Propagation in adult bees is best done with newly emerged bees, whose exoskeleton is still soft, making it easier for controlled injection. Using young bees also avoids any age-related variability in propagation and unintentional propagation of adult-acquired viruses.
1. Collect the bees in lots of 10-20 in queen cages, or similar containers.
2. Anaesthetize the bees for 1 minute with CO2 from a pressurized cylinder.
3. Make sure to bubble the CO2 gas through water to melt any CO2 micro-particles, which can be very injurious to bees. See the section 'Standard methods for immobilising, terminating, and storing adult Apis mellifera' in the BEEBOOK paper on miscellaneous methods (Human et al., 2013).
4. Proceed quickly to avoid excess anaesthesia for the bees and do not anaesthetize the bees more than once a day. Bees sometimes die from excess anaesthesia. This is especially important for infectivity tests, where the death of a bee is often a recorded experimental variable.
5. Inject 1~5 µl of virus suspension between the 2nd and 3rd integuments of the bee using a similar controlled-volume syringe set-up as for pupal propagation (see section 5.4.1.).
6. Incubate the inoculated bees in a hoarding cage at 30oC in 60-70% relative humidity with sufficient sterilized food and water (see the BEEBOOK paper on maintaining adult Apis mellifera workers in cages (Williams et al., 2013) for the appropriate amount of time for each virus (Table 3).
7. Include a control series with buffer-only inoculations, and a control series without inoculation, just incubation of the adult bees.