Gene transcripts encoding hypoxia-inducible factor (HIF) exhibit tissue- and muscle fiber type-dependent responses to hypoxia and hypercapnic hypoxia in the Atlantic blue crab, Callinectes sapidus

Hypoxia inducible factor (HIF) is a transcription factor that under low environmental oxygen regulates the expression of suites of genes involved in metabolism, angiogenesis, erythropoiesis, immune function, and growth. Here, we isolated and sequenced partial cDNAs encoding hif-α and arnt/hif-β from the Atlantic blue crab, Callinectes sapidus, an estuarine species that frequently encounters concurrent hypoxia (low O2) and hypercapnia (elevated CO2). We then examined the effects of acute exposure (1 hr) to hypoxia (H) and hypercapnic hypoxia (HH) on relative transcript abundance for hif-α and arnt/hif-β in different tissues (glycolytic muscle, oxidative muscle, hepatopancreas, gill, and gonads) using quantitative real-time RT-PCR. Our results indicate that hif-α and arnt/hif-β mRNAs were constitutively present under well-aerated normoxia (N) conditions in all tissues examined. Further, H and HH exposure resulted in both tissue-specific and muscle fiber type-specific effects on relative hif-α transcript abundance. In the gill and glycolytic muscle, relative hif-α mRNA levels were significantly lower under H and HH, compared to N, while no change (or a slight increase) was detected in oxidative muscle, hepatopancreas and gonadal tissues. H and HH did not affect relative transcript abundance for arnt/hif-β in any tissue or muscle fiber type. Thus, in crustaceans the HIF response to H and HH appears to involve changes in hif transcript abundance, with variation in hif-α and arnt/hif-β transcriptional dynamics occurring in both a tissue- and muscle fiber type-dependent manner.

Biochemical, metabolic and morphological responses of the intertidal gastropod Littorina littorea to ocean acidification and increase temperature

Future changes to the pH and temperature of the oceans are predicted to impact the biodiversity of marine ecosystems, particularly those animals that rely on the process of calcification. The marine intertidal gastropod Littorina littorea can be used as a model of intertidal organism for investigating the effects of ocean acidification and high temperature, alone and in combination because its ability to be quickly adapt against environmental stressor. In the first study a single species population of L. littorea was used to test for physiological and biochemical effects underpinning organismal responses to climate change and ocean acidification. Compared with control conditions, snails decreased metabolic rates by 31% in response to elevated pCO2 while by 15% in response to combined pCO2 and temperature. Decreased metabolic rates were associated with metabolic depression, a strategy to match oxygen demand and availability, and an increase in end-product metabolites in the tissue under acidified treatments, indicating an increased reliance on anaerobic metabolism. This study also showed that anthropogenic alteration of CO2 and temperature may also lead to plastic responses, a fundamental mechanism of many marine gastropods to cope environmental variability. At low pH and elevated temperature in isolation or combined showing lower shell growth than individuals kept under control conditions. Percentage change in shell length and thicknesses was also lower under acidified and temperature in isolation or combined than control condition, making shells were more globular and desiccation rates were higher. Further studies to broader latitudinal ranges for six populations of L. littorea showed that shell growth decreased in all six populations under elevated pCO2 compared to control snails particularly those at range edges. Elevated pCO2 also affected to the reduction of shell length and width that causing shell aspect ratio to increase across latitudinal gradients except individuals from Millport, UK. Percentage changes of aperture width and aperture area were also decrease under elevated pCO2 with greater reduction of aperture area were found at populations in the mid-ranges which is assumed this response might be linked to local adaptation of the individual to microclimatic conditions. This study also showed that metabolic rates were negatively affected by high pCO2 and show non-linear trend across latitudinal gradients in compared to individual kept under normal pCO2 conditions. Metabolomic analysis showed that two northern populations of Trondheim and TromsØ were distinct from other populations when exposed to low temperature (15 °C) with elevated pCO2 due to, in part, high concentrations of thymine, uracil, valine and lysine. A similar separation also occurred under medium (25 °C) and high (35 °C) temperature exposure in which one of northern population (Trondheim) was distinct from other populations and had lower concentrations of alanine, betaine and taurine while higher of valine. These results suggest that populations at northern latitudes may apply different ionic transport mechanisms under elevated pCO2 and elevated temperatures and those populations are likely to vary in terms of their physiological responses to this environmental challenge.

Tolerance of juvenile Mytilus galloprovincialis to experimental seawater acidification

Coastal ocean acidification is expected to interfere with the physiology of marine bivalves. In this work, the effects of acidification on the physiology of juvenile mussels Mytilus galloprovincialis were tested by means of controlled CO2 perturbation experiments. The carbonate chemistry of natural (control) seawater was manipulated by injecting CO2 to attain 2 reduced pH levels: −0.3 and −0.6 pH units as compared with the control seawater. After 78 d of exposure, we found that the absorption efficiency and ammonium excretion rate of juveniles were inversely related to pH. Significant differences among treatments were not observed in clearance, ingestion and respiration rates. Coherently, the maximal scope for growth and tissue dry weight were observed in mussels exposed to the pH reduction ΔpH = −0.6, suggesting that M. galloprovincialis could be tolerant to CO2 acidification, at least in the highly alkaline coastal waters of Ria Formosa (SW Portugal).

