physiology, can be affected by interactions with neighboring corals. This study employed small colonies (4 cm diameter) of Pocillopora verrucosa and Acropora hyacinthus configured in spatial arrays at 7 cm/s flow speed to test the hypothesis that ocean acidification (OA) alters interactions among them. Interaction effects were quantified for P. verrucosa using three measures of growth: calcification (i.e., weight), horizontal growth, and vertical growth. The study was carried out in May-June 2014 using corals from 10 m depth on the outer reef of Moorea, French Polynesia. Colonies of P. verrucosa were placed next to conspecifics or heterospecifics (A. hyacinthus) in arrangements of two or four colonies (pairs and aggregates) that were incubated at ambient and high pCO2 (1000 µatm) for 28 days. There was an effect of pCO2, and arrangement type on multivariate growth (utilizing the three measures of growth), but no interaction between the main effects. Conversely, arrangement and pCO2 had an interactive effect on calcification, with an overall 23 % depression at high pCO2 versus ambient pCO2 (i.e., pooled among arrangements). Within arrangements, there was a 34-45 % decrease in calcification for solitary and paired conspecifics, but no effect in conspecific aggregates, heterospecific pairs, or heterospecific aggregates. Horizontal growth was negatively affected by pCO2 and arrangement type, while vertical growth was positively affected by arrangement type. Together, our results show that conspecific aggregations can mitigate the negative effects of OA on calcification of colonies within an aggregation.

THE HERPESVIRUS NUCLEAR EGRESS COMPLEX COMPONENT, UL31, IS RECRUITED TO SITES OF DNA DAMAGE THROUGH POLY(ADP-RIBOSE) BINDING

The herpes simplex virus (HSV) UL31 gene encodes a conserved member of the herpesvirus nuclear egress complex that not only functions in the egress of DNA-containing capsids from the nucleus, but is also required for optimal viral genome expression, replication and packaging into capsids. Here, we report that the UL31 protein from HSV-2 and the orthologous protein, ORF69, from Kaposi's sarcoma-associated herpesvirus (KSHV) are recruited to sites of DNA damage. Recruitment of UL31 to sites of DNA damage occurred in HSV-2 infected cells, but did not require other viral proteins. The N-terminus of UL31 contains sequences resembling a poly(ADP-ribose) (PAR) binding motif. As protein poly-ADP ribosylation (PARylation) is a hallmark of the DNA damage response we examined the relationship between PARylation and UL31 recruitment to DNA damage. While the PAR polymerase (PARP)1/2 inhibitor, olaparib, prevented UL31 recruitment to damaged DNA, KU55933 inhibition of signaling through the ataxia telangiectasia mutated (ATM) DNA damage response pathway had no effect. These findings were further supported by experiments demonstrating direct and specific interaction between HSV-2 UL31 and PAR using purified components. Co-transfection with the viral kinase Us3, known to phosphorylate UL31, inhibited UL31 recruitment to DNA damage but also prevented the recruitment of other proteins recruited to DNA damage sites. The viral E3 ubiquitin ligase ICP0 was observed to co-localize with UL31 in transfected cells in a manner that is independent of the PAR-binding ability of UL31. However, inhibition of PARP1/2/3 did not reduce the ability of HSV-2 to replicate and we observed reduced PAR levels in the nuclei of infected cells. This study reveals a previously unrecognized function for UL31 orthologs and may suggest that the recognition of PAR by UL31 is coupled to the nuclear egress of herpesvirus capsids, influences viral DNA replication and packaging, or possibly modulates the DNA damage response mounted by virally infected cells.

Controller Time and Delay Costs - a Trade-off Analysis

Air traffic controller shortages remain a significant challenge in European ATM. Comparing different rules, we quantify the cost effectiveness of adding controller hours to Area Control Centre regulations to avert the delay cost impact on airlines. Typically, adding controller hours results in a net benefit. Distributions of delay duration and aircraft weight play an important role in determining the total cost of a regulation. Errors are likely to be incurred when analysing performance based on average delay values, particularly at the disaggregate level.