Identification of plastic-associated species in the Mediterranean Sea using DNA metabarcoding with Nanopore MinION

Abstract

Plastic debris in the ocean form a new ecosystem, termed ‘plastisphere’, which hosts a variety of marine organisms. Recent studies implemented DNA metabarcoding to characterize the taxonomic composition of the plastisphere in different areas of the world. In this study, we used a modified metabarcoding approach which was based on longer barcode sequences for the characterization of the plastisphere biota. We compared the microbiome of polyethylene food bags after 1 month at sea to the free-living biome in two proximal but environmentally different locations on the Mediterranean coast of Israel. We targeted the full 1.5 kb-long 16S rRNA gene for bacteria and 0.4–0.8 kb-long regions within the 18S rRNA, ITS, tufA and COI loci for eukaryotes. The taxonomic barcodes were sequenced using Oxford Nanopore Technology with multiplexing on a single MinION flow cell. We identified between 1249 and 2141 species in each of the plastic samples, of which 61 species (34 bacteria and 27 eukaryotes) were categorized as plastic-specific, including species that belong to known hydrocarbon-degrading genera. In addition to a large prokaryotes repertoire, our results, supported by scanning electron microscopy, depict a surprisingly high biodiversity of eukaryotes within the plastisphere with a dominant presence of diatoms as well as other protists, algae and fungi.

CARD-FISH in the Sequencing Era: Opening a New Universe of Protistan Ecology

“…A wide range of studies followed in various ecosystems such as marine, freshwaters, sediments, soils, and insect guts, to name a few ( Edgcomb et al, 2002 ; López-García et al, 2003 ; Romari and Vaulot, 2004 ; Lovejoy et al, 2006 ; Moon-van der Staay et al, 2006 ; Stoeck et al, 2006 ; Ohkuma and Brune, 2010 ; Piwosz and Pernthaler, 2010 ; Sauvadet et al, 2010 ; Scheckenbach et al, 2010 ; Stock et al, 2012 ; Medlin et al, 2017 ; Mukherjee et al, 2017 ). With the advent of sequencing technologies, Sanger sequencing of clone libraries has been replaced by high-throughput sequencing (HTS, also known as NGS – Next generation sequencing) techniques, such as already obsolete Ion torrent and 454 pyrosequencing ( Behnke et al, 2011 ; Bachy et al, 2013 ; Egge et al, 2013 , 2015 ; Georges et al, 2014 ; Balzano et al, 2015 ; Piwosz et al, 2018 ), currently the most often used Illumina MiSeq/HiSeq platforms ( Logares et al, 2014 ; de Vargas et al, 2015 ; Hu et al, 2016 ), and the most recent PacBio and Oxford Nanopore MinION platforms ( Orr et al, 2018 ; Davidov et al, 2020 ; Hatfield et al, 2020 ). HTS enables to process large number of samples at once at low cost, providing qualitative information on composition of protistan communities even up to the species level ( Amaral-Zettler et al, 2009 ; Stoeck et al, 2010 ; Logares et al, 2014 ).…”

“…The main strength of the FISH technique is that it is based on SSU rRNA gene phylogeny, and thus, it allows for the direct detection of phylotypes, the presence of which was identified in the samples by sequencing. Probes for novel lineages discovered with the HTS methods can be designed using full-length sequences of the 18S rRNA genes that are identical or have high similarities to short reads obtained via HTS ( Piwosz, 2019 ), obtaining full-length sequence with amplicon-based specific and general eukaryotic primers combined with Sanger sequencing ( Gimmler and Stoeck, 2015 ), or with Nanopore or PacBio long-read sequencing of amplicons ( Orr et al, 2018 ; Davidov et al, 2020 ; Hatfield et al, 2020 ). Regions of 18S rRNA unique to the selected, monophyletic group of interest can be then identified in an alignment and a robust phylogeny, at best a phylogenetic inference using the maximum likelihood (ML) criterion ( Felsenstein, 1981 ).…”

<em> </em>Current Research on Microbe-Plastic Interactions in the Marine Environment

“…Most plastisphere metabarcoding studies have targeted the overall eukaryotic fraction using the 18S rRNA gene as a marker 7,31,50,85,86,88,90,100,127,148,150 , but some have focussed particularly on the fungal plastisphere fraction 31,90 , in some cases employing fungal-specific ITS 148 or ITS2 31,87 marker genes.…”

“…Additional markers for marine microalgae (tufA) and metazoans (COI) have also been included on one occasion 148 . Taxa including (but not limited to) members of the diatoms, dinoflagellates, ciliates, fungi, brown, green and red algae, as well as a range of metazoan taxa are often reported as the dominant eukaryotes in the plastisphere (e.g., 7,12,85,86,88,100,127,148,150 ; see also the review by Rogers et al 197 ).…”

“…Indeed, diatoms are consistently described as an abundant and diverse group of colonisers on plastics, particularly during the early stages of biofilm formation 8,22,67,73,85,148,178 , while macro-and microscopic forms of metazoan taxa (e.g., bryozoans, crustaceans, molluscs, anthozoans, sponges, nematodes and annelids) often also make up a major proportion of the eukaryotic communities 12,85,86,100,127 likely, at least in part, because of their multicellular nature 127,178 . Apart from PCR-based methods, eukaryotic plastisphere members have been described in several publications using microscopy techniques 22,41,44,67,69,73,76,89,113,131 (Fig.…”