Structural and biochemical correlates of Na+,K+-ATPase driven ion uptake across the posterior gill epithelium of the true freshwater crab, Dilocarcinus pagei (Brachyura, Trichodactylidae).

To better comprehend the structural and biochemical underpinnings of ion uptake across the gills of true freshwater crabs, we performed an ultrastructural, ultracytochemical and morphometric investigation, and kinetically characterized the Na(+),K(+)-ATPase, in posterior gill lamellae of Dilocarcinus pagei. Ultrastructurally, the lamellar epithelia are markedly asymmetrical: the thick, mushroom-shaped, proximal ionocytes contain elongate mitochondria (41% cell volume) associated with numerous (≈14 µm² membrane per µm³cytoplasm), deep invaginations that house the Na(+),K(+)-ATPase, revealed ultracytochemically. Their apical surface is amplified (7.5 µm² µm⁻²)) by stubby evaginations whose bases adjoin mitochondria below the subcuticular space. The apical membrane of the thin, distal ionocytes shows few evaginations (1.6 µm² µm⁻²), each surrounding a mitochondrion, abundant in the cytoplasm below the subcuticular space; basolateral invaginations and mitochondria are few. Fine basal cytoplasmic bridges project across the hemolymph space, penetrating into the thick ionocytes, suggesting ion movement between the epithelia. Microsomal Na(+),K(+)-ATPase specific activity resembles marine crabs but is ≈5-fold less than in species from fluctuating salinities, and freshwater shrimps, suggesting ion loss compensation by strategies other than Na(+) uptake. Enzyme apparent K(+) affinity attains 14-fold that of marine crabs, emphasizing the relevance of elevated K(+) affinity to the conquest of fresh water. Western blotting and biphasic ouabain inhibition disclose two α-subunit isoforms comprising distinct functional isoenzymes. While enzyme activity is not synergistically stimulated by NH(4) (+) and K(+), each increases affinity for the other, possibly assuring appropriate intracellular K(+) concentrations. These findings reveal specific structural and biochemical adaptations that may have allowed the establishment of the Brachyura in fresh water.