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- 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.
- DNA-based methods as a powerful tool for the entomological authentication of honeyPublication . Honrado, Mónica; Quaresma, Andreia; Lopes, Ana; Pinto, M. Alice; Henriques, Dora; Amaral, Joana S.Honey is a food widely consumed worldwide and much appreciated for its nutritional, organoleptic and health properties. However, it is also considered one of the food products most prone to be adulterated in the EU. Up until now, honey authenticity addressed mainly the issue of sugars addition and botanical origin. Still, increased attention has recently been paid to honey entomological origin as it also relates to its geographical origin since honeybees carrying mitochondrial DNA (mtDNA) of distinct ancestries can be found across Europe. While in Portugal mtDNA of the autochthonous subspecies Apis mellifera iberiensis belongs to the African (A) lineage, in the northeastern part of Iberia African mitotypes are replaced by mitotypes of western European (M-lineage) ancestry. The native distribution of the M-lineage A. m. mellifera expands from the Pyrenees to Scandinavia and from the British Isles to the Ural Mountains while the C-lineage A. m. ligustica and A. m. carnica subspecies are naturally found in the Apennine and Balkan peninsulas, respectively [1]. Also, certain honeys holding the protected designation of origin (PDO) label should be produced by autochthonous A. mellifera subspecies, as mentioned in their EU geographical indications register.
- COI Metabarcoding as a Novel Approach for Assessing the Honey Bee Source of European HoneyPublication . Honrado, Mónica; Quaresma, Andreia; Henriques, Dora; Pinto, M. Alice; Amaral, Joana S.Honey is a widely consumed food product frequently subjected to adulteration, with the mislabeling of its botanical or geographical origin being one of the most common practices. Determining the entomological origin of honey is particularly challenging but of high relevance for ensuring its authenticity, especially for products with protected designation of origin (PDO) labels. This study presents a novel DNA metabarcoding approach targeting a highly informative 406 bp fragment of the cytochrome c oxidase I (COI) gene to differentiate among the three major mitochondrial lineages (A, M, and C) of honey bees (Apis mellifera L.) native to Europe. The target region was selected based on the calculated fixation index (FST), which is frequently used in Population Genetics as a measure of differentiation between populations. The approach was validated with 11 honey samples of known entomological origin and applied to 44 commercial honeys from 13 countries. The approach demonstrated high sensitivity, accurately identifying the entomological origin of honey, including samples produced by honey bees of varying ancestries, which could not be resolved by previous methods based on real-time PCR coupled with high-resolution melting (PCR-HRM) analysis. The results demonstrate the effectiveness of COI metabarcoding in verifying honey authenticity and highlight the predominance of C-lineage honey bees in the production of commercial honeys from northwestern Europe. This finding suggests a limited presence of the native M-lineage ancestry, underscoring the need for conservation efforts.
- Honey bee collected pollen for botanical identification via its2 metabarcoding: a comparison of preservation methods for citizen sciencePublication . Quaresma, Andreia; Brodschneider, Robert; Gratzer, Kristina; Gray, Alison; Keller, Alexander; Kilpinen, Ole; Rufino, José; Steen, Jozef van der; Vejsnaes, Flemming; Pinto, M. AliceWhile classical palynology has been the method of choice to assess botanical diversity of bee-collected pollen for multiple purposes, DNA metabarcoding is emerging as a powerful alternative being able to achieve high taxonomic identification accuracy. Moreover,DNA metabarcoding allows analysis of hundreds of samples in a single high-throughput sequencing run, therefore offering unprecedented scale in citizen science projects. Biases in metabarcoding can be introduced at any stage of sample processing and preservation is at the forefront of the pipeline. Hence, it is important to test how sample preservation influences quality and quantitative performance of pollen metabarcoding. While inmetabarcoding studies pollen has typically been preserved at −20°C (FRZ), this is not the best method to be applied by citizen scientists.
- Autent+ Desenvolvimento de abordagem inovadoras com vista à valorização e exploração do potencial de mercado do mel PortuguêsPublication . Amaral, Joana S.; Quaresma, Andreia; Rodrigues, Pedro João; Rufino, José; Henriques, Dora; Calaim, Luís; Gaspar, Albino; Pinto, M. AliceA FENAPICOLA candidatou-se, como proponente, a uma medida de investigação aplicada, tendo convidado o Instituto Politécnico de Bragança (IPB) como entidade parceira, envolvendo este último uma equipa multidisciplinar de 6 investigadores provenientes dos centros de investigação CIMO (Centro de Investigação de Montanha) e CEDRI (Centro de Investigação em Digitalização e Robótica Inteligente). Assim foi criado o projeto AUTENT+, um projeto financiado pelo Instituto de Financiamento da Agricultura e Pescas (IFAP), em resultado da candidatura submetida ao Plano Apícola Nacional (PAN) 2020/2022, medida 5.1 "Apoio a projetas de investigação aplicada”. O AUTENT + tem como principal objetivo a valorização do mel nacional como um produto autêntico e sustentável, através de abordagens que visam diferenciar, acrescentar valor e o potencial de mercado a este produto. Para tal, o projeto centra-se no desenvolvimento de metodologias inovadoras com vista à deteção de adulterações do mel, em particular no que respeita à origem botânica e entomológica/geográfica, e no desenvolvimento de ferramentas que permitam , de uma forma simples, informar o consumidor sobre as caraterísticas do produto que adquirem.
