A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research

The Proboscis Extension Response (PER) conditioning protocol, developed for the honey bee (Apis mellifera), provides an ecologically-relevant and easily quantifiable means for studying several different mechanisms of learning in many insect species.

The overall goal of this procedure is to show how to implement behavioral conditioning of a simple reflexive response to food in honeybees. This is accomplished by first capturing honeybees, restraining them in harnesses, feeding them a small amount of secret solution, and then allowing them to become accustomed to the restraint. Next beads are selected for conditioning that show sufficient motivation when their antennas are touched with sucrose.

During conditioning, odor presentation is paired with a sucrose reward to elicit the conditioned response. Next, unreinforced trials measure the presence or absence of the response, the latency of the response, and the duration of the response. The final step is data analysis and graphical presentation of the conditioning and test trials.

This technique has been successfully applied to studies of honeybee biology and health learning behavior is particularly sensitive to disruptive treatments such as pesticides, making it possible to assess the impact of these types of treatments on honeybee behavior at sub lethal levels. Visual demonstration of this method aids in understanding of the stimulus timing required for effective conditioning and the common errors that reduce effectiveness. An individual new to this method will struggle with the attention to detail required both the protocol and the surrounding conditions.

To begin collect worker bees from the entrance to the colony as they pause before departing or as they return from foraging. Capture only one bee per vial. Hold the vial opening horizontally or facing downward while fastening the lid to prevent escape.

Once the bees are collected, return to the lab and place a few of the vials at a time into an ice water slurry. Once a bee is immobile, remove the vial from the ice immediately to avoid overexposure and place the bee into a restraining harness. Restrain the bee with its dorsal thorax facing the cutaway portion of the holder and its head just above the top.

Gently press the bee so that its mouth parts extend beyond the edge of the holder. Slide a strip of duct tape in between the head and the thorax on the dorsal side and firmly attach the free end to the side of the holder. Make sure the tape on the harness is smooth and taut.

There should be no noticeable gaps between the front of the bee holder and tape. If there are gaps, the bee may not be able to extend his per bois properly or it may be able to escape. Allow the bees about 30 minutes to recover from harnessing before feeding.

Then in an area well away from the training area, feed each bee about three to four microliters of 0.5 molar sucrose during the interval between feeding and conditioning. Place the bees in a quiet area of the lab and in a plastic container with wet paper towels. During this time, a few minutes prior to beginning conditioning, Testa bes motivation to feed by touching their antennae with a small droplet of 0.5 molar sucrose solution.

Do not allow them to feed during this test. If they respond by extending their psis, they are probably sufficiently motivated to learn and can be used for the protocol. When ready, place the glass odor syringe in a modeling clay holder and adjust it so that the opening in the wide end is pointed directly at the bees and 10 E.The end of the syringe should be one to two centimeters from the bee securely placed the fitting connecting the cartridge to the Airstream tubing and valve system over the narrow end of the syringe.

At the beginning of each trial, place the harnessed bee on the peg in the conditioning arena with its antennae pointed directly toward the odor cartridge. Let the bee rest in the arena for 15 to 25 seconds before beginning the odor stimulus to allow it to become accustomed to its new surroundings. Press the start button to initiate the timing mechanism for odor delivery.

Monitor the bee's response to the odor after stimulus onset and before presentation of the unconditioned stimulus. Following each trial, allow the bee to rest in the conditioning area for another 15 to 25 seconds to allow for initial memory formation. Moving the bee too soon following the trial will significantly reduce the effectiveness of each conditioning trial.

To deliver the sucrose, hold the tip of the needle two to three centimeters from the bee while the odor is presented. Use the edge of the training arena to prevent shaking the syringe as this movement can distract the bee. A feeding signal will sound during the presentation of the odor stimulus.

As soon as the feeding signal sounds lightly touch the antennae until the bee extends as probos, then feed the bee. Timing between the presentation of the odor and the presentation of the sucrose is critical. Ideally, these two stimuli should overlap briefly.

If more than a few seconds elapses between presentation of the odor and presentation of the sucrose, the performance in conditioning will diminish. Following conditioning, use unreinforced test trials to assess how well the bees remember the conditioned association. First, set up a video camera on a tripod above the training area and focus the camera on the front of the bee's head so the antennae and probus are in sharp focus.

Make sure a small LED light, which indicates odor stimulus presentation is in an area behind the bee and where it is visible in the video, but not to the bee. This allows identification of the time of odor onset and offset during analysis. For the most consistent results, use freshly prepared odor cartridges for testing once everything is set up.

Begin testing by presenting the odor as shown earlier. Begin recording 20 seconds before presenting the odor and continue recording for at least 20 seconds afterwards. After testing, use video editing software to score each bee's response to the condition to stimulus.

Use a binary scoring system to score the psis extension response scoring the responses as either positive or negative. Score B'S response as positive. When the B extensus psis beyond the line made by connecting the tips of the opened mandibles only score a positive response when the B extends his prob bois after odor onset, but before odor offset score B'S response as negative when there is no extension of the prob Bois during the trial, or if the Pro Bois is extended after the offset of the odorant, other parameters can be collected such as the latency to extension and the number and total duration of the extension response responses to the condition.

Stimulus increased over subsequent conditioning trials. Responses to novel test odors, which differed from the stimulus in carbon chain length and or position of the carbon carbon were reduced proportionally to the degree of difference between the condition stimulus and the novel test odor. These data illustrate the variability and successful conditioning between new investigators.

Responses to octal differed depending on the group performing the testing. During test trials, the odor used as the condition stimulus elicited the strongest response. When octal was used as the condition stimulus, the response was stronger during testing when compared to methyl cyclone, a novel odor.

Similarly, methyl cyclone produced stronger responses when it was used as the conditioned stimulus. When backwards pairing was used, the conditioned response was reduced to near the level of the novel odor illustrating the importance of precise timing for successful conditioning After its development. This technique paved the way for researchers in the field of neuroscience to explore the neural bases of learning and memory and insect model organisms such as the honeybee.

Don't forget that working with insects, especially honeybees can be hazardous. Make sure you wear appropriate protective gear when working with these animals.