## 4 Discussion  
Results obtained from field and laboratory diagnostic procedures can give information about colony treatments against varroosis in a timely manner. Moreover, verifying the effectiveness of control measures helps beekeepers in choosing the veterinary medical product (VMP) suited to their conditions and management, as well as veterinarians who have the professional prerogative of being able to prescribe authorised veterinary medicines [23; 24]. Last but not least, results on varroa infestation levels are an important tool in breeding programs for varroa resistance [25]. Protocols were devised in order to have data that could allow making practical considerations also considering the apiary conditions. Despite all researchers using the same container (Figure 1), variable quantities of adult bees were obtained, probably depending on the nectar flow or the width of the cells of wax foundations or the subspecies of the bees, which could have influenced the weight of the samples (CC and DG2, Figure 2). The use of powdered sugar to dislodge mites from adult bees was effective, as also confirmed by other researchers [3; 4; 8; 10]. The hard shaking of the jar containing the powdered sugar has been proven in previous studies to be an important factor to improve the accuracy [26]. The use of alcohol solutions is effective in dislodging the mites [6], but sixty seconds of rapid agitation might not be feasible for large-scale beekeeping operations. The use of portable agitators can improve the accuracy and reduce the effort [27] and further improvements of the methodology, such as the immersion for 2 min in 91% isopropyl alcohol without shaking the sample, seem very promising alternatives [28]. Other methods used to evaluate varroa infestation, such as ether wash [29] or CO\({}_{2}\) test [11], were not considered in our trial for their lack of practicality, high costs or low efficiency in dislodging the mites. Concerning the dispersal mite infestation of the honey bee colonies (protocol 1), the number of mites collected with the powdered sugar roll method was not always statistically correlated with it and gave more accurate results in colonies that were weaker or less infested (Table 3). Natural mite mortality was related to the amount of dispersal mites only in one apiary (ITG1, Table 2), which was not treated with acaricides for two consecutive years. Correlation coefficients of natural mite fall were higher in strong colonies when the brood amount is lower and independently on hive infestation (Table 3). Spearman correlation (\(p\)-value) and coefficient of determination of the results gained as total mite infestation (protocol 2) was very good for powdered sugar roll method in \begin{table}
\begin{tabular}{c c c} \hline
**Colony Category** & **Powdered Sugar Roll** & **Natural Mite Fall** \\ \hline Weak & **0.77 (\(p<0.05\))** & **0.77 (\(p<0.05\))** \\ \hline Strong & 0.32 (\(p<0.05\)) & 0.30 (\(p<0.05\)) \\ \hline Low amount of brood & 0.58 (\(p<0.05\)) & 0.56 (\(p<0.05\)) \\ \hline High amount of brood & 0.44 (\(p<0.05\)) & **0.67 (\(p<0.05\))** \\ \hline Low infested & 0.44 (\(p<0.05\)) & 0.26 (\(p>0.05\)) \\ \hline High infested & 0.52 (\(p<0.05\)) & **0.68 (\(p<0.05\))** \\ \hline \end{tabular}
\end{table} 
Table 5: Spearman correlation between colonies classified according to strength, amount of brood and infestation class, and total infestation level obtained using different methods. Protocol 2.  
one apiary (CC), where infestation was significantly higher compared to other apiaries (Table 4). Natural mite fall count was correlated with the total amount of mites in three apiaries (Table 3), but the coefficient of determination was high only in two of them (JW1, CC). These results support previous findings where natural mite fall is strongly correlated with the total number of mites in the colonies [7]. There are also several factors that affect mite population increase in colonies (brood production, life cycle of worker and drone brood, reproductive potential of _V. destructor_ mite, etc.)