Combined metabolome and proteome analysis of the mantle tissue from Pacific oyster Crassostrea gigas exposed to elevated pCO2

Ocean acidification (OA) has been found to affect an array of normal physiological processes in mollusks, especially posing a significant threat to the fabrication process of mollusk shell. In the current study, the impact of exposure to elevated pCO2 condition was investigated in mantle tissue of Crassostrea gigas by an integrated metabolomic and proteomic approach. Analysis of metabolome and proteome revealed that elevated pCO2 could affect energy metabolism in oyster C. gigas, marked by differentially altered ATP, succinate, MDH, PEPCK and ALDH levels. Moreover, the up-regulated calponin-2, tropomyosins and myosin light chains indicated that elevated pCO2 probably caused disturbances in cytoskeleton structure in mantle tissue of oyster C. gigas. This work demonstrated that a combination of proteomics and metabolomics could provide important insights into the effects of OA at molecular levels.

Benthic and substrate cover data derived from a time series of photo-transect surveys for the Eastern Banks, Moreton Bay Australia, 2004-2015

This paper describes seagrass species and percentage cover point-based field data sets derived from georeferenced photo transects. Annually or biannually over a ten year period (2004-2015) data sets were collected using 30-50 transects, 500-800 m in length distributed across a 142 km**2 shallow, clear water seagrass habitat, the Eastern Banks, Moreton Bay, Australia. Each of the eight data sets include seagrass property information derived from approximately 3000 georeferenced, downward looking photographs captured at 2-4 m intervals along the transects. Photographs were manually interpreted to estimate seagrass species composition and percentage cover (Coral Point Count excel; CPCe). Understanding seagrass biology, ecology and dynamics for scientific and management purposes requires point-based data on species composition and cover. This data set, and the methods used to derive it are a globally unique example for seagrass ecological applications. It provides the basis for multiple further studies at this site, regional to global comparative studies, and, for the design of similar monitoring programs elsewhere.

Abundance and biomass of macrobenthos in the western part of the Black Sea during Mare Nigrum cruise S-RO2

Dataset containing macrobenthos data for samples collected during September 2008 in the North-West Black Sea (between 44°46' - 43°45' N latitude and 30° 11' - 29°35' E longitude). Macrobenthos sampling was done in 4 stations using a 0.14 m**2 Van Veen grab. Washing of the sample through two sieves - 1 mm and 0.25 mm mesh size; the material retained by the two sieves was examined at the binocular microscope; all animals were extracted, using fine tweezers and the species or group of species were identified and counted (in order to determine the density of populations); the larger organisms were measured and weighed (structure and biomass); for smaller organisms, the average wet weights inscribed in standard tables were used to calculate the biomass. Taxonomic identification was done at the GeoEcoMar by A. Teaca and T. Begun using the relevant taxonomic literature ( "Key-book for the identification of the Black Sea and Sea of Azov Fauna, 1968 -1972, Kiev - in Russian, V 1-4; BACESCU, M.C., MÜLLER, G. I., GOMOIU, M.-T., 1971). BACESCU, M.C., MÜLLER, G. I., GOMOIU, M.-T., 1971-Benthic ecological research to Black Sea. Comparative quantitative and qualitative analyse of pontic benthic fauna. Marine Ecology, 4, 1-357 (in Romanian). Key-book for the identification of the Black Sea and Sea of Azov Fauna, 1968 -1972, Kiev, V. 1-4 (in Russian).

