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.

Figure 1

Experiment location. (a) Top view. Inset shows location in Israel (b) open water location—side view. (c) Marina location—side view. OW open water location, m marina location.

Figure 2

Bacterial genera diversity and PE-specific species. (a) Beta-diversity of the bacterial genera and their distribution among the pooled samples (b) Shared and unique OTUs among the samples. In yellow—OTUs that were identified in both PE samples, but also in water samples. OTU counts refer to OTUs that corresponded to ≥ 2 reads. In red—OTUs that were unique to the PE samples. (c) The list of PE-specific bacterial taxa. Known petroleum and plastic-degrading genera are marked [according to their color in (a)]. OW open water samples, POW open water plastic samples, MW marina water samples, PM marina plastic samples.

Figure 3

Eukaryote taxa diversity. Beta-diversity of eukaryotic taxa and their distribution among the pooled samples. (a) 18S metabarcoding showing enrichment of diatoms (Bacillariophyceae) on PE—in light green. (b) tufA metabarcoding. (c) ITS metabarcoding (d) COI metabarcoding. OW open water samples, POW open water plastic samples, MW marina water samples, PM marina plastic samples.

Figure 4

Shared and unique eukaryotic OTUs among the samples. Venn diagrams are presented for each of the four eukaryotic barcodes: (a) 18S. (b) tufA. (c) ITS. (d) COI. In yellow—OTUs that were identified in both plastic samples, but also in water samples. In red—OTUs that were unique to both plastic samples. OW open water samples, POW open water plastic samples, MW marina water samples, PM pooled marina plastic samples, OTU counts refer to OTUs that corresponded to ≥ 2 reads. (e) List of plastic-specific eukaryote species and the number of their corresponding OTUs (in parentheses). In light green—diatoms, in dark green—green algae, in red—red algae, in brown—brown algae, in yellow—SARs clade protists (f) No shared OTUs were found among the four eukaryote barcodes.

Figure 5

SEM images of common eukaryotic taxa on polyethylene after 1 month. (a) Achnanthes sp. (b) Nevicula sp. (c) Fragilaria sp. (d) Licmophora sp. (e–i) Amphora sp. (j–l) Nitzschia sp. (m) Cylindrotheca closterium. (n) diatoms dug in the PE surface (white triangles). (o) Coccolithales sp. (p) Ulvales sp.