Percorrer por autor "Lopes, Ana Rita"
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- 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.
- 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.
- Colonisation patterns of Nosema ceranae in the Azores ArchipelagoPublication . Lopes, Ana Rita; Martín-Hernández, Raquel; Higes, Mariano; Segura, Sara K.; Henriques, Dora; Pinto, M. AliceNosema ceranae is a highly prevalent pathogen of Apis mellifera, which is distributed worldwide. However, there may still exist isolated areas that remain free of N. ceranae. Herein, we used molecular tools to survey the Azores to detect N. ceranae and unravel its colonisation patterns. To that end, we sampled 474 colonies from eight islands in 2014/2015 and 91 from four islands in 2020. The findings revealed that N. ceranae was not only present but also the dominant species in the Azores. In 2014/2015, N. apis was rare and N. ceranae prevalence varied between 2.7% in São Jorge and 50.7% in Pico. In 2020, N. ceranae prevalence increased significantly (p < 0.001) in Terceira and São Jorge also showing higher infection levels. The spatiotemporal patterns suggest that N. ceranae colonised the archipelago recently, and it rapidly spread across other islands, where at least two independent introductions might have occurred. Flores and Santa Maria have escaped the N. ceranae invasion, and it is remarkable that Santa Maria is also free of Varroa destructor, which makes it one of the last places in Europe where the honey bee remains naive to these two major biotic stressors.
- Distribution of pyrethroid and amitraz resistance in Varroa destructor mites from honey bee (Apis mellifera) colonies in PortugalPublication . Costa, Maíra; Yadró Garcia, Carlos A.; Rodrigues, Cláudia; Lopes, Ana Rita; Pérez-Pérez, Antonio; Martín Hernández, Raquel; Higes, Mariano; Pinto, M. Alice; Henriques, DoraHoney bees (Apis mellifera) are widely used for pollination and honey production worldwide. However, they face a serious threat from the ectoparasitic mite Varroa destructor. This parasite causes varroosis and transmits multiple viruses, such as the Deformed wing virus (DWVV), compromising the health and survival of colonies. While there are many acaricides commercially available to treat colonies against varroa, the recurrent use of certain molecules is leading to the development of varroa resistance. In Portugal, two classes of synthetic compounds are currently used: pyrethroids (fluvalinate and flumethrin) and formamidines (amitraz). However, the excessive and repeated use of acaricides has led to the development of resistance in mite populations. Pyrethroid resistance is linked to mutations in the Voltage-Gated Sodium Channel (VGSC) gene, with amino acid substitutions, at position 925 and 918. Amitraz resistance is associated with mutations in the Octopamine-like β-adrenergic receptor (Octβ2R). The amino acid changes include asparagine (N) to serine (S) at position 87 (N87S), observed in France; tyrosine (Y) to histidine (H) at position 215 (Y215H), in the USA; phenylalanine (F) to leucine (L) at position 290 (F290L), in Spain; and tyrosine (Y) to phenylalanine (F) at position 337 (Y337F), in Turkey. The distribution of resistance alleles has not yet been explored in Portugal. To fill this data gap, Varroa mites from honey bee colonies across various regions of the country were analyzed through DNA extraction, PCR with specific primers, and Sanger sequencing. The results confirmed the of widespread distribution of resistance alleles to pyrethroids at positions 925 and 918, like those observed in Spain. In contrast, preliminary results did not detect any amitraz resistance alleles, although the small sample size warrants caution in interpretation. This study represents an important step in understanding V. destructor resistance to chemical treatments in Portugal, providing essential data for monitoring acaricide resistance and supporting the development of more effective management strategies to help beekeepers better protect their colonies.
- Effectiveness of different traps and lures for coffee berry borer, Hypothenemus hampei (Ferrari, 1867) in Sao Tome IslandPublication . Carvalho, Maria Otília; Lopes, Ana Rita; Bento, Albino; Santos, Lucas; Guedes, Raul Narciso; Casquero, Pedro AntonioCoffee berry borer, Hypothenemus hampei (Ferrari, 1867), is a serious insect pest of organic coffee plantation in Sao Tome Island. To date, limited information regarding the seasonal phenology of this pest species on the islands limits the implementation of integrated pest management (IPM) programmes. As part of a coffee farmer training programme, three attractants were evaluated in red vs. transparent traps to assess olfactory and visual stimuli. The experiment was delineated in a split-block design with three types of attractants: commercial ethanol + 40 g of ripe Robusta coffee (A1), proportion 3:1 methanol and ethanol (A2), and commercial ethanol + 10 g of ground roasted Arabica coffee (A3); and two home- made transparent (D1) and red (D2) traps. The results showed that there was significant interaction between the trap model and the attractant for borer capture. The transparent trap baited with methanol and ethanol exhibited the best result with an average of 14.3 +/- 5.4 adults/trap/week. Transparent traps baited captured more borers and largest numbers of beetles were trapped late May through September. In short, home-made traps alone are not effective for controlling the coffee berry borer, but they are useful in monitoring this species.
