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>

<ol>
	<li>Michener, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The bees of the world. , (2000).</li><li>Corbet, S. A., Williams, I. H., Osborne, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bees+and+the+pollination+of+crops+and+wild+flowers+in+the+European+Community.">Bees and the pollination of crops and wild flowers in the European Community.</a> Bee world. 72 (2), 47-59 (1991).</li><li>Cameron, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+widespread+decline+in+North+American+bumble+bees.">Patterns of widespread decline in North American bumble bees.</a> Proceedings of the National Academy of Sciences. 108 (2), 662-667 (2011).</li><li>Goulson, D., Nicholls, E., Bot&#237;as, C., Rotheray, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bee+declines+driven+by+combined+stress+from+parasites,+pesticides,+and+lack+of+flowers.">Bee declines driven by combined stress from parasites, pesticides, and lack of flowers.</a> Science. 347 (6229), 1255957 (2015).</li><li>Jha, S., Stefanovich, L. E. V., Kremen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bumble+bee+pollen+use+and+preference+across+spatial+scales+in+human-altered+landscapes.">Bumble bee pollen use and preference across spatial scales in human-altered landscapes.</a> Ecological Entomology. 38 (6), 570-579 (2013).</li><li>Thomson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competitive+interactions+between+the+invasive+European+honey+bee+and+native+bumble+bees.">Competitive interactions between the invasive European honey bee and native bumble bees.</a> Ecology. 85 (2), 458-470 (2004).</li><li>Kleijn, D., Raemakers, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+retrospective+analysis+of+pollen+host+plant+use+by+stable+and+declining+bumble+bee+species.">A retrospective analysis of pollen host plant use by stable and declining bumble bee species.</a> Ecology. 89 (7), 1811-1823 (2008).</li><li>Harmon-Threatt, N. H., Kremen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bumble+bees+selectively+use+native+and+exotic+species+to+maintain+nutritional+intake+across+highly+variable+and+invaded+floral+resource+pools.">Bumble bees selectively use native and exotic species to maintain nutritional intake across highly variable and invaded floral resource pools.</a> Ecological Entomology. 40, 471-478 (2015).</li><li>Harmon-Threatt, N. H., Valpine, P., Kremen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+resource+preferences+of+a+native+bumblebee:+the+effects+of+availability+and+use-availability+models+on+preference+estimates.">Estimating resource preferences of a native bumblebee: the effects of availability and use-availability models on preference estimates.</a> Oikos. , (2016).</li><li>Martin, A. P., Carreck, N. M. L., Swain, J. L., Goulson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modular+system+for+trapping+and+mass-marking+bumblebees:+applications+for+studying+food+choice+and+foraging+range.">A modular system for trapping and mass-marking bumblebees: applications for studying food choice and foraging range.</a> Apidologie. 37, (2006).</li><li>Saifuddin, M., Jha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colony-level+variation+in+pollen+collection+and+foraging+preferences+among+wild-caught+bumble+bees.+(Hymenoptera:+Apidae).">Colony-level variation in pollen collection and foraging preferences among wild-caught bumble bees. (Hymenoptera: Apidae).</a> Environmental Entomology. 42 (2), 393-401 (2014).</li><li>Heinrich, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bumblebee Economics. , (2004).</li><li>Leonhart, S. D., Bluthgen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+same,+but+different:+pollen+foraging+in+honeybee+and+bumblebee+colonies.">The same, but different: pollen foraging in honeybee and bumblebee colonies.</a> Apidologie. 43, (2012).</li><li>Kriesell, L., Hilpert, A., Leonhardt, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+but+the+same:+bumblebee+species+collect+pollen+of+different+plant+sources+but+similar+amino+acid+profiles.">Different but the same: bumblebee species collect pollen of different plant sources but similar amino acid profiles.</a> Apidologie. 48, 102-116 (2017).</li><li>Al-Tikrity, W. S., Benton, A. W., Hillman, R. C., Clarke, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+the+amount+of+unsealed+brood+in+honeybee+colonies+and+their+pollen+collection.">The relationship between the amount of unsealed brood in honeybee colonies and their pollen collection.</a> Journal of Apicultural Research. 11 (1), 9-12 (1972).</li><li>Spaethe, J., Weidenm&#252;ller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+variation+and+foraging+rate+in+bumblebees+(Bombus+terrestris).">Size variation and foraging rate in bumblebees (Bombus terrestris).</a> Insectes Sociaux. 