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— In the present research project, we compare three different pollination types (viz., with insects, without insects, and solely with honeybees) of three widespread sweet cherry cultivars, with the aim of conducting measurements and drawing conclusions pertaining to the impact of pollinators on fruit set, yield, and fruit quality in commercial orchards. It included a total of three treatments: Isolation with anti-insect nets (IS), Isolation with cages and Honeybee Pollination (HB), and Open Pollination (OP). Recent research focuses mostly on the role that different types of pollinator have in sweet cherry pollination; however, they do not provide measurements of final yields. The applied experimental method, was fast, simple, of low labor cost, and yielded robust and valid results based upon statistical analysis that are easily comprehensible by the sweet cherry growers. From the results of the experiment, it is evident that the absence of pollinators is a restrictive factor in fruit production, not only in cross-pollinated but also in self-fertile varieties. The value of open pollination, during which cherry flowers are visited by native pollinators and honeybees is beyond doubt. Wind is not a means of transporting pollen to sweet cherry trees. Honeybees have proved to be effective managed pollinators and thus they represent an efficient approach that can ensure increased yields and large size fruits, when colonies placed in sweet cherry orchards.

Honeybee 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. EU, under the N° 09.200200/2021/864096/SER/ ENV.D.2 contract info:eu-repo/semantics/publishedVersion

Additional file 2: Supplementary materials and methods. Supplementary materials and methods describing in detail the datasets used, the laboratory methods, the bioinformatic pipeline, the SNP selection approach, and the sample classification using Machine Learning algorithms.

Additional file 3: Figure S1. PCA-Plots with the PCA-selected SNPs and 100 simulated individuals based on allele frequencies of the pools. (A) Using 300 SNPs, the evolutionary lineages M, C and O were well separated with the first two PCs, while lineage A can be differentiated with the third component (not shown). (B) Within the M-lineage, harboring only two European subspecies (A. m. mellifera and A. m. iberiensis), the first PC using 200 SNPs already contributes sufficient information. (C) In the O-lineage, four subspecies are represented that are separated with 600 SNPs using 4 PCs. (D) For lineage C that contains numerous subspecies (subspecies complex), 1100 SNPs were selected to obtain a better resolution.

Different brood interruption methods combined with chemical treatment may successfully remove most V. destructor from the honey bee colonies. Further, the timely brood reduction or break may positively impact nectar intake and is occasionally used by beekeepers during the main spring flow to increase honey production. However, the experience and data on the effect of brood break on honey production during the main summer flow are scarce or contradictory. To try to find an answer, several Varroa Task Force members started an experiment in 2021. The study takes place at 10 apiaries in 7 countries (Algeria, Croatia, Greece, Israel, Italy, Spain and Turkey). At each location, three different groups with at least 5 colonies per group were established. In two groups, queens were confined for 28 days in a small cage 28 (QC1) or 14 (QC2) days before the expected main summer honey harvest. The third group (C) of colonies had free laying queens to serve as a control group. In total 207 colonies are included in this year study. All colonies were evaluated for strength (number of frames with bees and brood) every 14 days after the experiment started. The net weight of extracted honey for each colony was measured. Adult bee infestation was measured at the beginning of the experiment and 42 days after the treatment of the control group. Queen caging groups were treated by OA trickling 28 days from the day of queens’ caging. The C group was treated by each partner using usual practices. At the beginning of experiment, there was no difference in colony strength between groups (ANOVA, F (2, 206) = 0.030, p = 0.971). A number of frames occupied with bees was significantly lower in QC groups comparing to C group 42 days after the caging but not 56 days after caging. Briefly, they started and finished with equal strength. Further, there was no significant difference in adult bee infestation at the beginning of the trial. However, at the last measurement (42 days after the treatment of QC1 and C group and 28 days after the treatment of QC2 group) significant differences are found (H = 12.825, p = 0.002). Namely, QC1 group had lower infestation comparing to C group. In the analysis of honey production, apiary and group as fixed factors were used in the model. Apiary as a factor had significant effect (GLM, F (9, 206) = 64.921, p < 0.001) with wide distribution of extracted honey between apiaries (average from 1.45 kg to 29.50 kg/apiary). Significant differences between groups are found (F (2, 206) = 4, 556, p=0.012), with group QC1 having lower amount of extracted honey comparing to QC2 and C groups. No significant differences were found between QC2 and C group. Preliminary results showed that QC1 group had a significantly lower final infestation, as well as honey production. On the other hand, QC2 group had the same amount of produced honey and final infestation as a control group. Measurements of colony strength before and after wintering period are planned.

