Name: Video: Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees
Uploaded: 2017-06-12T14:00:00.000+00:00
Duration: 10 min 19 s
Description: 6.6K Views. Virginia Tech. The loss of honey bee colonies presents a challenge to crop pollination services. Current pollinator protection practices warrant an alternative approach to minimize the contact of honey bees to harmful pesticides using repellent chemistries. Here, we provide detailed methods for a visual tracking protocol to screen deterrents for bees.

We would like to thank Dr. Thomas Kuhar for the use of the visual tracking software and equipment. We thank James Wilson and Scott O'Neal for their technical assistance.

1. Prepare Sugar-agarose Cubes
<ol>
	<li>Weigh out 8 g of sugar and place into a 50 mL Erlenmeyer flask.</li>
	<li>Fill the Erlenmeyer flask with 20 mL of de-ionized water. Dissolve the sugar by swirling the flask.</li>
	<li>Weigh 170 mg of agarose and add it to the sugar solution.</li>
	<li>Heat the sugar-agarose solution in a microwave on high for 25 s. Dissolve the agarose into the sugar solution.</li>
	<li>Allow the flask and sugar-agarose solution to cool. 
	NOTE: The flask should be cool to the touch, but do not allow the solution to solidify.
	<ol>
		<li>To prepare a sugar-agarose cube for the control treatment, pour the semi-cooled sugar-agarose solution into a weigh boat mold. 
		NOTE: The weigh boat mold has the dimensions 1.5 x 1.5 x 0.3 cm3.</li>
		<li>To prepare a sugar-agarose-compound cube for the repellent treatment, add the desired amount of compound to the semi-cooled solution (e.g., 1% DEET in sugar-agarose solution (v/v)). Swirl the flask to mix in the compound and then pour the solution into a weigh boat mold. 
		NOTE: Eight control molds and eight test molds will be prepared at this point. Control molds do not contain repellent.</li>
	</ol>
	</li>
	<li>Cool the sugar-agarose cubes in the molds in a refrigerator for 5-10 min. Remove the solidified sugar-agarose cubes from the weigh boat molds and place them into a plastic container with a moistened paper towel.</li>
	<li>Place the containers into a refrigerator for storage. The sugar-agarose cubes should be used within 7 days of preparation.</li>
</ol>
2. Programming the Video Tracking Software and Experimental Setup
<ol>
	<li>Under Experiment Settings in the Experiment Explorer bar (in the tracking software, see Materials Table), 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.
	<ol>
		<li>If the video recording needs to be centered, go into the settings of the camera and under the AOI controls and select the center X and center Y options.</li>
	</ol>
	</li>
	<li>Under Experiment Settings, change the number of arenas to 16. Under Tracked Features select Center-point detection.</li>
	<li>Select Arena Settings to setup the arena desired for the assay.
	<ol>
		<li>Place the 16 Petri dishes in a 4 x 4 pattern on top of a light box, positioned under the camera (Figure 1). 
		NOTE: These dishes are used to create the recording arena and are empty.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/55603/55603fig1.jpg" /> 
Figure 1: Petri dish arrangement on the light box. Petri dishes are arranged in a 4 x 4 block on top of the light box. This arrangement provides easy identification of the control and repellent treatments for the visual tracking protocol. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55603/55603fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>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 setup the arena.</li>
	<li>Select &#34;Create ellipse&#34; from the tool bar at the top of the Arena Settings screen and create a circle that matches the diameter of one of the petri dishes in the image grabbed. Place the Arena 1 marker into the circle (Figure 2).</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/55603/55603fig2.jpg" /> 
Figure 2: Screenshot of the Visual Tracking Software Arena Settings. The observation of diagonal stripes within the circle provides confirmation of the detection area in the circle. A Zone 1 marker is provided for each square and defines the target zone for each Petri dish. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55603/55603fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="6">
	<li>Select &#34;Zone Group 1&#34; located under Arena 1 in the right side tool panel. Select &#34;Create Rectangle&#34; from the tool bar at the top of the screen. Create a 30 x 30 pixel square using this and then position it at the center of the circle that was created in the previous step. Select &#34;Add Zone&#34; from the top tool bar and then click in the middle of the square. Move the Zone 1 marker so that it is in the square. 
	NOTE: The square created is the feeding zone and it is not a requirement that the feeding zone be exactly the same size as listed as long as it is slightly larger than the sugar-agarose cube.</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/55603/55603fig3.jpg" /> 
Figure 3: Screenshot of Completed Arena Settings. The completed arena settings should look like this example. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55603/55603fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="7">
	<li>Select &#34;Arena 1&#34; in the right side tool panel and then click duplicate complete arena. From the drop down menu select &#34;All Other Arenas&#34; and then click ok. Move the duplicated arena setups to the remaining petri dishes in the grabbed image (Figure 3).
	<ol>
		<li>Select &#34;Arena 1&#34; and then select &#34;Calibrate Scale&#34; on the top tool bar. Draw the calibration line across the diameter of the Arena 1 Petri dish. Change the diameter measurement to 9 cm (diameter of the Petri dishes being used). Select &#34;Validate Arena Settings&#34; on the right side tool panel.</li>
	</ol>
	</li>
	<li>Select &#34;Detection Settings&#34; on the control bar on the left.</li>
	<li>Select &#34;Detection Settings 1&#34; and then select &#34;Grey Scaling&#34; in the drop down menu located on the right side tool box. Under detection, set the range so that it is 0 to 83 (Figure 4A).</li>
