# Molecular Detection of _Acarapis woodi_ Using Hive Debris as Innovative and Non-Invasive Matrix
## 4 Discussion  
The results of the present study confirm the presence of _A. woodi_ in honey bee colonies in Italy and point out the potential underestimation of its presence in Italian apiaries. In fact, in this country, acarapisosis is now overshadowed by the exponential spread of the parasitic mite _Varroa destructor_. As a result, the presence of _A. woodi_ in several Italian regions is not regularly investigated. The prevalence of this parasite in Europe is relatively low: in Finland in 1991, it was slightly over 9%, rising to around 20% in the mid-1990s and then declining [8]. Similar observations were made in Greece where, over 10 years (1986 to 1995), infestation with _A. woodi_ showed a decreasing trend [9]. However, the data related to the prevalence of _A. woodi_ in these regions are outdated and often patchy. The prevalence of this mite in Spain in 2010-2011 (13-15.5%) increased compared to that in 2006-2007 (8.3-4%) [4; 10]. This change in prevalence was not related to the different techniques employed and variation in sampling area, and varroosis treatment could be responsible for the declined trend [10]. Recent reports show the disease has spread to new regions, mainly in Asia and in the eastern regions of Russia [11]. Another factor discouraging the diagnosis of _A. woodi_ in Italian apiaries is the time-consuming official method applied for its diagnosis. In fact, the conventional diagnostic method used in the surveillance program for detection of tracheal mites is the thoracic disk methods (TDM), which involves cutting a thoracic disc containing the prothoracic trachea for slide mounting and microscopic examination of the bee's tracheae [12; 13]. Although TMD has a good sensitivity, allowing for the detection of even low levels of infestation (below 5%), this method is very laborious and requires a high level of skill and concentration of the laboratory staff [13]. Guanine visualization and direct enzyme-linked immunosorbent assay (ELISA) are other methods available for the detection of tracheal mites [12]. The ELISA method is sensitive enough to detect tracheal mite infestation at very low levels but lacks specificity, as cross reactivity with other proteins present in the hemolymph and in the thoracic muscles can occur [12]. Indeed, guanine visualization is an indirect method based on detecting guanine, which is the main end product of nitrogen metabolism in the mite, but its presence is only in a negligible amount in bee excretions, which could lead to potential false-negative results [12; 14]. In this context, molecular techniques are more sensitive, as well as specific, especially in the case of low parasite loads. Sequencing of the amplified products of positive honey bee samples confirmed the infection by _A. woodi_ since these products had an identity of 95% with the COI sequence deposited in GenBank by [PERSON] et al. [10] from Spanish apiaries. By contrast, only one positive hive debris sample showed an identical COI sequence with those deposited in GenBank by [PERSON]. To the best of our knowledge, this is the first detection of _A. woodi_ in live debris sample. The importance of this matrix is due to its non-invasiveness because  
Figure 1: Gel electrophoresis of RT-PCR-amplified RNA analyzed in QIAxcel System, Qiagen. Lane 1–6, positive samples yielding amplicons of the expected size (180 bp); lane 7, positive control; lane 8, negative control; lane 9 DNA ladder. it does not imply killing honey bees; moreover, the use of molecular techniques is faster than the official methods previously described. This new sampling method may act as a pre-clinic indicator to detect the presence of the mite in the apiaries. The potential importance of _A. woodi_ infestation should not be underestimated. In fact, it is considered that in the UK, if 30% of bees in a colony are infested, the colony will die in the following spring. However, generally in Europe, a realistic threshold could be set at 10% [12].