Our study was conducted with the permission of United States Sugar Corporation. We want to thank R. Raid, S. Raid and T. Lang for their assistant. We also M. Stanback and three anonymous reviewers for suggestions that improved this manuscript.

1. Construction of Swarm Trap Boxes
NOTE: There are two alternate protocols for swarm trap construction included as steps 1.1 and 1.2. There are several additional ways trap boxes could be constructed. The following two methods are presented here because we have tested them in experiments and they have been successful. For either protocol, use 1.3 cm to 2 cm thick non-pressure treated plywood.
<ol>
	<li>Construction of Swarm Trap Protocol Option 1: Building a Trap Box
	<ol>
		<li>Cut six pieces of plywood 30 cm x 30 cm.</li>
		<li>Attach the four sides of the box together with nails or screws.</li>
		<li>Attach the bottom piece by nails or screws.</li>
		<li>Attach the top piece with screws or staples.
		<ol>
			<li>Do not use nails to secure the top. The top must be removable to get bees out and clean the box.</li>
		</ol>
		</li>
		<li>Drill a 2.5 cm hole in the middle of one face of the trap box 2.5 cm from the bottom edge.</li>
		<li>Apply primer, sealant, or paint to the outside of the trap box to protect wood from weather.
		<ol>
			<li>Do not apply any paint or sealant to the inside of the trap box. This could be repellant or toxic to the bees.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Construction of Swarm Trap Box Protocol Option 2: Use of a Standard Deep Hive Body
	<ol>
		<li>Obtain a standard 10 frame deep hive body from a local bee supply store.</li>
		<li>Cut a top and bottom from plywood each measuring 50.8 cm long by 41.3 cm wide.</li>
		<li>Attach the bottom piece by nails or screws.</li>
		<li>Attach the top piece with screws or staples.
		<ol>
			<li>Do not use nails to secure the top. The top must be removable to get bees out and clean the box.</li>
		</ol>
		</li>
		<li>Drill a 2.5 cm hole in the middle on one side of the trap box 2.5 cm from the bottom edge.</li>
		<li>Apply primer, sealant, or paint to the outside of the trap box to protect wood from weather.
		<ol>
			<li>Do not apply any paint or sealant to the inside of the trap box. This could be repellant or toxic to the bees.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Preparation of the Pheromone Lure
<ol>
	<li>Prepare a mixture of citral and geraniol at a 2:1 ratio.</li>
	<li>Place 1 ml of this mixture into a snap-top 1.5 ml polypropylene microcentrifugation tube.</li>
	<li>Wrap lures in aluminum foil to protect them from light. 
	Note: The cap of the centrifugation tube is punctured with a push pin when it is placed in the mounted swarm trap. 
	Note: Lures can be stored in a dark place at RT or for longer storage in a -10 &#176;C freezer.</li>
</ol>
3. Installation of Swarm Trap Boxes
NOTE: There are two alternate protocols for the installation of swarm trap boxes presented in steps 3.1 and 3.2.
<ol>
	<li>Installation of Swarm Trap Box Protocol Option 1: Installation on a Post
	<ol>
		<li>Place a 10 cm by 10 cm pressure treated wood fence post into the ground 10 m to 15 m from where the bird nest box is located.</li>
		<li>Construct a hanging board of 1.3 cm thick pressure treated wood measuring 20 cm by 20 cm.</li>
		<li>Align the hanging board in the middle of one side of the trap box, with 10 cm above the top of the box. Secure the board to the box with at least two nails or screws on either side of the board.</li>
		<li>Place the trap box on the post at a height (1.5 m to 3 m) that is safe and easy for removal when the trap box is colonized by bees. Screw the box to the post by attaching the hanging board. Place a screw approximately 5 cm below the top of the hanging board and screw in place.
		<ol>
			<li>Do not use nails because they are difficult to remove.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Installation of Swarm Trap Box Protocol Option 2: Installation on a Tree
	<ol>
		<li>Drill a 5 mm hole in the upper back corner of each side of the box.</li>
		<li>Thread a wire through the holes. 
		Note: If using this protocol it will be easier to put the wire through the holes prior to securing the top of the box.</li>
		<li>Select a tree approximately 10 m to 15 m away from where the bird nest box is located.
		<ol>
			<li>Do not place trap boxes in the same trees as bird boxes because when someone checks on the status of the bird box, trap boxes colonized by bees may be disturbed causing bees to become defensive.</li>
