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Journal of Insect Science logoLink to Journal of Insect Science
. 2024 Nov 20;24(6):3. doi: 10.1093/jisesa/ieae107

Bee cups 2.0: P-cups as single-use cages for honey bee (Hymenoptera: Apidae) experiments

Jay D Evans 1,, Zachary Lamas 2, Lindsey M Markowitz 3, Evan C Palmer-Young 4, Eugene V Ryabov 5,6, Dawn Boncristiani 7, Yan Ping Chen 8
Editor: Ellen Klinger
PMCID: PMC11576350  PMID: 39563067

Abstract

Honey bees and other pollinators face threats from pesticides, imperfect nutrition, and a diverse set of parasites and pathogens. Honey bees are also a research model for development, social behavior, microbiology, and aging. Tackling these questions requires a mix of in-hive and controlled laboratory experiments. We have perfected small-scale, inexpensive, disposable, and rearing arenas for honey bees that have proved useful for hundreds of bioassays with thousands of bees. We describe those arenas here, show their advantages over current hoarding cages, and provide videos demonstrating their many uses.

Keywords: Apis mellifera, bee pathology, virus, colony loss, toxicology

Introduction

Pesticides, disease, nutritional stress, and climate challenges provide genuine risks for honey bees and other pollinators, and research is needed to quantify and mitigate those risks. With a well-defined genome and decades of behavioral, disease, and ecological research, honey bees are also an important model species (Toth and Zayed 2021). Honey bee biology is rooted in their complex social structure, with a reproductive queen, thousands of female workers and, seasonally, reproductive males (drones). Consequently, key interactions between colony members, hive associates, food stores, and the hive environment take place at the level of entire colonies. However, detailed studies of honey bee biology often require reducing this hive environment to controlled, replicable, and economical units. At one extreme, honey bee biology can be studied at the levels of genes and their activities (Evans et al. 2013Morfin et al. 2023) or in assays using tissues or cells derived long ago from intact bees (Goblirsch and Adamczyk 2023). Many questions, however, must be answered using living bees that respond to challenges or stimuli. This need has led to the evolution of hoarding cages and chambers designed to host bees for days or weeks as part of experimental studies (Evans et al. 2009, Köhler et al. 2013, Kulincevic and Rothenbuhler 1975, Williams et al. 2013). Here we describe a significant advance in such contained experiments, namely, bee arenas built around single ‘Petri’ culture dishes that hold between 1 and 12 bees. In our hands, these ‘P-cups’ have performed well in thousands of bioassays, showing substantially reduced losses by accident, starvation, or escape of their subjects compared to current hoarding cages. They are also less costly and require a minimum of incubator and post-assay freezer space.

Experimental Design

Our primary criteria were that housing units be (i) single-use and sterile to avoid disease transfer, (ii) fully transparent to allow counting and measuring of bee behavior, (iii) inexpensive with widely available components, (iv) easily opened, and (v) favorable to bee health. Standard Petri dishes fit all these criteria. These dishes were outfitted with paper disks cut from standard office paper and sugar syrup feeders comprised of a single internal feeder (or multiple feeders for food choice experiments, i.e., Fig. 1A). The net cost per unit was less than $US 0.5 (ca. $0.2 for the Petri dish, $0.02 for paper, and from $0.02 to $0.3 for various feeding vessels). They are stackable, allowing a single ventilated incubator to house dozens of arenas (Fig. 1). Detailed protocols with variations are described below (also see https://www.youtube.com/@beeocene).

Fig. 1.

One image showing 18 dishes prepared for bees next to an image showing eight bees feeding in a dish.

A) P-cup with fondant feeders ready for bees. B) Worker bees engaged in a preference test of 3 nectar sources.

Needed Supplies

  • Laboratory incubator

  • Petri dishes (100 mm diameter × 15 mm high)

  • 16-ounce clear plastic cups with snap-on lids, e.g., Solo S-25883 cups with Solo S-25886.

  • 2.0 ml screw-cap centrifuge tubes, e.g., MTC Bio #C3172

  • 2-cm-thick plastic shim (we use 2 caps from 15 ml centrifuge tubes)

  • Office paper, e.g., SKILCRAFT Recycled Copy Paper, 92 Bright, 20 lb Bond Weight

  • Disposable Pasteur pipettes (e.g., Uline 5 ml transfer pipettes)

  • Caps for 15 ml centrifuge tubes

  • Icing fondant (available from specialty baking suppliers or beekeeping suppliers)

  • Sucrose syrup, 1:1 weight/volume with water or 2:1 w/v

  • Pollen (optional, use sterilized and locally sourced bee-collected pollen)

  • 40-quart Hobart mixing bowl (or similar steep-sided bowl to hold newly emerged bees while sorting into arenas)

  • Bleach solution

  • Pressurized CO2 canister, with regulator (for sedating older bees)

  • 4-liter stryofoam cooler (for sedating older bees)

Protocol

  1. Place frames (standard deep Langstroth frames, 24 cm × 482 mm) of emerging honey bees (Apis mellifera L.) inside a screened emergence box, then inside an incubator at 32 °C with high humidity (85%–90% RH) for 24 h.

