Bee cups 2.0:  P-Cups as single-use cages for honey bee experiments

doi:10.21203/rs.3.pex-2506/v1

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Bee cups 2.0: P-Cups as single-use cages for honey bee experiments

https://doi.org/10.21203/rs.3.pex-2506/v1

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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, rearing arenas for honey bees that have proved useful for hundreds of bioassays with thousands of bees. We describe those arenas here and provide links to videos demonstrating their many uses.

Environmental chemistry

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. Honey bee biology must remain rooted in their complex social structure, with a reproductive queen, thousands of female workers and, seasonally, reproductive males (drones). Interactions between colony members, hive associates, food stores, and the hive environment are critical for biology and colony fitness. However, detailed studies of key aspects of honey bee biology 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, in assays using tissues or cells derived long ago from intact bees. 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, Kohler et al., 2013, Kulincevic and Rothenbuhler, 1975, Williams et al., 2013). Here we describe a significant advance in such contained experiments, namely bee cups built around single ‘Petri’ culture dishes that hold between one 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.

As with the bee cups described by Evans et al. (2009), and vetted successfully by Huang et al. (2014), our primary criteria were that housing units be 1) single-use and sterile to avoid disease transfer, 2) fully transparent to allow counting and measuring of bee behavior, 3) inexpensive with widely available components, 4) easily opened, and 5) favorable to bee health. Standard 100mm x 15 mm plastic 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, Figure 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 tubes). They are stackable, allowing a single ventilated incubator to house dozens of arenas (Figure 1b). Detailed Methods with variations are described below and at the YouTube site https://www.youtube.com/@beeocene.

1. Establish frames of emerging bees in the incubator at 32^oC with high humidity for 24 hours.

2. Shake frame into a large metal mixing bowl to release newly-emerged bees into the bowl.

3. Gently collect 2-3 bees at a time by hand 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 (
https://youtu.be/i8CLniG6PL4).

4. Randomize plates to avoid pre-trial time differences, or source differences (if multiple colony sources are used, these should be mixed fully before dividing bees among trays).

5. Alternative 1: Inject individual bees with pathogens or feed multiple bees a pathogen suspension in feeder tubes.

6. Alternative 2: Bring adult worker bees or drones from the field by brushing into 16 oz. plastic cups, ca. 100 bees/cup. Place one cup into a small styrofoam cooler and infuse with CO₂ from a canister for 1-2 minutes. While bees are briefly narcotized, swiftly pick required number of bees and place into prepared arenas (https://youtu.be/QaBzpEs_9QU).

7. For each cup, place 4 ml of 1:1 w/v sucrose:water into a disposable Pasteur pipette bulb, cut to leave a 2 mm flange at the bulb opening. Bulbs are filled by drawing syrup, pointing bulb tip vertically to expel remaining air, then drawing syrup again until full (https://youtu.be/i8CLniG6PL4).

8. Alternative 1: Align bulbs so the bulb opening faces one direction for all arenas on a tray, then add a 3 cm shim under opposite side of plates to keep a slight downward slant for feeders, if feeders are meant to provide food for greater than four days.

9. Alternative 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 2.0ml cylindrical microcentrifuge tube (Figure 1A).

10. Place petri dishes on large flat deli trays into incubator at 32^oC, with humidifier or shallow tray of water.

11. 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.

12. Remove any dead bees from each dish with forceps and then freeze arenas with live bees at -80^oC, on deli tray and with feeding bulb in place.

13. Alternative 1: To collect frozen bees for analysis, remove survivors from arenas into 15 ml tubes as a group, then place back immediately back at -80^oC.

14. Alternative 2: Remove individual bees directly into 2 ml screwcap tubes with glass beads and buffer (https://youtu.be/V-K1a7SGjU0).

15. Alternative 3: Place arenas with live bees and feeders into -80^oC freezer for one hour. Take arenas out 3-4 at a time, and shake them horizontally, vigorously, 3-4 dishes at once, for 30 seconds. This will break bees apart and individual body parts (notably abdomens or heads in our case) which can be pooled by forceps into a screwcap tube for processing (https://youtu.be/VsOL8Cp4Su0). A total of 8 abdomens or 8 heads will fit into a 2 ml screwcap tube with extraction beads and buffer.

16. Process samples with 1) TriZol for RNA extraction (in which case buffer for beads or pestle disruption is from a commercial source, or homemade Guanidine thiocyanate buffer as in Evans et al., 2023) or 2) 500 ul 1X phosphate-buffered saline if destined for nano-luc quantification of virus loads or 3) any suitable DNA extraction buffer.

Evans, J.D.; Chen, Y.P.; Prisco, G.D.; Pettis, J.; Williams, V. Bee cups: Single-use cages for honey bee experiments. Journal of Apicultural Research 2009, 48, 300-302, doi:10.1080/00218839.2009.11101548.

Evans, J.D.; Schwarz, R.S.; Chen, Y.P.; Budge, G.; Cornman, R.S.; De la Rua, P.; de Miranda, J.R.; Foret, S.; Foster, L.; Gauthier, L. Standard methods for molecular research in Apis mellifera. Journal of Apicultural Research 2013, 52, 1-54.

Huang, S.K.; Csaki, T.; Doublet, V.; Dussaubat, C.; Evans, J.D.; Gajda, A.M.; Gregorc, A.; Hamilton, M.C.; Kamler, M.; Lecocq, A.; Muz, M.N.; Neumann, P.; Özkirim, A.; Schiesser, A.; Sohr, A.R.; Tanner, G.; Tozkar, C.Ö.; Williams, G.R.; Wu, L.; Zheng, H.; Chen, Y.P. Evaluation of cage designs and feeding regimes for honey bee (Hymenoptera: Apidae) laboratory experiments. Journal of Economic Entomology 2014, 107, 54-62.

Köhler, A.; Nicolson, S.W.; Pirk, C.W.W. A new design for honey bee hoarding cages for laboratory experiments. Journal of Apicultural Research 2013, 52, 12-14.

Kulincevic, J.M.; Rothenbuhler, W.C. Selection for resistance and susceptibility to in the honey bee. Journal of Invertebrate Pathology 1975, 25: 289-295.

Williams, G.R.; Alaux, C.; Costa, C.; Csáki, T.; Doublet, V.; Eisenhardt, D.; Fries, I.; Kuhn, R.; McMahon, D.P.; Medrzycki, P.; Murray, T.E.; Natsopoulou, M.E.; Neumann, P.; Oliver, R.; Paxton, R.J.; Pernal, S.F.; Shutler, D.; Tanner, G.; Van Der Steen, J.J.M.; Brodschneider, R. Standard methods for maintaining adult Apis mellifera in cages under in vitro laboratory conditions. Journal of Apicultural Research 2013, 52.

This work was supported by USDA-ARS, USDA-NIFA Pollinator Health Grants #2020-67013-31861 (JDE and YPC) and #2022-67013-36519 (JDE and EPY), and a grant from the Eva Crane Trust (EPY and JDE). We greatly appreciate the efforts of Kyle Grubbs and Eugene Ryabov in vetting and improving the described bioassays as part of their research. The authors declare no competing or conflicting interests.

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Bee cups 2.0: P-Cups as single-use cages for honey bee experiments

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