2.2 STUDY SITE CHARACTERIZATION AND SAMPLING
Sampling activities were performed in July 2019 in 19 sites located in
an urbanized area in the central portion of north Italy covering four
administrative provinces of northern Italy (i.e., Milano, Monza e della
Brianza, Lecco and Como) (Figure 1). In order to avoid non-independence,
the distance between sites were at least 2 km, above the foraging range
observed for these species (Biella et al., 2022). Sampling sites have
been selected to cover a gradient of growing urbanization ranging from
semi-natural areas composed of hay meadows close to forest patches to
the more urbanized context of the city center of Milan. To select
sampling sites impervious surfaces (i.e., concrete, building, and
asphalt) have been mapped using regional land-use cartography
(2018-DUSAF 6.0;https://www.dati.lombardia.it/Territorio/Dusaf-6-0-Uso-del-suolo-2018/7rae-fng6)
as explained in detail in (Tommasi et al., 2022), afterward sampling
sites have been chosen along a visible gradient of impervious cover.
In each site, five to six workers per species were collected with random
walks within a 50 m × 50 m plot using an entomological net. After
collection samples were stored at − 80 °C until further analyses.
Overall 192 individuals belonging to the investigated bumblebee species
(96 B. pascuorum and 96 B. terrestris ) have been subjected
to gut DNA extraction and multi-target parasite screening.
Floral resources were estimated in each site by counting the total
number of flowers within six squares 1 m × 1 m placed in flowering
spaces within the sampling area (see Tommasi et al., 2022 for a detailed
explanation about floral resources availability estimation). The data
were also used to estimate the diversity of the available floral
resources in each site and was calculated as Shannon diversity Index
(Shannon, 1948). The percentage of green habitat (i.e., meadows, forests
and urban green spaces) in the 1 km radius buffer surrounding each
sampling site have been calculated using the previously mentioned
land-use map (see Tommasi et al., 2022 for a detailed explanation about
land-use categorization). Specifically, the buffer size has been
selected according to the maximum foraging range expected for these
species (Knight et al. 2005; Greenleaf et al. 2007). Furthermore, green
habitat fragmentation has been estimated for each buffer surrounding the
sampling sites by calculating the Euclidean nearest neighbor distance
(ENN) of green habitat patches through the package landscape
metrics (Hesselbarth et al., 2019) in the software R (version
2022.12.0).
The distance of each sampling site from the closer honey bee hive and
the number of hives in the 1 km buffer surrounding the sites have been
calculated through the distance matrix function of QGIS (version
3.28.4). Honeybee hive locations have been obtained from the national
beekeeping database (BDA) upon request and released by the competence
office of each province involved in the present study. Variables
calculated for each sampling site are available in supporting
information Table S1.
[ FIGURE1]