## 3 Results
### Pathogen Spillover
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**Year** & **N\({}^{\circ}\) Virus** & _Apis mellifera_ & _Bombus terrestris_ & _Bombus pascuorum_ & _Anthidium florentium_ & _Megachile sculpturalis_ & _Osmia picornis_ \\ \hline  & 1 & ABPV (1) & ABPV (2) & & & \\
2020 & 2 & ABPV + BQCV (1) & & & & \\  & & CBPV + ABPV (1) & & & & \\  & 3 & CBPV + ABPV + BQCV & & & & \\  & & (1) & & & & \\  & 4 & CBPV + ABPV + DWV & CBPV + ABPV+ & & CBPV + ABPV + & & \\  & & + SBV (1) & & DWV + BQCV (1) & & DWV + BQCV (1) & & \\ \hline  & 1 & & & & & & \\
2021 & 2 & ABPV + DWV (3) & & & & & \\  & & & & & & & \\  & & & & & & & \\  & & & & & & & \\  & 3 & ABPV + CBPV + SBV (2) & & & & & \\  & & & (1) & & & & \\  & 4 & & & & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 11: Viral associations in the different species in 2020 and 2021. The numbers in parentheses refer to the number of times the association was found [ABPV = Acute Bee Paralysis Virus; CBPV = Chronic Bee Paralysis Virus; DWV = Deformed Wing Virus; BQCV = Black Queen Cell Virus; SBV = Sac Brood Virus; KBV = Kashmir Bee Virus; IAPV = Israeli Acute Paralysis Virus]. Figure 6: Graphical representation of the Linear Discriminant Analyses (LDA) determined by discriminant factors 1 (F1) and 2 (F2). With regard to the seasonal trends (Figure 7), and despite the limitations already mentioned in the methods, an expected trend characterized by a relatively higher incidence of positive samples, sometimes even at high titers, was observed during the summer months (June and July). However, positives were detected practically throughout the period, often at low titers, for ABPV and some sampling dates for CBPV and DWV. The trend was slightly different for BQCV, with several positive cases only in 2020, and for SBV, with only one positive case in 2020 and a constant level from May to August 2021. Regarding the electron microscopy (EM) results in 2020, immunoelectron microscopy for DWV identified 1 positive out of the 12 tested in one sample of a honeybee from modern park \"D\" with a high PCR titer \(>\)10\({}^{11}\) (Figure 8A), while the three DWV PCR positives with  
Figure 7: Seasonal trends of positivity for viruses during the two consecutive years (2020 and 2021) (Orange = Positive—P, Blue = Negative—N). titers of 10\({}^{3}\)-10\({}^{4}\) were not detected by EM. Similarly, the IEM results for ABPV was positive for two samples, one of _B. terrestris_ from modern park \"D\" (Figure 8B) and one from a honeybee from modern park \"C\" (Figure 8C), both with very high PCR titers (>10\({}^{12}\)). Of the nine IEM-negative samples, two were found to be positive for picornavirus-like particles with a low titer. In 2021, 2 IEM-positives out of the 21 tested were observed with medium and high titers (10\({}^{7}\) and 10\({}^{9}\)) in RT-PCR. Remarkably, they were all from honeybees in the same green area (modern park \"E\") at an interval of three months. Conversely, the four DWV positives, all from honeybees from three different green areas (two modern parks, \"D\" and \"E\", and roundabout \"F\"), with a low titer of 10\({}^{3}\)-10\({}^{4}\) in PCR, were not detected by EM. Among the 19 IEM-negative samples, isolated picornavirus-like particles with low titer (+/++) were visible in 3 samples. Out of these three samples, one--a _B. terrestris_ from modern park \"D\"--had a high titer for ABPV in RT-PCR (10\({}^{8}\)), and one--a _B. terrestris_ from roundabout \"F\"--was positive for both SBV and for ABPV at a low titer (10\({}^{4}\)). Finally, one sample--_O. bicornis_ from modern park \"D\"--was positive only by EM investigation and negative for all PCR, thus indicating the possibility of the presence of an unidentified virus. Concerning the detection of the _Nosema_ spp., all the samples were negative in 2020. In contrast, in 2021, 2 out of the 21 samples (9.52%) tested were found to be positive by microscopic examination (_A. mellifera_ from roundabout \"F\" and _An. forentinum_ from modern park \"E\", respectively). In both cases, the presence of _N.