Transport Physiology of Disease Vector Mosquitoes

描述（由申请人提供）：利用安虫的全基因组、形态简单性和幼虫中肠的细胞可及性。冈比亚，我们将创建一个综合的生理模型，整合分子，细胞和电化学机制上皮运输。特别是，我们希望了解管腔碱化的综合生理，这是幼虫消化和营养吸收的关键。我们假设碱化和营养吸收依赖于经上皮和纵向离子梯度，这是由幼虫中肠中初级H + v - atp酶和次级酸/碱转运体之间的特定空间分布和电化学相互作用产生的。我们在印第安人身上发现了8个基因。gambiae基因组编码了推测的酸/碱转运体，并设计了组织特异性cDNA文库。冈比亚幼虫，这大大提高了克隆效率。我们还开发了独特的纳米级分析方法，使我们能够以亚细胞分辨率分析上皮组织。

DESCRIPTION (provided by the applicant): 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.