DOI: 10.5061/dryad.z8w9ghx7m
title: Microbial colonization of microplastics in the Caribbean Sea

description: Microplastics in the ocean function as an artificial microbial reef, with
 unique and diverse communities of eukaryotic and bacterial microbiota
 colonizing its surface. It is not well understood if the communities in
 this biofilm, also termed “plastisphere”, are specific for the type of
 microplastic on which they develop. Here, we carried out a controlled
 6-week long in situ incubation experiment of six common plastic polymer in
 Bocas del Toro, Panama. The community composition of prokaryotes based on
 16S rRNA gene sequencing data, when judged under a null model analysis,
 show that neither plastic polymer type nor time exposed to the environment
 play a significant role in shaping biofilm communities. However, the null
 model analyses of eukaryotic communities based on 18S rRNA gene sequences
 reveal that they can be significantly influenced by plastic polymer type
 and time incubated. This was confirmed by  scanning electron
 microscopy, which allowed us to distinguish plastic-specific diatom
 communities by the end of the incubation period.
description: Microplastic pieces of varying plastic polymers were
 incubated <em>in situ</em> in a coastal bay in the Caribbean
 off of the coast of Bocas del Toro, Panama and sampled over a time series
 to assess biofilm formation. At each sampling point, 15 microplastic
 pieces of each plastic polymer type were randomly selected from their
 respective sachets and stored in ATL buffer at -20°C after proteinase K
 digestion prior to being transported and extracted for DNA as per the
 Qiagen DNeasy Blood and Tissue kit manufacturer’s protocol. The taxonomic
 composition of bacterial and eukaryotic communities were determined by the
 Illumina MiSeq 2x300 amplicon sequencing platform of 16S and 18S rRNA
 genes. PCR amplification was performed using primers 515F and 926R (Quince
 et al., 2011; Parada et al., 2016) to amplify the V4-V5 region of
 bacterial 16S rRNA genes and primers eukv4F and eukv4R (Stoeck et al.,
 2010) to amplify the V4 region of eukaryotic 18S rRNA genes.
 Microplastics were preserved in
 glutaraldehyde (Sigma-Aldrich, 5% (v/v)), cooled at 4°C for 2-8 hours,
 then transferred into 50% (v/v) ethanol in Phosphate Buffer Solution (PBS)
 and stored at -20℃ until further preparation and imaging at the ASU
 laboratory. Samples were then dehydrated through a graded ethanol series
 and critical-point dried. The dried samples were mounted on aluminum stubs
 and sputter-coated with 10-15 nm of gold-palladium (60/40). Images were
 generated using a TESCAN VEGA<sup>3</sup> scanning electron
 microscope (SEM) operated at 15kV. The diatom community, specifically,
 was taxonomically distinguished based on their morphology. We classified
 diatoms into 14 morphologically distinct groups denoted D1 through D14,
 with D14 comprising all “other” diatoms that were counted in low abundance
 (&lt;2). Five fields under SEM were counted, which amounted to 1-58
 cells for a taxonomic group depending on its density on the plastic piece.
 Each field had a SEM magnification of 1760x with working distances between
 13.36-14.02 mm, making each field approximately 200 µm<sup>2
 </sup>in size. Diatom density on the plastic surfaces was determined
 in duplicates for each plastic type and expressed in
 cells/mm<sup>2</sup>. 
description: Eukaryotic amplicon sequence
 variants (ASVs) classified as Metazoans were removed from the
 analyses. For the
 microscopy-derived diatom community data, D14, given its variability, was
 not used in any analyses.