Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

The overall goal of the following experiment is to quantify nutrients in pollen and bee bread, made by two honeybee subspecies and measure consumption of protein titers. This is achieved by presenting European and African bees with the same pollen source in a confined area and allowing the bees to store the pollen in their colonies as bee bread. Next, the bee bread is fed to newly emerged European and African bees in cages.

Then bees in the cages are sampled after four, seven, and 11 days of feeding, and the amount of bee bread remaining in each cage is measured to estimate consumption. Finally, the concentration of nutrients, including protein and amino acids in the pollen and bee bread made by European and Africanized bees are measured and compared between the races of bees. The results show that bee bread differs from pollen in protein and some amino acid concentrations, as well as hemolymph protein titers.

This method can help answer key questions in studies of honeybee nutrition, such as the role of pollen source on the nutrients in bee bread and how those nutrients are acquired by bees of different subspecies braces and genotypes. Demonstrating the techniques is Emily Watkins de Young, who is a student technician in my laboratory To begin after placing pollen traps on honeybee colonies, collect the pollen, use a coffee grinder to grind the pollen into a fine powder, establish five colonies, each of African honeybees or a HB and European honeybees, or EHB in an enclosed flight area or EFA, so that bees forage only on the pollen provided to prevent workers from drifting between EHB and A HB colonies. Divide the EFA into separate sections so that bees cannot cross between them into each section of the EFA place individual EHB or a HB colonies with 3, 500 to 4, 000 worker bees wax comb with nectar honey, immature brood, and empty comb feed ground pollen to colonies by placing a tray in each section of the EFA and spreading approximately 60 grams of pollen onto the trays.

In this way, forging bees can collect the pollen as curricular loads and store it in their colonies as bee bread to feed bees in cages. Place frames of the sealed worker brood from a HB and EHB colonies in separate emergence cages in an environmental room set at 32 to 24 degrees Celsius and 40%relative humidity. When the workers emerge in our approximately 24 hours old, it established 12 plexiglass bioassay cages and add either 100 newly emerged EHB or a HB worker bees to each cage, place a section of comb with a known number of either European bbr or EBB or Africanized B bread or A B, B cells in each cage to generate the following treatment conditions.

A HB fed A-B-B-E-H-B fed A B, BAHB Fed EBB and EHB Fed. EBB add vials of water and a 30%sucrose solution formulated by volume to each cage. Replace the sugar syrup and water vials daily for the 11 day study period.

Sample 10, newly emerged EHB, and A HB workers prior to placing them in the cages. Refer to these as day zero Bs and have them serve as a baseline for hemolymph protein concentrations. Remove 10 bees from each cage after they have fed on EBB or a B, B for four, seven, and 11 days.

Place the live bees on individual falcon tubes and set on ice packs. Select the subsample of four bees for analysis of hemolymph protein concentration after sampling bees on day 11, count the number of comb cells that still contain bee bread. Remove the remaining bee bread from the cells in each cage and store in separate micro centrifuge tubes by cage.

Keep the BBR samples at negative 80 degrees Celsius until analyzed for pH soluble protein concentration and amino acid content. To carry out protein analysis, take six samples of the pollen and a sample of EBB and A BB from each cage. Mix 20 milligrams of either pollen or bbr with one milliliter of 0.1 molar PBS vortex to mixture for 10 seconds and centrifuge at 571.2 G for one minute.

Remove a 25 microliter sample of the S supernatant and place it in a well of a 96 well flat bottom, E-I-A-R-I-A polystyrene plate. Replicate each sample in three wells. Heat the end of a 20 microliter capillary tube and pull the heated section until a needle-like point is formed.

Then draw hemolymph from bees collected from each cage by inserting the capillary tube into the right lateral portion of the thorax near the point of wing attachment. Collect additional lymph if needed by inserting the same tube into the membrane between the abdomen tur guides. Add one microliter of hemolymph to nine microliters of 0.1 molar PBS.

Use a Bradford protein assay kit and follow the manufacturer's instructions to determine the total soluble protein. Concentrations carry out amino acid analysis according to the text protocol as shown in these graphs, both the pH value and protein concentration were lower in bbr than in pollen. Both the EHB and A HB consumed more A BB than EBB as demonstrated here.

Levels of soluble protein in the hemolymph of A HB were significantly higher than EHB regardless of the type of bbr they consumed. These differences in hemolymph protein levels occurred even though EHB and A HB consumed similar amounts of each type of bbr. Of the 10 amino acids that were essential for honeybees, all but histamine were detected in the pollen.

In most cases, amino acid concentrations measured in bee bread were higher than in the pollen. For example, concentrations of leucine and thionine were about 60%higher in bbr compared with pollen and valine concentrations were about 25%higher. Alanine aspartic acid glutamine in levels also were higher in bbr than in pollen.

Amino acid concentrations did not defer greatly between A B, B and EBB with the exception of phenylalanine and cystine. Phenylalanine levels were about twice as high in A BB compared with either EBB or pollen. Cystine concentrations were lower in EBB compared with A, B, B or pollen.

After watching this video, you should have a good understanding of how to evaluate protein concentrations in bee bread, pollen, and honey bee hemolymph. With these techniques, you can determine if food storage and consumption and hemolymph protein concentrations are affected by genetic or environmental factors.