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- Comparative Analysis of DNA Extraction Methods for Individual Varroa destructorPublication . Costa, Maíra; Lopes, Ana; Yadró Garcia, Carlos A.; Vitrio, Nathalia; Gonçalves, Telma; Pinto, M. Alice; Henriques, DoraThe ectoparasitic mite Varroa destructor is one of the major honey bee threats and it is associated to population worldwide decline. Genetic analyses using the mtDNA of V. destructor are fundamental for establishing the taxonomy and distribution of the mites. Consequently, low-quality DNA can lead to inaccurate or inconsistent data, making genetic interpretation more challenging. In this study, we compared the concentration and quality of DNA extracted from individual female V. destructor using two different commercial kits, aiming to identify the optimal method for obtaining high-quality DNA. Total DNA was extracted from mites using both an automated and manual extracted method. In addition, manual kit extraction tested three incubation procedure (1h, 5h, and overnight). DNA concentration was quantified using three different instruments: the SpectroStarVR Nano LVis Plate spectrophotometer, the Quantus™ Fluorometer apparatus, and NanoDrop™. The manual extraction DNA concentration did not vary across incubation times and the concentration values varied between 0.240-0.545 ng/μl (Quantus), 0.72-4.49 ng/μl (spectrophotometer), and 0.0-1.47 ng/μl (NanoDrop). While extraction automatic approach yielded higher respectively 0.483-0.631 ng/μl, 20.33-9.0 ng/μl, and 5.3-6.8 ng/μl. In conclusion, the automated kit extraction seems to be the best extraction method since it produced the higher-concentration DNA using only one individual mite.
- Exploiting the mitogenomes of apis mellifera subspecies to develop an authentication tool to verify the entomological origin of mediterranean honeysPublication . 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.
- Estrutura populacional e estado de conservação das subespécies de Apis mellifera no Oriente Próximo e MédioPublication . Yadró Garcia, Carlos A.; Henriques, Dora; Honrado, Mónica; Amaral, Joana S.; Eissa, Asmaa Anwar; Haddad, Nizar; Obeidat, Wisam; Arruda, James; Lamghari, Fouad; Cilia, Giovanni; Martín-Hernández, Raquel; Nanetti, Antonio; Pinto, M. AliceA abelha melífera, Apis mellifera, é composta por 31 subespécies que se encontram distribuídas na Ásia, África e Europa. O objetivo deste trabalho é desvendar a estrutura populacional e verificar o estado de conservação de três subespécies do Médio Oriente, as quais têm sido pouco estudadas. Para isso, foi extraído o DNA a partir de tóraxes inteiros de machos de 329 amostras de A. m. lamarckii (Egito, 68 amostras), A. m. syriaca (Jordânia, 238 amostras) e A. m. jemenitica (Omã e Emirados Árabes Unidos, 23 amostras). Foram adicionadas 21 amostras de A. m. ligustica, que é uma subespécie amplamente utilizada pelos apicultores no mundo inteiro e por isso fonte de introgressão genética. O genoma completo das 329 amostras foi sequenciado na plataforma Illumina NovaSeq 600 tendo como objetivo uma cobertura de 20X. Os 329 genomas foram mapeados usando o genoma de referência Amel_HAv3.1 e foi implementada uma pipeline que garante a qualidade dos dados. No final, obteve-se um total de 4.030.485 de SNPs que foram usados na reconstrução da estrutura populacional com o ADMIXTURE e PCA. As amostras egípcias mostraram que apesar de terem alguma introgressão de A. m. ligustica, essa não é relevante e é variável (Q-values entre 1E-05 e 0.44), com a maior parte (97%) das amostras apresentando um valor médio de 0.07 ± 0.06 (Q-values, meia ± DP). A. m. syriaca apresenta uma estrutura complexa, tendo sido observados dois grupos distintos pelo PCA e três pelo ADMIXTURE. Relativamente seu ao estado de conservação, foram detetados 76 indivíduos com uma proporção considerável (Q-values entre 0.15 e 0.47) de introgressão com A. m. ligustica. No caso de A. m. jemenitica, foram observados dois cenários diferentes. Em Omã, todas as amostras estudadas mostraram ser puras. Por outro lado, apenas sete amostras dos Emirados Árabes Unidos foram classificadas como tal, enquanto as restantes mostraram proporções de introgressão semelhantes às do Egito. Estes resultados evidenciam o estado precário de integridade genética que estas subespécies apresentam nos locais estudados. No entanto, a existência de indivíduos que podem ser considerados puros para suas respetivas subespécies pode servir como ponto de partida para o desenvolvimento de planos de conservação.
