Transport Physiology of Disease Vector Mosquitoes

描述(由申请人提供)：利用完整的基因组以及幼虫中肠的形态简单和细胞可及性。冈比亚，我们将创建一个全面的生理模型，集成了上皮运输的分子、细胞和电化学机制。特别是，我们希望了解管腔碱化的综合生理学，这对幼虫的消化和养分吸收至关重要。我们推测，碱化和营养吸收依赖于跨上皮和纵向离子梯度，这种梯度是由幼虫中肠初级H V-ATPase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。我们已经在AN中发现了八个基因。冈比亚基因组，编码可能的酸/碱转运蛋白，并设计了组织特异性的cDNA文库。冈比亚幼虫，极大地提高了克隆效率。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。 为了探索蚊子中肠的综合生理学和功能基因组学，我们将完成四个特定的目标。目的1利用初步的生物信息学数据从An克隆酸/碱转运蛋白。冈比亚幼虫；基因产物将通过在非洲爪哇卵母细胞中转录表达的电化学分析进行评估。AIM 2将转运蛋白沿中肠定位于AN的整架。冈比亚幼虫与转录本特异性双氧合素-RNA探针的原位杂交。转运蛋白和H v V-ATPase的顶端/底端(极性)整合将通过免疫标记制剂的共聚焦显微镜来确定。目的3利用毛细管电泳法和离子选择性微电极测定特定中肠细胞跨基底膜/根尖膜的电化学势和磷酸化电位。用微电极测量完整和半完整AN的中肠跨膜电压。冈比亚幼虫。目的4用无创自参比离子选择微电极(SERIS LIX)和延时照相技术研究H V-ATPase与已识别的转运蛋白在中肠碱化中的电化学偶联机制、效率和作用。通过这项拟议的研究，将首次定义特定生理过程的分子和电化学机制的集成，这不仅对于了解病媒蚊子的中肠碱化，而且对于了解其他类型的运输上皮细胞也是至关重要的。由于这项工作是在疟疾媒介上进行的，因此它具有健康相关性，因为幼虫中肠是Bti、TMOF和其他杀幼虫的目标，是疟原虫与成虫-雌性中肠相互作用的APT模型。

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.