Rearing Honey Bee Larvae for Developmental Studies

Worker larvae were obtained from the honey bee (Apis mellifera ligustica) colonies at the Honey Bee Research Facility, School of Life Sciences, Arizona State University, Mesa, Arizona. Queens were confined to a fully drawn comb in an excluder cage (46 × 24 × 6 cm) as described by Peng et al. (1992 ). On the fifth day, the bees were shaken off the comb and the comb was brought into the grafting room to obtain 1.5–2 day old larvae.
Seven different larval diets were prepared by changing the sugar and water concentrations (Table 1). Sugars and the yeast extract were dissolved in distilled water and freshly thawed commercial royal jelly purchased from a local bee supply company was added to the mixture and mixed thoroughly on a shaker. The diets were divided into 2 ml centrifuge tubes and kept at -18° C in a freezer until they were used. The diets were thawed and brought to 34° C in a water bath just before feeding.
A total of 350 larvae were grafted; there were 7 treatment groups, 5 replicates, and 10 larvae in each replicate. The first day 5 aliquots of 200 mg food were placed in a polyethylene Petri dish (100 × 15 mm) and 10 larvae were grafted on each aliquot (Figure 1A). The Petri dishes were placed into a polyethylene tub (20 cm × 40 cm) containing 16% sulfuric acid, transferred into a humidity chamber, and kept there at 34° C and 90% RH. On the second day, 40 mg and the 3^rd day 80 mg of larval food/larvae were placed in new Petri dishes and the larvae were gently placed on top of the fresh food (Figure 1B). On the 4^th day, 120 mg and on the 5th day 180 mg of food/larvae was placed in Petri dishes and the larvae were transferred onto the food. On the 6th day larvae consumed most of the food, and they started depositing uric acid crystals on the dorsal side of the body. When uric acid crystals were observed, the larvae were removed from the feeding dishes, weighed, and transferred to a 100 × 15 mm Petri dishes lined with Kimwipes® tissue paper (Figure 1C).
The next day the old Kimwipes® tissue paper containing feces was removed and the larvae were gently transferred onto a clean tissue paper and kept at 34° C and 70% RH in the humidity chamber. At the end of the defecation stage larvae started spinning cocoons, and this was recorded as the spinning stage (Figure 1D). Dead or undeveloped pupae were removed from the Petri dishes, and pupae (Figure 1E) were kept in the humidity chamber until they completed development and became adults (Figure 1F). Bees were removed from the Petri dishes as soon as they become adults, weighed, inspected under a stereo microscope, and dissected to count the ovarioles.
The adult bees were classified as queen phenotypes if they completed the development in 15–16 days, had notches on the mandibles, curved stings, large spermathecae (1mm in diameter), and had no corbiculae; as worker phenotypes if they completed the development in 21–22 days, had rows of corbicular hairs, straight stings with barbs, and had mandibles without the notches; and as intercastes if they completed development between 17–20 days, had small notches on the mandibles, and/or undeveloped corbiculae.
Hive-reared A. mellifera served as controls. The brood comb from which larvae were grafted was removed from the colony 20 days after caging the queen and placed in an incubator. The next day newly-emerged bees were sampled, weighed, and dissected for ovariole counts.