4.2. Caging of queen
- Cage queen on an empty brood comb
- Confirm oviposition by visual inspection after few hours
- 72 hours after
confirmation of eggs --> Section 5.5. Grafting of
The developmental stages of honey bee larvae exhibit different susceptibilities towards pesticides and pathogens, and larvae should be exposed to pathogens or toxins in experiments at biologically relevant stages (Davis et al., 1988). If honey bees are to be reared from the very early larval stages, first instar larvae are needed. Two definitions of age are known: the biological age (larval stage) and the chronological age. To differentiate these two morphological parameters need to be investigated. During their development, honey bee larvae undergo five instars, and each instar lasts a different length of time (Bertholf, 1925). Head diameter (Rembold et al., 1980) and the developmental stages of the mouthpart, wing buds, leg buds and the gonapophyses (see drawings in Myser, 1954 and the BEEBOOK paper on miscellaneous research methods (Human et al., 2013)) are proper parameters to characterize each of the five instars. As the first instar stage lasts 14-20 hours, one should be aware that a larva that has reached the age of one day (24 hours post hatching from the egg) already has reached the second instar (Bertholf, 1925; Rembold et al., 1980). Larval age can also be determined by weighing (Rembold and Lackner, 1981; Vandenberg and Shimanuki, 1987; Davis et al., 1988). Weight of larvae can be confirmed using data of Wang (1965) and the BEEBOOK paper on miscellaneous research methods (Human et al., 2013): 12 hour old larvae have on average a weight of 0.36 ± 0.024 mg but this may depend on honey bee race. However, weighing will delay grafting and increase the risk of contamination and mortality.
Age of first instar larvae for grafting (for the grafting method, see the BEEBOOK paper on queen rearing (Büchler et al., 2013) is usually determined chronologically, and larvae younger than 12 hours after hatching are safely first instars. Chronological age can be controlled by caging the queen on a brood comb with a queen excluder cage (for information on obtaining brood and adult bees of known ages, see the BEEBOOK paper on miscellaneous research methods (Human et al., 2013)). Queens do not start oviposition immediately after caging, so onset of egg-laying has to be confirmed. It has to be taken into account that larvae hatch from eggs 66 to 93 hours after oviposition (Collins, 2004), hence general time schedules based on 72 hours need to be confirmed for every trial. We recommend that the queen lays eggs in large areas of the comb; larvae of the same age are usually found in ring-like areas on the comb. Releasing the queen from the excluder cage afterwards is important only for colony development and a fixed time cannot be given, as this strongly depends on the queen.
Instead of grafting, larvae can also be obtained using plastic queen cups mounted on commercially available artificial combs (Cupularve Nicotplast, France). On day four after caging the queen and depending on larval hatching, plastic cups containing first instar larvae can quickly be collected (Hendriksma et al., 2011a).