Background

1. Bee Propolis Virus ProjectPatricia Reeves, Morgan Manchester (Biology 160 Students) Kim Mogen, Brad Mogen (Biology Mentors)University of Wisconsin-River Falls, WI Results Methods The bees in which we conducted the experiments on, were provided by RenataBorba, a graduate student from the University of Minnesota studying honey bees. The samples were collected in September 2012, and frozen until used for the following methods of testing. Background Viruses There are varied beliefs on what the catalyst of CCD (Colony Collapse Disorder) is. Aside from the phorid fly, several other viruses are also crucial to the health of the honey bee, Apismellifera. The viruses that we tested our bees for are Deformed Wing Virus (DWV), Black Queen Cell Virus (BQCV), and Israeli Acute paralysis Virus (IAPV). DWV is a virus that was first isolated in the 1980s. It mutates the shape and size of A. mellifera’swingwhich hinders the ability to function inside and outside the hive, thus leading it to die. BQCV is a virus that infects the queen larvae , turns it black and kills it in the capped cell stage. IAPV is a virus originating from Israel that is considered the main culprit in CCD (2). The virus was imported to the U.S. from Australia. It renders A. mellifera motionless, and makes it so that A. melliferaeventually dies. RNA EXTRACTION: For RNA extraction, we used a plethora of materials that included: bee abdomen, lysis buffer, phenol, chloroform, and water. We crushed 5 bee abdomens, then added lysis buffer and mixed them to create a liquid enriched with RNA. After we extracted it, we added phenol then put it on the vortex. After chloroform was added, and the sample was shaken, we extracted the aqueous layers, put it on the vortex again, and then incubated the sample. It was then centrifuged to pellet RNA, we cleansed the pellet, centrifuged again, removed liquid and dried the liquid, then dissolved the pellet in water and incubated it. The US credits it production of honey to the European honey bee, or Apismellifera. Melli- meaning "honey" and ferre meaning "to bear", so its name is a literal translation of "honey bearing bee". These bees multitask by pollinating the food we eat and also collecting the nectar from those flowering plants and producing honey (1). The bees gather the nectar and store it behind their wings as they fly back to the hive. The worker bees than digest and regurgitate the nectar, breaking down the sugars from complex to simple, and place the newly made honey into cells in the hive. Once honey is done being collected, the bees fan their wings, creating a breeze, to reduce the water content and overall drying out the honey. They then seal the honey with "caps" made of wax. A recently investigated component to hive structure is propolis. Propolis is resin from trees used to seal up any unwanted holes in the hive. The reason to the research behind this substance is because it is believed to have health benefits for both humans and bees. It has antibacterial properties that is possibly creating healthier living environments for the bees. The researchers are going about this by having controlled hives and hives that promote propolis production, better known as propolis traps. These traps are made with particular gaps in between the frames. With these spaces, the bees will naturally want to produce extra propolis and possibly result in an overall healthier hive. In 2007, it had been brought to media attention that honey bee numbers where decreasing. This phenomenon has been dubbed CCD, or Colony Collapse Disorder. With bee colony populations at such low numbers, researchers have gone to work, trying to find a reason to the madness. The studies are showing that there are far too many factors contributing to the disappearances, but the basic conclusion is poor health among the bees. Table 1. The table shows the number of hives with control (no propolis), a propolis trap, or a propolis envelope that were positive for the virus (ΔCt>0), had low levels of the virus (ΔCt=-5 to 0), had very low levels of the virus (ΔCt=-10 to -5), and had no virus at all (ΔCt<-10). The viruses tested for were Black Queen Cell Virus (BQCV), Deformed Wing Virus (DWV), and Israeli Acute Paralysis Virus (IAPV). cDNASYNTHESIS: For cDNA synthesis, we diluted the RNA sample, then combined it with the cDNA reaction mix. It was placed in the Thermocycler, and the contaminating DNA was digested by DNAse enzyme. It was chilled on ice. After, it was rewarmed on the heat block. The final step was to add Superscript and put it back in the Thermocycler. Then frozen until the qPCR steps. Conclusion In conclusion, all three viruses appeared in the sample of bees provided for us. Of the controlled, trapped, and enveloped colonies, the bees that were affected the least were the bees that were in the trapped and enveloped colonies. REAL TIME PCR (qPCR):We mixed qPCR mix and cDNA in each well. Samples were capped and placed in PCR machine. A graph displaying the number of DNA synthesized was made, and from that graph we estimate threshold cycle (Ct). Finally, we used the Ct values to generate ΔCt. References 1.Cox-Foster, Diana. vanEngelsdorp, Dennis. 2009. Saving the Honeybee 2. "Bee Viruses." Beeologics. Beeologics LLC, n.d. Web. 9 Dec. 2013. <http://www.beeologics.com/colony-health/bee-viruses>. 3. "Honey Bee." Wikipedia. Wikimedia Foundation, 12 Aug. 2013. Web. 08 Dec. 2013.