Carbonic anhydrases and anion transport in mosquito midgut pH regulation.

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.

A GPI-linked carbonic anhydrase expressed in the larval mosquito midgut.

We have previously described the first cloning and partial characterization of carbonic anhydrase from larval Aedes aegypti mosquitoes. Larval mosquitoes utilize an alkaline digestive environment in the lumen of their anterior midgut, and we have also demonstrated a critical link between alkalization of the gut and carbonic anhydrase(s). In this report we further examine the nature of the previously described carbonic anhydrase and test the hypothesis that its pattern of expression is consistent with a role in gut alkalization. Additionally we take advantage of the recently published genome of the mosquito Anopheles gambiae to assess the complexity of the carbonic anhydrase gene family in these insects. We report here that the previously described carbonic anhydrase from Aedes aegypti is similar to mammalian CA IV in that it is a GPI-linked peripheral membrane protein. In situ hybridization analyses identify multiple locations of carbonic anhydrase expression in the larval mosquito. An antibody prepared against a peptide sequence specific to the Aedes aegypti GPI-linked carbonic anhydrase labels plasma membranes of a number of cell types including neuronal cells and muscles. A previously undescribed subset of gut muscles is specifically identified by carbonic anhydrase immunohistochemistry. Bioinformatic analyses using the Ensembl automatic analysis pipeline show that there are at least 14 carbonic anhydrase genes in the Anopheles gambiae genome, including a homologue to the GPI-linked gene product described herein. Therefore, as in mammals which similarly possess numerous carbonic anhydrase genes, insects require a large family of these genes to handle the complex metabolic pathways influenced by carbonic anhydrases and their substrates.