4.2.3. Obtaining newly emerged workers for laboratory experiments by caging queens
Newly emerged workers can also be collected from pre-selected brood frames that queens were previously restricted onto.
To obtain newly emerged workers from a frame that the queen was caged onto:
- Identify suitable source colonies, as discussed in section 4.1, and brood frames, as discussed by Crailsheim et al. (2012) in the in vitro larval rearing paper of the BEEBOOK. A frame previously used for brood production that is relatively new (i.e. not containing dark, soiled comb) and has adequate empty cells is most suitable, and will likely contain fewer pathogens and environmental contaminants. A frame from the source colony will likely be most successful for rearing known age cohorts of workers; however, one from a different colony can also be used. Number of empty cells available for egg laying will be determined by the number of individuals needed for experiments. Brood mortality of approximately 20 % should be expected (Fukuda and Sakagami, 1986).
- Locate the queen in the source colony and gently place her on the chosen brood frame by grasping her wings. A clip queen catcher cage can also be used to move her. Refer to the BEEBOOK paper by Human et al. (2013) for handling honey bees. Ensure that at least a few hundred workers are on the frame before the queen is moved. These workers can either be ones that were on the frame originally or ones brushed from another brood frame in the same colony that contains open brood. This will serve to calm her and will lessen the chances that she runs or flies, or is crushed during caging.
- Carefully place the frame, containing the queen and workers, in a queen excluder cage (Fig. 1), and seal it, ensuring the queen is not crushed. See section 5.2.1 for discussions on minimizing pathogen and environmental contaminant exposure when using cages.
- Place the caged frame in the broodnest, preferably between two brood frames containing eggs and larvae (Fig. 2). This will improve chances that the newly-laid eggs are accepted by the colony. Refer to Human et al. (2013) in the miscellaneous methods paper of the BEEBOOK for estimating developing worker bee age.
- After a defined period of time, remove the frame from the queen excluder cage and place it, with eggs and the queen, back into the colony in its previous position. Mark the frame with a permanent marker or a coloured drawing pin to help locate it in the future. The number of honey bees required for experiments will determine the length of time the queen is confined to the frame. Queens typically lay between 5-35 eggs per hour (Allen, 1960), and frames can be checked every 24 hours to determine if enough eggs have been laid by inspecting cells through the queen excluder cage with the aid of a flashlight. Refer to the miscellaneous methods paper of the BEEBOOK by Human et al. (2013) for identifying eggs. It is possible that the queen will not begin egg laying until a few hours after initial isolation. Queens should not be confined to the frame for more than 72 hours, or when the availability of cells for egg laying is low, to avoid significant disruption of egg-laying in the colony. Homogeneity of age of newly emerged bees will also determine the length the queen is restricted to the frame, although this can also be controlled for during regular removal of newly emerged adults from the frame.
- Remove the frame 19-20 days after initial queen restriction, just prior to adult emergence (Winston, 1987). The frames can be removed later if egg laying was significantly delayed, but care must be taken to prevent workers from emerging in the colony. Although a worker will usually emerge from a cell 21 days after an egg was laid, development time can vary between 20-28 days depending on environmental conditions such as temperature and nutrition (Winston, 1987).
- The frame and newly emerged adults can be subsequently handled according to #5, 6, and 7 of section 4.2.2.
Fig. 1. A brood frame containing workers, the queen, and many empty cells is being inserted into a queen excluder cage. Slits between 4.3 and 4.4 mm wide allow worker movement to and from the frame, but restrict queen passage.