		</ol>
		</li>
		<li>Secure the wire around the tree 1.5 m to 3 m off the ground. 
		Note: To protect the tree, a cambium saver can be used around the wire.
		<ol>
			<li>Make sure the box is level and secure so that it does not tip over. The box can be rested on a tree branch for added stability if available.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Addition of the Pheromone Lure
<ol>
	<li>Take a previously prepared lure from section 2 and puncture the top of the tube with a push pin.</li>
	<li>Place the lure inside the trap box on the bottom near the entrance after installation to ensure it does not fall out while installing the box.</li>
</ol>
5. Treatment of Bird Nest Box with Permethrin
<ol>
	<li>Select a permethrin product that is labeled for use in poultry or caged birds only.</li>
	<li>Follow label instructions for mixing and application 
	CAUTION: follow any personal protection guidelines on label recommendations.</li>
	<li>Spray the inside of the nest box entirely, until the point that the liquid runs off the sides of the box using a 3.7 L hand pump sprayer. 
	Note: Nest boxes can be treated prior to or after installation.</li>
</ol>
6. Removal of a Trap Box Occupied by Honey Bees
<ol>
	<li>Plug the entrance hole with a small towel. 
	CAUTION: wear proper protective garments to prevent stings from honey bees.
	<ol>
		<li>If swarm is to be killed, do so as soon as discovered. If swarm is to be utilized, allow bees to fully colonize trap boxes before attempting to remove them (usually about 2 weeks). If possible, remove occupied trap boxes at night (when all the bees are inside). The trap entrance can be plugged at night and then the box removed the following morning in daylight. 
		Note: Use a red filtered light when working around bees at night.</li>
	</ol>
	</li>
	<li>Unscrew the box from the post or cut the wire holding the box to the tree.</li>
	<li>Move the box to its new location (at least 4 km away) and pull the towel out from the entrance hole.</li>
</ol>
7. Destruction of Bee Colony in Trap Box
Note: In areas where it is illegal to keep Africanized honey bees, destruction of the colony may be warranted. If so, two protocols are presented here.
<ol>
	<li>Solarizing the Colony
	<ol>
		<li>Plug the entrance hole with a small towel. 
		CAUTION: wear proper protective garments to prevent stings from honey bees.</li>
		<li>Unscrew the box from the post or cut the wire holding the box to the tree.</li>
		<li>Enclose and seal the trap box completely in a plastic bag and leave in full sun for several days 
		CAUTION: wear proper protective garments to prevent stings from honey bees.</li>
		<li>Clean out the box by removing dead bees and comb.</li>
	</ol>
	</li>
	<li>Use of an Insecticide Spray
	<ol>
		<li>Use an insecticide spray labeled for use to kill bees to spray inside the trap box. 
		CAUTION: follow label instructions on insecticide and use appropriate personal protection equipment to prevent stings from honey bees.</li>
		<li>Plug the entrance and leave the box for 24 hr.</li>
		<li>Clean out the box by removing dead bees and comb. 
		Note: To reuse the trap box clean the inside with a soapy water solution to remove residual insecticide. Rinse well and allow to dry completely before reinstalling.</li>
	</ol>
	</li>
	<li>Use of an insecticide foam
	<ol>
		<li>Fill the entire cavity of the occupied trap with a detergent-based foam. (CAUTION: follow label instructions on insecticide and use appropriate personal protection equipment and wear proper protective garments to prevent stings from honey bees). 
		Note: The foam can be applied with a commercial pest control foam generator or a compressed air fire extinguisher. &#160; 
		Note: For using a fire extinguisher: Place 3.8 L of water into a 9.5 L fire extinguisher, add&#160; 190 ml of foaming agent (or dish washing detergent), seal the extinguisher, and pressurize it to 100-125&#160; pounds/sq. inch (psi).&#160; Just prior to use, agitate vigorously, and then apply the foam until the trap is overflowing. &#160; 
		Note: Foam kills the bees within 60 sec by entering the spiracles and blocking the trachea.</li>
		<li>Plug the entrance and leave the box for 24 hr.</li>
		<li>Clean out the box by removing dead bees and comb. 
		Note: To reuse the trap box clean the inside with a soapy water solution to remove residual insecticide. Rinse well and allow to dry completely before reinstalling.</li>
	</ol>
	</li>
</ol>

