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 emanations3. Further behavioral assays have indicated that a number of these compounds evoke behavioral responses in Ae. aegypti4,5,6,7, but precisely how these compounds are detected by the mosquito remains largely unknown. Recent research by our group has identified several human odorants that may be involved in Ae. aegypti host-seeking activity, though their roles have yet to be confirmed by further behavioral assays8. How these essential human odorants are decoded in the peripheral sensory system of Ae. aegypti has yet to be established.
Insects detect odorants through the chemosensory sensilla on their olfactory appendages. Inside each of the sensilla, different olfactory receptors, including odorant receptors (ORs), ionotropic receptors (IRs) and gustatory receptors (GRs), are expressed on the membrane of olfactory sensory neurons9. These ORs are responsible for sensing many odorants encountered by insects, especially the odors associated with food, hosts and mating partners10,11,12,13. A previous study focusing on deorphanizing the function of ORs in Anopheles gambiae using the Xenopus expression system coupled with a two-electrode voltage clamp has found that Anopheles ORs are specifically tuned to the aromatics that are the major components in human emanations14. A recent genome study identified up to 117 OR genes in Ae. aegypti15. Consequently, a way to systematically address the functions of these Aedes ORs in response to biologically or ecologically important odorants such as human odors or oviposition stimuli would provide useful information for those seeking to further understand the chemical ecology or neuroethology of Ae. aegypti.
The two-electrode voltage clamp (TEVC) technique was originally developed to examine the function of membrane ion channels in the mid-1990s16,17. Since then, TEVC has been used to investigate ORs from a number of different insect species14,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34. This functional examination of ORs has substantially contributed to answering important ecological questions in insects, including: 1) How do insects locate food sources? 2) How are they attracted by the volatile sex pheromones released by their mating partners? 3) How do they find a perfect oviposition site for their offspring? and 4) Are there any compounds, plant-derived or synthetic, that can efficiently protect humans from biting bugs? Answers to these questions are crucial for controlling important disease vectors such as mosquitoes.
A number of other approaches, including those based on the human embryonic kidney cell line 293 (HEK293), the Drosophila empty neuron system, zinc-finger nuclease, transcription activator-like effector nuclease, and the CRISPR/Cas9 gene editing system, have also been used in OR functional studies12,20,35,36,37. However, these techniques all require the skills of an experienced molecular biologist and involve multiple potentially confounding factors. TEVC/oocyte expression is capable of directly measuring odor-evoked receptor currents and ion conductance and has the added advantage of the speedy quick setup time required to express receptors from cRNA. In this study, we therefore used TEVC to examine the responses of one Ae. aegypti OR55 (AaegOR55) against several odorants with potential biological relevance, revealing that oocytes expressed with AaegOR55•AaegOrco showed a dose-dependent response to the human odorant benzaldehyde.
Protocol
Representative Results
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 …
Disclosures
The authors have nothing to disclose.
Acknowledgements
This project was supported by an award from the Alabama Agricultural Experiment Station (AAES) Multistate/Hatch Grants ALA08-045, ALA015-1-10026, and ALA015-1-16009 to N.L.
Materials
| 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 |
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