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Benefits of Propolis to Bee Health Final Report Marla Spivak University of Minnesota [email protected] We proposed two research objectives: 1) to test if there is a correlation between colony health and propolis collection in European-derived honey bees, and 2) to compare the immune systems of African- and European-derived colonies that have been enriched or deprived of a propolis envelope in the nest cavity. Objective 1. We placed propolis traps on colonies involved in long-term studies related to colony health: One set in colonies funded by the USDA- CAP study, and another a USDA study on the effects of high- and low-agricultural intensity landscapes on honey bee health in North Dakota. We could not collect data on propolis collection from ND study because the bees chewed up the mesh traps we (we tried a new type of mesh that clearly didn’t work). We did collect weights of propolis deposited in the traps in the USDA-CAP colonies, and found a correlation between colony population (frames of bees in colony) and amount of propolis deposited, but we are waiting for others in CAP team to finish analyzing colony health data before we can test if there is a correlation between propolis deposition and colony health. Thus, results are still forthcoming. Objective 2. We wanted to determine if the heavy propolis envelope in AHB colonies benefits the immune system as it does in European colonies. In our previous work, we found that individual bees in propolis-enriched colonies had lower abundance of at least two immune-related genes (meaning they were not expending energy on a costly immune response), and that this was likely due to a decrease in bacterial loads in the propolis-rich colonies (Simone et al, 2009, Evolution). This decreased physiological investment was the first clear evidence that the use of propolis acts as an external immune defense mechanism for bees. New graduate student, Renata Borba, from Brazil spent June and July 2011 at the Universidade de São Paulo in Ribeirão Preto, Brazil, in the labs of Dr. Ademilson Soares and Dra. Zila Simoes. She set up field colonies of Africanized bees that were made experimentally propolisrich or propolis-poor colonies, using propolis from Minnesota and green propolis from Brazil to enrich some colonies, and ethanol (the solvent for the propolis) for the control, propolis-poor colonies. She collected 7d old marked bees and extracted RNA for analysis of transcript levels of 7 antimicrobial peptides. Her data indicate that bacterial loads in colonies were low in both propolis-rich and poor AHB colonies. Specifically, the relative gene transcript levels for the general bacterial load (16S gene) in AHB colonies were the same in 7d old bees from the propolis-treated colonies compared to same-aged bees from the control colonies. Although was no significant difference was found in relative transcript levels of the antimicrobial peptide hymenoptaecin, we did a significant difference in transcript levels of AmEater, a gene involved in cellular immunity. In comparison with our previously published study, AHB appear to have lower bacterial loads in their colonies relative to European bees. But like in European colonies, propolis does benefit the immune system of AHB by decreasing costly investment in at least one antimicrobial peptide. Unfortunately, much of the RNA degraded in transit between Brazil and MN (the samples were maintained in RNAlater), so we did not have enough material to test other immune gene transcripts. This preliminary data warrants repeating on a larger scale, but all analyses will need to be conducted in Brazil to avoid having to transport the extracted RNA from bees. How are these findings applicable to beekeeping? We are finding that propolis within a bee colony is a vital component to bee health and immune system functioning. We are currently investigating whether the propolis must be a continuous envelope within the inner walls of the colony, or if it can be placed in a trap on top of the frames, or distributed in patches throughout the colony. If an envelope is critical, we can easily modify Langstroth boxes so that the lumber inside the boxes in unfinished, which will stimulate the bees to construct a natural propolis envelope. Figure 1. Colony of African bees in Brazil in which side walls of deep hive body were removed (slat by slat) to encourage bees to construct a thick ¼”- ½” wall of propolis. Bees leave entrance/ventiliation holes in the propolis envelope. These colonies comprised resin-rich colonies, in data below. Figure 2. Preliminary data on gene transcript levels of the 16S rRNA loci were measured as a generic indicator of the presence of eubacteria, normalized to the housekeeping gene actin. Six colony replicates were done for the two treatments. Sample size analyzed to date: control n=5 bees, propolis n= 14 bees. No significant differences determined by ANOVA (p = 0.75). Figure 3. Preliminary data on gene transcript levels for AmEater, a gene involved in cellular immunity, normalized to the housekeeping gene actin. Six colony replicates were done for the two treatments. Sample size analyzed to date: control n= 24 bees, propolis n= 32 bees. Significant differences determined by ANOVA (p = 0.014). Figure 4. Preliminary data on gene transcript levels for the immunopeptide hymenoptaecin normalized to the housekeeping gene actin. Six colony replicates were done for the two treatments. Sample size analyzed to date: control n= 30 bees, propolis n= 60 bees. No significant differences determined by ANOVA (p = 0.38).