	<li>Right click &#34;Detection Settings&#34; and make a new setting named &#34;Detection Settings 2&#34;. Make sure &#34;Grey Scaling&#34; is still selected, but do not change the other parameters. 
	NOTE: There will be large yellow circles in the video window over the arenas. This will aid in arranging the Petri dishes into the correct positions before recording (Figure 4B).</li>
</ol>
<img alt="Figure 4" src="/files/ftp_upload/55603/55603fig4.jpg" /> 
Figure 4: Screenshot of the Detection Settings 1 and 2. (A) Shows what the arena will look like with the grey scaling corrected for a honey bee subject in a petri dish. (B) Shows the arena detection area so that positioning of the petri dishes can be done between trials. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55603/55603fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="11">
	<li>Select &#34;Trial List&#34; in the Experimental Explorer and click &#34;Add Variable&#34; in the top tool bar. Name the user defined variable as &#34;Treatment&#34;. Select the &#34;Predefined Values&#34; drop down bar in the user-defined Treatment column and add C (control) and T (treatment) as Predefined Values. Select the &#34;Add Trials&#34; button located on the top tool bar. Add two trials, then for each arena select whether it is a control arena (C) or treatment arena (T) (Figure 5).</li>
</ol>
<img alt="Figure 5" src="/files/ftp_upload/55603/55603fig5.jpg" /> 
Figure 5: Screenshot of the Trial List. Labeling the arenas correctly is important here as the program uses the information here to separate the data to statistically analyze it. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55603/55603fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="12">
	<li>Select &#34;Data Profile&#34; under the Experiment Explorer panel. On the left hand column select &#34;Treatment&#34; located under the &#34;User-defined Independent Variables&#34; heading. This will add a &#34;Filter&#34; box into the area with the flow chart. Select &#34;C&#34; in the pop up box and then place the newly created filter in between the &#34;Start&#34; box and &#34;Result 1&#34; box. The flowchart arrows should adjust so that they point from the start box to the filter and finally to the result 1.
	<ol>
		<li>Repeat step 2.12, however this time select &#34;T&#34; for the filter and then select &#34;Result&#34; under the &#34;Common Elements&#34; section. Move the newly created boxes into the flowchart area and connect the boxes with arrows (Figure 6).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 6" src="/files/ftp_upload/55603/55603fig6.jpg" /> 
Figure 6: Screenshot of Data Profile. This shows how the flowchart should be set up to get the appropriate separation in the statistical analysis. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55603/55603fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="13">
	<li>Save the file.</li>
</ol>
3. Collect Honey Bee Individuals
<ol>
	<li>Put on protective clothing and select a hive to collect honey bees (mostly foragers).</li>
	<li>Remove the outer and inner cover of the hive. Use a hive tool to select a frame from the top hive body that does not contain brood. Gently lift the frame out of the hive box.</li>
	<li>Inspect the frame for the honey bee queen. If she is not there, sweep the honey bee workers from the frame into a container for transport. Collect enough individuals to be able to run two complete trials for each compound to be tested (16 individuals are utilized per trial with controls). 
	NOTE: The collected individuals should primarily be foragers; however, there may be other aged individuals such as nurse bees included in the collection.</li>
	<li>Make a note of the time when the honey bees were removed. Transfer them into a 9 cm x 7 cm x 9 cm plastic box containing air holes and place them into an incubator at 32 &#176;C and 70% relative humidity.</li>
	<li>Starve the honey bees for 15 h. 
	NOTE: This usually works best if the honey bees are collected the evening prior to being tested.</li>
</ol>
4. Conduct Visual Tracking Assay
<ol>
	<li>Start up the visual tracking program and open the saved experimental file that was created for this assay.</li>
	<li>Select &#34;Detection Settings 2&#34;.</li>
	<li>Place control sugar-agarose cubes into the centers of 8 of the 16 Petri dishes. Repeat this process with the sugar-agarose-compound (deterrent) cubes in the remaining 8 dishes.</li>
	<li>Use forceps to remove a single honey bee from the plastic box and place it into one of the control petri dishes.</li>
	<li>Repeat step 4.4 for the remaining 15 Petri dishes.</li>
	<li>Place all of the dishes on to the light box and position them so that the arena detection areas (shown on the computer screen when detection setting 2 is selected) fit into each of the petri dish arenas. Illuminate the light box from below by an array of LED lights set to the red spectrum. Surrounded the entire box and camera with a black plastic sheet to eliminate external light and reduce shadows within the arenas.</li>
	<li>After the petri dishes have been set, make sure &#34;Detection Settings 1&#34; is the selected setting. 
	NOTE: At this point the visual tracking software should be picking up only the honey bees within the petri dish arenas.</li>
	<li>Select &#34;Acquisition&#34; from the Experiment Explorer. Press the green &#34;Start Trial&#34; button located in the Acquisition Control pop up box on the right to begin recording.</li>
	<li>Run the assay for 10 min and then click the red &#34;Stop Trial&#34; button on the Acquisition Control pop up box to stop recording.</li>
	<li>Remove the individuals from the petri dishes and place them into a separate container so that they are not tested twice.</li>
	<li>Repeat steps 4.4-4.10 for the second trial on the same compound. 
	NOTE: This method can be modified to increase the number of honey bees per treatment as needed by the user.</li>
	<li>Select Statistics in the Experiment Explorer and then click calculate.</li>
	<li>Export the data to a data manager software and then analyze the data for significance using a using a one-way analysis of variance with a Tukey&#39;s post-test, and an unpaired t-test using preferred statistical software program.</li>
</ol>