Impact of long-term moderate hypercapnia and elevated temperature on the energy budget of isolated gills and branchial in vivo and in vitro enzyme capacities of Atlantic cod (Gadus morhua)

Effects of severe hypercapnia have been extensively studied in marine fishes, while knowledge on the impacts of moderately elevated CO2 levels and their combination with warming is scarce. Here we investigate ion regulation mechanisms and energy budget in gills from Atlantic cod acclimated long-term to elevated PCO2 levels (2500 µatm) and temperature (18 °C). Isolated perfused gill preparations established to determine gill thermal plasticity during acute exposures (10-22 °C) and in vivo costs of Na+/K+-ATPase activity, protein and RNA synthesis. Maximum enzyme capacities of F1Fo-ATPase, H+-ATPase and Na+/K+-ATPase were measured in vitro in crude gill homogenates. After whole animal acclimation to elevated PCO2 and/or warming, branchial oxygen consumption responded more strongly to acute temperature change. The fractions of gill respiration allocated to protein and RNA synthesis remained unchanged. In gills of fish CO2-exposed at both temperatures, energy turnover associated with Na+/K+-ATPase activity was reduced by 30% below rates of control fish. This contrasted in vitro capacities of Na+/K+-ATPase, which remained unchanged under elevated CO2 at 10 °C, and earlier studies which had found a strong upregulation under severe hypercapnia. F1Fo-ATPase capacities increased in hypercapnic gills at both temperatures, whereas Na+/K+ATPase and H+-ATPase capacities only increased in response to elevated CO2 and warming indicating the absence of thermal compensation under CO2. We conclude that in vivo ion regulatory energy demand is lowered under moderately elevated CO2 levels despite the stronger thermal response of total gill respiration and the upregulation of F1Fo-ATPase. This effect is maintained at elevated temperature.

Seawater acidification affects the physiological energetics and spawning capacity of the Manila clam Ruditapes philippinarum during gonadal maturation

Ocean acidification is predicted to have widespread implications for marine bivalve mollusks. While our understanding of its impact on their physiological and behavioral responses is increasing, little is known about their reproductive responses under future scenarios of anthropogenic climate change. In this study, we examined the physiological energetics of the Manila clam Ruditapes philippinarum exposed to CO2-induced seawater acidification during gonadal maturation. Three recirculating systems filled with 600 L of seawater were manipulated to three pH levels (8.0, 7.7, and 7.4) corresponding to control and projected pH levels for 2100 and 2300. In each system, temperature was gradually increased ca. 0.3 °C per day from 10 to 20 °C for 30 days and maintained at 20 °C for the following 40 days. Irrespective of seawater pH levels, clearance rate (CR), respiration rate (RR), ammonia excretion rate (ER), and scope for growth (SFG) increased after a 30-day stepwise warming protocol. When seawater pH was reduced, CR, ratio of oxygen to nitrogen, and SFG significantly decreased concurrently, whereas ammonia ER increased. RR was virtually unaffected under acidified conditions. Neither temperature nor acidification showed a significant effect on food absorption efficiency. Our findings indicate that energy is allocated away from reproduction under reduced seawater pH, potentially resulting in an impaired or suppressed reproductive function. This interpretation is based on the fact that spawning was induced in only 56% of the clams grown at pH 7.4. Seawater acidification can therefore potentially impair the physiological energetics and spawning capacity of R. philippinarum.

Data compilation on the biological response to ocean acidification: environmental and experimental context of data sets and related literature

The exponential growth of studies on the biological response to ocean acidification over the last few decades has generated a large amount of data. To facilitate data comparison, a data compilation hosted at the data publisher PANGAEA was initiated in 2008 and is updated on a regular basis (doi:10.1594/PANGAEA.149999). By January 2015, a total of 581 data sets (over 4 000 000 data points) from 539 papers had been archived. Here we present the developments of this data compilation five years since its first description by Nisumaa et al. (2010). Most of study sites from which data archived are still in the Northern Hemisphere and the number of archived data from studies from the Southern Hemisphere and polar oceans are still relatively low. Data from 60 studies that investigated the response of a mix of organisms or natural communities were all added after 2010, indicating a welcomed shift from the study of individual organisms to communities and ecosystems. The initial imbalance of considerably more data archived on calcification and primary production than on other processes has improved. There is also a clear tendency towards more data archived from multifactorial studies after 2010. For easier and more effective access to ocean acidification data, the ocean acidification community is strongly encouraged to contribute to the data archiving effort, and help develop standard vocabularies describing the variables and define best practices for archiving ocean acidification data.