- Projeto MITE- Varroa e vírus transmitidos: MonItorização de muTações e dEsenvolvimento de ferramentas moleculares inovadorasPublication . Henriques, Dora; Yadró Garcia, Carlos A.; Lopes, Ana; Costa, Maíra; Rufino, José; Martín-Hernández, Raquel; Higes, Mariano; Silva, Dinis; Pinto, M. AliceO ácaro ectoparasita varroa (Varroa destructor), que causa a doença varroose, e alguns dos vírus transmitidos, como o vírus das asas deformadas (Deformed wing vírus – DWV), são apontados como umas das mais importantes ameaças para a abelha melífera a nível global. O controle mais eficaz da varroa envolve o uso de acaricidas, sintéticos ou orgânicos. No entanto, o uso intensivo dos acaricidas sintéticos tem levado ao desenvolvimento de resistência da varroa ao tratamento em muitas regiões do mundo, o que tem causada uma maior perda de colónias. A base molecular de alguns dos mecanismos de resistência da varroa às moléculas sintéticas mais usadas (os piretroides formamidinas) foi descrita recentemente. Esta informação, quando associada a testes genéticos de fácil implementação, permite a monitorização das populações de varroa o que poderá ajudar na luta integrada contra a varroose. Ao contrário do que acontece com a varroa, para os vírus não há nenhum tratamento disponível. Em Portugal continental, não é conhecida a distribuição e prevalência dos vírus mais importantes das abelhas. No entanto, compreender a distribuição e disseminação das doenças é essencial ao desenvolvimento de estratégias adequadas ao seu controlo e contenção. Genericamente, neste projeto pretende-se verificar se existem em Portugal populações de varroa portadoras dos alelos que conferem resistência aos piretróides e ao amitraz, e em caso afirmativo estudar a sua distribuição geográfica. Pretende-se também modernizar o setor apícola ao desenvolver-se ferramentas moleculares inovadoras que possam ser facilmente usadas na luta integrada contra a varroose e concomitantemente na deteção dos vírus associados, como o DWV.
- A sequenciação de nova geração como uma abordagem promissora para a identificação da origem entomológica do melPublication . Honrado, Mónica; Henriques, Dora; Yadró Garcia, Carlos A.; Santos, Joana; Rufino, José; Medibees Consortium; Pinto, M. Alice; Amaral, Joana S.O mel é um alimento muito consumido e apreciado em todo o mundo pelas suas propriedades nutricionais e organoléticas, bem como pelos seus efeitos benéficos para a saúde. No entanto, é também considerado um dos alimentos mais suscetíveis de ser adulterado, quer pela mistura de mel de qualidade inferior, quer pela adição de açúcares, ou pela rotulagem incorreta da origem botânica e/ou geográfica, entre outras possíveis fraudes. Nos últimos anos, tem sido dada uma atenção crescente à origem entomológica do mel, uma vez que esta também está relacionada com a origem geográfica. No âmbito do projeto PRIMA “MEDIBEES” (https://medibees.org/), a sequenciação de nova geração (NGS) será utilizada com vista ao desenvolvimento de ferramentas moleculares que permitam identificar a origem entomológica de amostras de mel provenientes dos 8 países mediterrânicos do consórcio, de forma a diferenciar e valorizar méis produzidos por abelhas autóctones destes países. Com este objetivo, inicialmente procedeu-se à construção da base de dados das sequências de DNA mitocondrial das abelhas de modo a incluir 10 subespécies mediterrânicas das 4 linhagens maternas (A, M, C e O). Para tal, procedeu-se à extração de DNA e à respetiva sequenciação dos genomas completos, na plataforma Illumina Novaseq 6000, de um total de 1095 abelhas destes países. Posteriormente, utilizou-se o programa mitoZ 3.6 para fazer a montagem do genoma mitocondrial de cada uma das amostras, resultando na seleção de 283 sequências mitocondriais com boa montagem. Em seguida, foi utilizado o software MEGA 11, para realizar o alinhamento destas sequências. A informação obtida será posteriormente utilizada para a seleção de regiões com variantes (SNPs) informativos que possam ser usadas para o desenho de primers adequados e desenvolvimento de ferramentas para a identificação de méis produzidos por abelhas de diferentes linhagens mitocondriais e respetivas subespécies.
