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 ...

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+feral+honey+bees+(Apis+mellifera)+and+regent+parrots+(Polytelis+anthopeplus)+compete+for+nest+sites.">Do feral honey bees (Apis mellifera) and regent parrots (Polytelis anthopeplus) compete for nest sites.</a> Aust J Ecol. 19, 444-450 (1994).</li><li>Downs, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+nest+boxes+and+wild+Cape+Parrots+Poicephalus+robustus:+persistence+pays+off.">Artificial nest boxes and wild Cape Parrots Poicephalus robustus: persistence pays off.</a> Ostrich. 76, 222-224 (2005).</li><li>Seeley, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Nest+Cavity+Volume+by+the+Honey+Bee+(Apis+mellifera).">Measurement of Nest Cavity Volume by the Honey Bee (Apis mellifera).</a> Behav Ecol Sociobiol. 2, 201-227 (1977).</li><li>Schneider, S. S., DeGrandi-Hoffman, G., Smith, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+African+Honey+Bee:+Factors+contributing+to+a+successful+biological+invasion.">The African Honey Bee: Factors contributing to a successful biological invasion.</a> Annu Rev Entomol. 49, 351-376 (2004).</li><li>Seely, T. D., Visscher, P. K., Passino, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Group+decision+making+in+honey+bee+swarms.">Group decision making in honey bee swarms.</a> Behav Ecol Sociobiol. 94, 220-229 (2006).</li><li>Taylor, K. S., Waller, G. D., Crowder, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairment+of+a+classical+conditioning+response+of+the+honey+bee+(Apis+mellifera+L.)+by+sublethal+doses+of+synthetic+pyrethroid+insecticides.">Impairment of a classical conditioning response of the honey bee (Apis mellifera L.) by sublethal doses of synthetic pyrethroid insecticides.</a> Apidologie. 18, 243-252 (1987).</li><li>Ingram, E. M., Augustin, J., Ellis, M. D., Siegfried, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+sub-lethal+effects+of+orchard-applied+pyrethroids+using+viedo-tracking+software+to+quantify+honey+bee+behaviors.">Evaluating sub-lethal effects of orchard-applied pyrethroids using viedo-tracking software to quantify honey bee behaviors.</a> Chemosphere. 135, 272-277 (2015).</li><li>Rieth, J. P., Levin, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+repellent+effect+of+two+pyrethroid+insecticides+on+the+honey+bee.">The repellent effect of two pyrethroid insecticides on the honey bee.</a> Physiol Entomol. 13, 213-218 (1988).</li><li>Efstathion, C. A., Bardunias, P. M., Boyd, J. D., William, K. 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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 ne...

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 border="0" cellpadding="0" cellspacing="0" width="652">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<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. O...

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. ...

