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

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Abstract

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.

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All source data analysed is available in the supplemental material.

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Acknowledgements

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.

Funding

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).

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Authors and Affiliations

  1. Centro de Investigação de Montanha, Instituto Politécnico de Bragança, Campus de Santa Apolónia, 5300-253, Bragança, Portugal

    Andreia Quaresma & M. Alice Pinto

  2. Institute of Biology, University of Graz, Universitätsplatz 2, 8010, Graz, Austria

    Robert Brodschneider & Kristina Gratzer

  3. Department of Mathematics and Statistics, University of Strathclyde, Glasgow, UK

    Alison Gray

  4. Center for Computational and Theoretical Biology, Hubland Nord, Würzburg, Germany

    Alexander Keller

  5. Department of Bioinformatics, University of Würzburg, Am Hubland, BiocenterWürzburg, Germany

    Alexander Keller

  6. Danish Beekeepers Association, Sorø, Denmark

    Ole Kilpinen & Flemming Vejsnæs

  7. Research Centre in Digitalization and Intelligent Robotics (CeDRI), Instituto Politécnico de Bragança, Bragança, Portugal

    José Rufino

  8. Alveus AB Consultancy, Oisterwijk, Netherlands

    Jozef van der Steen

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  1. Andreia Quaresma
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  2. Robert Brodschneider
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  9. Flemming Vejsnæs
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  10. M. Alice Pinto
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Contributions

MAP, JvdS, RB, OK and FV conceived the ideas and designed the methodology. AQ performed the laboratorial metabarcoding work as well as all the analyses with assistance of MAP and AG. FV and OK performed the preliminary silica testing. Pollen sampling and sample preservation were carried out by RB, KG, OK and FV. AK and JR assembled the reference ITS2 database and assisted with bioinformatics. JvdS acquired funding. MAP and AQ wrote the original draft. All the authors contributed with data interpretation. All the authors critically revised and edited the manuscript for important intellectual content.

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Correspondence to M. Alice Pinto.

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Quaresma, A., Brodschneider, R., Gratzer, K. et al. Preservation methods of honey bee-collected pollen are not a source of bias in ITS2 metabarcoding. Environ Monit Assess 193, 785 (2021). https://doi.org/10.1007/s10661-021-09563-4

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