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The authors have nothing to disclose.

This visual tracking protocol provides a simple approach to screen chemical deterrents for honey bees in a relatively quick and easy manner. There are no recommended protocols for the laboratory testing of chemical deterrents for honey bees. Previous semi- and full-field studies have examined honey bee repellents14,15; however the described protocols are time consuming, labor intensive, and require additional facility resources outside of a general laboratory. This protocol was designed as a pre-requisite evaluation of chemical deterrents prior to semi- or full-field testing of such compounds with honey bees.
There are challenges when screening individual to evaluating chemical deterrents for honey bees outside of the hive. For example, honey bees are social insects that that rely on pheromones within the hive that affect behavior14. This protocol requires the use of individuals that no longer receive pheromone cues, in addition to starvation. Starvation is required to standardize the feeding responses of individual honey bees. The starvation time was determined by a 24 h time course study. It should be noted that starvation can have detrimental effects on the individual honey bees. For example, the honey bees become lethargic at 18 h after collection from the hive. Based on these observations, the honey bees were starved for 15 h after collection from the hive.
The critical steps involved with this protocol to avoid unsuccessful screening include: (1) conducting the tests after at least 12 h of starvation; (2) avoid conducting tests after starvation exceeds 18-19 h, as this decreases honey bee vigor; (3) replace the control individuals for each trial; and (4) manage of external light and control shadows within the arenas. Additionally, the Petri dishes should be replaced before screening a new compound screen. Occasionally, a honey bee will defecate within the Petri dish during recording. This usually does not interfere with the recording or data collection. All Petri dishes should be washed thoroughly after each screen to remove sugar-agarose and compound residues as well as honey bee feces.
This protocol is primarily designed to screen compounds for deterrence to honey bees, but can be easily adapted to discern deterrence in other insect species. A major benefit of using the visual tracking software is that it makes a full video recording for the screening of each compound. If there is a need to review and analyze each recording, the investigator can select the file of interest and quickly conduct the screen again with the same or new parameters. The visual tracking software also has the capability to detect individual insects smaller than a honey bee. However, this may require a reduced field of vision for the camera field and fewer arenas to be recorded in a single screen. The strength of this protocol is the ability to screen compounds within minutes for deterrent effects in a laboratory setting. As such, time and money could be saved by reducing a compound library of interest to a select number of candidates for field testing.

