We thank Colby Carpenter and Spencer Mathias for their assistance in 3D printing design. We thank Ellen Klinger for assistance in producing the photographic figures and Jonathan B. Koch for providing assistance with revisions. Funding was provided by the USDA-ARS-Pollinating Insect Biology, Management, and Systematics Research Unit.

1. Print pollen trap structures
<ol>
	<li>Download the appropriate STL file for the nest box that bumble bees are nesting in (e.g., Biobest or Koppert style hives, https://www.ars.usda.gov/pacific-west-area/logan-ut/pollinating-insect-biology-management-systematics-research/docs/pollen-traps/). The files are available to the public, free for download and modification by the end user.</li>
	<li>Open the STL file in the printer program. Follow printer manufacturer directions to build the four trap components. 
	NOTE: Allow approximately 3 h for the trap body to print, 2 h for catch basin to print and 30 min each for the filter and trap closure insert to print. Trap body size is 6 cm x 3.8 cm x 7 cm.</li>
</ol>
2. Pollen trap assembly
<ol>
	<li>Remove the support structures printed with the trap body and catch basin including those of the sieve structure of the trap body (Figure 1).</li>
	<li>Use a 3/16 in. (0.476 cm) drill bit mounted in a hand drill to clear any plastic strands crossing the raised edges of the pollen filter that might hinder a bee from moving through the filter holes. Use a box cutting razor blade and sandpaper to even out any bumps or raised edges on the flat side of the pollen filter.</li>
	<li>Place the pollen filter in the trap body by gently pushing the plastic filter through one side of the trap body. The filter will only fit in one way, as the left side of the trap has a larger opening to accommodate passage of the raised filter cones.
	<ol>
		<li>If the side slits are too small for the pollen filter to slide through smoothly, scrape enough plastic away from the slit in the trap body with a razor or other tool that can remove small portions of plastic at a time. Ensure that the pollen filter fits securely in place with no more than a 2 mm gap between the pollen filter and trap body.</li>
	</ol>
	</li>
	<li>Attach the catch basin to the trap body by sliding the raised edges on the bottom of the trap body into the groove on the top of the catch basin. The catch basin should be positioned directly underneath the sieve region of the trap body (Figure 1A&#8210;E).</li>
	<li>Make appropriate modifications by cutting or sanding the plastic to allow smooth placement and removal of the catch basin from the trap body. Placement of the catch basin will secure the pollen filter into place and it cannot be removed until the catch basin is removed, nor can the pollen filter be inserted while the catch basin is in place. 
	NOTE: If multiple hives are placed close to one another, providing each hive with a unique color combination of the trap body, catch basin and pollen filter structures in addition to deploying hives with varied orientation will help returning workers find their nests.</li>
</ol>
3. Bumble bee colony preparation
<ol>
	<li>Stop pollen feeding 24&#8210;48 h before deploying colonies. This will cause workers to use up any stored pollen and stimulate them to leave the nest in search of pollen.</li>
	<li>Prepare the hive trap body for installation by inserting a trap closure insert into the filter slot to prevent bees from escaping while installing the trap.</li>
	<li>Working under red light to prevent the bees from flying, lift the plastic nest box out of the cardboard outer box.</li>