The overall goal of this procedure is to prevent honey bee colonization of bird nest boxes by using insect repellent in the bird nest boxes and a pheromone lure in the swarm trap boxes. This method can help solve the conservation issue of Africanized honey bee hybrids competing for cavities with cavity-nesting birds. The main advantages of this technique are that it's safe for the birds, deters honey bees without the need to kill entire colonies or swarms and deters blood-feeding ectoparasites.We first had the idea for this method when we had to deal with an Africanized honey bee colony in a screech owl nest box here in South Florida. Visual demonstration of this method is critical as the implementation of this procedure varies according to the environmental and bee colony circumstances. Before placing the swarm trap box, add one milliliter of a two to one mixture of citral and geraniol into a 1.5 milliliter snap top polypropylene microcentrifugation tube and wrap the tube in aluminum foil to protect the lure from light.Then store the lure in a dark place at room temperature. To install the swarm trap on a post, fix a nine by nine centimeter pressure-treated 2.4 meter long post into the ground, 10 to 15 meters from where the bird nest box is located. Next, use at least two screws to secure a 1.3 centimeter thick 20 by 20 centimeter pressure-treated wooden hanging board in the middle of one side of the trap box.With the top of the board, set 10 centimeters above the top of the box. Then place a screw approximately five centimeters below where the top of the hanging board will rest and attach the hanging board to the post such that the trap box is 1.5 to three meters above the ground to allow the safe and easy removal of the box after colonization. To install the swarm trap box on a tree, first drill a five millimeter hole in the upper back corners of each side of the box and thread a wire through the holes.Next, select a tree approximately 10 to 15 meters away from where a bird nest box is located and secure the wire around the tree 1.5 to three meters off the ground. Then confirm that the box is level and will not tip over. When the swarm trap box has been secured, use a pushpin to puncture the cap of the tube of lure and place the lure at the bottom of the trap box near the entrance.Then use a 3.7 liter hand pump sprayer to coat the entire inside of the bird nest box with a freshly-prepared permethrin for poultry or caged birds until the liquid runs off the sides of the box. If the swarm is to be utilized, allow the bees to fully colonize the trap box for about two weeks. Then at night when all of the bees are inside, don the appropriate protective equipment and use a small towel to plug the trap box entrance.The next morning collect the box, move it to a new location at least four kilometers away and remove the towel. If destruction of the bee colony is warranted, as soon as the colony is discovered, plug the entrance hole with a small towel and remove the box. Seal the trap box completely in a plastic bag and leave the colony in the full sun.After several days remove the dead bees and comb. If the use of an insecticide is preferred, as soon as the colony is discovered, spray the inside of the trap box with an insecticide spray and plug the entrance with a small towel. After 24 hours, remove the dead bees and comb.Of the 77 swarms caught in this representative experiment, 75 were in swarm traps. Only two owl nest boxes were colonized, both of which were untreated. Significantly more bees chose swarm traps over untreated or treated owl boxes and several swarm trap boxes were colonized by bees more than once.Once mastered, this technique can be completed in under one hour if it is performed properly. While attempting this procedure it's important to remember to be safe. Always place the swarm trap boxes where they will be easy to remove and relocate them at night when the bees will not be active.This procedure can be modified to accommodate a wide range of bird species and habitats. After its development, this technique paved the way for researchers in the field of conservation to explore tools for managing the bee swarm colonization of bird nest boxes in recovery programs of endangered parrots. After watching this video you should have a good understanding of how to set up bee swarm trap boxes and to safely remove and relocate or destroy the bees that have colonized the trap box.Don't forget that working with Africanized honey bees can be extremely hazardous and that precautions such as wearing bee suits and gloves should always be taken while performing this procedure.

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8.5K ビュー.  University of Florida. The overall goal of this procedure is to prevent honey bee colonization of bird nest boxes by using insect repellent in the bird nest boxes and a pheromone lure in the swarm trap boxes. This method can help solve the conservation issue of Africanized honey bee hybrids competing for cavities with cavity-nesting birds. The main advantages of this technique are that it's safe for the birds, deters honey bees without the need to kill entire colonies or swarms and deters blood-feeding ectoparasite...

ビデオ: 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...

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The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis,&#160;while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown...

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a rapid method to confine and safely handle bees in the field

Author Spotlight: Novel Non-Lethal Method for Field Documentation and Sampling of At-Risk Bee Taxa

We demonstrate a tested method for safely handling field-collected bees. This method allows swift manipulation, identification, genetic sampling, and confirmation of plant-insect interactions via pollen collected while sampling. Easily adaptable, this approach offers a cost-effective, non-lethal means to study rare insect...

A Rapid Method to Confine and Safely Handle Bees in the Field

handling techniques to reduce stress in mice

Handling Techniques to Reduce Stress in Mice

This paper describes a handling technique in mice, the 3D-handling technique, which facilitates routine handling by reducing anxiety-like behaviors and presents details on two existing related techniques (tunnel and tail...

assessing agrochemical risk to mated honey bee queens

Assessing Agrochemical Risk to Mated Honey Bee Queens

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.

an instrumented pull test to characterize postural responses

An Instrumented Pull Test to Characterize Postural Responses

Impairment of postural reflexes, termed postural instability, is difficult to quantify. Clinical assessments such as the pull test suffer issues with reliability and scaling. Here, we present an instrumented version of the pull test to objectively characterize postural responses.

rna pull-down procedure to identify rna targets of a long non-coding rna

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

This RNA pull-down method allows identifying the RNA targets of a long non-coding RNA (lncRNA). Based on the hybridization of home-made, designed anti-sense DNA oligonucleotide probes specific to this lncRNA in an appropriately fixed tissue or cell line, it efficiently allows the capture of all RNA targets of the...

a murine model of group b streptococcus vaginal colonization

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic...

A Murine Model of Group B Streptococcus Vaginal Colonization

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 ...

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 ...

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.

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 ...