49 (2), 142-146 (2002).</li><li>Goodwin, R. M., Perry, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+pollen+traps+to+investigate+the+foraging+behaviour+of+honey+bee+colonies+in+kiwifruit.">Use of pollen traps to investigate the foraging behaviour of honey bee colonies in kiwifruit.</a> New Zealand Journal of Crop and Horticulture Science. 20 (1), 23-26 (1992).</li><li>Chua, C. K., Leong, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 3D PRINTING AND ADDITIVE MANUFACTURING: Principles and Applications (with Companion Media Pack) of Rapid Prototyping. , (2014).</li><li>Kearns, C. A., Inouye, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Techniques for Pollination Biologists. , (1993).</li><li>Velthuis, H. H., van Doorn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+century+of+advances+in+bumblebee+domestication+and+the+economic+and+environmental+aspects+of+its+commercialization+for+pollination.">A century of advances in bumblebee domestication and the economic and environmental aspects of its commercialization for pollination.</a> Apidologie. 37 (4), 421-451 (2006).</li><li>Moisan-Deserres, J., Girard, M., Chagnon, M., Fournier, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pollen+loads+and+specificity+of+native+pollinators+of+lowbush+blueberry.">Pollen loads and specificity of native pollinators of lowbush blueberry.</a> Journal of Economic Entomology. 107 (3), 1156-1162 (2014).</li><li>Medler, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nest+of+Bombus+huntii+Greene+(Hymenoptera:+Apidae).">A nest of Bombus huntii Greene (Hymenoptera: Apidae).</a> Entomological News. 70, 179-182 (1959).</li><li>Husband, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observation+on+colony+of+bumblebee+species+(Bombus+spp).">Observation on colony of bumblebee species (Bombus spp).</a> Great Lakes Entomologist. 10, 83-85 (1977).</li><li>Buttermore, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+of+successful+Bombus+terrestris++(L),+(Hymenoptera:+Apidae)+colonies+in+Southern+Tasmania.">Observations of successful Bombus terrestris (L), (Hymenoptera: Apidae) colonies in Southern Tasmania.</a> Australian Journal of Entomology. 36, 251-254 (1997).</li><li>Goulson, D., Peat, J., Stout, J. C., Tucker, J., Darvill, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+alloethism+in+workers+of+the+bumblebee,+Bombus+terrestris,+be+explained+in+terms+of+foraging+efficiency.">Can alloethism in workers of the bumblebee, Bombus terrestris, be explained in terms of foraging efficiency.</a> Animal Behaviour. 64 (1), 123-130 (2002).</li><li>Couvillon, M. J., Jandt, J. M., Duong, N. H. I., Dornhaus, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+of+worker+body+size+distribution+in+bumble+bee+(Bombus+impatiens)+colonies.">Ontogeny of worker body size distribution in bumble bee (Bombus impatiens) colonies.</a> Ecological Entomology. 35 (4), 424-435 (2010).</li><li>Russell, A. L., Morrison, S. J., Moschonas, E. H., Papaj, 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=Patterns+of+pollen+and+nectar+foraging+specialization+by+bumblebees+over+multiple+timescales+using+RFID.">Patterns of pollen and nectar foraging specialization by bumblebees over multiple timescales using RFID.</a> Scientific Reports. 7 (1), 1-13 (2017).</li><li>Hagbery, J., Nieh, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+lifetime+pollen+and+nectar+foraging+preferences+in+bumble+bees.">Individual lifetime pollen and nectar foraging preferences in bumble bees.</a> Naturwissenschaften. 99 (10), 821-832 (2012).</li><li>Baur, A., Strange, J. P., Koch, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foraging+economics+of+the+Hunt+bumble+bee,+a+viable+pollinator+for+commercial+agriculture.">Foraging economics of the Hunt bumble bee, a viable pollinator for commercial agriculture.</a> Environmental Entomology. 48 (4), 799-806 (2019).</li><li>Winter, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importation+of+non-native+bumble+bees+into+North+America:+potential+consequences+of+using+Bombus+terrestris+and+other+non-native+bumble+bees+for+greenhouse+crop+pollination+in+Canada,+Mexico,+and+the+United+States.">Importation of non-native bumble bees into North America: potential consequences of using Bombus terrestris and other non-native bumble bees for greenhouse crop pollination in Canada, Mexico, and the United States.</a> San Francisco. 33, (2006).</li><li>Ruz, L., Herrera, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preliminary+observations+on+foraging+activities+of+Bombus+dahlbomii+and+Bombus+terrestris+(Hym:+Apidae)+on+native+and+non-native+vegetation+in+Chile.">Preliminary observations on foraging activities of Bombus dahlbomii and Bombus terrestris (Hym: Apidae) on native and non-native vegetation in Chile.</a> Acta Horticulturae. 561, 165-169 (2001).</li></ol>

