Biblioteca Digital do IPB
Repositório de Publicações do Instituto Politécnico de Bragança
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A Computational Approach to Study Non-Synonymous Mutations
Publication . Bashir, Sana; Li, Fernanda; Shiraishi, Carlos S.H.; Yadró Garcia, Carlos A.; Barbosa, Daniela; Abreu, Rui M.V.; Rufino, José; Pinto, M. Alice; Henriques, Dora
Non-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.
Evaluating the consequences of plant protection products in the Western honey bee (Apis mellifera) genome
Publication . 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, Dora
The 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.
Uso de Acaricidas e risco de resistência da Varroa destructor em apiários de Portugal
Publication . Costa, Maíra; Rodrigues, Alves Cláudia; Yadró Garcia, Carlos A.; Lopes, Ana Rita; Pinto, M. Alice; Henriques, Dora
A Varroa destructor é um ácaro ectoparasita amplamente reconhecido como uma das principais ameaças à apicultura. Além de causar danos diretos às abelhas, atua como vetor de diversos vírus, comprometendo de forma significativa a saúde das colônias de Apis mellifera (abelha-europeia). Este estudo avaliou as práticas de manejo adotadas por apicultores portugueses no controle desse parasita, com base na análise de 96 questionários respondidos por 86 apicultores distribuídos em diferentes
regiões de Portugal. Observou-se uma predominância marcante da apicultura convencional, com uso majoritário de acaricidas sintéticos, especialmente produtos à base de amitraz, mencionados em 75% das respostas. Embora as recomendações oficiais apontem para dois tratamentos anuais, a maioria dos apicultores relatou realizar três ou mais, sendo que 53 afirmaram ter aumentado progressivamente a dosagem dos produtos ao longo do tempo, possivelmente em resposta à percepção de perda de
eficácia dos acaricidas. Apenas 42% realizam efetivamente a rotação de princípios ativos, sendo mencionada por alguns apicultores a alternância entre produtos com a mesma substância, o que não configura uma rotação adequada. Populações de V. destructor resistentes a piretroides e ao amitraz já foram documentadas em diversos países, associadas a mutações genéticas específicas. O uso contínuo dessas substâncias, sem rotatividade adequada, pode favorecer o surgimento de resistência. Esses
resultados reforçam a necessidade de adoção de estratégias de controle integradas e sustentáveis, que combinem o uso criterioso de acaricidas com métodos biotécnicos, visando manter a eficácia dos tratamentos e assegurar a viabilidade ecológica e econômica da apicultura.
Why single snp analyses fail: epistatic structural effects in honey bee CYP336A1
Publication . 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, Dora
Cytochrome 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 pressure
Publication . 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, Dora
Widespread 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.