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Owing to their foraging and honey production activities, honeybees form complex relationships with species across all domains, such as plants, viruses, bacteria and other hive pests, making honey a valuable biomonitoring tool for assessing their ecological niche. Thus, the application of honey shotgun metagenomics (SM) has paved the way for a detailed description of the species honeybees interact with. Nevertheless, SM bioinformatics tools and DNA extraction methods rely on resources not necessarily optimized for honey. In this study, we compared five widely used taxonomic classifiers using simulated species communities commonly found in honey. We found that Kraken 2 with a threshold of 0.5 performs best in assessing species distribution. We also optimized a simple NaOH\u2010based honey DNA extraction methodology (Direct\u2010SM), which profiled species seasonal variability similarly to an established column\u2010based DNA extraction approach (SM). Both approaches produce results consistent with melissopalinology analysis describing the botanical landscape surrounding the apiary. Interestingly, we detected a strong stability of the bacteria constituting the core and noncore gut microbiome across seasons, pointing to the potential utility of honey for noninvasive assessment of bee microbiota. Finally, the Direct\u2010SM approach to detect\n                    <jats:italic>Varroa<\/jats:italic>\n                    correlates well with the biomonitoring of mite infestation observed in hives. These observations suggest that Direct\u2010SM methodology has the potential to comprehensively describe honeybee ecological niches and can be tested as a building block for large\u2010scale studies to assess bee health in the field.\n                  <\/jats:p>","DOI":"10.1111\/1755-0998.13626","type":"journal-article","created":{"date-parts":[[2022,5,20]],"date-time":"2022-05-20T05:05:54Z","timestamp":1653023154000},"page":"2506-2523","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":18,"title":["Bee foraging preferences, microbiota and pathogens revealed by direct shotgun metagenomics of honey"],"prefix":"10.1111","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8118-5355","authenticated-orcid":false,"given":"Anastasios","family":"Galanis","sequence":"first","affiliation":[{"name":"Institute for Fundamental Biomedical Research (IFBR) BSRC \u2018Alexander Fleming\u2019  Vari Greece"},{"name":"Division of Animal and Human Physiology Department of Biology National and Kapodistrian University of Athens  Athens Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Philippos","family":"Vardakas","sequence":"additional","affiliation":[{"name":"Institute for Fundamental Biomedical Research (IFBR) BSRC \u2018Alexander Fleming\u2019  Vari Greece"},{"name":"Department of Apiculture Institute of Animal Science  Nea Moudania Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0005-8718","authenticated-orcid":false,"given":"Martin","family":"Reczko","sequence":"additional","affiliation":[{"name":"Institute for Fundamental Biomedical Research (IFBR) BSRC \u2018Alexander Fleming\u2019  Vari Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9260-8459","authenticated-orcid":false,"given":"Vaggelis","family":"Harokopos","sequence":"additional","affiliation":[{"name":"Institute for Fundamental Biomedical Research (IFBR) BSRC \u2018Alexander Fleming\u2019  Vari Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pantelis","family":"Hatzis","sequence":"additional","affiliation":[{"name":"Institute for Fundamental Biomedical Research (IFBR) BSRC \u2018Alexander Fleming\u2019  Vari Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5113-6192","authenticated-orcid":false,"given":"Efthimios M. 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