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).