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

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

Impact of long-term moderate hypercapnia and elevated temperature on the energy budget of isolated gills of Atlantic cod (Gadus morhua)

Effects of severe hypercapnia have been extensively studied in marine fishes, while the 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 (2,500 μatm) and temperature (18°C). Isolated perfused gill preparations were established to determine gill thermal plasticity during acute exposures (10-22°C) and in vivo costs of Na(+)/K(+)-ATPase activity and of 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 percent below rates of the control group. 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 more 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

Adjustments of molecular key components of branchial ion and pH regulation in Atlantic cod (Gadus morhua) in response to ocean acidification and warming.

Marine teleost fish sustain compensation of extracellular pH after exposure to hypercapnia by means of efficient ion and acid-base regulation. Elevated rates of ion and acid-base regulation under hypercapnia may be stimulated further by elevated temperature. Here, we characterized the regulation of transepithelial ion transporters (NKCC1, NBC1, SLC26A6, NHE1 and 2) and ATPases (Na(+)/K(+) ATPase and V-type H(+) ATPase) in gills of Atlantic cod (Gadus morhua) after 4weeks of exposure to ambient and future PCO2 levels (550μatm, 1200μatm, 2200μatm) at optimum (10°C) and summer maximum temperature (18°C), respectively. Gene expression of most branchial ion transporters revealed temperature- and dose-dependent responses to elevated PCO2. Transcriptional regulation resulted in stable protein expression at 10°C, whereas expression of most transport proteins increased at medium PCO2 and 18°C. mRNA and protein expression of distinct ion transport proteins were closely co-regulated, substantiating cellular functional relationships. Na(+)/K(+) ATPase capacities were PCO2 independent, but increased with acclimation temperature, whereas H(+) ATPase capacities were thermally compensated but decreased at medium PCO2 and 10°C. When functional capacities of branchial ATPases were compared with mitochondrial F1Fo ATP-synthase strong correlations of F1Fo ATP-synthase and ATPase capacities generally indicate close coordination of branchial aerobic ATP demand and supply. Our data indicate physiological plasticity in the gills of cod to adjust to a warming, acidifying ocean within limits. In light of the interacting and non-linear, dose-dependent effects of both climate factors the role of these mechanisms in shaping resilience under climate change remains to be explore

Mechanisms of acid base regulation and CO2 tolerance in marine fish: functional capacities, thermal interactions and mechanism-based modelling

Anthropogenic CO2 emissions threaten marine ecosystems by increasing water temperature and acidification. Fish generally seem to be less sensitive to elevated CO2 concentrations due to their efficient ion regulatory capacities. As temperature affects all ion regulatory processes, an increased sensitivity to elevated CO2 levels at the edges of the thermal window can be postulated. In this study, we are acclimating Atlantic cod to a combined setup of elevated PCO2 (390, 1120 and 3000 µatm) and temperatures (10, 15 and 18 °C). In isolated, perfused gill arches we determine the fractional costs of ion regulation, protein and RNA synthesis in relation to the global energy budget after acclimation and under acute exposure by the application of specific inhibitors. Acute warming increased overall gill oxygen consumption rates and fractional costs of the processes investigated, the latter most obvious when combined with hypercapnia. Furthermore, apical Na+/H+ exchangers seem to be essential for the response towards acidification. In isolated gill cells we fluorometrically track the changes of intracellular pH in gill cells after acute exposure to acidification to quantify cellular ion regulative processes and performance. Additionally, ion transport proteins are going to be inhibited to quantify their involvement in the entire regulation process. For the characterisation of the ion regulatory transcriptome and proteome we combine genetic, immunohistological and functional approaches. So far, genes of essential transporters involved in ion regulation have been isolated, and the expression of relevant transporters (NBC1, AE1, H+-ATPase, Na+/K+-ATPase) and the capacity of the Na+/K+-ATPase were found seasonally and population specific regulated. Measuring a portfolio of haematological and immunological endpoints the hypothesis is tested whether increased energy expenditure e. g. for ion regulation at elevated CO2 levels will suppress the immunocompetence at the edges of the cods thermal window. Together, all data will be integrated into a mechanistic model to mathematically formulate ion regulative capacities and its limitations in response to predicted climate scenarios

Microdeletions Including FMR1 in Three Female Patients with Intellectual Disability – Further Delineation of the Phenotype and Expression Studies

Fragile X syndrome (FXS) is one of the most common causes of intellectual disability/developmental delay (ID/DD), especially in males. It is caused most often by CGG trinucleotide repeat expansions, and less frequently by point mutations and partial or full deletions of the FMR1 gene. The wide clinical spectrum of affected females partly depends on their X-inactivation status. Only few female ID/DD patients with microdeletions including FMR1 have been reported. We describe 3 female patients with 3.5-, 4.2- and 9.2-Mb de novo microdeletions in Xq27.3-q28 containing FMR1. X-inactivation was random in all patients, yet they presented with ID/DD as well as speech delay, macrocephaly and other features attributable to FXS. No signs of autism were present. Here, we further delineate the clinical spectrum of female patients with microdeletions. FMR1 expression studies gave no evidence for an absolute threshold below which signs of FXS present. Since FMR1 expression is known to be highly variable between unrelated females, and since FMR1 mRNA levels have been suggested to be more similar among family members, we further explored the possibility of an intrafamilial effect. Interestingly, FMR1 mRNA levels in all 3 patients were significantly lower than in their respective mothers, which was shown to be specific for patients with microdeletions containing FMR1