DOI: 10.5061/dryad.sf7m0cggn
title: Maintenance of sodium ion balance in rainbow trout exposed to moderately acidic water is achieved through reduction of sodium efflux

description: In most freshwater fishes, exposure to acidic water decreases rates of
 active ion uptake and increases rates of passive ion efflux. To survive in
 low pH conditions, fish must possess mechanisms to maintain net ion flux
 rates close to zero. In this study, rainbow trout (Oncorhynchus mykiss)
 exposed to moderate acidity (pH = 4.5 or 5.5) in hard water demonstrated
 Na+ influx inhibition relative to fish held in control conditions (pH =
 8.0). However, Na+ efflux rates were significantly lower in fish exposed
 to low pH, which minimized the net loss of Na+. This represents a
 relatively novel mechanism utilized by an acid sensitive species to
 maintain ion balance in low pH and appeared to be a result of reduced gill
 paracellular permeability, though we did not observe any significant
 differences in the transcript abundance of 6 different tight junction
 genes in response to low pH exposure. Overall, we conclude that acid
 tolerance may be dictated by differences in efflux rates in fishes that,
 like the rainbow trout, possess acid sensitive mechanisms of ion
 absorption.
description: # Maintenance of sodium ion balance in rainbow trout exposed to moderately
 acidic water is achieved through reduction of sodium efflux acid_trout.csv
 \[Access this dataset on
 Dryad]([https://doi.org/10.5061/dryad.sf7m0cggn](https://doi.org/10.5061/dryad.sf7m0cggn)) Dataset in "acid_trout.csv" contains data presented in figures 1, 2, 3, and 5, and also includes mass for each fish (see below for unit descriptions for the presented data). Data in rows 2 to 85 were obtained in Experimental Series 1 [fish_id = [1, 84] Data in rows 86-121 were collected in Experimental Series 2 [fish_id = [113, 148] Data in rows 122-156 were collected in Experimental Series 3 [fish_id = [157, 192] Description of the data and file structure for "acid_trout.csv" Column labels: fish_id = Fish ID used to organize multiple endpoints measured in the same fish (e.g. Series 1 and 3) ph = pH treatment time = duration of the pH exposure (h) sodium_influx = Na+ influx (umol/g/h); presented in Fig. 1A; empty cells in rows 86-156 are a result of this parameter only being measured in Series 1 (fish_id = [1, 84]) sodium_efflux = Na+ efflux (umol/g/h); presented in Fig. 1B; empty cells in rows 86-156 are a result of this parameter only being measured in Series 1 (fish_id = [1, 84]) sodium_netflux = Na+ net flux (umol/g/h); presented in Fig. 1C; empty cells in rows 86-156 are a result of this parameter only being measured in Series 1 (fish_id = [1, 84]) body_sodium = Tissue [Na+] (umol/g); presented in Fig. 1D; empty cells in rows 86-156 are a result of this parameter only being measured in Series 1 (fish_id = [1, 84]) potassium_netflux = K+ net flux (umol/g/h); presented in Fig. 2B; empty cells in rows 86-156 are a result of this parameter only being measured in Series 1 (fish_id = [1, 84]) chloride_netflux = Cl- net flux (umol/g/h); presented in Fig. 2A; empty cells in rows 86-156 are a result of this parameter only being measured in Series 1 (fish_id = [1, 84]) ammonia_netflux = Ammonia net flux (umol/g/h); presented in Fig. 2C; empty cells in rows 86-156 are a result of this parameter only being measured in Series 1 (fish_id = [1, 84]) peg_clearance = PEG-4000 clearance rate (uL/g/h); presented in Fig. 3A; empty cells in rows 2-84 and 122-156 are a result of this parameter only being measured in Series 2 (fish_id = [113, 148]) drinking rate = DATA NOT PRESENTED IN PAPER; empty cell M99 is a missing data point = n/a blood_ph = PCO2-Corrected Blood pH; presented in Fig. 5D; empty cells in rows 2-121 are a result of this parameter only being measured in Series 3 (fish_id = [157, 192]) plasma_bicarb = PCO2-Corrected Plasma [HCO3-] (mmol/L); presented in Fig. 5E; empty