A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (Bombus terrestris)

The overall goal of this procedure is to assess the gustatory sensitivity of adult worker bumble bees towards nutrients and toxins. With this method, we can measure the natural feeding responses of bees in fine detail. This will allow us to identify the bee's ability to detect both nutrients and toxins in solution.

The main advantage of this technique is that it provides several response measures, such as the amount of food bees consume in a short period and the duration of the feeding bout. This has several advantages in comparison to existing go-no-go methods, such as a proboscis extension reflex protocol. Assays of behavior provide valuable insights into neuronal mechanisms.

This assay will provide a controlled way of studying the freely moving bees responses to toxic compounds in food. A visual demonstration of this method is really critical, as the preparation and the presentation of the solutions can be difficult to learn. They require a really precise delivery of the solutions at the correct time.

This demonstration uilizes Bombus terrestris audax from commercially reared colonies. Two hours before the start of the experiment, collect individual worker bumble bees using a plastic vial with a perforated plastic stopper. To begin, transfer vials of individually collected bees to a dark environment and starve them for two to four hours.

Bees are tested in a holding tube. The holding tube is a modified 15 milliliter centrifuge tube with a four millimeter hole drilled at the tip and a piece of steel mesh fixed inside. Next, fix two pieces of cardboard to the sides of the polystyrene holding tube cradle to shield the bee from visual stimuli during the experiment.

After the starvation period, transfer one bee from the vial into a holding tube. Then, fix the holding tube to the polystyrene cradle using dental wax. Next, position a digital microscopic camera.

Attach the camera to a suitable computer setup for recording. Then, position the camera five centimeters above the tip of the holding tube. Finally, adjust the holding tube so that at least 18 milliliters of the tip is within the video frame.

The test is broken into two phases:the pretest, where a drop of sucrose is presented, and the test phase, where the solution of interest is presented. Before the start of the experiment, connect a six centimeter-long silicone tube with a one millimeter inner diameter via a male adapter to another six centimeter length of silicone tubing but with a four millimeter inner diameter. This will function like a pipette bulb.

Now, set up the test solutions. First, prepare a 100 microliter microcapillary tube with the test solution, then scan the tube with a flatbed scanner at 600 dpi. Next, connect the microcapillary tube to the silicone tube.

Now, insert the capillary into a micro-manipulator holder, such as a modified one milliliter syringe. Then use the manipulator to get the capillary in a ready position. Begin video recording after the habituation period.

In this study, the proboscis activity is recorded at 26.7 frames per second with 25X magnification. Secondly, prepare a syringe with 500 millimolar sucrose dissolved in DI water. Then attach the syringe to a female adapter.

Now, start the test. First, present the syringe with a sucrose droplet of about 3.5 microliters at the holding tube tip to motivate the bee to extend its proboscis. Give the bee up to five minutes to consume the sucrose.

If the droplet is not fully consumed, remove the bee from the experiment. Once the bee finishes the droplet, immediately remove the syringe. Then, position the microcapillary five to 10 millimeters from the holding tube tip and gently squeeze the tubing as needed to keep the solution at the tip of the microcapillary tube.

Run the test for two minutes, starting at the first contact of the proboscis with the solution. After two minutes, remove the microcapillary and scan it again to measure the volume of test solution consumed by the bee. Feeding responses to one molar sucrose, one molar sucrose with one millimolar quinine and pure deionized water were compared.

The continued proboscis activity was scored during the first two minutes of the tests. A feeding bout is defined as contact between the extended proboscis and the solution without disruption to contact for intervals of five seconds or greater. The cumulative duration of the feeding bouts with sucrose containing quinine was significantly reduced in comparison to sucrose alone but not to deionized water alone.

Similarly, the treatments have a significant effect on the frequency of feeding bouts during the test phase. Likewise, the speed of proboscis retraction differed significantly between treatments. Bumble bees retracted their proboscis away from the test solution significantly faster after the first contact with sucrose containing quinine than with sucrose or deionized water alone.

The total consumption of the test solution was measured after the test phase. Bees ate significantly less of the sucrose solution containing quinine than pure sucrose solution. The response to quinine was not significantly different to deionized water.

Once mastered, this technique can be done in two to three hours with approximately eight bees if it's performed correctly and all the bees respond to the solution of interest. After watching this video, you should have a good understanding of the value of this method for providing data about the types of substances that bees can detect in food. While attempting this method, it's really important not to spill any solution into the holding tube and to not leak any of the solution of interest out of the microcapillary tube.

We hope that this technique will allow researchers in the field of gustation to further investigate how the bees'taste system incurs information about nutrients and toxins.