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- Genetic variation of detoxification genes: from genes to proteinsPublication . Henriques, Dora; Li, Fernanda; Bashir, Sana; Quaresma, Andreia; Lopes, Ana Rita; Taliadoros, Demetris; Webster, Matthew; Shiraishi, Carlos S.H.; Yadró Garcia, Carlos A.; Abreu, Rui M.V.; Rufino, José; Rosa-Fontana, Annelise; Verbinnen, Gilles; Graaf, Dirk C. de; De Smet, Lina; Pinto, M. AliceHoney bees (Apis mellifera) are exposed to natural and synthetic xenobiotics, requiring genetic adaptations for survival. Several gene families have been implicated in insect pesticide resistance, including cytochrome P450s, glutathione-S-transferases (GSTs), esterases, and uridine diphosphate (UDP)-glycosyltransferases. This study investigates genetic variation in these detoxification gene families and predicts the structural and functional effects of non-synonymous SNPs (single nucleotide polymorphisms) on protein structure and function. We analyzed SNPs mapped to these detoxification genes extracted from over 1,500 whole genomes representing 15 subspecies and the four main honey bee lineages: M, C, A, and O. Functional annotation and variant effects were predicted using SnpEff. Allele frequencies and each SNP’s fixation index (FST) were calculated per population and evolutionary lineage. Bioinformatics and molecular modeling techniques were employed to evaluate non-synonymous SNPs’ structural and functional consequences. Protein structures were generated from FASTA files using AlphaFold3, converted from mmCIF to PDB format, and visualized in PyMOL. Functional site predictions were performed using Proteins Plus, and molecular dynamics simulations were conducted in YASARA to assess stability and conformational changes in proteins. Our results indicate many non-synonymous SNPs in some subspecies, such as A. m. jemenitica and A. m. intermissa. The genes with the highest number of non-synonymous mutations belong to the CYP family, particularly Probable cytochrome P450 6a14 and CYP9Q1. Conversely, genes such as Cytochrome P450 6k1 and Methyl farnesoate epoxidase exhibit no non-synonymous SNPs. By understanding intraspecific genetic variation, we move closer to reliably predicting how honey bee populations will respond to pesticide exposure.
- Diversity patterns of P450 and ABC transporter genes in 17 honey bee subspeciesPublication . Li, Fernanda; Rosa-Fontana, Annelise; Yadró Garcia, Carlos A.; Rufino, José; Verbinnen, Gilles; Graaf, Dirk C. de; De Smet, Lina; 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 and ATP-binding cassette sub-family F member 1that contain 46 and 41 genes, respectively. Here, the sequences of P450 monooxygenases and ABC transporters 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.
- Large Scale Mitochondrial Evidence of C-lineage Dominance in European Honey BeesPublication . Li, Fernanda; Lopes, Ana Rita; Costa, Maíra; Henriques, Dora; Quaresma, Andreia; Yadró Garcia, Carlos A.; Albo, Alexandre; Čereškienė, Laima Blažytė; Brodschneider, Robert; Brusbardis, Valters; Carreck, Norman L.; Charistos, Leonidas; Chlebo, Robert; Coffey, Mary F.; Dahle, Bjørn; Danneels, Ellen; Dobrescu, Constantin; Dupleix-Marchal, Anna; Filipi, Janja; Gajda, Anna; Gratzer, Kristina; Groeneveld, Linn Fenna; Hatjina, Fani; Johannesen, Jes; Kolasa, Michal; Körmendy-Rácz, János; Kovačić, Marin; Kristiansen, Preben; Martikkala, Maritta; McCormack, Grace P.; Martín-Hernández, Raquel; Pavlov, Borce; Poirot, Benjamin; Pietropaoli, Marco; Radev, Zheko; Raudmets, Aivar; René-Douarre, Vincent; Roessink, Ivo; Škerl, Maja Ivana Smodiš; Soland, Gabriele; Titera, Dalibor; Van der Steen, Jozef; Varnava, Andri; Vejsnæs, Flemming; Fedoriak, Mariia M.; Zarochentseva, Oksana; Webster, Matthew T.; Graaf, Dirk C. de; Pinto, M. AliceIn Europe, distribution of the several endemic honey bee (Apis mellifera) subspecies has suffered