Percorrer por autor "Rosa-Fontana, Annelise"
A mostrar 1 - 10 de 10
Resultados por página
Opções de ordenação
- Assessing pesticide impact on honeybee gut microbiota: a call for microbial diversity as an environmental risk assessment endpointPublication . Rosa-Fontana, Annelise; Aguado, Daniel; Martín Hernández, Raquel; Higes, Mariano; Henriques, Dora; Pinto, M. AliceA recent roadmap for integrating environmental microbiota into risk assessments under the European Food Safety Authority (EFSA) remit has been published. The honeybee gut microbiota has emerged as a promising avenue to protect bees against stressors. Honeybees exhibit a stable core microbiota, and dysbiosis may serve as an indicator of adverse conditions. We investigated the gut microbiota of newly emerged “Apis mellifera iberiensis” workers exposed to a single concentration of the insecticide flupyradifurone (FPF, 36 ppm). The control groups included pure syrup (negative control, NC) and syrup supplemented with 1% acetone (acetone control, AC). Laboratory trials followed official guidelines (OECD No. 245). The abdomen of each bee was separated from the thorax, and DNA extraction was performed individually. Full-length 16s rRNA amplicon metagenomic was sequenced through PacBio sequel II (HiFi/CCS mode). The Shannon diversity index was used to analyze honeybee gut microbiota composition across experimental groups. Our results revealed a significant increase in bacterial community diversity (Shannon index, P = 0.003) after ten days of chronic exposure to FPF. This effect was more pronounced when compared to the AC group (P = 0.003) than to the NC group (P = 0.03). These findings demonstrate that FPF disrupts the honeybee gut microbiota. This study represents the first characterization of honeybee gut microbiota strictly adhering to OECD guidelines without modifications or adaptations. Furthermore, we have provided new insights into pesticide risk assessment, highlighting an overlooked aspect of bee health assessment. We propose integrating this approach into pesticide risk assessments by using diversity indices as comparative parameters. Specifically, we advocate for the inclusion of honeybee gut microbiota dysbiosis as a sublethal effect in the initial screening phase of risk assessments (laboratory-based assays) and as a key parameter for evaluating pollinator health.
- Bee3Pomics: utilização das “Omics” no estudo dos efeitos dos pesticidas na abelha melíferaPublication . Henriques, Dora; Yadró Garcia, Carlos A.; Lopes, Ana; Rufino, José; Rosa-Fontana, Annelise; Pinto, M. AliceOs pesticidas podem ter efeitos adversos em organismos não alvo, tais como os insetos polinizadores. Para estudar esses efeitos, são realizadas avaliações de risco quando novas moléculas são homologadas. A abelha melífera (Apis mellifera) tem sido usada como organismo modelo nessas avaliações. No entanto, o impacto da variação genética intraespecífica na sensibilidade toxicológica ainda é desconhecido. As 'omics' prometem ser uma ferramenta útil para abordar esse problema. Este projeto tem dois grandes objetivos. Primeiro, pretende-se utilizar mais de 2000 genomas de 11 das 31 subespécies de abelhas descritas para estudar a diversidade genética nos genes de detoxificação (famílias P450, glutationa-S-transferases, carboxilesterases, UDPglucuronosiltransferase, transportadores ABC). Em segundo lugar, pretende-se compreender os efeitos moleculares da exposição aos pesticidas. Para isso, serão coletados dados de pesticidas de 315 apiários distribuídos pelos 27 países da União Europeia. Indivíduos desses mesmos apiários serão sequenciados, e os dados genómicos serão integrados com os dados dos pesticidas através de testes de Associação Genética- Ambiente (GEA). As variantes resultantes desta análise, quando não sinónimas, serão validadas por modelação de proteínas.
- Bioinformatics pipeline to evaluate patterns of diversity in detoxification genes in the Western honey bee (Apis mellifera)Publication . Barbosa, Daniela; Li, Fernanda; Bashir, Sana; Lopes, Ana Rita; Yadró Garcia, Carlos A.; Quaresma, Andreia; Rufino, José; Rosa-Fontana, Annelise; Verbinnen, Gilles; de Graaf, Dirk C.; De Smet, Lina; Taliadoros, Demetris; Webster, Matthew; 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.
- 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.
- 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. 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 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.
- Genetic Diversity of Detoxification Genes in 18 Honey Bee SubspeciesPublication . Li, Fernanda; Barbosa, Daniela; Bashir, Sana; Moreira de Sá, Leandro; Yadró Garcia, Carlos A.; Rufino, José; Rosa-Fontana, Annelise; Verbinnen, Gilles; de Graaf, Dirk; de Smet, Lina; Taliadoros, Demetris; Webster, Matthew; Pinto, M. Alice; Henriques, DoraThe honey bees (Apis mellifera) is a key pollinator that is exposed to a wide array of xenobiotics, both natural (plant allelochemicals) and synthetic (pesticides), while foraging or through contaminated food within the hive. These compounds have both lethal and sub-lethal effects, impairing foraging activity and negatively affecting bee development and colony health. Similar to other insects, honey bees rely on detoxification pathways to metabolise xenobiotics into less toxic or more readily excretable forms. This process is a key mechanism underlying insecticide resistance and is influenced by genetic variation. Therefore, investigating polymorphisms in detoxification-related genes is a promising approach to predict species-specific responses to pesticide exposure. Five major gene families are involved in xenobiotic detoxification: cytochrome P450 monooxygenases (CYPs), carboxyl/cholinesterases (CCEs), glutathione Stransferases (GSTs), ATP-binding cassette transporters (ABCs), and uridine 5′-diphospho-glucuronosyltransferases (UGTs). In this study, we examined the genomic detoxification inventory of over 1,600 individuals representing 18 A. mellifera subspecies representing the four main evolutionary lineages. For each lineage and subspecies, single-nucleotide polymorphism (SNP) loci were identified within these genes, allele frequency and FST (fixation index) were calculated. Additionally, all variants were annotated to assess their potential impact on protein function. Findings from this study have the potential to inform breeding and conservation strategies by identifying populations more vulnerable to chemical stressors, ultimately supporting honey bee health in changing environments.
