A Step-by-Step Guide to Mosquito Electroantennography

Summary

The present article details a step-by-step protocol for successful and low noise electroantennograms in several genera of mosquitoes, including both females and males.

Abstract

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when acquiring a blood-meal. To locate a host to feed on, mosquitoes rely on a wide range of sensory cues, including visual, mechanical, thermal, and olfactory. The study details a technique, electroantennography (EAG), that allows researchers to assess whether the mosquitoes can detect individual chemicals and blends of chemicals in a concentration-dependent manner. When coupled with gas-chromatography (GC-EAG), this technique allows to expose the antennae to a full headspace/complex mixture and determines which chemicals present in the sample of interest, the mosquito can detect. This is applicable to host body odors as well as plant floral bouquets or other ecologically relevant odors (e.g., oviposition sites odorants). Here, we described a protocol that permits long durations of preparation responsiveness time and is applicable to both female and male mosquitoes from multiple genera, including Aedes, Culex, Anopheles, and Toxorhynchites mosquitoes. As olfaction plays a major part in mosquito-host interactions and mosquito biology in general, EAGs and GC-EAG can reveal compounds of interest for the development of new disease vector control strategies (e.g., baits). Complemented with behavioral assays, the valence (e.g., attractant, repellent) of each chemical can be determined.

Introduction

Mosquitoes are the deadliest organisms on earth, claiming the lives of more than one million people per year and place more than half the world population at risk of exposure to the pathogens they transmit, while biting¹. These insects rely on a wide range of cues (i.e., thermal, visual, mechanical, olfactory, auditory) to locate a host to feed on (both plant and animal), for mating and oviposition, as well as to avoid predators at both the larval and adult stages^2,3. Among these senses, olfaction plays a critical role in the above mentioned behaviors, in particular for medium to long-range detection of odorant molecules^2,3. Odors emitted by a host or an oviposition site are detected by various specific olfactory receptors (e.g., GRs, ORs, IRs) located on the mosquito palps proboscis, tarsi, and antennae^2,3.

As olfaction is a key component of their host-seeking (plant and animal), mating and oviposition behaviors, it thus constitutes an ideal target to study to develop new tools for mosquito control⁴. Research on repellents (e.g., DEET, IR3535, picaridin) and baits (e.g., BG sentinel human lure) is extremely prolific⁵, but because of the current challenges in mosquito control (e.g., insecticide resistance, invasive species), it is essential to develop new efficient control methods informed by the mosquito biology.

Many techniques (e.g., olfactometer, landing assays, electrophysiology) have been used to assess the bioactivity of compounds or mixtures of compounds in mosquitoes. Among them, electroantennography (or electroantennograms (EAGs)) can be used to determine whether the odorants are detected by the mosquito antennae. This technique was initially developed by Schneider⁶ and has been used in many different insect genera since then, including moths^7,8,9, bumblebees^10,11, honeybees^12,13, and fruit flies^14,15 to name a few. Electroantennography has also been employed using various protocols, including single or multiple antennae in mosquitoes^{16,17,18,19,20,21,22,23,24,25}.

Mosquitoes are relatively small and delicate insects with rather thin antennae. While performing EAGs on larger insects such as moths or bumblebees is relatively easy because of their larger size and thicker antennae, conducting EAGs in mosquitoes can be challenging. In particular, maintaining a good signal-to-noise ratio and a lasting responsive preparation are two major requirements for data reproducibility and reliability.

The step-by-step guide to low noise EAGs proposed here directly offers solutions to these limitations and make this protocol applicable to several mosquito species from various genera, including Aedes, Anopheles, Culex, and Toxorhynchites, and describes the technique for both females and males. Electroantennography offers a quick yet reliable way to screen and determine bioactive compounds that can then be leveraged in bait development after valence has been determined with behavioral assays.

