LAUSR

Abstract The entomological source of honey is linked to its origin and may substantially contribute to the end products' price. For example, honey from native and/or non-domesticated species, such as Asian Apis dorsata and Apis cerana, is more valued and achieves higher prices compared to the honey from managed colonies of A. mellifera. Such types of honey are often subject to various fraudulent practices, such as mislabelling of the origin or admixing with lower-cost and low-quality honey which does not match the label declaration. The entomological origin may be determined through DNA-based techniques. Here, we report on new DNA-based tools for the correct identification of the DNA signatures of three economically important honeybee species: Apis mellifera, Apis cerana, and Apis dorsata. Initially, we constructed EPIC markers for a nuclear single-copy adenine nucleotide translocase (ANT) gene to explore the properties of interspecific variation in honeybee tissue samples. For confirmation of the samples’ identity, we used widespread mitochondrial tRNALeu – cox2 marker. Based on successful sequencing and phylogenetic analyses, we identified a short interspecifically variable part in the ANT gene for which we designed PCR primers that allow the successful amplification of fragmented DNA from sources like honey. Amplified fragments were differentially double digested by restriction endonucleases MnlI and HpaI, which resulted in specific restriction patterns observed on agarose gel electrophoresis. Verification of the approach was performed with 25 tissue samples and tested additionally with 20 honey samples and 1 royal jelly sample. Our results demonstrate the correct assignment of the samples to their entomological origin on the interspecific level. Furthermore, PCR amplification using the designed EPIC marker and double restriction protocol was demonstrated as useful in detecting mixed samples as well as two mislabelled honey samples claimed to belong to Asian honeybee Apis cerana. Additionally, EPIC markers are also useful in detecting heterozygosity, for example, in tracing paternal and maternal lines in contrast to abundantly used mitochondrial tRNALeu – cox2, which is inherited via maternal line only and may thus significantly contribute to the resolving of paternal relationships. We show that our method is not limited to honey but may also be applied to other honeybee products, such as royal jelly.