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Honey environmental DNA reveals entomological fingerprints through dual mitochondrial cytochrome c oxidase subunit 1 (COI) and cytochrome b (CYTB) metabarcoding


Abstract

Honey carries environmental DNA (eDNA) from organisms encountered by honey bees and can therefore encode an “entomological fingerprint” of agricultural and forest landscapes. We compared two metabarcoding assays targeting mitochondrial regions (cytochrome c oxidase subunit 1, COI; and cytochrome b, CYTB) to profile plant-sucking insects (Hemiptera) in honey samples from Italy and Türkiye, including citrus blossom, polyfloral, and honeydew honey samples. We designed a new short CYTB primer pair optimised for Aphididae and evaluated it alongside our previously published Hemiptera-COI assay. Across 3.51 million reads, our pipeline assigned 1.25 million COI and 1.48 million CYTB reads to Neoptera. CYTB resolved more taxa per sample (mean 7.9 families, 36 species) than COI (4.5 families, 11.7 species) and yielded a higher proportion of species-level assignments (98.3% vs. 43.7%). COI captured Metcalfa pruinosa (Flatidae) where abundant—often dominating polyfloral honey samples—whereas CYTB revealed fine-scale diversity of aphids relevant to citrus orchards and conifer/oak forests. Short-amplicon haplotype screening recovered multiple COI haplotypes for M. pruinosa, Thelaxes suberi, Cinara cedri and Aphis gossypii, suggesting potential for population monitoring from honey eDNA. Our results show that combining complementary metabarcodes mitigates primer and database biases and enhances landscape-scale inference, honey authentication, and surveillance of invasive or pest hemipterans.

Data availability

The datasets generated during the current study showing alignments of CYTB and COI primers with Hemiptera sequences are available in Zenodo with the following DOI: https://doi.org/10.5281/zenodo.19183389 . The sequence datasets generated and analysed during the current study are available in the EMBL-EBI European Nucleotide Archive (ENA) repository http://www.ebi.ac.uk/ena , with the project accession number PRJEB102161.

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Acknowledgements

We thank beekeepers who provided honey samples, in particular Nicola Mele and Zeid Nabulsi.

Funding

This research was funded by the University of Bologna 2023–2025 RFO programs, by Regione Emilia-Romagna, BEE-RER-3 projects—CUP E37G22000030007—del Regolamento (UE) No. 1308/2013 (OCM Apicoltura), and by the Italian Ministry of Agriculture, Food Sovereignty, and Forestry (MASAF)—project ItaHoneyDNA—CUP J39I24001640006. KENJ has been supported by a Fulbright Fellowship.

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LF designed the study, interpreted the results, drafted the manuscript, provided funds and supervised the project. AR organised laboratory analyses, compiled data, coordinated the sampling and drafted the manuscript. SB performed bioinformatic analyses, curated the internal databases, carried out data analyses and drafted the manuscript. VT performed laboratory analyses and drafted the manuscript. KENJ performed laboratory analyses and revised the manuscript. AÖK performed laboratory analyses, collected samples and metadata. GS, VJU and FB provided metadata, tested primer pairs, and elaborated data. All authors read and approved the final manuscript.

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Correspondence to
Luca Fontanesi.

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Ribani, A., Bovo, S., Taurisano, V. et al. Honey environmental DNA reveals entomological fingerprints through dual mitochondrial cytochrome c oxidase subunit 1 (COI) and cytochrome b (CYTB) metabarcoding.
Sci Rep (2026). https://doi.org/10.1038/s41598-026-46493-y

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  • DOI: https://doi.org/10.1038/s41598-026-46493-y

Keywords

  • Apiculture
  • Beekeeping
  • DNA fingerprint
  • eDNA
  • Hemiptera
  • Honeydew


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