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1 Biology, McMaster University, Hamilton, Canada
* To whom correspondence should be addressed. E-mail: craigpm{at}mcmaster.ca.
Little is known regarding the ionoregulatory abilities of zebrafish exposed to softwater despite the popularity of this model organism for physiology and aquatic toxicology. We examined genomic and nongenomic changes to gills of zebrafish as they were progressively acclimated from moderately hard freshwater to typical softwater over 7 days and held in softwater for another 7 days. Gills were sampled daily and mRNA expression levels of gill Na+/K+ATPase (NKA) 
1a subunit, epithelium calcium channel (ECaC), carbonic anhydrase-1 & 2 (CA-1; CA-2), Na+, H+-exchanger (NHE-2), V-type proton (H+)-ATPase and copper transport protein (CTR-1) were quantified by real-time PCR. Changes in enzyme activities of gill NKA were determined and protein levels of NKA and ECaC were quantified by Western blotting. Levels of mRNA for ECaC increased 4-fold after day 6, with an associated increase in ECaC protein levels after 1 week in softwater. CA-1 and CA-2 exhibited a 1.5- and 6-fold increase in gene expression on day 6 and 5, respectively. Likewise, there was a 5 fold increase in NHE-2 expression after day 6. Suprisingly, CTR-1 mRNA showed a large transient increase (over 3-fold) on day 6 while H+-ATPase mRNA did not change. These data demonstrate a high degree of phenotypic plasticity in zebrafish gills exposed to an ion-poor environment. This not only enhances our understanding of ionoregulatory processes in fish but also highlights the need for proper experimental design for studies involving pre-acclimation to softwater (e.g. metal toxicity).
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