Assessment of muscular energy metabolism and heat shock response of the green abalone Haliotis fulgens (Gastropoda: Philipi) at extreme temperatures combined with acute hypoxia and hypercapnia.

The interaction between ocean warming, hypoxia and hypercapnia, suggested by climate projections, may push an organism earlier to the limits of its thermal tolerance window. In a previous study on juveniles of green abalone (Haliotis fulgens), combined exposure to hypoxia and hypercapnia during heat stress induced a lowered critical thermal maximum (CTmax), indicated by constrained oxygen consumption, muscular spams and loss of attachment. Thus, the present study investigated the cell physiology in foot muscle of H. fulgens juveniles exposed to acute warming (18 °C to 32 °C at +3 °C day-1) under hypoxia (50% air saturation) and hypercapnia (~1000 µatm PCO2), alone and in combination, to decipher the mechanisms leading to functional loss in this tissue. Under exposure to either hypoxia or hypercapnia, citrate synthase (CS) activity decreased with initial warming, in line with thermal compensation, but returned to control levels at 32 °C. The anaerobic enzymes lactate and tauropine dehydrogenase increased only under hypoxia at 32 °C. Under the combined treatment, CS overcame thermal compensation and remained stable overall, indicating active mitochondrial regulation under these conditions. Limited accumulation of anaerobic metabolites indicates unchanged mode of energy production. In all treatments, upregulation of Hsp70 mRNA was observed already at 30 °C. However, lack of evidence for Hsp70 protein accumulation provides only limited support to thermal denaturation of proteins. We conclude that under combined hypoxia and hypercapnia, metabolic depression allowed the H. fulgens musculature to retain an aerobic mode of metabolism in response to warming but may have contributed to functional loss.

Microsatellite markers for the notothenioid fish Lepidonotothen nudifrons and two congeneric species.

BACKGROUND: Loss of genetic variability due to environmental changes, limitation of gene flow between pools of individuals or putative selective pressure at specific markers, were previously documented for Antarctic notothenioid fish species. However, so far no studies were performed for the Gaudy notothen Lepidonotothen nudifrons. Starting from a species-specific spleen transcriptome library, we aimed at isolating polymorphic microsatellites (Type I; i.e. derived from coding sequences) suitable to quantify the genetic variability in this species, and additionally to assess the population genetic structure and demography in nototheniids. RESULTS: We selected 43,269 transcripts resulting from a MiSeq sequencer run, out of which we developed 19 primer pairs for sequences containing microsatellite repeats. Sixteen loci were successfully amplified in L. nudifrons. Eleven microsatellites were polymorphic and allele numbers per locus ranged from 2 to 17. In addition, we amplified loci identified from L. nudifrons in two other congeneric species (L. squamifrons and L. larseni). Thirteen loci were highly transferable to the two congeneric species. Differences in polymorphism among species were detected. CONCLUSIONS: Starting from a transcriptome of a non-model organism, we were able to identify promising polymorphic nuclear markers that are easily transferable to other closely related species. These markers can be a key instrument to monitor the genetic structure of the three Lepidonotothen species if genotyped in larger population samples. When compared with anonymous loci isolated in other notothenioids, i.e. Type II (isolated from genomic libraries), they offer the possibility to test how the effects of occurring environmental change influence the population genetic structure in each species and subsequently the composition of the entire ecosystem.

Response of branchial Na(+)/K(+) ATPase to changes in ambient temperature in Atlantic cod (Gadus morhua) and whiting (Merlangius merlangus).

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.

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 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.

A first insight into the spleen transcriptome of the notothenioid fish Lepidonotothen nudifrons: Resource description and functional overview.

In this study, we describe a de novo sequencing and assembly of the spleen transcriptome of Lepidonotothen nudifrons, a notothenioid fish widely distributed around the Antarctic Peninsula and the Scotia Arc. Sequences were generated on an Illumina MiSeq system and assembled to a total of 112,477 transcripts. Putative functional annotation was possible for more than 34% of the transcripts. This data will be relevant for future studies targeting the erythrocyte turnover, oxygen transport mechanism and immune system, which are key functional traits to investigate cold adaptation and thermal sensitivity of Antarctic notothenioids.

Mitochondrial function in Antarctic nototheniids with ND6 translocation.