The European honey bee, Apis melliferaL., is an economic and agriculturally important insect that provides pollination services that are valued at more than $200 billion globally1. In the United States and Europe, honey bee colony numbers have been declining. The United States has lost ca. 60% of managed honey bee colonies from 1947-2008 whereas Europe has lost ca. 27% from 1961-20072,3. There are a number of factors that might be responsible for the increased number of colony losses, including but not limited to, parasite infestations, pathogen infections, beekeeping practices, and pesticide use2-4.
Honey bees may be exposed to pesticides via two main pathways. Pesticide exposure outside of the hive can occur when foraging individuals come into contact with crops that have been sprayed with chemicals for protection from pests. Pesticide exposure within the hive can occur when beekeepers utilize chemicals to control in-hive pests and pathogens, such as mites, bacteria, and microsporidia4. Pesticide residues have been identified within wax, pollen, and honey bee samples from 24 apiaries in the United States and Canada5,6. Effects of pesticide contact to honey bees include acute toxicity as well as sub-lethal effects such as paralysis, disorientation, and behavioral and health changes1,7. As modern agriculture requires the use of pesticides to maintain high crop yields, these chemicals will continue to be relied upon in the future2. In order to better protect honey bees from pesticide exposures, there is a need for the development of new protocols and regulations5. One possible approach for protection is the use of repellents to reduce the exposure of honey bees to pesticides while foraging for food.
Insect repellents (IRs) have typically been used as personal bite protection measures against arthropod disease vectors8. The most widely used and successful IR, developed more than 60 years ago, is DEET8,9. It is considered to be the gold standard for insect repellent testing and is used by the World Health Organization and Environmental Protection Agency as a positive control for novel repellent screening10. Additionally, DEET has been found to disperse honey bees from a threat to their colony11. Current attributes associated with personal IRs include: (1) lasting effect against a broad number of arthropods; (2) non-irritating to the user when applied to the skin or clothing; (3) odorless or pleasant odor; (4) no effect on clothing; (5) no oily appearance when applied to skin and to withstand sweating, washing, and wiping by the user; (6) no effect on commonly used plastics; and (7) chemically stable and affordable for widespread use12. A repellent used for honey bees would only need a few of these attributes such as lasting effects, non-irritating to applicators, odorless or pleasant odor, chemically stable and affordable for widespread use, and non-toxic to honey bees. However, before exploring these attributes in depth, a method for screening compounds for repellency/deterrence in a high-throughput manner is needed. Here, we describe a protocol for a laboratory assay to screen compounds for the deterrence of honey bees, an important step in determining repellency. The following protocol is modified from a previous study describing a visual tracking method to assess the sublethal effects of pesticides on honey bees13. However, this protocol differs in that it is designed to measure the effects of candidate repellents that might deter honey bees from pesticide-treated crops. There are no recommended protocols for the laboratory testing of chemical deterrents for honey bees and, thus, this protocol provides a simple approach to screen such compounds.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 mL Erlenmeyer flask</td>
			<td style="width:71px;">Kimax</td>
			<td style="width:119px;">26500-50</td>
			<td style="width:167px;">used for making the sugar/agarose cubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sugar</td>
			<td style="width:71px;">Kroger</td>
			<td style="width:119px;"></td>
			<td>any similar product will sufffice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Deionized water</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>acquired in house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose</td>
			<td style="width:71px;">Apex</td>
			<td style="width:119px;">20-102</td>
			<td>used for making the sugar/agarose cubes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mold for agarose cubes (Weigh Boat)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>any mold that will provide the researcher with a 1.5 X 1.5 X 0.3 cm sugar/agarose cube will suffice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EthoVision XT</td>
			<td style="width:71px;">Noldus</td>
			<td style="width:119px;"></td>
			<td>visual tracking software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">633 nm LEDs</td>
			<td style="width:71px;">Cyron</td>
			<td>HTP904E</td>
			<td>These lights were placed into a constructed light box to illuminate the arenas from below.&#160; The box was a simple wooden structure with a frosted plastic/plexi glass cover that allowed the light to disperse upwards without any glare.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laptop or PC</td>
			<td style="width:71px;">Dell</td>
			<td style="width:119px;">Inspiron One 2305</td>
			<td>necessary for video tracking software. Any pc device capable of runnin tbe visual tracking software will suffice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bee Keeping protective clothing</td>
			<td style="width:71px;">Dadant &#38; Sons Inc</td>
			<td style="width:119px;">V0126</td>
			<td>any protective hood and jacket will suffice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hive tool</td>
			<td style="width:71px;">Dadant &#38; Sons Inc</td>
			<td style="width:119px;">M00757</td>
			<td>used to open honey bee hive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Container for honey bees</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>any container suitable for housing and storing honey bees will suffice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Featherweight forceps narrow tip</td>
			<td style="width:71px;">Bioquip</td>
			<td align="right" style="width:119px;">4748</td>
			<td>used to select individual honey bees</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">9 cm (diameter) petri dish</td>
			<td style="width:71px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">S01778</td>
			<td>arena used to contain individual honey bees during video tracking</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Recording Device (Camera)</td>
			<td style="width:71px;">Basler</td>
			<td style="width:119px;">acA-1300-60gm</td>
			<td>any device that can record the subject clearly and transfer the file to a computer will suffice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GraphPad Prism</td>
			<td style="width:71px;">Graphpad</td>
			<td style="width:119px;"></td>
			<td>any statistical software package will suffice</td>
		</tr>
	</tbody>
</table>

