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First published online November 2, 2007
Journal of Experimental Biology 210, 3919-3930 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.008342

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Cloning and characterization of AgCA9, a novel -carbonic anhydrase from Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) larvae

Kristin E. Smith, Leslie A. VanEkeris and Paul J. Linser^*

The Whitney Laboratory for Marine Biology, University of Florida, 9505 Ocean Shore Boulevard, Saint Augustine, FL 32080, USA

View larger version (78K): [in this window] [in a new window]   Fig. 1. Alignment of AgCA9 with AgCA6, AgCA10 and AgCA11, formally cloned by the Linser laboratory. Also included are Homo sapiens CAII (HCAII) and Drosophila melanogaster CAI (DrosCAI). Alignment was performed in ClustalX and visualized using Genedoc. Similarity groups were enabled using the default scoring matrix, Blosum 62 matrix. Regions with high similarity are highlighted in blue (100%). Regions of lesser similarity are highlighted in red (>80%) and yellow (>60%). The three histidines essential for -CA activity are indicated with a star (HCAII his-94, his-96, his-119). The black bars indicate antigenic sites against which antibodies (5340 and 5563) were generated.

View larger version (4K): [in this window] [in a new window]   Fig. 2. qPCR was used to measure expression of AgCA9 mRNA in alimentary canal regions – gastric caeca (GC), anterior midgut (AMG), posterior midgut (PMG), Malpighian tubules (MT) and rectum – relative to whole alimentary canal. All values were normalized to an 18s RNA endogenous control. `Whole' was artificially set to a value of 1.0 in this graph, with all other samples expressed as a proportion of `whole'. Results indicate that AgCA9 mRNA is significantly more abundant in the rectum samples (*P<0.001 as determined by one-way analysis of variance). Error bars indicate standard deviation between three individual qPCR runs.

View larger version (78K): [in this window] [in a new window]   Fig. 3. In situ hybridization was used to detect mRNA in whole larval alimentary canals and carcass. A full-length DIG-labeled RNA probe was used to detect AgCA9 mRNA in whole-mount larvae. Antisense probe generated intense staining in the GC, the transitional region between the anterior and posterior midguts (indicated by arrow) and the rectum of the larval alimentary canal, with weaker staining seen in the cells of the PMG (A). Carcass showed the most intense staining in the muscle fibers and fat body (D). Sense probe did not significantly stain any areas of the alimentary canal (B) or carcass (C). For abbreviations, see Fig. 2 legend. Scale bars: 400 µm.

View larger version (70K): [in this window] [in a new window]   Fig. 4. The specificity of the affinity-purified antibody 5563 was verified using a western blot (A) and immunohistochemistry (B–E). Part A shows the results of an antibody adsorption assay. The left panel of the image is the Fast Green staining of total protein to ensure equal loading. Antibody was pre-incubated with peptide prior to detection of protein. The western blot illustrates the detection of protein with pre-immune IgY (lane 1), affinity-purified antibody 5563 IgY (lane 2) and peptide-blocked IgY (lane 3). Images B–E illustrate immunohistochemistry results obtained using affinity-purified antibody 5563 IgY (E) and peptide-blocked IgY (B,C,D) in longitudinal sections of whole larvae embedded in paraffin. The images were generated on a laser confocal microscope (Leica SP2). B and C are images of the same section of larval alimentary canal (without the rectum) with two different fluorochromes (FITC = green = AgCA9; TRITC = red = Na+/K+-ATPase). D and E are images of the rectum positioned such that the anterior end is to the left. Na+/K+-ATPase immunostaining was used as a counter-stain to better visualize the regions of the alimentary canal in images C–E. The pre-adsorbed antibody (B–D) failed to detect AgCA9 protein compared with antibody not treated with peptide (E; also Fig. 6A). Apparent exoskeleton staining is non-specific (B,C; arrows). For abbreviations, see Fig. 2 legend. Scale bars, 150 µm (B,C); 75 µm (D,E).

