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

Ohio State University, The, Columbus OH

Investigators

Abstract

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.