Back to search

MILJØFORSK-Miljøforskning for en grønn samfunnsomstilling

Environmental Barcoding of Aquatic Invertebrates

Alternative title: Miljøstrekkoding av ferskvannsinvertebrater

Awarded: NOK 4.5 mill.

Freshwater ecosystems represent an irreplaceable natural resource and are heavily influenced by human activities. These ecosystems hold almost 100 000 species, 6% of all described diversity. Many of these species are not used in monitoring programs since the identification of cryptic, diverse and taxonomically difficult organism-groups (e.g. invertebrates) often is both costly and challenging. However, by using short, standardised DNA fragments and Next Generation Sequencing (NGS) techniques, species in environmental- and bulk samples can be effectively identified. This method can potentially revolutionize the way we assess and monitor biodiversity in freshwater ecosystems and DNA metabarcoding can become an effective tool for Norway and other European countries seeking to fulfill their commitment to the European Commission Water Framework Directive. The field of DNA metabarcoding has seen tremendous progress in methodology over the last decade, and protocols for actual implementation as well as regional reference libraries are currently being developed and rigorously tested. International collaboration is key to this development, in Europe fore-fronted by the EU COST-Action DNAqua-net (http://dnaqua.net/). In EBAI we wish to advance such protocols by analysis of environmental and historical samples from the reference watershed Atna and Lake Jonsvatnet in Central Norway. Our aim is to develop general best-practice procedures to maximize the effectiveness and accuracy of species-level identification at minimum cost using next generation sequencing techniques. We have tested how different filter types, filter preservation and pre filtration influence the recorded diversity through analysis of environmental DNA in water samples. Our results show that filters preserved on silica-gel or in lysis buffer give most consistent results, and that mixed cellulose nitrate filters reflect more consistent invertebrate communities than polyethersulfone filters. Pre-filtration had no significant influence on the community of species recorded from eDNA in water (Majaneva et al. 2018a). Sorting invertebrates from bulk samples can be a very time consuming and resource demanding. We have therefore tested if different methods for DNA extraction of unsorted bulk samples of macro invertebrates will influence which species that can be recorded. Among the tested extraction kits, Qiagen's Power Plant Pro kit gave the mos consistent results, and values that correlated with sample size. This DNA extraction method also was most robust against the influence of PCR inhibiting substances from lake sediments (Majaneva et al. 2018b). Our results show that the method of DNA preservation and isolation influences the conclusions on which organisms that are present in a water shed. The preferred method is dependent on water quality as well as the presence of trace substances in samples that influence molecular analysis. In the last part of our project we have compared identification of benthic invertebrates by traditional methods with identification using DNA metabarcoding of the same samples. We have further compared these results with those based on identification of invertebrates in eDNA (water) samples from the same sites. Final results of these analyses are expected during autumn 2019. Like similar studies of other communities (e.g. Sun et al. 2019), we expect that DNA metabarcoding detects species that are difficult to identify using morphology, but that some species also only are detected with traditional methods. As part of this project, we have written a report to the Norwegian Environment Agency where we summarize the current state of knowledge in DNA metabarcoding of invertebrates in freshwater habitats. The report will be published by the NTNU University Museum in the autumn 2019 (Ekrem & Majaneva 2019). References Ekrem, T. & Majaneva, M. 2019. DNA-metastrekkoding til undersøkelser av invertebrater i ferskvann. ? NTNU Vitenskapsmuseet naturhistorisk notat 2019-x: 1-xx. Majaneva M, Diserud OH, Eagle SHC, Boström E, Hajibabaei M, Ekrem T (2018a) Environmental DNA filtration techniques affect recovered biodiversity. Scientific Reports 8, 4682. doi: 10.1038/s41598-018-23052-8. Majaneva M, Diserud OH, Eagle SHC, Hajibabaei M, Ekrem T (2018b) Choice of DNA extraction method affects DNA metabarcoding of unsorted invertebrate bulk samples. Metabarcoding and Metagenomics 2: e26664. doi: 10.3897/mbmg.2.26664 Sun, Z., M. Majaneva, E. Sokolova, S. Rauch, S. Meland & T. Ekrem, 2019. DNA metabarcoding adds valuable information for management of biodiversity in roadside stormwater ponds. Ecology and Evolution 00: 1? 11. doi: 10.1002/ece3.5503.

Prosjektet har: 1. Bidratt til større forståelse om hvordan miljø-DNA i ferskvann kan isoleres og analyseres. 2. Bidratt til kunnskap om ulike ekstraksjonsprotokoller for DNA fra bulkprøver av makroinvertebrater. 3. Styrket samarbeidet mellom NTNU Vitenskapsmuseet og nasjonale og internasjonale partnere. 4. Tilrettelagt for aktiv deltakelse i EUs COST-aksjon DNAqua-Net. 5. Gitt grunnlag for kunnskapsrapport til Miljødirektoratet om DNA-metastrekkoding av invertebrater i ferskvann. 6. Gitt kompetanse til å delta i et internasjonalt forskningsprosjekt finansiert av Nordisk ministerråd. 7. Gitt muligheten til å utvikle et fagområde ved NTNU som vil være viktig for fremtidig miljøovervåkning. 8. Lagt grunnlaget for bruk av metodikk i to PhD-grader, en mastergrad og to bachelorgrader med prosjektdeltakere som medveiledere. 9. Kommunisert resultater og kunnskap til allmenhet og fagfeller.

The possibility to identify species in environmental samples using short, standardised DNA fragments and Next Generation Sequencing (NGS) techniques has the potential to revolutionize the way we assess and monitor biodiversity in freshwater ecosystems. Here we suggest an experimental set-up to advance protocols for environmental barcoding through analysis of recent and historical samples from the reference watershed Atna and Lake Jonsvatnet in Central Norway. Our aim is to develop general best-practice procedures to maximise the effectiveness and accuracy of species-level identification at minimum cost using next generation sequencing techniques. By doing so in an international framework and including a post-doctoral position, we wish to promote knowledge transfer and capacity building among the partnering institutions and ensure recruitment of qualified personnel to this important area of nature research and management. Through collaboration with the Norwegian Environment Agency we will ensure that results are disseminated to relevant user groups.

Publications from Cristin

Funding scheme:

MILJØFORSK-Miljøforskning for en grønn samfunnsomstilling