: Taking advantage of the complete genome as well as the morphological simplicity and cellular accessibility of larval midgut in An. gambiae, we will create a comprehensive physiological model that integrates molecular, cellular, and electrochemical mechanisms of epithelial transport. In particular, we wish to understand the integrative physiology of lumen alkalinization, which is critical for larval digestion and nutrient uptake. We postulate that alkalinization and nutrient uptake depend upon transepithelial and longitudinal ion gradients, which are generated by specific spatial distribution and electrochemical interaction between primary H + V-ATPases and secondary acid/base transporters in the larval midgut. We have identified eight genes in the An. gambiae genome, which encode the putative acid/base transporters and designed tissue-specific cDNA library from An. gambiae larvae, which dramatically increased the cloning efficiency. We have also developed unique nanoscale analytical approaches, which allow us to analyze epithelial tissue with subcellular resolution. To explore the integrative physiology and functional genomics of the mosquito midgut, we will complete four specific aims.
Aim 1 uses preliminary bioinformatics data to clone acid/base transporters from An. gambiae larvae; the gene products will be evaluated by electrochemical analysis of transcript expression in Xenopus oocytes.
Aim 2 localizes the transporters along the midgut in whole-mounts of An. gambiae larvae using in situ hybridization with transcript-specific dioxygenin-RNA probes. The apical/basal (polar) integration of the transporters and H v V-ATPases will be determined by confocal microscopy of immuno-labeling preparations.
Aim 3 seeks to determine electrochemical motive forces across basal/apical membranes and phosphorylation potentials in specified midgut cells using capillary electrophoresis and ion selective microelectrodes. Trans-membrane voltages will be measured with microelectrodes in midgut of intact and semi-intact An. gambiae larvae.
Aim 4 examines the mechanism, efficiency, and role of electrochemical coupling between H + V-ATPase and identified transporters in midgut alkalinization using non-invasive self-referencing ion-selective microelectrodes (SERIS LIX) and time-lapse photography of a lumen alkalinization profile in vivo. With this proposed study, integration of the molecular and electrochemical mechanism of a specific physiological process will be defined for the first time, which is crucial not only for understanding midgut alkalinization in disease vector mosquitoes but other types of transporting epithelia as well. Since the work is to be done on a malaria vector, it has health relevance because the larval midgut is the target for Bti, TMOF, and other larvicides and is an apt model for the plasmodium interaction with the adult-female midgut.