Fish of the suborder Notothenioidei have successfully radiated into the Southern Ocean and today comprise the dominant fish sub-order in Antarctic waters in terms of biomass and species abundance. During evolution in the cold and stable Antarctic climate, the Antarctic lineage of notothenioids developed several unique physiological adaptations, which make them extremely vulnerable to the rapid warming of Antarctic waters currently observed. Only recently, a further phenomenon exclusive to notothenioid fish was reported: the translocation of the mitochondrial gene encoding the NADH Dehydrogenase subunit 6 (ND6), an indispensable part of complex I in the mitochondrial electron transport system.This study investigated the potential physiological consequences of ND6 translocation for the function and thermal sensitivity of the electron transport system in isolated liver mitochondria of the two nototheniid species Notothenia coriiceps and Notothenia rossii, with special attention to the contributions of complex I (NADH DH) and complex II (Succinate DH) to oxidative phosphorylation. Furthermore, enzymatic activities of NADH:Cytochrome c Oxidoreductase and Cytochrome C Oxidase were measured in membrane-enriched tissue extracts.During acute thermal challenge (0-15°C), capacities of mitochondrial respiration and enzymatic function in the liver could only be increased until 9°C. Mitochondrial complex I (NADH Dehydrogenase) was fully functional but displayed a higher thermal sensitivity than the other complexes of the electron transport system, which may specifically result from its unique amino acid composition, revealing a lower degree of stability in notothenioids in general. We interpret the translocation of ND6 as functionally neutral but the change in amino acid sequence as adaptive and supportive of cold stenothermy in Antarctic nototheniids. From these findings, an enhanced sensitivity to ocean warming can be deduced for Antarctic notothenioid fish.

Acclimation of ion regulatory capacities in gills of marine fish under environmental hypercapnia.

The preservation of ion balance and pH despite environmental fluctuations is essential for the maintenance of vital cellular functions. While several ion transporters contribute to acid-base regulation in fish, the involvement and expression of key transporters under hypercapnia remain to be established. Here, two members of the HCO(3)(-) transporter family (Na(+)/HCO(3)(-) cotransporter NBC1 and Cl(-)/HCO(3)(-) exchanger AE1) were described for the first time in gills of marine fish. Benthic eelpout Zoarces viviparus were acclimated to 10,000 ppm CO(2). Hypercapnia did not affect whole animal oxygen consumption over a period of 4 days. During a time series of 6 wk NBC1 mRNA levels first decreased by about 40% (8 to 24 h) but finally increased about threefold over control. mRNA expression of AE1 decreased transiently by 50% at day 4 but recovered to control levels only. Reduced mRNA levels were also found for two Na(+)/H(+) exchangers (NHE1A, NHE1B) during the first days (by 50-60% at days 1 and 2), followed by restoration of control levels. This pattern was mirrored in a slight decrease of NHE1 protein contents and its subsequent recovery. In contrast, Na(+)-K(+)-ATPase mRNA and protein contents, as well as maximum activity, rose steadily from the onset of hypercapnia, and reached up to twofold control levels at the end. These results indicate shifting acclimation patterns between short- and long-term CO(2) exposures. Overall, ion gradient-dependent transporter mRNA levels were transiently downregulated in the beginning of the disturbance. Upregulation of NBC1 on long timescales stresses the importance of this transporter in the hypercapnia response of marine teleosts. Long-term rearrangements include Na(+)-K(+)-ATPase at higher densities and capacities, indicating a shift to elevated rates of ion and acid-base regulation under environmental hypercapnia.

Molecular characterisation and expression of Atlantic cod (Gadus morhua) myoglobin from two populations held at two different acclimation temperatures.

Much previous research has demonstrated the plasticity of myoglobin concentrations in both cardiac and skeletal myocytes in response to hypoxia and training. No study has yet looked at the effect of thermal acclimation on myoglobin in fish. Atlantic cod (Gadus morhua) from two different populations, i.e. the North Sea and the North East Arctic, were acclimated to 10 and 4 degrees C. Both the myoglobin mRNA and myoglobin protein in cod hearts increased significantly by up to 3.7 and 2.3 fold respectively as a result of acclimation to 4 degrees C. These increments were largest in the Arctic population, which in earlier studies have been shown to possess cold compensated metabolic demands at low temperatures. These metabolic demands associated with higher mitochondrial capacities may have driven the increase in cardiac myoglobin concentrations, in order to support diffusive oxygen supply. At the same time the increase in myoglobin levels may serve further functions during cold acclimation, for example, protection of the cell against reactive oxygen species, and scavenging nitric oxide, thereby contributing to the regulation of mitochondrial volume density.