Description: Oxygen equilibrium curves have been widely used to understand oxygen transport in numerous organisms. A major challenge has been to monitor oxygen binding characteristics and concomitant pH changes as they occur in vivo, in limited sample volumes. Here we report a technique allowing highly resolved and simultaneous monitoring of pH and blood pigment saturation in minute blood volumes. We equipped a gas diffusion chamber with a broad range fibre optic spectrophotometer and a micro-pH optode and recorded changes of pigment oxygenation along PO2 and pH gradients to test the setup. Oxygen binding parameters derived from measurements in only 15 µl of haemolymph from the cephalopod Octopus vulgaris showed low instrumental error (0.93%) and good agreement with published data. Broad range spectra, each resolving 2048 data points, provided detailed insight into the complex absorbance characteristics of diverse blood types. After consideration of photobleaching and intrinsic fluorescence, pH optodes yielded accurate recordings and resolved a sigmoidal shift of 0.03 pH units in response to changing PO2 from 0-21 kPa. Highly resolved continuous recordings along pH gradients conformed to stepwise measurements at low rates of pH changes. In this study we showed that a diffusion chamber upgraded with a broad range spectrophotometer and an optical pH sensor accurately characterizes oxygen binding with minimal sample consumption and manipulation. We conclude that the modified diffusion chamber is highly suitable for experimental biologists who demand high flexibility, detailed insight into oxygen binding as well as experimental and biological accuracy combined in a single set up.

Description: In the eurythermal cuttlefish Sepia officinalis, performance depends on hearts that ensure systemic oxygen supply over a broad range of temperatures. We therefore aimed to identify adjustments in energetic cardiac capacity and underlying mitochondrial function supporting thermal acclimation and adaptation that could be crucial for the cuttlefish's competitive success in variable environments. Two genetically distinct cuttlefish populations were acclimated to 11, 16 and 21°C. Subsequently, skinned and permeabilised heart fibres were used to assess mitochondrial functioning by means of high-resolution respirometry and a substrate-inhibitor protocol, followed by measurements of cardiac citrate synthase and cytosolic enzyme activities. Temperate English Channel cuttlefish had lower mitochondrial capacities but larger hearts than subtropical Adriatic cuttlefish. Warm acclimation to 21°C decreased mitochondrial complex I activity in Adriatic cuttlefish and increased complex IV activity in English Channel cuttlefish. However, compensation of mitochondrial capacities did not occur during cold acclimation to 11°C. In systemic hearts, the thermal sensitivity of mitochondrial substrate oxidation was high for proline and pyruvate but low for succinate. Oxygen efficiency of catabolism rose as temperature changed from 11 to 21°C via shifts to oxygen-conserving oxidation of proline and pyruvate and via reduced relative proton leak. The changes observed for substrate oxidation, mitochondrial complexes, relative proton leak and heart mass improve energetic efficiency and essentially seem to extend tolerance to high temperatures and reduce associated tissue hypoxia. We conclude that cuttlefish sustain cardiac performance and, thus, systemic oxygen delivery over short- and long-term changes of temperature and environmental conditions by multiple adjustments in cellular and mitochondrial energetics.

Description: Water parameters in the 2 years before spawning of F0 (08.02.2016-06.03.2018) and during larval and juvenile phase of F1: Larval period until 17.05.2018 (48 dph, 900 dd) and 01.06.2018 (63 dph, ~900 dd) for warm and cold life condition respectively, for the juveniles until 28.09.2018 (180 dph, ~4000 dd) and 12.02.2019 (319 dph, ~5100 dd) for warm and cold conditioned fish respectively. Means ± s.e. over all replicate tanks per condition. Temperature (Temp.), pH (free scale), salinity, oxygen and total alkalinity (TA) were measured weekly in F1 and monthly in F0; sea water (SW) measurements were conducted in 2017 and 2018. Water parameters during larval and early juvenile phase of F0: Larval period until (45 dph, 900 dd, 06.12.2013), early juveniles until 1.5 years. Means ± s.e.m. over all measurements per condition (triplicate tanks in larvae, single tanks in juveniles). Temperature (Temp.) and pH (NBS scale) were measured daily. pH (total scale), salinity, phosphate, silicate and total alkalinity (TA) were measured once at the beginning and once at the end of the larval phase and 9 times during juvenile phase; PCO2 was calculated with CO2sys; A–Ambient PCO2, D1000 –ambient + 1000 µatm CO2, L – Larvae, J – Juveniles.