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O., Hurley, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+nest+cavities+by+Africanized+and+European+honey+bees.">Selection of nest cavities by Africanized and European honey bees.</a> Apidologie. 26, 467-475 (1995).</li><li>Eadie, J., Sherman, P., Semel, B., Casro, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conspecific+Brood+Parasitism,+Population+Dynamics,+and+the+Conservation+of+Cavity-Nesting+Birds.">Conspecific Brood Parasitism, Population Dynamics, and the Conservation of Cavity-Nesting Birds.</a> In: Behavioral Ecology and Conservation Biology. , 306-340 (1998).</li><li>Newton, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+nest+sites+in+limiting+the+numbers+of+hole-nesting+birds:+a+review.">The role of nest sites in limiting the numbers of hole-nesting birds: a review.</a> Biol Conserv. 70, 265-276 (1994).</li><li>White, T. H., Abreu-Gonz&#225;lez, W., Toledo-Gonz&#225;lez, M., Torres-B&#225;ez, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+the+field:+artificial+nest+cavities+for+Amazona+parrots.">From the field: artificial nest cavities for Amazona parrots.</a> Wildl Soc Bull. 33, 756-760 (2005).</li><li>Vaughan, C., Nemeth, N., Marineros, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecology+and+management+of+natural+and+artificial+Scarlet+Macaw+(Ara+macao)+nest+cavities+in+Costa+Rica.">Ecology and management of natural and artificial Scarlet Macaw (Ara macao) nest cavities in Costa Rica.</a> Ornitol Neotrap. 14, 381-396 (2003).</li><li>WCS Guatemala Program. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+interventions+to+conserve+Scarlet+Macaws+as+flagships+for+the+Maya+Biosphere+Reserve,+Guatemala.">Integrated interventions to conserve Scarlet Macaws as flagships for the Maya Biosphere Reserve, Guatemala.</a> 2009-2010 Report to BBC Wildlife Fund. , (2011).</li><li>Fish and Wildlife Service. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Puerto Rican Parrot (Amazona vittata) 5-Year Review: Summary and Evaluation. , (2008).</li><li>Berkunsky, I., DiazLuque, J. 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The authors have nothing to disclose.

