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Carbonic anhydrase in the midgut of larval Aedes aegypti: cloning, localization and inhibition

Maria del Pilar Corena¹, Theresa J. Seron¹, Herm K. Lehman², Judith D. Ochrietor¹, Andrea Kohn¹, Chingkuang Tu³ and Paul J. Linser^1,*

¹ The Whitney Laboratory, University of Florida, Saint Augustine, FL 32080, USA,
² Department of Biology, Hamilton College, Clinton, NY 13323, USA and
³ Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL 32611, USA

View larger version (51K): [in a new window]   Fig. 1. Effect of inhibition of carbonic anhydrase on the pH of culture medium of fourth-instar larvae of Aedes aegypti. Mosquito larvae typically alkalize the medium in which they are reared (Stobbart, 1971). (A) Six culture wells each containing five fourth-instar larvae incubated for 5 h in medium containing 0.003 % Bromothymol Blue. The blue color is retained, indicating a pH greater than 7.6. (B) The same as A, except that each well also contains a different concentration of the specific carbonic anhydrase inhibitor methazolamide ranging from 10–6 to 10–3 mol l–1 from left to right. A yellow color indicates a pH below 7.6.

View larger version (81K): [in a new window]   Fig. 2. Effect of methazolamide on the alkalization of the midgut using the Bromothymol Blue (BTB) assay of pH within living, but isolated, gut tissue. Gut tubes were dissected after pre-loading with BTB and then incubated for 5 h in hemolymph substitute (Clark et al., 1999) in the absence (A) or presence (B) of 10–6 mol l–1 methazolamide. The loss of blue coloration in B shows that the internal pH of the gut lumen has dropped below 7.6. Scale bar, 600 µm.

View larger version (7K): [in a new window]   Fig. 3. Relative activity of carbonic anhydrase in different pooled segments of the midgut of larval Aedes aegypti. Midguts were dissected from early fourth-instar larvae and separated into gastric caeca (GC), anterior midgut (AM), posterior midgut (PM) and Malpighian tubules (MT). The relative activity of carbonic anhydrase was measured using the O18 mass spectrometry method (Silverman and Tu, 1986) normalized to total protein content. The activity of the anterior midgut was lower than that of the water blank and, thus, is set as ‘zero’ activity. Values are means + S.E.M., N=3.

View larger version (72K): [in a new window]   Fig. 4. Carbonic anhydrase from the midgut of larval Aedes aegypti. (A) Alignment (BLAST) of the predicted amino acid sequence of Aedes aegypti cDNA (A-CA) (GenBank accession number AF395662) with several known -carbonic anhydrases. Regions of exact homology across all species are highlighted in blue (100 %); regions with less homology are highlighted in red (>75 %) and green (>50 %). Similar amino acid residues have been included in the shading. (B) A homology tree comparing A-CA and several vertebrate -carbonic anhydrases (DNAman software).

View larger version (68K): [in a new window]   Fig. 5. Comparison of the extrapolated amino acid sequences of A-CA with six putative dipteran carbonic anhydrase genes identified in the Drosophila melanogaster gene data bases. (A) An alignment of A-CA with the amino acid sequences of the six Drosophila melanogaster genes (with accession numbers listed) identified through bioinformatics searching. Regions of exact homology across all species are highlighted in blue (100 %); regions with less homology are highlighted in red (>75 %) and green (>50 %). Similar amino acid residues were included in the shading. (B) A homology tree comparison of these dipteran carbonic anhydrases.

View larger version (84K): [in a new window]   Fig. 6. Polymerase chain reaction (PCR) analysis of Aedes aegypti cDNA (A-CA)-like sequences in cDNA-amplified RNA samples taken from regions of the midgut of larval Aedes aegypti. PCR was performed using exact primers for the cloned A-CA. Equal quantities of total RNA were amplified and analyzed from anterior midgut (lane 2), gastric caeca (lane 3), posterior midgut (lane 4), whole gut (lane 5), Malpighian tubules (lane 6) and a water template control (lane 7). Note the primary product in caeca and posterior midgut samples at the expected size of approximately 894 nucleotides. Also note the absence of this band from other gut regions but the appearance of bands of higher molecular masses. Lane 1 is a 100 base pair (bp) molecular mass ladder.

View larger version (165K): [in a new window]   Fig. 7. Hansson’s histochemistry of whole, dissected midgut from early fourth- instar larvae of Aedes aegypti viewed at low (A) and high (B,C) magnification. Intense dark staining is observed in the gastric caeca (GC) and the posterior midgut (PM), indicating carbonic anhydrase activity. Large, relatively unstained cells are also evident in the posterior midgut (A,C). The posterior-most extensions of the lobes of the gastric caeca exhibit relatively low levels of reaction product, indicating lower levels of the enzyme in these cells relative to other cells of the caeaca. Scale bars: 350 µm (A), 250 µm (B), 200 µm (C).

View larger version (67K): [in a new window]   Fig. 8. In situ hybridization localization of mRNA for Aedes aegypti cDNA (A-CA). (A) An isolated larval mosquito gut probed with DIG-labeled cRNA for A-CA. Strong labeling was observed in the posterior midgut (PM) and gastric caeca (GC). (B) Isolated midgut reacted with the sense (control) cRNA for A-CA. Scale bars, 200 µm.