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. aegypti^4,5,6,7, 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 partners^10,11,12,13. 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-1990s^16,17. Since then, TEVC has been used to investigate ORs from a number of different insect species^{14,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34}. 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 studies^{12,20,35,36,37}. 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

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 Unit) at 25 ± 2°C and a photoperiod of 12: 12 (L:D) h (lights on 8 am).
2. Anesthetize ~800 4-6-day old female mosquitoes without blood feeding using CO₂. Cut the mosquito antennae (olfactory tissues) under a microscope (40x) using a pair of scissors and collect in a 1.5 mL centrifuge tube kept on dry ice.
   NOTE: The collected antennae can be stored in a freezer at -80 °C or be used immediately.

2. OR Cloning from Antennae of Ae. aegypti

1. Extract total RNA from the antennae using a commercially available total RNA kit following the manufacturer’s protocol.
2. Digest the total RNA using the commercial DNA-free kit in order to remove DNase and other ions following the manufacturer’s protocol.
3. Synthesize cDNA from 1.5 µg of DNA-free RNA using the Oligo d(T)20-primed SuperScript IV First-Strand Synthesis System^13,30.
4. Design PCR primers for the OR genes according to the sequences available at VectorBase (https://www.vectorbase.org/) and the special requirements of the enzyme cutting site. Add a Kozak sequence (GCCACC), which initiates translation in most eukaryotic organisms, between the cutting site and the start codon in each forward primer³⁰.
5. Clone the coding sequences of the OR genes by PCR using gene-specific primers containing restriction endonuclease sites and the Kozak sequence following the manufacturer’s protocol.
6. Detect PCR products through a 1% agarose gel and purify using a commercial gel extraction kit following the manufacturer’s protocol.
7. Perform recombinant plasmid construction through cloning of PCR products into a pT7Ts vector after digestion by restriction enzymes following the manufacturer’s protocol.
8. Verify the recombinant plasmids by Sanger sequencing and confirm using the VectorBase database.

3. cRNA Synthesis

1. Linearize the constructed plasmids with specific restriction enzyme(s).
   NOTE: Plasmid DNA must be linearized with a restriction enzyme downstream of the gene to be transcribed.
2. Use the linearized plasmids to synthesize cRNAs with a commercial T7 kit following a previous description^13,30.
3. Premix cRNA of OR and Orco (1:1) in a centrifuge tube and aliquot the mixed cRNA into a 2 μL volume (250 ng/µL/each) for each PCR tube. Store the aliquoted cRNA at -80 °C before use.

4. Xenopus Oocyte Collection

NOTE: The procedure is following the instruction of Schneider et al.³⁸.

1. Anesthetize Xenopus laevis for 20-30 min in 1 L of ultra-purified water with 1 g of ethyl m-aminobenzoate methanesulfonate salt (Figure 1A). Pinch one of the frog’s toes using fingers; if there is no response, the frog is sufficiently anesthetized and ready for surgery. Transfer the anesthetized frog on the ice bed.
2. The surgery
   1. Rub the frog ventral area with 10% povidone-iodine solution and wipe the solution with clean cotton balls. Repeat this step 2 times.
   2. Make a small abdominal incision (10-15 mm, Figure 1B) with a disposable scalpel. Penetrate skin, fascia-muscle layer separately. The incision is in the lower part of the abdomen, lateral to the midline.
      NOTE: Do not injury internal organs.
   3. Grasp parts of ovarian lobes with forceps and pull to the exterior gently. Lift the ovary and cut it off at the body wall level with scissors. Repeat several times until get enough ovarian materials (~200 eggs, Figure 1C). Transfer the ovarian materials into washing buffer (96 mM NaCl, 2 mM KCl, 5 mM MgCl₂•6H₂O, 5 mM HEPES sodium salt, 10 µg/mL gentamycin, pH 7.6).
   4. Close the surgery wounds (both muscle and skin) with absorbable sutures that typically degrade and fall out naturally in 10-14 days.
   5. After each surgery, place the frog in 1 L of recovery solution (containing 3.4 g of canning & pickling salt and 2 g of instant ocean sea salt) until it wakes up and then return it back to the frog facility.
      NOTE: Up to 4 surgeries can be performed on each frog. The operation interval of each frog should be more than 2 months.
3. Enzymatic defolliculated digestion and oocytes culturing
   1. Separate the oocytes (~200 eggs, Figure 1C) into small pieces.
   2. Transfer all oocytes into a digestion solution, including 10 mL of washing buffer (Section 4.2.3) and 2 mg/mL collagenase B for 40-60 min at ambient temperature in order to remove follicular cell layers. Examine the samples under a microscope to determine whether 80% of the oocytes have been fully defolliculated; if not, check every 10 min till most of the oocytes are ready.
   3. Rinse the oocytes with 1x Ringer’s solution (96 mM NaCl, 2 mM KCl, 5 mM MgCl₂·6H₂O, 5 mM HEPES sodium salt, pH 7.6), washing buffer (Section 4.2.3), modified Barth’s saline (78 mM NaCl, 1 mM KCl, 0.33 mM Ca(NO₃)₂·4H₂O, 0.41 mM CaCl₂·2H₂O, 0.82 mM MgSO₄·7H₂O, 2.4 mM NaHCO₃, 10 mM HEPES sodium salt, 1.8 mM sodium pyruvate, 10 µg/mL gentamycin, 10 µg/mL streptomycin, pH 7.6) in sequence (5 times for each buffer, using 10 mL each time). Rinsing the oocytes extensively with buffers to remove immature and poor-quality oocytes.
   4. Harvest oocytes at stage V-VI according to Dumont’s classification. Transfer health/good quality oocytes at stage V-VI with a clear white/dark division and no visually obvious damage (Figure 1D) into a 100 mm x 15 mm Petri dish containing modified Barth’s saline (Section 4.3.3). Discard the bad quality oocytes (Figure 1E). Maintain digested oocytes in insect growth chambers at 18 °C for about 24 hours before microinjection.

