ABSTRACT In vertebrate salt-secreting epithelia, Na + moves passively down an electrochemical gradient via a paracellular pathway. We assessed how this pathway is modified to allow Na + secretion in hypersaline environments. Mummichogs ( Fundulus heteroclitus ) acclimated to hypersaline [2× seawater (2SW), 64‰] for 30 days developed invasive projections of accessory cells with an increased area of tight junctions, detected by punctate distribution of CFTR (cystic fibrosis transmembrane conductance regulator) immunofluorescence and transmission electron miscroscopy of the opercular epithelia, which form a gill-like tissue rich in ionocytes. Distribution of CFTR was not explained by membrane raft organization, because chlorpromazine (50 μmol l −1 ) and filipin (1.5 μmol l −1 ) did not affect opercular epithelia electrophysiology. Isolated opercular epithelia bathed in SW on the mucosal side had a transepithelial potential ( V t ) of +40.1±0.9 mV ( N =24), sufficient for passive Na + secretion (Nernst equilibrium voltage≡ E Na =+24.11 mV). Opercular epithelia from fish acclimated to 2SW and bathed in 2SW had higher V t of +45.1±1.2 mV ( N =24), sufficient for passive Na + secretion ( E Na =+40.74 mV), but with diminished net driving force. Bumetanide block of Cl − secretion reduced V t by 45% and 29% in SW and 2SW, respectively, a decrease in the driving force for Na + extrusion. Estimates of shunt conductance from epithelial conductance ( G t ) versus short-circuit current ( I sc ) plots (extrapolation to zero I sc ) suggested a reduction in total epithelial shunt conductance in 2SW-acclimated fish. In contrast, the morphological elaboration of tight junctions, leading to an increase in accessory-cell–ionocyte contact points, suggests an increase in local paracellular conductance, compensating for the diminished net driving force for Na + and allowing salt secretion, even in extreme salinities.

Studies of euryhaline crustaceans have identified conserved osmoregulatory adaptions allowing hyper-osmoregulation in dilute waters. However, previous studies have mainly examined decapod brachyurans with marine ancestries inhabiting estuaries or tidal creeks on a seasonal basis. Here, we describe osmoregulation in the atyid Halocaridina rubra , an endemic Hawaiian shrimp of freshwater ancestry from the islands' anchialine ecosystem (coastal ponds with subsurface freshwater and seawater connections) that encounters near-continuous spatial and temporal salinity changes. Given this, survival and osmoregulatory responses were examined over a wide salinity range. In the laboratory, H. rubra tolerated salinities of ~0–56‰, acting as both a hyper- and hypo-osmoregulator and maintaining a maximum osmotic gradient of ~868 mOsm kg −1 H 2 O in freshwater. Furthermore, hemolymph osmolality was more stable during salinity transfers relative to other crustaceans. Silver nitrate and vital mitochondria-rich cell staining suggest all gills are osmoregulatory, with a large proportion of each individual gill functioning in ion transport (including when H. rubra acts as an osmoconformer in seawater). Additionally, expression of ion transporters and supporting enzymes that typically undergo upregulation during salinity transfer in osmoregulatory gills (i.e. Na + /K + -ATPase, carbonic anhydrase, Na + /K + /2Cl − cotransporter, V-type H + -ATPase and arginine kinase) were generally unaltered in H. rubra during similar transfers. These results suggest H. rubra (and possibly other anchialine species) maintains high, constitutive levels of gene expression and ion transport capability in the gills as a means of potentially coping with the fluctuating salinities that are encountered in anchialine habitats. Thus, anchialine taxa represent an interesting avenue for future physiological research.

SUMMARY Bull sharks, Carcharhinus leucas , are one of only a few species of elasmobranchs that live in both marine and freshwater environments. Osmoregulation in euryhaline elasmobranchs is achieved through the control and integration of various organs (kidney, rectal gland and liver) in response to changes in environmental salinity. However, little is known regarding the mechanisms of ion transport in the gills of euryhaline elasmobranchs and how they are affected by osmoregulatory challenges. This study was conducted to gain insight into the branchial ion and acid-base regulatory mechanisms of C. leucas by identifying putative ion transporters and determining whether their expression is influenced by environmental salinity. We hypothesised that expression levels of the Na + /K + -ATPase (NKA) pump, Na + /H + exchanger 3 (NHE3), vacuolar-type H + -ATPase (VHA) and anion exchanger pendrin (PDN) would be upregulated in freshwater (FW) C. leucas . Immunohistochemistry was used to localise all four ion transporters in gills of bull sharks captured in both FW and estuarine/seawater (EST/SW) environments. NHE3 immunoreactivity occurred in the apical region of cells with basolateral NKA expression whereas PDN was apically expressed in cells that also exhibited basolateral VHA immunoreactivity. In accordance with our hypotheses, quantitative real-time PCR showed that the mRNA expression of NHE3 and NKA was significantly upregulated in gills of FW-captured C. leucas relative to EST/SW-captured animals. These data suggest that NHE3 and NKA together may be important in mediating branchial Na + uptake in freshwater environments, whereas PDN and VHA might contribute to Cl - /HCO 3 - transport in marine and freshwater bull shark gills.

SUMMARY In the gills of freshwater teleost fishes, vacuolar proton-ATPase (V-H + -ATPase) is found on the apical membrane of pavement and chloride (Na + /K + -ATPase-rich) cells, and is an important transporter for energizing Na + uptake and H + excretion. In the gills of elasmobranch fishes, the V-H + -ATPase has not been extensively studied and its expression in freshwater individuals has not been examined. The goals of this study were to examine the effects of environmental salinity on the expression of V-H + -ATPase in the gills of an elasmobranch (the Atlantic stingray, Dasyatis sabina ) and determine if V-H + -ATPase and Na + /K + -ATPase are expressed in the same cells. We found that gills from freshwater stingrays had the highest relative abundance of V-H + -ATPase and greatest number of V-H + -ATPase-rich cells, using immunoblotting and immunohistochemistry, respectively. When freshwater animals were acclimated to sea water for 1 week, V-H + -ATPase abundance and the number of V-H + -ATPase-rich cells decreased significantly. Atlantic stingrays from seawater environments were characterized by the lowest expression of V-H + -ATPase and least number of V-H + -ATPase-rich cells. In contrast to teleost fishes, localization of V-H + -ATPase in freshwater stingray gills was not found in pavement cells and occurred on the basolateral membrane in cells that are presumably rich in mitochondria. In freshwater stingrays acclimated to sea water and seawater stingrays, V-H + -ATPase localization appeared qualitatively to be stronger in the cytoplasm, which may suggest the transporter was stored in vesicles. Using a double-immunolabeling technique, we found that V-H + -ATPase and Na + /K + -ATPase occurred in distinct cells, which suggests there may be two types of mitochondrion-rich cells in the elasmobranch gill epithelium. Based on these findings, we propose a unique model of NaCl and acid–base regulation where the V-H + -ATPase-rich cells and Na + /K + -ATPase-rich cells are the sites of Cl – uptake/HCO 3 – excretion and Na + uptake/H + excretion, respectively.