video tracking protocol to screen deterrent chemistries for honey bees

The European honey bee, Apis mellifera L., is an economically and agriculturally important pollinator that generates billions of dollars annually. Honey bee colony numbers have been declining in the United States and many European countries since 1947. A number of factors play a role in this decline, including the unintentional exposure of honey bees to pesticides. The development of new methods and regulations are warranted to reduce pesticide exposures to these pollinators. One approach is the use of repellent chemistries that deter honey bees from a recently pesticide-treated crop. Here, we describe a protocol to discern the deterrence of honey bees exposed to select repellent chemistries. Honey bee foragers are collected and starved overnight in an incubator 15 h prior to testing. Individual honey bees are placed into Petri dishes that have either a sugar-agarose cube (control treatment) or sugar-agarose-compound cube (repellent treatment) placed into the middle of the dish. The Petri dish serves as the arena that is placed under a camera in a light box to record the honey bee locomotor activities using video tracking software. A total of 8 control and 8 repellent treatments were analyzed for a 10 min period with each treatment was duplicated with new honey bees. Here, we demonstrate that honey bees are deterred from the sugar-agarose cubes with a compound treatment whereas honey bees are attracted to the sugar-agarose cubes without an added compound.

A visual tracking protocol was developed to record the amount of time the honey bees spent in a target zone with either sugar-agarose (control treatment) or sugar-agarose-compound cube (deterrent treatment). The recorded time was analyzed using a statistical software program and the mean time spent &#177; standard error in the target zone is reported as a bar graph. DEET, the gold standard for insect repellent/deterrent testing, was used in this protocol as a positive control. The honey bees provided with a sugar-agarose cube (negative control) spent 343 &#177; 26 s in the target zone whereas the honey bees provided with a sugar-agarose-DEET (repellent) spent 16 &#177; 4 s in the target zone (Figure 7). DEET significantly reduced the amount of time spent by the honey bees in the target zone by ca. 95% compared to that of the control treatment.
Compounds that were of interest to determine deterrence to honey bees were then screened through this protocol. Figure 8A represents a compound that does not deter honey bees from the food source in the target zone. The mean time spent by honey bees in the target zone in control dishes was ca. 352 &#177; 60 s, compared to ca. 282 &#177; 43 s for honey bees within petri dishes that had sugar-agarose cubes infused with compound A. Figure 8B represents a compound other than DEET having similar deterrence effects on the individual honey bees. Honey bees within in the control petri dishes remained in the target zone for a mean time of ca. 493 &#177; 31 s, compared to ca. 23 &#177; 3 s for honey bees within petri dishes containing a sugar-agarose cube infused with compound B. These results validate the use of this protocol for screening of chemical deterrents for honey bees. Prior to running this protocol with compounds of interest, it may be necessary to conduct a time-course trial to determine the amount of starvation time for the honey bees.
<img alt="Figure 7" src="/files/ftp_upload/55603/55603fig7.jpg" /> 
Figure 7: Example Results from the Visual Tracking Software Deterrence Protocol Positive Control DEET. Honey bees are collected from a hive in the evening and removal time is recorded. They are then transferred into a plastic container containing air holes. The box is placed into an incubator set at 32 &#176;C and held overnight for 15 h. The following morning, individuals are placed into petri dishes containing either a control sugar-agarose cube or a DEET-infused sugar-agarose cube. The deterrence protocol described is then run. The results shown in this figure are typical for the repellency gold standard-DEET. From this figure we see that the average amount of time a starved honey bee will spend in the feeding zone with a control cube (ca. 343 s) is significantly greater (P &#60;0.0001) than the average time an individual spends in the feeding zone with a cube impregnated with DEET (ca. 16 s). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55603/55603fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/55603/55603fig8.jpg" /> 
Figure 8: Example Results from Visual Tracking Software Tested Compounds. (A) Represents data that shows the mean time spent by individuals in the target zone in control dishes (ca. 352 s) is not significantly difference from the mean time spent in the target zone in tested dishes with compound A (ca. 282 s). (B) Represents data showing significant differences in mean time spent in the target zone between control cubes (ca. 493 s) and compound B-infused cubes (ca. 23 s). An unpaired t-test was run to determine significance (P &#60;0.0001; DF 15). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55603/55603fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees at JoVE.com

Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees

The loss of honey bee colonies presents a challenge to crop pollination services. Current pollinator protection practices warrant an alternative approach to minimize the contact of honey bees to harmful pesticides using repellent chemistries. Here, we provide detailed methods for a visual tracking protocol to screen deterrents for bees.

