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- Development of LAMP Primers for the Detection of Pyrethroid Resistance Mutations in Varroa destructorPublication . Costa, Maíra; Yadró García, Carlos A.; Lopes, Ana; Bejaoui, Mohamed Khalil; Almeida, Jhennifer; Correia, Lucas; Sánchez, Sara; Li, Fernanda; Pinto, M. Alice; Henriques, DoraVarroa destructor is one of the main threats to Apis mellifera L., directly affecting colony health and contributing to their global decline. Control of this mite is traditionally achieved using acaricides, with pyrethroids such as tau-fuvalinate and fumethrin being the most used, acting on voltage-gated sodium channels (VGSC). However, the intensive use of these compounds by beekeepers has led to the emergence of resistance, associated with mutations at residues 918 and 925 of the VGSC gene [1]. Traditional methods for detecting these mutations, such as PCR, TaqMan and RT-PCR, are eective but require expensive laboratory equipment. In this context, Loop-mediated Isothermal Amplification (LAMP) is a promising alternative, offering rapid and cost-effective detection without the need for thermal cycling [2]. LAMP is based on the use of a set of four to six primers, including two inner primers (FIP and BIP), two outer primers (F3 and B3), and optionally two loop primers (LoopF and LoopB), which are introduced to accelerate the amplification reaction [3]. This study aimed to develop specific LAMP primers, using the NEB LAMP software, for the detection of the main mutations associated with Varroa destructor resistance to pyrethroids in Portugal. The predictive efficiency, specificity, and thermodynamic properties of the designed primers were assessed using BLAST, eLAMP, and OligoAnalyzer tools, considering qPCR parameters. This work successfully identified specific primer sets, including loop primers, for the detection of the mutation at position 925, which may be used in future experimental validations for rapid diagnostic applications.
- Diversity patterns of P450 genes in 17 honey bee subspeciesPublication . Li, Fernanda; Yadró Garcia, Carlos A.; Rufino, José; Rosa-Fontana, Annelise; Verbinnen, Gilles; Graaf, Dirk C.; Smet, Lina de; Pinto, M. Alice; Henriques, DoraHoney bees (Apis mellifera) inhabit a vast geographical range, spanning diverse natural and agricultural ecosystems. They are exposed to different levels and types of natural (such as plant allelochemicals) and synthetic (such as pesticides) xenobiotics within this range. Several genes have been implicated in the resistance of insects to pesticides, including the P450 monooxygenases superfamily that contains 46 genes. Here, the sequences of P450 monooxygenases from >1500 individuals representing 17 subspecies of the four honey bee main lineages will be analyzed. The functional annotation and effects of each variant will then be predicted using SnpEff and the allele frequency and FST (fixation index) of each SNP per population and evolutionary lineages will be calculated. It is expected to have highly differentiated SNPs among the different subspecies/lineages.
- Bioinformatics pipeline to evaluate patterns of diversity in detoxification genes in Western Honey BeePublication . Barbosa, Daniela; Li, Fernanda; Bashir, Sana; Lopes, Ana; Yadró García, Carlos A.; Quaresma, Andreia; Rufino, José; Rosa-Fontana, Annelise; Verbinnen, Gilles; Graaf, Dirk C.; Smet, Lina de; Pinto, M. Alice; Henriques, DoraThe Western honey bee, Apis mellifera, displays significant genetic diversity in detoxification genes, which is pivotal for environmental adaptation and resilience. Herein, we developed a bioinformatics pipeline to investigate patterns of diversity in these genes, focusing on single nucleotide polymorphisms (SNPs) across A. mellifera populations, with variant annotation performed using both snpEff and the Variant Effect Predictor (VEP). Our pipeline integrates GATK, VCFtools, PLINK, bcftools, snpEff, and VEP to process genomic data systematically. Regions of interest were defined in a BED file for variant filtering. Using GATK, SNPs were extracted from a VCF file and conversion to PLINK format for population genetics analyses. Variants were filtered by minor allele frequency (MAF) and population differentiation (FST index) to identify SNPs with considerable. Variants were annotated with snpEff and VEP to predict functional impacts, enabling a comparative analysis of their annotation consistency and depth. Custom scripts were developed to map SNPs to detoxification genes, quantify SNP density, and integrated gene descriptions and lineage data. The resulting data were visualized using a combination of and generate different graphs using ggplot2 and chromoMap for chromossomal maps. Quality control steps were applied through the pipeline ensuring data reliability. Our findings reveal distinct SNP patterns in detoxification genes, highlighting candidate SNPs associated with A. mellifera subspecies-specific adaptations. The comparison of snpEff and VEP annotations provides insights into their strengths and limitations, which can help optimize software selection for genomic studies. This pipeline offers a reproducible framework for studying genetic diversity in A. mellifera that is adaptable to other species, advancing conservation and evolutionary genomics.
- 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.
- Exploring population structure and adaptation in honey bee subspecies from southern glacial refugia: A. M. Iberiensis and A. M. LigusticaPublication . Yadró Garcia, Carlos A.; Henriques, Dora; Cilia, Giovanni; Rufino, José; Martín-Hernández, Raquel; Nanetti, Antonio; Pinto, M. AliceGlacial refugia harbor populations with complex diversity patterns. In honey bees, the Iberian and Italian Peninsulas served as two of the most important glacial refugia in Europe. Here, we analyzed whole genomes generated from drones to infer population structure, genetic diversity, and the molecular basis of the local adaptation for the two native subspecies of these Peninsulas: A. m. iberiensis (N=86; M-lineage) and A. m. ligustica (N=225; C-lineage). For A. m. iberiensis, Admixture analysis revealed a strong cline between two genetic backgrounds from Southwest to Northeast and no C-lineage introgression was detected. For A. m. ligustica, introgression with A. m. carnica occurred in Central and Southern Italy (median q-valuecarnica=0.069; IQR=0.187), away from the natural hybridization zone in Northeastern Italy where higher admixture proportions were detected (median q-valuecarnica=0.229; IQR=0.262). A. m. mellifera introgression was detected especially in the Northwest (median q-valuemellifera 0.053; IQR=0.030), and with lower values in Central and Southern Italy (median q-valuemellifera 0.014; IQR=0.041). A. m. iberiensis showed higher diversity when compared to A. m. ligustica: π (πiberiensis=0.325, πligustica= 0.245, p-value<0.001); He (Heiberiensis=0.319, Heligustica=0.319; p-value<0.001) but lower relatedness (IBD kinshipiberiensis=0.002, IBD kinshipligustica=0.014; p-value<0.001). Selection signatures were detected and cross-validated using PCAdapt, SAMBADA, and RDA. SNPs with q-adjusted p-values < 0.01 detected by at least two methods were considered strong candidates. For A. m. ligustica, 133 candidate SNPs annotated to 125 genes were detected by all three methods, including dnaJ homolog subfamily C member 9, nephrin, and the diuretic hormone receptor, and these were correlated with precipitation. For A. m. iberiensis, 528 SNPs annotated to 527 genes were detected, and these included proteins related to heat-shock response, such as Cyp40 and rrp45. While no common candidate SNPs were detected between both subspecies, 20 common genes containing candidate SNPs were detected, such as 4-coumarate-CoA ligase 1, CPR9, and alpha-mannosidase 2.
- 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.
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