Teleosts inhabiting fresh water (FW) depend upon ion-absorptive ionocytes to counteract diffusive ion losses to the external environment. A Clc Cl^- channel family member, Clc-2c, was identified as a conduit for basolateral Cl^- transport by Na⁺/Cl^- cotransporter 2 (Ncc2)-expressing ionocytes in stenohaline zebrafish (Danio rerio). It is unresolved whether Clc-2c/clc-2c is expressed in euryhaline species and how extrinsic and/or intrinsic factors modulate branchial clc-2c mRNA. Here, we investigated whether environmental salinity, prolactin (Prl) and osmotic conditions modulate clc-2c expression in euryhaline Mozambique tilapia (Oreochromis mossambicus). Branchial clc-2c and ncc2 mRNAs were enhanced in tilapia transferred from seawater (SW) to FW, whereas both mRNAs were attenuated upon transfer from FW to SW. Next, we injected hypophysectomized tilapia with ovine prolactin (oPrl) and observed a marked increase in clc-2c from saline-injected controls. To determine whether Prl regulates clc-2c in a gill-autonomous fashion, we incubated gill filaments in the presence of homologous tilapia Prls (tPrl₁₇₇ and tPrl₁₈₈). By 24 h, tPrl₁₈₈ stimulated clc-2c expression ~5-fold from controls. Finally, filaments incubated in media ranging from 280 to 450 mosmol/kg for 3 and 6 h revealed that extracellular osmolality exerts a local effect on clc-2c expression; clc-2c was diminished by hyperosmotic conditions (450 mosmol/kg) compared with isosmotic controls (330 mosmol/kg). Our collective results suggest that hormonal and osmotic control of branchial clc-2c contributes to the FW adaptability of Mozambique tilapia. Moreover, we identify for the first time a regulatory link between Prl and a Clc Cl^- channel in a vertebrate.

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