Rapid blood acid-base regulation by European sea bass (Dicentrarchus labrax) in response to sudden exposure to high environmental CO2

Fish in coastal ecosystems can be exposed to acute variations in CO2 that can approach 1 kPa CO2 (10,000 μatm). Coping with this environmental challenge will depend on the ability to rapidly compensate the internal acid-base disturbance caused by sudden exposure to high environmental CO2 (blood and tissue acidosis); however, studies about the speed of acid-base regulatory responses in marine fish are scarce. We observed that upon exposure to ~1 kPa CO2, European sea bass (Dicentrarchus labrax) completely regulate erythrocyte intracellular pH within ~40 minutes, thus restoring haemoglobin-O2 affinity to pre-exposure levels. Moreover, blood pH returned to normal levels within ~2 hours, which is one of the fastest acid-base recoveries documented in any fish. This was achieved via a large upregulation of net acid excretion and accumulation of HCO3− in blood, which increased from ~4 to ~22 mM. While the abundance and intracellular localisation of gill Na+/K+-ATPase (NKA) and Na+/H+ exchanger 3 (NHE3) remained unchanged, the apical surface area of acid-excreting gill ionocytes doubled. This constitutes a novel mechanism for rapidly increasing acid excretion during sudden blood acidosis. Rapid acid-base regulation was completely prevented when the same high CO2 exposure occurred in seawater with experimentally reduced HCO3− and pH, likely because reduced environmental pH inhibited gill H+ excretion via NHE3. The rapid and robust acid-base regulatory responses identified will enable European sea bass to maintain physiological performance during large and sudden CO2 fluctuations that naturally occur in coastal environments. Summary statement European sea bass exposed to 1 kPa (10,000 μatm) CO2 regulate blood and red cell pH within 2 hours and 40 minutes, respectively, protecting O2 transport capacity, via enhanced gill acid excretion.


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Increased CO2 in aquatic environments, or environmental hypercapnia, 54 causes significant physiological challenges for water breathing animals including 55 fish. As environmental CO2 increases, there is a corresponding rise in CO2 within 56 the fish's blood, which in turn induces a decrease in blood pH. This condition is 57 referred to as a respiratory acidosis, and depending on its magnitude, can disrupt 58 multiple homeostatic processes including gas exchange (Crocker and Cech Jr,59 The gill ionocytes of marine fish excrete H + using apical Na + -H + exchangers 105 (NHEs) driven by basolateral Na + /K + -ATPase (NKAs) (Brauner et    Hypercapnia exposure 144 Individual sea bass were moved to isolation tanks (~20 L) and left to acclimate 145 overnight for a minimum of 12 hours before exposure to hypercapnia. During the 146 acclimation period isolation tanks were fed by the RAS at a rate of ~4 L min -1 ; 147 with overflowing water recirculated back to the RAS. After overnight acclimation, 148 hypercapnia exposure began by switching inflow of water from low CO2 control 149 conditions to high CO2 seawater delivered from a header tank (~150 L) in which 150 pCO2 levels had already been increased to ~1 kPa using an Aqua Medic pH 151 computer (AB Aqua Medic GmbH). The pH computer maintained header tank 152 pCO2 levels using an electronic solenoid valve which fed CO2 to a diffuser in the 153 header tank if pH rose above 6.92 and stopped CO2 flow if pH dropped below 154 6.88. Additionally, to reduce CO2 fluctuation in isolation tanks during exposures, 155 the gas aerating each tank was switched from ambient air to a gas mix of 1% 156 CO2, 21% O2 and 78% N2 (G400 Gas mixing system, Qubit Biology Inc.). During 157 exposures overflowing water from each isolation tank recirculated to the header 158 tank creating an isolated experimental system of ~250 L. The experimental 159 system was maintained at 14°C using a heater/chiller unit (Grant TX150 R2, 160 Grant Instruments) attached to a temperature exchange coil in the header tank.

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To characterise the time course of acid-base regulation sea bass were exposed 162 to hypercapnia (~1 kPa CO2) for either ~10 minutes, ~40 minutes, or ~135 163 minutes before measurements were taken. pH of isolation tanks was monitored 164 with a separate pH probe and matched the header tank ~5 minutes after initial 165 exposure. Measurements of an additional group of sea bass were obtained at 166 normocapnic CO2 levels (~0.05 kPa CO2) to act as a pre-exposure control 167 (hereafter this group is referred to as time = 0). At the time of sampling 168 measurements of seawater pH (NBS scale), temperature and salinity, as well as 169 samples of seawater to measure total CO2 (TCO2)/Dissolved Inorganic Carbon 170 (DIC), were taken from each isolation tank. DIC analysis was conducted using a 171 custom built system described in detail by Lewis et al. (2013). Measurements of 172 pH, salinity, temperature and DIC were then input into CO2SYS (Pierrot et al.,173 2006) to calculate pCO2 and total alkalinity (TA) based on the equilibration 174 constants refitted by Dickson and Millero (1987).

