Application of DNA metabarcoding in North Sea soft bottom monitoring
Soft-bottom animals bigger than 1 mm, also known as macrobenthos, are good indicators for environmental quality. Consequently, soft sediment samples are taken all around the North Sea in the framework of EU directives such as MSFD, environmental impact assessments or follow-up of long-term trends. Despite these different objectives, the overall processing of the samples is largely similar. Macrobenthos individuals are sorted, counted and identified under a stereo-microscope by taxonomic experts. This is a time-consuming process (up to 3 days for one sample), and accuracy depends on the skills of the identifier. DNA-based approaches, where species can be distinguished based on their unique DNA sequence (i.e. DNA barcode), may speed up and complement the traditional methods for species identification, but require standardization of laboratory protocols and data processing to make results comparable across different North sea countries. Moreover, it is of paramount importance to link DNA-based profiles with the long history of morphological data collection to be able to study long term shifts in faunal communities.
Within GEANS, we aim to optimize and harmonise DNA-based methods across the North Sea region for macrobenthos. Reading the DNA barcodes from the bulk samples prior to sorting (i.e. metabarcoding) may allow more time- and cost-efficient monitoring that is less dependent on identification skills of the taxonomic expert at hand.
The soft-bottom pilot includes three different types of case studies chosen based on stakeholders’ needs in the different countries:
1) Impact of marine aggregate extraction on the soft sediment ecosystem
Within this case study, DNA-based methods will be used to identify macrobenthos in sites experiencing different intensities of marine aggregate extraction in the Belgian part of North Sea. Marine aggregate extraction in Belgium is a federal competence that belongs to the FPS Economy and is regulated by the Law of 13 June 1969. The aim of this law, and the subsequent Royal Decrees is to sustainably regulate marine aggregate extraction. Monitoring on macrobenthic communities is performed to assess the impact of marine aggregate extraction. Monitoring data are then translated into clear and simple information for sound management of the environment using biological indices e.g. Benthic Ecosystem Quality Index(BEQI) or Benthic Quality Index, but also using multivariate methods, species richness and density. In this case study, traditional morphological analyses will be run in parallel to DNA-based (both bulk and eDNA metabarcoding) methods to calculate cost and time benefits. In addition, we aim to find a DNA-based alternative where the output of the DNA-based method is used as input for biological indices. Ultimately, we aim to link the long term morphological time series with these new DNA-based approaches.
2) MSFD Monitoring in the North Sea
Traditional macrobenthos haps core-sampling in the soft-bottom sediment of the Danish North Sea typically covers in the order of about 10-10 of the habitat of interest. Thus, rare species are seldom included or they are poorly quantified. In contrast, DNA molecules of macrobenthos species are released in the water column and in the sediment where they can reside for many months. New sequencing technologies now allow to study DNA molecules directly from the environment (so-called eDNA). Within this case study, eDNA-based methods will be compared with the traditional macrobenthos haps core-sampling of soft sediment by sampling intact sediment cores of ca. 7 cm with a diameter of ca. 2 cm from the core samples in the Danish, German and Belgian parts of the North Sea. DNA will be extracted directly from the sediment and a metabarcoding approach with universal eukaryote primers will be determined.
Specific aims are to:
- Compare small-scale (between samples) patterns of selected macrobenthos species as obtained from eDNA and sampling of live specimens.
- Compare large-scale (basin-wise) distribution pattern of selected macrobenthos species as obtained from eDNA and live specimens
- Explore causes of observed mismatch between live specimens and their eDNA footprint in marine offshore sediments
- Establish proxies (e.g. M-OTU) for benthic biodiversity measures
More than 200 sediment samples were collected from Danish stations during April 23-25 2019.
3) Long-term soft-bottom monitoring station
Norderney belongs to Senckenberg´s Long Term Ecological Research (LTER) North Sea Benthos Observatory. Five stations are located at water depths between 10 and 20 m north of the island of Norderney (German Wadden Sea). Since 1978, macrobenthos sampling has been carried out on board of the RV “Senckenberg” using a 0.2 m2 Van Veen grab, a single grab is taken at each station. Samples are sieved over 0.63 mm mesh size and fixed in 4% buffered formaldehyde. Species number, abundance and biomass are determined.
In total, 196 species have been found in the area since 1978, dominated by crustaceans, polychaetes and molluscs. Similarly, long term macrobenthos sampling (since 1985) has been undertaken at four stations spread over the Belgian part of the North Sea. Here over 400 species have been found, dominated as well by polychaetes, crustaceans and molluscs. Up to now the identification of species is done by traditional taxonomic analyses but within this case study molecular and traditional taxonomic methods will be run in parallel.