A Wind Tunnel for Odor Mediated Insect Behavioural Assays

This method can answer key questions in the field of chemical ecology, such as identifying behaviorally active components. The main advantages of this technique is that we can release odors by many different means, and we can monitor the whole behavioral repertoire of the insect. Prepare the glass tubes with gauze, using a rubber band.

Separate 10 insects into the prepared glass tubes, and cover the remaining end with plastic snap caps. Allow the insects to acclimatize to the temperature, light conditions, and humidity of the wind tunnel room for at least two hours. Here the sprayer protocol is demonstrated to prepare odor sources.

Fill the one-milliliter, gastight syringe with a synthetic odor mix, and insert the syringe into the syringe pump. Connect the syringe tip to the fluorinated ethylene propylene tubing using tubing adapters. Now, start the syringe pump, followed by the broadband ultrasonic generator.

The release of the aerosol from the sprayer can be confirmed by point a light from below the release point. Position the glass tube with the insects onto a holder 180 centimeters downwind from the odor visual source, and 30 centimeters from the ground. The capped end should point upwind.

To start the protocol, open the cap and start the timer. Observe the flight pattern, and pay special attention to the flight characteristics and upwind orientation. Shown here, is an oriented flight without landing.

Oriented flight with landing is shown here, on a natural odor source, which is included as a control. A closeup of a take off can be seen here, as well as a closeup of a landing on a natural odor source. Score the flight performance according to predefined behavior categories, such as take off, oriented flight over a short distance, oriented flight over a longer distance, and landing.

Collect the insects landing on the walls of the wind tunnel, outside the odor plume, and replace them back onto the holder. Use a separate syringe, dedicated to pure ethanol, for all cleaning. Run the sprayer with 96%ethanol for a minimum of 10 minutes between the treatments, to clean the inside of the tubes and nozzle.

Clean the syringes and the tip of the nozzle with 96%ethanol, after use. Upon completion, wash all metal and glass hardware with ethanol and water, and leave until dry. Heat all metal and glass hardware to 300 degrees Celsius for six hours, to remove any contaminants.

Visually guided odor responses of blow flies are shown here. Details of flight behavior with and without a visual stimuli were studied. There was a significant effect of visual cue on landing response, but not on the other behavioral steps.

A comparison of landing responses of pea moths to pea plants and the corresponding volatile collections are presented here. Similar landings were recorded towards both types of cues. A synthetic blend of pea flower volatiles resulted in slightly lower landings, as compared to natural flower volatiles.

Here is shown the effect of plant volatile backgrounds on apple fruit moth response to synthetic blends. Odor dispensers with a complex blend and a simple blend resulted in similar upwind attraction, with a clean background. However, when embedded in an apple volatile background, the moths preferred the complex blend.

When attempting this procedure, it's important to remember that the flight pattern is not uniform. It varies a lot across insect orders. Blow flies are strong fliers and have a wider casting movements, compared to the moths.

The wind tunnel facilitates numerous methods for releasing odors into the wind tunnel, depending on the question at hand. We can release from odor dispensers, from authentic sources, synthetic mixes, and headspace collections. Also, a specific odor can be released on top of a background odor, we can use different visual stimuli, and we can perform two-choice bioassays.

Wind tunnel experiments are important tools in identifying behaviorally active compounds and chemicals, both in applied research, as well as in basic research. A good example is if you want to develop tools for pest control.