Assessing distribution of Didemnum vexillum in Scotland using environmental DNA

Materials and Methods

Water sampling and DNA extraction

The survey included 24 sites in the Loch Creran and the wider Lynn of Lorn (including Loch Linnhe and Loch Etive) and 34 sites in the wider Firth of Clyde areas. At each site, ten 0.5 L water samples (within the top 50 cm of the water column) were collected using individual sterile Gosselin HDPE plastic bottles (Fisher Scientific) during August to October 2019 (Supplementary Table S1). At each site, two sampling points were selected and five replicate water samples were collected from each point respectively. Water samples were filtered on site after collection, using sterile 50 mL syringes (Fisher Scientific) and 0.22 µm Sterivex filters (Merck Milipore). Each day one blank “in field” filtering control, consisting of 0.5 L of sterile seawater (collected from an offshore location and autoclaved in the laboratory), was processed alongside the field samples. Filters were kept on ice in dark, inside an insulated polystyrene box, and stored at −20ºC within 12–24 h of collection.

Environmental DNA was extracted from filters using DNeasy Blood and Tissue extraction kit (Qiagen) following the protocol described by Spens et al. (2017). DNA was eluted in 100 µL of AE buffer (Qiagen) and stored at −80 ^oC. A negative extraction control, containing the same extraction reagents as the field and blank samples, was included in each extraction batch.

Detection of Didemnum vexillum eDNA using quantitative real-time PCR (qPCR)

To detect D. vexillum eDNA in collected water samples, a species-specific real time PCR (qPCR) assay and relevant methodologies described in Matejusova et al. (2021) were applied. Reactions were prepared in a UV-cabinet located in a dedicated, positive-pressure clean room. The DNA template was added in a UV-cabinet located in a separate pre-PCR room. Cabinets and all pipettes were cleaned before and after each use with 1% sodium hypochlorite solution (Fisher Scientific), followed by 10% Microsol 4 decontaminant (Mettler Toledo, Anachem). Field samples, blank field controls and extraction controls were run in triplicates on the QuantStudio 5 Real time PCR System (Applied Biosystems). Each plate also contained a minimum of three non-target controls (NTC). Presence of PCR inhibition in the DNA extracted from water samples was tested on 2 ml of DNA using Exogenous Internal Positive Control Reagents (Applied Biosystems).

To calculate a copy number of D. vexillum target in the field samples, a standard curve, generated using the synthetic construct (www.amsbio.com) containing D. vexillum qPCR amplicon, was prepared. The standard curve range spanned between 5.548 × 10⁵ and 0.555 copies reaction⁻¹ and the target was amplified in triplicate for each concentration alongside samples and controls. The mean copy number of the target was calculated for all water samples showing a positive amplification of D. vexillum eDNA (Ct<41) in at least two out of the three technical replicates (Wilcox et al., 2013; Agersnap et al., 2017). At the sampling site, D. vexillum status, inferred from the eDNA amplified, was classified as: (1) detected, (2) suspected and (3) not detected (see Matejusova et al., 2021 for more details). An additional status category, (4) inconclusive, was considered for sampling sites where multiple water samples showed an amplification of the target species eDNA in only one out of three technical replicates respectively. The mean copy number of target reaction^-1 was calculated using the R package “ednar”¹ (https://github.com/alexd106/ednar).

Mapping

Maps of the sampled sites were generated using QGIS software version 3.20.2 (QGIS.org, 2021). Latitude and longitude point data of D. vexillum eDNA detection were transformed to Europe Albers Equal Area Conic coordinates (ESRI: 102013) and plotted onto a 1:25000 scale layer of the Scottish coastline Map data: Land Cover for Scotland Data (Macaulay Land Use Research Institute, 1993). Labels were edited for clarity using Inkscape software version 0.92 (Inkscape Project, 2020).