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Introduction

Protocol

Representative Results

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Acknowledgements

Materials

References

Biology

A Step-by-Step Guide to Mosquito Electroantennography

Published: March 10th, 2021

DOI:

10.3791/62042

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 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

  1. World Health Organization. World health statistics 2019: monitoring health for the SDGs, sustainable development goals. World Health Organization. , (2019).
  2. Takken, W. The role of olfaction in host-seeking of mosquitoes: a review. International Journal of Tropical Insect Science. 12 (1-2-3), 287-295 (1991).
  3. Zwiebel, L. J., Takken, W. Olfactory regulation of mosquito-host interactions. Insect Biochemistry and Molecular Biology. 34 (7), 645-652 (2004).
  4. Potter, C. J. Stop the biting: targeting a mosquito's sense of smell. Cell. 156 (5), 878-881 (2014).
  5. Paluch, G., Bartholomay, L., Coats, J. Mosquito repellents: a review of chemical structure diversity and olfaction. Pest Management Science. 66 (9), 925-935 (2010).
  6. Schneider, D. Electrophysiological investigation on the antennal receptors of the silk moth during chemical and mechanical stimulation. Experientia. 13 (2), 89-91 (1957).
  7. Raguso, R. A., Light, D. M., Pickersky, E. Electroantennogram responses of Hyles lineata (Sphingidae: Lepidoptera) to volatile compounds from Clarkia breweri (Onagraceae) and other moth-pollinated flowers. Journal of Chemical Ecology. 22 (10), 1735-1766 (1996).
  8. Schweitzer, E. S., Sanes, J. R., Hildebrand, J. G. Ontogeny of electroantennogram responses in the moth, Manduca sexta. Journal of Insect Physiology. 22 (7), 955-960 (1976).
  9. Martel, V., Anderson, P., Hansson, B. S., Schlyter, F. Peripheral modulation of olfaction by physiological state in the Egyptian leaf worm Spodoptera littoralis (Lepidoptera: Noctuidae). Journal of Insect Physiology. 55 (9), 793-797 (2009).
  10. Spaethe, J., Brockmann, A., Halbig, C., Tautz, J. Size determines antennal sensitivity and behavioral threshold to odors in bumblebee workers. Naturwissenschaften. 94 (9), 733-739 (2007).
  11. Suchet, C., et al. Floral scent variation in two Antirrhinum majus subspecies influences the choice of naïve bumblebees. Behavioral Ecology and Sociobiology. 65 (5), 1015-1027 (2011).
  12. De Jong, R., Pham-Delègue, M. H. Electroantennogram responses related to olfactory conditioning in the honeybee (Apis mellifera ligustica). Journal of Insect Physiology. 37 (4), 319-324 (1991).
  13. Patte, F., Etcheto, M., Marfaing, P., Laffort, P. Electroantennogram stimulus-response curves for 59 odourants in the honeybee, Apis mellifica. Journal of Insect Physiology. 35 (9), 667-675 (1989).
  14. Alcorta, E. Characterization of the electroantennogram in Drosophila melanogaster and its use for identifying olfactory capture and transduction mutants. Journal of Neurophysiology. 65 (3), 702-714 (1991).
  15. Park, K. C., Ochieng, S. A., Zhu, J., Baker, T. C. Odor discrimination using insect electroantennogram responses from an insect antennal array. Chemical Senses. 27 (4), 343-352 (2002).
  16. Du, Y. J., Millar, J. G. Electroantennogram and oviposition bioassay responses of Culex quinquefasciatus and Culex tarsalis (Diptera: Culicidae) to chemicals in odors from Bermuda grass infusions. Journal of Medical Entomology. 36 (2), 158-166 (1999).
  17. Costantini, C., et al. Electroantennogram and behavioural responses of the malaria vector Anopheles gambiae to human-specific sweat components. Medical and Veterinary Entomology. 15 (3), 259-266 (2001).
