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First published online June 15, 2007
Journal of Experimental Biology 210, 2320-2332 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.005041

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Salinity-stimulated changes in expression and activity of two carbonic anhydrase isoforms in the blue crab Callinectes sapidus

Laetitia Serrano, Kenneth M. Halanych and Raymond P. Henry^*

Department of Biological Sciences, 101 Life Science Building, Auburn University, Auburn, AL 36849, USA

View larger version (47K): [in this window] [in a new window]   Fig. 1. Nucleotide and deduced amino acid sequences of CasCAg (A) and CasCAc (B) cDNA from Callinectes sapidus gills (GenBank EF375491 and GenBank EF375490, respectively). Both sequences are numbered at the end of each line. The grey shading corresponds to forward and reverse specific primers used to performed qPCR. Asterisks indicate stop codon. Putative N-glycosylation motifs are boxed. Putative polyadenylylation signal is underlined.

View larger version (253K): [in this window] [in a new window]   Fig. 2. Multiple alignment of translated amino acid sequences of Callinectes sapidus CasCAg and CasCAc isoforms (GenBank EF375491 and GenBank EF375490, respectively) with partial CA isoforms sequences from the crab Carcinus maenas (CamCA1 and CamCA2) (Henry et al., 2003) and with other well-characterized -CA isoforms published in GenBank from invertebrates such as the insects Aedes aegypti (AeaCA: AF395662) and Anopheles gambiae (AngCA: DQ518576) and from vertebrates such as the batrachia Xenopus laevis (XelCA: BC042287), the agnatha Petromyzon marinus (DQ157849), the teleostei Tribolodon hakonensis (TrhCA: AB055617) and the mammal Homo sapiens (HosCAI: M33987; HosCAII: NM000067; HosCAIII: NM005181; HosCAIV: NM000717; HosCAV: L19297; HosCAVI: NM001215; HosCAVII: BC033865). Intensity of shading indicates degree of similarity: dark background: 100% agreement; medium dark background: >50% agreement; light background: >30% agreement. Symbols under sequences: + indicate the 36 putative active site residues; z indicates the zinc-binding histidine residues; black circles localize the active-site hydrogen bond network; and the most common proton shuttle group is boxed (Hewett-Emmett and Tashian, 1996).

View larger version (5K): [in this window] [in a new window]   Fig. 3. Hemolymph (solid line) and diluted seawater (broken line) osmolality (mOsm kg H2O-1) for Callinectes sapidus acclimated to 35 p.p.t. (t=0 h) and transferred to 15 p.p.t. for 28 days (d). Values are means ± s.e.m., N=4–12.

View larger version (7K): [in this window] [in a new window]   Fig. 4. CA activity (µmol CO2 mg protein-1 min-1) in anterior (G3, white bars) and posterior (G7, dark bars) gills of Callinectes sapidus acclimated to 35 p.p.t. (t=0) and at various times after being transferred to 15 p.p.t. Values are means ± s.e.m., N=6–18. Letters above bars indicate statistical comparisons within G7 across the time course: two columns not sharing at least one same letter are statistically different (P<0.05, two-way ANOVA, Tukey's post-hoc test). There were no significant differences among G3.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Relative expression of CasCAg (A) and CasCAc (B) mRNA in anterior (G3, white bars) and posterior (G7, black bars) gills of Callinectes sapidus acclimated to 35 p.p.t. (t=0) and at various times after being transferred to 15 p.p.t. Values are means ± s.e.m., N=4–14. Letters above bars indicate statistical comparisons within G7 across the time course: two columns not sharing at least one same letter are statistically different (P<0.05, two-way ANOVA, Tukey's and Fisher LSD's post-hoc tests). There were no significant differences among G3.

View larger version (9K): [in this window] [in a new window]   Fig. 6. Relative amounts of CasCAg and CasCAc mRNA transcripts in (A) anterior (G3) and (B) posterior (G7) gills of Callinectes sapidus acclimated to 35 p.p.t. (t=0) and at various times after being transferred to 15 p.p.t. Values are means ± s.e.m., N=4–5.

View larger version (10K): [in this window] [in a new window]   Fig. 7. Relative Na+/K+-ATPase -subunit (NAK) expression in anterior (G3, white bars) and posterior (G7, black bars) gills of Callinectes sapidus acclimated to 35 p.p.t. (t=0) and at various times after being transferred to 15 p.p.t. Values are means ± s.e.m., N=4–14. Letters above bars indicate statistical comparisons within G7 across the time course: two columns not sharing at least one same letter are statistically different (P<0.05, two-way ANOVA, Fisher LSD's post-hoc test). There were no significant differences among G3.

View larger version (8K): [in this window] [in a new window]   Fig. 8. Relative arginine kinase (AK) mRNA expression in anterior (G3, white bars) and posterior (G7, black bars) gills of Callinectes sapidus acclimated to 35 p.p.t. (t=0) and at various times after being transferred to 15 p.p.t. Values are means ± s.e.m., N=4–5. Letters above bars indicate statistical comparisons within G7 across the time course: two columns not sharing at least one same letter are statistically different (P<0.05, two-way ANOVA, Fisher LSD's post-hoc test). There were no significant differences among G3.