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First published online December 3, 2004
Journal of Experimental Biology 207, 4623-4631 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01333

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Ion-motive ATPases and active, transbranchial NaCl uptake in the red freshwater crab, Dilocarcinus pagei (Decapoda, Trichodactylidae)

Dirk Weihrauch^1,*, John Campbell McNamara², David W. Towle³ and Horst Onken⁴

¹ Department of Animal Physiology, University of Osnabrueck, 49076 Osnabrueck, Germany
² Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, Ribeirão Preto 14040-901, São Paulo, Brasil
³ Mount Desert Island Biological Laboratory, Salsbury Cove, ME 04672, USA
⁴ School of Biological Sciences, Washington State University, Pullman, WA 99164-4236, USA

View larger version (50K): [in a new window]   Fig. 1. Agarose gel showing template-dependent, product abundance during the logarithmic phase of PCR amplification. 2 µg total RNA, isolated from the posterior gills of Dilocarcinus pagei, were employed in the reverse transcription assay. Different volumes (0.25, 0.5 or 1 µl) of the resulting cDNA were used to perform 23 PCR amplification cycles. Lane M is a DNA ladder.

View larger version (75K): [in a new window]   Fig. 2. Photomicrographs of transverse, 0.5 µm thick, epoxy-resin sections taken through the anterior and posterior gill lamellae of the freshwater, trichodactylid crab, Dilocarcinus pagei. (A) Microanatomy of a typical, medial lamella from anterior gill no. 4, showing the identical, very thin (2–5 µm) epithelia (e), symmetrically arranged on both sides of the lamella, separated by the ample hemolymph space (h), which contains a discontinuous septum (s) and hemocytes. Both epithelia consist of the very attenuated, lateral, apical flanges (f) that emerge from the pillar cell perikarya (pc) underlying the fine cuticle (c). Bar, 20 µm. (B) Architectural organization of the dense, osmiophilic, central region of a typical, medial lamella from posterior gill no. 7, revealing the highly asymmetrical nature of the distal (d) and proximal (p) epithelia underlying the thickened cuticle (c). The well-developed, distal epithelium measures 3–10 µm in thickness and consists exclusively of the extensive, apical flanges (f) of the pillar cells (pc), populated by numerous apical vesicles (v) and invaginations. The dense, proximal epithelial cells measure 18–20 µm in thickness and are characterized by large, spherical nuclei, numerous basal and apical invaginations (i), and a few apical vesicles. The lamellar septum is absent, and the distal pillar cell perikarya traverse the hemolymph space, abutting directly on the opposing, thick, proximal epithelial cells (arrows). Bar, 20 µm.

View larger version (49K): [in a new window]   Fig. 3. Alignment of the deduced partial amino acid sequence for Dilocarcinus pagei Na+/K+-ATPase -subunit with representative invertebrate and vertebrate sequences, including blue crab Callinectes sapidus (AF327439), fruit fly Drosophila melanogaster (AY069184), zebrafish Danio rerio (NM_131688) and rat Rattus norvegicus (M28647). Alignment was produced using Multalin (Corpet, 1988) and GeneDoc software. Blue color denotes identical amino acids (aa) in all sequences; green, identical aa in four of the five sequences; yellow, identical aa in three of the five.

View larger version (32K): [in a new window]   Fig. 4. Alignment of the deduced partial amino acid sequence for Dilocarcinus pagei V-type H+-ATPase B-subunit with representative invertebrate and vertebrate sequences, including green shore crab Carcinus maenas (AF189779), tobacco hornworm Manduca sexta (X64354), steelhead trout Oncorhynchus mykiss (AAD33861 and rat Rattus norvegicus (NM_057213). Alignment was produced using Multalin (Corpet, 1988) and GeneDoc software. Red color denotes identical amino acids in all sequences; yellow, identical aa in three of the five.

View larger version (19K): [in a new window]   Fig. 5. Phylogenetic relationships among partial nucleotide sequences for the V-type H+-ATPase B-subunit obtained from different brachyuran crabs. An unrooted tree was produced using Multalin (Corpet, 1988) and Drawtree (Felsenstein, 1993).

View larger version (35K): [in a new window]   Fig. 6. Semi-quantitative reverse transcriptase/polymerase chain reaction (RT-PCR) analysis of Na+/K+-ATPase -subunit and V-type H+-ATPase B-subunit mRNA abundance in gill homogenates from Dilocarcinus pagei. In all experiments, PCR was performed using 1 µl cDNA, which was 5% of the cDNA reverse transcribed from 2 µg total RNA. The primer pairs NAK 10F/NAK 16R and DiloHATF1/DiloHATR1 were employed to amplify the Na+/K+-ATPase -subunit and V-type H+-ATPase B-subunit, respectively. (A) Demonstration of template-dependent quantification of Na+/K+-ATPase -subunit (left panels) and V-type H+-ATPase B-subunit (right panels) in anterior and posterior gills of D. pagei. (B) Graphs corresponding to data in A, showing the digitized pixel densities of the PCR products. Blue diamonds, anterior gills; red circles, posterior gills. Values are means ± S.E.M. (N=3).

View larger version (14K): [in a new window]   Fig. 7. Representative time-course of the short-circuit current (Isc) across a distal split lamella from a posterior gill of Dilocarcinus pagei. The vertical current deflections are the responses to 4 mV voltage pulses, and are directly proportional to the transepithelial conductance (Gte, see separate scale bar). Experimental stages: 1, addition of internal ouabain (2 mol l–1); 2, ouabain washout; 3, symmetrical addition of DMSO (0.1%); 4, external concanamycin (10 µmol l–1); 5, internal concanamycin (10 µmol l–1); 6, washout of DMSO and concanamycin, and internal addition of NaCN (5 mmol l–1); 7, NaCN washout.