Before ocean acidification: calcifier chemistry lessons

Ocean Acidification (OA) has been an important research topic for a decade. Scientists have focused on how the predicted 56% decline in the seawater carbonate ion () concentration will dramatically impair the ability of calcifiers, ranging from coccolithophores to shellfish, to form calcium carbonate (CaCO₃) structures, and the implications of the reduced carbonate saturation state (Ω) for increased dissolution of such structures. However, most published OA studies have overlooked a fundamental issue: most calcifying organisms do not rely on carbonate from seawater to calcify; they use either bicarbonate () or metabolically-produced CO₂. The ability of important primary (corals, coralline seaweeds and coccolithophores) and secondary (mollusks) producers to modify their local carbonate chemistry suggests that the primary threat to them from OA is by dissolution rather than impaired calcification. Here, we draw on calcification research from an era before OA and combine it with recent studies that question the source of the carbonate ion, to provide new insights into how OA might affect calcifying organisms. Organismal modification of local carbonate chemistry may enable calcifiers to successfully form calcareous structures despite OA.

Impacts of ocean acidification on respiratory gas exchange and acid–base balance in a marine teleost, Opsanus beta

The oceanic carbonate system is changing rapidly due to rising atmospheric CO₂, with current levels expected to rise to between 750 and 1,000 μatm by 2100, and over 1,900 μatm by year 2300. The effects of elevated CO₂ on marine calcifying organisms have been extensively studied; however, effects of imminent CO₂ levels on teleost acid–base and respiratory physiology have yet to be examined. Examination of these physiological processes, using a paired experimental design, showed that 24 h exposure to 1,000 and 1,900 μatm CO₂ resulted in a characteristic compensated respiratory acidosis response in the gulf toadfish (Opsanus beta). Time course experiments showed the onset of acidosis occurred after 15 min of exposure to 1,900 and 1,000 μatm CO₂, with full compensation by 2 and 4 h, respectively. 1,900-μatm exposure also resulted in significantly increased intracellular white muscle pH after 24 h. No effect of 1,900 μatm was observed on branchial acid flux; however, exposure to hypercapnia and HCO₃ ⁻ free seawater compromised compensation. This suggests branchial HCO₃ ⁻ uptake rather than acid extrusion is part of the compensatory response to low-level hypercapnia. Exposure to 1,900 μatm resulted in downregulation in branchial carbonic anhydrase and slc4a2 expression, as well as decreased Na⁺/K⁺ ATPase activity after 24 h of exposure. Infusion of bovine carbonic anhydrase had no effect on blood acid–base status during 1,900 μatm exposures, but eliminated the respiratory impacts of 1,000 μatm CO₂. The results of the current study clearly show that predicted near-future CO₂ levels impact respiratory gas transport and acid–base balance. While the full physiological impacts of increased blood HCO₃ ⁻ are not known, it seems likely that chronically elevated blood HCO₃ ⁻ levels could compromise several physiological systems and furthermore may explain recent reports of increased otolith growth during exposure to elevated CO₂

Response of two marine bacterial isolates to high CO2 concentration

Experimental results related to the effects of ocean acidification on planktonic marine microbes are still rather inconsistent and occasionally contradictory. Moreover, laboratory or field experiments that address the effects of changes in CO₂ concentrations on heterotrophic microbes are very scarce, despite the major role of these organisms in the marine carbon cycle. We tested the direct effect of an elevated CO₂ concentration (1000 ppmv) on the biomass and metabolic rates (leucine incorporation, CO₂ fixation and respiration) of 2 isolates belonging to 2 relevant marine bacterial families, Rhodobacteraceae (strain MED165) and Flavobacteriaceae (strain MED217). Our results demonstrate that, contrary to some expectations, high pCO₂ did not negatively affect bacterial growth but increased growth efficiency in the case of MED217. The elevated partial pressure of CO₂ ( pCO₂) caused, in both cases, higher rates of CO₂ fixation in the dissolved fraction and, in the case of MED217, lower respiration rates. Both responses would tend to increase the pH of seawater acting as a negative feedback between elevated atmospheric CO₂ concentrations and ocean acidification.