- 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.
- Honeybee gut microbiota as an emerging endpoint for pesticide risk assessmentsPublication . Rosa-Fontana, Annelise; Aguado-López, Daniel; Jabal-Uriel, Clara; Martín-Hernández, Raquel; Higes, Mariano; Pinto, M. Alice; Henriques, Dora; Tosi, Simone; Rodríguez Gomez, Juan MiguelA recent roadmap for the integration of environmental microbiotas in risk assessments under the European Food Safety Authority (EFSA) remit has been published. Healthy honeybee gut microbiota has emerged as a promising avenue to protect bees against stressors. Honeybees exhibit a consistent core microbiota, and dysbiosis, as part of a multiple stressor system, may be an indicator of adverse scenarios. We therefore investigated the honeybee gut microbiota of Apis mellifera carnica workers exposed to a single concentration of the insecticide flupyradifurone (FPF, 36ppm). The laboratory trials were carried out in accordance with official protocols (OECD Nº 245). The abdomen of each bee was separated from the thorax, and DNA extraction was performed individually. Full-length 16s rRNA amplicon metagenomic was sequenced through PacBio sequel II (HiFi/CCS mode). The absolute abundance of four bacterial genera constituting the core honeybee microbiota unveiled a Lactobacillus-dominated gut in both treated and non-treated bees. Treated bees exhibited a twofold increase in the bacterial load of Snodgrassella, contrasting with a 50% reduction in the Bifidobacterium load and the complete absence of Gilliamella as compared to the untreated bees. Our findings revealed that FPF disrupted the honeybee gut microbiota. We have developed a new approach, overlooked in risk assessments studies so far, to assess the impact of pesticides bee health until now. Thus, we propose its use as a novel endpoint in pesticide risk assessments. Current risk assessments are performed in a tiered approach, i. e., moving from laboratory assays (first screening) to semi field and field studies, and require no sublethal effect assessments. We therefore advocate for the inclusion of honeybee gut microbiota dysbiosis as a sublethal effect in the first screening step of risk assessments, and as a key parameter to assess pollinator’s health.
- Honeybee gut microbiota is an imperative endpoint for pesticide risk assessmentPublication . Rosa-Fontana, Annelise; Aguado-López, Daniel; Uriel Clara, Jabal; Martín-Hernández, Raquel; Higes, Mariano; Pinto, M. Alice; Henriques, Dora; Tosi, Simone; Rodríguez Gomez, Juan MiguelIn nature, honeybee workers acquire their stable gut microbial community by the 7th day post-emergence, with older bees transmitting microorganisms to younger bees in the comb, thereby establishing natural microbial diversity. In contrast, younger caged bees sampled for laboratory trials (OECD Guideline No. 245) are in contact with older bees for only a few hours. Newly emerged bees harbor minimal to no bacteria, potentially resulting in lower diversity, richness, and bacterial loads in their gut. However, this method best simulates the natural state within a controlled environment.Existing studies have modified standardized protocols to simulate the microbiota present in the honeybee digestive tract within the hive environment. A common approach involves diluting the gut contents of forager bees and incorporating this into the diet of caged bees. In our trials, we strictly adhered to OECD Guideline No. 245 (Chronic Oral Toxicity Test; 10-Day Feeding), exposing newly emerged Apis mellifera carnica workers to a single concentration of the insecticide flupyradifurone (FPF, 36 ppm). The standard reference dimethoate (1 ppm) and control groups (pure food and food + acetone) were also included. DNA was extracted individually from the bee abdomens, and full-length 16S rRNA amplicon metagenomics were sequenced using PacBio Sequel II (HiFi/CCS mode). The absolute abundance of four bacterial genera comprising the core honeybee microbiota revealed a Lactobacillus-dominated gut in both treated and untreated bees. Treated bees exhibited a twofold increase in the bacterial load of Snodgrassella, contrasting with a 50% reduction in Bifidobacterium and the complete absence of Gilliamella compared to untreated bees. Our findings demonstrate that FPF significantly disrupts the honeybee gut microbiota. This study presents, for the first time, the composition of the gut microbiota in honeybees strictly subjected to the OECD guideline without modifications or adaptations. Results from OECD-based tests already meet reliability requirements for risk assessments. Therefore, following OECD standards strictly illuminate three distinct advantages: (1) streamlining the process leading to a ring test, (2) reducing variations introduced by external factors potentially brought into hives by foraging bees, and (3) reducing bacterial diversity in lab-tested bees, thereby facilitating the establishment of acceptable fluctuations in microbiota composition. We have developed a new approach, overlooked in risk assessments studies so far, to assess the impact of pesticides on bee health. We propose adopting this approach as a new endpoint in pesticide risk assessments. Specifically, we advocate for the inclusion of honeybee gut microbiota dysbiosis as a sublethal effect in the first screening step of risk assessments, and as a key parameter to assess pollinator's health. We will present a summary of the most relevant bacteria for bee health, alongside fluctuations in the microbiota and diversity indices. Additionally, we will provide recommendations on the most suitable indicators for assessing gut microbiota dysbiosis.
- 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.