**5. Materials and Methods**

#### *5.1. Honeybee Collection and DNA Isolation*

Forager honeybees were recognized as bees returning to the hive and captured at the hive entrance about the midday. Forager honeybees from Poland were collected from one location in Lublin [51◦15- N 22◦34- E] each month from April to September 2018 (PL1-PL6). Forager honeybees from UK were collected from the roof of the Fogg Building of Queen Mary University, London (UK2) [51◦52- N 0◦03- W] and in the garden of the Natural History Museum, London (UK1) [51◦29- N 0◦10- W] in July 2019. Greek (GR1, GR2) samples of forager honeybees were collected in November 2017 from two colonies inhabiting the garden of The Agricultural University of Athens [37◦59- N 23◦42- E]. Forager honeybees from Spain (ES1, ES2) were collected in November 2017 from experimental colonies located at Marchamalo (Centro Apícola y Agroambiental de Marchamalo (CIAPA-IRIAF), Marchamalo, Spain [40◦68- N 3◦21- W]. Thai samples of forager bees consisting of both western honeybee (*Apis mellifera*) and Asian honeybee (*A. cerana*) were collected in February 2018 in the proximity of Chiang Mai University [18◦50- 98◦58- E], *A. mellifera* samples were marked TAI1 and TAI2, and *A. cerana* as TAI3 and TAI4. Genomic DNA was extracted from whole honeybees using QIAamp DNA Kit according to manufacturer's instructions. Isolates were sent to the Biobank, Poland for NGS analysis.

#### *5.2. NGS*

NGS sequencing and the analysis of the 16S rRNA bacterial gene amplicon was based on the V3-V4 region and the ITS2 eukaryotic region for bee DNA samples. Amplicon libraries, were prepared using the *16S Metagenomic Sequencing Library Preparation*, *Preparing 16S Ribosomal RNA Gene Amplicons for the Illumina MiSeq System* (Illumina® San Diego, CA, USA) protocol. Information about primers sequences, PCR conditions is shown in Supplementary Materials Table S5.

All data are available at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA686953 (Submission Registration date: 21 December 2020).

#### *5.3. Positive, Negative Control*

The positive quality control for the V3-V4 region of the 16s rRNA gene was the DNA isolate derived from an ear swab. For the ITS2 region, it was DNA isolated from the *Saccharomyces cerevisiae* strain. PCR grade water was the negative quality control for both kinds of amplicons.
