First published online May 15, 2009
Journal of Experimental Biology 212, 1662-1671 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.028084
Carbonic anhydrases and anion transport in mosquito midgut pH regulation
Paul J. Linser*,
Kristin E. Smith,
Terri J. Seron and
Marco Neira Oviedo
The University of Florida Whitney Laboratory, 9505 Ocean Shore Boulevard,
St Augustine, FL 32080, USA
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Fig. 1. (A) A living fourth instar Anopheles gambiae larva that had been
fed pH sensitive dye (m-cresol purple) to highlight the gut luminal
pH gradient. The structure and compartments of the alimentary canal are
outlined in black. (B) A stylized diagram of the gut with the anterior to the
left. The brackets above indicate the extent of each region, and the
approximate pH of each is indicated (modified from
Neira Oviedo et al., 2008 ).
GC, gastric caeca; AMG, anterior midgut; PMG, posterior midgut; HG, hindgut;
MT, Malpighian tubules; CM, caecal membrane; PT, peritrophic matrix.
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Fig. 2. Confocal immunofluorescence microscopy to identify GPI-linked carbonic
anhydrase 10 (CA10) in isolated whole-mount preparations of Anopheles
gambiae fourth instar larval gut. (A) A low magnification view (maximum
projection of a stack of images) of the anterior half of the midgut. CA10 is
labeled in green and muscle actin (phalloidin) in red. This panel is an
overlay of the two color channels and therefore red indicates peripheral
muscles of the gut and yellow indicates simultaneous labeling for CA10 and
muscle actin. Note lateral complexes of muscles that are double labeled and
central muscles (in this view dorsal) that are labeled for actin only. (B) A
single color channel (green; CA10) at high magnification. Arrow indicates
specific muscles that label for CA10. (C) The same view but with only the red
color channel (phalloidin; muscle) shown. (D) Cell nuclei labeled with DRAQ5
(blue; nuclei). (E) An overlay of all three color channels demonstrating that
specific gut muscles are labeled for CA10 and others are not. (F) A single
plane of a stack of images at higher magnification, showing the muscle cell
surface location of the CA10 labeling (green). GC, gastric caeca; AMG,
anterior midgut. Arrows in B,C and E indicate the same point in the
CA10-positive muscles (modified from Seron
et al., 2004). Scale bars, 80 µm (A); 20 µm (B–E); 30
µm (F).
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Fig. 3. Molecular phylogenetic analysis of carbonic anhydrase (CA) gene sequences
from Homo sapiens (black), Anopheles gambiae (green),
Aedes aegypti (blue) and Drosophila melanogaster (red)
(Smith et al., 2007).
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Fig. 4. Confocal immunofluorescence microscopy to identify AgCA9 in sections and
whole mounts of Anopheles gambiae larval gut tissues. (A) A
longitudinal section of a fourth instar larva labeled for AgCA9 (green) and
Na+/K+-ATPase (red). Note prominent AgCA9 labeling in
the cells and lumen of the gastric caeca, the ectoperitrophic fluid and the
anterior portion of the rectum. (B,C) Two views of a whole-mount preparation
of larval rectum showing AgCA9 (green; B) and
Na+/K+-ATPase (C). (D) A high magnification
cross-section of the rectum with the anterior to the right showing the
mutually exclusive labeling for CA-9 (green) and
Na+/K+-ATPase (red). The dorsal anterior rectum (DAR)
cells are indicated by the arrow. (E) A high magnification view of an isolated
rectum with the DAR cells (arrow) labeled for AgCA9 and the external rectal
musculature labeled in red (phalloidin). GC, gastric caeca; AMG, anterior
midgut; PMG, posterior midgut; MT, Malpighian tubules. All arrows indicate the
DAR cells. Scale bars, 150 µm (A–C), 75 µm (D,E).
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Fig. 5. Analyses of the members of the SLC4A family of anion
exchangers/transporters. (A) A molecular phylogenetic tree of the annotated
members of this gene family from Anopheles gambiae (green), Aedes
aegypti (blue), Drosophila melanogaster (red) and Homo
sapiens (black). The human genes include putative and confirmed splice
variants of several genes. Similarly, several putative splice variants of the
Drosophila genes are included. The tree was generated using MrBayes,
1.5 million iterations and the JTT amino acid substitution model. All nodes
represent at least 95% probability but the branch lengths are arbitrary. (B)
Hydropathy plots generated for Drosophila NDAE1 (black) and
Anopheles AgAE1 (blue). (C) The hypothetical secondary structure of
one putative splice variant of AgAE1.
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Fig. 6. A DNA microarray-based analysis of regionalized expression of the three
annotated SLC4A gene family members in Anopheles gambiae larvae.
Whole larval gut expression (W) is compared with transcriptome levels in the
gastric caeca (GC), anterior midgut (AMG), the posterior midgut (PMG) and the
Malpighian tubule/hindgut (HG) (Neira
Oviedo et al., 2008). A color scale indicates the transcriptome
levels.
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Fig. 7. Partial model of CA distribution in gut (upper drawing) and physiological
impact (lower drawing). Blue arrows indicate hypothetical diffusion of
metabolic CO2, only indicated in AMG cells which lack an
 -CA. V, V-ATPase; Na/K, Na+/K+-ATPase; NHA,
Na+/H+ antiporter; CA, carbonic anhydrase.
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© The Company of Biologists Ltd 2009