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><tbody><tr><td>MakerBot Replicator+</td><td>MakerBot</td><td>Model PABH65</td><td></td></tr><tr><td>MakerBot Tough Material</td><td>PLA Filament</td><td></td><td>various colors</td></tr><tr><td>Nest Box</td><td>Biobest</td><td></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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5.2K Views.  Utah State University. We presenteren een niet-dodelijk en geautomatiseerd mechanisme om stuifmeel te verzamelen van hommel(Bombus)werknemers die terugkeren naar een bijenkorf. Instructies voor het produceren, voorbereiden, installeren en gebruiken van de apparaten zijn inbegrepen. Door gebruik te maken van 3D-geprinte objecten was de aanpassing aan het ontwerp tijdig, efficiënt en zorgde voor een snelle doorlooptijd voor het testen.

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

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

Introduction of the invasive Africanized honey bee (AHB) into the Neotropics is a serious problem for many cavity nesting birds, specifically parrots. These bees select cavities that are suitable nest sites for birds, resulting in competition. The difficulty of removing bees and their defensive behavior makes a prevention protocol necessary. Here, we describe a push-pull integrated pest management protocol to deter bees from inhabiting bird boxes by applying a bird safe insecticide, permethrin, to repel bees from nest boxes, while simultaneously attracting them to pheromone-baited swarm traps. Shown here is an example experiment using Barn Owl nest boxes. This protocol successfully reduced colonization of Barn Owl nest boxes by Africanized honey bees. This protocol is flexible, allowing adjustments to accommodate a wide range of bird species and habitats. This protocol could benefit conservation efforts where AHB are located.

Preventing colonization of bird nest boxes by invasive Africanized honey bees is important for conservation efforts of nest-site limited birds. We provide an integrated pest management approach to &#34;push&#34; bees away from nest boxes with a repellant insecticide, permethrin, and &#34;pull&#34; them toward pheromone baited swarm traps.

Introduction of the invasive Africanized honey bee (AHB) into the Neotropics is a serious problem for many cavity nesting birds, specifically parrots. ...

Environment

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

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

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

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

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 ...

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 ...

Low-Cost Automated Flight Intercept Trap for the Temporal Sub-Sampling of Flying Insects Attracted to Artificial Light at Night

Sampling methods are selected depending on the targeted species or the spatial and temporal requirements of the study. However, most methods for passive sampling of flying insects have a poor temporal resolution because it is time-consuming, costly and/or logistically difficult to perform. Effective sampling of flying insects attracted to artificial light at night (ALAN) requires sampling at user-defined time points (nighttime only) across well-replicated sites resulting in major time and labor-intensive survey effort or expensive automated technologies. Described here is a low-cost automated intercept trap that requires no specialist equipment or skills to construct and operate, making it a viable option for studies that require temporal sub-sampling across multiple sites. The trap can be used to address a wide range of other ecological questions that require a greater temporal and spatial scale than is feasible with previous trap technology.

To study the impacts of artificial light at night (ALAN) on nocturnal flying insects, sampling needs to be confined to nighttime. The protocol describes a low-cost automated flight intercept trap that allows researchers to sample at user-defined periods with increased replication.

Sampling methods are selected depending on the targeted species or the spatial and temporal requirements of the study. However, most methods for passive ...

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

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

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

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

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

Neuroscience

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

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

The Proboscis Extension Response (PER) conditioning protocol, developed for the honey bee (Apis mellifera), provides an ecologically-relevant and easily quantifiable means for studying several different mechanisms of learning in many insect species.

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

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

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

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

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

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

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

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

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

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

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 ...

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

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