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Blood sampling and analysis 176 Following hypercapnia exposures (Table 1)  The gill irrigation tank used was filled with water from the header tank and 180 maintained at an appropriate CO2 level by aeration with the same gas mix feeding 181 the isolation tanks. The water chemistry of the gill irrigation chamber was 182 measured following the same procedures outlined for the isolation chambers, 183 with one DIC sample taken at the end of blood sampling (Table S1). based on Boutilier et al. (1984Boutilier et al. ( , 1985. Haemoglobin (Hb) content of 10 µL of 199 whole blood was also assessed by the cyanmethemoglobin method (after    Acid-base relevant fluxes (µmol kg -1 h -1 ) were then calculated using the  sampled for blood acid-base measurements as detailed previously. The water 265 chemistry of isolation boxes (Table S2) and gill irrigation chambers (Table S3) 266 was measured at the time of blood sampling.

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Gill sampling 268 Gill tissue was sampled from sea bass exposed to ambient CO2 conditions (n 269 = 5) and to hypercapnia for ~135 minutes (n=5, taken immediately after the flux 270 measurements) in normal TA seawater (Table S4). Mean water chemistry 271 conditions during flux measurements (Table 2) and experienced by sea bass prior 272 to gill sampling (Table S4)  t-test (control response < CO2-exposed response). for ~10 minutes (Fig. 1A, D). Following this initial acidosis sea bass completely 400 restored blood pH to control levels after ~135 minutes (Fig. 1A, D). Blood pH   This was driven by a switch from a small apparent HCO3excretion to a large 429 apparent HCO3uptake ( Fig. 2A). There were no significant differences in TAmm 430 excretion (Fig. 2B). minutes of exposure to hypercapnia (Fig. 3A, B exposure levels (Fig. 3B).
450 Sea bass exposed to hypercapnia also experienced a ~25% increase in 451 haemoglobin levels (Fig. 3D), at ~10 minutes and ~ 135 minutes compared to

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To test the influence of environmental availability of [HCO3 -] on acid-base 467 regulation, a group of sea bass were exposed to hypercapnia in low alkalinity 468 seawater. These sea bass were unable to compensate for a respiratory acidosis 469 when exposed to acute hypercapnia for ~135 minutes (Fig. 4A). Blood pH was accumulate HCO3when exposed to environmental hypercapnia for ~135 475 minutes (Fig. 4C). Indeed, the 11.8 mM increase in plasma [HCO3 -] (95% CI = 476 10.6-12.9 mM) followed the predicted non-bicarbonate buffering line (Fig. 4D). between European sea bass in control conditions (n = 10, Time = 0), exposed to 480 hypercapnia for ~10 minutes (n = 8) in normal (~2800 µM) total alkalinity (TA) 481 seawater, exposed to hypercapnia for ~135 minutes in normal (~2800 µM) TA seawater (n = 9), and exposed to hypercapnia for ~135 minutes in low (~200 µM) 483 TA seawater (n =8). Significant differences between parameters at each time  regulatory response after exposure to 1 kPa CO2 (Fig. 8). Of these species, only 647 the Japanese amberjack (Seriola quinqueradiata) was able to restore blood pHe 648 faster than sea bass (~60 min vs ~135 min; Fig. 8B). The remaining four species 649 regulated blood pHe between 3 and 24 h post CO2 exposure ( Figure 8C, D, E, F). Overall, our study highlights the capacity of European sea bass to rapidly (2 701 hours) regulate blood and erythrocyte acid-base status and O2 transport capacity 702 upon exposure to a pronounced and sudden increase in environmental CO2 703 levels. Sea bass' ability to rapidly upregulate H + excretion appears to be mediated 704 via the increased exposure of NHE3-containing apical surface area of gill 705 ionocytes, rather than changes in NHE3 or NKA protein abundance or 706 localisation. Additionally, sea bass erythrocyte pHi is regulated even more rapidly 707 than blood pH (40 minutes), which enables them to quickly restore the affinity of 708 haemoglobin for O2, and therefore blood O2 transport capacity during exposure 709 to elevated CO2. In conjunction, these acid-base regulatory responses will 710 minimise the impact of pronounced and rapidly fluctuating CO2 in their natural 711 environments, and so may prevent disruption of energetically costly activities 712 such as foraging or digestion, and may make sea bass more resilient to impacts 713 of hypoxia and additional stressors during acute periods of hypercapnia. This is 714 an avenue where we believe further research effort is necessary.