ALBERT

Description: 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 4 weeks of exposure to ambient and future PCO2 levels (550 μatm, 1200 μatm, 2200 μatm) 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 explored.

Description: CO2-driven seawater acidification has been demonstrated to enhance intestinal bicarbonate secretion rates in teleosts, leading to an increased release of CaCO3 under simulated ocean acidification scenarios. In this study, we investigated if increasing CO2 levels stimulate the intestinal acid–base regulatory machinery of Atlantic cod (Gadus morhua) and whether temperatures at the upper limit of thermal tolerance stimulate or counteract ion regulatory capacities. Juvenile G. morhua were acclimated for 4 weeks to three CO2 levels (550, 1200, and 2200 μatm) covering present and near-future natural variability, at optimum (10°C) and summer maximum temperature (18°C), respectively. Immunohistochemical analyses revealed the subcellular localization of ion transporters, including Na+/K+-ATPase (NKA), Na+/H+-exchanger 3 (NHE3), Na+/HCO−3 cotransporter (NBC1), pendrin-like Cl−/HCO−3 exchanger (SLC26a6), V-type H+-ATPase subunit a (VHA), and Cl− channel 3 (CLC3) in epithelial cells of the anterior intestine. At 10°C, proteins and mRNA were generally up-regulated for most transporters in the intestinal epithelium after acclimation to higher CO2 levels. This supports recent findings demonstrating increased intestinal HCO−3 secretion rates in response to CO2 induced seawater acidification. At 18°C, mRNA expression and protein concentrations of most ion transporters remained unchanged or were even decreased, suggesting thermal compensation. This response may be energetically favorable to retain blood HCO−3 levels to stabilize pHe, but may negatively affect intestinal salt and water resorption of marine teleosts in future oceans.

Description: 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 were 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.

Description: CO2-driven seawater acidification has been demonstrated to enhance intestinal bicarbonate secretion rates in teleosts, leading to an increased release of CaCO3 under simulated ocean acidification scenarios. In this study, we investigated if increasing CO2 levels stimulate the intestinal acid–base regulatory machinery of Atlantic cod (Gadus morhua) and whether temperatures at the upper limit of thermal tolerance stimulate or counteract ion regulatory capacities. Juvenile G. morhua were acclimated for 4 weeks to three CO2 levels (550, 1200, and 2200 μatm) covering present and near-future natural variability, at optimum (10°C) and summer maximum temperature (18°C), respectively. Immunohistochemical analyses revealed the subcellular localization of ion transporters, including Na+/K+-ATPase (NKA), Na+/H+-exchanger 3 (NHE3), Na+/HCO−3 cotransporter (NBC1), pendrin-like Cl−/HCO−3 exchanger (SLC26a6), V-type H+-ATPase subunit a (VHA), and Cl− channel 3 (CLC3) in epithelial cells of the anterior intestine. At 10°C, proteins and mRNA were generally up-regulated for most transporters in the intestinal epithelium after acclimation to higher CO2 levels. This supports recent findings demonstrating increased intestinal HCO−3 secretion rates in response to CO2 induced seawater acidification. At 18°C, mRNA expression and protein concentrations of most ion transporters remained unchanged or were even decreased, suggesting thermal compensation. This response may be energetically favorable to retain blood HCO−3 levels to stabilize pHe, but may negatively affect intestinal salt and water resorption of marine teleosts in future oceans.

Description: The maintenance of ion and pH homeostasis despite changes in ambient temperature is crucial for ectothermic organisms. Thermal sensitivity of Na+/K+ ATPase mRNA expression, protein expression and activity was determined in gills of North Sea cod (NC) and Northeastern Arctic cod (NEAC), acclimated for 6 weeks at 4 and 10 °C and compared to field samples of North Sea cod (sNC), acclimatized to early spring (4 °C) and summer (18 °C) conditions. The same analyses were conducted in gills of the confamiliar whiting, acclimated at 4 and 10 °C. Branchial Na+/K+ ATPase capacities remained uncompensated at functional and protein levels in NC and NEAC at both acclimation temperatures. Na+/K+ ATPase mRNA expression in NEAC acclimated at 10 °C was about twofold higher compared to NC, indicating some population-specific differentiation at this level. Lower Na+/K+ ATPase capacities in gills of warm-acclimatized sNC at common assay temperatures indicate thermal compensation between seasonal extremes, and post-translational modifications contributed to this mitigation at high assay temperature. Together, cod compensates Na+/K+ ATPase capacities on the warm edge of the thermal window and below 4 °C, respectively. In contrast, whiting Na+/K+ ATPase capacities were cold compensated at 4 °C, supported by 1.5-fold higher mRNA and protein expression. Besides, capacities were lower in whiting compared to NC and NEAC at optimum temperature, which may be advantageous in terms of reduced maintenance cost, but at temperatures ≤4 °C, compensation may represent an energy trade-off to maintain homeostasis. The species-specific response of gadid Na+/K+ ATPase indicates certain threshold temperatures beyond which compensation of the pump is elicited, possibly related to the different biogeography of these species.

Description: 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.

Description: 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.