  18. Collins, L. E., Blackwell, A. Electroantennogram studies of potential oviposition attractants for Toxorhynchites moctezuma and T. amboinensis mosquitoes. Physiological Entomology. 23 (3), 214-219 (1998).
  19. Seenivasagan, T., Sharma, K. R., Sekhar, K., Ganesan, K., Prakash, S., Vijayaraghavan, R. Electroantennogram, flight orientation, and oviposition responses of Aedes aegypti to the oviposition pheromone n-heneicosane. Parasitology Research. 104 (4), 827-833 (2009).
  20. Puri, S. N., Mendki, M. J., Sukumaran, D., Ganesan, K., Prakash, S., Sekhar, K. Electroantennogram and behavioral responses of Culex quinquefasciatus (Diptera: Culicidae) females to chemicals found in human skin emanations. Journal of Medical Entomology. 43 (2), 207-213 (2014).
  21. Cooperband, M. F., McElfresh, J. S., Millar, J. G., Carde, R. T. Attraction of female Culex quinquefasciatus Say (Diptera: Culicidae) to odors from chicken feces. Journal of Insect Physiology. 54 (7), 1184-1192 (2008).
  22. Dekker, T., Ignell, R., Ghebru, M., Glinwood, R., Hopkins, R. Identification of mosquito repellent odours from Ocimum forskolei. Parasites & Vectors. 4 (1), 183 (2011).
  23. Choo, Y. M., et al. Reverse chemical ecology approach for the identification of an oviposition attractant for Culex quinquefasciatus. Proceedings of the National Academy of Sciences. 115 (4), 714-719 (2018).
  24. Wolff, G. H., Lahondère, C., Vinauger, C., Riffell, J. A. Neuromodulation and differential learning across mosquito species. bioRxiv. , 755017 (2019).
  25. Lahondère, C., et al. The olfactory basis of orchid pollination by mosquitoes. Proceedings of the National Academy of Sciences. 117 (1), 708-716 (2020).
  26. Beyenbach, K., Masia, R. Membrane conductances of principal cells in Malpighian tubules of Aedes aegypti. Journal of Insect Physiology. 48, 375-386 (2002).
  27. Oesterle, A. . The Pipette Cookbook. , (2018).
  28. R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing. , (2018).
  29. Afify, A., Betz, J. F., Riabinina, O., Lahondère, C., Potter, C. J. Commonly used insect repellents hide human odors from Anopheles mosquitoes. Current Biology. 29 (21), 3669-3680 (2019).
  30. Martin, F., Riveron, J., Alcorta, E. Environmental temperature modulates olfactory reception in Drosophila melanogaster. Journal of Insect Physiology. 57 (12), 1631-1642 (2011).
  31. Qiu, Y. T., Gort, G., Torricelli, R., Takken, W., van Loon, J. J. Effects of blood-feeding on olfactory sensitivity of the malaria mosquito Anopheles gambiae: application of mixed linear models to account for repeated measurements. Journal of Insect Physiology. 59 (11), 1111-1118 (2013).
  32. Taylor, B., Jones, M. D. R. The circadian rhythm of flight activity in the mosquito Aedes aegypti (L.): the phase-setting effects of light-on and light off. Journal of Experimental Biology. 51 (1), 59-70 (1969).
  33. Eilerts, D. F., VanderGiessen, M., Bose, E. A., Broxton, K., Vinauger, C. Odor-specific daily rhythms in the olfactory sensitivity and behavior of Aedes aegypti mosquitoes. Insects. 9 (4), 147 (2018).
  34. Krishnan, B., Dryer, S. E., Hardin, P. E. Circadian rhythms in olfactory responses of Drosophila melanogaster. Nature. 400 (6742), 375-378 (1999).
  35. Pelletier, J., Guidolin, A., Syed, Z., Cornel, A. J., Leal, W. S. Knockdown of a mosquito odorant-binding protein involved in the sensitive detection of oviposition attractants. Journal of Chemical Ecology. 36 (3), 245-248 (2010).

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