In situ analysis of pH gradients in mosquito larvae using noninvasive, self-referencing, pH-sensitive microelectrodes

The alkaline environment, pH approximately 11, in the anterior midgut lumen of mosquito larvae is essential for normal nutrition and development. The mechanism of alkalization is, however, unknown. Although evidence from immu nohistochemistry, electron microscopy and electrophysiology suggests that a V-ATPase is present in the basal membranes of the epithelial cells, its ph ysiological role in the alkalization process has not been demonstrated. To investigate a possible role of the V-ATPase in lumen alkalization, pH gradi ents emanating from the hemolymph side of the midgut in semi-intact mosquit o larvae were measured using non-invasive, self-referencing, ion-selective microelectrodes (SERIS), Large Hf concentration gradients, with highest con centrations close to the basal membrane (outward [H+] gradients), were foun d in the anterior midgut, whereas much smaller gradients, with concentratio ns lowest close to this membrane (inward [H+] gradients), were found in the gastric caeca and posterior midgut, Similar region-specific pH gradients, with consistent anterior-to-posterior profiles, were observed in individual s of two Aedes species, Aedes aegypti from semi-tropical Florida and Aedes canadensis from north-temperate Massachusetts. The gradients remained in a steady state for up to 6h, the maximum duration of the recordings. Bafilomycin A(1) (10(-5), 10(-7)mol l(-1)) on the hemolymph side greatly di minished the [H+] gradients in the anterior midgut but had no effect on the gradients in the gastric caecum and posterior midgut, These physiological data are consistent with the previous findings noted above. Together, they support the hypothesis that a basal, electrogenic Hf V-ATPase energizes lum inal alkalization in the anterior midgut of larval mosquitoes.