The European honey bee, Apis mellifera L., is an economically and agriculturally important pollinator that generates billions of dollars annually. Honey ...

video-tracking-protocol-to-screen-deterrent-chemistries-for-honey-bees

Nanyang Technological University

Research

JoVE Journal

Environment

6.6K Views.  Virginia Tech. The loss of honey bee colonies presents a challenge to crop pollination services. Current pollinator protection practices warrant an alternative approach to minimize the contact of honey bees to harmful pesticides using repellent chemistries. Here, we provide detailed methods for a visual tracking protocol to screen deterrents for bees.

Video: Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees

A Technique to Screen American Beech for Resistance to the Beech Scale Insect (Cryptococcus fagisuga Lind.)

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed.&#160;The disease is initiated by feeding activities of the invasive beech scale insect, Cryptococcus fagisuga, which creates entry points for infection by one of the Neonectria species of fungus.&#160;Without scale infestation, there is little opportunity for fungal infection. Using scale eggs to artificially infest healthy trees in heavily BBD impacted stands demonstrated that these trees were resistant to the scale insect portion of the disease complex1.&#160;Here we present a protocol that we have developed, based on the artificial infestation technique by Houston2, which can be used to screen for scale-resistant trees in the field and in smaller potted seedlings and grafts.&#160;The identification of scale-resistant trees is an important component of management of BBD through tree improvement programs and silvicultural manipulation.

A Technique to Screen American Beech for Resistance to the Beech Scale Insect Cryptococcus fagisuga Lind.

Beech bark disease is initiated by feeding activities of the beech scale insect that create fungal entry points in the bark.&#160;Trees that are resistant to the scale insect are also disease resistant.&#160;Here we present the protocol we have developed to screen individual beech trees for beech scale resistance.

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed.&#160;The ...

Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as plant-soil feedbacks (PSF) have large effects on plant performance, and create environmental heterogeneity that depends on the community composition. Understanding the importance of PSF for plant community assembly necessitates understanding of the role of heterogeneity in PSF, in addition to mean PSF effects. Here, we describe a protocol for manipulating plant-induced soil heterogeneity. Two example experiments are presented: (1) a field experiment with a 6-patch grid of soils to measure plant population responses and (2) a greenhouse experiment with 2-patch soils to measure individual plant responses. Soils can be collected from the zone of root influence (soils from the rhizosphere and directly adjacent to the rhizosphere) of plants in the field from conspecific and heterospecific plant species. Replicate collections are used to avoid pseudoreplicating soil samples. These soils are then placed into separate patches for heterogeneous treatments or mixed for a homogenized treatment. Care should be taken to ensure that heterogeneous and homogenized treatments experience the same degree of soil disturbance. Plants can then be placed in these soil treatments to determine the effect of plant-induced soil heterogeneity on plant performance. We demonstrate that plant-induced heterogeneity results in different outcomes than predicted by traditional coexistence models, perhaps because of the dynamic nature of these feedbacks. Theory that incorporates environmental heterogeneity influenced by the assembling community and additional empirical work is needed to determine when heterogeneity intrinsic to the assembling community will result in different assembly outcomes compared with heterogeneity extrinsic to the community composition.

Understanding the role of environmental heterogeneity in species coexistence has typically focused on types of heterogeneity that are extrinsic to the community&#8217;s species composition. We provide novel detailed methods for creating soil heterogeneity treatments using soils subject to plant-soil feedback conditioning, or heterogeneity intrinsic to the community composition.

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as ...

A Protocol for Conducting Rainfall Simulation to Study Soil Runoff

Rainfall is a driving force for the transport of environmental contaminants from agricultural soils to surficial water bodies via surface runoff. The objective of this study was to characterize the effects of antecedent soil moisture content on the fate and transport of surface applied commercial urea, a common form of nitrogen (N) fertilizer, following a rainfall event that occurs within 24&#160;hr&#160;after fertilizer application. Although urea is assumed to be readily hydrolyzed to ammonium and therefore not often available for transport, recent studies suggest that urea can be transported from agricultural soils to coastal waters where it is implicated in harmful algal blooms. A rainfall simulator was used to apply a consistent rate of uniform rainfall across packed soil boxes that had been prewetted to different soil moisture contents. By controlling rainfall and soil physical characteristics, the effects of antecedent soil moisture on urea loss were isolated. Wetter soils exhibited shorter time from rainfall initiation to runoff initiation, greater total volume of runoff, higher urea concentrations in runoff, and greater mass loadings of urea in runoff. These results also demonstrate the importance of controlling for antecedent soil moisture content in studies designed to isolate other variables, such as soil physical or chemical characteristics, slope, soil cover, management, or rainfall characteristics. Because rainfall simulators are designed to deliver raindrops of similar size and velocity as natural rainfall, studies conducted under a standardized protocol can yield valuable data that, in turn, can be used to develop models for predicting the fate and transport of pollutants in runoff.

