Browsing by Author "Steen, Jozef van der"
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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; Steen, Jozef van der; Varnava, Andri; Vejsnæs, Flemming; Webster, Matthew T.; Fedoriak, Mariia M.; Zarochentseva, Oksana; Graaf, Dirk C.; 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.
- 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.
- 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.
- 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. AliceDNA metabarcoding is emerging as a powerful method for botanical identification of bee-collected pollen, allowing 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 the first step of the pipeline. Hence, it is important to test whether the pollen preservation method influences metabarcoding performance. While in metabarcoding studies pollen has typically been preserved at −20°C, this is not the best method to be applied by citizen scientists. Here, we compared the freezing method (FRZ) with ethanol (EtOH), silica gel (SG) and room temperature (RT) in 87 pollen samples collected from hives in Austria and Denmark.
- 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.
- INSIGNIA: um projeto de monitorização ambiental de pesticidas através da utilização da abelha melliferaPublication . Pinto, M. Alice; Amaral, Joana S.; Baveco, Hans; Biron, David G.; Brodschneider, Robert; Brusbardis, Valters; Carreck, Norman L.; Charistos, Leonidas; Coffey, Mary F.; Fernandez-Alba, Amadeo; Formato, Giovanni; Graaf, Dirk C.; Gratzer, Kristina; Gray, Alison; Hatjina, Fani; Kasiotis, Konstantinos; Kilpinen, Ole; Pietropaoli, Marco; Roessink, Ivo; Rufino, José; Vejsnæs, Flemming; Steen, Jozef van derINSIGNIA ("cItizeN Science InvestiGatioN for pestIcIcides in Apicultutarl products"; https://www.insignia-bee,eu/) é um projecto financiado pela agência "Directorate General for Health and Food Safety" da Comissão Europeia, e que teve início em Outubro de 2018. O consórcio INSIGNIA é coordenado por Jozef van der Steen e integra 16 instituições parceiras de 12 países Europeus, entre as quais está o Centro de Investigação e Montanha (CIMO) do Instituto Politécnico de Bragança (IPB).
- Introducing the INSIGNIA project: environmental monitoring of pesticide use through honey beesPublication . Steen, Jozef van der; Brodschneider, Robert; Gratzer, Kristina; Bieszczad, Sarah; Hatjina, Fani; Charistos, Leonidas; Carreck, Norman L.; Gray, Alison; Pinto, M.Alice; Amaral, Joana S.; Rufino, José; Quaresma, Andreia; Roessink, Ivo; Baveco, Hans; Formato, Giovanni; Pietropaoli, Marco; Kasiotis, Konstantinos; Anagnostopoulos, Christ; Zafeiraki, Effrosyni; Fernandez-Alba, Amadeo; Eulderink, Caroline; Vejsnæs, Flemming; Kilpinen, Ole; Coffey, Mary F.; Biron, David G.; Brusbardis, Valters; Graaf, Dirk C.INSIGNIA aims to design and test an innovative, non-invasive, scientifically proven citizen science environmental monitoring protocol for the detection of pesticides by honey bees. It is a 30-month pilot project initiated and financed by the EC (PP-1-1-2018; EC SANTE). The study is being carried out by a consortium of specialists in honey bees, apiculture, statistics, analytics, modelling, extension, social science and citizen science from twelve countries. Honey bee 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. Honey bee colonies forage over a circle of 1 km radius, increasing to several km if required, depending on the availability and attractiveness of food. All material collected is accumulated in the hive.
- Large Scale Mitochondrial Evidence of C-lineage Dominance in European Honey BeesPublication . Li, Fernanda; Lopes, Ana; Costa, Maíra; Henriques, Dora; Quaresma, Andreia; Yadró García, Carlos A.; Albo, Alexandre; Čereškienė, Laima Blažytė; 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; Poirot, Benjamin; Pietropaoli, Marco; Radev, Zheko; Raudmets, Aivar; René-Douarre, Vincent; Roessink, Ivo; Škerl, Maja Ivana Smodiš; Soland, Gabriele; Titera, Dalibor; Steen, Jozef van der; Varnava, Andri; Vejsnæs, Flemming; Fedoriak, Mariia M.; Zarochentseva, Oksana; Webster, Matthew T.; Graaf, Dirk C.; Pinto, M. AliceIn Europe, distribution of the several endemic honey bee (Apis mellifera) subspecies has suffered a considerable shift in the last century. In particular, beekeepers tend to favour subspecies of Eastern European ancestry (C-lineage), such as the Italian honey bee (A. m. ligustica), due to their perceived docility and high honey production. As a result, large scale migratory beekeeping and trade of C-lineage queens have exposed the native European honey bees to introgression and replacement, jeopardizing their genetic integrity and locally adapted traits. The maternally-inherited and highly polymorphic mitochondrial intergenic region tRNAleu-cox2 is routinely used for the assessment of honey bee diversity and introgression at large geographical scales. In this study, we conducted a survey on tRNAleu-cox2 variation in more than 1300 colonies from 33 European countries to assess current status of mitochondrial diversity patterns in Europe. Total genomic DNA was extracted from the bee thorax followed by PCR amplification of the tRNAleu-cox2 region and sequencing. The haplotypes were identified using alignments in MEGA 11. A clear dominance of C-lineage haplotypes was found (65%). The most prevalent C-lineage haplotype was C2 (54%) followed by C3 (23%) and C1(22%). Contrary to this trend, in Portugal, Spain and Ireland, less than 10% of the colonies exhibit the C-haplotype. Furthermore, the analysed apiaries in isolated protected areas (n=7) revealed a high proportion of colonies of western European (M-lineage) ancestry (80%) with M4 as the most frequent haplotype. African haplotypes (A lineage) were also found, albeit at lower frequencies, and were mainly concentrated in the Iberian Peninsula (9%). Intensive queen breeding and migratory beekeeping is homogenizing the gene pool of European bee populations. This survey of honey bee maternal diversity across Europe highlights the alarming dominance of C-lineage haplotypes and underscores the importance of conservation apiaries, as they have effectively preserved the autochthonous M-lineage subspecies in different countries. This work was conducted in the framework of the project Better-B, funded by the European Union, the Swiss State Secretariat for Education, Research, and Innovation, and UK Research and Innovation under the UK government's Horizon Europe funding guarantee (grant number 10068544).
