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

Abstract

Fish in coastal ecosystems can be exposed to acute variations in CO2 of between 0.2 and 1 kPa CO2 (2000-10,000 µatm). Coping with this environmental challenge will depend on the ability to rapidly compensate for 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 sudden exposure to ∼1 kPa CO2, European sea bass (Dicentrarchus labrax) completely regulate erythrocyte intracellular pH within ∼40 min, thus restoring haemoglobin-O2 affinity to pre-exposure levels. Moreover, blood pH returned to normal levels within ∼2 h, 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 mmol l-1. 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, probably 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.

Keywords: Gill plasticity; Hypercapnia; Ionocytes; O2 transport; Respiratory acidosis.

Fig. 1.

Changes in blood chemistry between European sea bass in control conditions and after exposure to ∼0.9 kPa CO₂ in normal alkalinity and low alkalinity seawater. Fish were exposed to control conditions [∼0.05 kPa CO₂, total alkalinity (TA) ∼2800 µmol l⁻¹, time=0, n=10], or ∼0.9 kPa CO₂ for ∼10 min (n=8), ∼40 min (n=9) and ∼135 min (n=9) in normal alkalinity (TA ∼2800 µmol l⁻¹) seawater and ∼135 min in low alkalinity (TA ∼200 µmol l⁻¹) seawater. (A) Blood pH, (B) plasma P_CO2 and (C) plasma HCO₃⁻. Box plots show median (horizontal line), upper and lower quartiles (box) and 1.5× interquartile range (whiskers); circles are individual data points. Significant differences between control fish and fish exposed to ∼0.9 kPa CO₂ in normal TA conditions are indicated by different lowercase letters at each time point (A: Dunn's test, P<0.05; B: pairwise comparison using Benjamini–Hochberg correction, P<0.05; C: pairwise comparison of least square means, P<0.05). Significant differences between sea bass exposed to ∼0.9 kPa CO₂ for ∼135 min in normal TA seawater (black) and low TA seawater (red) are indicated by asterisks (*P<0.05 or ***P<0.001) (A: two-sample t-test, P<0.001; B: two-sample t-test, P<0.05; C: Welch's t-test, P<0.001). (D) Combined changes of all three acid–base parameters are expressed as a pH/HCO₃⁻/P_CO2 diagram for sea bass in normal TA and low TA at different time points (blue dashed line indicates estimated non-bicarbonate blood buffer line based on equations from Wood et al., 1982). Values represent means±s.e.m.

Fig. 2.

Changes in flux measurements between European sea bass in control conditions and after ∼135 min exposure to hypercapnia. Fish were exposed to control conditions (0.06 kPa CO₂, n=7) or to ∼135 min hypercapnia (∼0.77 kPa CO₂, n=8). (A) Excretion of HCO₃⁻, (B) excretion of total ammonia (T_amm) and (C) net acid–base flux. Significant differences in parameters are indicated by different lowercase letters (Student's t-test, P<0.05).

Fig. 3.

Changes in oxygen transport capacity between European sea bass in control conditions (∼0.05 kPa CO₂, time=0) and after exposure to ∼0.9 kPa CO₂ for ∼10, ∼40 and ∼135 min. (A) Erythrocyte intracellular pH (pH_i), (B) haemoglobin (Hb)–O₂ binding affinity (P₅₀), (C) haematocrit (Hct) and (D) Hb level. Significant differences between parameters at each time point are indicated by different lowercase letters (A,C,D: pairwise comparisons of least square means, P<0.05; B: Dunn's test, P<0.05).

Fig. 4.

Comparison of plasma ion concentrations between European sea bass in control conditions and exposed to hypercapnia in normal or low TA seawater. Fish were exposed to control conditions (n=7, time=0), or hypercapnia for ∼10 min (n=8), ∼40 min (n=9) or ∼135 min (n=7) in normal (∼2800 µmol l⁻¹) TA seawater or for ∼135 min in low (∼200 µmol l⁻¹) TA seawater (n=6). (A) Plasma [Cl⁻] and (B) plasma [Na⁺]. Significant differences between [Cl⁻] in sea bass in normal TA seawater at each time point are indicated by different lowercase letters (pairwise comparison of least squares means, P<0.05). The significant difference between [Cl⁻] in sea bass exposed to hypercapnia for ∼135 min in normal TA and low TA seawater is indicated by asterisks (two sample t-test, ***P<0.001). No significant differences were observed in [Na⁺]. Insets in A and B show correlation between plasma [Cl⁻] and [HCO₃⁻] and between plasma [Na⁺] and [H⁺], respectively. τ and P-values represent results of Kendall's tau correlation. Shaded area represents 95% confidence interval (CI) of linear regression between measures. For insets and measurements taken after ∼135 min of exposure to hypercapnia, the colour indicates the TA treatment (i.e. black, normal TA; red, low TA).

Fig. 5.

Comparison of Na⁺/K⁺-ATPase (NKA) and Na⁺/H⁺ exchanger 3 (NHE3A) protein levels in European sea bass in control conditions and exposed to hypercapnia. (A) European sea bass gill ionocytes express abundant basolateral NKA (Ai, red) and apical NHE3 (Aii, green). (B–D) Comparison of gill ionocytes between fish exposed to control conditions (∼0.06 kPa CO₂; B) and ∼0.77 kPa CO₂ for ∼135 min (C) revealed no changes in intracellular localisation, but hypercapnia-exposed fish had significantly wider apical surface area (D) (n=5 per treatment, one-tailed t-test, P=0.001). The purple line in B and C denotes the slice at which Bi and Ci were imaged. Nuclei (blue) are stained with DAPI. MV, microvilli; AP, apical pit; N, nuclei.

Fig. 6.

Comparison of blood pH/HCO₃⁻/P_CO2 across marine teleost species. (A) European sea bass, Dicentrarchus labrax (present study), (B) Japanese amberjack, Seriola quinqeradiata (Hayashi et al., 2004; re-plotted raw data provided by personal communication with Dr Atsushi Ishimatsu, Can Tho University), (C) Japanese flounder, Paralichthys olivaceus (Hayashi et al., 2004), (D) conger eel, Conger (Toews et al., 1983), (E) coho salmon, Oncorhynchus kisutch (Perry, 1982), and (F) Atlantic cod, Gadus morhua (Larsen et al., 1997). The corresponding blood pH and HCO₃⁻ of each species ∼2 h after 1 kPa CO₂ exposure is indicated to allow direct comparisons with European sea bass. Times below the relevant point indicate when blood pH was not statistically different from pre-exposure levels for each species. The time course of the acid–base response after 2 h is indicated by a dashed black line. The dashed blue line is an approximated non-HCO₃⁻ buffer line based on the mean Hct of sea bass from the present study and calculated using the equation for rainbow trout from Wood et al. (1982).