A DNA (meta)barcoding approach to assess changes in seabed ecosystems related to human- induced pressures Devriese LisaA, De Backer AnneliesA, Maes SaraA, Van Hoey GertA, Haegeman AnneliesB, Ruttink TomB, Wittoeck JanA, Hillewaert HansA, De Tender CarolineA, Hostens KrisA A Institute for Agricultural and Fisheries Research (ILVO), Animal Sciences Unit, Aquatic environment and quality, Ankerstraat 1, 8400 Oostende, Belgium B Institute for Agricultural and Fisheries Research (ILVO), Plant Sciences Unit, Growth and Development, Caritasstraat 39, 9090 Melle, Belgium Contact: [email protected] I. Aim II. Methodology Macrobenthos communities Development of a DNA metabarcoding • Amplicon sequencing of the V4 fragment of the 18S rDNA using Illumina technology pipeline to assess marine benthic DNA extracts of individual species and artificial mixtures of various species biodiversity in the North Sea. • Sanger sequencing of 4 DNA barcode amplicons: COI (313-319 bp), COI (655-661 bp), V4 18S rDNA (370-582 bp), V7-V8 18S rDNA (281-592 bp) Evaluation of DNA metabarcoding to assess effects of sand extraction on Bacterial communities bacterial species composition in the • Amplicon sequencing of the V3-V4 fragment of the 16S rDNA using Illumina technology Belgian part of the North Sea. (De Tender et al., 2015) DNA extracts of sediment samples (Buiten Ratel sand bank) IV. Macrobenthos III. Macrobenthos Evaluation of the Sanger sequencing Amplicon sequencing, e.g. V4 18S rDNA effectiveness of the barcoding primers (V4 18S) on Taxonomic resolution Species identification OK individual species Failed sequencing of the barcoding Sequence not in public database Identification Genus level and artificial mix primers: Identified as other species * Failed PCR samples. Phylum Class COI COI 18S 18S large small V4 V7 – V8 Mix Mix Mollusca Bivalvia Abra alba Donax vittatus * * samples DNA Good taxonomic Ensis directus Limecola balthica Kurtiella bidentata resolution, except Fabulina fabula Unique Asbjornsenia pygmaea Unique for species of the Mya truncata Spisula solida samples DNA Gastropoda Buccinum undatum * * Detected order Amphipoda. Crepidula fornicata * Not detected Nassarius reticulatus Annelida Polychaeta Aonides oxycephala Aphrodita aculeata Capitella (C. capitata) Chaetozone setosa * * Cirratulidae (Cirratulus) Eteone longa * V. Bacteria VI. Conclusion Eunereis longissima Glycera alba * Glycera tridactyla Lanice conchilega Amplicon sequencing: V3-V4 16S rDNA DNA barcode reference database Lagis koreni * Lumbrineris futilis * Our aim is to establish a complete Mediomastus fragilis Magelona johnstoni Bacterial communities on sediment in relation Nephtys assimilis North Sea DNA barcode reference Nephtys caeca to the impact of sand extraction activities: Nephtys cirrosa database for bacteria and Nephtys hombergii Nephtys longosetosa Phylum level Notomastus latericeus Acidobacteria macrobenthos, in close cooperation Ophelia borealis Actinobacteria Bacteroidetes Owenia fusiformis Chloroflexi with other institutes along the Pholoe baltica Cyanobacteria Proteobacteria Phyllodoce mucosa Gemmatimonadetes Lentisphaerae North Sea Poecilochaetus serpens Planctomycetes Malmgrenia lunulata Proteobacteria are the Scolelepis bonnieri Unasigned – other Verrucomicrobia Scoloplos armiger dominant Spiophanes bombyx Macrobenthos communities Spio Sthenelais boa classes of Echinodermata Ophiuroidea Acrocnida brachiata Taxonomic resolution can be Ophiura ophiura bacterial Ophiura albida increased by combining multiple Echinoidea Echinocardium cordatum Echinocyamus pusillus communties at Psammechinus miliaris barcodes Arthropoda Malacostraca Abludomelita obtusata Control Medium High Buiten Ratel. Bathyporeia elegans Bathyporeia guilliamsoniana Leucothoe incisa Control site Next step: Megaluropus agilis Medium impact Microprotopus maculatus High impact Evaluation of DNA metabarcoding Nototropis falcatus Nototropis swammerdamei Pariambus typicus Sand extraction pipeline for real (sieved) sediment Medium impact Pontocrates arenarius Siphonocetes kroyeranus activities lead samples Tryphosa nana High impact Urothoe brevicornis Urothoe poseidonis to shifts in the Diastylis bradyi Iphinoe trispinosa bacterial Bacterial communities Cnidaria Anthozoa Sagartia troglodytes Arthropoda Malacostraca Gastrosaccus spinifer DNA metabarcoding revealed that Palaemon serratus community Processa modica Thia scutellata composition. disturbance of the seabed affects Pestarella tyrrhena Chordata Leptocardii Branchiostoma lanceolatum bacterial communities Barcode sequences for 70 most Control site Bacterial assemblage composition abundant macrobenthos species were could be an interesting tool to Dredging intensity: added to ILVO DNA barcode reference High impact: >10000 m³/ha measure changes in marine database. Medium impact: 1000-5000 m³/ha ecosystems We thank the aggregate extraction industry through the Sand Fund for financing this research. Shiptime on RV Belgica was provided by Belspo and RBINS OD Nature. De Tender, C. A.; Devriese, L. I.; Haegeman, A.; Maes, S.; Ruttink, T.; Dawyndt, P. Bacterial community profiling of plastic litter in the Belgian part of the North Sea. Environ Sci Technol. 2015, 49, 9629-9638.