5. cRNA Microinjection and Expression of Odorant Receptors (ORs) and OR Co-receptors (Orco) in Xenopus Oocytes

1. Put a metal matrix into the 100 mm x 15 mm Petri dish, adding modified Barth’s saline (Section 4.3.3) until the matrix is submerged. Transfer oocytes on the matrix, and discard the oocytes that do not meet the size. Arrange the oocytes with the vegetal poles on the upper side (Figure 1F).
2. Pull a fresh glass capillary (1.0 mm OD x 0.5 mm ID, length = 100 mm) for each sample using a micropipe puller (Heat = 525, Pull = 50, Velocity = 50, Time = 250) and sharpen with a micropipette beveler at an angle of 25°. Mount the capillary tube on the nanoliter injector.
3. Before injection, remove one tube of premixed cRNAs of OR and Orco (Section 3.3) from the freezer and place it on ice until completely dissolved. Fill the capillary tube with the OR/Orco mixture by pressing the Fill button of nanoliter injector.
4. Inject 10 ng (5ng OR + 5ng Orco; ~20 nL) of the premixed cRNA for each oocyte with a nanoliter injector at a RNAse-free station. (Figure 1F).
   NOTE: Before injection, we press the EMPTY button to discharge little mixture to ensure the capillary tube is not blocked. Waiting for about 5 seconds to let the cRNAs enter the oocyte entirely.
5. After injection, store oocytes on a sterile 24-well cell culture plate with modified Barth’s saline at 18°C for 3-7 days before whole-cell recording.

6. Whole-cell Current Recording using a Two-electrode Voltage-clamp system (Figure 2)

1. Before the two-electrode Voltage-clamp recording, dissolve the individual odorant compounds in dimethyl sulfoxide (DMSO) to obtain a series of stock solutions (100 μg/μL). Dilute these stock solutions with 1x ND96 buffer (91 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂·2H₂O, 2 mM MgCl₂·6H₂O, 5 mM HEPES sodium salt, pH 7.6) 1000-fold to obtain a set of working solutions (0.1 μg/μL).
2. Ensure that everything on the TMC vibration isolation table, including the metal shield, microscope, light source, and waste pump, is carefully grounded to reduce the effect of external electrical noise on the whole-cell signal recording.
3. Prepare microelectrodes by pulling a glass capillary (1.20 mm OD x 0.94 mm ID, length = 750 mm) using a micropipe puller (Heat = 525, Pull = 50, Velocity = 50, Time = 250). Fill the capillaries with 3 M KCl. Chloridize the Ag/AgCl wires for 2-5 min in 10% chlorine bleach until they turn a dark grey color (pale grey Ag/AgCl wires need to be chloridized).
4. Turn on the machine by switching on the Oocyte Clamp and Digidata Digitizer (Figure 3A). Open the software (e.g., Clampex 10.3) and click the Record button. Turn on the switch for the 1x ND96 buffer (Section 6.1) and the pump for the waste buffer.
5. Conduct a pre-test by inserting microelectrodes into the flowing buffer and then pressing the V_m Electrode Test button and the V_e Electrode Test button to check the resistance of the microelectrodes. V_m should be < 40 mV and V_e should be < 40 μA. Switch off the 1x ND96 buffer (Section 6.1) flow and the waste pump after the pretest.
6. Place each oocyte in the perfusion chamber (Figure 2) filled with 1x ND96 buffer (Section 6.1) and gently position the oocytes with the vegetal poles facing up by the glass caterpillar. Insert the electrodes slowly into the oocyte (Figure 3B). Adjust the V_m parameter to 0 and then the V_e parameter to 0. Change the Clamp button from OFF to FAST. Adjust the Gain button to ~ -80 mV.
7. Check the value of V_e, which should be constant. If not, there is a leak in at least one of the two microelectrodes. If this happens, replace any faulty components with new capillaries.
8. Record whole-cell currents from the injected oocytes with an oocyte clamp at a holding potential of ~−80 mV (Figure 2).
9. Perform data acquisition and analysis.
   1. Resume the flow of the 1x ND96 buffer (Section 6.1) and turn on the waste pump.
   2. Odorant solution application via the perfusion system: switch off the 1x ND96 buffer (Section 6.1) and flow the odorant solution for 10 seconds. Then record the peak value. To obtain the response of the OR to the test odorant, subtract the baseline value from the peak value.
   3. Stop the flow of odorant solution and switch to the 1x ND96 buffer (Section 6.1). Wash the oocyte using the buffer until the current trace recovers from the previous odorant stimulation. The oocyte is then ready for stimulation by another odorant solution. Generally, one oocyte can be used to detect 20-30 chemical stimulations.
      NOTE: When testing the concentration-dependent response, the lowest concentration should always be applied first before moving on to the higher concentrations.

Results

Using the single sensillum recording (SSR) technique, we recently pinpointed human odorants thought to be important for Ae. aegypti host-seeking behavior⁸. However, the molecular mechanism driving the process of sensing human odorants in the peripheral sensory system of Ae. aegypti remains unknown. ORs play an important role in odorant ligand detection in most insects^10,11,12. To pe...

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