a considerable shift in the last century. In particular, beekeepers tend to favour subspecies of Eastern European ancestry (C-lineage), such as the Italian honey bee (A. m. ligustica), due to their perceived docility and high honey production. As a result, large scale migratory beekeeping and trade of C-lineage queens have exposed the native European honey bees to introgression and replacement, jeopardizing their genetic integrity and locally adapted traits. The maternally-inherited and highly polymorphic mitochondrial intergenic region tRNAleu-cox2 is routinely used for the assessment of honey bee diversity and introgression at large geographical scales. In this study, we conducted a survey on tRNAleu-cox2 variation in more than 1300 colonies from 33 European countries to assess current status of mitochondrial diversity patterns in Europe. Total genomic DNA was extracted from the bee thorax followed by PCR amplification of the tRNAleu-cox2 region and sequencing. The haplotypes were identified using alignments in MEGA 11. A clear dominance of C-lineage haplotypes was found (65%). The most prevalent C-lineage haplotype was C2 (54%) followed by C3 (23%) and C1(22%). Contrary to this trend, in Portugal, Spain and Ireland, less than 10% of the colonies exhibit the C-haplotype. Furthermore, the analysed apiaries in isolated protected areas (n=7) revealed a high proportion of colonies of western European (M-lineage) ancestry (80%) with M4 as the most frequent haplotype. African haplotypes (A lineage) were also found, albeit at lower frequencies, and were mainly concentrated in the Iberian Peninsula (9%). Intensive queen breeding and migratory beekeeping is homogenizing the gene pool of European bee populations. This survey of honey bee maternal diversity across Europe highlights the alarming dominance of C-lineage haplotypes and underscores the importance of conservation apiaries, as they have effectively preserved the autochthonous M-lineage subspecies in different countries. This work was conducted in the framework of the project Better-B, funded by the European Union, the Swiss State Secretariat for Education, Research, and Innovation, and UK Research and Innovation under the UK government's Horizon Europe funding guarantee (grant number 10068544).
- Evaluating the consequences of plant protection products in the Western honey bee (Apis mellifera) genomePublication . Barbosa, Daniela; Li, Fernanda; Bashir, Sana; Yadró Garcia, Carlos A.; Taliadoros, Demetris; Webster, Matthew; Rufino, José; Roessink, Ivo; Buddendorf, Bas; van der Steen, Jozef; Murcia, Maria; Fernández-Alba, Amadeo Rodríguez; Pinto, M. Alice; Henriques, DoraThe Western honey bee (Apis mellifera)is a key model organism for evaluating risks from Plant Protection Products (PPPs). Despite well-documented impacts of PPPs on honey bees survival and behaviour, their molecular consequences remain underexplored. Herein, over 472 whole genomes from samples collected across Europe in the framework of the project Better-B were used to investigate the genetic basis of PPP exposure through Genomic-Environment Association (GEA) analysis. By integrating genomic data from honey bee colonies with processed PPP exposure data collected in the framework of the project INSIGNIAEU and from the PEST-CHEMGRIDS database as well as from environmental datasets from CORINE land cover, this study used SAMBADA (a spatial analysis tool) and Redundancy Analysis (RDA) scripts to identify candidate genes potentially linked to pesticide stress. This research addresses a knowledge gap, offering a pathway to mitigate PPPrelated molecular effects and support sustainable beekeeping, and can inform breeding programs to bolster honey bee resilience. Ultimately, this work advances our understanding of PPP impacts at the molecular level, fostering resilience in a key pollinator essential for global food security.
