Name: video tracking protocol to screen deterrent chemistries for honey bees
Uploaded: 2017-06-12T14:00:00
Description: Watch this Scientific Journal Video about Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees at JoVE.com

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 ...

<ol>
	<li>Gallai, N., Salles, J. M., Settele, J., Vaissi&#232;re, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Economic+valuation+of+the+vulnerability+of+world+agriculture+confronted+with+pollinator+decline.">Economic valuation of the vulnerability of world agriculture confronted with pollinator decline.</a> Ecol Econ. 68 (3), 810-821 (2009).</li><li>van Engelsdorp, D., Meixner, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+historical+review+of+managed+honey+bee+populations+in+Europe+and+the+United+States+and+the+factors+that+may+affect+them.">A historical review of managed honey bee populations in Europe and the United States and the factors that may affect them.</a> J Invertebr Pathol. 103, S80-S95 (2010).</li><li>Aizen, M. A., Harder, L. 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A., Frazier, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Levels+of+Miticides+and+Agrochemicals+in+North+American+Apiaries:+Implications+for+Honey+Bee+Health.">High Levels of Miticides and Agrochemicals in North American Apiaries: Implications for Honey Bee Health.</a> PLoS ONE. 5 (3), (2010).</li><li>Li, Y., Kelley, R. A., Anderson, T. D., Lydy, M. 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H., Hebert, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+repellents:+Historical+perspectives+and+new+developments.">Insect repellents: Historical perspectives and new developments.</a> J Am Acad Dermatol. 58 (5), 865-871 (2008).</li><li>Dickens, J. C., Bohbot, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini+review:+Mode+of+action+of+mosquito+repellents.">Mini review: Mode of action of mosquito repellents.</a> Pestic Biochem Phys. 106 (3), 149-155 (2013).</li><li>Lawrence, K. L., Achee, N. L., Bernier, U. R., Mundal, K. D., Benante, J. 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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. Th...

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 b...

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 ...

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