Protocol

1. Saline solution preparation

1. Prepare the saline in advance and store in the fridge.
2. Follow Beyenbach and Masia²⁶ to prepare the solution.
   ​NOTE: Saline recipe in mM: 150.0 NaCl, 25.0 HEPES, 5.0 glucose, 3.4 KCl, 1.8 NaHCO₃, 1.7 CaCl₂, and 1.0 MgCl₂. The pH is adjusted to 7.1 with 1 M NaOH. Do not add glucose or sucrose to the preparation at this time to increase shelf storage. Add the needed quantity to the saline right before running the EAGs (around 50 mL per experiment).

2. Odor preparation and storage

1. Prepare the odorant mixtures or single compound dilutions in advance in 1.5 mL amber vials and store at -20 °C to prevent compound degradation.
   NOTE: Concentrations will depend on the test to be conducted. 0.1% or 1% are commonly used to determine whether a compound can be detected or not. For a dose-response curve, prepare serial dilutions of a given chemical and test them from that the lowest to the highest concentrations.
2. Prepare the dilutions in water, ethanol, hexane, paraffin oil, or mineral oil, depending on the solubility of the tested chemical.
3. Make sure to prepare a solvent control (a vial containing only the solvent) for the experiment.
4. Remove the odorants from the freezer 30 min prior to starting the experiments to allow them to thaw. Vortex each vial prior to use to mix well the chemical and the solvent.
5. Pipette 10 µL of solution on to a piece of filter paper (0.5 cm x 2 cm) loaded inside a labeled glass syringe or Pasteur pipette.
6. Load each compound or mixture in a specific Pasteur pipette or syringe to prevent contamination.
   NOTE: Load 10 min before starting the experiment so the odor can diffuse in the syringe but not for longer to prevent degradation. Let the Pasteur pipette or syringe remain capped at this time to allow a good diffusion of the chemical before the experiment starts.
7. After each EAG run, dispose the piece of the filter paper and replace it with a new one to prevent the paper from getting oversoaked and risking needle blockage. Replace the needles regularly (every 10 runs).

3. Mosquito separation

1. Isolate the mosquitoes on the day of the experiments.
2. Use mosquitoes that are at least 6 days old on the day of the experiments to increase the chances that the females are mated to enhance their response to host-related odorants.
   NOTE: Adjust mosquito age at the time of the test depending on the project. Check and harmonize the physiological status (e.g., blood-fed, starved, never previously fed, etc.).
3. Starve the mosquitoes up to 12 h (i.e., no access to sugar) to increase their motivation and sensitivity.
4. Place the mosquito container in the fridge (4 °C) until they stop flying so individuals can easily be delicately transferred to single cups with forceps.
   NOTE: Species with a higher tolerance to cold can be put down using a CO₂ fly pad. Ensure that the mosquitoes do not stay on it for a long time to prevent desiccation, which would decrease the responsiveness of the mosquito EAG preparation.
5. Store the cups containing single mosquitoes at room temperature before the EAGs are performed and discard any mosquitoes that may not be used during the day.

