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First published online February 29, 2008
Journal of Experimental Biology 211, 957-968 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.012021
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Cationic pathway of pH regulation in larvae of Anopheles gambiae

Bernard A. Okech*, Dmitri Y. Boudko{dagger}, Paul J. Linser and William R. Harvey{ddagger}

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


Figure 1
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  		Fig. 1. A living An. gambiae larva that was fed m-Cresol Purple dye illustrates the well known anterior to posterior pH gradient along the larval mosquito alimentary canal. The pH is mildly alkaline in gastric caeca (GC), increases to high values in the anterior midgut (AMG), starts to drop in central midgut, returns to mildly alkaline in posterior midgut (PMG) and becomes neutral at the posteriormost region of the hindgut. SG, salivary gland; CR, cardia; MT, Malpighian tubule; RG, rectum.

 

Figure 2
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  		Fig. 2. Western blots of mosquito larval membrane proteins separated on SDS–PAGE and probed with a polyclonal antibody to subunit B of the H+ V-ATPase (A), a monoclonal antibody to subunit {alpha} of the Na+/K+ P-ATPase (B), and polyclonal epitope-specific antibodies to AgNHA1 (C) and AgNAT8 (D). The arrows indicate the band of protein that is recognized by each antibody. The nitrocellulose membrane was cut into strips and probed with the different antibodies leading to differences in the backgrounds of the blots.

 

Figure 3
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  		Fig. 3. Max projection images of immunolocalized transport proteins in the alimentary canal of larval An. gambiae. (A,B) The localization of AgNHA1 (red) and H+ V-ATPase (red), respectively. (C) The colocalization of AgNHA1 (red) with Na+/K+-ATPase (green). (D) The colocalization of H+ V-ATPase (red) with Na+/K+ P-ATPase (green). Ventral view of the alimentary canal so DAR cells are not visible. The yellow color in C,D results from the colocalization of AgNHA1 and H+ V-ATPase (both red) with Na+/K+ P-ATPase (green).

 

Figure 4
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  		Fig. 4. Max projection images at higher magnification than in Fig. 3 of regions of the alimentary canal showing the location of transport proteins. (A) Posterior cap cells and other scattered cells (white arrows) in the gastric caeca are labeled intensely with the H+ V-ATPase antibody (red) whereas the rest of the caecal cells are stained with Na+/K+ P-ATPase antibody (green). (B) The DAR cells are labeled with H+ V-ATPase antibody (red). (C) The rest of the rectum is labeled with Na+/K+ P-ATPase antibody (green). (D) H+ V-ATPase (red) and Na+/K+ P-ATPase (green) are colocalized in the rectum. (E) AgNHA1 (red) and Na+/K+ P-ATPase (green) are also colocalized in the rectum; the DAR cells have a greenish background. (F,H) Na+/K+ P-ATPase is present on the dorsal side of the rectum with the conspicuous absence of staining in the DAR cells. (G) In a ventral view of the rectum, Na+/K+ P-ATPase labeling (green) is widespread. (A,D,E) The yellow color results from the colocalization of AgNHA1 or H+ V-ATPase (both red) with Na+/K+ P-ATPase (green). (I) H+ V-ATPase is present on the apical membranes of the principal cells in the Malpighian tubules (MT). (J,K) AgNHA1 and the Na+/K+ P-ATPase (green) are present in the entire nervous system. Shown here are the trilobed thoracic ganglion (TTG; J) and abdominal ganglion (AG; K). Scale bars in A and B (for B–H), 150 µm; in I (for I–K), 100 µm.

 

Figure 5
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  		Fig. 5. Immunolocalization of transport proteins in longitudinal sections of mosquito alimentary canal from various regions. H+ V-ATPase (red) and Na+/K+ P-ATPase (green) antibodies labeled sections of the gastric caeca (GC; A), anterior midgut (AMG; B), posterior midgut (PMG; C,D), a Malpighian tubule (MT; white arrow in D) and rectum (G). The apical membrane of posterior midgut region is labeled with AgNHA1 antibody (red; E) and a nutrient amino acid transporter, AgNAT8 antibody (red; F). The yellow color in A,D,G results from the colocalization of H+ V-ATPase (red) with Na+/K+ P-ATPase (green). The nuclei are labeled blue with DRAQ. Scale bar, 100 µm.

 

Figure 6
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  		Fig. 6. Model illustrating the role of membrane transport proteins in pH regulation as well as in H+, Na+ and K+ recycling and amino acid absorption. The localization and colocalizations of H+ V-ATPase, AgNAT8, AgNHA1 and Na+/K+ P-ATPase provide insight into these processes in anterior and posterior midgut cells. The rectum is divided into two functional parts based on the immunolocalization of the transport proteins; postulated interactions between these and yet to be identified proteins are discussed in the text. The shades of pink or orange indicate transport proteins localized in this study; shades of green indicate proteins localized in other studies and white indicates postulated but unidentified proteins. The key points are (1) that H+ V-ATPase is basal and Na+/K+ P-ATPase is apical in anterior midgut, (2) that H+ V-ATPase and AgNAT8 constitute a functional NHE (NHEV-NAT) in the apical membrane of posterior midgut cells, and (3) that AgNHA1 recycles H+ in conjunction with H+ V-ATPase and recycles Na+ in conjunction with AgNAT8 in the apical membrane of posterior midgut cells.

 




© The Company of Biologists Ltd 2008