- Maternal origin of honeybee (Apis mellifera) colonies from across EuropePublication . Lopes, Ana; Costa, Maíra; Tounakti, Sahar; Henriques, Dora; Quaresma, Andreia; Yadró Garcia, Carlos A.; Albo, Alexandre; Blažytė-Čereškienė, Laima; Broodschneider, Robert; Brusbardis, Valters; Carreck, Norman L.; Charistos, Leonidas; Chlebo, Robert; Coffey, Mary F.; Danneels, Ellen; Dobrescu, Constantin; Filipi, Janja; Gajda, Anna; Galea, Thomas; Gratzer, Kristina; Hatjina, Fani; Johannesen, Jes; Körmendy-Rácz, János; Kovačić, Marin; Preben, Kristiansen; Martikkala, Maritta; Martín-Hernández, Raquel; Pietropaoli, Marco; Poirot, Benjamin; Radev, Zheko; Raudmets, Aivar; Douarre, Vincent; Rodriguez-Flores, Maria Shantal; Roessink, Ivo; Ivana, Maja; Škerl, Maja Ivana Smodiš; Titera, Dalibor; Steen, Jozef van der; Varnava, Andri; Vejsnæs, Flemming; Webster, Matthew T.; Graaf, Dirk C.; Pinto, M. AliceWorldwide commercial beekeeping poses a threat to the native origin of the honeybee (Apis mellifera), with beekeepers favouring subspecies of Eastern European C-lineage ancestry, due to their docile behaviour and high honey production traits. In many parts of western and northern Europe, queens of Western European M-lineage ancestry have been massively replaced by queens of C-lineage ancestry, and this has led to the development of conservation programs aiming at recovering native lines. The maternally-inherited mitochondrial DNA (mtDNA), particularly the intergenic region tRNAleu-cox2, has been the marker of choice for assessing honey bee variation at large geographical scales. Herein, we will show the results of the mtDNA analysis of over 850 colonies collected across 28 European countries. These samples were subjected to DNA extraction, followed by PCR, and Sanger sequencing. The analysis of the sequences was conducted in Mega 11. The results indicated that, apart from Portugal, Spain, and the conservation centres in France and Denmark, where the colonies exhibited African or M haplotypes, the remaining countries are dominated by colonies of C-lineage maternal ancestry. In conclusion, this unprecedented mtDNA analysis conducted across Europe underscores the worrying dominance of C-lineage genetic variation, highlighting the urgent need for strategic conservation efforts to preserve the native genetic diversity of Apis mellifera.
- Pollen identification by its2 metabarcoding: curation of the sequences retrieved from genbank to build a reference databasePublication . Quaresma, Andreia; Keller, Alexander; Rufino, José; Steen, Jozef van der; Pinto, M. AliceA powerful way of studying the quality of the environment is by examining the pollen collected by honey bees (Apis mellifera) as it contains information on available plant sources, spatial and temporal floral diversity, as well as on chemical contaminants. This entails botanical identification of pollen which has typically been addressed by classical palynology, a costly approach that often provides low taxonomic resolution, is time-consuming, labour intensive, and requires plant taxonomy expertise. However, with high-throughput sequencing becoming increasingly affordable, pollen metabarcoding is gaining momentum, and it is a promising alternative to classical palynology. But one of the main drawbacks of pollen metabarcoding is the lack of good quality reference databases for the barcode of choice. BCdatabaser (Keller et al. 2020) was developed to automatically generate a standardized database for the ITS2 barcode from the primary sequence database GenBank. While using BCdatabaser to construct an ITS2 reference database for identification of bee-collected pollen, we noticed several misidentified sequences retrieved from GenBank, which would impact identification accuracy. There were two types of problems: plant sequences that were assigned to the wrong plant species and fungi sequences that were identified as plants. To overcome these issues, we developed scripts in bash and R to curate an ITS2 reference database for pollen identification purposes. These scripts allowed us to identify the Fungi sequences retrieved from GenBank for subsequent removal from the database, to perform a pairwise alignment of all the sequences using vsearch v2.14.1 (Rognes et al., 2016) and, then to remove all the sequences with low identity percentage using an iteration process in R v4.1.2. The database curation is automated therefore enabling easy update of the ITS2 database to take advantage of the new sequences that are regularly deposited in GenBank.