A rainfall simulator was used to apply a consistent rate of uniform rainfall to packed soil boxes in a study of the fate and transport of urea, a nonpoint source environmental contaminant. Under uniform soil and rainfall conditions, antecedent soil moisture content exerted strong control over urea loss in surface runoff.

Rainfall is a driving force for the transport of environmental contaminants from agricultural soils to surficial water bodies via surface runoff. The ...

A Push-pull Protocol to Reduce Colonization of Bird Nest Boxes by Honey Bees

Introduction of the invasive Africanized honey bee (AHB) into the Neotropics is a serious problem for many cavity nesting birds, specifically parrots. These bees select cavities that are suitable nest sites for birds, resulting in competition. The difficulty of removing bees and their defensive behavior makes a prevention protocol necessary. Here, we describe a push-pull integrated pest management protocol to deter bees from inhabiting bird boxes by applying a bird safe insecticide, permethrin, to repel bees from nest boxes, while simultaneously attracting them to pheromone-baited swarm traps. Shown here is an example experiment using Barn Owl nest boxes. This protocol successfully reduced colonization of Barn Owl nest boxes by Africanized honey bees. This protocol is flexible, allowing adjustments to accommodate a wide range of bird species and habitats. This protocol could benefit conservation efforts where AHB are located.

Preventing colonization of bird nest boxes by invasive Africanized honey bees is important for conservation efforts of nest-site limited birds. We provide an integrated pest management approach to &#34;push&#34; bees away from nest boxes with a repellant insecticide, permethrin, and &#34;pull&#34; them toward pheromone baited swarm traps.

Introduction of the invasive Africanized honey bee (AHB) into the Neotropics is a serious problem for many cavity nesting birds, specifically parrots. ...

A Protocol for Collecting and Constructing Soil Core Lysimeters

Leaching of nutrients from land applied fertilizers and manure used in agriculture can lead to accelerated eutrophication of surface water. Because the landscape has complex and varied soil morphology, an accompanying disparity in flow paths for leachate through the soil macropore and matrix structure is present. The rate of flow through these paths is further affected by antecedent soil moisture. Lysimeters are used to quantify flow rate, volume of water and concentration of nutrients leaching downward through soils. While many lysimeter designs exist, accurately determining the volume of water and mass balance of nutrients is best accomplished with bounded lysimeters that leave the natural soil structure intact. 
Here we present a detailed method for the extraction and construction of soil core lysimeters equipped with soil moisture sensors at 5 cm and 25 cm depths. Lysimeters from four different Coastal Plain soils (Bojac, Evesboro, Quindocqua and Sassafras) were collected on the Delmarva Peninsula and moved to an indoor climate controlled facility. Soils were irrigated once weekly with the equivalent of 2 cm of rainfall to draw down soil nitrate-N concentrations. At the end of the draw down period, poultry litter was applied (162 kg TN ha-1) and leaching was resumed for an additional five weeks. Total recovery of applied irrigation water varied from 71% to 85%. Nitrate-N concentration varied over the course of the study from an average of 27.1 mg L-1 before litter application to 40.3 mg L-1 following litter application. While greatest flux of nutrients was measured in soils dominated by coarse sand (Sassafras) the greatest immediate flux occurred from the finest textured soil with pronounced macropore development (Quindocqua).

A detailed method for extraction and assembly of intact soil core lysimeters and their use for study of leachate and associated loss of nutrients from surface applied poultry litter is demonstrated.

Leaching of nutrients from land applied fertilizers and manure used in agriculture can lead to accelerated eutrophication of surface water. Because the ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

Evaluating the Effect of Environmental Chemicals on Honey Bee Development from the Individual to Colony Level

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be brought back to the beehive after the bees have foraged on flowers that have been sprayed with pesticides. Pesticide contaminated food can be exchanged between workers which then feed larvae and therefore can potentially affect the development of honey bees. Thus, residual pesticides in the environment can become a chronic damaging factor to honey bee populations and gradually lead to colony collapse. In the presented protocol, honey bee feeding methods are described and applied to either an individual honey bee or to a colony. Here, the insect growth regulator (IGR) pyriproxyfen (PPN), which is widely used to control pest insects and is harmful to the development of honey bee larvae and pupae, is used as the pesticide. The presenting procedure can be applied to other potentially harmful chemicals or honeybee pathogens for further studies.

