Heterologous Expression and Functional Analysis of Aedes aegypti Odorant Receptors to Human Odors in Xenopus Oocytes

Summary

A protocol is presented that functionally characterizes mosquito ORs in response to human odors using a Xenopus oocyte expression system coupled with a two-electrode voltage clamp, providing a powerful new technique for exploring the responses of mosquitoes ORs to exposure to human odors.

Abstract

The mosquito Aedes aegypti (Linnaeus), a vector of many important human diseases including yellow fever, dengue fever and Zika fever, shows a strong preference for human hosts over other warm-blooded animals for blood meals. Olfactory cues play a critical role for mosquitoes as they explore their environment and seek a human host to obtain blood meals, thus transmitting human diseases. Odorant receptors (ORs) expressed in the olfactory sensory neurons are known to be responsible for the interaction of mosquito vectors with human odors. To gain deeper insights into Ae. aegypti’s olfactory physiology and investigate their interactions with humans at the molecular level, we used an optimized protocol of Xenopus Oocytes heterologous expression to functionally analyze Ae. aegypti odorant receptors in response to human odors. Three example experiments are presented: 1) Cloning and synthesizing cRNAs of ORs and odorant receptor co-receptor (Orco) from four to six days old Ae. aegypti antennae; 2) Microinjection and expression of ORs and Orco in Xenopus oocytes; and 3) Whole-cell current recording from Xenopus oocytes expressing mosquito ORs/Orco with a two-electrode voltage-clamp. These optimized procedures provide a new way for researchers to investigate human odor reception in Aedes mosquitoes and reveal the underlying mechanisms governing their host-seeking activity at a molecular level.

Introduction

The yellow fever mosquito Ae. aegypti can transmit many deadly diseases including yellow fever, dengue fever and Zika fever, causing tremendous distress and loss of life. Mosquitoes make use of multiple cues such as CO₂, skin odor, and body heat to locate their hosts¹. Given that both humans and other warm-blooded animals produce CO₂ and have similar body temperatures, it seems likely that female Ae. aegypti rely primarily on skin odor for host discrimination². This creates a complex picture, however, with one early study isolating more than 300 compounds from human skin emanations....

Protocol

The protocol for this procedure, the Care and Use of Laboratory Animals, is approved and monitored (Auburn University’s Institutional Animal Care and Use Committee: approved protocol # 2016-2987).

NOTE: Custom gene synthesis is a viable alternative to cloning for mosquito OR genes.

1. Mosquito and Olfactory Appendages (Antennae) Collection

1. Maintain Ae. aegypti mosquitoes (obtained from Dr. James Becnel, USDA, ARS, Mosquito and Fly Research.......

Discussion

TEVC is a classic technique that is widely used to examine the function of membrane receptors. Although a detailed protocol has already been published⁴³ that shares considerable similarity with the procedure presented here, the proposed method here introduces some important modifications. For example, here, the cRNA of both OR and Orco are premixed and aliquoted into small volume samples immediately after synthesis and stored at -80 °C until use rather than mixing them separately on the .......

Materials

Name	Company	Catalog Number	Comments
24-well cell culture plate	CytoOne	CC7682-7524	Used for oocyte culture
African clawed frog	Nasco	LM00535	Used to harvest Xenopus oocytes
Ag/AgCl wire electrode	Warner Instruments	64-1282	Used for microelectrodes
Clampex 10.3	Axon	N.A.	Used for signal recording
Clampfit 10.3	Axon Instruments Inc.	N.A.	Used for data analysis
Collagenase B	Sigma	11088815001	Used for oocyte digestion
Digidata Digitizer	Axon CNS	Digidata 1440A	Used for data acquisition
E.Z.N.A. Plasmid DNA Mini kit	Omega	D6942-01	Used for plasmid preparation
Ethyl-M-aminobenzoate methanesulfonate salt	Sigma	886-86-2	Used for anesthetizing frogs
Glass capillary	FHC	30-30-1	Used for microinjection
Glass capillary	Warner Instruments	64-0801	Used for preparing microelectrodes
GyroMini Nutating Mixer	Labnet	S0500	Used for oocyte digestion
Insect Growth Chambers	Caron Products	model 6025	Used for oocyte incubation
Leica Microscope	Leica	S6 D	Used for cutting mosquito antennae
Light Source	Schott	A20500	Providing light sources for observation
Magnetic stand	Narishige	GJ-1	Used to hold the reference electrode
Micromanipulator	Leica	115378	Used for minor movement of electrode
Micropipe puller	Sutter	model P-97	Used to pull capillaries
Micropipette beveler	Sutter	model BV-10	Used to sharpen capillaries
mMESSAGE mMACHINE T7 kit	Invitrogen	AM1344	Used for synthesizing cRNA
Nanoject II Auto-Nanoliter Injector	Drummond	3-000-204	Used for microinjection
Oligo d(T)20-primed SuperScript IV First-Strand Synthesis System	Invitrogen	18091050	Used for synthesizing cDNA
Olympus Microscope	Olympus	SZ61	Used for microinjection
One Shot TOP10 Chemically Competent E. coli cells	Invitrogen	C404003	Used for transformation
Oocyte clamp amplifier	Warner Instruments	model OC-725C	Used for TEVC recording
QIAquick gel extraction kit	Qiagen	28704	Used for gel purification
TMC Vibration Isolation Table	TMC	63-500	Used for isolating the vibration from the equipment
TURBO DNA-free kit	Invitrogen	AM1907	Used to remove DNase and other ions in RNA