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.

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			<td height="21" style="height:21px;width:216px;">Citral (ACROS Organics)</td>
			<td style="width:117px;">Fisher Scientific</td>
			<td style="width:119px;">AC11044-1000</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Geraniol (ACROS Organics)</td>
			<td style="width:117px;">Fisher Scientific</td>
			<td>AC41090-1000</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PermaCap CS</td>
			<td style="width:117px;">BASF</td>
			<td style="width:119px;"></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>

Watch this Scientific Journal Video about A Push-pull Protocol to Reduce Colonization of Bird Nest Boxes by Honey Bees at JoVE.com

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. 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 "push" bees away from nest boxes with a repellant insecticide, permethrin, and "pull" them toward pheromone baited swarm traps.

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

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3 . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high&#8208;resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high&#8208;throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.

More than half of proteins are small proteins (molecular mass &#60;200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of ...

Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity

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

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

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

A Protocol for Conducting Rainfall Simulation to Study Soil Runoff

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

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

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

A Protocol for Collecting and Constructing Soil Core Lysimeters

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

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

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

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 ...

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

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

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

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

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

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

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

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

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

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Measuring Hypopharyngeal Gland Acinus Size in Honey Bee (Apis mellifera) Workers

The nurse hypopharyngeal glands produce the protein fraction of the worker and royal jelly that is fed to developing larvae and queens. These paired glands that are located in the head of the bee are highly sensitive to the quantity and quality of pollen and pollen substitutes that the nurse bee consumes. The glands get smaller when nurses are fed deficient diets and are large when they are fed complete diets. Because nurse hypopharyngeal gland size is a robust indicator of nurse nutrition, it is essential that those studying honey bee nutrition know how to measure these glands. Here, we provide detailed methods for dissecting, staining, imaging, and measuring nurse bee hypopharyngeal glands. We present comparisons of unstained and stained tissue and data that were used to study the impact of pollen on gland size. This method has been used to test how diet impacts hypopharyngeal gland size but has further use for understanding the role of these glands in hive health.

Measuring Hypopharyngeal Gland Acinus Size in Honey Bee Apis mellifera Workers

Hypopharyngeal gland acinus size is a robust measure of nurse honey bee nutrition. Here, we provide a detailed protocol for dissecting, staining, imaging, and measuring nurse bee hypopharyngeal gland acini.

The nurse hypopharyngeal glands produce the protein fraction of the worker and royal jelly that is fed to developing larvae and queens. These paired ...

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

Summary

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Chapters in this video

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity

A Protocol for Conducting Rainfall Simulation to Study Soil Runoff

A Protocol for Collecting and Constructing Soil Core Lysimeters

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

Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees

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

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

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Measuring Hypopharyngeal Gland Acinus Size in Honey Bee (Apis mellifera) Workers