Description: 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.

Description: The world's oceans are acidifying and warming as a result of increasing atmospheric CO2 concentrations. The thermal tolerance of fish greatly depends on the cardiovascular ability to supply the tissues with oxygen. The highly oxygen-dependent heart mitochondria thus might play a key role in shaping an organism's tolerance to temperature. The present study aimed to investigate the effects of acute and chronic warming on the respiratory capacity of European sea bass (Dicentrarchus labrax L.) heart mitochondria. Broodstock fish were caught in the Gulf of Morbihan, France. Larvae were raised at the aquaculture facility Aquastream (Ploemeur-Lorient, France) and obtained at 2 dph (20 January 2016). European sea bass were reared in the laboratory in six ocean acidification and warming (OAW) conditions: two temperatures (warm and cold life condition) and three PCO2 conditions (control, Δ500 and Δ1000). Conditions were chosen to follow the predictions of the IPCC for the next 130 years: ΔT = 5°C and ΔPCO2 = 500 and 1000 µatm, following RCP 6.0 and RCP 8.5 respectively. The fish were reared under these conditions from 3 dph (days post hatch) until mitochondrial respiration measurements at 3700 to 4100 dd (degree days, 183–199 dph and 234–249 dph in warm and cold life conditioned fish, respectively). During the experimental period, fish of all three PCO2 conditions of the respective temperature were used for mitochondrial respiration measurements on permeabilized heart fibres. Fish were not fed for 2 days prior to the experiments. Two batches of eight fish each were processed per day. Juveniles were randomly caught from their tanks and anesthetized with MS-222. Mass, fork length and body length were directly determined with a precision balance (Mettler, Columbus, OH, USA) and a calliper, to the nearest 0.01 g and 0.01 mm, respectively. Afterwards, fish were killed by a cut through the neck, and the heart was completely dissected from the fish, followed by excavation and permeabilization of the ventricle. Tissue from a whole ventricle was used for respiration measurements in each respiration chamber of the oxygraphs and respiration rates were normalized to ventricle mass. During the permeabilization step, the livers and the carcasses of the fish were weighed to calculate the hepatosomatic index (HSI) and condition factor (K). Mitochondrial respiration of the permeabilized heart fibres was measured using four Oroboros Oxygraph-2K respirometers with DatLab 6 software (Oroboros Instruments, Innsbruck, Austria). Permeabilized fibers have the advantage of resembling the living state as closely as possible, while still allowing control of the supply of substrates and inhibitors to the mitochondria (Saks et al., 1998; Pesta and Gnaiger, 2012). Measurements were conducted at 15 and 20°C for all treatments to determine the effect of acute temperature changes on mitochondrial metabolism in vitro. A standard substrate–uncoupler–inhibitor titration protocol was employed to measure the respiration rates of the different complexes. Residual respiration after antimycin A addition was used to correct all mitochondrial respiration rates.

Description: 1) Our study addresses the role of non-genetic and genetic inheritance in shaping the adaptive potential of populations under a warming ocean scenario. We used a combined experimental approach (transgenerational plasticity and quantitative genetics) to partition the relative contribution of maternal vs. paternal (additive genetic) effects to offspring body size (a key component of fitness), and investigated a potential physiological mechanism (mitochondrial respiration capacities) underlying whole organism growth/size responses. 2) In very early stages of growth (up to 30 days), offspring body size of marine sticklebacks benefited from maternal transgenerational plasticity (TGP): offspring of mothers acclimated to17°C were larger when reared at 17°C, and offspring of mothers acclimated to 21°C were larger when reared at 21°C. The benefits of maternal TGP on body size were stronger and persisted longer (up to 60 days) for offspring reared in the warmer (21°C) environment, suggesting that maternal effects will be highly relevant for climate change scenarios in this system. 3) Mitochondrial respiration capacities measured on mature offspring (F1 adults) matched the pattern of TGP for juvenile body size, providing an intuitive mechanistic basis for the maternal acclimation persisting into adulthood. Size differences between temperatures seen at early growth stages remained in the F1 adults, linking offspring body size to maternal inheritance of mitochondria. 4) Lower maternal variance components in the warmer environment were mostly driven by mothers acclimated to ambient (colder) conditions, further supporting our tenet that maternal effects were stronger at elevated temperature. Importantly, all parent-offspring temperature combination groups showed genotype x environment (GxE) interactions, suggesting that reaction norms have the potential to evolve. 5) To summarise, transgenerational plasticity and genotype x environment interactions work in concert to mediate impacts of ocean warming on metabolic capacity and early growth of marine sticklebacks. TGP can buffer short-term detrimental effects of climate warming and may buy time for genetic adaptation to catch up, therefore markedly contributing to the evolutionary potential and persistence of populations under climate change.