	<li>Locate the plastic nest entrance at the front of the nest. There are two styles of entrance depending on the nest supplier: Koppert-style boxes (step 3.5) and Biobest-style boxes (step 3.6).</li>
	<li>For Koppert-style hive entrances, mount the pollen trap onto the nest entrance by pulling up on the entrance tab until both entrance holes are open.
	<ol>
		<li>Insert the two tubes of the pollen trap into the entrance holes, ensuring that the sieve of the pollen trap is on the bottom. Gently push down on the plastic entrance tab to secure the pollen trap in place.</li>
	</ol>
	</li>
	<li>To install the pollen trap into Biobest-style hive entrances, use a flat head screwdriver to gently pry the plastic entrance device from the nest box. Insert the pollen trap into the nest entrance holes until the pollen trap is firmly against the nest (Figure 1E).
	<ol>
		<li>Secure the trap to the nest using tape or quick dry glue where the trap contacts the nest box if needed.</li>
	</ol>
	</li>
	<li>Return the plastic nest box to the cardboard box. Cardboard may need to be cut away to accommodate the pollen trap.</li>
</ol>
4. Deployment of nests
<ol>
	<li>Place nest boxes into the study area. Provide cover to protect from precipitation and anchorage for wind as these can adversely affect the quality and quantity of pollen that is collected. Provide hives placed in greenhouses with adequate sun cover to reduce overheating.</li>
	<li>Remove the trap closure insert from the trap body to allow bees to forage freely so that foragers can orient themselves with the surrounding area and the location of their nest. Orientation flight time should be complete in 24 h under normal conditions.</li>
</ol>
5. Pollen collection
<ol>
	<li>To engage the trap, slide the pollen filter into the filter slot, ensuring that it is securely in place.</li>
	<li>Install the catch basin by sliding it onto the trap body from the front until it is fully closed. If the catch basin is excessively loose or falls off the trap body, use a rubber band to secure it to the trap body.</li>
	<li>Observe bees entering and exiting the pollen trap at first deployment to ensure the pollen filter holes are large enough to accommodate the bees.
	<ol>
		<li>If workers are unable to pass through the pollen filter, remove the filter and then use drill bits larger than 3/16 in. (0.476 cm) to increase the holes sizes. Do so in a sequential manner, increasing hole diameter 1/32 in. (0.079 cm) each time, as holes that are too large will not collect any pollen.</li>
		<li>Once bees are able to pass through the filter, continue to observe the entrance to ensure pollen is being removed upon re-entry. 
		NOTE: Bees should have some difficulty moving though the pollen filter holes, especially the first few times they pass through. If they pass through too easily, the pollen may not be dislodged from the corbiculae.</li>
	</ol>
	</li>
	<li>After the designated period of pollen collection, slide and remove catch basin from the trap body.</li>
	<li>Process the pollen loads according to your experimental design.</li>
	<li>Remove the pollen filter to allow workers to forage freely until the next period of pollen collection. The trap body may remain attached to the hive for the duration of the experiment. 
	NOTE: Pollen traps may be engaged for as long as the researcher desires to collect pollen from a colony. However, deployment of pollen traps for over 24 h in a week may result in starvation of brood in a hive and retard colony development.</li>
</ol>