- Challenges in varroosis control: preliminary investigation of amitraz resistance in varroa destructor in PortugalPublication . Costa, Maíra; Lopes, Ana; Yadró Garcia, Carlos A.; Gonçalves, Mariana Lousada; Coelho, Liliana; Pires, Sancia; Pinto, M. Alice; Henriques, DoraVarroosis is a disease caused by the ectoparasitic mite Varroa destructor, identified as one of the most significant global threats to the honey bee (Apis mellifera). The most effective control of this mite is through synthetic or organic acaricides. However, the excessive and repeated use of synthetic acaricides has led to the development of resistance. Amitraz is a synthetic pesticide commonly used in the control of V. destructor, but resistance to this compound has been observed. Previous studies observed a substitution of asparagine by serine at position 87 (N87S) of the Octβ2R gene, associated with amitraz resistance in France, and a substitution of tyrosine by histidine at position 215 (Y215H), associated with amitraz resistance in the USA. Building upon this knowledge, we aim to implement the first screening in Portugal for mutations associated with V. destructor resistance to amitraz. Unlike several European countries and the USA, Portugal lacks information regarding gene variation implicated in V. destructor resistance to amitraz, as well as allelic frequencies and their geographical distribution. To investigate the resistance mechanism, primers were developed to amplify the two known target regions of amitraz in V. destructor. DNA was extracted from individual female varroa mites using a commercial extraction kit, and the obtained DNA was PCR-amplified with the developed primers, followed by Sanger sequencing. With the knowledge obtained, we hope to assist beekeepers in selecting the most suitable acaricide to manage V. destructor in their apiaries and gain a deeper understanding of amitraz resistance in Portugal.
- Insights into population structure and conservation status of north African honey beesPublication . Yadró Garcia, Carlos A.; Henriques, Dora; Haider, Yamina; Eissa, Asmaa Anwar; Aglagane, Abdessamad; Rufino, José; Nanetti, Antonio; Martín-Hernández, Raquel; Pinto, M. AliceThe population structure of North African A. mellifera subspecies has been overlooked. Here, WG generated from drones were analyzed to assess population structure and the conservation status of three A-lineage subspecies: A. m. lamarckii (Egypt, 68 samples), A. m. intermissa (Algeria, 69 samples), and A. m. sahariensis (Algeria, 68 samples; Morocco, 26 samples). Population structure was assessed by SNMF and PCA. For A. m. lamarckii, variable C-lineage introgression was detected (median q-valueC-lineage=0.068; IQR=0.074) and PCA showed a well-grouped cluster slightly shifted towards C-lineage. Moroccan samples showed a highly compact group close to, but distinct from, the Algerian samples, and without signals of C-lineage introgression. In Algeria, only 28 A. m. sahariensis samples showed high purity values for this subspecies, and the remaining 48 samples showed variable introgression from A. m. intermissa (q-valueintermissa=0.127; 0.021). A more concerning scenario was found for A. m. intermissa, in which only 17 samples were classified as pure A. m. intermissa, 21 samples showed variable A. m. sahariensis introgression (q-valuesahariensis=0.125; 0.016), and the remaining 32 samples showed to be pure A. m. sahariensis. In the PCA analysis, a large portion of Algerian samples formed a cluster containing individuals of both subspecies. The remaining Algerian samples formed five well-defined isolated clusters: three containing A. m. sahariensis samples and two containing A. m. intermissa samples. SNMF runs at K=7 for the Algerian samples also recovered the existence of these high-purity isolated clusters. While most A. m. intermissa samples were located in the northern part of Algeria, some were located farther south. For A. m. sahariensis, a large portion of the samples were located close to the Mediterranean coast. These results suggest that, in addition to the C-lineage introgression (especially in A. m. lamarckii), the geographical delimitation originally described by Ruttner does not exist anymore in North Africa.