  2. Shake the frame against a large metal mixing bowl to release newly emerged bees into a metal mixing bowl. The bowl is cleaned by wiping with 10% bleach between trials.

  3. Gently collect 2–3 bees at a time by hand (wearing lab gloves that are exchanged between trials) and drop into a Petri dish lined with a cut paper circle for a total of 8 bees/arena or as many as needed for replication (Supplementary Video S1, settingUpPCups.mp4).

Alternative 3.1: Inject individual bees with pathogens (e.g., viruses, Boncristiani et al. 2021) or feed multiple bees a pathogen suspension in feeder tubes.

Alternative 3.2: Bring adult worker bees or drones from the field by brushing into 16 oz. plastic cups with snap-on lids, ca. 150 bees/cup. Place 1 cup into a small styrofoam cooler and infuse with CO2 from a canister for 1 min, then wait for 90 s. While bees are briefly narcotized, swiftly pick the required number of bees by hand and place them into prepared arenas (Supplementary Video S2, Beecollection.mp4).

Alternative 3.3: Honey bee pupae can be removed directly from brood cells prior to eclosion and placed into arenas. Since the food is on the ground level, naïve bees can feed once they are active.

  • 4. For each cup, prepare a feeder by cutting the edge of a 15 ml centrifuge tube lid to form a gateway 1 cm across (Fig. 1A). Place a 1 g ball of icing fondant in this lid prior to placing bees in the arena.

Alternative 4.1: Place 4 ml of 1:1 w/v sucrose:water into a disposable Pasteur pipette bulb that was cut to leave a 2 mm flange at the bulb opening. Bulbs are filled by drawing syrup, pointing the bulb tip vertically to expel remaining air, and then drawing syrup again until full (Supplementary Video S1, SettingupPcups.mp4).

Alternative 4.2: To allow for accurate measurements of food consumption, place 1.5 ml of heavy syrup (2:1 w/v sugar/syrup) into a skirted 2.0 ml cylindrical microcentrifuge tube (Fig. 1B). Dishes can remain on the level and viscous syrup will remain in the tube. We also explored quantifying consumption using fondant feeders, by placing ca. 500 mg fondant in the detached lids of 1.7 ul microcentrifuge tubes, weighing these feeders before placing them into arenas housing 8 bees, and then weighing each feeder individually after a 7-day trial. Weight loss ranged from 52 to 348 mg across arenas (mean 182 mg), suggesting that, even with the confounding effects of dehydration, this is a viable method for measuring relative consumption.

  • 5. To better mimic the nutritional and microbial nutrition of newly emerged bees, a small (1 g) patty of pollen collected in a hive pollen trap, mixed 1:1 with sugar syrup (1:1 water sucrose v/w) can be placed in the arena.

  • 6. Place Petri dishes on large deli trays into the incubator. If using liquid feeders, align bulbs so the bulb opening faces one direction for all arenas on a tray, then add a 2 cm shim under the tray opposite the open-bulb side of plates to keep a slight downward slant for feeders (this can be achieved by using 2 caps from 15 ml centrifuge tubes, for example).

  • 7. Assess mortality daily (ideally) or as needed. Arenas with proper 4 ml feeders should maintain 8–10 bees for 10 days prior to feeder replenishment, while arenas with 1 g fondant will support the same number of bees for over 2 weeks, without any additional water.

  • 8. Remove any dead bees from each dish with forceps and then freeze arenas with live bees at −80 °C.

Alternative 8.1: To collect bees periodically for analysis, remove individual bees directly using forceps and place into 2 ml screw-cap tubes with glass beads and buffer (Supplementary Video S3, Collectingbeesfromcups.mp4).

Alternative 8.2: Place arenas with live bees and feeders into −80 °C freezer for 1 h. Take arenas out 3–4 at a time, and shake them horizontally, vigorously, 3–4 dishes at once, for 30 s. This will break bees apart and individual body parts (notably abdomens or heads in our case) can be pooled by forceps into a screw-cap tube for processing (Supplementary Video S4, Frozenshakingofbees.mp4). A total of 8 abdomens or 8 heads will fit into a 2 ml screw-cap tube with extraction beads and buffer.

  • 9. Process samples with suitable extraction methods (RNA, DNA, chemiluminescence, e.g., Evans et al. 2022) for desired post-trial data.