Description: Thermal tolerance windows serve as a powerful tool for estimating the vulnerability of marine species and their life stages to increasing temperature means and extremes. However, it remains uncertain to which extent additional drivers, such as ocean acidification, modify organismal responses to temperature. This study investigated the effects of CO2-driven ocean acidification on embryonic thermal sensitivity and performance in Atlantic cod, Gadus morhua, from the Kattegat. Fertilized eggs were exposed to factorial combinations of two PCO2 conditions (400 µatm vs. 1100 µatm) and five temperature treatments (0, 3, 6, 9 and 12 °C), which allow identifying both lower and upper thermal tolerance thresholds. We quantified hatching success, oxygen consumption (MO2) and mitochondrial functioning of embryos as well as larval morphometrics at hatch and the abundance of acid?base-relevant ionocytes on the yolk sac epithelium of newly hatched larvae. Hatching success was high under ambient spawning conditions (3-6 °C), but decreased towards both cold and warm temperature extremes. Elevated PCO2 caused a significant decrease in hatching success, particularly at cold (3 and 0 °C) and warm (12 °C) temperatures. Warming imposed limitations to MO2 and mitochondrial capacities. Elevated PCO2 stimulated MO2 at cold and intermediate temperatures, but exacerbated warming-induced constraints on MO2, indicating a synergistic interaction with temperature. Mitochondrial functioning was not affected by PCO2. Increased MO2 in response to elevated PCO2 was paralleled by reduced larval size at hatch. Finally, ionocyte abundance decreased with increasing temperature, but did not differ between PCO2 treatments. Our results demonstrate increased thermal sensitivity of cod embryos under future PCO2 conditions and suggest that acclimation to elevated PCO2 requires reallocation of limited resources at the expense of embryonic growth. We conclude that ocean acidification constrains the thermal performance window of embryos, which has important implication for the susceptibility of cod to projected climate change.

Description: Oceans are experiencing increasing acidification in parallel to a distinct warming trend in consequence of ongoing climate change. Rising seawater temperatures are mediating a northward shift in distribution of Atlantic cod (Gadus morhua), into the habitat of polar cod (Boreogadus saida), that is associated with retreating cold water masses. This study investigates the competitive strength of the co-occurring gadoids under ocean acidification and warming (OAW) scenarios. Therefore, we incubated specimens of both species in individual tanks for 4 months, under different control and projected temperatures (polar cod: 0, 3, 6, 8 °C, Atlantic cod: 3, 8, 12, 16 °C) and PCO2 conditions (390 and 1170 µatm) and monitored growth, feed consumption and standard metabolic rate. Our results revealed distinct temperature effects on both species. While hypercapnia by itself had no effect, combined drivers caused nonsignificant trends. The feed conversion efficiency of normocapnic polar cod was highest at 0 °C, while optimum growth performance was attained at 6 °C; the long-term upper thermal tolerance limit was reached at 8 °C. OAW caused only slight impairments in growth performance. Under normocapnic conditions, Atlantic cod consumed progressively increasing amounts of feed than individuals under hypercapnia despite maintaining similar growth rates during warming. The low feed conversion efficiency at 3 °C may relate to the lower thermal limit of Atlantic cod. In conclusion, Atlantic cod displayed increased performance in the warming Arctic such that the competitive strength of polar cod is expected to decrease under future OAW conditions.