Preservation methods of honey bee-collected pollen are not a source of bias in ITS2 metabarcoding

Pollen 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.We are deeply indebted to Susana Lopes and Maria Magalhães, from CIBIO—Research Centre in Biodiversity and Genetic Resources—InBIO Associate Laboratory, for their time devoted to library preparation and sequencing in the MiSeq. AQ acknowledges the PhD scholarship (DFA/BD/5155/2020) funded by FCT. This work was funded by the Health and Food Safety Directorate General, European Commission through the project INSIGNIA—Environmental monitoring of pesticide use through honeybees SANTE/E4/SI2.788418-SI2.788452- INSIGINIA-PP-1–1-2018. Fundação para a Ciência e a Tecnologia (FCT) provided financial support by national funds (FCT/MCTES) to CIMO (UIDB/00690/2020).info:eu-repo/semantics/publishedVersio

Honey bee collected pollen for botanical identification via its2 metabarcoding: a comparison of preservation methods for citizen science

DNA 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.AQ acknowledges the PhD scholarship (DFA/BD/5155/2020) funded by FCT. This study was funded by INSIGNIA “Environmental monitoring of pesticides use through honey bees” (SANTE/E4/SI2.788418-SI2.788452).info:eu-repo/semantics/publishedVersio

Semi-automated sequence curation for reliable reference datasets in ITS2 vascular plant DNA (meta-)barcoding

One of the most critical steps for accurate taxonomic identification in DNA (meta)-barcoding is to have an accurate DNA reference sequence dataset for the marker of choice. Therefore, developing such a dataset has been a long-term ambition, especially in the Viridiplantae kingdom. Typically, reference datasets are constructed with sequences downloaded from general public databases, which can carry taxonomic and other relevant errors. Herein, we constructed a curated (i) global dataset, (ii) European crop dataset, and (iii) 27 datasets for the EU countries for the ITS2 barcoding marker of vascular plants. To that end, we first developed a pipeline script that entails (i) an automated curation stage comprising five filters, (ii) manual taxonomic correction for misclassified taxa, and (iii) manual addition of newly sequenced species. The pipeline allows easy updating of the curated datasets. With this approach, 13% of the sequences, corresponding to 7% of species originally imported from GenBank, were discarded. Further, 259 sequences were manually added to the curated global dataset, which now comprises 307,977 sequences of 111,382 plant species.AQ acknowledges the PhD scholarship (2020.05155.BD), funded by the Portuguese Foundation for Science and Technology (FCT). This work was developed in the framework of INSIGNIA – Environmental monitoring of pesticide use through honeybees (SANTE/E4/SI2.788418-SI2.788452-INSIGINIA-PP-1-1-2018) and INSIGNIA-EU - Preparatory action for monitoring of environmental pollution using honey bees (Procurement procedure ENV/2021/OP/0014 of 28-09-2021). FCT provided financial support by national funds (FCT/MCTES) to CIMO (UIDB/00690/2020 and UIDP/00690/2020) and SusTEC (LA/P/0007/2021).info:eu-repo/semantics/publishedVersio

Challenges and perspectives for beekeeping in Ethiopia. A review

International audienceThe honey bee is an important fruit and vegetable pollinator and a producer of honey and other hive products. Beekeeping is a sustainable and high-potential activity for local communities and especially for the rural poor to gain additional income through non-timber forest products, does not require much land or high starting costs, maintains biodiversity and increases crop yields. Ethiopia is one of the top ten honey and beeswax producers in the world, but plays only a minor role in the international honey trade. Unlike large-scale beekeepers using modern techniques found in most leading honey-producing countries, the majority of Ethiopian beekeepers are small-scale producers practicing traditional beekeeping. In this article, we summarize the knowledge on Ethiopian beekeeping, honey bees, honey bee pests, marketing strategies, cultural aspects and major challenges of beekeeping. Furthermore, we used FAOSTAT data to calculate a pollination gap in order to draw the attention of stakeholders and decision-makers to bees and their importance in pollination and sustainable rural development. In regard to forage, we compiled 590 bee forage plants and their flowering times as a supplement to the article. This review outlines the following major points: (1) Ethiopia is a top honey and beeswax producer mainly for the domestic market; (2) Equipment for traditional beekeeping is easily accessible but brings disadvantages (gender gap, limitations in hive management and lower honey yield), while transitional and modern systems require certain beekeeping skills and higher starting costs; (3) Colony numbers increased by 72% from 1993 to 2018 and crop areas needing pollination by 150%; (4) Honey yield per hive and number of beehives managed per area of bee-pollinated crops increased by 20% and 28%, respectively; (5) Pesticide use has been increasing and there is a lack in pesticide use education. Recommendations to realize Ethiopia’s tremendous apicultural potential are discussed

Honey bee collected pollen for botanical identification via its2 metabarcoding: a comparison of preservation methods for citizen science

While 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.info:eu-repo/semantics/publishedVersio

Bio-Monitoring of environmental pollution using the citizen science approach

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 contractinfo:eu-repo/semantics/publishedVersio