Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees (Apis mellifera)

Our protocol demonstrates how to produce honey bee virus particles en masse for their use in controlled, high-throughput bioassays for rapid screening of viral treatment effectiveness. The assay uses easy-to-culture bee pupae rather than cell culture, reducing the labor of obtaining experimental subjects. The virus can then be used to produce predictable mortality curves and repeatable bioassays.

There's been substantial interest in understanding how so-called honey bee viruses affect a larger host range. This virus production technique can be paired with other pollinator systems to test the infectivity. For mass bee extraction by larval self-removal, cage a honey bee queen on an empty, drawn-out Langstroth frame and return her to her colony.

After allowing the queen to lay eggs on the frame for 24 hours, check the frame to ensure that most of the comb cells contain newly laid eggs and release the queen. Exactly 192 hours after caging the queen, remove the frame from the colony and brush off any adult bees. Upon return to the laboratory, place the frame in an incubator set to the internal conditions of a hive and thoroughly clean containers of the same height and width of the larva-filled frame with sequential soap and water, bleach, and ethanol washes.

Line the bottom of the containers with several layers of paper towels and add several overlapping layers of thinner absorbent material. When the containers are ready, place the frame onto the container's focal larvae side down and use a piece of tented aluminum foil to cover the container and frame. After overnight incubation, carefully lift individual wipes from the top layer of each container to allow the larvae to be gently poured onto a transfer tray.

Then, use blunt, soft forceps to spread the larvae across the surface of the trays without touching and place the tray covered with tented foil into the incubator until the larvae pupate and mature to the wide-eye stage. For virus injection of the pupae, mix the virus particles of interest at the appropriate concentration in sterile PBS in a 15-milliliter tube and attach an injector apparatus to a manual multipipette. To test the injector efficacy, load the injector with 100 microliters of water and dispense the water in one-microliter doses.

If the injections are accurate, load 100 microliters of the virus particle solution into the injector and gently grasp one pupa along the thorax, applying just enough pressure to force internal fluids into the abdomen to make the tergite divisions more apparent. Stabilizing the arm holding the syringe against the lab bench for support, insert the needle just into the cuticle between the third and fourth abdominal tergites and inject one microliter of virus solution. When all the pupae have been injected, re-cover the tray with tented foil before returning the pupae to the incubator for another three to five days.

At the end of the incubation, pool the pupae by colony source in individual 50-milliliter conical centrifuge tubes and vortex to homogenize the contents. To perform a viral feeding bioassay, plug the feeder holes of clean cages with an appropriately sized centrifuge tube and partially fill 15-milliliter centrifuge tubes with freshly prepared feeding solution. Then, invert the tubes and use a thumbtack to poke one to two holes around the tips.

Next, brush all of the newly emerged bees from their emergence boxes into a collection receptacle lightly coated around the edges with vegetable oil and gently mix the bees by hand. Transfer 35 individual bees into a single, greased cup and place the contents of the cup into an acrylic cage. To prepare the virus inoculum, mix an appropriate quantity of thawed, concentrated virus particles with 30%sucrose solution in one sterile container per treatment type and add 600 microliters of virus solution onto individual inoculum trays.

Then, carefully insert each inoculum tray into their corresponding cages, taking care to not accidentally release any bees. When all of the inocula have been consumed, replace the centrifuge tubes from the top feeder hole with the prepared feeder tubes and record the mortality within each cage at 12 hour intervals for the first 72 hours of the experiment and every 24 hours thereafter. To remove dead bees, slide the cage door up just far enough to allow a pair of alcohol-sterilized forceps to be inserted to scoop out the dead bees.

Depending on the design of the experiment, place live specimens from each cage into individual centrifuge tubes on dry ice to allow sampling for viral titer measurements. In this representative larger-virus harvesting effort, every pupa was initially injected with an approximately 95%Israeli acute paralysis virus inoculum. Although 10 out of the 16 colony samples involved in these extractions contained highly pure virus, other samples varied in their virus proportion, with some even being dominated by other viruses, such as deformed wing virus.

As illustrated in the table, threshold cycle values can be used as a predictor of proportion. Comparing the dose response-survival curves of honeybees fed the same virus particles during two different years reveals that, despite identical testing parameters, the trials conducted in 2018 and 2019 produced noticeably different survival responses in all but the control treatment groups, which received no virus in their sucrose inoculum. Honeybee responses to viral infection can vary greatly depending on the season, hive, or even inoculum distribution within individual cages.

Therefore, large sample sizes are critical for confirming treatment effects. Using stocks of virus particles, the experiment can be expanded to include injections of adult honeybee workers, queens, or drones, or to other species of pollinators to assess the host range. Infectious stocks of virus can be used to test how the infection interacts with other factors, like diet, chemical exposure, or social environments.