Herein we present a method to feed pesticide contaminated food to both an individual honey bee and a beehive colony. The procedure evaluates the pesticide effect on individual honey bees by in vivo feeding of basic larval diet and also on the natural condition of beehive colony.

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be ...

Development of New Methods for Quantifying Fish Density Using Underwater Stereo-video Tools

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and estimating fish abundance. We developed and implemented a rotating stereo-video camera tool that covers a full 360 degrees of sampling, which maximizes sampling effort compared to stationary camera tools. A variety of studies have detailed the ability of static, stereo-camera systems to obtain highly accurate and precise measurements of fish; the focus here was on the development of methodological approaches to quantify fish density using rotating camera systems. The first approach was to develop a modification of the metric MaxN, which typically is a conservative count of the minimum number of fish observed on a given camera survey. We redefine MaxN to be the maximum number of fish observed in any given rotation of the camera system. When precautions are taken to avoid double counting, this method for MaxN may more accurately reflect true abundance than that obtained from a fixed camera. Secondly, because stereo-video allows fish to be mapped in three-dimensional space, precise estimates of the distance-from-camera can be obtained for each fish. By using the 95% percentile of the observed distance from camera to establish species-specific areas surveyed, we account for differences in detectability among species while avoiding diluting density estimates by using the maximum distance a species was observed. Accounting for this range of detectability is critical to accurately estimate fish abundances. This methodology will facilitate the integration of rotating stereo-video tools in both applied science and management contexts.

We describe a new method for counting fishes, and estimating relative abundance (MaxN) and fish density using rotating stereo-video camera systems. We also demonstrate how to use distance from camera (Z distance) to estimate species-specific detectability.

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and ...

Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by environmental organochlorine pesticides (OCPs) in hepatocytes. HepG2 cells were exposed to &#946;-hexachlorocyclohexane (&#946;-HCH) for 24 h at an equivalent dose of internal exposure in general population. Ultrastructure in hepatocytes was examined by transmission electron microscopy (TEM) to show the damage of mitochondria. Mitochondrial function was further evaluated by mitochondrial fluorescence intensity, adenosine 5'-triphosphate (ATP) levels, oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP) in HepG2 cells incubated with &#946;-HCH. The mitochondria fluorescence intensity after stained by mitochondrial green fluorescent probe was observed with a fluorescence microscopy. The luciferin-luciferase reaction was used to determine ATP levels. The MMP was detected by the cationic dye JC-1 and analyzed under flow cytometry. OCR was measured with an extracellular flux analyzer. In summary, these protocols were used in detecting mitochondrial function in hepatocytes with to investigate mitochondria damages.

Understanding the influence of environmental organochlorine pesticides (OCPs) on mitochondrial function in hepatocytes is important in exploring the mechanism of OCPs causing metabolic disorders. This paper presents detailed methods on detecting hepatic mitochondrial function.

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by ...

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide regulation studies. The risk of exposure to fungicide residues is likely to be especially high if the spray occurs on, or near host plants while the bees are collecting pollen to provision their nests. For species of Osmia that consume pollen from a select group of plants (oligolecty), the inability to use pollen from non-host plants can increase their risk factor for fungicide-related toxicity. This manuscript describes protocols used to successfully rear oligolectic mason bees, Osmia ribifloris sensu lato, from egg to prepupal stage within cell culture plates under standardized laboratory conditions. The in vitro-reared bees are subsequently used to investigate the effects of fungicide exposure and pollen source on bee fitness. Based on a 2 &#215; 2 fully crossed factorial design, the experiment examines the main and interactive effects of fungicide exposure and pollen source on larval fitness, quantified by prepupal biomass, larval developmental time, and survivorship. A major advantage of this technique is that using in vitro-reared bees reduces natural background variability and allows the simultaneous manipulation of multiple experimental parameters. The described protocol presents a versatile tool for hypotheses testing involving the suite of factors affecting bee health. For conservation efforts to be met with significant, lasting success, such insights into the complex interplay of physiological and environmental factors driving bee declines will prove to be critical.

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

Fungicide sprays on flowering plants may expose solitary bees to high concentrations of pollen-borne fungicide residues. Using laboratory-based experiments involving in vitro-reared bee larvae, this study investigates the interactive effects of consuming fungicide-treated pollen derived from host and non-host plants.

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide ...