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

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="http://cloudfront.jove.com/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="http://cloudfront.jove.com/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="http://cloudfront.jove.com/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="http://cloudfront.jove.com/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="http://cloudfront.jove.com/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="http://cloudfront.jove.com/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. 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><tbody><tr><td>50 mL Erlenmeyer flask</td><td>Kimax</td><td>26500-50</td><td>used for making the sugar/agarose cubes</td></tr><tr><td>Sugar</td><td>Kroger</td><td></td><td>any similar product will sufffice</td></tr><tr><td>Deionized water</td><td></td><td></td><td>acquired in house</td></tr><tr><td>Agarose</td><td>Apex</td><td>20-102</td><td>used for making the sugar/agarose cubes</td></tr><tr><td>Mold for agarose cubes (Weigh Boat)</td><td></td><td></td><td>any mold that will provide the researcher with a 1.5 cm x 1.5 cm x 0.3 cm sugar/agarose cube will suffice</td></tr><tr><td>EthoVision XT</td><td>Noldus</td><td></td><td>visual tracking software</td></tr><tr><td>633 nm LEDs</td><td>Cyron</td><td>HTP904E</td><td>These lights were placed into a constructed light box to illuminate the arenas from below.&amp;nbsp; 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><td>Laptop or PC</td><td>Dell</td><td>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><td>Bee Keeping protective clothing</td><td>Dadant &amp;amp; Sons Inc</td><td>V0126</td><td>any protective hood and jacket will suffice</td></tr><tr><td>Hive tool</td><td>Dadant &amp;amp; Sons Inc</td><td>M00757</td><td>used to open honey bee hive</td></tr><tr><td>Container for honey bees</td><td></td><td></td><td>any container suitable for housing and storing honey bees will suffice</td></tr><tr><td>Featherweight forceps narrow tip</td><td>Bioquip</td><td>4748</td><td>used to select individual honey bees</td></tr><tr><td>9 cm (diameter) Petri dish</td><td>Fisher Scientific&amp;nbsp;</td><td>S01778</td><td>arena used to contain individual honey bees during video tracking</td></tr><tr><td>Recording Device (Camera)</td><td>Basler</td><td>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><td>GraphPad Prism</td><td>Graphpad</td><td></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...

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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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Environment

6.6K Views.  Virginia Tech.  Het verlies van honingbijkolonies is een uitdaging om de bestuivingsdiensten te bestuderen. Huidige beschermingspraktijken van de bestuderende instanties garanderen een alternatieve aanpak om het contact van honingbijen tegen schadelijke pesticiden met behulp van afweermiddelen te beperken. Hier bieden we gedetailleerde methodes voor een visuele tracking protocol om afschermingen voor bijen te screenen. 

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

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. ...

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 ...

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker bees spend the first half of their life inside the dark hive, where they are nursing the larvae or building the regular hexagonal combs for food (e.g. pollen or nectar) and brood3. The antennae are extraordinary multisensory feelers and play a pivotal role in various tactile mediated tasks4, including hive building5 and pattern recognition6. Later in life, each single bee leaves the hive to forage for food. Then a bee has to learn to discriminate profitable food sources, memorize their location, and communicate it to its nest mates7. Bees use different floral signals like colors or odors7, 8, but also tactile cues from the petal surface9 to form multisensory memories of the food source. Under laboratory conditions, bees can be trained in an appetitive learning paradigm to discriminate tactile object features, such as edges or grooves with their antennae10, 11, 12, 13. This learning paradigm is closely related to the classical olfactory conditioning of the proboscis extension response (PER) in harnessed bees14. The advantage of the tactile learning paradigm in the laboratory is the possibility of combining behavioral experiments on learning with various physiological measurements, including the analysis of the antennal movement pattern.

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L.

In this protocol we show how to condition harnessed honey bees to tactile stimuli and introduce a 2D motion capture technique for analyzing the kinematics of fine-scale antennal sampling pattern.

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker ...

Neuroscience

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

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

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.

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.

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). ...

Radio Frequency Identification and Motion-sensitive Video Efficiently Automate Recording of Unrewarded Choice Behavior by Bumblebees

We present two methods for observing bumblebee choice behavior in an enclosed testing space. The first method consists of Radio Frequency Identification (RFID) readers built into artificial flowers that display various visual cues, and RFID tags (i.e., passive transponders) glued to the thorax of bumblebee workers. The novelty in our implementation is that RFID readers are built directly into artificial flowers that are capable of displaying several distinct visual properties such as color, pattern type, spatial frequency (i.e., &#8220;busyness&#8221; of the pattern), and symmetry (spatial frequency and symmetry were not manipulated in this experiment). Additionally, these visual displays in conjunction with the automated systems are capable of recording unrewarded and untrained choice behavior. The second method consists of recording choice behavior at artificial flowers using motion-sensitive high-definition camcorders. Bumblebees have number tags glued to their thoraces for unique identification. The advantage in this implementation over RFID is that in addition to observing landing behavior, alternate measures of preference such as hovering and antennation may also be observed. Both automation methods increase experimental control, and internal validity by allowing larger scale studies that take into account individual differences. External validity is also improved because bees can freely enter and exit the testing environment without constraints such as the availability of a research assistant on-site. Compared to human observation in real time, the automated methods are more cost-effective and possibly less error-prone.

This video describes Radio-Frequency Identification (RFID) and motion-sensitive video recording methods to monitor choice behavior by bumblebees.

We present two methods for observing bumblebee choice behavior in an enclosed testing space. The first method consists of Radio Frequency Identification ...

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

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from flowering plants. Only a few studies have described the gustatory responses of bees toward toxins in food, and these experiments have mainly used the proboscis extension response on restrained honey bees. Here, a new behavioral assay is presented for measuring the feeding responses of freely-moving, individual worker bumble bees to nutrients and toxins. This assay measures the amount of solution ingested by each bumble bee and identifies how tastants in food influence the microstructure of the feeding behavior.
The solutions are presented in a microcapillary tube to individual bumble bees that have been previously starved for 2-4 hr. The behavior is captured on digital video. The fine structure of the feeding behavior is analyzed by continuously scoring the position of the proboscis (mouthparts) from video recordings using event logging software. The position of the proboscis is defined by three different behavioral categories: (1) proboscis is extended and in contact with the solution, (2) proboscis is extended but not in contact with the solution and (3) proboscis is stowed under the head. Furthermore the speed of the proboscis retracting away from the solution is also estimated.
In the present assay the volume of solution consumed, the number of feeding bouts, the duration of the feeding bouts and the speed of the proboscis retraction after the first contact is used to evaluate the phagostimulatory or the deterrent activity of the compounds tested.
This new taste assay will allow researchers to measure how compounds found in nectar influence the feeding behavior of bees and will also be useful to pollination biologists, toxicologists and neuroethologists studying the bumble bee&#39;s taste system.

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

A novel behavioral assay is described for investigating the short term gustatory responses of the mouthparts of freely-moving bumble bees (Bombus terrestris) toward nutrients and toxins in solution.

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from ...

Preparation of Single-cohort Colonies and Hormone Treatment of Worker Honeybees to Analyze Physiology Associated with Role and/or Endocrine System

Honeybee workers are engaged in various tasks related to maintaining colony activity. The tasks of the workers change according to their age (age-related division of labor). Young workers are engaged in nursing the brood (nurse bees), while older workers are engaged in foraging for nectar and pollen (foragers). The physiology of the workers changes in association with this role shift. For example, the main function of the hypopharyngeal glands (HPGs) changes from the secretion of major royal jelly proteins (MRJPs) to the secretion of carbohydrate-metabolizing enzymes. Because worker tasks change as the workers age in typical colonies, it is difficult to discriminate the physiological changes that occur with aging from those that occur with the role shift. To study the physiological changes in worker tissues, including the HPGs, in association with the role shift, it would be useful to manipulate the honeybee colony population by preparing single-cohort colonies in which workers of almost the same age perform different tasks. Here we describe a detailed protocol for preparing single-cohort colonies for this analysis. Six to eight days after single-cohort colony preparation, precocious foragers that perform foraging tasks earlier than usual appear in the colony. Representative results indicated role-associated changes in HPG gene expression, suggesting role-associated HPG function. In addition to manipulating the colony population, analysis of the endocrine system is important for investigating role-associated physiology. Here, we also describe a detailed protocol for treating workers with 20-hydroxyecdysone (20E), an active form of ecdysone, and methoprene, a juvenile hormone analogue. The survival rate of treated bees was sufficient to examine gene expression in the HPGs. Gene expression changes were observed in response to 20E- and/or methoprene-treatment, suggesting that hormone treatments induce physiological changes of the HPGs. The protocol for hormone treatment described here is appropriate for examining hormonal effects on worker physiology.

Here we describe our detailed protocol for the preparation of single-cohort honeybee colonies&#160;&#8211; a useful tool for analyzing the role-associated worker physiology. We also describe detailed protocols for treating workers with juvenile hormone and ecdysone to evaluate the involvement of these hormones in the regulation of worker behavior and/or physiology.

Honeybee workers are engaged in various tasks related to maintaining colony activity. The tasks of the workers change according to their age (age-related ...

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize and to study. Therefore, bees have long been a favored model amongst neurobiologists and neuroethologists for studying the neural basis of social and natural behavior. It is important, however, that the experimental techniques used to study bees do not interfere with the behaviors being studied. Because of this, it has been necessary to develop a range of techniques for pharmacological manipulation of honey bees. In this paper we demonstrate methods for treating restrained or free-flying honey bees with a wide range of pharmacological agents. These include both noninvasive methods such as oral and topical treatments, as well as more invasive methods that allow for precise drug delivery in either systemic or localized fashion. Finally, we discuss the advantages and disadvantages of each method and&#160;describe common hurdles and how to best overcome them. We conclude with a discussion on the importance of adapting the experimental method to the biological questions rather than the other way around.

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

This manuscript describes several protocols for administering pharmacological agents to honey bees, including simple noninvasive methods for free-flying bees, as well as more invasive variants that allow precise localized treatment of restrained bees.

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize ...

SwarmSight: Real-time Tracking of Insect Antenna Movements and Proboscis Extension Reflex Using a Common Preparation and Conventional Hardware

Many scientifically and agriculturally important insects use antennae to detect the presence of volatile chemical compounds and extend their proboscis during feeding. The ability to rapidly obtain high-resolution measurements of natural antenna and proboscis movements and assess how they change in response to chemical, developmental, and genetic manipulations can aid the understanding of insect behavior. By extending our previous work on assessing aggregate insect swarm or animal group movements from natural and laboratory videos using the video analysis software SwarmSight, we developed a novel, free, and open-source software module, SwarmSight Appendage Tracking (SwarmSight.org) for frame-by-frame tracking of insect antenna and proboscis positions from conventional web camera videos using conventional computers. The software processes frames about 120 times faster than humans, performs at better than human accuracy, and, using 30 frames per second (fps) videos, can capture antennal dynamics up to 15 Hz. The software was used to track the antennal response of honey bees to two odors and found significant mean antennal retractions away from the odor source about 1 s after odor presentation. We observed antenna position density heat map cluster formation and cluster and mean angle dependence on odor concentration.

This protocol describes steps for using the novel software, SwarmSight, for frame-by-frame tracking of insect antenna and proboscis positions from conventional web camera videos using conventional computers. The free, open-source software processes frames about 120 times faster than humans and performs at better than human accuracy.

Many scientifically and agriculturally important insects use antennae to detect the presence of volatile chemical compounds and extend their proboscis ...

Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees

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