Alkalinization by chloride/bicarbonate pathway in larval mosquito midgut

Abstract

The midgut of mosquito larvae maintains a specific lumen alkalinization profile with large longitudinal gradients (pH ≈ 3 units⋅mm⁻¹) in which an extremely alkaline (pH ≈ 11) anterior midgut lies between near-neutral posterior midgut and gastric cecum (pH 7–8). A plasma membrane H⁺ V-ATPase energizes this alkalinization but the ion carriers involved are unknown. Capillary zone electrophoresis of body samples with outlet conductivity detection showed a specific transepithelial distribution of chloride and bicarbonate/carbonate ions, with high concentrations of both anions in the midgut tissue: 68.3 ± 5.64 and 50.8 ± 4.21 mM, respectively. Chloride was higher in the hemolymph, 57.6 ± 7.84, than in the lumen, 3.51 ± 2.58, whereas bicarbonate was higher in the lumen, 58.1 ± 7.34, than the hemolymph, 3.96 ± 2.89. Time-lapse video assays of pH profiles in vivo revealed that ingestion of the carbonic anhydrase inhibitor acetazolamide and the ion exchange inhibitor DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid), at 10⁻⁴ M eliminates lumen alkalinization. Basal application of these inhibitors in situ also reduced gradients recorded with self-referencing pH-sensitive microelectrodes near the basal membrane by ≈65% and 85% respectively. Self-referencing chloride-selective microelectrodes revealed a specific spatial profile of transepithelial chloride transport with an efflux maximum in anterior midgut. Both acetazolamide and DIDS reduced chloride effluxes. These data suggest that an H⁺ V-ATPase-energized anion exchange occurs across the apical membrane of the epithelial cells and implicate an electrophoretic Cl⁻/HCO exchanger and carbonic anhydrase as crucial components of the steady-state alkalinization in anterior midgut of mosquito larvae.