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Supplementary Material and Methods
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Experimental setup and sample collection
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Surface sediment (top 20 cm) was collected from the mouth of the Plym Estuary
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(50°22’17.22’’N, 04°06’34.45’’W) in the South West of the UK. The sediment was sieved
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through a 1mm mesh to remove large fauna, stones and other debris. In order to
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contaminate the sediment with oil, 375 g was air dried before 20 g of IFO-180 crude oil was
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dissolved in 100 mL hexane and mixed with the dry sediment. The oil-contaminated
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sediment was incubated overnight with continuous airflow, and after the hexane had
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evaporated was mixed with 1125 g of fresh sediment. The contaminated sediment was
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maintained for 2 months submerged in a continuous flow seawater aquarium at 15°C and in
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the dark before experimental setup. Oil-contaminated sediment (450 g) was transferred to
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individual plastic tubes (length 18 cm, diameter 6 cm) that were sealed at one end
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(Supplementary Figure 1). The tubes were maintained submerged in continuous flow
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seawater aquaria at 15°C and in the dark. After 7 days, 2 adult H. diversicolor with the same
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length and weight were added to 3 experimental cores and 3 cores were used as controls
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(i.e. no bioturbation). The polychaetes formed gallery burrows within 2-3 days and remained
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active throughout the experimental period (Supplementary Figure 1).
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After 30 days, a single sediment sample (4.75 cm3) was taken from each core
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containing H.diversicolor from a burrow-enriched area of the sediment. The sediment was
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collected 5 cm below the sediment water interface using plastic mini-cores (0.95 cm2 surface
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area, 5 mL syringe with the end cut off), these samples are referred to as “burrows”. In cores
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without H.diversicolor sediments were sampled in the same way and are referred to as “un-
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bioturbated sediments”. The sampled sediment was transferred into 2 mL micro-centrifuge
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tubes and stored at -80oC until further analysis.
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DNA extraction
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DNA was extracted using the DNeasy extraction kit (Qiagen) with minor modifications.
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Sediment (0.25 g) was weighed into tubes containing glass beads (0.5 g, 100–300 μm
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diameter, MPBIO lysing matrix B). ATL lysis buffer (Qiagen) (600 μL) was added to the
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tubes. The tubes were then placed in a bead beater (Mini-Bead Beater 1, BioSpec) and
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beadbeated at maximum speed (48 rpm) for 1 minute. The tubes were incubated at 56°C for
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10 minutes before being beadbeated for another 1 minute and a final incubation at 56°C for a
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further 10 minutes. The tubes were spun (10,000 rpm, 30s), and to remove humic
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acids/inhibitors the supernatant added to a QIA-shredder column (Qiagen) containing 0.1 g
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Polyvinylpolypyrrolidone (PVPP), incubated at room temperature for 10 minutes and finally
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spun (12,000 g, 30s). The rest of the protocol was carried out according to the
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manufacturer’s instructions. The DNA was re-suspended in elution buffer and quantified
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using a Nanodrop® ND-1000 spectrophotometer (Thermo-scientific).
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RNA extraction and cDNA synthesis
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Sediment (0.25g) was weighed into tubes containing 0.5g glass beads (100–300 μm,
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MPBIO) and 1mL TRI Reagent® (Ambion) before bead beating. Samples were heated at
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60°C for 10 min before 600 μL of supernatant was transferred to a new tube containing
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100µl 1-bromo-3-chloro-propane and vortexed. The tubes were centrifuged to separate the
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organic and aqueous phases before the aqueous phase was transferred to a QIA-shredder
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column containing PVPP as described above. The filtrate was precipitated with 2-propanol
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(equal volume) and sodium acetate (1/10 volume) for 1 hr at -20°C before the RNA pellet
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was isolated and washed by centrifugation. The RNA pellet was resuspended in 100 μL
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RNAse-free water and further cleaned using the RNeasy kit (Qiagen) according to the
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manufacturer’s instructions. DNAse treatment was performed using RQ1 RNase-Free
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DNase (Promega) according to the manufactures instructions. Control PCRs confirmed the
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presence of only RNA. cDNA generation was performed using an Omniscript RT kit (Qiagen)
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in accordance with manufacturer’s instructions.