- Correction to: Invasion genetics of the Asian hornet Vespa velutina nigrithorax in Southern EuropePublication . Quaresma, Andreia; Henriques, Dora; Godinho, Joana; Maside, Xulio; Bortolotti, Laura; Pinto, M. AliceIn the original publication of the article, the fourth author’s family name and affiliations were published incorrectly. The correct author’s name and affiliations are given in this correction. The original article has been corrected.
- Identification of botanical origin of bee-collected mixed pollen samples: a comparison between palynological and DNA metabarcoding methodsPublication . Quaresma, Andreia; Steen, Jozef van der; Amaral, Joana S.; Biron, David G.; Brodschneider, Robert; Brusbardis, Valters; Carreck, Norman L.; Formato, Giovanni; Gratzer, Kristina; Hatjina, Fani; Kilpinem, Ole; Pietropaoli, Marco; Rufino, José; Vejsnaes, Flemming; Pinto, M. AliceIdentification of botanical origin of mixed pollen samples has several applications, including unraveling plant-pollinator interactions, determining botanical origin of honey, monitoring allergy-related airborne pollen sources, or even monitoring pesticide use in crops. These applications have typically been addressed using light microscopy, a costly approach that often provides low taxonomic resolution. However, with high-throughput sequencing (HTS) becoming increasingly affordable, DNA metabarcoding is emerging as a promising alternative to classical palynology. In addition to be time- and cost-effective for large sample sizes, metabarcoding has the potential to allow identification of pollen mixtures at the species level. However, before it can be widely employed in pollen analysis, the reliability of this molecular tool must be appraised. Herein, we compared the two approaches on 61 bee-collected pollen samples from eight European countries. The samples were homogenized and split into two sub-samples. One set of 61 sub-samples was analyzed by palynology experts from the “Institut für Bienenkunde”, Germany, and the other one was subjected to HTS, using ITS2 as the barcode, in the labs of CIMO and CIBIO. Comparisons of the relative abundances at the family level show no significant differences (P ≥ 0.1057, Wilcoxon signed-rank test) and high correlation values (0.2736 ≤ r ≤ 1.000, Pearson’s correlation) between the two approaches. The highest correlation values were observed for Italian samples (0.7245 ≤ r ≤ 0.9842; global r = 0.8958) and the lowest for Greek samples (0.0266 ≤ r ≤ 0.9703; global r = 0.5149). These results suggest that ITS2 metabarcoding offers a reliable alternative to classical palynology and this approach is now being employed in the European project INSIGNIA (https://www.insignia-bee.eu/), which is developing a standard protocol for using the honey bee as a tool for environmental monitoring.
- Preservation methods of honey bee-collected pollen are not a source of bias in ITS2 metabarcodingPublication . Quaresma, Andreia; Brodschneider, Robert; Gratzer, Kristina; Gray, Alison; Keller, Alexander; Kilpinen, Ole; Rufino, José; Steen, Jozef van der; Vejsnæs, Flemming; Pinto, M. AlicePollen metabarcoding is emerging as a powerful tool for ecological research and offers unprecedented scale in citizen science projects for environmental monitoring via honey bees. Biases in metabarcoding can be introduced at any stage of sample processing and preservation is at the forefront of the pipeline. While in metabarcoding studies pollen has been preserved at − 20 °C (FRZ), this is not the best method for citizen scientists. Herein, we compared this method with ethanol (EtOH), silica gel (SG) and room temperature (RT) for preservation of pollen collected from hives in Austria and Denmark. After ~ 4 months of storage, DNAs were extracted with a food kit, and their quality and concentration measured. Most DNA extracts exhibited 260/280 absorbance ratios close to the optimal 1.8, with RT samples from Austria performing slightly worse than FRZ and SG samples (P < 0.027). Statistical differences were also detected for DNA concentration, with EtOH samples producing lower yields than RT and FRZ samples in both countries and SG in Austria (P < 0.042). Yet, qualitative and quantitative assessments of floral composition obtained using high-throughput sequencing with the ITS2 barcode gave non-significant effects of preservation methods on richness, relative abundance and Shannon diversity, in both countries. While freezing and ethanol are commonly employed for archiving tissue for molecular applications, desiccation is cheaper and easier to use regarding both storage and transportation. Since SG is less dependent on ambient humidity and less prone to contamination than RT, we recommend SG for preserving pollen for metabarcoding. SG is straightforward for laymen to use and hence robust for widespread application in citizen science studies.
