1. Obtaining Beebread from AHB and EHB Colonies

1. Place pollen traps on honey bee colonies and collect pollen. Grind the pollen into a fine powder (similar to pollen shed from anthers) using a coffee grinder.
2. Establish 5 colonies each of AHB and EHB in an enclosed flight area (EFA) so that bees forage only on the pollen provided. To prevent workers from drifting between EHB and AHB colonies, divide the EFA into separate sections so that bees cannot cross between them. Place individual EHB or AHB colonies with 3,500-4,000 worker bees, wax comb with nectar, honey, immature brood and empty comb in each section of the EFA.
   NOTE: The colonies do not have stored pollen when established. The rate that pollen is stored can be increased by not including a laying queen in the colonies.
3. Feed ground pollen to colonies by placing a tray with the pollen in each section of the EFA. Spreading about 60 g of pollen on each tray so that foraging bees can collect it as corbicular loads and store the pollen in their colonies as beebread. Continue providing fresh pollen on each tray daily for 3 weeks.
4. Refer to beebread from the European colonies as European beebread (EBB), and from Africanized colonies as Africanized beebread (ABB).

2. Feeding Bees in Cages

1. Place frames of sealed worker brood from AHB and EHB colonies in separate emergence cages in an environmental room set at 32-34 °C and 40% relative humidity..
2. When the workers emerge and are about 24 hr old, establish 12 Plexiglas bioassay cages (dimensions = 11.5 x 7.5 x 16.5 cm³) and add either 100 newly emerged EHB or 100 newly emerged AHB worker bees to each cage. Place a section of comb with a known number of either EBB or ABB cells (24-30 cells per cage) in each cage to generate the following treatment combinations: AHB fed ABB, EHB fed ABB, AHB fed EBB and EHB fed EBB. (4 treatments; 6 cages per treatment; 24 cages in total).
3. Add vials of water and a 50% honey and water solution formulated by volume to each cage. Refill the honey and water vials daily for the 11 day study period.

3. Sampling Worker Bees and Beebread and Estimating Consumption

1. Sample 10 newly emerged EHB and AHB workers prior to placing them in the cages. Refer to these as Day 0 bees and have them serve as a baseline for hemolymph protein concentrations.
2. Remove 10 bees from each cage after they fed on EBB or ABB for 4, 7, and 11 days.
3. Place the live bees in individual microcentrifuge tubes and set on ice packs. Select a subsample of four bees for analysis of hemolymph protein concentration.
4. After sampling bees on Day-11, count the number of comb cells that still contain beebread. This is a relative measure of beebread consumption.
5. Remove the remaining beebread from the cells in each cage and store in separate microcentrifuge tubes according to cage. Keep the beebread samples at -80 °C until analyzed for pH, soluble protein concentration, and amino acid content.

4. Estimating pH of Pollen and Beebread

1. Take six random 0.3 g samples of the pollen fed to bees in the EFA and dissolve it in 300 µl of distilled water. Measure the pH using a waterproof double junction pH spear with an accuracy of +0.01.
2. Take 0.3 g sample of beebread that remained after the 11-day feeding period in each cage. Dissolve the beebread in 300 µl of distilled water and measure pH as described for pollen (4.1).

5. Protein Analysis

1. Take six samples of the pollen and a sample of EBB and ABB from each cage. Store samples at -20 °C until analyzed for soluble protein concentration.
2. Mix 20 mg of either pollen or beebread with 1,000 µl of 0.1 M phosphate buffer solution (PBS).
3. Vortex the mixture for 10 sec and centrifuge at 571.2 x g for 1 min.
4. Remove a 10 µl sample of the supernatant and place in wells of a 96 well flat bottom EIA/RIA polystyrene plate. Replicate each sample in three wells.
5. Draw hemolymph from bees collected from each cage by inserting a 20 μl capillary tube (that had been heated and pulled to a needle-sharp point) into the right lateral portion of the thorax near the point of attachment of the wings. Collect additional hemolymph, if needed, by inserting the same tube into the membrane between the abdominal tergites.
6. Add 1 μl of hemolymph to 9 μl of 0.1 M PBS. Store the hemolymph solution at -20 °C until analysis for soluble protein.
7. Determine total soluble protein concentrations in pollen, beebread, and hemolymph samples using a commercial Bradford protein assay kit. Follow the manufacturer’s instructions.
8. Establish a standard curve to estimate soluble protein concentration in the samples by measuring protein absorbance with known protein concentrations in bovine serum albumin (BSA). Measure protein absorbance at 595 nm using a spectrophotometer.

6. Amino Acid Analysis

1. Pool individual samples from comb cells of each colony to create a representative sample of EBB and ABB for analysis.
2. Take a 50 mg pollen or beebread sample weighed into autosampler vials, and add 1 ml of distilled water to the vial, along with 100 µl of a 50 ng/μl internal standard solution consisting of d₄-alanine, d₂₃-lauric acid, ¹³C₆-glucose and d₃₉-arachidiac acid.
3. Cap the sample and sonicate for 5 min.
4. Condition an HLB cartridge by adding 1 ml of methanol, equilibrated by adding 1 ml of distilled water followed by the addition of 1 ml of the beebread or pollen sample. Wash the cartridge with 1 ml of 5.0% MeOH/H₂O and elute with 1 ml of 80% MeOH/H₂O.
5. Evaporate the sample to dryness under a stream of nitrogen. Reconstitute the sample with 50 µl of Pyridine and 100 µl of N,O-Bis (trimethylsilyl)trifluoroacetamide + Trimethylchlorosilane (BSTFA + TMCS).
6. Cap and incubate the sample at 70 °C for 30 min.
7. Allow the sample to cool and transfer it to a clean autosampler vial.
8. Cap and place the sample into a Mass Selective Detector interfaced to a gas chromatograph to analyze the samples both for volatile compounds and organic acids. Separate the sugar and organic acids following TMS derivatization with BSTFA + TMCS using a column (30 m x 0.25 mm i.d.) with a 1.0 µm film thickness.
9. Set the column oven at 50 C for 2 min, then increase the temperature linearly to 290 °C at 5 C/min. and hold for 7 min. Set the GC injector and GC/MS interface to 250 °C and 290 °C, respectively.
   1. Use Helium as a carrier at a flow rate of 1.0 ml/min. Set the MS source temperature to 230 °C.
10. Tune and calibrate the mass spectrometer daily with Perfluorotributylamine (PFTBA). Use a 1 µl injection of PFTBA in the full scan (35-700 amu) positive ion mode to obtain data on the presence and concentrations of amino acids.