Carbonic anhydrases and anion transport in mosquito midgut pH regulation

Abstract

Mosquito larvae use a digestive strategy that is relatively rare in nature. The anterior half of the larval mosquito midgut has a luminal pH that ranges between 10.5 and 11.5. Most other organisms, both large and small, initiate digestion in an acid medium. The relative uniqueness of the highly alkaline digestive strategy has been a long-standing research focus in larval lepidopterans. More recently, the disease vector potential of mosquitoes has fueled specific interest in larval mosquito biology and the alkaline digestive environment in the midgut. The probable principle anion influencing the highly alkaline gut lumen is bicarbonate/carbonate. Bicarbonate/carbonate is regulated at least in part by the activity of carbonic anhydrases. Hence, we have focused attention on the carbonic anhydrases of the mosquito larva. Anopheles gambiae, the major malaria mosquito of Africa, is an organism with a published genome which has facilitated molecular analyses of the 12 carbonic anhydrase genes annotated for this mosquito. Microarray expression analyses, tissue-specific quantitative RT-PCR, and antibody localization have been used to generate a picture of carbonic anhydrase distribution in the larval mosquito. Cytoplasmic, GPI-linked extracellular membrane-bound and soluble extracellular carbonic anhydrases have been located in the midgut and hindgut. The distribution of the enzymes is consistent with an anion regulatory system in which carbonic anhydrases provide a continuous source of bicarbonate/carbonate from the intracellular compartments of certain epithelial cells to the ectoperitrophic space between the epithelial cells and the acellular membrane separating the food bolus from the gut cells and finally into the gut lumen. Carbonic anhydrase in specialized cells of the hindgut (rectum) probably plays a final role in excretion of bicarbonate/carbonate into the aquatic environment of the larva. Detection and characterization of classic anion exchangers of the SLC4A family in the midgut has been problematic. The distribution of carbonic anhydrases in the system may obviate the requirement for such transporters, making the system more dependent on simple carbon dioxide diffusion and ionization via the activity of the enzyme.

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.

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).

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).

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).

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.

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.

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 α-CA. V, V-ATPase; Na/K, Na⁺/K⁺-ATPase; NHA, Na⁺/H⁺ antiporter; CA, carbonic anhydrase.