As a highly successful invasive insect17, AHB constitute a major competitor for cavity-nesting birds throughout their range. The protocol presented here is designed to reduce the colonization rates of bird nest boxes by AHB. The main components of this design include 1) the use of a repellant and bird-safe insecticide, permethrin; 2) The provision of an alternate nesting site for bees; and 3) the use of an attractant pheromone inside trap boxes. Bees usually make the &#34;right&#34; decision when choosing a new nest site18. Making trap boxes more attractive than bird nest boxes will increase the likelihood of the former being chosen. Specifically, trap boxes should have a smaller entrance hole located closer to the floor than the ceiling23. Finally, higher boxes tend to be preferred by honey bees. However, because AHB are far less choosey about nest sites than EHB2, the pheromone is the most important component of attractiveness24.
This protocol can be adapted to fit a variety of situations. For areas with a very high density of honey bees, one can install two traps per nest box. For cliff nesting birds, traps can be hoisted into place parallel to a nest cavity or above and below. Trap boxes can be placed on pulley systems for easier removal in areas that are hard to reach. Africanized honey bees have a swarming season and an absconding season that varies with location. Swarming usually coincides with the peak blooming period of flowers. Absconding occurs when the entire colony of bees abandon a nest site, usually due to a dearth or deterioration of the nest site. It is important to predict when these seasons are. Because the timing and frequency of swarming and absconding varies and is often not reported in the literature, one should contact local beekeepers or agricultural agencies for information. This will allow for more targeted application of permethrin and/or lures. Additionally, a management plan is essential for the success of this protocol. Occupied trap boxes need to be monitored regularly (at least every three weeks) and relocated or destroyed. The maintenance of available trap boxes for bees to colonize is important for the protocol to be effective. The overall goal is not to reduce the population of bees in an area, but rather to give them alternative nest sites.
Safety should always be a priority. For example, even though bees generally prefer higher nest sites, we tend to place trap boxes lower to the ground to facilitate their safe removal. Trap boxes should be placed in locations that will not place a biologist checking the status of a bird nest box in danger. Personal protective equipment (bee suit or jacket and bee gloves) should be worn by anyone removing a trap box with bees inside.
We used a microencapsulated permethrin spray formulation in this study because it is effective for 60 days. Other formulations are emulsified concentrates (EC) and dusts. Dust formulations are difficult to apply to tops and sides of boxes, where scout bees spend the majority of time performing assessment walks. Although EC formulations are common and easy to apply, they remain effective for only two weeks. The use of the natural product pyrethrum is not recommended as it is photosensitive and is only effective for a few days. Permethrin is the insecticide of choice because is it relatively non-toxic for birds and humans. It does not persist in the environment, is repellant to bees and lasts longer than the natural derived chemical. For these reasons permethrin spray formulation is recommended.
Attempts to keep AHB out of areas has failed throughout the Americas. Trapping near ports in Florida, delayed the arrival of AHB, but by 2007 they were established throughout the southern part of the state. Countries that are dominated by AHB, have simply given up and resorted to learning how to manage AHB. In areas where managing AHB is not illegal, bees that move into trap boxes can easily be removed and utilized for beekeeping. They can be relocated to an apiary for management or given to local beekeepers. Incorporating a local beekeeper to assist in the project by removing colonized boxes can benefit both parties. The beekeeper gets free bees and the biologists have one less task to concern themselves with.
This push-pull method greatly reduced colonization of Barn Owl nest boxes by AHB. The defensive nature of AHB and the difficulty of removing swarms from nest boxes makes a prevention protocol essential. This protocol can be used for any cavity nesting bird as well as cavity nesting mammals. It could prove to be an important conservation tool in the recovery of many endangered parrots throughout the Caribbean, South America, Africa and even Australia where the recently introduced Asian honey bee (Apis cerana) is quickly spreading.

