Towards honey authentication: assessing European honey entomological origin by a molecular identification approach
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abstract
Following the European Union (EU) legislation, honey should be produced by the western honey bee,
Apis mellifera. Across Europe, 10 different A. mellifera subspecies can be found, comprising 3 different
lineages (A, M and C) based on mtDNA [1]. In general, honey bees occupy allopatric geographical
ranges according to their evolutionary lineages, allowing to establish an entomological origin for honey
produced in different EU countries. Additionally, several honeys with protected designation of origin
(PDO) detail the subspecies traditionally used in their production [2]. While numerous works focused on
the botanical and/or geographical authenticity of honey, only a few have attempted its entomological
authentication. For that purpose, DNA-based methods have been considered as the most suitable tools
since they allow the unequivocal species identification. So far, only few works described the use of DNAbased
methods to establish the entomological origin of honey [3,4] and those were focused on different
species of honey bees, including Meliponini and/or Trigonini stingless bees. To our knowledge, this is the
first attempt to distinguish among different European honey bee subspecies commonly used in honey
production, with further application to honey authentication.
In this work, DNA markers were developed for the differentiation of A. mellifera subspecies DNA in
honey. For this purpose, individuals of A. m. iberiensis lineage A (n=22) from Portugal and Spain (n=5), A.
m. iberiensis lineage M from Spain (n=7), A. m. mellifera lineage M from France, Netherlands, Scotland
and Norway (n=7), A. m. ligustica lineage C from Italy (n=4), A. m. carnica lineage C from Croacia and
Serbia (n=4) and commercial Buckfast lineage C bees (n=10) were tested. Different sets of primers were
designed targeting the cytochrome oxidase I gene. The specificity and sensitivity of the designed primers
were assayed by qualitative polymerase chain reaction (PCR). Species-specific primers successfully
allowed the identification of A. m. iberiensis lineage A by end-point PCR. The use of real-time PCR coupled
with High Resolution Melting analysis allowed the separation of A. mellifera honey bee subspecies in different clusters according to their lineages. The developed methodologies were applied to the analysis
of authentic honey samples from Portugal (produced by A. m. iberiensis lineage A), Spain (produced
by A. m. iberiensis lineage M), and Italy (produced by A. m. ligustica lineage C), allowing its successful
entomological origin identification.
This work has been supported by FCT (Fundação para a Ciência e Tecnologia)
through project UID/QUI/50006/2013 – POCI/01/0145/FEDER/007265 with financial support from FCT/
MEC through national funds and co-financed by FEDER, under the Partnership Agreement PT2020 and
by the project NORTE-01-0145-FEDER-000011. S. Soares and J. Costa are grateful to FCT grants (SFRH/
BPD/102404/2014 and SFRH/BD/75091/2010) financed by POPH-QREN (subsidized by FSE and MCTES).
