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

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.