Since the accidental introduction of more than two dozen African honey bee queens into Sao Paulo, Brazil in 19571, these insects have spread throughout South and Central America and up into the southern United States, usurping and replacing the less well adapted European honey bees (EHB) in the feral population. These hybrid Africanized honey bees (AHB) have traits that put them in conflict with both humans and wildlife. They exhibit highly defensive behavior, reproduce very fast, and are less selective about where they make a home1,2.
Although nest boxes have long been used by ornithologists to enhance breeding by cavity-nesting birds3,4, their use has been shown to be especially effective for enhancing the breeding of threatened and endangered parrots in the tropics5,6. Unfortunately, Africanized honey bees often choose these same nest boxes&#160;&#8211; sometimes even killing the occupants. Additionally, these AHB pose a significant threat to biologists when they are conducting nest studies. In areas of South America where AHB have been established, several parrot conservation projects have seen their artificial nest boxes occupied by AHB7-10. Competition for nest boxes with honey bees is widespread and is not confined to parrots. Many other bird species both in the New and Old World are affected11-15. Without efforts to mitigate the threat of these invasive AHB, recent successes in parrot conservation could be lost.
Current practices for managing competition from bees includes the use of repeated, high doses of permethrin7, manually removing swarms, closing nest entrances immediately after breeding season8, or the application of chemicals to the outsides of nest boxes15. The labor involved and the danger to biologists render these strategies less than optimal. Development of a prevention protocol is warranted.
Honey bee colonies multiply via a process known as swarming, when the old queen leaves the colony with up to three fourths of the worker bees16. Africanized honey bees&#39; high reproductive rate allows them to swarm more frequently (four to eight times per year) than EHB (who swarm once or twice per year)17. Moreover, AHB may swarm throughout the year rather than during a particular season like EHB. Consequently, AHB can quickly occupy most of the suitable nesting sites in a given area.
Honey bee house hunting behavior can be utilized to influence their choice of nest site. A small fraction of bees in a swarm fly off to locate and inspect new potential nest sites. Several potential nest sites are scouted for simultaneously. These scout bees return to the swarm and perform dances to indicate the location of the site. The stronger the dancing is for a site, the faster the number of scout bees builds up at that site18. Therefore, bees dance stronger for better quality sites, compared to sites that are mediocre or poor. Once a quorum threshold is reached, the swarm takes off and flies to the new chosen home site18.
Sub-lethal contact doses of the insecticide permethrin hamper a scout bee&#39;s ability to orient and reduce its activity level19,20. An application of permethrin inside nest boxes at first should be repellant to scout bees21, and overtime as the chemical degrades, scout bees exposed to permethrin during their assessment of the box, should be rendered less able to perform strong recruitment dances, decreasing the amount of scout bees recruited to the site. A more suitable (ideal cavity volume, small entrance hole located at the bottom, and a pheromone attractant) alternate nest site is provided in the form of a trap box for the bees. This excellent trap site should reach a quorum faster than the insecticide treated bird box leading the swarm to choose the trap box over the bird box.
As an example, we describe an experiment to reduce colonization of Barn Owl artificial nest boxes by AHB in South Florida sugarcane fields. Our objective was to test the effectiveness of using a push-pull method that will &#34;push&#34; bees away from owl nest boxes treated with an insecticide and &#34;pull&#34; them toward pheromone baited swarm traps.

<table><tbody><tr><td>Citral (ACROS Organics)</td><td>Fisher Scientific</td><td>AC11044-1000</td><td></td></tr><tr><td>Geraniol (ACROS Organics)</td><td>Fisher Scientific</td><td>AC41090-1000</td><td></td></tr><tr><td>PermaCap CS</td><td>BASF</td><td></td><td>controlled release permethrin</td></tr></tbody></table>

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.

A previous small pilot push-pull study conducted in Brazil showed swarm traps were effective and reduced bee occupation of parrot nest boxes22. To determine if the push-pull method can be effective at a larger scale to reduce colonization of bird nest boxes by AHB, we conducted a field study in sugarcane fields in southern Florida, USA. Sixty Barn Owl nest boxes were installed and each was paired with a swarm trap located 15 m away. Every swarm trap received a pheromone lure. Odd numbered owl boxes were treated with a microencapsulated permethrin spray insecticide every 60 days and even numbered nest boxes were left as untreated controls. Nest boxes and swarm traps were checked weekly for the presence or absence of 1) scout bees and 2) bee colonies.
Boxes and traps were monitored from March 2014 to July 2015. Of the 77 swarms caught, 75 were in swarm traps (Figure 1). Only two owl nest boxes were colonized during this experiment; both were untreated. We allowed bees to remain in trap boxes for a minimum of two weeks before we relocated them 4 km away to an apiary. To reduce the chances of bees absconding (leaving the trap box) we let them stay in the trap box for an additional two weeks before they were transferred to a new hive box. Honey comb and debris was scraped out of the trap box, it was remounted in the experimental site and a new lure was placed inside. Significantly more bees chose swarm traps over untreated owl boxes (X2(df =1) = 29.2, P &#60;0.0001) and treated owl boxes (X2(df=1) = 36.1, P &#60;0.0001) as determined by Pearson&#39;s chi-square test with Yates&#39; continuity correction. Several trap boxes were colonized by bees more than once, 12 trap boxes were occupied twice by bees, five boxes were occupied three times and two boxes were occupied four times. For statistical analysis, occupancy per trap box was only counted once. Scents from previous bee colonies in a nest site can attract new colonies23 so likely previously occupied trap boxes were more attractive than unoccupied boxes. Although several treated owl nest boxes were scouted by honey bees, no swarms moved into these boxes. At a nearby control site (containing 30 Barn owl boxes) honey bees colonized 63% of boxes. We found that the use of swarm traps greatly reduced the number of bee colonies occupying owl nest boxes. No treated nest boxes were colonized, suggesting that permethrin is effective at keeping AHB from colonizing nest boxes. However, swarm traps alone were effective, therefore application of permethrin could be used only at critical time periods (i.e., bee swarm season and just prior to birds&#39; breeding season) rather than applying throughout the year.
<img alt="Figure 1" src="/files/ftp_upload/53950/53950fig1.jpg" /> 
Figure 1. Number of boxes colonized by honey bees. Number of Africanized honey bee swarms that colonized either trap boxes or untreated barn owl nest boxes over a 17 month time period (March of 2014 to July of 2015) in sugarcane fields of southern Florida. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53950/53950fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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. ...

