## 2 Materials and Methods
### Test Protocols  
In order to gather data about the number of mites in dispersal and reproductive phase inside the honey bee colonies and to correlate that information with the infestation level obtained with the samples, two protocols were prepared:  
* Protocol 1. Dispersal mite infestation assessment  
First, the natural mite fall was assessed for 10 days [1]. On day 10, the honey bee colony strength was estimated [17] and a sample of adult bees from each colony was collected. The samples were analysed with the powdered sugar roll and a subsequent soapy solution wash technique, as described above. The dispersal mite infestation was estimated with the application of an oxalic acid treatment [18] performed on day 10. The oxalic acid solution was prepared using 1 litre of water to dissolve 100 grams of oxalic acid dihydride pure crystals and 1 kg of sucrose and administered to the colonies with a syringe, trickling 5 mL for each intercomb space occupied by bees. The amount of dispersal mites fallen after the treatment was assessed by using sticky or oily label sheets on the bottom boards, replaced every second day (or adapted to local conditions). The counts were performed for five days in a row [1].  
A total of 2 researchers (ITG and NA) participated in the trials. Four apiaries in Croatia and Algeria with a total of 60 colonies were involved.  
* Protocol 2. Total mite infestation assessment  
As for protocol 1, the first step was to assess the natural mite fall for 10 days. On day 10, colony strength was estimated with the Liebefeld method, and a sample of bees from each colony was collected on day 10 and analysed by the powdered sugar roll and soapy solution wash techniques, as described before. The total mite infestation of the colonies was estimated by counting the mite fall on bottom boards from day 10 to day 40. During that period, the queen was caged for 25 days (API.MO.BRU. Campodoro-Italy). Then, at the end of the queen caging period, in absence of brood, the test colonies were treated with oxalic acid (as previously described) [19]. Mite counts on bottom boards were performed until 5 days after the oxalic acid treatment. The sum of the mites fallen during the queen caging period, plus the mites fallen after the treatment, represent the total number of mites in the colony.  
A total of three researchers (DG, JW, CC) participated in the trials. Five apiaries with a total of 91 colonies were involved.  
Statistical analysis  
A one-way ANOVA model was used to test the differences between the apiaries in terms of colony population size (adult bees and brood). For infestation level, [PERSON]'s test was used for equality variances, and [PERSON]'s ANOVA was applied.  
The statistical differences between the sampling methods were analysed by Kruskal-Wallis test [20]. To verify how many mites were dislodged with the powdered sugar roll method, [PERSON]'s tau coefficient [21] was used to measure the ordinal association between the number of mites found after the powdered sugar shaking and the number of mites found with the soapy solution washing.  
Spearman correlation test [22] was applied to evaluate the methods. Comparisons were made between:  
* 10-day natural mite fall and dispersal varroa population (protocol 1) or total varroa population (protocol 2)
* mites found with the powdered sugar roll method and dispersal varroa population (protocol 1) or total varroa population (protocol 2)  
With a stepwise regression analysis, it was possible to verify if the factors: number of adult bees, brood, apiary, and tester were significant in the analysis.  
Thus, colonies were divided according to the second quartile (median) into two groups in terms of three features: amount of adult bees (weak or strong colonies); amount of brood (low or high); infestation level (low or high infested colonies). Spearman correlation test was applied to evaluate the methods used in both protocols.  
Analysis was performed using XLSTAT(tm) (Addinsoft, Paris, France) and Statistica(r) ver. 13 (StatSoft, Hamburg, Germany) software.