We examined the involvement of mitochondria-rich (MR) cells in ion uptake through gill epithelia in freshwater-adapted killifish Fundulus heteroclitus, by morphological observation of MR cells and molecular identification of the vacuolar-type proton pump

narrow physiological range, equivalent to about one-third seawater osmolality. The gills, kidney and intestine are important osmoregulatory organs in fishes, creating ionic and osmotic gradients between the body fluid and external environments (Evans, 1993). It is well established that gill mitochondria-rich (MR) cells, or chloride cells, are responsible for salt secretion in seawater-adapted fish. In freshwateradapted teleosts, by contrast, active ion absorption from a hyposmotic environment is necessary to compensate for the constant diffusional loss of ions through the gill epithelia (McCormick, 1995). The killifish Fundulus heteroclitus is a euryhaline species that can be adapted to a wide range of salinities (Griffith, 1974; Hardy, 1978). Killifish branchial MR cells are larger in freshwater than in seawater (Katoh et al., 2001). By contrast, in most fish examined so far, seawater-type MR cells are more developed in terms of cell size, extension of the tubular system, density of mitochondria and Na+/K+-ATPase activity (Langdon and Thorpe, 1985; Richman et al., 1987; McCormick, 1995; Uchida et al., 1996, 2000; Sasai et al., 1998). In freshwater-adapted killifish, the apical membrane of branchial MR cells show projections with microvilli that expand the apical surface area, suggesting active ion absorption through MR cells (Katoh et al., 2001). The Na+/H+-exchanger (NHE) in the apical membrane of gill MR cells has been advocated as the major pathway for Na+ uptake and H+ excretion in freshwater teleosts. However, it is now considered less likely that Na+ uptake occurs via NHE, since the driving force for such uptake is lacking in this model (Lin and Randall, 1993). Meanwhile, an alternative model incorporating the vacuolar-type proton pump (V-ATPase) and a conductive Na+ channel has been proposed as the Na+absorbing mechanism. In this model, V-ATPase in the apical 793 The Journal of Experimental Biology 206, 793-803 © 2003 The Company of Biologists Ltd doi:10.1242/jeb.00159