- A Computational Approach to Study Non-Synonymous MutationsPublication . Bashir, Sana; Li, Fernanda; Shiraishi, Carlos S.H.; Yadró Garcia, Carlos A.; Barbosa, Daniela; Abreu, Rui M.V.; Rufino, José; Pinto, M. Alice; Henriques, DoraNon-synonymous SNPs (Single Nucleotide Polymorphisms) result in amino acid substitutions within proteins. While some may have minimal impact on protein structure and function, others can significantly alter stability, conformation, and biological activity. Therefore, it is crucial to predict how SNP-induced changes affect protein structure and function. Here, we developed a novel pipeline that integrates bioinformatics and molecular modeling techniques to evaluate the structural and functional consequences of a non-synonymous SNP in a protein. Initially, Protein Plus was used to predict the potential active sites, which will help to determine whether a mutation is located within the functional binding regions. Identification of active sites is necessary to prioritize mutations that may alter protein structure and function. PolyPhen-2 and I-Mutant were used to evaluate the functional impact of mutations and the thermodynamic stability of the proteins. Next, to visualize the structural changes, AlphaFold3 was used to generate highconfidence 3D models for wild and mutant proteins. Finally, molecular dynamics (MD) simulations were conducted using YASARA to explore the dynamic behavior and stability of both mutant and wild-type proteins. MD simulations provide valuable insight into structural flexibility, potential conformational shifts, and the overall impact of the mutation on protein function. This computational pipeline provides a detailed framework to evaluate the structural and energetic consequences of non-synonymous mutations.
- An unprecedented large-scale survey of honey bee mitochondrial diversity in Europe: c-lineage dominance and the need for conservation effortsPublication . Li, Fernanda; Costa, Maíra; Lopes, Ana Rita; Gonçalves, Telma; Henriques, Dora; Quaresma, Andreia; Yadró Garcia, Carlos A.; Albo, Alexandre; Blažytė-Čereškienė, Laima; Brodschneider, Robert; Brusbardis, Valters; Carreck, Norman L.; Charistos, Leonidas; Chlebo, Robert; Coffey, Mary F.; Dahle, Bjørn; Danneels, Ellen; Dobrescu, Constantin; Dupleix-Marchal, Anna; Filipi, Janja; Gajda, Anna; Gratzer, Kristina; Groeneveld, Linn Fenna; Hatjina, Fani; Johannesen, Jes; Kolasa, Michal; Körmendy-Rácz, János; Kovačić, Marin; Kristiansen, Preben; Martikkala, Maritta; McCormack, Grace P.; Martín-Hernández, Raquel; Pavlov, Borce; Pietropaoli, Marco; Poirot, Benjamin; Radev, Zheko; Raudmets, Aivar; René-Douarre, Vincent; Roessink, Ivo; Škerl, Maja Ivana Smodiš; Soland-Reckeweg, Gabriele; Titera, Dalibor; Van der Steen, Jozef; Varnava, Andri; Vejsnæs, Flemming; Webster, Matthew T.; Fedoriak, Mariia M.; Zarochentseva, Oksana; Graaf, Dirk C. de; Pinto, M. AliceEurope is home to ten Apis mellifera subspecies, which belong to three mitochondrial lineages: the Western European (M), Eastern European (C), and African (A). However, the long-standing human-mediated movement of queens, primarily of C-lineage ancestry, has threatened the genetic integrity of many of these native subspecies through introgression and replacement. This has led to the establishment of conservation programs to recover the native lines in some European countries. The maternally-inherited mitochondrial DNA (mtDNA), particularly the highly polymorphic intergenic region tRNAleu-cox2, has been the marker of choice for assessing honey bee variation and introgression at large geographical scales. Herein, we will show the results of the tRNAleu-cox2 variation obtained from over 1200 colonies sampled across the range of the ten subspecies and covering 33 European countries. These revealed that apart from a few countries (Portugal, Spain, and Ireland) and isolated protected populations, European populations are predominantly dominated by C-lineage haplotypes, and many native subspecies exhibit a signature of C-derived introgression. In conclusion, this unprecedented survey of honey bee diversity across Europe underscores the concerning dominance of C-lineage genetic variation, highlighting the urgent need for strategic conservation efforts to preserve the native genetic diversity of Apis mellifera.
