From Hive to Lab: Could “P-Cup” Arenas Redefine Honey Bee Research?

Pesticides weaken honey bees’ immune systems, diseases spread rapidly through hives, and poor nutrition from dwindling wildflower habitats leaves bees vulnerable. Add to this the unpredictable swings of a changing climate, and the challenges facing honey bee populations become clear. At the same time, researchers turn to these industrious insects for insights into social behavior, aging, and microbiology, using their intricate hive dynamics as a window into larger biological systems.

However, conducting controlled and replicable experiments on honey bee (Apis mellifera) biology presents researchers with many challenges, especially due to bees’ complex social structure and simultaneous interactions within each hive. While researchers often use tools like hoarding cages and chambers to house bees during experiments, these methods often fall short in terms of cost, efficiency, and ensuring bee survival.

Researchers in the Bee Research Laboratory at the United States Department of Agriculture’s Agricultural Research Service in Beltsville, Maryland, recently developed a small-scale, inexpensive, and disposable rearing arena for honey bees that improves upon existing hoarding cage methods. As detailed in a report published in November in the open-access Journal of Insect Science, the researchers developed “P-cup” arenas and performed various tests to compare their design and effectiveness to traditional hoarding cages.

Using 100 millimeter Petri dishes as tiny, transparent, single-use arenas for housing honeybees, the team equipped each “P-cup” with a paper disk, sugar syrup feeders, and sometimes fondant feeders to support bee nutrition. “There have been lots of designed houses for honey bees, scaling down from full-sized hives to wooden and, lately, plastic arenas like [the ones we used in the study],” says Jay Evans, Ph.D., a lead scientist in the Bee Research Laboratory at the USDA-ARS. “We mainly wanted a sterile space since we were studying disease.” The team also wanted a home that could confine the bees at a typical density, even when they were following just a few bees at once.

The experts collected newly emerged bees from frames placed in an incubator. They transferred the bees into the Petri dishes by hand, ensuring sterile conditions between trials. They also provided the bees with either fondant (for longer-term feeding) or sucrose syrup (for controlled feeding experiments). The team kept the arenas in an incubator to maintain appropriate temperature and humidity levels.

While other researchers have used disposable cups for years, the ones the team developed were much smaller and had a higher throughput. But, according to Evans: “Our best breakthrough was in the feeders. Feeders on the roof of traditional hoarding cages can be inaccessible to some bees and can either over-drip or cavitate and run dry. These are basically two-dimensional arenas, so all the bees can reach the food, even newly emerged bees or bees that are less healthy.”

The researchers injected some bees with pathogens or used field-caught bees that they briefly sedated with carbon dioxide before sorting them into arenas. They monitored the bees’ health and recorded mortality daily. After the trial period, the team processed and analyzed the bees for further study using RNA, DNA, or chemiluminescence techniques.

The study’s findings showed that the P-cup system significantly improved bee survival rates and experimental efficiency. In trials with newly emerged bees, just over 4 percent died over 12 days, a much lower mortality rate than researchers have previously seen in traditional setups. This finding suggests the system has strong viability for long-term studies.

Even when injected with the deformed wing virus (DWV), over 95 percent of bees survived for at least 96 hours, showing that the P-cup system supports robust bee health despite experimental stressors. Evans says he was surprised by how long the bees lived, noting that even small groups survived without additional water sources. He suggested that future studies incorporate elements like a “jungle gym” to further assess the impact of viral infections on behavior.

Researchers at the United States Department of Agriculture’s Agricultural Research Service have developed a small-scale, inexpensive, and disposable rearing arena for honey bees—using simple Petri dishes—that improves upon existing methods. (Video originally published supplemental to Evans et al 2024, Journal of Insect Science)

The P-cup system also had practicality perks. First of all, the fondant feeders outperformed liquid feeders, keeping bees alive for weeks without maintenance and eliminating the risk of messy leaks. Researchers also had the chance to streamline experiments by running multiple trials in small incubators, effortlessly studying temperature effects on disease. Evans also highlights the setup’s value for testing new bee medicines, enabling cost-effective, high-replication trials with ease. The team has already used them to screen hundreds of possible antiviral drugs.

“These studies will probably always have to be followed up with ‘real-colony’ studies given the complexities of nutrition and social interactions in large colonies,” says Evans. “But these approaches will certainly play a role in screening for the factors that have big effects on bee health, from pesticides to viruses, and for monitoring behavior interactions among workers in a way that can be recorded and repeated.”

The team is exploring automated methods to track long-term bee behaviors in P-cup arenas to study the effects of virus infections, building on advances from colleague Zachary Lamas, Ph.D., a researcher in Evans’ lab, and work at the University of Illinois Bee Lab. “While our benign virus assay did not cause mortality, we have been able to start using these to measure mortality caused by a more virulent paralysis virus and they are easily monitored daily for dead and dying bees,” says Evans. “We think these arenas will be a good addition to replicated studies of pesticide and disease impacts.”