- Exploiting the mitogenomes of Apis mellifera subspecies to authenticate the origin of Mediterranean honeysPublication . Honrado, Mónica; Henriques, Dora; Santos, Joana; Yadró Garcia, Carlos A.; Medibees Consortium; Pinto, M. Alice; Amaral, Joana S.As defined by the Codex Alimentarius, honey is the natural sweet substance produced by honeybees from the nectar of plants.1 This natural product is widely appreciated but is also considered one of the foods most prone to adulteration. The increasing demand for monofloral honey and those with protected designation of origin (PDO) has led to increased fraud by mislabeling botanical and geographical origin.2 Verifying the geographical origin of honey is a challenging endeavor. Recently, attention has been paid to the entomological origin, as it aligns with the geographical patterns of honeybee subspecies. The Mediterranean region is a hot spot of Apis mellifera subspecific diversity shaped by thousands of years of evolution. Although contemporary human-mediated movements of queens have impacted the native subspecific distribution, several PDO honeys specify the subspecies that produce those honeys, thus offering a unique avenue for authentication. As part of the European PRIMA project MEDIBEES, 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, the DNA of 1280 honeybees representing 16 subspecies and the four lineages (A.m. sahariensis, A.m. intermisa, 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. caucasica, A.m. meda, A.m. anatoliaca, A.m. syriaca, A.m. jemenitica, A.m. lamarcki) was extracted, and their whole genomes were sequenced. The MitoZ software was used to assemble the mitochondrial genomes, resulting in 769 mitochondrial genomes successfully assembled. Subsequently, each of these genomes was aligned individually with a reference genome using MEGA software, and mitogenomes not specific to Apis mellifera were discarded. Of these, only the mitogenomes corresponding to the native ancestry were retained, resulting in a final set of 355 mitogenomes in the database. A phylogenetic analysis was conducted with the final 355 mitochondrial sequences, revealing four distinct clusters corresponding to the four maternal lineages. This dataset was used for calculating the fixation index (FST) pairwise values, and a sliding window of 400 bp was used to identify single nucleotide polymorphisms (SNPs) that effectively differentiate (FST>0.98) the four lineages, enabling the identification of promising regions for primer design. This work resulted in the discovery of three promising regions for discriminating the four maternal lineages: one in the COI gene, one in the ND1 gene, and one in the CYTB gene (Fig. 1).
