If you like spending time outdoors, you’re probably quite familiar with being ridden with mosquito bites by the end of the day (especially in North Carolina in the middle of Summer). That’s why many people utilize insecticides either directly on themselves or in the environment where they’re outside. Insecticides are also used to control medically and agriculturally important pests, which is why it’s important to ensure that they aren’t toxic to animals or humans. The widely used insecticides, for this purpose, are pyrethroids.
Pyrethroids are the synthetic version of the naturally occurring pyrethrum insecticide extracted from Chrysanthemum flower species. This class of insecticide is used globally to control several insect species, most especially, those that are vectors of life-threatening diseases such as malaria, yellow fever, and dengue fever. However, the consistent use of pyrethroids has resulted in pyrethroid resistance in many insect species. This resistance has been linked to mutations in the voltage-gated sodium channel (VGSC) gene which are targets of pyrethroids. The Dong Lab investigates and identifies mutations in the sodium channel genes of mosquitoes responsible for pyrethroid resistance.
VGSCs are responsible for the transmission of electrical signals across the membranes of excitable cells, such as neurons, through the opening and closing of their gates in response to membrane depolarization. Pyrethroid kills mosquitoes by prolonging the opening (activation) of sodium channels and inhibiting closing (inactivation), causing a continuous depolarization of the cell which leads to insect paralyzes and death. This mechanism of action of pyrethroids is known as a knockdown mechanism. Mutations in the VGSC gene prevent this mechanism from occurring because the channels are no longer sensitive to pyrethroid. This ‘fight-back’ mechanism is referred to as knockdown resistance. Multiple sodium channel gene mutations have been identified in mosquito populations all over the world. My project aims to study a VGSC mutation, F174I, recently discovered in Aedes aegypti mosquito populations in Florida. The F174I mutation has the amino acid Phenylalanine (F) replaced by Isoleucine (I) at position 174 of the VGSC gene.
For this project, I will be using molecular and electrophysiological techniques to collect data. First, primers are designed based on the mutation site and are sent to a biotech company for synthesis. Once the primers are diluted and prepared, it’s run through PCR with a VGSC template in order to get many copies of VGSC with the F174I mutation. This process is followed by bacteria transformation, plasmid isolation, linearization, and cRNA synthesis. The cRNA is injected into Xenopus (frog) oocytes for the expression of the VGSC protein. Oocytes of frogs are good for use because of their large size and high fidelity of translation. The properties of the expressed protein are studied using the two-electrode voltage-clamp technique. The oocytes are thereafter exposed to pyrethroid to observe sensitivity or resistance. This project hopes to add to the body of knowledge on VGSC mutations conferring pyrethroid resistance in mosquitoes.