- Development of LAMP Primers for the Detection of Pyrethroid Resistance Mutations in Varroa destructorPublication . Costa, Maíra; Yadró Garcia, Carlos A.; Lopes, Ana Rita; 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 effective 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.
- An unparalleled survey of honey bee genetic diversity in Europe – insights from wing shape, nuclear whole genome, and mitochondrial DNA dataPublication . Pinto, M. Alice; Li, Fernanda; Lopes, Ana Rita; Taliadoros, Demetris; Costa, Maíra; Yadró Garcia, Carlos A.; Cunha, Larissa; Henriques, Dora; Martin Hernandez, Giselle; Albo, Alexandre; Blažytė-Čereškienė, Laima; Brodschneider, Robert; Brusbardis, Valters; Carreck, Norman; Charistos, Leonidas; Chlebo, Robert; Cillia, Giovanni; Coffey, Mary F.; Dahle, Bjorn; Danneels, Ellen; Dobrescu, Constantin; Filipi, Janja; Gajda, Anna; Gratzer, Kristina; Groeneveld, Linn; Hatjina, Fani; Johannesen, Jes; Kolasa, Michal; Körmendy-Rácz, János; Kovačić, Marin; Kristiansen, Preben; Dupleix-Marchal, Anna; Martikkala, Maritta; McCormack, Grace; Martín-Hernández, Raquel; Nanetti, Antonio; Pavlov, Borce; Pietropaoli, Marco; Poirot, Benjamin; Radev, Zheko; Raudmets, Aivar; René-Douarre, Vincent; Roessink, Ivo; Škerl, Maja Ivana Smodiš; Soland, Gabriele; Titera, Dalibor; Van der Steen, Jozef; Varnava, Andri; Vejsnæs, Flemming; Graaf, Dirk C. de; Webster, MathewEurope is home to 10 subspecies of Apis mellifera, classified into four main lineages sensu Ruttner: M (western and northern European), C (southeastern European), A (African), and O (Middle Eastern). However, large-scale movements of honey bee genetic material—driven by transhumance and, more importantly, queen trading—has intensified gene flow, particularly from the C-lineage subspecies A. m. carnica and A. m. ligustica, threatening the genetic integrity of several subspecies and blurring the genetic boundaries between subspecies. Here, we analyzed over 1,200 recently collected samples from apiaries in 33 countries using a multi-marker approach: maternally inherited mitochondrial DNA (tRNAleu-cox2 intergenic region), nuclear genome-wide single nucleotide polymorphisms (SNPs), and wing shape (DeepWings©) to provide the most updated and extensive account of honey bee genetic variation in Europe. Our results reveal a strong dominance of C-lineage ancestry across Europe, with noteworthy introgression signals in island subspecies and A. m. mellifera populations outside conservation apiaries. Notably, all three markers consistently captured these broad introgression patterns. This unprecedented survey highlights a worrying trend of genetic homogenization in European honey bee populations. Moreover, the widespread prevalence of C-lineage genetic introgression underscores the need for conservation efforts to preserve the diversity shaped by evolution, which is crucial for adaptation to growing environmental challenges.