cells in rows 2-121 are a result of this parameter only being measured in Series 3 (fish_id = [157, 192]) plasma_sodium = Plasma [Na+] (mmol/L); presented in Fig. 5B; empty cells in rows 2-121 are a result of this parameter only being measured in Series 3 (fish_id = [157, 192]) plasma_chloride = Plasma [Cl-] (mmol/L); presented in Fig. 5C; empty cells in rows 2-121 are a result of this parameter only being measured in Series 3 (fish_id = [157, 192]) muscle_sodium = Muscle [Na+] (mmol/L); presented in Fig. 5A; empty cells in rows 2-121 are a result of this parameter only being measured in Series 3 (fish_id = [157, 192]) Data for blood_ph, plasma_bicarb, plasma_sodium, plasma_chloride, and muscle_sodium were obtained from the same individuals (same fish_id label) as those in "tj_genes.csv" and "tj_ct_refgenes.csv" Note that n/a = not available Code/Software Figure generation and statistical analyses from data presented in "acid_trout.csv" were executed using the scripts found in "zimmer_cjfas_20241007_330PM.R" using R Studio version 325 programming language (version 4.3.2) in RStudio (2023.12.1, Build 402). \============================================== tj_genes.csv \[Access this dataset on Dryad]([https://doi.org/10.5061/dryad.sf7m0cggn](https://doi.org/10.5061/dryad.sf7m0cggn)) Dataset in "tj_genes.csv" contains gene expression data presented in figure 4 for each gene, organized by fish_id, time (duration exposure), and ph (pH treatment). Description of the data and file structure for "tj_genes.csv" Data presented in columns cldn8_2, cldn28, oclna, oclnb, zo1, and marveld2a are relative transcript abundance values (unitless) for cldn 8.2 (Fig. 4A), cldn28b (Fig. 4B), oclna (Fig. 4C), oclnb (Fig. 4D), tjp1b (Fig. 4E), and marveld2a (Fig. 4F). Relative transcript abundance was calculated from raw ct values using the ddCt method (Livak and Schmittgen, 2001; see paper for reference), normalizing the expression of target genes to the reference gene ef1a that did not vary significantly as a function of time of pH (see manuscript and file "tj_ct_refgenes.csv" for raw ct values of reference genes that were analyzed in this study). Transcript abundance was expressed relative to the abundance measured in fish exposed to pH 8.0 for 24 h. Data in "tj_genes.csv" were obtained from the same individuals (same fish_id) as those presented in "acid_trout.csv" and "tj_ct_refgenes.csv" Code/Software Figure generation and statistical analyses from data presented in "tj_genes.csv" were executed using the scripts found in "zimmer_cjfas_20241007_330PM.R" using R Studio version 325 programming language (version 4.3.2) in RStudio (2023.12.1, Build 402). \============================================== tj_ct_refgenes.csv \[Access this dataset on Dryad]([https://doi.org/10.5061/dryad.sf7m0cggn](https://doi.org/10.5061/dryad.sf7m0cggn)) Dataset in "tj_ct_refgenes.csv" contains raw ct values for two reference genes (ef1a and 18s) and their geometric mean, that were all tested to verify a stable reference gene. The expression of ef1a was ultimately used as the reference gene in the ddCt calculations that generated the relative transcript abundance data in "tj_genes.csv" that is presented in Fig. 4 of the paper. Description of the data and file structure for "tj_genes.csv" Data presented in columns ef1a and 18S are raw Ct values for these two reference genes, organized by fish_id that corresponds to the individuals presented in both "tj_genes.csv" and "acid_trout.csv". Column "genorm" is the geometric mean of the Ct values for ef1a and 18S for each individual fish_id. Code/Software Statistical analyses of data presented in "tj_ct_refgenes.csv" were executed using the scripts found in "zimmer_cjfas_20241007_330PM.R" using R Studio version 325 programming language (version 4.3.2) in RStudio (2023.12.1, Build 402) and the result of the statistical analyses is presented in text under "Analytical techniques and calculations".