- Bio-Monitoring of environmental pollution using the citizen science approachPublication . Steen, Jozef van der; Amaral, Joana S.; Baveco, Hans; Blanco Muñoz, Patricia; Brodschneider, Robert; Brusbardis, Valters; Buddendorf, Bas; Carreck, Norman L.; Danneels, Ellen; Charistos, Leonidas; Graaf, Dirk C.; Díaz Galiano, Francisco José; Fernandez-Alba, Amadeo; Ferrer-Amate, Carmen; Formato, Giovanni; Gómez Ramos, María José; Gratzer, Kristina; Gray, Alison; Hatjina, Fani; Henriques, Dora; Kasiotis, Konstantinos; Kilpinen, Ole; Lopes, Ana; Martínez Bueno, María Jesús; Murcia-Morales, María; Pietropaoli, Marco; Pinto, M. Alice; Quaresma, Andreia; Rufino, José; Roessink, Ivo; Vejsnæs, Flemming; Zafeiraki, EffrosyniHoneybee colonies are excellent bio-samplers of biological material such as nectar, pollen, and plant pathogens, as well as non-biological material such as pesticides or airborne contamination. The INSIGNIA-EU project aims to design and test an innovative, non-invasive, scientifically proven citizen science environmental monitoring protocol for the detection of pesticides, microplastics, heavy metals, and air pollutants by honey bee colonies http://insignia-eu.eu. In the pilot INSIGNIA project (2018-2021), a protocol was developed and tested for citizen-science-based monitoring of pesticides using honeybees. As part of the project, biweekly pollen was obtained from sentinel apiaries over a range of European countries and landscapes and analysed for botanical origin, using state-of-theart molecular techniques such as metabarcoding. An innovative non-biological matrix, the “APIStrip”, was also proved to be very efficient for detecting the residues of 273 agricultural pesticides and veterinary products, both authorized and unauthorized. The data collected are used to develop and test a spatial modelling system aimed at predicting the spatiallyexplicit environmental fate of pesticides and honeybee landscape-scale pollen foraging, with a common underlying geo-database containing European land-use and land-cover data (CORINE), the LUCAS database (landcover) supplemented with national data sets on agricultural and (semi-) natural habitats. After a call by the European Commission, a new 2 years project was granted aiming to present a comprehensive pan-European environmental pollution monitoring study with honey bees. Although pesticides used in agriculture, are a known hazard due to their biological activity, other pollutants, have even been recognized as such, for which we have not been aware of their impact for many years. An example is air pollution which increased while our societies industrialized and is currently regarded as the single largest environmental health risk in Europe (https://www.eea.europa.eu/). Unfortunately, other pollutants such as heavy metals, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, airborne particulate matter, and microplastics have also reached our environment. The outcome of this project will provide the first standardized EU-wide monitoring of all types of environmental pollutants with honey bee colonies. The project is funded by the EU, under the N° 09.200200/2021/864096/SER/ ENV.D.2 contract.
- Patterns of Vespa velutina invasion in western Iberia and Italy as revealed by mitochondrial and microsatellite markersPublication . Quaresma, Andreia; Pinto, M. Alice; Henriques, DoraThe Yellow-legged or Asian hornet, Vespa velutina nigrithorax, is naturally distributed in Southeast Asia, and it is the first successful exotic Vespidae predator to be accidently introduced in Europe. In 2004, the first individual was reported in France, probably from China and, in the last decade, it spread rapidly through the French territory and to other European countries. In the Iberian Peninsula it was reported for the first time in Spain, in 2010, and in Portugal, in 2011. In 2012, two individuals were reported in the Italian region of Liguria. Using a population genetics framework, the goal of this study was to test the genetic patterns of colonization of this invasive honey bee predator in the Atlantic side of Iberia and in Italy. A total of 246 individuals, each representing a single colony, were collected across the invaded areas in Portugal (190), Spain (45), and Italy (11). Additionally, a dataset containing samples from France, Vietnam, South Korea, Indonesia and two provinces of China provided by Arca et al. (2015) was used as a reference for testing hypothesis about the origin of the invasion and the expansion patterns. The genetic variability was assessed using 16 microsatellite loci and the mitochondrial DNA (mtDNA) cytochrome C oxidase I (COI). The mtDNA analysis revealed the presence of a single haplotype in Iberia and Italy, which has been also reported for France and the UK. Microsatellite analysis showed a low genetic diversity in these populations, as it is expected from an expansion of an invasive species. Population structure analysis showed that the European populations separate in two clusters: (i) one that contains the populations from France and Italy, and (ii) one with the Portuguese individuals. The Spanish population has individuals in both clusters, while the Portuguese population showed some migrants that cluster with the French individuals. Thus, it was demonstrated that the expansion of the Asian hornet is different in the three countries. Spain and Italy showed a natural spread of the hornet, whereas the Portuguese population showed a human-mediated diffusion together with a natural spread of the Asian hornet.