For electroantennography, using mosquitoes in a specific and consistent physiological status is critical to obtain reliable results. The step-by-step protocol allows for lasting electroantennographic recordings in mosquitoes. The method can be used in several mosquito species, and in both males and females.
The electroantennographic technique can also be transposed to other insect models, including beetles, flies, kissing bugs, and ants. To begin, remove the 1.5 milliliter amber vials containing the odorant mixtures and a solvent control from the 20 degree Celsius freezer in which they're stored to prevent degradation. Next, pipette out 10 microliters of the odorant solution onto a piece of filter paper loaded inside a labeled glass syringe.
Isolate the mosquitoes on the day of the experiment. Working under the microscope, gently break the tip of two borosilicate capillaries with filaments using a pair of forceps. Before running the electroantennography, or EAG experiment, ensure that the electrode holders are clear inside, with no borosilicate debris.
Then perform chloridization by soaking the silver wires of the electrode holders in pure bleach for five minutes, until the wires turn dark, matte gray. Then rinse the wires. Loosen the rubber stopper and use a 20 gauge needle to fill the inside of the capillary with saline solution.
Rinse the capillaries and insert the wires in the two capillaries. Keep the tip of the wire less than one millimeter from the tip of the capillary. Pass the capillary through the rubber ring inside the electrode holder without breaking it and gently tighten the rubber stopper, after verifying that no air bubbles are present.
Use the capillary with the wider opening on the reference electrode holder and the smaller opening on the recording electrode holder. Leave the two mounted electrode holders on a wet cleaning wipe to prevent the tip from drying out until ready to mount the head. Ensure that the air table is up.
There is no blockage in the airline and the air is on. Verify that the tank of medical air is full to avoid changing it in the middle of the experiment and confirm bubbles in the humidifier. Turn on the computers, the software applications, and the valve power supply.
After verifying the internet connection, deliver a control pulse to verify that the valve delivering the pulses is functional. Place an aluminum plate on ice with a piece of wet cleaning wipe over it and put a small dollop of electrode gel in a corner. Place a mosquito cup on ice to let the mosquito cool down.
Clip the tip of each antenna of the mosquito with micro scissors. Use forceps to drag the mosquito next to the electrode gel dollop and dip each antenna's tip gently in the gel. Using forceps, pull the mosquito antennae out, while maintaining them next to each other.
Chop the head of the mosquito using micro scissors. Gently dip the tip of the reference electrode in the gel and place it in contact with the neck, letting the head stick to it. Place the reference head electrode on a micro manipulator.
Connect both electrode holders to the amplifier. Use the micro manipulator to place the recording electrode as close as possible to the antenna tips. Then insert the antenna tips in the recording electrode.
Place the airline tubing close to the mosquito head preparation, at one centimeter. Turn on the amplifier and the noise reducer and ensure that the baseline signal is not noisy. Once the noise level is satisfactory, insert the first odor syringe in the airline hole to perform the test and close the Faraday cage.
Then click record on the EAG software to deliver the pulse. Measure EAG responses as amplitude in millivolts and then proceed with the next odor or concentration. The experiment successfully demonstrates that mosquito species can exhibit variable olfactory responses to different chemicals.
There was an absence of response to the mineral oil, which was used as a negative control. Error in the results due to electrical noise can be reduced by grounding the elements to the Faraday cage, using alligator clips. The response threshold to the same odorant can vary in magnitude for different species of mosquitoes.
For example, Toxorhynchites rutilus septentrionalis mosquitoes produce very large EAGs, compared to Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus. A large deflection in response to the positive control, benzaldehyde, was noticed. While there was a lack of response to the negative control, mineral oil.
These average antennal responses are shown in a bar diagram. Additionally, the threshold of detecting a specific chemical for each mosquito species was determined by presenting the antenna, the increasing concentrations of the chemical, and plotting a dose response curve. It is important to start the recording as soon as possible after the head has been chopped and mounted, to ensure great responsiveness of the preparation.
EAG allows to determine whether the mosquito antennae respond to a given chemical at a given concentration. However, it does not allow for determining the balance of this chemical. For example, whether it's an attractant or repentance.
