# Evaluating the Efficacy of Common Treatments Used for _Vairimorpha_ (_Nosema_) spp. [PERSON]
## 2 Materials and Methods  
Experiment 1: Laboratory Cage Study for Ewalting Efficacy of Fumagilin-B, Nozevit Plus, and Honey-B-Healthy(R) against Vairimorpha ceranae  
In July 2014, capped brood combs were obtained from honey bee colonies at Oregon State University apiaries (Corvallis, OR, USA). We placed the combs in an incubator under simulated hive conditions (33 \({}^{\circ}\)C, 55% RH) to facilitate adult worker bee emergence. Twenty-four hours later, we gently brushed newly emerged bees into a large container and mixed them gently by hand. After the bees were mixed, we placed 250 individual bees inside cylindrical wire cages (63.06 cm\({}^{3}\)) and returned them to the incubator, as per [19]. The caged bees were immediately provided with ad libitum access to a glass vial containing 25 mL of a _V. ceranae_ spore/50% sucrose solution dosed at 40,000 spores/bee. The vials were covered with two layers of cheesecloth and then secured, inverted, and placed upon the top of the cage. Each cage also contained 25 g of finely ground wildflower pollen mixed with a 33% sucrose solution in a 2:1 (weight/volume) ratio. Prior to the experiment, the wildflower pollen was sent to the USDA National Science Laboratory (Gastonia, NC, USA) for pesticide analysis to assess the possible presence of pesticide residues. We found 34 ppb of fluvalinate, 3.7 ppb of chlorfenopyr, 2.6 ppb of trifuralin, and trace concentrations of all other pesticides that were tested for. The results of this pesticide residue analysis are presented as Supplementary Table S1 in the Supplemental Materials.  
The spore concentration of the _V. ceranae_ solution was formulated and purified by centrifugation following the methods of [24]. Briefly, the contents of infected gut samples were collected and centrifuged at 5000 rpm for five minutes at room temperature to produce a pellet of spores. After discarding the supernatant, the pellet was resuspended in distilled water by vortexing. This process was repeated 2-3 times. DNA analysis was performedusing the methods of [25] to confirm that only _V. ceranae_ spores were present in the inoculum. Once the 25 mL solution containing _V. ceranae_ was completely consumed, the caged bees were provided ad libitum access to water and 50% sucrose syrup (weight/volume). Three days after inoculation of bees with _V. ceranae_ spores, we provided 25 mL of sucrose syrup containing one of four treatments to the cages. There were five cage replicates per treatment for the following four treatments: Fumagilin-B, Nozevit Plus, Honey-B-Healthy(r), and a negative control (provided only 50% sucrose syrup). We prepared all treatments in 50% sucrose syrup according to the product label. Once the bees completely consumed the 25 mL of treated sucrose syrup, the caged bees were again given ad libitum access to water and sucrose syrup. In all, there were four treatments and five replicates (cages) of each treatment, for a total of 20 cages.  
Once a week, we measured consumption of the pollen by bees and replaced unconsumed pollen with fresh pollen mixed with sucrose solution as described previously. Bee mortality was recorded every other day and dead bees were removed at the time of diet replacement for convenience. We measured consumption of both water and sucrose solution and replaced them on alternate days. At 16 and 23 days after spore inoculation, we removed 25 bees at random from each experimental cage for infection analysis. The abdomens of the bees were used to estimate _V. ceranae_ prevalence (presence or absence of spores) and intensity (# of spores/bee) by light microscopy techniques as described by [26]. Each bee abdomen was checked individually for _V. ceranae_ infection.