*2.1. Sampling*

Dead honeybee sampling and shipment were carried out by o fficial veterinarians of the Local Health Service specifically trained in beekeeping, in the presence of the beekeeper who issued the notification. The o fficial veterinarians operated in accordance with the abovementioned guidelines, defining the working protocol with all monitoring details, to further standardize the procedure across the di fferent apiaries and beekeepers. Samples consisted of 250–1000 dead honeybees, collected in suitably sealed, properly identified containers. Optionally, samples of comb, bee bread, and vegetable matrices (most frequently leaves, corn seedlings, maize) were also collected. The samples were individually packed in plastic sampling bags to avoid cross contamination, properly identified and the proper storage was guaranteed by immediate freezing after collection. Dead honeybees were collected at the hive entrance or from the ground in front of the hive, bee bread was taken directly from the comb as well as honey. All beehives were opened and clinically inspected in order to evaluate the size of colonies and to estimate the possible impact of the bee killing incidents on the colony itself. Vegetable matrices were collected in the immediate vicinity of the hive or from the near crops treated with pesticides. All samples were stored at −20 ◦C until delivery to the territorially competent Veterinary Institute (IZS) and until toxicological analysis. Samples considered in the present work were delivered to our laboratory between 2015 and 2019. Figure 1 depicts the location of the sampling points in Italy for all matrices and mortality events by year.

**Figure 1.** *Cont*.

**Figure 1.** Location of positive and negative samples for each officially reported honeybee mortality event in Italy from 2015 to 2019.