- Why single snp analyses fail: epistatic structural effects in honey bee CYP336A1Publication . Li, Fernanda; Lima, Daniela; Bashir, Sana; Yadró Garcia, Carlos A.; Graaf, Dirk C. de; De Smet, Lina; Verbinnen, Gilles; Rosa-Fontana, Annelise; Rufino, José; Martín-Hernández, Raquel; Pinto, M. Alice; Henriques, DoraCytochrome P450 enzymes are central to pesticide metabolism and resistance, yet how these proteins diversify substrate specificity while maintaining catalytic function remains poorly understood. A genome-wide analysis of CYP336A1 (a nicotine-metabolizing P450) across 1467 Apis mellifera males from 25 countries spanning the Mediterranean, Middle East, Europe, and Cuba revealed an intricate haplotype architecture. Despite the detection of only 28 single-nucleotide variants (SNPs), 45 distinct haplotypes were detected for CYP336A1. Among these, 23 haplotypes carried at least four SNPs, and four harboured more than 10. A five-SNP haplotype (D202G; M207I; I222V; V226I; Q238K) dominated at 36% frequency, far exceeding the next most common single-SNP haplotype (D262N, 9%). Interestingly, this dominant haplotype was completely absent from the Iberian Peninsula, North Africa, and Oman and, consequently, from five A. mellifera subspecies: iberiensis, intermissa, jemenitica, mellifera and sahariensis. To investigate the functional impact of the identified variants, individually and in combination, we used in sillico protein structural approaches. Protein models were generated with trRosetta, validated with MolProbity, and evaluated using TM-score and RMSD via TM-Align. Structural modelling revealed remarkable fold congruency: the enzyme encoded by the five-SNP haplotype retained a near-identical fold as compared to the wild-type enzyme (TM-score = 0.998, RMSD = 0.34 Å), as did a rarer 13-SNP haplotype (2%) (TM-score = 0.998, RMSD = 0.38 Å). Individual SNPs also produced minimal backbone displacement (0.32–0.54 Å), suggesting that P450 diversification proceeds through subtle structural adjustments rather than major disruption. Moreover, most SNPs clustered within substrate-recognition regions, whereas catalytic residues remained invariant across haplotypes, demonstrating a partitioning between substrate-recognition/binding evolution and preservation of catalytic machinery. Importantly, single-variant effects cannot predict multi-variant haplotype outcomes. As such, heavy reliance on individual SNPs for pesticide risk assessment may misestimate real metabolic capacity.
- Landscape-scale genomic responses of the western honey bee (Apis mellifera) to pesticide pressurePublication . Lima, Daniela; Li, Fernanda; Yadró Garcia, Carlos A.; Taliadoros, Demetris; Webster, Matthew; Rufino, José; Roessink, I.; Buddendorf, Bas; Van der Steen, Jozef; Murcia-Morales, María; Fernández-Alba, Amadeo R.; Graaf, Dirk C. de; Lopes, Ana Rita; Pinto, M. Alice; Henriques, DoraWidespread pesticide use associated with intensive agriculture has been proven to impact the Western honey bee (Apis mellifera). However, despite evidence of declines in survival, development, foraging efficiency, and overall colony health, the genomic underpinning of this abiotic stressor is largely unknown. This study involved 102 whole-genome sequences from honey bee colonies from across Europe, sampled under the Better-B project, to search for genetic correlations to pesticide exposure using Genomic-Environment Association (GEA) analyses. The environmental exposure data were collected within the framework of the INSIGNIA-EU project, which used APIStrips (in-hive pesticide-adsorbing strips) to quantify pesticide residues brought into the hive by foraging in many of the sampled colonies. The APISTRIP data were complemented by modelled exposure grids from PEST-CHEMGRIDS and agriculture zones from the CORINE Land Cover. Employing three complementary GEA approaches-SAMBADA (spatial analysis tool), LFMM (latent factor mixed models), and RDA (Redundancy Analysis)—we identified SNPs in the bee’s genome significantly correlated with agricultural pressure and pesticide use. Notably, several genes with known roles in detoxification and stress response, including venom carboxylesterase6 and CYP336A1, were highlighted. These variants point to molecular pathways targeted by agrochemical toxicity, offering insights into the consequences of pesticide use. Such findings may, in the future and upon further validation, help refine risk assessment frameworks, support the development of resilience-oriented breeding programs, and promote sustainable apicultural practices.