Transport Physiology of Disease Vector Mosquitoes

描述（由申请人提供）：利用完整的基因组以及形态简单和细胞可及性的幼虫中肠在An。冈比亚，我们将创建一个综合的生理模型，整合上皮运输的分子，细胞和电化学机制。特别是，我们希望了解管腔碱化，这是幼虫消化和营养吸收的关键综合生理。我们推测，碱化和营养吸收依赖于transepithelial和纵向离子梯度，这是由特定的空间分布和初级H + V-ATP酶和次级酸/碱转运蛋白在幼虫中肠之间的电化学相互作用产生的。我们已经确定了八个基因在一个。gambiae基因组中编码酸/碱转运蛋白的基因，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。 为了探索蚊子中肠的整合生理学和功能基因组学，我们将完成四个具体目标。目的1利用初步的生物信息学数据克隆安氏杆菌的酸/碱转运蛋白。冈比亚幼虫;基因产物将通过非洲爪蟾卵母细胞中转录物表达的电化学分析来评估。目的2定位了中肠转运蛋白的沿着分布。冈比亚幼虫使用原位杂交与转录特异性doxygenin-RNA探针。将通过免疫标记制备物的共聚焦显微镜测定转运蛋白和Hv V-ATP酶的顶端/基底（极性）整合。目的3旨在利用毛细管电泳和离子选择性微电极确定特定中肠细胞中跨基底/顶膜的电化学动力和磷酸化电位。将在完整和半完整的An中肠中使用微电极测量跨膜电压。冈比亚幼虫。目的4利用非侵入性自参比离子选择性微电极（SERIS LIX）和体内肠腔碱化曲线的延时摄影，研究H + V-ATP酶和已鉴定的转运蛋白之间的电化学偶联在中肠碱化中的机制、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整合，这不仅对于了解疾病媒介蚊子中肠碱化，而且对于其他类型的运输上皮细胞也至关重要。由于这项工作是在疟疾病媒上进行的，因此它具有健康相关性，因为幼虫中肠是Bti、TMOF和其他杀幼虫剂的靶标，并且是疟原虫与成虫-雌性中肠相互作用的适当模型。

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.