Browsing by Author "Carreck, Norman L."
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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.
- Genetic integrity of the Dark European honey bee (Apis mellifera mellifera) from protected populations: a genome-wide assessment using SNPs and mtDNA sequence dataPublication . Pinto, M. Alice; Henriques, Dora; Chávez-Galarza, Julio; Kryger, Per; Garnery, Lionel; Zee, Romée van der; Dahle, Bjørn; Soland-Reckeweg, Gabriele; De la Rúa, Pilar; Dall'Olio, Raffaele; Carreck, Norman L.; Johnston, J. SpencerThe recognition that the Dark European honey bee, Apis mellifera mellifera, is increasingly threatened in its native range has led to the establishment of conservation programmes and protected areas throughout western Europe. Previous molecular surveys showed that, despite management strategies to preserve the genetic integrity of A. m. mellifera, protected populations had a measurable component of their gene pool derived from commercial C-lineage honey bees. Here we used both sequence data from the tRNAleu-cox2 intergenic mtDNA region and a genome-wide scan, with over 1183 single nucleotide polymorphisms (SNPs), to assess genetic diversity and introgression levels in several protected populations of A. m. mellifera, which were then compared with samples collected from unprotected populations. MtDNA analysis of the protected populations revealed a single colony bearing a foreign haplotype, whereas SNPs showed varying levels of introgression ranging from virtually zero in Norway to about 14% in Denmark. Introgression overall was higher in unprotected (30%) than in protected populations (8%), and is reflected in larger SNP diversity levels of the former, although opposite diversity levels were observed for mtDNA. These results suggest that, despite controlled breeding, some protected populations still require adjustments to the management strategies to further purge foreign alleles, which can be identified by SNPs.
- 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.
- Introducing the INSIGNIA project: Environmental monitoring of pesticides use through honey beesPublication . Carreck, Norman L.; Amaral, Joana S.; Anagnostopoulos, Christ; Baveco, Hans; Bieszczad, Sarah; Biron, David G.; Brodschneider, Robert; Brusbardis, Valters; Charistos, Leonidas; Coffey, Mary F.; Eulderink, Caroline; Fernández-Alba, A.R.; Formato, Giovanni; Graaf, Dirk C.; Gratzer, Kristina; Gray, Alison; Hatjina, Fani; Kasiotis, Konstantinos; Kilpinen, Ole; Murcia-Morales, Maria; Pietropaoli, Marco; Pinto, M. Alice; Quaresma, Andreia; Roessink, Ivo; Rufino, José; Vejsnæs, Flemming; Zafeiraki, Effrosyni; Van der Steen, J.INSIGNIA aims to design and test an innovative, non-invasive, scientifically proven citizen science environmental monitoring protocol for the detection of pesticides via honey bees. It is a pilot project initiated and financed by the European Commission (PP-1-1-2018; EC SANTE). The study is being carried out by a consortium of specialists in honey bees, apiculture, chemistry, molecular biology, 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 about 1 km radius, increasing to several km if required depending on the availability and attractiveness of food. All material collected is concentrated in the hive, and the honey bee colony can provide four main matrices for environmental monitoring: bees, honey, pollen and wax. For pesticides, pollen and wax are the focal matrices. Pollen collected in pollen traps will be sampled every two weeks to record foraging conditions. During the season, most of pollen is consumed within days, so beebread can provide recent, random sampling results. On the other hand wax acts as a passive sampler, building up an archive of pesticides that have entered the hive. Alternative in-hive passive samplers will be tested to replicate wax as a “pesticide-sponge”. Samples will be analysed for the presence of pesticides and the botanical origin of the pollen using an ITS2 DNA metabarcoding approach. Data on pollen and pesticides will be then be combined to obtain information on foraging conditions and pesticide use, together with evaluation of the CORINE database for land use and pesticide legislation to model the exposure risks to honey bees and wild bees. All monitoring steps from sampling through to analysis will be studied and tested in four countries in year 1, and the best practices will then be ring-tested in nine countries in year 2. Information about the course of the project and its results and publications will be available in the INSIGNIA website www.insignia-bee.eu.
- Its2 metabarcoding: a promising approach for identification of botanical origin of bee-collected pollenPublication . Quaresma, Andreia; Van der Steen, Joseph; Amaral, Joana S.; Biron, David G.; Brodschneider, Robert; Brusbardis, Valters; Carreck, Norman L.; Mary, Frances Coffey; Formato, Giovanni; Graaf, Dirk C.; Gratzer, Kristina; Hatjina, Fani; Kilpinem, Ole; Keller, Alexander; Laget, Dries; Pietropaoli, Marco; Rufino, José; Vejsnaes, Flemming; Pinto, M. AliceBee products have long been used in human’s diet and their consumption has increasingly been recognized has beneficial for human’s health. One such product is pollen, which is a particularly interesting food as it contains bioactive compounds and all the essential amino-acids needed by humans. However, the composition of bee-collected pollen depends on the environment where the visited plants grow (e.g.: climatic conditions, soil type) and, above all, on the plant species [1]. Therefore, identification of the botanical origin of bee-collected pollen is important for a fuller characterization of this food product. Until recently, pollen identification has been carried out 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 light microscopy. 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 relative abundances obtained by the two approaches on 108 bee-collected pollen samples from 10 European countries. To that end, the 108 samples were first homogenized and split into two identical sub-sets. One sub-set was analysed 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. Pairwise comparisons of the relative abundances at the family level of the 108 samples show no significant differences (P ≥ 0.1057, Wilcoxon signed-rank test) and high correlation values (0.2736 ≤ r ≤ 0.9842, 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 = 0.4929). Despite, the few outliers, which can be improved by further optimization of the protocols, these results suggest that ITS2 metabarcoding promises to be a reliable alternative to light microscopy. This molecular 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.
- 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.