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16S rRNA and 18S rRNA transcript quantification
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For bacteria qPCR, the primers of Suzuki et al (2000) were used (Forward BACT1369F 5´-
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CGG TGA ATA CGT TCY CGG-3´, Reverse PROK1492R 5´-GGW TAC CTT GTT ACG
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ACT T-3´) and for eukaryotes those of Zhu et al (2005) (Forward EUK345f 5´-
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AAGGAAGGCAGCAGGCG-3´, Reverse EUK499r 5´-CACCAGACTTGCCCTCYAAT-3´).
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qPCR was carried out using the Sensi-FAST SYBR Q-PCR kit (Bioline) in 10µl reactions
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containing 5 μL of sensi-fast master mix, 0.25 μL of each primer (final concentration 0.4 μM),
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1 μL of cDNA template and 4 μL nuclease free water. A Qiagen rotor gene3000 (Qiagen)
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was used to perform the reactions. Cycling conditions were an initial denaturation of 94°C
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for 3 min, then 40 cycles of 94°C for 10s, annealing for 15s at 59°C for bacteria or 60°C for
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eukaryotes, elongation and acquisition of fluorescence data at 72°C for 20s. Standard curves
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for Q-PCR were constructed using known amounts of purified target template generated by
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PCR amplification of the target gene from genomic DNA from either Escherichia coli or
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Saccharomyces cerevisiae. Transcript copy numbers were back calculated to water content
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corrected sediment weights (see below).
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Sediment water content
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Sediment (0.25g) was sub-sampled from each sample for DNA/RNA analysis and weighed
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into pre-combusted (550°C for 8 hours) glass petri-dishes. The dishes were placed in a
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drying oven at 60°C overnight and reweighed. Water content (%) was calculated as:
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𝑊𝑎𝑡𝑒𝑟 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 (%) = (1 −
Dry weight sediment + dish (g)
) × 100
Wet weight sediment + dish (g)
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16S rRNA/18S rRNA amplicon sequencing and bioinformatics
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Bacteria 16S rRNA and eukaryote 18S rRNA gene amplicon sequencing was carried as
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previously described (Taylor et al., 2014; Taylor and Cunliffe, 2014). In summary, the V4
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variable region of the 16S rRNA gene was amplified using the PCR primers 515F and 806R
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(Caporaso et al., 2011), and the following PCR conditions: 94°C for 3 minutes, followed by
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28 cycles of 94°C for 30 seconds, 53°C for 40 seconds and 72°C for 1 minute, and a final
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elongation step at 72°C for 5 minutes. The V9 variable region of the 18S rRNA gene was
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amplified using PCR primers 1391F (Lane, 1991) and EukB (Medlin et al., 1988). The same
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PCR conditions were used except the annealing temperature was changed to 57°C.
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Sequencing of amplified 16S rRNA and 18S rRNA genes was performed on an Ion Torrent
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PGM (Life technologies) according to manufactures instructions.
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Sequences were analysed using the QIIME software package (Caporaso et al., 2010)
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as previously described for 16S rRNA (Taylor et al., 2014) using the Greengenes database
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(DeSantis et al., 2006) and 18S rRNA (Taylor and Cunliffe, 2014) using the SILVA database
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(Quast et al., 2013) as a reference. In brief, quality filters were used to remove short
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(<250bp bacteria, <150bp eukaryotes) and low quality reads (average phred score <25).
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Chimeras were then identified and removed. Operational taxonomic units (OTUs) were
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defined at 97% similarity and classified against the reference databases.
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Hydrocarbon analysis
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After 30 days, the H. diversicolor were removed and the experimental cores sacrificed for
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hydrocarbon analysis. The sediment was homogenised before hydrocarbon analysis using
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standard protocols. In summary, aliphatic and aromatic hydrocarbons were extracted in
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hexane/acetone and analysed using GC-FID. PAHs were extracted in
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hexane/acetone/triethylamine and analysed using GC-MS.
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Statistical analyses
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Significant differences in hydrocarbon concentrations and Q-PCR results were determined
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by t-tests using the SPSS statistics software package (IBM). Permutational Multivariate
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Analysis of Variance (PERMANOVA) was performed with 999 permutations in QIIME using
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UniFrac distance matrices and OTU tables as inputs to investigate differences in community
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composition at the OTU level. In order to establish if there were significant differences
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between burrows and sediment in the relative abundance of specific eukaryote and bacteria
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groups, data were converted from percentages to arcsin values before t-test analysis using
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SPSS.
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References
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Caporaso, J.G., Lauber, C.L., Walters, W.A., Berg-Lyons, D., Lozupone, C.A., Turnbaugh,
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P.J. et al. (2011) Global patterns of 16S rRNA diversity at a depth of millions of
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