Detecting V. ceranae Infection Presence
Approximately eight honeybee and eight bumblebee individuals per visit
to each field site were randomly selected to test for the presence or
absence of V. ceranae infection (target N per species per site =
16; total honeybee, n = 75; bumblebee, n = 86; Appendix S1: Table S4).
Only sites with a minimum of eight bees total were included in the
analysis. When less than eight individuals of each species were
collected during one of the two visits to a site, infection was tested
in all individuals collected (Appendix S1: Table S4). The selected
bumblebees were predominantly Bombus impatiens , but also included
single individuals from Bombus fervidus, Bombus bimaculatus, andBombus pensylvanicus species that were all collected from a
single field site visit that had relatively low Bombus impatiensabundance (Site E, Visit 1). Ultimately, we modeled the binary presence
or absence of V. ceranae in individual honeybees and bumblebees
and all sites had a minimum of eight samples tested for V.
ceranae presence per host species.
Abdominal contents were dissected from each sample using sterilized
forceps and immediately placed on dry ice. Half of the abdomen was
placed in a microcentrifuge tube for DNA analysis, and the other half
was stored for reference. DNA was extracted using the DNeasy Blood &
Tissue Kit (Qiagen, Germantown, MD, USA) following the manufacturer’s
instructions for tissue samples. Following extraction, DNA purity and
concentration were quantified using Nanodrop 2000 software (Thermo
Fisher Scientific, Waltham, MA, USA). One sample with a nucleic acid
concentration less than 10 ng/µL was removed from the study due to
insufficient DNA extraction (a honeybee from Site E, Visit 1).
To ensure adequate extraction of bee DNA, polymerase chain reactions
(PCR) were conducted on all samples using A. mellifera 18S rRNA
gene primers, which produced bands at 784 bp (Cardinal et al. 2010).
Sequences for these bands were confirmed via Sanger sequencing. To
determine presence or absence of V. ceranae infection in each
sample, PCR was conducted with V. ceranae -positive and
H2O negative controls using the primers Nosema-F
(5′-CGGATAAAAGAGTCCGTTACC-3′) and Nosema-R (5′-TGAGCAGGGTTCTAGGGAT-3′)
for the V. ceranae large subunit ribosomal RNA gene (GenBank
Accession No: DQ486027; Chen et al. 2008). Details on the PCR procedure
can be found in Appendix S2. A subset of samples was selected for Sanger
sequencing to confirm the identification of V. ceranae (GenBank
Accession Numbers: bee 18S rRNA, OQ545564–OQ545565 and V.
ceranae large subunit rRNA, OQ550096–OQ550100).