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The authors have nothing to disclose.

Collection of pollen from bumble bee colony entrances can allow for a variety of ecological and agricultural studies. Identifying the floral sources from which bumble bees collect pollen provides valuable information and insight into the diversity of plants that contribute to a colony&#8217;s overall diet19. Identifying the pollen source has implications for both agricultural production and studies of ecosystem services in wild lands12,20....

Bumble bees (Bombus spp.) are large robust insects that are found across the temperate, alpine, and arctic regions of the world1. They are important to&#160;plant communities&#160;and provide important pollination service for the agricultural crops that they visit2. Recent declines in the&#160;abundance and distribution of several species has brought their importance as pollinators to the forefront of&#160;public&#160;awareness3. Researchers have identified several stressors that are likely contributing to population&#160;declines including a lack of diverse and abundant floral resources on which bumble bees forage4. Identifying which&#160;plant species bumble bees forage from allows researchers and land managers to understand how bumble bees may be responding to changes in resource availability, competition, and&#160;anthropogenic disturbances5,6.
Studies investigating the pollen foraging preferences of bumble bees are often conducted by researchers catching individual bees foraging at flowers, and then removing the corbicular pollen loads from specimens for further processing and identification7,8,9,10. While this method provides insight into how a species or an assemblage of bumble bee species utilizes the resources in an area7, it is time intensive and potential differences in preferences among hives cannot be discerned without additional molecular analyses to identify colony of origin of the foraging bee11.
For some studies of foraging dynamics, it is desired to conduct the studies at individual colonies; however, wild bumble bee nests are generally located underground or at ground level making them difficult to locate12. Commercially produced bumble bee hives provide researchers greater access and better experimental control and the removal of pollen off workers is still primarily conducted by capturing foragers as they return to the hive and manually removing their corbicular pollen loads13,14. The removal of pollen by hand from the corbicula of a bee is time intensive with a low hourly yield of pollen especially at hive entrances where the rate of returning pollen foragers may be low. Additionally, manually removing pollen from bees can result in stings from disturbed workers.
Pollen traps have been used for experimental removal of pollen from honey bees for decades15; yet, a passive method for removing pollen from bumble bees has not been developed. The primary obstacle in developing a mechanism to remove pollen from returning forager bumble bees is the large variation of worker sizes that exist in a bumble bee colony16. Honey bee pollen traps are effective largely because honey bee worker size does not vary much. Additionally, these traps require only minor manipulations after installation and don&#8217;t require bees to be sacrificed17. This is achieved using screens or plastic surfaces that dislodge the pollen off of the hind legs of workers as they return to the hive. These traps remove only a portion of the pollen loads from returning foragers and the various designs of those result in varied efficiencies at pollen collection. As the pollen is removed from the bee legs, it falls through a screen and into a collection basin to which the bees have no access, so that the researcher can remove it with only minor disturbance to the hive.
The purpose of the present study is to adapt the techniques used for collecting pollen from honey bee hives and apply them to bumble bee nests using 3D printed structures and test the trap designs on colonies of Bombus huntii. The design process followed the assumptions that the traps should be inexpensive to produce, adaptable to a variety of bumble bee species, cause minimal harm or disturbance to the bees, and that the rate of pollen removal should exceed hand collection of pollen. Three-dimensional printing technology is versatile, easily accessible, and a cost-effective tool allowing researchers to replicate and modify objects for specific purposes18. The technique presented here instructs the user to build pollen traps and attach them onto commercially available bumble bee colonies. The traps are not designed to be use with wild colonies. These traps passively remove the corbicular pollen loads from the hind legs of pollen carrying bumble bees as they return to their nest boxes.

<table border="0" cellpadding="0" cellspacing="0" style="width:461px;" width="462">
	<colgroup>
		<col />
		<col span="2" />
		<col />
	</colgroup>
	<tbody>
		<tr height="35">
			<td height="35" style="height:35px;width:71px;">MakerBot Replicator+</td>
			<td style="width:87px;">MakerBot</td>
			<td style="width:87px;">Model PABH65</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:71px;">MakerBot Tough Material</td>
			<td style="width:87px;">PLA Filament</td>
			<td style="width:87px;"></td>
			<td>various colors</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:71px;">Nest Box</td>
			<td style="width:87px;">Biobest</td>
			<td style="width:87px;"></td>
			<td>Not sold publicly without bee purchase</td>
		</tr>
	</tbody>
</table>

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.

Eight different pollen filter designs were tested to determine their efficacy and efficiency at removing corbicular pollen loads from returning bumble bee workers. All designs were successful at removing at least of one corbicular pollen load from a returning forager. However, some were found to slow workers from leaving or entering the hive or failed to remove pollen loads (Table 1). Pollen traps with various filters were tested sequentially on 4 laboratory reared colonies of B. huntii Greene f...

Watch this Scientific Journal Video about A 3D Printed Pollen Trap for Bumble Bee Bombus Hive Entrances at JoVE.com

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.

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.  ...

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Research

JoVE Journal

Biology

5.1K Views.  Utah State University. 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.

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

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Associated Chromosome Trap for Identifying Long-range DNA Interactions

Genetic information encoded by DNA is organized in a complex and highly regulated chromatin structure. Each chromosome occupies a specific territory, that may change according to stage of development or cell cycle. Gene expression can occur in specialized transcriptional factories where chromatin segments may loop out from various chromosome territories, leading to co-localization of DNA segments which may exist on different chromosomes or far apart on the same chromosome. The Associated Chromosome Trap (ACT) assay provides an effective methodology to identify these long-range DNA associations in an unbiased fashion by extending and modifying the chromosome conformation capture technique. The ACT assay makes it possible for us to investigate mechanisms of transcriptional regulation in trans, and can help explain the relationship of nuclear architecture to gene expression in normal physiology and during disease states.