4. Electrode holder and capillary preparation

1. Capillary pulling, preparation, and storage
   1. Use borosilicate capillaries with filaments (I.D: 0.78 mm, O.D: 1 mm). Pull them depending on the equipment²⁷.
      NOTE: Store the pulled capillaries in a Petri dish. Place the Petri dish on to pieces of wax or unscented modeling clay to prevent them from moving and breaking.
   2. Before running the EAG experiment, gently break the tip of 2 capillaries with a pair of forceps under the microscope.
      NOTE: Ensure that one is slightly bigger than the other to fit either the neck (larger capillary) or the tips of the antennae (smaller capillary). Ensure that the cut is clean with no crack present on the capillary wall. This requires patience and practice.
   3. If still intact, reuse these capillaries after rinsing with de-ionized (DI) water after the experiment is over. Remove the excess water by gently applying a cleaning wipe against the tip. Place back in storage Petri dish. If the tip is crooked, discard the capillary.
2. Electrode holder and capillary mounting
   1. Label the two electrode holders as "recording" and "reference" by using pieces of lab tape of different colors. This will help guide the mounting of the mosquito head and electrodes.
   2. Ensure that the electrode holders are clear inside and that no borosilicate debris are present.
   3. Chloridization: Soak the silver wires of the electrode holders in pure bleach for about 5 min. The wires turn from shiny light grey to matte dark grey.
   4. Loosen the rubber stopper and fill the inside of the capillary with 10% saline solution using a 20 G needle.
   5. Fill the borosilicate capillary with the saline solution using a syringe. Ensure that no bubbles are present in neither the electrode holder nor the pulled capillary.
      ​NOTE: To reduce the chances of having bubbles in the capillary, keep pushing saline in the capillary while gently pulling the needle out and use capillaries with a filament. It is possible to load the capillaries with a solution composed of 1:3 electrode gel and saline solution. This can help prevent evaporation of the saline and can be particularly useful when learning and practicing EAGs, as the experimenter will need more time to complete the different steps.
   6. After soaking, rinse the silver wires with DI water and insert them in the two capillaries. Ensure that the tip of the wire is less than 1 mm from the tip of the capillary. Ensure that the capillary passes the rubber ring inside the electrode holder without breaking. Gently tighten the rubber stopper. Verify that no air bubbles are present.
   7. Use the capillary with the wider opening on the reference electrode holder (neck), and the smaller opening on the recording electrode holder (antennae).
   8. Leave the two mounted electrode holders on a wet cleaning wipe to prevent the tip from drying out until ready to mount the head.

5. EAG rig preparation (Figure 1)

1. Ensure that the air table is up, that there is no blockage in the airline. Ensure that the tank of medical air is still full to avoid changing it in the middle of the experiment. Make sure that there are bubbles in the humidifier.
2. Air and pulse delivery system
   1. Turn on the medical air gas tank.
   2. Check the level of the two flowmeters.
      ​NOTE: The flowmeter controlling the main air current bathing the preparation during the whole experiment should be at 140 mL/min and the other related to the odor pulse should read 15 mL/min.
3. If doing GC-EAD, turn on the machine, gas tanks and create/load the file/method.
4. Turn on the computers, the software applications, valve power supply, and verify the internet connection for the software application to work.
   1. Software application: A short script can be written to deliver the pulse.
   2. EAG software: use any electrophysiology software.
   3. Implement the parameters in the software (e.g., amplifier, duration of recording, duration of the pulses, etc.).
5. Deliver a control pulse to verify that the valve delivering the pulses is functional.
6. Set the power supply at 5.2 V. Verify the amplifier parameters.
   NOTE: The parameters used for the data presented here are: low cut-off filter of 0.1 Hz; high cut-off filter of 500 Hz; Gain of x100.

6. Mosquito head preparation and mounting (Figure 2)

1. Place an aluminum plate on ice and place a piece of wet cleaning wipe over it.
2. Put a small dollop of electrode gel in a corner.
3. Place a mosquito cup on ice and let the mosquito cool down for a couple of minutes, or until it is stops flying.
   NOTE: Some species are cold-resistant and might require a quick anesthesia over a CO₂ fly pad to go down. The least the mosquito stays on either, the better.
4. Place the mosquito on its back and clip the tip of each antenna (only a small portion of the last segment) with micro scissors.
5. Use forceps to drag the mosquito next to the electrode gel dollop and dip each antenna's tip gently in the gel. Avoid dipping more than just the very last segment in the electrode gel.
6. Using forceps, pull the mosquito antennae out while maintaining them next to each other. Let them come out together from the gel. Make sure that the antennae do not touch the cleaning wipe's surface, or they might separate.
7. Place the mosquito on its side and chop the head using micro scissors or a razor blade.
   NOTE: Once the head is chopped, proceed quickly to the next steps and to the EAG rig to start the recordings. The preparation should stay responsive for about 30 min.
8. Take the reference electrode and gently deep the tip in the gel. Keep in contact with neck tissues and let the head stick on it.
9. Move the electrode holders under the EAG microscope and view through the microscope to place the head (i.e., reference) electrode on a micromanipulator. Ensure that the antennae are at the center.
10. Grab the recording electrode, place it in front of the antennae tips. Move and align it as close as possible to the tips using the micromanipulator. Using the microscope, move the tip of the recording electrode towards the antennae.
11. Connect both electrode holders to the amplifier before inserting the tips to prevent them from moving after insertion.
12. Insert the antennae tips in the recording electrode. Ensure that they just contact the saline and the electrode gel and are visible by transparency through the capillary. The antenna goes in by the "suction effect".
13. Adjust the position of the head and tips with forceps under the microscope, if needed.
14. Place the airline tubing close to the mosquito head preparation (distance: 1 cm).
    NOTE: If the head falls off, return to the dissection station and remount the head or prepare a new one if it has been lost or if it has been more than 5 min since the head was cut off. A good connection between the capillary and the neck/antennae is essential for low noise and reliable recording. Ideally, the antennae tips will be at less than 1 mm from the wire of the recording electrode once inserted.
15. Turn off the light source, if used.
16. Position the vacuum line near the mosquito head preparation (distance: 20 cm) and align with the main airline.
    ​NOTE: The vacuum will help remove chemicals surrounding the head preparation after the stimulus, which could lead to EAG responses after the pulses were applied.

