Browsing by Author "Rosa-Fontana, Annelise"
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- 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 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.
- 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.; 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 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.; 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.
- 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.