## 2 Materials and Methods
### Viral Investigations
#### 2.5.2 Molecular Analyses  
Each sample was homogenized by maintaining the rate of 1-1.5 individuals in 1 mL of physiological saline depending on the amount of matrix available (Table 3). The total RNA was purified from 100 \(\upmu\)L (for ABPV/CBPV/DWN) and from 230 \(\upmu\)L (for SBV/BQCV/IAPV/KBV) of clarified homogenate using the RNeasy Mini Kit (Qiagen, Mississauga, ON, Canada). The RNA were recovered in 40 \(\upmu\)L of the elution buffer. DNA extraction was also performed on the samples using GenEluteTM Bacterial Genomic DNA Kits (Sigma-Aldrich, Buchs, Switzerland) with a lysozyme pre-incubation step. Complementary DNA synthesis and amplification reactions were performed using different amplification kits and sets of primers depending on the pathogen under investigation (Table 4). \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline \multirow{2}{*}{**Year**} & \multirow{2}{*}{**Date**} & \multirow{2}{*}{**Green Area**} & \multirow{2}{*}{**Species**} & \multirow{2}{*}{\
\begin{tabular}{c} **N\({}^{\circ}\)** \\ **Collected** \\ \end{tabular} } & \multicolumn{2}{c}{**N.**} & \multirow{2}{*}{**Weight**} & \multirow{2}{*}{
\begin{tabular}{c} **Volume** \\ **(mL)** \\ \end{tabular} } \\  & & & & & **Corrected** & & **ForPCR** & & **(mL)** \\ \hline \multirow{8}{*}{2020} & 8 July 2020 & Modern park E & _A. mellifera_ & 10 & 3 & 0.31 g & 2 \\  & 8 July 2020 & Modern park E & _B. terrestris_ & 10 & 3 & 0.60 g & 2 \\  & 8 July 2020 & Modern park E & _An. forentinuum_ & 10 & 3 & 0.47 g & 2 \\  & 17 July 2020 & Modern park D & _A. mellifera_ & 10 & 3 & 0.30 g & 2 \\  & 17 July 2020 & Modern park D & _B. terrestris_ & 10 & 3 & 0.60 g & 2 \\  & 17 July 2020 & Modern park D & _An. forentinuum_ & 10 & 3 & 0.81 g & 2 \\  & 23 July 2020 & Modern park C & _A. mellifera_ & 10 & 3 & 0.23 g & 2 \\  & 23 July 2020 & Modern park C & _An. forentinuum_ & 10 & 3 & 0.38 g & 2 \\  & 30 July 2020 & Historic city park A & _A. mellifera_ & 10 & 3 & 0.29 g & 2 \\  & 30 July 2020 & Historic city park A & _B. terrestris_ & 10 & 3 & 0.56 g & 2 \\  & 13 July 2020 & Modern park E & _B. pascuorum_ & 10 & 3 & 0.43 g & 2 \\  & 14 September 2020 & Modern park E & _A. Mellifera_ & 10 & 3 & 0.35 g & 2 \\ \hline \multirow{8}{*}{2021} & 8 March 2021 & Modern park E & _A. mellifera_ & 7 & 5 & 0.44 g & 5 \\  & 12 March 2021 & Modern park E & _O. bicornis_ & 10 & 5 & 0.43 g & 5 \\  & 18 March 2021 & Modern park E & _A. mellifera_ & 9 & 5 & 0.51 g & 5 \\  & 18 March 2021 & Historic city park B & _O. bicornis_ & 10 & 5 & 0.65 g & 5 \\  & 7 May 2021 & Modern park D & _A. mellifera_ & 10 & 5 & 0.39 g & 5 \\  & & Modern park D & _O. bicornis_ & 10 & 5 & 0.45 g & 5 \\  & 24 June 2021 & Modern park E & _A. mellifera_ & 10 & 5 & 0.47 g & 5 \\  & & Modern park E & _An. forentinuum_ & 10 & 5 & 0.89 g & 5 \\  & 24 June 2021 & Roundabout F & _A. mellifera_ & 10 & 5 & 0.41 g & 5 \\  & & Roundabout F & _An. forentinuum_ & 10 & 5 & 0.67 g & 5 \\
2021 & Roundabout G & _A. mellifera_ & 10 & 5 & 0.36 g & 5 \\  & 1 July 2021 & Historic city park B & _M. sculpturalis_ & 10 & 5 & 1.69 g & 5 \\  & 2 July 2021 & Modern park D & _A. mellifera_ & 10 & 5 & 0.46 g & 5 \\  & 2 August 2021 & Modern park D & _An. forentinuum_ & 10 & 5 & 0.85 g & 5 \\  & 2 August 2021 & Modern park E & _An. forentinuum_ & 10 & 5 & 0.93 g & 5 \\  & 2 August 2021 & Roundabout F & _An. forentinuum_ & 10 & 5 & 1.02 g & 5 \\  & 2 August 2021 & Roundabout F & _B. terrestris_ & 7 & 5 & 0.90 g & 5 \\  & 3 August 2021 & Modern park D & _B. terrestris_ & 8 & 5 & 0.97 g & 5 \\  & 3 August 2021 & Historic city park B & _M. sculpturalis_ & 10 & 5 & 1.79 g & 5 \\  & 5 August 2021 & Historic city park A & _B. terrestris_ & 8 & 5 & 1.05 g & 5 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Summary of the samples collected and analyzed for virus presence in the seven green areas (Historic city parks: A, B; Modern parks: C, D, E; Roundabout: F, G) during the two consecutive years of surveys (2020 and 2021).