7. Recordings

1. After insertion of the tips of the antennae, turn on the amplifier and the noise reducer. Observe the baseline signal and ensure that it is not noisy.
   NOTE: Observe whether large oscillations in the electrical signal are present. Adjust the position of the head and antennae tips as needed until the signal is clean. Use alligator clips to ground anything that introduces noise to the Faraday cage or air table. A baseline signal of less than 0.01 mV in amplitude is ideal for detecting and discriminating minute EAG responses.
2. Once the noise level is satisfying, insert the first odor syringe to test in the airline hole.
3. Close the Faraday cage. Do not stay in front of the preparation, to reduce noise.
4. Click on Record on EAG software.
5. Deliver the pulse(s) using the software application.
   NOTE: The number and duration of the pulses will vary depending on the experiments. Here, single 1 s. pulses per odorant have been used. Odorants were separated by 45 s.
6. Note the response of the mosquito antennae in the lab notebook.
   NOTE: If the odorant is detected by the mosquito antennae, a clear deflection in the signal is observed (see Figure 3A).
7. Proceed with the next odor or concentration. Do not forget to randomize the presentation of odorants unless a dose-response curve is performed.
   NOTE: A negative control and a positive control should be used in the experiments. This will ensure that the responses observed are indeed olfactory responses and not due to mechanical or electric noise.
8. At the end of the recording, apply a positive control to verify that the antennae are still responsive.
   NOTE: Use 0.1% or 1% benzaldehyde as all mosquito species tested so far have been responsive to this compound.
9. Proceed with the next mosquito preparation.

8. Cleaning

1. Turn off the amplifier, the noise reducer, the airline, and the computer.
2. Return the odorants to the freezer.
3. Remove the filter papers from the glass syringes and clean with 100% ethanol if residue is visible on the walls. Let dry on a cleaning wipe overnight.
4. Clean the electrode holders with DI water to remove any possible trace of salt. Dry by gently applying against a piece of cleaning wipe.
5. Place mosquito remains in the freezer and dispose 24 h later.
   ​NOTE: If working with infected mosquitoes, follow safety requirements at your institution.

9. Data analyses

1. Measure either manually or automatically the EAG responses.
   NOTE: The EAG amplitude (-mV) is measured here. Average if multiple pulses have been applied for each compound. Depending on the software used, EAGs can be automatically detected and measured. However, it is essential to inspect each response individually to verify the shape of the response and assess the possible carry-over, delayed response, etc. The ideal EAG response is aligned with the pulse, shows a clear deflection, and is repeatable between mosquito preparations (Figure 3).
2. Present the raw data to show minimal variability, low noise signal, and clear responses (Figure 3B).
   NOTE: The data can also be normalized (e.g., Z-score). The negative control value (e.g., mineral oil) (i.e., the baseline) can be subtracted from the response and, if not, should be presented in the figures. A positive control should also be presented.
3. Perform statistical analysis using any statistics software²⁸.

