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A Step-by-Step Guide to Mosquito Electroantennography

Published: March 10th, 2021



1Department of Biochemistry; The Fralin Life Science Institute; The Global Change Center; Department of Entomology and the Center for Emerging Zoonotic and Arthropod-Borne Pathogens, Virginia Polytechnic Institute and State University

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.

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.

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 stages2,3. Among these senses, olfaction plays a critical role in the above mentioned behaviors, in particular for medium to long-r....

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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 r.......

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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.......

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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.......

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I am grateful to Dr. Clément Vinauger and Dr. Jeffrey Riffell for helpful discussions. The following reagents were obtained through BEI Resources, NIAID, NIH: Anopheles stephensi, Strain STE2, MRA-128, contributed by Mark Q. Benedict; Aedes aegypti, Strain ROCK, MRA-734, contributed by David W. Severson; Culex quinquefasciatus, Strain JHB, Eggs, NR-43025. The author thanks Dr. Jake Tu, Dr. Nisha Duggal, Dr. James Weger and Jeffrey Marano for providing Culex quinquefasciatus and Anopheles stephensi (strain: Liston) mosquito eggs. Aedes albopictus and Toxorhynchites rutilus septentrionalis are derived f....

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Name Company Catalog Number Comments
Air table Clean Bench TMC 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 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 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 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
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 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

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