Collaborative Research: Na,K,Cl Cotransporters in Mosquito Epithelial Transport - Connecting Molecules to Physiology

这项研究探索了蚊子在其生命周期中面临的具有挑战性的条件下用来调节盐分和水平衡的机制。大多数蚊子幼虫生活在淡水中，必须吸收盐分才能抵消盐分从体内扩散出去的趋势。相比之下，成年雌性蚊子在进食含盐血后必须排出大量盐分。这个项目的具体重点是三种蚊子蛋白质，它们有助于将钠、钾和氯化物等盐带进或带出体内。其中一种蛋白质与人类和其他脊椎动物中的蛋白质密切相关，这些蛋白质对盐分和水的平衡非常重要。因此，研究蚊子版本的这种蛋白质可能会揭示脊椎动物和昆虫共有的盐分运输蛋白质的基本特性。另外两种蛋白质只在蚊子和其他昆虫中发现，还没有在任何动物身上发现。更好地了解这些新的昆虫蛋白质可能对制定控制蚊子和其他害虫的策略特别有用，因为有可能在不影响脊椎动物蛋白质的情况下针对它们。这项研究项目将为研究人员开发新的工具，以评估这些蛋白质在蚊子体内的位置，它们携带什么盐，以及哪些化学物质会干扰它们的功能。此外，三名本科生和一名研究生将获得密切指导的研究经验。蚊子必须根据它们的生命阶段、性别和环境而不同地分泌和吸收离子。黄热病蚊虫埃及伊蚊的三种蛋白与脊椎动物的Na+依赖的阳离子氯化物共转运蛋白(CCCS)序列相似，CCCS参与离子的吸收和分泌。这项工作将生产关键试剂，并改进必要的程序，以便开始将蚊子CCCS的分子特性与其运输功能和整个动物的生理作用联系起来。第一个目标是开发识别和区分三种蚊子CCC的异构体特异性抗体。这些抗体的选择性将通过比较它们在不同组织和发育阶段、通过RNA干扰沉默特定CCC表达的蚊子以及注射编码特定CCC的CRNAs的非洲爪哇卵母细胞中的反应性来评估。第二个目标是确定每个蚊子CCC在非洲爪哇卵母细胞中的功能异源表达。CCCS的功能表达将使用标准的放射性同位素摄取分析和新开发的非放射性方法进行评估。拟议工作的结果也可能有助于确定新的分子靶标，以帮助开发用于控制昆虫病媒和农业害虫的新杀虫剂。三名本科生和一名研究生将接受指导研究培训，主要调查人员将启动社区推广计划。

This research explores the mechanisms that mosquitoes use to regulate their salt and water balance under the challenging conditions they face during their lifecycles. Most mosquito larvae live in freshwater and must absorb salt to counteract the tendency for salt to diffuse out of their bodies. In contrast, adult female mosquitoes must expel large amounts of salt after they engorge on a salty blood meal. The specific focus of this project is on three mosquito proteins that help carry salts such as sodium, potassium, and chloride into or out of the body. One of these proteins is closely related to proteins in humans and other vertebrate animals that are important to salt and water balance. Thus, studying the mosquito version of this protein may uncover fundamental properties of salt-transporting proteins that are shared between vertebrates and insects. The other two proteins are only found in mosquitoes and other insects, and have not been characterized in any animal. Gaining a better understanding of these novel insect proteins may be especially useful in developing strategies to control mosquitoes and other insect pests, since it may be possible to target them without affecting vertebrate proteins. This research project will develop new tools for researchers to assess where these proteins are located in the mosquito body, what salts they carry, and what chemicals can interfere with their function. Additionally, three undergraduate students and one graduate student will receive closely mentored research experiences. Mosquitoes must secrete and absorb ions differently depending on their life stage, sex, and environment. Three proteins from the yellow fever mosquito Aedes aegypti have sequence similarity to vertebrate Na+-dependent cation-chloride cotransporters (CCCs), which participate in both ion absorption and secretion. This work will produce critical reagents and refine procedures that are necessary to begin linking the molecular properties of the mosquito CCCs to their transport functions and whole-animal physiological roles. The first objective is to develop isoform-specific antibodies that recognize and differentiate among each of the three mosquito CCCs. The selectivity of the antibodies will be evaluated by comparing their reactivity among different mosquito tissues and developmental stages, mosquitoes in which expression of a specific CCC has been silenced by RNA interference, and in Xenopus oocytes injected with cRNAs encoding a particular CCC. The second objective is to confirm functional heterologous expression of each mosquito CCC in Xenopus oocytes. Functional expression of the CCCs will be evaluated using standard radioisotope uptake assays and newly developed non-radioactive methods. Findings from the proposed work may also be useful in identifying novel molecular targets to aid in the development of new insecticides for the control of insect disease vectors and agricultural pests. Three undergraduate students and one graduate student will receive mentored research training and the principal investigators will initiate community outreach programs.