3.2.1.1. Maintaining mites on adult honey bees

Mites can be maintained on bees in hoarding cages (see section ‘Cages to keep bees in vitro in the laboratory’ in the BEEBOOK paper on maintaining adult bees in vitro (Williams et al., 2013)). A temperature of around 33 ºC and RH of 60-70 % is also adapted for mites. Mortality can be high in case phoretic mites are used. Their age is unknown at the time of collection and the variability in their life expectancy is therefore high. However, mites will commonly survive for 1 week or longer in bee cages established in this way. It is recommended using mite-tight cages or keeping each cage in a dish to avoid any escaped mites from entering another cage of a different treatment group. This procedure can be used in acaricide toxicity in vitro assays, in assays where the investigator is attempting to trace the movement of a compound (such as dsRNA) from a bee food (sugar water, pollen patties, etc.), through bees, and into mites, or in other similar assays.

 A common method for establishing in vitro studies with varroa-infested adult honey bees involves individually selecting worker bees carrying mites from combs/frames in colonies and placing them in cups or cages. This method is relatively time-consuming and can be particularly difficult for researchers with limited bee experience. An alternative method includes the separate collection of bees and mites followed by the infestation of the bees with the collected mites. Mites can be transferred onto the caged bees using a brush or, if mites were collected in a container, they can be introduced in a cage with bees. The mites readily spread across the bees within minutes. A primary benefit of the latter method is that it is feasible for a single, inexperienced experimenter to accomplish quickly. Additionally, this method obviates the need for maintaining colonies with high varroa populations (which are preferred when infested bees are chosen individually from frames, see section 4.6. ‘Breeding mites in colonies’).

Collecting bees and mites:

1.     Collect worker bees from frames and place in cages as described in section ‘Cages to keep bees in vitro in the laboratory’ of the BEEBOOK paper on maintaining adult bees in vitro (Williams et al., 2013)).

The number of bees placed in each cage can vary depending on experimental requirements.

2.     Collect mites using the methods described in section 3.1. ‘Collecting mites’.

In vitro infestation of bees with varroa:

3.     Add 5-10 mites to a small (5 cm) Petri dish containing a filter paper circle wetted with 1:1 sugar syrup (by volume) or water.

4.     Place the Petri dish with mites on the bottom of the cage containing worker bees.

5.     Tap the cage lightly against the laboratory bench or table to cause the bees to drop from the top of the cage to the bottom and contact the mites.

This artificial infestation procedure relies on the questing behaviour of mites, which readily attach to bees that contact them. The sugar syrup impregnated in the filter paper will keep the bees for an increased duration at the bottom of the cage to give more time for mites to find a host.

6.     Remove the Petri dish after all of the mites have attached to bees.

7.     Repeat steps 5-6 until the desired number of mites has been added to the cage. Partitioning the mite additions, rather than trying to add them all to the cup at one time, results in a more even distribution of mites on bees. Be aware that some bees will inevitably carry multiple mites and some bees will have no mites.

More mites should be added to each cage than are needed for the experiment. For example, add 30 mites to a cage when the experiment calls for the live recovery of 25 mites later in the study. This is necessary because some mites will fail to attach to any bee. Regardless, the number of varroa mites per cage or per bee can vary based on the predetermined experimental criteria.

8.     Add a sugar syrup feeder and water supply to the cage (see section ‘Cages to keep bees in vitro in the laboratory’ in the BEEBOOK paper on maintaining adult bees in vitro (Williams et al., 2013)) after the mites have been added.

Bees must stay hydrated and fed to ensure mite survival.

If keeping mites on brood is possible in Eppendorf tubes (see section 3.2.1.2. ‘Maintaining mites on honey bee brood’), keeping mites on adult bees works better in hoarding cages. Ventilation within the Eppendorf tubes is poor and the health of the bees kept in them decreases rapidly.