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Nanyang Technological University

Research

JoVE Journal

Environment

8.6K Views.  University of Florida.  Het voorkomen van kolonisatie van vogel nestkasten door invasieve Africanized honingbijen is belangrijk voor de pogingen tot instandhouding van het nest van het terrein beperkt vogels. Wij bieden een geïntegreerde plaagbestrijding benadering van "push" bijen weg van nestkasten met een afweermiddel insecticide, permethrine, en "pull" hen in de richting van feromonen aas zwerm vallen. 

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

Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered &#34;bee-safe,&#34; there is mounting evidence that fungicide residues in pollen are associated with bee declines (for both honey and bumble bee species). While the mechanisms remain relatively unknown, researchers have speculated that bee-microbe symbioses are involved. Microbes play a pivotal role in the preservation and/or processing of pollen, which serves as nutrition for larval bees. By altering the microbial community, it is likely that fungicides disrupt these microbe-mediated services, and thereby compromise bee health. This manuscript describes the protocols used to investigate the indirect mechanism(s) by which fungicides may be causing colony decline. Cage experiments exposing bees to fungicide-treated flowers have already provided the first evidence that fungicides cause profound colony losses in a native bumble bee (Bombus impatiens). Using field-relevant doses of fungicides, a series of experiments have been developed to provide a finer description of microbial community dynamics of fungicide-exposed pollen. Shifts in the structural composition of fungal and bacterial assemblages within the pollen microbiome are investigated by next-generation sequencing and metagenomic analysis. Experiments developed herein have been designed to provide a mechanistic understanding of how fungicides affect the microbiome of pollen-provisions. Ultimately, these findings should shed light on the indirect pathway through which fungicides may be causing colony declines.

Microbial consortia within bumble bee hives enrich and preserve pollen for bee larvae. Using next generation sequencing, along with laboratory and field-based experiments, this manuscript describes protocols used to test the hypothesis that fungicide residues alter the pollen microbiome, and colony demographics, ultimately leading to colony loss.

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered ...

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 ...

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.

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 ...

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). ...

Neuroscience

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 ...

Monitoring Colony-level Effects of Sublethal Pesticide Exposure on Honey Bees

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment methods. Here we describe methods to measure the quantities of adult bees, brood, and food resources by weighing hives and hive parts, by photographing frames, and by installing hives on scales and with internal sensors. Data from these periodic evaluations are then combined with running average and daily detrended data on hive weight and internal hive temperature. The resulting datasets have been used to detect colony-level effects of imidacloprid applied in a sugar syrup as low as 5 parts per billion. The methods are objective, require little training, and provide permanent records in the form of sensor output and photographs.

Sublethal doses of pesticides may affect colonies in ways that are difficult to detect using only periodic or visual methods. Methods to measure total adult bee mass, brood, and food resources by weighing hives and hive parts, photographing frames, and installing sensors, are provided. Data analysis is also addressed.

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment ...

