Effects of Propolis use on honey bee health Becky DeValk, Kiana Dusek, Mikayla Hanrahan, Megan Poulos, (Biology 160 students), Brad Mogen and Kim Mogen 1 (Biology mentors) 1 University of Wisconsin-River Falls, WI Introduction Research has shown that an increase of propolis production in Apis melifera, (the honey bee) hives have a beneficial effect on the honey bee health. Bees produce propolis to fill in the cracks and crevices in their hives and to also keep out predators. But, as well as making their hive smoother and keeping intruders out, it is said to have antibacterial properties. “Research shows that propolis exhibits significant antibacterial and antifungal (and most of them antiviral) activity [1].” In this experiment, there were three types of hives used. One type was a control hive, the second type had a propolis trap and the third type had a propolis envelope. A propolis trap covers the top of the hives and stimulates the bee to produce more propolis. The propolis envelope covers all four walls of the hive and stimulates an even higher production of propolis. Honey Bees, are a keystone species. They pollinate crops, which gives us food to eat. Bees pollinate 1/3 of our crops and without them most of our produce would not be available for consumption. But on the devastating side of their beneficial effect, there are 3 major viruses depleting our honey bee population. DWV, IAPV and BQCV are the 3 major culprits of this depletion. In our experiment, we are using different methods to detect the amount of viruses in the honey bee samples. “Deformed Wing Virus (DWV) is a honeybee viral pathogen either persisting as an unapparent infection or resulting in wing deformity [2].” Some of the drastic effects of DWV are “pupae death and adult bees emerging with deformed wings, a bloated, shortened abdomen, and discoloration. These bees are not viable and die soon after emergence [3].” Israeli Acute Paralysis Virus (IAPV) “was first described in 2004 in Israel, where severe bee mortality has inflicted heavy losses on Israeli apiculture (beekeeping) [6].” Recently, “the presence of IAPV has been strongly correlated with a new syndrome of honey bee losses observed in the United States, called the Colony Collapse Disorder (CCD)” [6]. The third virus, “Black Queen Cell Virus, BQCV, was first founded in Australia, it is found to be the most common killer of the queen larvae [4].” “Symptoms of BQCV are limited to queen larvae. The immature bee dies and turns black after its cell is sealed [5].” We believe the beneficial effects of propolis will have a favorable outcome on the amount of virus found in the honey bee hive population. In other words, we hypothesize if the propolis production in the hives is increased, through traps and envelopes, then the amount of viruses in the population of honey bees will decrease. Materials and Methods The honey bees tested were collected by Renata Borba in September of 2012 from hives located near the University of Minnesota and frozen until used for testing. To analyze the honey bees for virus levels, the RNA must be extracted from the abdomen. To extract this RNA, the abdomens must be removed and crushed before adding a lysis buffer containing Guanidinium and Ammonium thiocynate to denature all proteins, thus freeing the RNA. Next, phenol is added to further denature proteins and added chloroform pulls the proteins away from the RNA. To activate these chemicals, a series of incubation and centrifugation must occur before three distinct layers appear with the top layer containing RNA free of contaminant proteins. The last step in extracting RNA is adding isopropyl alcohol to precipitate the RNA for easy collection. The pellet of RNA is then dissolved into RNase free water and stored at -80°C until later use. The next part of detecting viruses in honey bees is to synthesize complementary DNA from the RNA previously extracted In order to digest any contaminating DNA, a cDNA reaction mix containing DNase I, DNase buffer, random primers, RNaseOut, and dNTP mix must be added to the RNA and run through a thermocycler, thus allowing the primers to bind to the RNA. A heat block must occur before adding a Superscript mix containing Superscript reverse transcriptase enzyme, buffer, and Dithiothreitol and running through the thermocycler once more. Finally, to quantify the virus RNA in the honey bee samples, real time qPCR is used and data can be analyzed to calculate virus amounts. In Figure 3, The graph of Israeli Acute Paralysis Virus displays that both collections of bees show the relatively same amount of virus. Though, colony 18 shows the most amount of virus out of all colonies. Conclusion In conclusion, we were able to detect the three viruses in most of the bees, whether individual or pooled. In our experiment, we were unable to firmly say which method of testing, individual or pooled, was more effective. We had a range from no results to having a large amount of virus identified. Our hypothesis stated that if the propolis production in the hives was increased, by using traps and envelopes, then the amount of viruses in the population of honey bees would decrease. After thorough analysis of our data, we conclude that our hypothesis was incorrect because in this experiment propolis production had no correlation to the amount of virus present in the honey bees. Acknowledgements We thank the Biology 160 Bee students for their tedius work and Renata Borba for raising and collecting the bees. We want to thank Renata for allowing us to participate in her research. We also want to thank Dr. Mogen for guiding us through this experiment. References 1. A Kujumgieva, I Tsvetkovaa, Yu Serkedjievaa, V Bankovab, R Christovb, S Popovb Antibacterial, antifungal and antiviral activity of propolis of different geographic origin. Journal of Ethnopharmacology, 64(3), Constanze Yue and Elke Genersch Deformed Wing Virus. Journal of General Virology, 103, Retrieved December 5th, Joachim R. de Mirandaa, Elke Generschb Deformed wing virus. Journal of Invertebrate Pathology. 4. M Benjeddou, N Leat, M Allsopp, S Davison Detection of Acute Bee Paralysis Virus and Black Queen Cell Virus from Honeybees by Reverse Transcroptase PCR. Applied and Environmental Microbiology, 67(5), Retrieved December 6, Entomology Department of Univeristy of Georgia. Honey Bee Disorders: Viral Diseases. Retrieved December 6, 2013, from The Univeristy of Georgia, College of Agricultural and Environmental Sciences: 6. Philippe Blanchard, Frank Schurr, Olivier Celle, Nicolas Cougoule, Patrick Drajnudel, Richard Thiéry, Jean-Paul Faucon, Magali Ribière. (2008). Journal of Invertebrate Pathology. First detection of Israeli acute paralysis virus (IAPV) in France, a dicistrovirus affecting honeybees (Apis mellifera), 99(3), Retrieved December 10th, 2013, from * Positive numbers indicate a high amount of virus while a negative number means a low amount of virus detected.. Results In Figure 1, the graph of Black Queen Cell Virus shows that in colonies 6, 13, 28, 30, 33, and 36 only the pooled 20 bees had little to no virus. While colony 23 indicated that only the individual 5 bees showed little virus. Colony 18 and 22 shows that no virus was detected at all in either groups. In colonies 7, 14, 17, and 26 both of the collections showed very low traces of virus. In Figure 2, the graph of Deformed Wing Virus illustrations that only colony 6 detected a small amount of virus from the pooled group. While, colonies 18 and 36 only show amounts in the individual groups. The only two colonies with high amounts of virus discovered in them are 14 and 17.* The remaining colonies show recognition of virus in both groups of bees. Figure 1: qPCR results showing the Ct values which indicate the amount of BQCV present Figure 2: qPCR results showing the Ct values which indicate the amount of BQCV present Figure 3: qPCR results showing the Ct values which indicate the amount of BQCV present