Observing Honey Bees Immune Response to Exposure of Porpolis Kayla Pierce, Abby Hennen, Sean Seczko, Michael Xung University of Wisconsin-River Falls, WI Honey Bees are a Keystone Species The honey bee, Apis mellifera, is a keystone species. Keystone Species are species that have particularly strong effects on the composition or survival of the communities; removal of a keystone species would result is a dramatic impact on the community that the species inhabited [1]. As a keystone species, the honey bees play a fundamental role in supporting the entire community including the human community. While honey bees may not be the most abundant member of the community as of recently because of the life threating disease that the honey bees have been infected by. The honey bees are responsible for the pollination of our fruits, vegetables, nuts and flowers. By losing the honey bees it will have a huge impact on the way we live and what we eat [2]. Discovering honey bees natural defense: Propolis Propolis is a complex resin that honey bees collect from trees and is used to seal crack in the hive. Our hypothesis is that honey bees exposed to proplis will have a better immune system then the honey bees with less exposure. Background of Honey Bees Viruses DWV: Deformed wing virus, deformed wing virus is an RNA virus. Can cause wing deformity and premature death in honeybees. They found DWV on the parasitic mite varroa destructor suggesting that the mite may be involved in the transmission of DWV. The virus multiplies slowely which permits the infected individual to survive to adulthood. Positive strand RNA virus that was initially isolated from adult honeybees from Japan infested with the parasitic mite varroa destructor [3]. BQCV: Black Queen Cell Virus, The main symptoms for BAQV consist of blackened cell walls of sealed queen cells, containing dead pro pupae. Diseased larvae have a pale yellow appearance and tough sac like skin, much like sacbrood. Symptoms of BQCV are limited to queen larvae. The immature dies and turns black after its cell is sealed. There may be an association between BQCV and Nosema disease [4]. IAPV: Israeli Acute Paralysis Virus, transmitted by Varroa destructor which is an active vector of the virus. Has the ability to kill both pupae and adult bees.CCD and IAPV are linked. Of those colonies that suffered from CCD all had IAPV present while healthy colonies did not have IAPV. IAPV was present while imported from Australian and in royal jelly from China. A Little known virus that sets bees wings shivering and eventually causes paralysis. IAPV afflicted bees are typically found dead outside their hives. The presence of IAPV was found to be the best indicator for CCD but its possible that the virus is infecting bees whole they are vulnerable at state [5]. qPCR Add 19 uL of qPCR mix to the wells of the 96-well plate, in triplicate for each sample, testing the same PCR primers. Add 1 µL of cDNA toe each well. Place them in a real time qPCR machine. Set the machine to: 50oC for 2 minutes, 95oC for 2 minutes, 45 cycles: 95oC 20 seconds, 60oC for 30 seconds, and 72oC for 80 seconds. At the end of the cycles, a graphical indication of how much DNA was synthesized in each cycle. From the graph produced the reader can estimate the threshold cycle. The Ct values can be determined by the formula: ΔCt=Ct(actin)-Ct(DWV). Conclusion After analyzing our final research we found that our hypothesis was incorrect. We hypothesized that honey bees exposed to propolis would have a better immune system. In conclusion that the propolis is inconclusive in our results, the propolis didn’t have a great enough effect to conclude that propolis is substance that helps the bees immune system. Acknowledgements We thank the BIOL 160 BEE students for their work and the local beekeepers for trapping bees for testing. References 1.Copyright © 2007 Pearson Education Inc., Publishing as Pearson Benjam. 2.Spivak, Marla, Dr. "Research : Bee Lab : Department of Entomology : University of Minnesota. "Research : Bee Lab : Department of Entomology : University of Minnesota. University of Minnesota, 15 Nov Web. 09 Dec "Honey Bee Health." — COLOSS. Plone Foundation, n.d. Web. 10 Dec "Entomology." Honey Bee Program. N.p., n.d. Web. 10 Dec Budge, Giles, and Joachin R. De Miranda. "The Acute Bee Paralysis Virus–Kashmir Bee Virus–Israeli Acute Paralysis Virus Complex." The Acute Bee Paralysis Virus–Kashmir Bee Virus–Israeli Acute Paralysis Virus Complex. N.p., Jan Web. 10 Dec Methods RNA extraction collect the abdomens of the 5 bees in a Kaypak bag, then add Buffer. Then proceed to crush the bees. Transfer to a micro centrifuge tube, centrifuged at 10,000g for 8 minutes at 4oC. After centrifuge, transfer all but any precipitated debris. After, under the fume hood, add 380 uL phenol to the tube containing the supernatant. Vortex, then incubate for 10 minutes at 95oC. Cool on ice for 5-10 minutes and add 200 uL of chloroform. Centrifuge at 10,000g for 15 minutes at 4oC. The aqueous layer contains the RNA. Without disturbing the other layers, transfer the top layer to another tube. Add 500 uL of isopropyl alcohol to the tube. Vortex then incubate the sample at 4oC. Centrifuge the tube again at 10,000g for 10 minutes at 4o C. After centrifuging, there should be a pellet at the bottom. Remove all the liquid but the pellet. Wash the RNA pellet once by adding 70% ethanol to the tube and vortex briefly. Centrifuge again at 7,000g for 5 minutes at 4oC. Pour the supernatant out and centrifuge for the last time, remove extra liquid. Dry the pellet for 5-20 minutes. Dissolve the pellet in RNase- free water by incubating for 10 minutes at 60oC. Store the dissolved pellet for the next lab at -80oC. cDNA Place 2.9 uL of cDNA reacton mix into a.5 mL microcentrifuge tube. Add 8 uL of your RNA to the tube and mix gently. Place the tube into the thermocycler programed for: a) 37oC for 1 hour, b) 75oC for 10 minutes, c) 4oC for 2 minutes, and d) 70oC for 10 minutes. Place the tube on ice. Place the tube in the microcentrifuge and spin for 30 Seconds. Place the tubes in the 42oC heat block for 2 minutes. Add 4µL of superscript mix to the tube. Place the thermocycler again for: a) 42oC for 50 minutes, b) 70oC for 15 minutes, and c) 4oC to hold. The samples will be stored in the freezer until the next lab.