Results

To demonstrate survivability, frames of developing worker bees sourced from April 2024 were placed into an incubator at 32 °C with humidity bolstered by a 200 cm × 200 cm tray of water. Bees from 3 to 24 h of age were placed into 24 P-cup arenas holding fondant feeders (step 3). Arenas contained single bees (n = 8), 2 bees (n = 8 cups), 4 bees (n = 8 cups), or 8 bees (n = 8 cups). Bees were monitored daily for 12 days, after which a total of 4 bees (4/90 = 4.4%) had died (1 each from 2- and 4-bee arenas and 2 bees from one 8-bee arena).

In a second trial with fondant feeders, 196 bees were injected with a dose of 10^5 copies of the cloned deformed wing virus (Evans et al. 2022). At 96 h (when bees were collected for viral analysis) survivorship was 95.4% (187/196). Some of this observed mortality likely reflected the trauma of injection. Bees were active throughout and were easily counted directly on trays or photographed for future screening (Fig. 2).

Fig. 2.

One image showing 24 experimental P-cups with bees, next to image showing stacks of P-cups with bees inside an incubator.

A) Dishes of worker honey bees with fondant feeders. B) Stacked set of 120 P-cups with syrup feeders on a slight slant, as part of a bee medicines trial.

Discussion

When optimized, mortality in these arenas over the course of 4 or more days is lower than in cup studies with large cohorts of bees (e.g., Huang et al. 2014). We have successfully maintained bees in P-cups with both fondant and liquid feeders for 21-day trials, supplementing the liquid feeders every 5–7 days, while never supplementing the fondant feeders. Overall, the fondant method offered more stability, higher survivorship, and less maintenance than the liquid feeders, with some bees surviving 35 days without changing the feeder. Chief advantages of the P-cup protocol over existing hoarding cages are higher survival, the ease of counting and tracking bees, the ability to execute dozens of independent trials in a single incubator, and the low costs of readily available materials.

Supplementary Material

ieae107_suppl_Supplementary_Videos_S1
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ieae107_suppl_Supplementary_Videos_S2
Download video file (51.2MB, mp4)
ieae107_suppl_Supplementary_Videos_S3
Download video file (2.8MB, mp4)
ieae107_suppl_Supplementary_Videos_S4
Download video file (2.2MB, mp4)

Acknowledgments

We greatly appreciate the efforts of Kyle Grubbs in vetting and improving the described bioassays as part of their research. The authors declare no conflicts or competing interests.

Contributor Information

Jay D Evans, United States Department of Agriculture, Agricultural Research Service, Bee Research Laboratory, Beltsville, MD 20705, USA.

Zachary Lamas, United States Department of Agriculture, Agricultural Research Service, Bee Research Laboratory, Beltsville, MD 20705, USA.

Lindsey M Markowitz, United States Department of Agriculture, Agricultural Research Service, Bee Research Laboratory, Beltsville, MD 20705, USA.

Evan C Palmer-Young, United States Department of Agriculture, Agricultural Research Service, Bee Research Laboratory, Beltsville, MD 20705, USA.

Eugene V Ryabov, United States Department of Agriculture, Agricultural Research Service, Bee Research Laboratory, Beltsville, MD 20705, USA; Cell and Molecular Science, James Hutton Institute, Dundee, Great Britain.

Dawn Boncristiani, United States Department of Agriculture, Agricultural Research Service, Bee Research Laboratory, Beltsville, MD 20705, USA.

Yan Ping Chen, United States Department of Agriculture, Agricultural Research Service, Bee Research Laboratory, Beltsville, MD 20705, USA.

Author contributions

Jay Evans (Conceptualization [lead], Data curation, Funding acquisition [equal], Resources [lead], Validation [equal], Writing—original draft [lead]), Evan Palmer-Young (Investigation, Writing—review & editing [supporting]), Eugene Ryabov (Conceptualization, Writing—review & editing [supporting]), Zach Lamas (Conceptualization, Validation [supporting]), Lindsey Markowitz (Investigation, Writing—original draft [supporting]), Dawn Boncristiani (Conceptualization, Investigation, Validation [supporting]), and Yanping Chen (Conceptualization, Data curation [supporting], Writing—review & editing [equal])

Funding

This work was supported by United States Department of Agriculture (USDA)-Agricultural Research Service (ARS), USDA-NIFA Pollinator Health Grants #2020-67013-31861 (J. D. E. and Y. P. C.) and #2022-67013-36519 (J. D. E. and E. P. Y.), and a grant from the Eva Crane Trust (E. P. Y. and J. D. E.). None of the funders influenced the study or interpretations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ieae107_suppl_Supplementary_Videos_S1
Download video file (5.3MB, mp4)
ieae107_suppl_Supplementary_Videos_S2
Download video file (51.2MB, mp4)
ieae107_suppl_Supplementary_Videos_S3
Download video file (2.8MB, mp4)
ieae107_suppl_Supplementary_Videos_S4
Download video file (2.2MB, mp4)

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