ハチミツの抑止化学物質をスクリーニングするためのビデオトラッキングプロトコル

The overall goal of this procedure is to evaluate chemical deterrents for honey bees. This method can help answer key questions in the field of entomology, such as what chemicals manipulate honey bee behavior away from a food source. The main advantage of this protocol is that it provides a simple approach to screen chemical deterrents for honey bees in a relatively quick and easy manner.

To prepare a sugar-agarose cube for the control treatment, pour a semi-cooled sugar-agarose solution into a weigh boat mold. For the repellent treatment, add the desired amount of compound to the semi-cooled agarose solution. In this demonstration, a final concentration of 1%DEET is used.

Swirl the flask to mix in the compound and then pour the solution into a weigh boat mold. After cooling the sugar-agarose cubes, remove the solidified cubes from the weigh boat molds and place them into a plastic container with a moistened paper towel. Place the containers into a refrigerator for storage.

In the tracking software, navigate to experiment settings in the experiment explorer bar, located on the left of the screen. Make sure that the correct camera is selected and that the video recording is centered on the Petri dish arenas. If the video recording needs to be centered, go into the settings of the camera, under the area of interest controls and select the Center X and Center Y options.

Under experiment settings, change the number of arenas to 16. Under tracked features, select center point detection. Then select arena settings to set up the arena desired for the assay.

Place the 16 Petri dishes in a four by four pattern on top of a light box positioned under the camera. In the arena settings, use the grab background button located on the right side tool panel to take a picture of the 16 Petri dishes. This will be used as the template to set up the arena.

Select create ellipse from the toolbar at the top of the arena settings screen and create a circle that matches the diameter of one of the Petri dishes in the grabbed image. Place the arena one marker into the circle. Then select zone group one, located under arena one in the right side tool panel.

Select create rectangle from the toolbar at the top of the screen. Create a 30 by 30 pixel square using this tool. And then position it at the center of the circle that was created in the previous step.

Select add zone from the top toolbar and then click in the middle of the square. Move the zone one marker so that it is in the square. Next, select arena one in the right side tool panel and then click duplicate complete arena.

From the dropdown menu, select all other arenas and then click OK.Move the duplicated arena setups to the remaining Petri dishes in the grabbed image. Then select arena one and select calibrate scale on the top toolbar. Draw the calibration line across the diameter of the arena one Petri dish.

Change the diameter measurement to nine centimeters. Select validate arena settings on the right side tool panel. Next, select detection settings on the control bar on the left.

Select detection settings one and then select gray scaling in the dropdown menu located in the right side tool box. Under detection, set the range so that it is zero to 83. Right click detection settings and make a new setting named Detection Settings Two.

Make sure gray scaling is still selected but do not change the other parameters. Select trial list in the experimental explorer and click add variable in the top tool bar. Name the user defined variable as Treatment.

Select the predefined values dropdown bar in the user defined treatment column and add control and treatment as predefined values. Click the add trials button located at the top toolbar and add two trials. Then for each arena, select whether it is a control arena or a treatment arena.

Next, select data profile under the experiment explorer panel. On the left hand column, select treatment, located under the user defined independent variables heading. This will add a filter box into the area with the flowchart.

Select C in the popup box and then place the newly created filter in between the start box and the result one box. The flowchart arrows should adjust so that they point from the start box to the filter and finally to the result one. Repeat this step, however this time, select T for the filter and then select result under the common elements section.

Move the newly created boxes into the flowchart area and connect the boxes with arrows, save the file. Start up the visual tracking program and open the saved experimental file that was created for this assay. Select detection settings two.

Then place control sugar-agarose cubes into the centers of eight of the 16 Petri dishes. Repeat this process with the sugar-agarose compound cubes in the remaining eight dishes. Use forceps to remove a single honey bee from the plastic box and place it into one of the control Petri dishes.

Repeat this step for the remaining 15 Petri dishes. Place all of the dishes onto the light box. Illuminate the light box from below by an array of LED lights set to the red spectrum and position them so that the arena detection areas fit into each of the Petri dish arenas.

Surround the entire box and camera with a black plastic sheet to eliminate external light and reduce shadows within the arenas. After the Petri dishes have been set, make sure detection settings one is the selected setting. At this point, the visual tracking software should be picking up only the honey bees within the Petri dish arenas.

Then select acquisition from the experiment explorer. Press the green start trial button located in the acquisition control popup box on the right to begin recording. Run the assay for 10 minutes and then click the red stop trial button on the acquisition control popup box to stop recording.

Representative results of the effect of DEET on the feeding behavior of individual honey bees are shown here. The individuals exposed to the control cubes spent an average of 343 seconds in the feeding zone, compared to 16 seconds for individuals exposed to cubes with DEET. Here representative results of the effects of two different compounds on the feeding behavior of individual honey bees are shown.

Individuals exposed to a non-deterrent compound spent an average of 282 seconds in the feeding zone, as compared to the controls, which spent around 352 seconds there. Exposure to a deterrent compound results in individuals spending a significantly less amount of time, around 23 seconds, in the feeding zone. As compared to the controls that spent an average of 493 seconds in the zone.

Once mastered, this technique can be done in 20 minutes if it is performed properly. While attempting this procedure, it is important to remember to prepare everything in advance and starve the honey bees for an adequate amount of time. After watching this video you should have a good understanding of how to determine the effects of a compound that is infused into a food source on individual honey bees using video tracking software.

Following this procedure, other methods like near field and field assays can be performed in order to answer additional questions concerning a compound's effect at the colony level. After its development, this technique paved the way for researchers in the field of entomology to explore novel chemistries and their effects on feeding behavior in honey bees. Don't forget that working with compounds can be extremely hazardous and precautions, such as wearing personal protection equipment, should always be taken while performing this procedure.