Impacts of seawater acidification on mantle gene expression patterns of the Baltic Sea blue mussel: implications for shell formation and energy metabolism

Marine organisms have to cope with increasing CO₂ partial pressures and decreasing pH in the oceans. We elucidated the impacts of an 8-week acclimation period to four seawater pCO₂ treatments (39, 113, 243 and 405 Pa/385, 1,120, 2,400 and 4,000 μatm) on mantle gene expression patterns in the blue mussel Mytilus edulis from the Baltic Sea. Based on the M. edulis mantle tissue transcriptome, the expression of several genes involved in metabolism, calcification and stress responses was assessed in the outer (marginal and pallial zone) and the inner mantle tissues (central zone) using quantitative real-time PCR. The expression of genes involved in energy and protein metabolism (F-ATPase, hexokinase and elongation factor alpha) was strongly affected by acclimation to moderately elevated CO₂ partial pressures. Expression of a chitinase, potentially important for the calcification process, was strongly depressed (maximum ninefold), correlating with a linear decrease in shell growth observed in the experimental animals. Interestingly, shell matrix protein candidate genes were less affected by CO₂ in both tissues. A compensatory process toward enhanced shell protection is indicated by a massive increase in the expression of tyrosinase, a gene involved in periostracum formation (maximum 220-fold). Using correlation matrices and a force-directed layout network graph, we were able to uncover possible underlying regulatory networks and the connections between different pathways, thereby providing a molecular basis of observed changes in animal physiology in response to ocean acidification.

Acute extracellular acid–base disturbance in the burrowing sea urchin Brissopsis lyrifera during exposure to a simulated CO2 release

We tested the hypothesis that as infaunal organisms are regularly exposed to elevated CO₂, burrowing sea urchins will demonstrate a lower sensitivity to massive CO₂ release than has previously been recorded for epifaunal organisms. Infaunal urchins Brissopsis lyrifera were exposed to CO₂ acidified sea water (nominal pH 7.8 (control), 7.3, 6.5 and 5.9; T = 10 °C, S = 34) for 12 h and aspects of their extracellular acid–base balance measured every 2 h. In common with epifaunal urchins B. lyrifera exhibited an uncompensated respiratory acidosis in its extracellular fluid, but was more sensitive to CO₂ acidification than epifaunal urchins. The lower extracellular pH of B. lyrifera may indicate a higher metabolism than epifaunal urchins and this could explain the heightened sensitivity of this species to elevated CO₂. Thus, the results of this present study do not support our original hypothesis. Instead we suggest an alternative hypothesis that as infaunal organisms are exposed naturally to high levels of CO₂, they may already be closer to the limits of their physiological performance. Thus any further CO₂ increase could compromise their function. As a result of this sensitivity, infaunal urchins may be more at risk from an accidental release of CO₂ from geological sub-seabed storage sites, or from the deliberate injection of CO₂ into deep water masses, than their epifaunal counterparts.

Characteristics of digestive enzymes of calanoid copepod species from different latitudes in relation to temperature, pH and food

In calanoid copepods it is poorly understood how enzymatic activities and patterns are affected by abiotic and biotic factors. Such knowledge, however, is crucial to assess metabolic functioning and performance of organisms in different habitats. Therefore, our study focuses on digestive enzyme activities in relation to temperature, pH and food in the Arctic species Calanus glacialis and in Centropages hamatus and Temora longicornis from the North Sea. Enzyme activities were measured over a range from 0 to 70 °C (lipases/esterases, proteinases) and pH 5 to 9 (proteinases). In all species, relative proteinases activity peaked at 40/50 °C and pH 6; relative lipases/esterases activity peaked at 30 °C. Between 0 and 20 °C, lipase activity of C. glacialis was higher (40-70% of maximum) than that of the boreal copepods (25-64%), which suggests thermal adaptation of the lipid metabolism in the polar species. Incubating C. glacialis with the diatom Thalassiosira weissflogii showed (i) that enzyme activities increased especially in the alkaline range and (ii) that enzyme patterns, revealed by gel electrophoresis, differed from that of starving individuals, indicating that feeding induced enzyme expression. Such studies, linking abiotic and biotic conditions to enzyme functioning, can help elucidating the capacity of copepods to respond to environmental changes.

Major cellular and physiological impacts of ocean acidification on a reef building coral

As atmospheric levels of CO₂ increase, reef-building corals are under greater stress from both increased sea surface temperatures and declining sea water pH. To date, most studies have focused on either coral bleaching due to warming oceans or declining calcification due to decreasing oceanic carbonate ion concentrations. Here, through the use of physiology measurements and cDNA microarrays, we show that changes in pH and ocean chemistry consistent with two scenarios put forward by the Intergovernmental Panel on Climate Change (IPCC) drive major changes in gene expression, respiration, photosynthesis and symbiosis of the coral, Acropora millepora, before affects on biomineralisation are apparent at the phenotype level. Under high CO₂ conditions corals at the phenotype level lost over half their Symbiodinium populations, and had a decrease in both photosynthesis and respiration. Changes in gene expression were consistent with metabolic suppression, an increase in oxidative stress, apoptosis and symbiont loss. Other expression patterns demonstrate upregulation of membrane transporters, as well as the regulation of genes involved in membrane cytoskeletal interactions and cytoskeletal remodeling. These widespread changes in gene expression emphasize the need to expand future studies of ocean acidification to include a wider spectrum of cellular processes, many of which may occur before impacts on calcification.