The associated chromosome trap (ACT) assay is a novel unbiased method for identifying long-range DNA interactions. The characterization of long range DNA interactions will allow us to determine the relationship of nuclear architecture to gene expression in both normal physiology and in diseased states.

Genetic information encoded by DNA is organized in a complex and highly regulated chromatin structure.  Each chromosome occupies a specific territory, ...

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best established model to study how plasticity in lifespan and aging is explained by social factors.
The worker caste of honey bees includes nurse bees, which tend the brood, and forager bees, which collect nectar and pollen. Previous work has shown that brain functions and flight performance senesce more rapidly in foragers than in nurses. However, brain functions can recover, when foragers revert back to nursing tasks. Such patterns of accelerated and reversed functional senescence are linked to changed metabolic resource levels, to alterations in protein abundance and to immune function. Vitellogenin, a yolk protein with adapted functions in hormonal control and cellular defense, may serve as a major regulatory element in a network that controls the different aging dynamics in workers.
Here we describe how the emergence of nurses and foragers can be monitored, and manipulated, including the reversal from typically short-lived foragers into longer-lived nurses. Our representative results show how individuals with similar chronological age differentiate into foragers and nurse bees under experimental conditions. We exemplify how behavioral reversal from foragers back to nurses can be validated. Last, we show how different cellular senescence can be assessed by measuring the accumulation of lipofuscin, a universal biomarker of senescence.
For studying mechanisms that may link social influences and aging plasticity, this protocol provides a standardized tool set to acquire relevant sample material, and to improve data comparability among future studies.

In honey bee workers, aging depends on social behaviors rather than on chronological age. Here we show how worker-types with very different aging patterns can be obtained and analyzed for cellular senescence.

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best ...

Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees collect and convert to beebread by placing colonies in a specially constructed enclosed flight area. Methods were developed to analyze the protein and amino acid composition of the pollen and beebread. We also describe how consumption of the beebread was measured and methods used to determine adult worker bee hemolymph protein titers after feeding on beebread for 4, 7 and 11 days after emergence. Methods were applied to determine if genotype affects the conversion of pollen to beebread and the rate that bees consume and acquire protein from it. Two subspecies (European and Africanized honey bees; EHB and AHB respectively) were provided with the same pollen source. Based on the developed methods, beebread made by both subspecies had lower protein concentrations and pH values than the pollen. In general, amino acid concentrations in beebread made by either EHB or AHB were similar and occurred at higher levels in beebread than in pollen. Both AHB and EHB consumed significantly more of the beebread made by AHB than by EHB. Though EHB and AHB consumed similar amounts of each type of beebread, hemolymph protein concentrations in AHB were higher than in EHB. Differences in protein acquisition between AHB and EHB might reflect environmental adaptations related to the geographic region where each subspecies evolved. These differences could contribute to the successful establishment of AHB populations in the New World because of the effects on brood rearing and colony growth.

Here are methods to quantify nutrients in pollen before and after its conversion to beebread by two subspecies of honeybees. We describe techniques to measure beebread consumption and resulting protein titers in both subspecies.

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees ...

Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination services, as well as an important model social insect used to study memory and learning, aging, and more. Here we describe a detailed protocol for the dissection of the dorsal abdominal wall of a bee in order to visualize its dorsal vessel, which serves the role of the heart in the insect. A successful dissection will expose a functional heart that, under the proper conditions, can maintain a steady heartbeat for an extended period of time. This allows the investigator to manipulate heart rate through the application of cardiomodulatory compounds to the dorsal vessel. By using either a digital microscope or a microscope equipped with a digital camera, the investigator can make video recordings of the dorsal vessel before and after treatment with test compounds. The videos can then be scored at a time convenient to the user in order to determine changes in heart rate, as well as changes in the pattern of heartbeats, following treatment. The advantages of this protocol are that it is relatively inexpensive to set up, easy to learn, requires little space or equipment, and takes very little time to conduct.

The abdominal dorsal vessel of the honey bee and other insects serves as the functional equivalent of the mammalian heart and plays an important role in nutrient transport, waste removal, immune function, and more. Here we describe a protocol for the visualization and pharmacological manipulation of bee heart rate.

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination ...

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 ...

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 ...