Additional file 4: Figure S2. Visualization using a t-SNE manifold plot of the 1988 honey bee samples from the pool sequencing individually genotyped for 4094 SNPs. Samples have been color-coded according to the pool name which represents subspecies and country of origin as listed in Table 1.

This guideline is a general guideline for the application of honey bee colonies for bio-monitoring and how to organise an Apicultural citizen science study. The study objective determines the toolbox for the beekeepers. This guideline has been designed and tested for its practical and scientific merits in the INSIGNIA study 2018-2021. This resulted in a best practice guide for Apicultural citizen science studies for bio-monitoring with honey bee colonies.The supporting INSIGNIA studies, reports, and publications are available on theINSIGNIA website (https://www.insignia-bee.eu) - publications. This website will bemaintained for seven (7) years after the end of the INSIGNIA study 2018-2021.

INSIGNIA ("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). Um dos objetivos do projeto INSIGNIA é desenvolver um protocolo para monitorização ambiental através do pólen. O protocolo será aplicado num contexto de ciência do cidadão, pelo que os apicultores serão parte ativa no processo de amostragem ambiental através da colheita bimensal de pólen nas suas colmeias. As amostras de pólen serão analisadas relativamente a resíduos de pesticidas (autorizados e não autorizados) e medicamentos de uso veterinário. Adicionalmente, serão determinados parâmetros botânicos das misturas de pólen (número de espécies e abundância relativa) recorrendo a técnicas de metagenómica e sequenciação de nova geração, estando esta tarefa sob responsabilidade do CIMO. info:eu-repo/semantics/publishedVersion

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. The honey bee colony can provide four main matrices for environmental monitoring: bees, honey, pollen and wax. Because of the non-destructive remit of the project, for pesticides, pollen is the focal matrix and used as trapped pollen and beebread in this study. Although beeswax can be used as a passive sampler for pesticides, this matrix is not being used in INSIGNIA because of its polarity dependent absorbance, which limits the required wide range of pesticides to be monitored. Alternatively, two innovative non-biological matrices are being tested: i) the “Beehold tube”, a tube lined with the generic absorbent polyethylene-glycol PEG, through which hive-entering bees are forced to pass, and ii) the “APIStrip” (Absorbing Pesticides In-hive Strips) with a specific pesticide absorbent which is hung between the bee combs. Beebread and pollen collected in pollen traps are being sampled every two weeks to be analysed for pesticide residues and to record foraging conditions. Trapped pollen provides snapshots of the foraging conditions and contaminants on a single day. During the active season, the majority of beebread is consumed within days, so beebread provides recent, random sampling results. The Beehold tube and the APIStrips are present throughout the 2-weeks sampling periods in the beehive, absorbing and accumulating the incoming contaminants. The four matrices i.e. trapped pollen, beebread, Beehold tubes and APIStrips will be analysed for the presence of pesticides. The botanical origin of trapped pollen, beebread and pollen in the Beehold tubes will also be determined with an innovative molecular technique. Data on pollen and pesticide presence will 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 rigorously 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, its results and publications will be available on the INSIGNIA website www.insignia-bee.eu and via social media: on Facebook (https://www.facebook.com/insigniabee.eu/); Instagram insignia_bee); and Twitter (insignia_bee). Although the analyses of pesticide residues and pollen identification will not be completed until December 2019, in my talk I will present preliminary results of the Year 1 sampling. info:eu-repo/semantics/publishedVersion

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. info:eu-repo/semantics/publishedVersion