However, environmental CO2 variation cannot be the sole driver for enhanced acidCbase regulatory capacities in all species. with commercial pellet (Horizon 80, Skretting) with a supplement of chopped frozen mussel (with a dose of 100?mg?l?1 benzocaine. Anaesthetic was introduced into the isolation box without any visual or physical disturbance to sea bass, thus eliminating capture or handling stress prior to sampling. When sea bass were moderately anaesthetised (no response to physical stimuli and cessation of gill ventilation), they were immediately transferred to a gill irrigation tank (made up of 40?mg?l?1 benzocaine to maintain anaesthesia) in which gill ventilation was artificially maintained by a micro-pump. Once gill water flow was stable (gill operculum just open and exhalant water flow just visualised), blood was sampled via caudal vessel puncture using a heparinised 1?ml syringe. The gill irrigation tank used was filled with water from the header tank and maintained at an appropriate is the volume of water (l) in the isolation tank (after the initial sample is taken), is the mass of the sea bass (kg), is the duration of the flux period (h), and [and 93?kDa in rabbit and planes allowed us to assess intracellular localisation, and to identify the image slice that captured the entire apical surface (typically, the second slice from the top of the cell). Next, the ionocyte apical surface area (identified by NHE3 immunofluorescence signal) was quantified using FIJI (Schindelin et al., 2012). The apical surface area for control and hypercapnia-exposed fish (tests were conducted on IKK-16 least-square means generated by package emmeans (https://CRAN.R-project.org/package=emmeans), with Tukey adjusted testing. Measurements of blood pH and comparisons made with Dunn’s test from package FSA (https://github.com/droglenc/FSA), using BenjaminiCHochberg corrections for multiple comparisons. As a result of unusually high measurements of plasma [Cl?] and [Na+] in some samples, a ROUT test was conducted (and plane visualisation, we found no evidence of NHE3 intracellular localisation (Fig.?5B,Bi,C,Ci). Blind analysis revealed the ionocyte’s apical surface area (based on the NHE3 signal) significantly increased, almost doubling from IKK-16 3.340.17 to 6.360.49?m2, after exposure to 135?min of hypercapnia (one-tailed (Blondeau-Bidet et al., 2019). This is puzzling because they used the same species, comparable environmental salinity and the same antibodies as in our study. Additionally, that study was able to detect NHE3 mRNA in gills from the same specimens. We do not know the reasons for this discrepancy between positive gene expression but a lack of immunostaining for the protein itself, but we speculate this might be due to differences between populations, feeding and exercise regimes, immunostaining protocols, or reagents, potentially coupled with a disconnect between mRNA and protein expression. To investigate the mechanisms used by sea bass to enhance acid excretion, we examined whether changes in gill NKA and NHE3 occur after acute (135?min) exposure IKK-16 to hypercapnia. Gill NKA and NHE3 protein abundance did not change, ruling out increased protein synthesis as the mechanism responsible for the observed upregulation in acid excretion; this is not surprising considering the short time frame of our experiments. We also examined the potential translocation of pre-existing NKA and NHE3 to the ionocyte basolateral and apical membranes, respectively. Such mechanisms upregulate acidCbase regulatory ion transport in elasmobranchs (Roa et al., MST1R 2014; Tresguerres et al., 2005, 2006, 2007b) and hagfish (Parks et al., 2007; Tresguerres et al., 2007a); however, NKA and NHE3 protein abundance in the gill membrane fraction of European sea bass gills was also unchanged, ruling out NKA and NHE3 translocation in our experiments. Finally, we hypothesised that sea bass could have remodelled the apical membrane of.