Experiment: Elevated temperature and PCO2 affect enzyme activities in differentially oxidative tissues of Notothenia rossii

Mitochondrial plasticity plays a central role in setting the capacity for acclimation of aerobic metabolism in ectotherms in response to environmental changes. We still lack a clear picture if and to what extent the energy metabolism and mitochondrial enzymes of Antarctic fish can compensate for changing temperatures or PCO2 and whether capacities for compensation differ between tissues. We therefore measured activities of key mitochondrial enzymes (citrate synthase (CS), cytochrome c oxidase (COX)) from heart, red muscle, white muscle and liver in the Antarctic fish Notothenia rossii after warm- (7 °C) and hypercapnia- (0.2 kPa CO2) acclimation vs. control conditions (1 °C, 0.04 kPa CO2). In heart, enzymes showed elevated activities after cold-hypercapnia acclimation, and a warm-acclimation-induced upward shift in thermal optima. The strongest increase in enzyme activities in response to hypercapnia occurred in red muscle. In white muscle, enzyme activities were temperature-compensated. CS activity in liver decreased after warm-normocapnia acclimation (temperature-compensation), while COX activities were lower after cold- and warm-hypercapnia exposure, but increased after warm-normocapnia acclimation. In conclusion, warm-acclimated N. rossii display low thermal compensation in response to rising energy demand in highly aerobic tissues, such as heart and red muscle. Chronic environmental hypercapnia elicits increased enzyme activities in these tissues, possibly to compensate for an elevated energy demand for acid-base regulation or a compromised mitochondrial metabolism, that is predicted to occur in response to hypercapnia exposure. This might be supported by enhanced metabolisation of liver energy stores. These patterns reflect a limited capacity of N. rossii to reorganise energy metabolism in response to rising temperature and PCO2.

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.

Differential impacts of elevated CO2 and acidosis on the energy budget of gill and liver cells from Atlantic cod

Ocean acidification impacts fish and other marine species through increased seawater PCO2 levels (hypercapnia). Knowledge of the physiological mechanisms mediating effects in various tissues of fish is incomplete. Here we tested the effects of extracellular hypercapnia and acidosis on energy metabolism of gill and liver cells of Atlantic cod. Exposure media mimicked blood conditions in vivo, either during normo- or hypercapnia and at control or acidic extracellular pH (pHe). We determined metabolic rate and energy expenditure for protein biosynthesis, Na+/K+-ATPase and H+-ATPase and considered nutrition status by measurements of metabolic rate and protein biosynthesis in media with and without free amino acids (FAA). Addition of FAA stimulated hepatic but not branchial oxygen consumption. Normo- and hypercapnic acidosis as well as hypercapnia at control pHe depressed metabolic stimulation of hepatocytes. In gill cells, acidosis depressed respiration independent of PCO2 and FAA levels. For both cell types, depressed respiration was not correlated with the same reduction in energy allocated to protein biosynthesis or Na+/K+-ATPase. Hepatic energy expenditure for protein synthesis and Na+/K+- ATPase was even elevated at acidic compared to control pHe suggesting increased costs for ion regulation and cel- lular reorganization. Hypercapnia at control pHe strongly reduced oxygen demand of branchial Na+/K+-ATPase with a similar trend for H+-ATPase. We conclude that extracellular acidosis triggers metabolic depression in gill and metabolically stimulated liver cells. Additionally, hypercapnia itself seems to limit capacities for metabolic usage of amino acids in liver cells while it decreases the use and costs of ion regulatory ATPases in gill cells.

Seawater carbonate chemistry, sample density and Strongylocentrotus purpuratus size, filtering and respiration rate during experiments, 2011

Anthropogenic CO2 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 pCO2 (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 pCO2, while body length corrected feeding rates did not differ between larvae from both treatments. Calculating scope for growth illustrates that larvae raised under high pCO2 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.