- Evolution of amitraz resistance in varroa destructor: historical assessment of the F290L mutation in iberian populationsPublication . Costa, Maíra; Pérez-Pérez, António; Santos, Ana Clara; Prado, Rafaela; Lopes, Doralinda; Lopes, Ana Rita; Yadró Garcia, Carlos A.; Martín-Hernández, Raquel; Higes, Mariano; Pinto, M. Alice; Henriques, DoraVarroa destructor is one of the main parasites of the honey bee (Apis mellifera), causing significant colony losses worldwide. The control of this mite often relies on acaricides, with amitraz being among the most widely used products in Portugal and Spain over the last decade. However, the continuous and persistent use of the same active substance exerts strong selective pressure on parasite populations, potentially favouring alleles associated with treatment resistance. Recently, a point mutation resulting in a phenylalanine-to-leucine substitution at position 290 (F290L) was identified in V. destructor populations from Spain, putatively associated with amitraz resistance. This mutation has been detected at high frequency in recent samples, suggesting resistance may have evolved due to sustained amitraz use. To assess whether the F290L mutation was already present before the widespread amitraz adoption, DNA extraction and sequencing analysis will be performed on mites collected since 2006, prior to the massive use of this treatment in Iberian beekeeping. This approach will clarify whether the allele frequency of F290L increased because of the selective pressure exerted by amitraz or if it already existed in high frequencies in earlier populations. This study will contribute to a better understanding of the evolution of resistance and to the development of more effective monitoring and management strategies to combat V. destructor.
- From the popular tRNAleu-COX2 intergenic region to the mitogenome: insights from diverse honey bee populations of Europe and North AfricaPublication . Henriques, Dora; Chávez-Galarza, Julio; Quaresma, Andreia; Neves, Cátia J.; Lopes, Ana Rita; Costa, Cecilia; Costa, Filipe Oliveira; Rufino, José; Pinto, Maria A.The tRNAleu-COX2 intergenic region of the mitochondrial DNA has been used for assessing diversity in honey bee (Apis mellifera L.) populations worldwide. However, differential mutation rates in different partitions of the mitogenome may produce incongruent results. In this study, we sequenced 123 mitogenomes of 7 subspecies from lineages A, M, and C. This allowed generating a comprehensive dataset to investigate the phylogenetic and phylogeographic congruence among the mitogenome, individual genes, and the tRNAleu-COX2 region.We showed that the diversity patterns inferred fromthe tRNAleu-COX2 marker are not fully paralleled by those obtainedwith the mitogenome and the individual genes; while the three lineages are supported by these, the African sub-lineages and the haplotypes are not. Thus, conclusions drawn from the tRNAleu-COX2 region need to be taken with caution and this marker may not be appropriate to infer phylogenetic relationships between honey bee colonies.
- 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.
- Genomic DNA extraction from honey bee (Apis mellifera) queen spermathecal contentPublication . Yadró, Carlos A.; Lopes, Ana Rita; Henriques, Dora; Musin, Eduard; Wegener, Jakob; Pinto, M. AliceGenetic analysis of the honey bee spermathecal content can be particularly useful to provide an estimate of the genetic diversity and purity of the surrounding populations. Here we compared the concentration and quality of DNA extracted from queen spermatheca using four commercial kits to determine the best method to obtain DNA suitable for single nucleotide polymorphism genotyping by next-generation sequencing. The four kits were tested with different adjustments in the lysis incubation time, use of RNA-carrier, elution conditions and number of re-elutions. Only the use of QIAamp DNA Microkit with 3 h of lysis incubation, the addition of RNA-carrier and multiple re-elutions produced a DNA concentration over the required threshold.
- High prevalence of the F290L amitraz-resistance allele in varroa destructor populations from PortugalPublication . Costa, Maíra; Sánchez, Sara; Lopes, Ana Rita; Yadró Garcia, Carlos A.; Pérez-Pérez, Antonio; Martín-Hernández, Raquel; Higes, Mariano; Pinto, M. Alice; Henriques, DoraThe Western honey bee (Apis mellifera) has a crucial role in pollination and apicultural production but faces a major threat from the ectoparasitic mite Varroa destructor. This parasite causes varroosis and acts as a vector for multiple viruses, undermining colony health and survival. Chemical control relies mainly on two classes of synthetic acaricides: pyrethroids (fluvalinate and flumethrin) and formamidines (amitraz). However, the repeated and prolonged use of these compounds has promoted the development of resistance in V. destructor populations. Amitraz resistance has been associated with mutations in the Octopamine-like β-adrenergic receptor (Octβ2R), including N87S (France), Y215H (USA), F290L (Spain), and Y337F (Turkey). Until now, the distribution of these resistance alleles had not been investigated in Portugal. To address this gap, mites collected from different regions of the country were analysed through DNA extraction, PCR with specific primers, and Sanger sequencing. The results revealed an unexpectedly high frequency (98.3%) of the F290L allele, which has also been reported in Spanish populations, suggesting a strong potential selective pressure resulting from the prolonged use of amitraz. This atypically high frequency raises important questions regarding the origin and evolutionary trajectory of this resistance allele in Portugal. Therefore, a retrospective analysis of mite samples collected before the widespread adoption of amitraz is proposed. This approach will clarify whether the F290L allele was already present in ancestral V. destructor populations or whether its current prevalence arose from recent selective pressure. Integrating this data will be essential for understanding the evolutionary dynamics of resistance and for supporting the development of more effective monitoring and management strategies against this parasite.