Nucleic acids released by organisms and isolated from environmental substrates are increasingly being used for molecular biomonitoring. While environmental DNA (eDNA) has received attention recently, the potential of environmental RNA as a biomonitoring tool remains less explored. Several recent studies using paired DNA and RNA metabarcoding of bulk samples suggest that RNA might better reflect “metabolically active” parts of the community. However, such studies mainly capture organismal eDNA and eRNA. For larger eukaryotes, isolation of extra-organismal RNA will be important, but viability needs to be examined in a field-based setting. In this study we evaluate (a) whether extra-organismal eRNA release from macroeukaryotes can be detected given its supposedly rapid degradation, and (b) if the same field collection methods for eDNA can be applied to eRNA. We collected eDNA and eRNA from water in lakes where fish community composition is well documented, enabling a comparison between the two nucleic acids in two different seasons with monitoring using conventional methods. We found that eRNA is released from macroeukaryotes and can be filtered from water and metabarcoded in a similar manner as eDNA to reliably provide species composition information. eRNA had a small but significantly greater true positive rate than eDNA, indicating that it correctly detects more species known to exist in the lakes. Given relatively small differences between the two molecules in describing fish community composition, we conclude that if eRNA provides significant advantages in terms of lability, it is a strong candidate to add to the suite of molecular monitoring tools.

Sampling was conducted at the IISD Experimental Lakes Area (IISD-ELA), a remote research and monitoring facility in north-western Ontario, Canada. We sampled two lakes in summer and autumn of 2017 and repeated the summer and autumn sampling in five lakes in 2018. There are 14 species of fish across all the study lakes (mean 8, range 6-10 species per lake). All lakes have overlapping community compositions, including Salvelinus namaycush, a cold-water top predator, in every lake. Water samples were taken at six depths, dispersed vertically throughout the water column at the deepest centre point of each lake. The sampling points were distributed at six evenly spaced intervals, but because the lakes were different depths, absolute measurements differ between the lakes. One 500 ml water sample was taken per depth (for a total of 6 samples per lake per season) using an electrical pump and Jayflex PVC tubing (Winnipeg Johnston Plastics, MB, Canada) secured to a weight. In total, 84 water samples were taken throughout the entire study (6 samples x 2 lake states x 7 lake replicates). Water was filtered onto 47 mm 0.7 μm pore GF/F filters using an electric vacuum pump and filtering manifold (Pall Corporation, ON, Canada). The filters were divided into two with scissors which had been bleached, rinsed and autoclaved. Half of each filter was immediately stored in screw-cap tubes at -20 ⁰C. The other half was preserved in 370μl RLT buffer (Qiagen) with 1% β-mercaptoethanol and then frozen at -20°C for RNA analysis. One negative control of 500 ml distilled water was stored in the cooler and filtered in the same way as the field samples for each lake. In total, 84 eDNA and 84 eRNA samples were taken across the entire study. Filters were shipped on dry ice to McGill University, Montréal for molecular analysis.

DNA was extracted from filters using the Qiagen Blood and Tissue kit following the manufacturer’s instructions except that 370μl of ATL buffer was added to the filter used in an initial overnight incubation step. After incubation, 325μl of fluid was transferred into an autoclaved microtube for the downstream extraction protocol. RNA was extracted from the filter halves preserved in RLT and β-mercaptoethanol. Extractions of eRNA were performed from the first half of the filter using the Qiagen RNEasy Mini kit with some modifications to accommodate the filter. Filters were vortexed for 20 seconds and centrifuged in the RLT/ β-mercaptoethanol buffer for 3 minutes at 14,000rpm. A total of 325μl of this buffer was mixed with 325μl ethanol and the rest of the procedure followed the kit protocol intended for extracting total RNA from animal cells. The eRNA was resuspended in two elutions of 30μl RNAse free water to give a final volume of 60μl.

To avoid DNA contamination in the eRNA samples, DNA was digested from 20µl eRNA extracts with the DNA-free™ DNA Removal Kit (ThermoFisher Scientific) following the manufacturer’s instructions and using 2µl DNase I Buffer, 1µl rDNase I and 2µl DNase Inactivation reagent. RNA samples were checked for residual contaminating DNA using PCR amplification using the MiFish-U primers tagged with Illumina sequencing adapters (Miya et al., 2015). These primers target a hypervariable region of the 12S rRNA locus (163-185bp in length). We used the following PCR chemistry: 7.4µl nuclease free water (Qiagen), 1.25µl 10X buffer (Genscript), 1 mM MgCl2 (ThermoFisher Scientific), 0.2mM GeneDirex dNTPs, 0.05mg bovine serum albumen (ThermoFisher Scientific), 0.25mM each primer, 1U taq (Genscript) and 2µl DNA in a final volume of 12.5µl. We followed a touchdown thermocycling protocol which we have found reduces the amount of non-specific amplification (bacterial taxa) at this locus: 95°C for 3 minutes, 12 cycles of touchdown PCR (98°C for 20 seconds, 66°C for 15 seconds decreasing by 0.2°C each time, 72°C for 15 seconds) followed by 28 cycles with an annealing temperature of 64°C, 72°C extension for 5 minutes. We then visualised the resulting PCR products on 1% agarose gels stained with SYBR Safe. No residual contaminating DNA was found in eRNA samples. A total of 10μl of sample was therefore reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription kit (ThermoFisher Scientific) in 20µl reactions following the kit instructions.

We then amplified the cDNA and eDNA in triplicate 12.5µl reactions following the MiFish-U PCR protocol laid out above, and checked amplification using 1% agarose gels with SYBR Safe. We then combined the triplicate reactions into one sample and performed a cleanup with AMPure beads using a ratio of 1 part PCR product to 1.2 parts beads. Cleaned amplicons were then dual-indexed using the Nextera v2 DNA indexes, and cleaned again with a ratio of 1 part PCR product to 0.8 parts beads. The second clean-up was performed using a Biomek FX Laboratory liquid handling Automation Workstation (Beckman Coulter, CA). Samples were equimolarised to 3ng/μl and sent for sequencing at Génome Québec, Montréal. Samples were sequenced using 2 x 250bp paired end sequencing with an Illumina MiSeq.

We used custom scripts to remove adapters, merge paired sequences, check quality and generate amplicon sequencing variants (ASVs). Samples were received as demultiplexed fastq files from Génome Québec. Non-biological nucleotides were removed (primers, indices and adapters) using cutadapt. Paired reads were merged using PEAR. Quality scores for sequences were analysed with FASTQC. Amplicon sequencing variants (ASVs) were generated using the UNOISE3 package, which uses a denoising pipeline to remove sequencing error and to cluster sequences into single variants (100% similarity). The full pipeline is available from https://github.com/CristescuLab/YAAP. After ASVs were generated, we assigned taxonomy using BLAST+ and BASTA, a last common ancestor algorithm. We used a custom reference database which contained only fish known to exist in the Lake of the Woods region (Ontario, CA), downloaded from the NCBI database on 12 August 2018.

See ReadMe file.