- Genomic insights into middle eastern honey bee subspecies: population structure and genetic integrityPublication . Henriques, Dora; Yadró Garcia, Carlos A.; Honrado, Mónica; Amaral, Joana S.; Muz, Mustafa Necati; Muz, Dilek; Haddad, Nizar; Al Shagour, Banan; Obeidat, Wisam; Hosri, Chadi; Arab, Alireza; Arruda, James; Lamghari, Fouad; Rufino, José; Martín-Hernández, Raquel; Nanetti, Antonio; Pinto, M. AliceThe genetic patterns of Middle Eastern A. mellifera subspecies have been understudied, hindering a comprehensive understanding of honey bee evolutionary history. Here, we studied the genetic integrity of five Middle Eastern subspecies across a broad geographical range: Turkey (A. m. anatoliaca, N=97; A. m. caucasia, N=75; A. m. syriaca, N=18), Jordan and Lebanon (A. m. syriaca, N=238 and N=29), Iran (A. m. meda, N=75), Oman, and the UAE (A. m. jemenitica, N=13 and N=10). ADMIXTURE and PCA analyses were conducted on SNPs detected from whole-genomes. Our findings reveal concerning conservation statuses for many populations/subspecies. In A. m. caucasia and A. m. anatoliaca, only 10 and 28 samples, respectively, were pure (introgression < 90%). In the A. m. caucasia range, 60 samples were hybrids of A. m. caucasia, A. m. syriaca, and A. m. ligustica. In the A. m. anatoliaca range, 69 samples showed high hybridization degrees with A. m. syriaca, and A. m. caucasia. Only six samples in the Turkish range of the A. m. syriaca range were identified as pure, while the rest were also hybrids. All samples from Jordan and Lebanon showed variable A. m. ligustica introgression. In Iran, 23 samples were classified as pure A. m. meda. The rest showed introgression primarily due to A. m. ligustica and A. m. caucasia. In the UAE, two main groups were identified: the first comprised hybrids of A. m. jemenitica, A. m. lamarckii and A. m. ligustica, and the second group mainly consisted of hybrids of A. m. lamarckii and A. m. ligustica. Oman stands out as the sole location where all samples were identified as pure A. m. jemenitica. This study indicates widespread hybridization across various regions and underscores the urgent need for targeted conservation efforts for Middle Eastern subspecies.
- Distribution of Alleles Linked to Pyrethroid and Amitraz Resistance in Varroa destructor across PortugalPublication . Costa, Maíra; Yadró Garcia, Carlos A.; Lopes, Ana; Quaresma, Andreia; Rodrigues, Claúdia; Pinto, M. Alice; Henriques, DoraVarroa destructor, an ectoparasitic mite of honey bees (Apis mellifera), is a significant threat to apiculture by causing varroosis and transmitting dangerous viruses such as the deformed wings virus. This mite can be controlled by the use of synthetic or organic acaricides. Currently, in Portugal, two classes of synthetic compounds are used: pyrethroids (fluvalinate and flumethrin), and formamidines (amitraz). However, excessive and repeated use of acaricides has led to resistance in mite populations. Resistance to pyrethroids is primarily associated with mutations in the voltage-gated sodium channel gene, and involves an amino acid change at position 925, where a leucine (L) is found in the wild type. At this position, three alleles have been described that confer Varroa resistance to pyrethroids, where leucine is replaced by valine (L925V), isoleucine (L925I), or methionine (L925M). A novel mutation at position 918 was described in a population in the Valencian Community in Spain, where the amino acid methionine, generally found at position 918, was replaced by leucine (M918L). For amitraz, resistance is linked to mutations in the Octβ2R gene, involving a substitution of asparagine by serine at position 87 (N87S), found in France, and a substitution of tyrosine by histidine at position 215 (Y215H), found in the USA. However, the distribution of these resistance-associated alleles remains unexplored in Portugal. To address this gap, we sampled Varroa mites from honey bee colonies from different locations in Portugal, and used a PCR assay with mutation-specific primers, followed by Sanger sequencing of the amplicons. The results confirmed pyrethroid resistance alleles at positions 925 and 918 in the Portuguese populations. In contrast, amitraz-resistance alleles remained undetected, although this result should be interpreted with caution as the sample size was reduced. These findings represent a step forward for understanding the status of Varroa resistance in Portugal, providing baseline data for the development of more effective, region-specific management strategies. Further research with larger sample sizes is essential to confirm these preliminary observations.