View larger version (22K): [in this window] [in a new window]   Fig. 5. A Western blot was used to determine AgCA9 protein localization in larval alimentary canal regions including GC, AMG, PMG, MT and rectum. The left panel of the figure is the Fast Green staining of total protein to ensure equal loading. The strongest AgCA9 signal was seen in both the GC and rectum. CA protein expression was less abundant in the MT and minimal in the AMG and PMG. AgCA9 is indicated by an arrow. For abbreviations, see Fig. 2 legend.

View larger version (104K): [in this window] [in a new window]   Fig. 6. Immunohistochemistry was performed to detect AgCA9 and Na+/K+-ATPase protein in longitudinal sections of whole larvae embedded in paraffin. The images were generated on a laser confocal microscope (Leica SP2). The anterior end of the larvae is towards the left. B–D are images of the same section with the three different fluorochromes (FITC = green = AgCA9; TRITC = red = Na+/K+-ATPase; Cy5 = blue = DRAQ-5 nuclear stain, respectively). AgCA9 protein detection was variable in the epithelial cells of the GC but was consistently seen in the proteinaceous matrix that fills the GC lumen and lines the alimentary canal within the ectoperitrophic space (ectoperitrophic fluid) (A). Na+/K+-ATPase was used as a counter-stain to identify the regions of the alimentary canal (A,C,D). Na+/K+-ATPase is localized to the apical membrane in the cells of the AMG (C, open arrow) and to the basal membrane in the cells of the PMG (C, arrow). In the vicinity of the switch from apical to basal localization is the beginning of the TR (A, arrow; C, arrowhead). AgCA9 detection in the transitional region reveals an association with the periphery of the nucleus (B,D; e.g. arrows). Nuclear localization was confirmed by counterstaining with the nuclear stain DRAQ-5 (D, blue). For abbreviations, see Fig. 2 legend. Scale bars: 150 µm (A); 172.2 µm (B–D).

View larger version (83K): [in this window] [in a new window]   Fig. 7. Immunohistochemistry of AgCA9 in Malpighian tubule (MT) sections. The confocal image demonstrates punctate AgCA9 protein localization within the principal cells of the MT (green). Stellate cells (e.g. arrows) do not appear to contain AgCA9 protein. The nuclei were detected using nuclear stain DRAQ-5 (blue). Scale bar: 47.62 µm.

View larger version (107K): [in this window] [in a new window]   Fig. 8. Immunohistochemistry of AgCA9 protein in whole-mount larvae (A,C,D) and longitudinal paraffin sections of larvae (B). The figure shows laser confocal microscope images of An. gambiae larval rectum immuno-stained with chicken antibodies 5340 (green; A,D) and 5563 (green; B) and mouse monoclonal antibodies against Na+/K+-ATPase (red; B,C). The ileum is indicated at the anterior end of the rectum (A,C,D). AgCA9 protein localized to the DAR cells exclusively (A,B,D; arrow). The banding pattern seen in the DAR cells of whole mounts of the rectum is due to the circumferential muscles of the rectum. This is better seen in D, which shows muscle stained with phalloidin (red). Na+/K+-ATPase localized to the entire rectum, with the exception of the DAR cells (B,C). DAR cells are indicated with an arrow in each image. Scale bars: 150 µm (A,C); 75 µm (B,D).

View larger version (60K): [in this window] [in a new window]   Fig. 9. A phylogenetic analysis was generated for members of the CA family in Homo sapiens (black), Drosophila melanogaster (red), Aedes aegypti (blue) and Anopheles gambiae (green) genomes. Alignments were created using ClustalW and trimmed and visualized using Genedoc. Phylogeny was prepared using MrBayes, with the JTT amino acid substitution model and 1.5 million iterations. Trees were visualized using Treeview. The tree suggests that amplification of insect CAs most likely took place after the divergence of deuterostomes and protostomes as the insect CAs overwhelmingly clustered together. CA-RPs are conserved between protostomes and deuterostomes and are likely to be ancient proteins. Interestingly, AgCA9 (asterisk) and its Drosophila and Aedes homologues were the sole insect CA that clusters with human CAs, indicating that it most closely resembles the primal CA from which other CAs branched.