Results

Electroantennography is a powerful tool to determine whether a chemical or blend of chemicals is detected by an insect antenna. It can also be used to determine the detection threshold for a given chemical using a gradual increase of concentration (i.e., dose curve response, Figure 4B). Moreover, it is useful to test the effects of repellent on the response to host-related odors²⁹.

Positive and negative controls should always be used in EAG...

Discussion

Olfactory mediated behaviors are affected by many factors, including physiological (e.g., age, time of day) and environmental (e.g., temperature, relative humidity)³⁰. Thus, when conducting EAGs, it is essential to use insects that are in the same physiological status (i.e., monitoring for age, starving, mating)³¹ and to also maintain a warm and humid environment around the preparation to avoid desiccation. A temperature around 25 °C is ideal and 60% to 80% humidity fo...

Materials

Name	Company	Catalog Number	Comments
Air table Clean Bench	TMC	https://www.techmfg.com/products/labtables/cleanbench63series/accessoriess	Noise reducer
Analog-to-digital board	National Instruments	BNC-2090A
Benchtop Flowbuddy Complete	Genesee Scientific	59-122BC	To anesthesize mosquitoes
Borosillicate glass capillary	Sutter Instrument	B100-78-10	To make the recording and references capillaries
Chemicals	Sigma Aldrich	Benzaldehyde: 418099-100 mL; Butyric acid: B103500-100mL; 1-Hexanol: 471402-100mL; Mineral oil: M8410-1L	Chemicals used for the experiments presented here
CO2	Airgas or Praxair	N/A	To anesthesize mosquitoes
Cold Light Source	Volpi	NCL-150
Disposable syringes	BD	1 mL (309628)  / 3 mL (309657)
Electrode cables	World Precision Instruments	5371
Electrode gel salt free	Parkerlabs	12-08-Spectra-360
Faraday cage	TMC	https://www.techmfg.com/products/electric-and-magnetic-field-cancellation/faradaycages	Noise reducer
Flowmeters	Bel-art	65 mm (H40406-0010) / 150 mm (H40407-0075)	One of each
GCMS vials and caps	Thermo-fisher scientific	2-SVWKA8-CPK	To prepare odorant dilutions
Glass syringes (Fortuna)	Sigma Aldrich	Z314307	For odor delivery to the EAG prep
Humbug	Quest Scientific	http://www.quest-sci.com/	Noise reducer
2 mm Jack Holder, Narrow, 90 deg., With Wire	A-M Systems	675748	Electrode holder
Magnetic bases	Kanetec	MB-FX	x 2
MATLAB + Toolboxes	Mathworks	https://www.mathworks.com/products/matlab.html	For delivering the pulses
Medical air	Airgas or Praxair	N/A	For main airline
Microscope	Nikkon	SMZ-800N
Micromanipulators Three-Axis Coarse/Fine Compact Micromanipulator	Narishige	MHW-3	x 2
Microelectrode amplifier with headstage	A-M Systems	Model 1800
Mosquito rearing supplies	Bioquip	https://www.bioquip.com/Search/WebCatalog.asp
Needles	BD	25G (305127) / 21G (305165)
Pasteur pipettes	Fisher Scientific	13-678-6A	For odor delivery to the EAG prep
PTFE Tubing of different diameters	Mc Master Carr	N/A	To connect solenoid valve, flowmeter, airline ect.
30V/5A DC Power Supply	Dr. Meter	PS-305DM
R version 3.5.1	R project	https://www.r-project.org/	For data analyses
Relay for solenoid valve	N/A	Custom made
Silver wire 0.01”	A-M Systems	782500
Solenoid valve (3-way)	The Lee Company	LHDA0533115H
WinEDR software	Strathclyde Electrophysiology Software	WinEDR V3.9.1	For EAG recording
Whatman paper	Cole Parmer	UX-06648-03	To load chemical in glass syringe / Pasteur pipette