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First published online September 16, 2005
Journal of Experimental Biology 208, 3627-3636 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01820
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Induction of branchial ion transporter mRNA expression during acclimation to salinity change in the euryhaline crab Chasmagnathus granulatus

Carlos M. Luquet1,2,*, Dirk Weihrauch3, Mihaela Senek4 and David W. Towle5,{dagger}

1 Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pab. II, Ciudad Universitaria, C1428EHA Buenos Aires, Argentina
2 CONICET (Consejo Nacional de Investigaciones Cientificas y Tecnicas), Rivadavia 1917, C1033AAJ Buenos Aires, Argentina
3 Department of Animal Physiology, University of Osnabrueck, 49076 Osnabrueck, Germany
4 College of the Atlantic, Bar Harbor, ME 04609, USA
5 Mount Desert Island Biological Laboratory, Salsbury Cove, ME 04672, USA



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  		Fig. 1. Amplification of target transporter and housekeeping cDNAs from Chasmagnathus granulatus gill by conventional PCR. Expected sizes of amplification products are indicated in parentheses: lane 1, DNA ladder; lane 2, arginine kinase (1088 bp); lane 3, DNA ladder; lane 4, V-type H+-ATPase B-subunit (390 bp); lane 5, Na+/K+/2Cl- cotransporter (2100 bp); lane 6, Na+/K+-ATPase {alpha}-subunit (703 bp); lane 7, DNA ladder. Sizes of ladder standards are indicated at the right; each gel image was adjusted to give parallel ladder bands.

 


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  		Fig. 2. Partial nucleotide and translated amino acid sequences of cDNAs amplified from Chasmagnathus granulatus gill, shown with identifications derived from BLASTX analysis. (A) arginine kinase; (B) V-type H+-ATPase B-subunit; (C) Na+/K+/2Cl- cotransporter; (D) Na+/K+-ATPase {alpha}-subunit. GenBank accession numbers are given in parentheses. Locations of primers used in quantitative PCR are indicated in blue.

 


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  		Fig. 3. Multiple alignment of translated amino acid sequences of transporter and housekeeping cDNAs from Chasmagnathus granulatus with corresponding fragments obtained from GenBank, indicated by species names and accession numbers. Blue background, 100% agreement; green background, 75-80% agreement, yellow background, 50-67% agreement. (A) Arginine kinase: Chasmagnathus granulatus (present study, AF233357), Eriocheir sinensis (AAF43437), Apis mellifera (AF023619), Octopus vulgaris (AB042331); (B) V-type H+-ATPase B-subunit: Chasmagnathus granulatus (present study, AF189783), Eriocheir sinensis (AAF08284), Xenopus laevis (AAH46738), Caenorhabditis elegans (AAF60418), Manduca sexta (AAS38817); (C) Na+/K+/2Cl- cotransporter: Chasmagnathus granulatus (present study, AF548368), Carcinus maenas (AAG62044), Manduca sexta (AAA75600), Squalus acanthias (AAM74966), Mus musculus (AAH38612); (D) Na+/K+-ATPase {alpha}-subunit: Chasmagnathus granulatus (present study, AF548369), Eriocheir sinensis (AAG39936), Apis mellifera (XP_392363), Octopus rubescens (AAQ72761), Xenopus laevis (AAH43743). Asterisks indicate position markers between the numbered sites.

 


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  		Fig. 4. Quantitative PCR analysis of mRNA transcript abundance in anterior (3, 4, 5) and posterior (6, 7, 8) gills of Chasmagnathus granulatus following transfer from 30{per thousand} salinity to 2{per thousand}, normalized to the mean of posterior gills from crabs acclimated to 30{per thousand} seawater (zero-time value). (A) Arginine kinase; (B) V-type H+-ATPase B-subunit; (C) Na+/K+/2Cl- cotransporter; (D) Na+/K+-ATPase {alpha}-subunit. Means and standard errors were calculated from gills 3, 4 and 5 (anterior) and gills 6, 7 and 8 (posterior), with triplicate determinations for each gill preparation. Results of statistical analysis by ANOVA and post hoc comparisons are presented in the text.

 


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  		Fig. 5. Quantitative PCR analysis of mRNA transcript abundance in anterior (3, 4, 5) and posterior (6, 7, 8) gills of Chasmagnathus granulatus following transfer from 30{per thousand} salinity to 45{per thousand}, normalized to the mean of posterior gills from crabs acclimated to 30{per thousand} seawater (zero-time value). (A) Arginine kinase; (B) V-type H+-ATPase B-subunit; (C) Na+/K+/2Cl- cotransporter; (D) Na+/K+-ATPase {alpha}-subunit. Means and standard errors were calculated from gills 3, 4 and 5 (anterior) and gills 6, 7 and 8 (posterior), with triplicate determinations for each gill preparation. Results of statistical analysis by ANOVA and post hoc comparisons are presented in the text.

 




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