Biology

A 3D Printed Pollen Trap for Bumble Bee (Bombus) Hive Entrances

To verify the plant sources from which bumble bees forage for pollen, individuals must be collected to remove their corbicular pollen loads for analysis. This has traditionally been done by netting foragers at nest entrances or on flowers, chilling the bees on ice, and then removing the pollen loads from the corbiculae with forceps or a brush. This method is time and labor intensive, may alter normal foraging behavior, and can result in stinging incidents for the worker performing the task. Pollen traps, such as those used on honey bee hives, collect pollen by dislodging corbicular pollen loads from the legs of workers as they pass through screens at the nest entrance. Traps can remove a large quantity of pollen from returning forager bees with minimal labor, yet to date no such trap is available for use with bumble bee colonies. Workers within a bumble bee colony can vary in size making size selection of entrances difficult to adapt this mechanism to commercially reared bumble bee hives. Using 3D printing design programs, we created a pollen trap that successfully removes the corbicular pollen loads from the legs of returning bumble bee foragers. This method significantly reduces the amount of time required by researchers to collect pollen from bumble bee foragers returning to the colony. We present the design, results of pollen removal efficiency tests, and suggest areas of modifications for investigators to adapt traps to a variety of bumble bee species or nest box designs.

A 3D Printed Pollen Trap for Bumble Bee Bombus Hive Entrances

We present a non-lethal and automated mechanism to collect pollen from bumble bee (Bombus) workers returning to a hive. Instructions for producing, preparing, installing and using the devices are included. By using 3D-printed objects, modification to the design was timely, efficient and allowed for quick turnaround for testing.

To verify the plant sources from which bumble bees forage for pollen, individuals must be collected to remove their corbicular pollen loads for analysis.  ...

Collection and Identification of Pollen from Honey Bee Colonies

Researchers often collect and analyze corbicular pollen from honey bees to identify the plant sources on which they forage for pollen or to estimate pesticide exposure of bees via pollen. Described herein is an effective pollen-trapping method for collecting corbicular pollen from honey bees returning to their hives. This collection method results in large quantities of corbicular pollen that can be used for research purposes. Honey bees collect pollen from many plant species, but typically visit one species during each collection trip. Therefore, each corbicular pollen pellet predominantly represents one plant species, and each pollen pellet can be described by color. This allows the sorting of samples of corbicular pollen by color to segregate plant sources. Researchers can further classify corbicular pollen by analyzing the morphology of acetolyzed pollen grains for taxonomic identification. These methods are commonly used in studies related to pollinators such as pollination efficiency, pollinator foraging dynamics, diet quality, and diversity. Detailed methodologies are presented for collecting corbicular pollen using pollen traps, sorting pollen by color, and acetolyzing pollen grains. Also presented are results pertaining to the frequency of pellet colors and taxa of corbicular pollen collected from honey bees in five different cropping systems.

We describe methods for collecting corbicular pollen from honey bees as well as protocols for color sorting, acetolysis, and microscope slide preparation of pollen for taxonomic identification. In addition, we present pellet color and taxonomic diversity of corbicular pollen collected from five cropping systems using pollen traps.

Researchers often collect and analyze corbicular pollen from honey bees to identify the plant sources on which they forage for pollen or to estimate ...

Assessing Agrochemical Risk to Mated Honey Bee Queens

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not accurately capture the effects of agrochemical exposure on honey bee queens. As the sole producer of fertilized eggs inside a honeybee colony, the queen is arguably the most important single member of a functioning colony unit. Therefore, understanding how agrochemicals affect queen health and productivity should be considered a critical aspect of pesticide risk assessment. Here, an adapted method is presented to expose honey bee queens and worker queen attendants to agrochemical stressors administered through a worker diet, followed by tracking egg production in the laboratory and assessing first instar eclosion using a specialized cage, referred to as a Queen Monitoring Cage. To illustrate the method&#39;s intended use, results of an experiment in which worker queen attendants were fed diet containing sublethal doses of imidacloprid and effects on queens were monitored are described.

This protocol was developed to enhance the understanding of how agrochemicals affect honey bee (Apis mellifera) reproduction by establishing methods to expose honey bee queens and their worker caretakers to agrochemicals in a controlled, laboratory setting and carefully monitoring their relevant responses.

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not ...

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

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