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Exploiting the mitogenomes of apis mellifera subspecies to develop an authentication tool to verify the entomological origin of mediterranean honeys

Publication . Honrado, Mónica; Henriques, Dora; Santos, Joana; Yadró Garcia, Carlos A.; Martín-Hernández, Raquel; Nanetti, Antonio; González, Amelia Virginia; Al Shagour, Banan; Hosri, Chadi; Farrugia, Dylan; Giovanni, Cilia; Zammit Mangion, Marion; Muz, Mustafa Necati; Haddad, Nizar; Galea, Thomas; Haider, Yamina; Obeidat, Wisam; Aglagane, Abdessamad; Arab, Alireza; Varnava, Andri; Eissa, Asmaa Anwar; Muz, Dilek; Hatjina, Fani; Lamghari, Fouad; Arruda, James; Caristos Caristos, Leonidas; Pinto, M. Alice; Amaral, Joana S.

Honey is highly susceptible to adulteration. Currently, the assessment of its geographical origin remains one of the most difficult tasks, which is typically performed by melyssopalynology. Recently, the attention has shifted towards indirect approaches such as the entomological origin based on geographical distribution patterns of honey bee subspecies. Although queens’ trade has impacted the natural subspecies distribution, honeys produced with autochthonous bees or bearing a Protected Designation of Origin specifying the producing honey bee subspecies, offer a unique avenue for authentication. In the MEDIBEES project, we aim to develop a DNA-metabarcoding approach to authenticate honey's entomological origin focusing on mitochondrial lineages A, M, C, and O. To achieve this goal, the DNA from 1251 honey bees representing 16 subspecies (A.m. sahariensis, A.m. intermissa, A.m. siciliana, A.m. ruttneri, A.m. iberiensis, A.m. ligustica, A.m. macedonica, A.m. adami, A.m. cecropia, A.m. cypria, A.m. caucasia, A.m. meda, A.m. anatoliaca, A.m. syriaca, A.m. jemenitica, A.m. lamarcki) was extracted and the whole genome sequenced. From those, 740 mitogenomes were assembled using the MitoZ software. The quality of the assembled mitogenome was assessed by aligning all the sequences using MEGA and 348 samples were deleted. Finally, a phylogenetic analysis was conducted to eliminate non-local subspecies, resulting in a total of 326 mitogenomes. This dataset was used for calculating the fixation index (FST) pairwise values, and a sliding window of 400bp was used to identify single nucleotide polymorphisms that effectively differentiate (FST>0.98) the four lineages, enabling the identification of promising regions for primer design. In this study, three regions were identified that discriminate the four maternal lineages while showing an appropriate length for metabarcoding, namely in the COI, ND1 gene, and CYTB genes.

2024Conference object

Open access

Identification of the entomological origin of European honey by high resolution melting analysis of a COI mini-barcode

Publication . Honrado, Mónica; Lopes, Ana; Pinto, M. Alice; Amaral, Joana S.

Honey is widely consumed worldwide and highly appreciated for its organoleptic, nutritional and health properties. Honey is also considered one of the foods most prone to be adulterated, either by admixing of honey with lower quality, by sugars’ addition, or by origin mislabelling, among other possible frauds. Recently, great attention has been paid to the development of techniques for authenticating honey through its entomological origin, which is also related to its geographical origin, since bees carrying mitochondrial DNA (mtDNA) from distinct ancestries can be found throughout Europe. Moreover, consumers are increasingly concerned with ethical and environmental issues, paying attention to issues such as the protection of biodiversity and the mode of production. For these reasons, the development of methodologies to authenticate the entomological origin of honey contributes not only to assure consumers rights and avoid unfair competition by the identification of frauds, but also to promote and valorise autochthonous honeybee subspecies. In this work, a one-step approach based on HRM analysis of a 150 bp fragment of the COI gene was developed to establish the entomological origin of honey by discriminating A, M and C mtDNA lineages and differentiating a SNP associated with a high frequency of C1 or C2 mitotypes in the Italian honey bee A. m. ligustica and the Carniolan honey bee A. m. carnica. The method showed high analytical performance and was able to successfully identify the entomological origin of honeys of known origin obtained from research apiaries/beekeepers. Therefore, it was applied to 44 commercial honeys from different countries. It confirmed the entomological authenticity of French PDO honeys that should be produced by the Corse ecotype A. m. mellifera. For the remaining honeys, the results were also in good agreement with the declared geographical origin. This method is also capable of indicating the mixture of honeys produced by honey bees of different lineages, although not allowing to identify the lineages or mitotypes in the mixture. This was the case of three honeys from Slovenia that did not cluster with C2 mitotype A. m. carnica as expected, suggesting the mixture of honeys produced by honeybees of different mitotypes.

2022Conference object

Open access