Vertical and horizontal environmental DNA (eDNA) patterns of fish in a shallow and well-mixed North Sea area

Study Extent

The integration of eDNA metabarcoding into monitoring programs provides valuable information about fish community structures. Despite the growing body of evidence supporting the method's effectiveness in distinguishing fine-scale eDNA signals, there is a limited understanding of eDNA distribution in shallow, well-mixed environments, especially related to sampling depth. We analyzed 167 samples collected from the surface and bottom water at 17 locations of the Belgian Part of the North Sea (BPNS) and compared this to beam trawl catch data collected simultaneously at the same locations.

Sampling

1.Sampling was carried out between September 26 and October 7, 2022 at the Belgium Part of the North Sea (BPNS) with the research vessel Belgica. Water samples were collected approximately 1 m above the seafloor at 17 locations covering the distribution of the three distinct fish communities (coastal, transition and offshore) (Figure 1). At twelve locations, water was sampled both at the surface and bottom to study the vertical distribution pattern of eDNA. A rosette equipped with 10 L Niskin bottles was deployed to collect the water samples: bottom samples were taken 1 meter above the seabed and surface samples 1 meter below the sea surface. Prior to deployment, the rosette was held below the surface for three minutes to allow the Niskin bottles and the CTD to equilibrate with the surrounding water mass. To minimize the potential disturbance of eDNA caused by the downward movement of the rosette, the surface samples were collected before the bottom samples. Five biological replicates were collected at each location and depth, with each replicate corresponding to a single Niskin bottle. From each Niskin bottle, 2 L seawater was pre-filtered through a 200 µm sterilized nylon mesh to remove bigger particles and organisms that can potentially cause clogging during the subsequent filtering process. Filtering of the samples was conducted onboard in a dedicated lab for eDNA analyses, right after the sample collection. The samples were filtered through a 0.45 µm Sterivex-HV filter (Millipore SterivexTM, PVDF, with Luer outlet) using a peristaltic pump (Masterflex L/S® Variable Speed Pump HV-77910-75) until the filter was close to clogging. 2. Quality control Negative control samples were collected in the field and laboratory environments. Negative field controls were taken by collecting commercial source water from the Niskin bottles after they were carefully rinsed using commercial source water, also using the prefilter. Negative filter controls were included by filtering source water over a blanco 0.45 µm Sterivex filter. Negative extraction controls were included by applying the extraction protocol on blanco 0.45 µM Sterivex filters. Negative PCR controls were included by replace the extracted eDNA with 3 µl of UltraPure™ water.

Method steps

1. 1. eDNA extraction and Library preparation. After overnight incubation with the lysisbuffer at 56°C, the eDNA was extracted using the DNeasy Blood and Tissue kit (Qiagen). Library preparation The genetic analysis was based on 12S. A one-step amplification protocol was used in triplicate using fusion primers (Sigma Aldrich), which contained the template specific primer sequence and a unique barcode tag of 6 to 10 nucleotides. After amplification, the PCR replicates were pooled and purified using magnetic CleanNGS beads (CleanNA). After purification, the three 12S libraries were quality checked on the BioAnalyzer. The eDNA libraries were sequenced with Illumina MiSeq, 2 x 300 bp. 2. Bioinformatic processing The quality of the raw Illumina MiSeq sequencing reads was verified with FASTQC v0.11.9. The paired-end reads were then reorientated, demultiplexed and trimmed by using cutadapt. After demultiplexing, DADA2 was used for denoising, dereplication, merging, and removing of chimeric reads from the demultiplexed sequences. The taxonomic assignment of the resulting ASV sequences was performed against a custom made reference database using RDP classifier in DADA2 with a minimum bootstrapping support of 80. ASVs that remained unassigned at species level with RDP were successively run with BLASTn v2.12.0 against the custom made reference databases and the GenBank nucleotide database (from march 2023). After taxonomic assignment, the count table was cleaned by using MicroDecon using the field, filter, DNA extraction and PCR negative control samples.

Belgian part of the North Sea